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  • 1.
    Andersson, Dan I.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Biological roles of translesion synthesis DNA polymerases in eubacteria2010In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 77, no 3, p. 540-548Article, review/survey (Refereed)
    Abstract [en]

    Biological systems are strongly selected to maintain the integrity of their genomes by prevention and repair of external and internal DNA damages. However, some types of DNA lesions persist and might block the replication apparatus. The universal existence of specialized translesion synthesis DNA polymerases (TLS polymerases) that can bypass such lesions in DNA implies that replication blockage is a general biological problem. We suggest that the primary function for which translesion synthesis polymerases are selected is to rescue cells from replication arrest at lesions in DNA, a situation that, if not amended, is likely to cause an immediate and severe reduction in cell fitness and survival. We will argue that the mutagenesis observed during translesion synthesis is an unavoidable secondary consequence of this primary function and not, as has been suggested, an evolved mechanism to increase mutation rates in response to various stresses. Finally, we will discuss recent data on additional roles for translesion synthesis polymerases in the formation of spontaneous deletions and in transcription-coupled TLS, where the coupling of transcription to TLS is proposed to allow the rescue of the transcription machinery arrested at DNA lesions.

  • 2.
    Ausmees, Nora
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wahlstedt, Helene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Bagchi, Sonchita
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Elliot, Marie A.
    Buttner, Mark J.
    Flärdh, Klas
    SmeA, a small membrane protein with multiple functions in Streptomyces sporulation including targeting of a SpoIIIE/FtsK-like protein to cell division septa2007In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 65, no 6, p. 1458-1473Article in journal (Refereed)
    Abstract [en]

    Sporulation in aerial hyphae of Streptomyces coelicolor involves profound changes in regulation of fundamental morphogenetic and cell cycle processes to convert the filamentous and multinucleoid cells to small unigenomic spores. Here, a novel sporulation locus consisting of smeA (encoding a small putative membrane protein) and sffA (encoding a SpoIIIE/FtsK-family protein) is characterized. Deletion of smeA-sffA gave rise to pleiotropic effects on spore maturation, and influenced the segregation of chromosomes and placement of septa during sporulation. Both smeA and sffA were expressed specifically in apical cells of sporogenic aerial hyphae simultaneously with or slightly after Z-ring assembly. The presence of smeA-like genes in streptomycete chromosomes, plasmids and transposons, often paired with a gene for a SpoIIIE/FtsK- or Tra-like protein, indicates that SmeA and SffA functions might be related to DNA transfer. During spore development SffA accumulated specifically at sporulation septa where it colocalized with FtsK. However, sffA did not show redundancy with ftsK, and SffA function appeared distinct from the DNA translocase activity displayed by FtsK during closure of sporulation septa. The septal localization of SffA was dependent on SmeA, suggesting that SmeA may act as an assembly factor for SffA and possibly other proteins required during spore maturation.

  • 3.
    Bagchi, Sonchita
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Tomenius, Henrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Belova, Lyubov M.
    Ausmees, Nora
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Intermediate filament-like proteins in bacteria and a cytoskeletal function in Streptomyces2008In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 70, no 4, p. 1037-1050Article in journal (Refereed)
    Abstract [en]

    Actin and tubulin cytoskeletons are conserved and widespread in bacteria. A strikingly intermediate filament (IF)-like cytoskeleton, composed of crescentin, is also present in Caulobacter crescentus and determines its specific cell shape. However, the broader significance of this finding remained obscure, because crescentin appeared to be unique to Caulobacter. Here we demonstrate that IF-like function is probably a more widespread phenomenon in bacteria. First, we show that 21 genomes of 26 phylogenetically diverse species encoded uncharacterized proteins with a central segmented coiled coil rod domain, which we regarded as a key structural feature of IF proteins and crescentin. Experimental studies of three in silico predicted candidates from Mycobacterium and other actinomycetes revealed a common IF-like property to spontaneously assemble into filaments in vitro. Furthermore, the IF-like protein FilP formed cytoskeletal structures in the model actinomycete Streptomyces coelicolor and was needed for normal growth and morphogenesis. Atomic force microscopy of living cells revealed that the FilP cytoskeleton contributed to mechanical fitness of the hyphae, thus closely resembling the function of metazoan IF. Together, the bioinformatic and experimental data suggest that an IF-like protein architecture is a versatile design that is generally present in bacteria and utilized to perform diverse cytoskeletal tasks.

  • 4.
    Berghoff, Bork A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Justus Liebig Univ, Inst Mikrobiol & Mol Biol, D-35392 Giessen, Germany..
    Hoekzema, Mirthe
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Aulbach, Lena
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wagner, Gerhart E. H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Two regulatory RNA elements affect TisB-dependent depolarization and persister formation2017In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 103, no 6, p. 1020-1033Article in journal (Refereed)
    Abstract [en]

    Bacterial survival strategies involve phenotypic diversity which is generated by regulatory factors and noisy expression of effector proteins. The question of how bacteria exploit regulatory RNAs to make decisions between phenotypes is central to a general understanding of these universal regulators. We investigated the TisB/IstR-1 toxin-antitoxin system of Escherichia coli to appreciate the role of the RNA antitoxin IstR-1 in TisB-dependent depolarization of the inner membrane and persister formation. Persisters are phenotypic variants that have become transiently drug-tolerant by arresting growth. The RNA antitoxin IstR-1 sets a threshold for TisB-dependent depolarization under DNA-damaging conditions, resulting in two sub-populations: polarized and depolarized cells. Furthermore, our data indicate that an inhibitory 5 UTR structure in the tisB mRNA serves as a regulatory RNA element that delays TisB translation to avoid inappropriate depolarization when DNA damage is low. Investigation of the persister sub-population further revealed that both regulatory RNA elements affect persister levels as well as persistence time. This work provides an intriguing example of how bacteria exploit regulatory RNAs to control phenotypic heterogeneity.

  • 5.
    Bernander, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    The cell cycle of Sulfolobus2007In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 66, no 3, p. 557-562Article, review/survey (Refereed)
    Abstract [en]

    Much of the current information about the archaeal cell cycle has been generated through studies of the genus Sulfolobus. The overall organization of the cell cycle in these species is well understood, and information about the regulatory principles that govern cell cycle progression is rapidly accumulating. Exciting progress regarding the control and molecular details of the chromosome replication process is evident, and the first insights into the elusive crenarchaeal mitosis and cytokinesis machineries are within reach.

  • 6.
    Björkman, Johanna
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Samuelsson, Patrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium1999In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 31, no 1, p. 53-58Article in journal (Other (popular science, discussion, etc.))
    Abstract [en]

    Many mutations in rpsL cause resistance to, or dependence on, streptomycin and are restrictive (hyperaccurate) in translation. Dependence on streptomycin and hyperaccuracy can each be reversed phenotypically by mutations in either rpsD or rpsE. Such compensatory mutations have been shown to have a ram phenotype (ribosomal ambiguity), increasing the level of translational errors. We have shown recently that restrictive rpsL alleles are also associated with a loss of virulence in Salmonella typhimurium. To test whether ram mutants could reverse this loss of virulence, we have isolated a set of rpsD alleles in Salmonella typhimurium. We found that the rpsD alleles restore the virulence of strains carrying restrictive rpsL alleles to a level close to that of the wild type. Unexpectedly, three out of seven mutant rpsD alleles tested have phenotypes typical of restrictive alleles of rpsL, being resistant to streptomycin and restrictive (hyperaccurate) in translation. These phenotypes have not been previously associated with the ribosomal protein S4. Furthermore, all seven rpsD alleles (four ram and three restrictive) can phenotypically reverse the hyperaccuracy associated with restrictive alleles of rpsL. This is the first demonstration that such compensations do not require that the compensating rpsD allele has a ribosomal ambiguity (ram) phenotype.

  • 7. Bouet, Jean-Yves
    et al.
    Nordström, Kurt
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Lane, David
    Plasmid partition and incompatibility: the focus shifts2007In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 65, no 6, p. 1405-1414Article, review/survey (Refereed)
    Abstract [en]

    The mitotic apparatus that a plasmid uses to ensure its stable inheritance responds to the appearance of an additional copy of the plasmid's centromere by segregating it from the pre-existing copies: if the new copy arises by replication of the plasmid the result is partition, if it arrives on a different plasmid the result is incompatibility. Incompatibility thus serves as a probe of the partition mechanism. Coupling of distinct plasmids via their shared centromeres to form mixed pairs has been the favoured explanation for centromere-based incompatibility, because it supports a long-standing assumption that pairing of plasmid replicas is a prerequisite for their partition into daughter cells. Recent results from molecular genetic and fluorescence microscopy studies challenge this mixed pairing model. Partition incompatibility is seen to result from various processes, including titration, randomized positioning and a form of mixed pairing that is based on co-activation of the same partition event rather than direct contact between partition complexes. The perspectives thus opened onto the partition mechanism confirm the continuing utility of incompatibility as an approach to understanding bacterial mitosis. The results considered are compatible with the view that direct pairing of plasmids is not essential to plasmid partition.

  • 8.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bergman, Jessica M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Autoregulation of the tufB operon in Salmonella2016In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 100, no 6, p. 1004-1016Article in journal (Refereed)
    Abstract [en]

    In Salmonella enterica and related species, translation elongation factor EF-Tu is encoded by two widely separated but near-identical genes, tufA and tufB. Two thirds of EF-Tu is expressed from tufA with the remaining one third coming from tufB. Inactivation of tufA is partly compensated by a doubling in the amount of EF-TuB but the mechanism of this up-regulation is unknown. By experimental evolution selecting for improved growth rate in a strain with an inactive tufA we selected six different noncoding or synonymous point mutations close to the tufB start codon. Based on these results we constructed a total of 161 different point mutations around the tufB start codon, as well as tufB 3'-truncations, and measured tufB expression using tufB-yfp transcriptional and translational fusions. The expression data support the presence of two competing stem-loop structures that can form in the 5'-end of the tufB mRNA. Formation of the 'closed' structure leads to Rho-dependent transcriptional termination of the tufB mRNA. We propose a model in which translational speed is used as a sensor for EF-Tu concentration and where the expression of tufB is post-transcriptionally regulated. This model describes for the first time how expression of the most abundant Salmonella protein is autoregulated.

  • 9.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Co-evolution with recombination affects the stability of mobile genetic element insertions within gene families of Salmonella2018In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 108, no 6, p. 697-710Article in journal (Refereed)
    Abstract [en]

    Bacteria can have multiple copies of a gene at separate locations on the same chromosome. Some of these gene families, including tuf (translation elongation factor EF-Tu) and rrl (ribosomal RNA), encode functions critically important for bacterial fitness. Genes within these families are known to evolve in concert using homologous recombination to transfer genetic information from one gene to another. This mechanism can counteract the detrimental effects of nucleotide sequence divergence over time. Whether such mechanisms can also protect against the potentially lethal effects of mobile genetic element insertion is not well understood. To address this we constructed two different length insertion cassettes to mimic mobile genetic elements and inserted these into various positions of the tuf and rrl genes. Wemeasured rates of recombinational repair that removed the inserted cassette and studied the underlying mechanism. Our results indicate that homologous recombination can protect the tuf and rrl genes from inactivation by mobile genetic elements, but forinsertions within shorter gene sequences the efficiency of repair is very low. Intriguingly, we found that physical distance separating genes on the chromosome directly affects the rate of recombinational repair suggesting that relative location will influence the ability of homologous recombination to maintain homogeneity.

  • 10.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wrande, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Liljas, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Fitness-compensatory mutations in rifampicin-resistant RNA polymerase2012In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 85, no 1, p. 142-151Article in journal (Refereed)
    Abstract [en]

    Mutations in rpoB (RNA polymerase beta-subunit) can cause high-level resistance to rifampicin, an important first-line drug against tuberculosis. Most rifampicin-resistant (RifR) mutants selected in vitro have reduced fitness, and resistant clinical isolates of M. tuberculosis frequently carry multiple mutations in RNA polymerase genes. This supports a role for compensatory evolution in global epidemics of drug-resistant tuberculosis but the significance of secondary mutations outside rpoB has not been demonstrated or quantified. Using Salmonella as a model organism, and a previously characterized RifR mutation (rpoB R529C) as a starting point, independent lineages were evolved with selection for improved growth in the presence and absence of rifampicin. Compensatory mutations were identified in every lineage and were distributed between rpoA, rpoB and rpoC. Resistance was maintained in all strains showing that increased fitness by compensatory mutation was more likely than reversion. Genetic reconstructions demonstrated that the secondary mutations were responsible for increasing growth rate. Many of the compensatory mutations in rpoA and rpoC individually caused small but significant reductions in susceptibility to rifampicin, and some compensatory mutations in rpoB individually caused high-level resistance. These findings show that mutations in different components of RNA polymerase are responsible for fitness compensation of a RifR mutant. 

  • 11. Dorazi, Robert
    et al.
    Götz, Dorothee
    Munro, Stacey
    Bernander, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    White, Malcolm F.
    Equal rates of repair of DNA photoproducts in transcribed and non-transcribed strands in Sulfolobus solfataricus2007In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 63, no 2, p. 521-529Article in journal (Refereed)
    Abstract [en]

    The nucleotide excision repair (NER) pathway removes bulky lesions such as photoproducts from DNA. In both bacteria and eukarya, lesions located in transcribed strands are repaired significantly faster than those located in non-transcribed strands due to damage signalling by stalled RNA polymerase molecules: a phenomenon known as transcription-coupled repair (TCR). TCR requires a mechanism for coupling the detection of stalled RNA polymerase molecules to the NER pathway, provided in bacteria by the Mfd protein. In the third domain of life, archaea, the pathway of NER is not well defined, there are no Mfd homologues and the existence of TCR has not been investigated. In this report we looked at rates of removal of photoproducts in three different operons of the crenarchaeon Sulfolobus solfataricus following UV irradiation. We found no evidence for significantly faster repair in the transcribed strands of these three operons. The rate of global genome repair in S. solfataricus is relatively rapid, and this may obviate the requirement for a specialized TCR pathway. Significantly faster repair kinetics were observed in the presence of visible light, consistent with the presence of a gene for photolyase in the genome of S. solfataricus.

  • 12.
    Ettema, Thijs J. G.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Lindas, Ann-Christin
    Hjort, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Poplawski, Andrzej B.
    Kaessmann, Henrik
    Grogan, Dennis W.
    Kelman, Zvi
    Andersson, Anders F.
    Pelve, Erik A.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Rolf Bernander (1956-2014): pioneer of the archaeal cell cycle Obituary2014In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 92, no 5, p. 903-909Article in journal (Refereed)
    Abstract [en]

    On 19 January 2014 Rolf (Roffe') Bernander passed away unexpectedly. Rolf was a dedicated scientist; his research aimed at unravelling the cell biology of the archaeal domain of life, especially cell cycle-related questions, but he also made important contributions in other areas of microbiology. Rolf had a professor position in the Molecular Evolution programme at Uppsala University, Sweden for about 8 years, and in January 2013 he became chair professor at the Department of Molecular Biosciences, The Wenner-Gren Institute at Stockholm University in Sweden. Rolf was an exceptional colleague and will be deeply missed by his family and friends, and the colleagues and co-workers that he leaves behind in the scientific community. He will be remembered for his endless enthusiasm for science, his analytical mind, and his quirky sense of humour.

  • 13.
    Ettema, Thijs J. G.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Molecular Evolution.
    Lindås, Ann-Christin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Molecular Evolution.
    Bernander, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Molecular Evolution.
    An actin-based cytoskeleton in archaea2011In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 80, no 4, p. 1052-1061Article in journal (Refereed)
    Abstract [en]

    In eukaryotic and bacterial cells, spatial organization is dependent upon cytoskeletal filaments. Actin is a main eukaryotic cytoskeletal element, involved in key processes such as cell shape determination, mechanical force generation and cytokinesis. We describe an archaeal cytoskeleton which forms helical structures within Pyrobaculum calidifontis cells, as shown by in situ immunostaining. The core components include an archaeal actin homologue, Crenactin, closely related to the eukaryotic counterpart. The crenactin gene belongs to a conserved gene cluster denoted Arcade (actin-related cytoskeleton in Archaea involved in shape determination). The phylogenetic distribution of arcade genes is restricted to the crenarchaeal Thermoproteales lineage, and to Korarchaeota, and correlates with rod-shaped and filamentous cell morphologies. Whereas Arcadin-1, -3 and -4 form helical structures, suggesting cytoskeleton-associated functions, Arcadin-2 was found to be localized between segregated nucleoids in a cell subpopulation, in agreement with possible involvement in cytokinesis. The results support a crenarchaeal origin of the eukaryotic actin cytoskeleton and, as such, have implications for theories concerning the origin of the eukaryotic cell.

  • 14.
    Hammarlöf, Disa L
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mutants of the RNA-processing enzyme RNase E reverse the extreme slow-growth phenotype caused by a mutant translation factor EF-Tu2008In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 70, no 5, p. 1194-1209Article in journal (Refereed)
    Abstract [en]

    Salmonella enterica with mutant EF-Tu (Gln125Arg) has a low level of EF-Tu, a reduced rate of protein synthesis and an extremely slow growth rate. Eighty independent suppressor mutations were selected that restored normal growth. In some cases (n = 7) suppression was due to mutations in tufA but, surprisingly, in most cases (n = 73) to mutations in rne, the gene coding for RNase E. These rne mutations alone had only modest effects on growth rate. Fifty different suppressor mutations were isolated in rne, all located in or close to the N-terminal endonucleolytic half of RNase E. Steady state levels of several mRNAs were lower in the mutant tuf strain but restored to wild-type levels in the tuf-rne double mutant. In contrast, the half-lives of mRNAs were unaffected by the tuf mutation. We propose a model where the tuf mutation causes the ribosome following RNA polymerase to pause, possibly in a codon-specific manner, exposing unshielded nascent message to RNase E cleavage. Normal growth rate can be restored by increasing EF-Tu activity or by reducing RNase E activity. Accordingly, RNase E is suggested to act at two distinct stages in the life of mRNA: early, on the nascent transcript; late, on the complete mRNA.

  • 15. He, Lin
    et al.
    Söderbom, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wagner, Gerhart
    Binnie, Uta
    Binns, Nigel
    Masters, Millicent
    PcnB is required for the rapid degradation of RNAI, the antisense RNA that controls the copy number of ColE1-related plasmids1993In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 9, no 6, p. 1131-1142Article in journal (Refereed)
    Abstract [en]

    The replication of ColE1-related plasmids is controlled by an unstable antisense RNA, RNAI, which can interfere with the successful processing of the RNAII primer of replication. We show here that a host protein, PcnB, supports replication by promoting the decay of RNAI. In bacterial strains deleted for PcnB a stable, active form of RNAI, RNAI*, which appears to be identical to the product of 5'-end processing by RNAase E, accumulates. This leads to a reduction in plasmid copy number. We show, using a GST-PcnB fusion protein, that PcnB does not interfere with RNAI/RNAII binding in vitro. The fusion protein, like PcnB, has polyadenylating activity and is able to polyadenylate RNAI (and also another antisense RNA, CopA) in vitro.

  • 16. Hjort, K
    et al.
    Bernander, R
    Cell cycle regulation in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius.2001In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 40, no 1, p. 225-34Article in journal (Refereed)
    Abstract [en]

    The regulation and co-ordination of the cell cycle of the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius was investigated with antibiotics. We provide evidence for a core regulation involving alternating rounds of chromosome replication and genome segregation. In contrast, multiple rounds of replication of the chromosome could occur in the absence of an intervening cell division event. Inhibition of the elongation stage of chromosome replication resulted in cell division arrest, indicating that pathways similar to checkpoint mechanisms in eukaryotes, and the SOS system of bacteria, also exist in archaea. Several antibiotics induced cell cycle arrest in the G2 stage. Analysis of the run-out kinetics of chromosome replication during the treatments allowed estimation of the minimal rate of replication fork movement in vivo to 250 bp s-1. An efficient method for the production of synchronized Sulfolobus populations by transient daunomycin treatment is presented, providing opportunities for studies of cell cycle-specific events. Possible targets for the antibiotics are discussed, including topoisomerases and protein glycosylation.

  • 17.
    Hjort, Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Nicoloff, Hervé
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Unstable tandem gene amplification generates heteroresistance (variation in resistance within a population) to colistin in Salmonella enterica2016In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 102, no 2, p. 274-289Article in journal (Refereed)
    Abstract [en]

    Heteroresistance, a phenomenon where subpopulations of a bacterial isolate exhibit different susceptibilities to an antibiotic, is a growing clinical problem where the underlying genetic mechanisms in most cases remain unknown. We isolated colistin resistant mutants in Escherichia coli and Salmonella enterica serovar Typhimurium at different concentrations of colistin. Genetic analysis showed that genetically stable pmrAB point mutations were responsible for colistin resistance during selection at high drug concentrations for both species and at low concentrations for E. coli. In contrast, for S. Typhimurium mutants selected at low colistin concentrations, amplification of different large chromosomal regions conferred a heteroresistant phenotype. All amplifications included the pmrD gene, which encodes a positive regulator that up-regulates proteins that modify lipid A, and as a result increase colistin resistance. Inactivation and over-expression of the pmrD gene prevented and conferred resistance, respectively, demonstrating that the PmrD protein is required and sufficient to confer resistance. The heteroresistance phenotype is explained by the variable gene dosage of pmrD in a population, where sub-populations with different copy number of the pmrD gene show different levels of colistin resistance. We propose that variability in gene copy number of resistance genes can explain the heteroresistance observed in clinically isolated pathogenic bacteria.

  • 18.
    Holmqvist, Erik
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Unoson, Cecilia
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Reimegård, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wagner, Gerhart E. H.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    A mixed double negative feedback loop between the sRNA MicF and the global regulator Lrp2012In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 84, no 3, p. 414-427Article in journal (Refereed)
    Abstract [en]

    Roughly 10% of all genes in Escherichia coli are controlled by the global transcription factor Lrp, which responds to nutrient availability. Bioinformatically, we identified lrp as one of several putative targets for the sRNA MicF, which is transcriptionally downregulated by Lrp. Deleting micF results in higher Lrp levels, while overexpression of MicF inhibits Lrp synthesis. This effect is by antisense; mutations in the predicted interaction region relieve MicF-dependent repression of Lrp synthesis, and regulation is restored by compensatory mutations. In vitro, MicF sterically interferes with initiation complex formation and inhibits lrp mRNA translation. In vivo, MicF indirectly activates genes in the Lrp regulon by repressing Lrp, and causes severely impaired growth in minimal medium, a phenotype characteristic of lrp deletion strains. The double negative feedback between MicF and Lrp may promote a switch for adequate Lrp-dependent adaptation to nutrient availability. Lrp adds to the growing list of transcription factors that are targeted by sRNAs, thus indicating that perhaps the majority of all bacterial genes may be directly or indirectly controlled by sRNAs.

  • 19.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Error-prone EF-Tu reduces in vivo enzyme activity and cellular growth rate1991In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 5, no 3, p. 623-630Article in journal (Refereed)
    Abstract [en]

    Mutations in Salmonella typhimurium encoding error-prone EF-Tu (tufA8, tufB103) enhance translational error levels and also cause a reduced growth rate. The relative changes in error level and growth rate are inversely related and dependent on the status of the two tuf genes. Possible causes of the reduced growth rate were investigated. Several important parameters with the potential to alter growth rate (the EF-Tu-ribosome interaction, the in vivo elongation rate and the processivity of translation), are all relatively unaffected by the tuf mutations. The small reduction in processivity observed in some strains is not quantitatively related to the growth rate reduction. Instead, the error-enhancing mutations are associated with a large reduction in the specific activity of a test protein, β-galactosidase, suggesting by inference that the reduced growth rate is a consequence of the synthesis of error-containing proteins.

  • 20.
    Kirsebom, Leif A
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    RNase P: a 'Scarlet Pimpernel'1995In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 17, no 3, p. 411-420Article, review/survey (Refereed)
    Abstract [en]

    RNase P is responsible for the maturation of the 5'-termini of tRNA molecules in all cells studied to date. This ribonucleoprotein has to recognize and identify its cleavage site on a large number of different precursors. This review covers what is currently known about the function of the catalytic subunit of Escherichia coli RNase P, M1 RNA, and the protein subunit, C5, in particular with respect to cleavage-site selection. Recent genetic and biochemical data show that the two C residues in the 3'-terminal CCA sequence of a precursor interact with the enzyme through Watson-Crick base-pairing. This is suggested to result in unfolding of the amino acid acceptor-stem and exposure of the cleavage site. Furthermore, other close contact points between M1 RNA and its substrate have recently been identified. These data, together with the two existing three-dimensional structure models of M1 RNA in complex with its substrate, establish a platform that will enable us to seek an understanding of the underlying mechanism of cleavage by this elusive enzyme.

  • 21.
    Koshla, Oksana
    et al.
    Ivan Franko Natl Univ Lviv, Dept Genet & Biotechnol, 4 Hrushevskoho St, UA-79005 Lvov, Ukraine.
    Yushchuk, Oleksandr
    Ivan Franko Natl Univ Lviv, Dept Genet & Biotechnol, 4 Hrushevskoho St, UA-79005 Lvov, Ukraine.
    Stash, Iryna
    Ivan Franko Natl Univ Lviv, Dept Genet & Biotechnol, 4 Hrushevskoho St, UA-79005 Lvov, Ukraine.
    Dacyuk, Yuriy
    Ivan Franko Natl Univ Lviv, Dept Phys Earth, 4 Hrushevskoho St, UA-79005 Lvov, Ukraine.
    Myronovskyi, Maksym
    Helmholtz Inst Pharmaceut Res, Saarland Campus,Bldg C2-3, D-66123 Saarbrucken, Germany.
    Jager, Gunilla
    Umea Univ, Dept Mol Biol, 6K Och 6L, S-90197 Umea, Sweden.
    Sussmuth, Roderich D.
    Tech Univ Berlin, Inst Chem, Str 17 Juni 124-TC2, D-10623 Berlin, Germany.
    Luzhetskyy, Andriy
    Helmholtz Inst Pharmaceut Res, Saarland Campus,Bldg C2-3, D-66123 Saarbrucken, Germany.
    Bystrom, Anders
    Umea Univ, Dept Mol Biol, 6K Och 6L, S-90197 Umea, Sweden.
    Kirsebom, Leif
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ostash, Bohdan
    Ivan Franko Natl Univ Lviv, Dept Genet & Biotechnol, 4 Hrushevskoho St, UA-79005 Lvov, Ukraine.
    Gene miaA for post-transcriptional modification of tRNAXXA is important for morphological and metabolic differentiation in Streptomyces2019In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 112, no 1, p. 249-265Article in journal (Refereed)
    Abstract [en]

    Members of actinobacterial genus Streptomyces possess a sophisticated life cycle and are the deepest source of bioactive secondary metabolites. Although morphogenesis and secondary metabolism are subject to transcriptional co-regulation, streptomycetes employ an additional mechanism to initiate the aforementioned processes. This mechanism is based on delayed translation of rare leucyl codon UUA by the only cognate tRNA(UAA)(Leu) (encoded by bldA). The bldA-based genetic switch is an extensively documented example of translational regulation in Streptomyces. Yet, after five decades since the discovery of bldA, factors that shape its function and peculiar conditionality remained elusive. Here we address the hypothesis that post-transcriptional tRNA modifications play a role in tRNA-based mechanisms of translational control in Streptomyces. Particularly, we studied two Streptomyces albus J1074 genes, XNR_1074 (miaA) and XNR_1078 (miaB), encoding tRNA (adenosine(37)-N6)-dimethylallyltransferase and tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase respectively. These enzymes produce, in a sequential manner, a hypermodified ms(2)i(6)A37 residue in most of the A36-A37-containing tRNAs. We show that miaB and especially miaA null mutant of S. albus possess altered morphogenesis and secondary metabolism. We provide genetic evidence that miaA deficiency impacts translational level of gene expression, most likely through impaired decoding of codons UXX and UUA in particular.

  • 22.
    Koskiniemi, Sanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pränting, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gullberg, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Näsvall, Joakim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Activation of cryptic aminoglycoside resistance in Salmonella enterica2011In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 80, no 6, p. 1464-1478Article in journal (Refereed)
    Abstract [en]

    Aminoglycoside resistance in bacteria can be acquired by several mechanisms, including drug modification, target alteration, reduced uptake and increased efflux. Here we demonstrate that increased resistance to the aminoglycosides streptomycin and spectinomycin in Salmonella enterica can be conferred by increased expression of an aminoglycoside adenyl transferase encoded by the cryptic, chromo-somally located aadA gene. During growth in rich medium the wild-type strain was susceptible but mutations that impaired electron transport and conferred a small colony variant (SCV) phenotype or growth in glucose/glycerol minimal media resulted in activation of the aadA gene and aminoglycoside resistance. Expression of the aadA gene was positively regulated by the stringent response regulator guanosine penta/tetraphosphate ((p) ppGpp). SCV mutants carrying stop codon mutations in the hemA and ubiA genes showed a streptomycin pseudo-dependent phenotype, where growth was stimulated by streptomycin. Our data suggest that this phenotype is due to streptomycin-induced readthrough of the stop codons, a resulting increase in HemA/UbiA levels and improved electron transport and growth. Our results demonstrate that environmental and mutational activation of a cryptic resistance gene can confer clinically significant resistance and that a streptomycin-pseudo-dependent phenotype can be generated via a novel mechanism that does not involve the classical rpsL mutations.

  • 23.
    Larsson Hammarlöf, Disa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Bergman, Jessica M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Garmendia, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Turnover of mRNAs is one of the essential functions of RNase E2015In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 98, no 1, p. 34-45Article in journal (Refereed)
    Abstract [en]

    RNase E is an essential bacterial endoribonuclease with a central role in processing tRNAs and rRNA, and turning over mRNAs. Previous studies in strains carrying mutations in the rne structural gene have shown that tRNA processing is likely to be an essential function of RNase E but have not determined whether mRNA turnover is also an essential function. To address this we selected extragenic suppressors of temperature-sensitive mutations in rne that cause a large increase in mRNA half-life at the non-permissive temperature. Fifteen suppressors were mapped to three different loci: relBE (toxin-antitoxin system); vacB (RNase R); and rpsA (ribosomal protein S1). Each suppressor class has the potential to interact with mRNA and each restores wild-type levels of mRNA turnover but does not reverse the minor defects in tRNA and rRNA processing. RelE toxin is especially interesting because its only known activity is to cleave mRNAs in the ribosomal A-site. The relBE suppressor mutations increase transcription of relE, and controlled overexpression of RelE alone was sufficient to suppress the rne ts phenotype. Suppression increased turnover of some major mRNAs (tufA, ompA) but not all mRNAs. We propose that turnover of some mRNAs is one of the essential functions of RNase E.

  • 24.
    Lind, Peter A
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Tobin, Christina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Berg, Otto G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Molecular Evolution.
    Kurland, Charles G
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Compensatory gene amplification restores fitness after inter-species gene replacements2010In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 75, no 5, p. 1078-1089Article in journal (Refereed)
    Abstract [en]

    Genes introduced by gene replacements and other types of horizontal gene transfer (HGT) represent a significant presence in many archaeal and eubacterial genomes. Most alien genes are likely to be neutral or deleterious upon arrival and their long-term persistence may require a mechanism that improves their selective contribution. To examine the fate of inter-species gene replacements, we exchanged three native S. typhimurium genes encoding ribosomal proteins with orthologues from various other microbes. The results show that replacement of each of these three genes reduces fitness to such an extent that it would provide an effective barrier against inter-species gene replacements in eubacterial populations. However, these fitness defects could be partially ameliorated by gene amplification that augmented the dosage of the heterologous proteins. This suggests that suboptimal expression is a common fitness constraint for inter-species gene replacements, with fitness costs conferred by either a lower expression level of the alien protein compared with the native protein or a requirement for an increased amount of the alien protein to maintain proper function. Our findings can explain the observation that duplicated genes are over-represented among horizontally transferred genes, and suggest a potential coupling between compensatory gene amplification after HGT and the evolution of new genes.

  • 25.
    Macvanin, Mirjana
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Johanson, Urban
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Fusidic acid-resistant EF-G perturbs the accumulation of ppGpp2000In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 37, no 1, p. 98-107Article in journal (Refereed)
    Abstract [en]

    Reductions in growth rate caused by fusidic acidresistantEF-G mutants in Salmonella typhimuriumcorrelate strongly with increased mean cell size. This isunusual because growth rate and cell size normallycorrelate positively. The global transcription regulatormoleculeppGpp has a role in co-ordinating growth rateand division, and its basal level normally correlatesinversely with cell size at division.Weshowthat fusidicacid-resistant EF-G mutants have perturbed ppGppbasal levels during steady-state growth and perturbedinduced levels during starvation. One mutation, fusA1,associatedwith the slowest growth rate and largest cellsize, causes a reduction in the basal level of ppGpp toone-third of that found in the wild-type strain. OtherfusA mutants with intermediate or wild-type growthrates and cell sizes have either normal or increasedbasal levels of ppGpp. There is an inverse relationshipbetween the basal level of ppGppin vivo and the degreeto which translation dependent on mutant EF-G isinhibited by ppGpp in vitro. This enhanced interactionbetween mutant EF-G and ppGpp correlates with anincreased KM for GTP. Our results suggest that mutantEF-G modulates the production of ppGpp by the RelA(PSI) pathway. In conclusion, fusidic acid-resistant EFGmutations alter the level of ppGpp and break thenormal relationship between growth rate and cell sizeat division. It would not be surprising if other phenotypesassociated with these mutants, such as loss ofvirulence, were also related to perturbations in ppGpplevels effected through altered transcription patterns.

  • 26. Mazza, P.
    et al.
    Noens, E. E.
    Schirner, K.
    Grantcharova, Nina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mommaas, A. M.
    Koerten, H. K.
    Muth, G.
    Flärdh, Klas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    van Wezel, G. P.
    Wohlleben, W.
    MreB of Streptomyces coelicolor is not essential for vegetative growth but is required for the integrity of aerial hyphae and spores2006In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 60, no 4, p. 838-852Article in journal (Refereed)
    Abstract [en]

    MreB forms a cytoskeleton in many rod-shaped bacteria which is involved in cell shape determination and chromosome segregation. PCR-based and Southern analysis of various actinomycetes, supported by analysis of genome sequences, revealed mreB homologues only in genera that form an aerial mycelium and sporulate. We analysed MreB in one such organism, Streptomyces coelicolor. Ectopic overexpression of mreB impaired growth, and caused swellings and lysis of hyphae. A null mutant with apparently normal vegetative growth was generated. However, aerial hyphae of this mutant were swelling and lysing; spores doubled their volume and lost their characteristic resistance to stress conditions. Loss of cell wall consistency was observed in MreB-depleted spores by transmission electron microscopy. An MreB-EGFP fusion was constructed to localize MreB in the mycelium. No clearly localized signal was seen in vegetative mycelium. However, strong fluorescence was observed at the septa of sporulating aerial hyphae, then as bipolar foci in young spores, and finally in a ring- or shell-like pattern inside the spores. Immunogold electron microscopy using MreB-specific antibodies revealed that MreB is located immediately underneath the internal spore wall. Thus, MreB is not essential for vegetative growth of S. coelicolor, but exerts its function in the formation of environmentally stable spores, and appears to primarily influence the assembly of the spore cell wall.

  • 27.
    Michalska, Karolina
    et al.
    Argonne Natl Lab, Midwest Ctr Struct Genom, Argonne, IL USA; Argonne Natl Lab, Struct Biol Ctr, Biosci Div, Argonne, IL USA.
    Nhan, Dinh Quan
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA USA.
    Willett, Julia L. E.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA USA; Univ Minnesota, Dept Microbiol & Immunol, Minneapolis, MN USA.
    Stols, Lucy M.
    Argonne Natl Lab, Midwest Ctr Struct Genom, Argonne, IL USA.
    Eschenfeldt, William H.
    Argonne Natl Lab, Midwest Ctr Struct Genom, Argonne, IL USA.
    Jones, Allison M.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA USA.
    Nguyen, Josephine Y.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA USA.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala Univ, Dept Cell & Mol Biol, Uppsala, Sweden.
    Low, David A.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA USA; Univ Calif Santa Barbara, Biomol Sci & Engn Program, Santa Barbara, CA USA.
    Goulding, Celia W.
    Univ Calif Irvine, Dept Mol Biol & Biochem, Irvine, CA USA; Univ Calif Irvine, Pharmaceut Sci, Irvine, CA USA.
    Joachimiak, Andrzej
    Argonne Natl Lab, Midwest Ctr Struct Genom, Argonne, IL USA; Argonne Natl Lab, Struct Biol Ctr, Biosci Div, Argonne, IL USA; Univ Chicago, Dept Biochem & Mol Biol, Chicago, IL USA.
    Hayes, Christopher S.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA USA; Univ Calif Santa Barbara, Biomol Sci & Engn Program, Santa Barbara, CA USA.
    Functional plasticity of antibacterial EndoU toxins2018In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 109, no 4, p. 509-527Article in journal (Refereed)
    Abstract [en]

    Bacteria use several different secretion systems to deliver toxic EndoU ribonucleases into neighboring cells. Here, we present the first structure of a prokaryotic EndoU toxin in complex with its cognate immunity protein. The contact‐dependent growth inhibition toxin CdiA‐CTSTECO31 from Escherichia coli STEC_O31 adopts the eukaryotic EndoU fold and shares greatest structural homology with the nuclease domain of coronavirus Nsp15. The toxin contains a canonical His‐His‐Lys catalytic triad in the same arrangement as eukaryotic EndoU domains, but lacks the uridylate‐specific ribonuclease activity that characterizes the superfamily. Comparative sequence analysis indicates that bacterial EndoU domains segregate into at least three major clades based on structural variations in the N‐terminal subdomain. Representative EndoU nucleases from clades I and II degrade tRNA molecules with little specificity. In contrast, CdiA‐CTSTECO31 and other clade III toxins are specific anticodon nucleases that cleave tRNAGlu between nucleotides C37 and m2A38. These findings suggest that the EndoU fold is a versatile scaffold for the evolution of novel substrate specificities. Such functional plasticity may account for the widespread use of EndoU effectors by diverse inter‐bacterial toxin delivery systems.

  • 28.
    Moraleda-Munoz, Aurelio
    et al.
    Univ Granada, Fac Ciencias, Dept Microbiol, Avda Fuentenueva S-N, E-18071 Granada, Spain.
    Marcos-Torres, Francisco Javier
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Univ Granada, Fac Ciencias, Dept Microbiol, Avda Fuentenueva S-N, E-18071 Granada, Spain.
    Perez, Juana
    Univ Granada, Fac Ciencias, Dept Microbiol, Avda Fuentenueva S-N, E-18071 Granada, Spain.
    Munoz-Dorado, Jose
    Univ Granada, Fac Ciencias, Dept Microbiol, Avda Fuentenueva S-N, E-18071 Granada, Spain.
    Metal-responsive RNA polymerase extracytoplasmic function (ECF) sigma factors2019In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 112, no 2, p. 385-398Article, review/survey (Refereed)
    Abstract [en]

    In order to survive, bacteria must adapt to multiple fluctuations in their environment, including coping with changes in metal concentrations. Many metals are essential for viability, since they act as cofactors of indispensable enzymes. But on the other hand, they are potentially toxic because they generate reactive oxygen species or displace other metals from proteins, turning them inactive. This dual effect of metals forces cells to maintain homeostasis using a variety of systems to import and export them. These systems are usually inducible, and their expression is regulated by metal sensors and signal-transduction mechanisms, one of which is mediated by extracytoplasmic function (ECF) sigma factors. In this review, we have focused on the metal-responsive ECF sigma factors, several of which are activated by iron depletion (FecI, FpvI and PvdS), while others are activated by excess of metals such as nickel and cobalt (CnrH), copper (CarQ and CorE) or cadmium and zinc (CorE2). We focus particularly on their physiological roles, mechanisms of action and signal transduction pathways.

  • 29.
    Nagaev, Ivan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Björkman, Johanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Andersson, Dan I
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Biological cost and compensatory evolution in fusidic acid-resistant Staphylococcus aureus2001In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 40, no 2, p. 433-439Article in journal (Refereed)
    Abstract [en]

    Fusidic acid resistance resulting from mutations in elongation factor G (EF-G) of Staphylococcus aureus is associated with fitness costs during growth in vivo and in vitro. In both environments, these costs can be partly or fully compensated by the acquisition of secondary intragenic mutations. Among clinical isolates of S. aureus, fusidic acid-resistant strains have been identified that carry multiple mutations in EF-G at positions similar to those shown experimentally to cause resistance and fitness compensation. This observation suggests that fitness-compensatory mutations may be an important aspect of the evolution of antibiotic resistance in the clinical environment, and may contribute to a stabilization of the resistant bacteria present in a bacterial population.

  • 30.
    Nicoloff, Hervé
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lon protease inactivation, or translocation of the lon gene, potentiate bacterial evolution to antibiotic resistance2013In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 90, no 6, p. 1233-1248Article in journal (Refereed)
    Abstract [en]

    Previous work demonstrated that selection for Escherichia coli mutants with low antibiotic resistance frequently resulted in co-selection of lon mutations and that lon(-) mutants evolved higher-level resistance faster than a lon(+) strain. Here we show that lon mutation causes a very low multidrug resistance by inducing the AcrAB-TolC pump via stabilization of the acrAB transcriptional activators MarA and SoxS, which are substrates of the Lon protease. Fast evolution of lon(-) mutants towards higher resistance involves selection of frequent next-step mutations consisting of large duplications including acrAB and the mutated lon gene. Resistance results from the combined effects of acrAB duplication and lon mutation increasing dosage of efflux pump. In contrast, when acrAB duplication occurs as the first step mutation, increased Lon activity caused by lon(+) co-duplication mitigates the effect of acrAB duplication on resistance, and faster evolution towards higher resistance is not observed. As predicted, when the functional lon gene is relocated far from acrAB to prevent their co-duplication, first-step acrAB duplication confers higher resistance, which then allows selection of frequent next-step mutations and results in faster evolution towards higher resistance. Our results demonstrate how order of appearance of mutations and gene location can influence the rate of resistance evolution.

  • 31.
    Olsson, Jan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Evolutionary Biology.
    Dasgupta, Santanu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Nordström, Kurt
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Eclipse period during replication of plasmid R1: Contributions from Structural events and from Copy-Number-control system2003In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 50, no 1, p. 291-301Article in journal (Refereed)
    Abstract [en]

    The eclipse period (the time period during which a newly replicated plasmid copy is not available for a new replication) of plasmid R1 in Escherichia coli was determined with the classic Meselson-Stahl density-shift experiment. A mini-plasmid with the wild-type R1 replicon and a mutant with a thermo-inducible runaway-replication phenotype were used in this work. The eclipses of the chromosome and of the wild-type plasmid were 0.6 and 0.2 generation times, respectively, at temperatures ranging from 30 degrees C to 42 degrees C. The mutant plasmid had a similar eclipse at temperatures up to 38 degrees C. At 42 degrees C, the plasmid copy number increased rapidly because of the absence of replication control and replication reached a rate of 350-400 plasmid replications per cell and cell generation. During uncontrolled replication, the eclipse was about 3 min compared with 10 min at controlled replication (the wild-type plasmid at 42 degrees C). Hence, the copy-number control system contributed significantly to the eclipse. The eclipse in the absence of copy-number control (3 min) presumably is caused by structural requirements: the covalently closed circular plasmid DNA has to regain the right degree of superhelicity needed for initiation of replication and it takes time to assemble the initiation factors.

  • 32.
    Paintdakhi, Ahmad
    et al.
    Yale Univ, Microbial Sci Inst, West Haven, CT 06516 USA.;Yale Univ, Howard Hughes Med Inst, New Haven, CT 06520 USA..
    Parry, Bradley
    Yale Univ, Microbial Sci Inst, West Haven, CT 06516 USA.;Yale Univ, Howard Hughes Med Inst, New Haven, CT 06520 USA.;Yale Univ, Dept Mol Cellular & Dev Biol, New Haven, CT 06520 USA..
    Campos, Manuel
    Yale Univ, Microbial Sci Inst, West Haven, CT 06516 USA.;Yale Univ, Howard Hughes Med Inst, New Haven, CT 06520 USA.;Yale Univ, Dept Mol Cellular & Dev Biol, New Haven, CT 06520 USA..
    Irnov, Irnov
    Yale Univ, Microbial Sci Inst, West Haven, CT 06516 USA.;Yale Univ, Howard Hughes Med Inst, New Haven, CT 06520 USA.;Yale Univ, Dept Mol Cellular & Dev Biol, New Haven, CT 06520 USA..
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Surovtsev, Ivan
    Yale Univ, Microbial Sci Inst, West Haven, CT 06516 USA.;Yale Univ, Howard Hughes Med Inst, New Haven, CT 06520 USA.;Yale Univ, Dept Mol Cellular & Dev Biol, New Haven, CT 06520 USA..
    Jacobs-Wagner, Christine
    Yale Univ, Microbial Sci Inst, West Haven, CT 06516 USA.;Yale Univ, Howard Hughes Med Inst, New Haven, CT 06520 USA.;Yale Univ, Dept Mol Cellular & Dev Biol, New Haven, CT 06520 USA.;Yale Univ, Sch Med, Dept Microbial Pathogenesis, New Haven, CT 06510 USA..
    Oufti: an integrated software package for high-accuracy, high-throughput quantitative microscopy analysis2016In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 99, no 4, p. 767-777Article in journal (Refereed)
    Abstract [en]

    With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re-emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single-cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand-alone, open-source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non-diffraction-limited fluorescence signals and is scalable for high-throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post-processing analysis, makes the software broadly accessible to users irrespective of their computational skills.

  • 33. Paulander, Wilhelm
    et al.
    Maisnier-Patin, Sophie
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Multiple mechanisms to ameliorate the fitness burden of mupirocin resistance in Salmonella typhimurium2007In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 64, no 4, p. 1038-1048Article in journal (Refereed)
    Abstract [en]

    We examined how the fitness costs of mupirocin resistance caused by mutations in the chromosomal isoleucyl–tRNA synthetase gene (ileS) can be ameliorated. Mupirocin-resistant mutants were isolated and four different, resistance-conferring point mutations in the chromosomal ileS gene were identified. Fifty independent lineages of the low-fitness, resistant mutants were serially passaged to evolve compensated mutants with increased fitness. In 34/50 of the evolved lineages, the increase in fitness resulted from additional point mutations in isoleucine tRNA synthetase (IleRS). Measurements in vitro of the kinetics of aminoacylation of wild-type and mutant enzymes showed that resistant IleRS had a reduced rate of aminoacylation due to altered interactions with both tRNAIle and ATP. The intragenic compensatory mutations improved IleRS kinetics towards the wild-type enzyme, thereby restoring bacterial fitness. Seven of the 16 lineages that lacked second-site compensatory mutations in ileS, showed an increase in ileS gene dosage, suggesting that an increased level of defective IleRS compensate for the decrease in aminoacylation activity. Our findings show that the fitness costs of ileS mutations conferring mupirocin resistance can be reduced by several types of mechanisms that may contribute to the stability of mupirocin resistance in clinical settings.

  • 34.
    Paulsson, Johan
    et al.
    Harvard Univ, Dept Syst Biol, Boston, MA 02115 USA..
    El Karoui, Meriem
    Harvard Univ, Dept Syst Biol, Boston, MA 02115 USA.;Univ Edinburgh, Inst Cell Biol, Sch Biol Sci, Edinburgh EH9 3JR, Midlothian, Scotland..
    Lindell, Monica
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The processive kinetics of gene conversion in bacteria2017In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 104, no 5, p. 752-760Article in journal (Refereed)
    Abstract [en]

    Gene conversion, non-reciprocal transfer from one homologous sequence to another, is a major force in evolutionary dynamics, promoting co-evolution in gene families and maintaining similarities between repeated genes. However, the properties of the transfer - where it initiates, how far it proceeds and how the resulting conversion tracts are affected by mismatch repair - are not well understood. Here, we use the duplicate tuf genes in Salmonella as a quantitatively tractable model system for gene conversion. We selected for conversion in multiple different positions of tuf, and examined the resulting distributions of conversion tracts in mismatch repair-deficient and mismatch repair-proficient strains. A simple stochastic model accounting for the essential steps of conversion showed excellent agreement with the data for all selection points using the same value of the conversion processivity, which is the only kinetic parameter of the model. The analysis suggests that gene conversion effectively initiates uniformly at any position within a tuf gene, and proceeds with an effectively uniform conversion processivity in either direction limited by the bounds of the gene.

  • 35.
    Pelve, Erik A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Lindås, Ann-Christin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Knöppel, Anna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Mira, Alex
    Bernander, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Four chromosome replication origins in the archaeon Pyrobaculum calidifontis2012In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 85, no 5, p. 986-995Article in journal (Refereed)
    Abstract [en]

    Replication origins were mapped in hyperthermophilic crenarchaea, using high-throughput sequencing-based marker frequency analysis. We confirm previous origin mapping in Sulfolobus acidocaldarius, and demonstrate that the single chromosome of Pyrobaculum calidifontis contains four replication origins, the highest number detected in a prokaryotic organism. The relative positions of the origins in both organisms coincided with regions enriched in highly conserved (core) archaeal genes. We show that core gene distribution provides a useful tool for origin identification in archaea, and predict multiple replication origins in a range of species. One of the P. calidifontis origins was mapped in detail, and electrophoretic mobility shift assays demonstrated binding of the Cdc6/Orc1 replication initiator protein to a repeated sequence element, denoted Orb-1, within the origin. The high-throughput sequencing approach also allowed for an annotation update of both genomes, resulting in the restoration of open reading frames encoding proteins involved in, e.g., sugar, nitrate and energy metabolism, as well as in glycosylation and DNA repair.

  • 36.
    Pelve, Erik A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Lindås, Ann-Christin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Martens-Habbena, Willm
    de la Torre, José R.
    Stahl, David A.
    Bernander, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Cdv-based cell division and cell cycle organization in the thaumarchaeon Nitrosopumilus maritimus2011In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 82, no 3, p. 555-566Article in journal (Refereed)
    Abstract [en]

    Cell division is mediated by different mechanisms in different evolutionary lineages. While bacteria and euryarchaea utilize an FtsZ-based mechanism, most crenarchaea divide using the Cdv system, related to the eukaryotic ESCRT-III machinery. Intriguingly, thaumarchaeal genomes encode both FtsZ and Cdv protein homologues, raising the question of their division mode. Here, we provide evidence indicating that Cdv is the primary division system in the thaumarchaeon Nitrosopumilus maritimus. We also show that the cell cycle is differently organized as compared to hyperthermophilic crenarchaea, with a longer pre-replication phase and a shorter post-replication stage. In particular, the time required for chromosome replication is remarkably extensive, 15-18 h, indicating a low replication rate. Further, replication did not continue to termination in a significant fraction of N. maritimus cell populations following substrate depletion. Both the low replication speed and the propensity for replication arrest are likely to represent adaptations to extremely oligotrophic environments. The results demonstrate that thaumarchaea, crenarchaea and euryarchaea display differences not only regarding phylogenetic affiliations and gene content, but also in fundamental cellular and physiological characteristics. The findings also have implications for evolutionary issues concerning the last archaeal common ancestor and the relationship between archaea and eukaryotes.

  • 37.
    Pettersson, B M Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kirsebom, Leif A
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    tRNA accumulation and suppression of the bldA phenotype during development in Streptomyces coelicolor2011In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 79, no 6, p. 1602-1614Article in journal (Refereed)
    Abstract [en]

    Streptomyces coelicolor undergoes distinct morphological changes as it grows on solid media where spores differentiate into vegetative and aerial mycelium that is followed by the production of spores. Deletion of bldA, encoding the rare tRNA(Leu) UAA, blocks development at the stage of vegetative mycelium formation. From previous data it appears that tRNA(Leu) UAA accumulates relatively late during growth while two other tRNAs do not. Here, we studied the expression of 17 different tRNAs including bldA tRNA, and the RNA subunit of the tRNA processing endoribonuclease RNase P. Our results showed that all selected tRNAs and RNase P RNA increased with time during development. However, accumulation of bldA tRNA and another rare tRNA(Leu) isoacceptor started at an earlier stage compared with the other tRNAs. We also introduced the bldA tRNA anticodon (UAA) into other tRNAs and introduced these into a bldA deletion strain. In particular, one such mutant tRNA derived from the tRNA(Leu) CAA isoacceptor suppressed the bldA phenotype. Thus, the bldA tRNA scaffold is not critical for function as a regulator of S. coelicolor cell differentiation. Further substitution experiments, in which the 5'- and 3'-flanking regions of the suppressor tRNA were changed, indicated that these regions were important for the suppression.

  • 38.
    Pränting, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Escape from growth restriction in small colony variants of Salmonella typhimurium by gene amplification and mutation2011In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 79, no 2, p. 305-315Article in journal (Refereed)
    Abstract [en]

    Antibiotic resistance in bacteria is generally associated with fitness costs that often can be reduced by second-site compensatory mutations. Here, we examined how a protamine-resistant small colony variant of Salmonella typhimurium adapts to the growth reduction conferred by a resistance mutation in hemC (encoding a haem-biosynthesis enzyme). We show that adaptation occurs in a multi-step process where fitness is successively increased. Thus, the initial adaptive response was selection for an unstable gene amplification of the mutant hemC gene that provided a small fitness increase. Fitness was increased further by a mutation that restored HemC function in one gene copy, relaxing selection for the amplification. Subsequently, the amplification segregated back to the haploid state and even higher fitness. The end result was in most cases mutant strains with a hemC sequence different from that of the wild-type strain. These findings suggest that gene amplification facilitates adaptive evolution. A higher gene dosage increases the target size for compensatory mutations and improves fitness of the cell, thereby allowing an increase in the population size, further increasing the probability of a subsequent stable mutation. Our results provide a novel genetic basis for growth compensation in small colony variants.

  • 39. Reimann, Julia
    et al.
    Lassak, Kerstin
    Khadouma, Sunia
    Ettema, Thijs J. G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Yang, Nuan
    Driessen, Arnold J. M.
    Klingl, Andreas
    Albers, Sonja-Verena
    Regulation of archaella expression by the FHA and von Willebrand domain-containing proteins ArnA and ArnB in Sulfolobus acidocaldarius2012In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 86, no 1, p. 24-36Article in journal (Refereed)
    Abstract [en]

    The ability of microorganisms to sense and respond to sudden changes in their environment is often based on regulatory systems comprising reversible protein phosphorylation. The archaellum (former: archaeal flagellum) is used for motility in Archaea and therefore functionally analogous to the bacterial flagellum. In contrast with archaellum-mediated movement in certain members of the Euryarchaeota, this process, including its regulation, remains poorly studied in crenarchaeal organisms like Sulfolobus species. Recently, it was shown in Sulfolobus acidocaldarius that tryptone limiting conditions led to the induction of archaella expression and assembly. Here we have identified two proteins, the FHA domain-containing protein ArnA and the vWA domain-containing protein ArnB that are involved in regulating archaella expression in S. acidocaldarius. Both proteins are phosphorylated by protein kinases in vitro and interact strongly in vivo. Phenotypic analyses revealed that these two proteins are repressors of archaella expression. These results represent the first step in understanding the networks that underlie regulation of cellular motility in Crenarchaeota and emphasize the importance of protein phosphorylation in the regulation of cellular processes in the Archaea.

  • 40.
    Roy, Saumendra P.
    et al.
    Department of Molecular Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Rahman, Mohammad M.
    Di Yu, Xiao
    Department of Molecular Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Tuittila, Minna
    Knight, Stefan D.
    Department of Molecular Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Zavialov, Anton V.
    Department of Molecular Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Crystal structure of enterotoxigenic Escherichia coli colonization factor CS6 reveals a novel type of functional assembly2012In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 86, no 5, p. 1100-1115Article in journal (Refereed)
    Abstract [en]

    Coli surface antigen 6 (CS6) is a widely expressed enterotoxigenic Escherichia coli (ETEC) colonization factor that mediates bacterial attachment to the small intestinal epithelium. CS6 is a polymer of two protein subunits CssA and CssB, which are secreted and assembled on the cell surface via the CssC/CssD chaperone usher (CU) pathway. Here, we present an atomic resolution model for the structure of CS6 based on the results of X-ray crystallographic, spectroscopic and biochemical studies, and suggest a mechanism for CS6-mediated adhesion. We show that the CssA and CssB subunits are assembled alternately in linear fibres by the principle of donor strand complementation. This type of fibre assembly is novel for CU assembled adhesins. We also show that both subunits in the fibre bind to receptors on epithelial cells, and that CssB, but not CssA, specifically recognizes the extracellular matrix protein fibronectin. Taken together, structural and functional results suggest that CS6 is an adhesive organelle of a novel type, a hetero-polyadhesin that is capable of polyvalent attachment to different receptors.

  • 41. Shcherbakov, Dmitri
    et al.
    Akbergenov, Rashid
    Matt, Tanja
    Sander, Peter
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Böttger, Erik C
    Directed mutagenesis of Mycobacterium smegmatis 16S rRNA to reconstruct the in-vivo evolution of aminoglycoside resistance in Mycobacterium tuberculosis2010In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 77, no 4, p. 830-40Article in journal (Refereed)
    Abstract [en]

    Summary Drug resistance in Mycobacteriumtuberculosis is a global problem, with major consequences for treatment and public health systems. As the emergence and spread of drug-resistant tuberculosis epidemics is largely influenced by the impact of the resistance mechanism on bacterial fitness, we wished to investigate whether compensatory evolution occurs in drug resistant clinical isolates of M. tuberculosis. By combining information from molecular epidemiology studies of drug resistant clinical M. tuberculosis isolates with genetic reconstructions and measurements of aminoglycoside susceptibility and fitness in M. smegmatis, we have reconstructed a plausible pathway for how aminoglycoside resistance develops in clinical isolates of M. tuberculosis. Thus, we show by reconstruction experiments that base changes in the highly conserved A-site of 16S rRNA that (i) cause aminoglycoside resistance, (ii) confer a high fitness cost and (iii) destabilize a stem-loop structure, are associated with a particular compensatory point mutation that restores rRNA secondary structure and bacterial fitness, while maintaining to a large extent the drug resistant phenotype. The same types of resistance and associated mutations can be found in M. tuberculosis in clinical isolates, suggesting that compensatory evolution contributes to the spread of drug-resistant tuberculosis disease.

  • 42.
    Singh, Bhupender
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Nitharwal, Ram Gopal
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Ramesh, Malavika
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pettersson, B. M. Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Kirsebom, Leif A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Dasgupta, Santanu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Asymmetric growth and division in Mycobacterium spp.: compensatory mechanisms for non-medial septa2013In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 88, no 1, p. 64-76Article in journal (Refereed)
    Abstract [en]

    Mycobacterium spp., rod-shaped cells belonging to the phylum Actinomycetes, lack the Min- and Noc/Slm systems responsible for preventing the placement of division sites at the poles or over the nucleoids to ensure septal assembly at mid-cell. We show that the position for establishment of the FtsZ-ring in exponentially growing Mycobacterium marinum and Mycobacterium smegmatis cells is nearly random, and that the cells often divide non-medially, producing two unequal but viable daughters. Septal sites and cellular growth disclosed by staining with the membrane-specific dye FM4-64 and fluorescent antibiotic vancomycin (FL-Vanco), respectively, showed that many division sites were off-centre, often over the nucleoids, and that apical cell growth was frequently unequal at the two poles. DNA transfer through the division septum was detected, and translocation activity was supported by the presence of a putative mycobacterial DNA translocase (MSMEG2690) at the majority of the division sites. Time-lapse imaging of single live cells through several generations confirmed both acentric division site placement and unequal polar growth in mycobacteria. Our evidence suggests that post-septal DNA transport and unequal polar growth may compensate for the non-medial division site placement in Mycobacterium spp.

  • 43. Singh, Vinayak
    et al.
    Dhar, Neeraj
    Pató, János
    Kolly, Gaëlle S
    Korduláková, Jana
    Forbak, Martin
    Evans, Joanna C
    Székely, Rita
    Rybniker, Jan
    Palčeková, Zuzana
    Zemanová, Júlia
    Santi, Isabella
    Signorino-Gelo, François
    Rodrigues, Liliana
    Vocat, Anthony
    Covarrubias, Adrian Suarez
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Rengifo, Monica G
    Johnsson, Kai
    Mowbray, Sherry
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Buechler, Joseph
    Delorme, Vincent
    Brodin, Priscille
    Knott, Graham W
    Aínsa, José A
    Warner, Digby F
    Kéri, György
    Mikušová, Katarína
    McKinney, John D
    Cole, Stewart T
    Mizrahi, Valerie
    Hartkoorn, Ruben C
    Identification of aminopyrimidine-sulfonamides as potent modulators of Wag31-mediated cell elongation in mycobacteria.2017In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 103, no 1, p. 13-25Article in journal (Refereed)
    Abstract [en]

    There is an urgent need to discover new anti-tubercular agents with novel mechanisms of action in order to tackle the scourge of drug-resistant tuberculosis. Here, we report the identification of such a molecule - an AminoPYrimidine-Sulfonamide (APYS1) that has potent, bactericidal activity against M. tuberculosis. Mutations in APYS1-resistant M. tuberculosis mapped exclusively to wag31, a gene that encodes a scaffolding protein thought to orchestrate cell elongation. Recombineering confirmed that a Gln201Arg mutation in Wag31 was sufficient to cause resistance to APYS1, however, neither overexpression nor conditional depletion of wag31 impacted M. tuberculosis susceptibility to this compound. In contrast, expression of the wildtype allele of wag31 in APYS1-resistant M. tuberculosis was dominant and restored susceptibility to APYS1 to wildtype levels. Time-lapse imaging and scanning electron microscopy revealed that APYS1 caused gross malformation of the old pole of M. tuberculosis, with eventual lysis. These effects resembled the morphological changes observed following transcriptional silencing of wag31 in M. tuberculosis. These data show that Wag31 is likely not the direct target of APYS1, but the striking phenotypic similarity between APYS1 exposure and genetic depletion of Wag31 in M. tuberculosis suggests that APYS1 might indirectly affect Wag31 through an as yet unknown mechanism.

  • 44.
    Tubulekas, Ioannis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Growth and translation elongation rate are sensitive to the concentration of EF-Tu1993In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 8, no 4, p. 761-770Article in journal (Refereed)
    Abstract [en]

    We have used quantitative immunoblotting to estimate the amount of EF-Tu in a variety of S. typhimurium strains with wild-type, mutant, insertionally inactivated or plasmid-borne tuf genes. In the same strains we have measured translation elongation rate, exponential growth rate and the level of nonsense codon readthrough. In the wild-type strain, at moderate to fast growth rates, our data show that EF-Tu makes up 8–9% of total cell protein. Strains with either of the tuf genes insertionally inactivated have 65% of the wild-type EF-Tu level, irrespective of which tuf gene remains active, or whether that gene is wild-type or a kirromycin-resistant mutant. Strains with only one active tuf gene have reduced growth and translation elongation rates. From the magnitude of the reduction in elongation rate relative to the level of EF-Tu we calculate that in glucose minimal medium the in vivo saturation level of wild-type ribosomes by ternary complexes is only 63%. Strains with a ribosome mutation causing a poor interaction with ternary complex are non-viable on minimal medium when the level of EF-Tu is reduced.

  • 45.
    Unoson, Cecilia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wagner, Gerhart E. H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    A small SOS-induced toxin is targeted against the inner membrane in Escherichia coli2008In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 70, no 1, p. 258-70Article in journal (Refereed)
    Abstract [en]

    We previously reported on an SOS-induced toxin, TisB, in Escherichia coli and its regulation by the RNA antitoxin IstR-1. Here, we addressed the mode of action of TisB. By placing the tisB reading frame downstream of a controllable promoter on a plasmid, toxicity could be analysed in the absence of the global SOS response. Upon induction of TisB, cell growth was inhibited and plating efficiency decreased rapidly. The onset of toxicity correlated with a drastic decrease in transcription, translation and replication rates. Cellular RNA was degraded, but in vitro experiments showed that TisB did not affect translation or transcription directly. Thus, these effects are downstream consequences of membrane damage: TisB is predicted to be hydrophobic and membrane spanning, and Western analyses demonstrated that this peptide was strictly localized to the cytoplasmic membrane fraction. Membrane damage and cell killing under tisB multicopy expression are also seen by live/death staining and the formation of ghost cells. This is reminiscent of another toxin, Hok of plasmid R1, which also targets the membrane. The biological significance of the istR/tisB locus is still elusive; deletion of the entire locus gave no fitness phenotype in competition experiments.

  • 46. van Biesen, T
    et al.
    Söderbom, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wagner, E G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Frost, L S
    Structural and functional analyses of the FinP antisense RNA regulatory system of the F conjugative plasmid1993In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 10, no 1, p. 35-43Article in journal (Refereed)
    Abstract [en]

    The efficiency of conjugation of F-like plasmids is regulated by the FinOP fertility inhibition system. The transfer (tra) operon is under the direct control of the TraJ transcriptional activator which, in turn, is negatively regulated by FinP, an antisense RNA, and FinO, a 22 kDa protein. Recently, FinO has been shown to extend the chemical stability of FinP in vivo in the absence of traJ mRNA. The in vitro secondary structures of both the FinP and TraJ RNAs were determined by the use of single- and double-strand-specific nucleases; both RNAs were found to have double stem-loop structures that are complementary to each other and, therefore, FinP RNA and TraJ RNA have the potential to form a duplex with each other. This was verified by in vitro binding experiments. The reaction was shown to be biomolecular with an apparent rate constant (kapp) of 5 x 10(5)M-1s-1, a value that is similar to those found for other natural antisense RNA systems. Preliminary evidence for the in vivo formation of the FinP-TraJ RNA duplex was obtained by primer extension of the traJ mRNA; the presence of both FinO and FinP was required to cause a dramatic reduction in the steady-state level of traJ mRNA, perhaps as a result of RNase III degradation of the resulting RNA duplex.

  • 47.
    Virtanen, Petra
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wäneskog, Marcus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Class II contact‐dependent growth inhibition (CDI) systems allow for broad‐range cross‐species toxin delivery within the Enterobacteriaceae family2019In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 111, no 4, p. 1109-1125Article in journal (Refereed)
    Abstract [en]

    Contact‐dependent growth inhibition (CDI) allows bacteria to recognize kin cells in mixed bacterial populations. In Escherichia coli, CDI mediated effector delivery has been shown to be species‐specific, with a preference for the own strain over others. This specificity is achieved through an interaction between a receptor‐binding domain in the CdiA protein and its cognate receptor protein on the target cell. But how conserved this specificity is has not previously been investigated in detail. Here, we show that class II CdiA receptor‐binding domains and their Enterobacter cloacae analog are highly promiscuous, and can allow for efficient effector delivery into several different Enterobacteriaceae species, including Escherichia, Enterobacter, Klebsiella and Salmonella spp. In addition, although we observe a preference for the own receptors over others for two of the receptor‐binding domains, this did not limit cross‐species effector delivery in all experimental conditions. These results suggest that class II CdiA proteins could allow for broad‐range and cross‐species growth inhibition in mixed bacterial populations.

  • 48. Westra, Edze R
    et al.
    Pul, Ümit
    Heidrich, Nadja
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Jore, Matthijs M
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Stratmann, Thomas
    Wurm, Reinhild
    Raine, Amanda
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mescher, Melina
    van Heereveld, Luc
    Mastop, Marieke
    Wagner, Gerhart E. H
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Schnetz, Karin
    van der Oost, John
    Wagner, Rolf
    Brouns, Stan J. J.
    H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO2010In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 77, no 6, p. 1380-1393Article in journal (Refereed)
    Abstract [en]

    The recently discovered prokaryotic CRISPR/Cas defence system provides immunity against viral infections and plasmid conjugation. It has been demonstrated that in Escherichia coli transcription of the Cascade genes (casABCDE) and to some extent the CRISPR array is repressed by heat-stable nucleoid-structuring (H-NS) protein, a global transcriptional repressor. Here we elaborate on the control of the E. coli CRISPR/Cas system, and study the effect on CRISPR-based anti-viral immunity. Transformation of wild-type E. coli K12 with CRISPR spacers that are complementary to phage Lambda does not lead to detectable protection against Lambda infection. However, when an H-NS mutant of E. coli K12 is transformed with the same anti-Lambda CRISPR, this does result in reduced sensitivity to phage infection. In addition, it is demonstrated that LeuO, a LysR-type transcription factor, binds to two sites flanking the casA promoter and the H-NS nucleation site, resulting in derepression of casABCDE12 transcription. Overexpression of LeuO in E. coli K12 containing an anti-Lambda CRISPR leads to an enhanced protection against phage infection. This study demonstrates that in E. coli H-NS and LeuO are antagonistic regulators of CRISPR-based immunity.

  • 49.
    Zorzet, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pavlov, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Nilsson, Annika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Error-prone initiation factor 2 mutations reduce the fitness cost of antibiotic resistance2010In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, ISSN 20132454, Vol. 75, no 5, p. 1299-1313Article in journal (Refereed)
    Abstract [en]

    Mutations in the fmt gene (encoding formyl methionine transferase) that eliminate formylation of initiator tRNA (Met-tRNA(i)) confer resistance to the novel antibiotic class of peptide deformylase inhibitors (PDFIs) while concomitantly reducing bacterial fitness. Here we show in Salmonella typhimurium that novel mutations in initiation factor 2 (IF2) located outside the initiator tRNA binding domain can partly restore fitness of fmt mutants without loss of antibiotic resistance. Analysis of initiation of protein synthesis in vitro showed that with non-formylated Met-tRNA(i) IF2 mutants initiated much faster than wild-type IF2, whereas with formylated fMet-tRNA(i) the initiation rates were similar. Moreover, the increase in initiation rates with Met-tRNA(i) conferred by IF2 mutations in vitro correlated well with the increase in growth rate conferred by the same mutations in vivo, suggesting that the mutations in IF2 compensate formylation deficiency by increasing the rate of in vivo initiation with Met-tRNA(i). IF2 mutants had also a high propensity for erroneous initiation with elongator tRNAs in vitro, which could account for their reduced fitness in vivo in a formylation-proficient strain. More generally, our results suggest that bacterial protein synthesis is mRNA-limited and that compensatory mutations in IF2 could increase the persistence of PDFI-resistant bacteria in clinical settings.

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