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  • 1.
    Abdulkarim, Farhad
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Ehrenberg, Måns
    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.
    Mutants of EF-Tu defective in binding aminoacyl-tRNA1996In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 382, no 3, p. 297-303Article in journal (Refereed)
    Abstract [en]

    Five single amino acid substitution variants of EF-Tu from Salmonella typhimurium were tested for their ability to promote poly(U)-translation in vitro. The substitutions are Leu120Gln, Gln124Arg and Tyr160 (Asp or Asn or Cys). They were selected by their kirromycin resistant phenotypes and all substitutions are in domain I at the interface between domains I and III of the EF-Tu · GTP configuration. The different EF-Tu variants exhibit a spectrum of phenotypes. First, k(cat)/K(M) for the interaction between ternary complex and the programmed ribosome is apparently reduced by the substitutions Leu120Gln, Gln124Arg and Tyr160Cys. Second, this reduction is caused by a defect in the interaction between these EF-Tu variants and aminoacyl-tRNA during translation. Third, in four cases out of five the affinity of the complex between EF-Tu · GTP and aminoacyl-tRNA is significantly decreased. The most drastic reduction is observed for the Gln124Arg change, where the association constant is 30-fold lower than in the mild-type case. Fourth, missense errors are increased as well as decreased by the different amino acid substitutions. Finally, the dissociation rate constant (k(d)) for the release of GDP from EF-Tu is increased 6-fold by the Tyr160Cys substitution, but remains unchanged in the four other cases. These results show that the formation of ternary complex is sensitive to many different alterations in the domain I-III interface of EF-Tu.

  • 2.
    Abdulkarim, Farhad
    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.
    Homologous recombination between the tuf genes of Salmonella typhimurium1996In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 260, no 4, p. 506-522Article in journal (Refereed)
    Abstract [en]

    The genes coding for the translation factor EF-Tu, tufA and tufB are separated by over 700 kb on the circular chromosome of Salmonella typhimurium. The coding regions of these genes have 99% identity at the nucleotide level in spite of the presumed ancient origin of the gene duplication. Sequence comparisons between S. typhimurium and Escherichiacoli suggest that within each species the two tuf genes are evolving inconcert. Here we show that each of the S. typhimurium tuf genes cantransfer genetic information to the other. In our genetic system thetransfers are seen as non-reciprocal, i.e. as gene conversion events.However, the mechanism of recombination could be reciprocal, with sisterchromosome segregation and selection leading to the isolation of aparticular class of recombinant. The amount of sequence informationtransferred in individual recombination events varies, but can be close tothe entire length of the gene. The recombination is RecABCD-dependent,and is opposed by MutSHLU mismatch repair. In the wild-type, this typeof recombination occurs at a rate that is two or three orders of magnitudegreater than the nucleotide substitution rate. The rate of recombinationdiffers by six orders of magnitude between a recA and a mutS strain.Mismatch repair reduces the rate of this recombination 1000-fold. The rateof recombination also differs by one order of magnitude depending onwhich tuf gene is donating the sequence selected for. We discuss threeclasses of model that could, in principle, account for the sequencetransfers: (1) tuf mRNA mediated recombination; (2) non-allelic reciprocalrecombination involving sister chromosomes; (3) non-allelic geneconversion involving sister chromosomes, initiated by a double-strandbreak close to one tuf gene. Although the mechanism remains to bedetermined, the effect on the bacterial cells is tuf gene sequencehomogenisation. This recombination phenomenon can account for theconcerted evolution of the tuf genes.

  • 3.
    Abdulkarim, Farhad
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Liljas, Lars
    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.
    Mutations to kirromycin resistance occur in the interface of domains I and III of EF-Tu.GTP1994In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 352, p. 118-122Article in journal (Refereed)
    Abstract [en]

    The antibiotic kirromycin inhibits protein synthesis by binding to EF-Tu and preventing its release from the ribosome after GTP hydrolysis.We have isolated and sequenced a collection of kirromycin resistant tuf mutations and identified thirteen single amino acid substitutions at sevendifferent sites in EF-Tu. These have been mapped onto the 3D structures of EF-Tu’GTP and EF-Tu.GDP. In the active GTP form of EF-Tu themutations cluster on each side of the interface between domains I and III. We propose that this domain interface is the binding site for kirromycin.

  • 4.
    Abdulkarim, Farhad
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Tuohy, TMF
    Buckingham, RH
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Missense substitutions lethal to essential functions of EF-Tu1991In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 73, no 12, p. 1457-1464Article in journal (Refereed)
    Abstract [en]

    We have used a simple selection and screening method to isolate function defective mutants of EF-Tu. From 28 mutants tested, 12 different missense substitutions, individually lethal to some essential function of EF-Tu, were identified by sequencing. In addition we found a new non-lethal missense mutation. The frequency of isolation of unique mutations suggests that this method can be used to easily isolate many more. The lethal mutations occur in all three structural domains of EF-Tu, but most are in domain II. We aim to use these mutants to define functional domains on EF-Tu.

  • 5.
    Andersson, Dan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Björkman, Johanna
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Antibiotikaresistens: är den reversibel?1998In: Smittskydd: Smittskyddsinstitutets tidskrift, ISSN 1401-0690, Vol. 4, no 1, p. 3-5Article, book review (Other academic)
  • 6.
    Andersson, Dan I.
    et al.
    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.
    Antibiotic resistance and its cost: is it possible to reverse resistance?2010In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 8, no 4, p. 260-271Article, review/survey (Refereed)
    Abstract [en]

    Most antibiotic resistance mechanisms are associated with a fitness cost that is typically observed as a reduced bacterial growth rate. The magnitude of this cost is the main biological parameter that influences the rate of development of resistance, the stability of the resistance and the rate at which the resistance might decrease if antibiotic use were reduced. These findings suggest that the fitness costs of resistance will allow susceptible bacteria to outcompete resistant bacteria if the selective pressure from antibiotics is reduced. Unfortunately, the available data suggest that the rate of reversibility will be slow at the community level. Here, we review the factors that influence the fitness costs of antibiotic resistance, the ways by which bacteria can reduce these costs and the possibility of exploiting them.

  • 7.
    Andersson, Dan I.
    et al.
    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.
    Effects of antibiotic resistance on bacterial fitness, virulence and transmission2008In: Evolutionary biology of bacterial and fungal pathogens / [ed] Baquero, Fernando Nombela, Cesar Cassell, Gail H., Washington DC: ASM Press , 2008, p. 307-318Chapter in book (Refereed)
  • 8.
    Andersson, Dan I
    et al.
    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.
    Evolution of antibiotic resistance at non-lethal drug concentrations.2012In: Drug resistance updates, ISSN 1368-7646, E-ISSN 1532-2084, Vol. 15, no 3, p. 162-172Article in journal (Refereed)
    Abstract [en]

    Human use of antimicrobials in the clinic, community and agricultural systems has driven selection for resistance in bacteria. Resistance can be selected at antibiotic concentrations that are either lethal or non-lethal, and here we argue that selection and enrichment for antibiotic resistant bacteria is often a consequence of weak, non-lethal selective pressures - caused by low levels of antibiotics - that operates on small differences in relative bacterial fitness. Such conditions may occur during antibiotic therapy or in anthropogenically drug-polluted natural environments. Non-lethal selection increases rates of mutant appearance and promotes enrichment of highly fit mutants and stable mutators.

  • 9.
    Andersson, Dan I.
    et al.
    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.
    Gene amplification and adaptive evolution in bacteria2009In: Annual Review of Genetics, ISSN 0066-4197, E-ISSN 1545-2948, Vol. 43, p. 167-195Article, review/survey (Refereed)
    Abstract [en]

    Gene duplication-amplification (GDA) processes are highly relevant biologically because they generate extensive and reversible genetic variation on which adaptive evolution can act. Whenever cellular growth is restricted, escape from these growth restrictions often occurs by GDA events that resolve the selective problem. In addition, GDA may facilitate subsequent genetic change by allowing a population to grow and increase in number, thereby increasing the probability for subsequent adaptive mutations to occur in the amplified genes or in unrelated genes. Mathematical modeling of the effect of GDA on the rate of adaptive evolution shows that GDA will facilitate adaptation, especially when the supply of mutations in the population is rate-limiting. GDA can form via several mechanisms, both RecA-dependent and RecA-independent, including rolling-circle amplification and nonequal crossing over between sister chromatids. Due to the high intrinsic instability and fitness costs associated with GDAs, they are generally transient in nature, and consequently their evolutionary and medical importance is often underestimated.

  • 10.
    Andersson, Dan I.
    et al.
    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.
    Microbiological effects of sublethal levels of antibiotics2014In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 12, no 7, p. 465-478Article, review/survey (Refereed)
    Abstract [en]

    The widespread use of antibiotics results in the generation of antibiotic concentration gradients in humans, livestock and the environment. Thus, bacteria are frequently exposed to non-lethal (that is, subinhibitory) concentrations of drugs, and recent evidence suggests that this is likely to have an important role in the evolution of antibiotic resistance. In this Review, we discuss the ecology of antibiotics and the ability of subinhibitory concentrations to select for bacterial resistance. We also consider the effects of low-level drug exposure on bacterial physiology, including the generation of genetic and phenotypic variability, as well as the ability of antibiotics to function as signalling molecules. Together, these effects accelerate the emergence and spread of antibiotic-resistant bacteria among humans and animals.

  • 11.
    Andersson, Dan I
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Muller's ratchet decreases fitness of a DNA-based microbe1996In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 93, no 2, p. 906-907Article in journal (Refereed)
    Abstract [en]

    Muller proposed that an asexual organism will inevitably accumulate deleterious mutations, resulting in an increase of the mutational load and an inexorable, ratchet-like, loss of the least mutated class [Muller, H.J. (1964) Mutat. Res. 1, 2-9]. The operation of Muller's ratchet on real populations has been experimentally demonstrated only in RNA viruses. However, these cases are exceptional in that the mutation rates of the RNA viruses are extremely high. We have examined whether Muller's ratchet operates in Salmonella typhimurium, a DNA-based organism with a more typical genomic mutation rate. Cells were grown asexually under conditions expected to result in high genetic drift, and the increase in mutational load was determined. S. typhimurium accumulated mutations under these conditions such that after 1700 generations, 1% of the 444 lineages tested had suffered an obvious loss of fitness, as determined by decreased growth rate. These results suggest that in the absence of sex and with high genetic drift, genetic mechanisms, such as back or compensatory mutations, cannot compensate for the accumulation of deleterious mutations. In addition, we measured the appearance of auxotrophs, which allowed us to calculate an average spontaneous mutation rate of approximately 0.3-1.5 x 10(-9) mutations per base pair per generation. This rate is measured for the largest genetic target studied so far, a collection of about 200 genes.

  • 12.
    Andersson, Dan I.
    et al.
    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.
    Persistence of antibiotic resistance in bacterial populations2011In: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 35, no 5, p. 901-911Article, review/survey (Refereed)
    Abstract [en]

    Unfortunately for mankind, it is very likely that the antibiotic resistance problem we have generated during the last 60 years due to the extensive use and misuse of antibiotics is here to stay for the foreseeable future. This view is based on theoretical arguments, mathematical modeling, experiments and clinical interventions, suggesting that even if we could reduce antibiotic use, resistant clones would remain persistent and only slowly (if at all) be outcompeted by their susceptible relatives. In this review, we discuss the multitude of mechanisms and processes that are involved in causing the persistence of chromosomal and plasmid-borne resistance determinants and how we might use them to our advantage to increase the likelihood of reversing the problem. Of particular interest is the recent demonstration that a very low antibiotic concentration can be enriching for resistant bacteria and the implication that antibiotic release into the environment could contribute to the selection for resistance.

  • 13.
    Andersson, Dan I
    et al.
    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.
    Selection and Transmission of Antibiotic-Resistant Bacteria2017In: Microbiology Spectrum, ISSN 2165-0497, Vol. 5, no 4, article id UNSP MTBP-0013-2016Article in journal (Refereed)
    Abstract [en]

    Ever since antibiotics were introduced into human and veterinary medicine to treat and prevent bacterial infections there has been a steady selection and increase in the frequency of antibiotic resistant bacteria. To be able to reduce the rate of resistance evolution, we need to understand how various biotic and abiotic factors interact to drive the complex processes of resistance emergence and transmission. We describe several of the fundamental factors that underlay resistance evolution, including rates and niches of emergence and persistence of resistant bacteria, time- and space-gradients of various selective agents, and rates and routes of transmission of resistant bacteria between humans, animals and other environments. Furthermore, we discuss the options available to reduce the rate of resistance evolution and/or transmission and their advantages and disadvantages.

  • 14.
    Andersson, Dan I.
    et al.
    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.
    Kubicek-Sutherland, Jessica Z.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mechanisms and consequences of bacterial resistance to antimicrobial peptides2016In: Drug resistance updates, ISSN 1368-7646, E-ISSN 1532-2084, Vol. 26, p. 43-57Article, review/survey (Refereed)
    Abstract [en]

    Cationic antimicrobial peptides (AMPs) are an intrinsic part of the human innate immune system. Over 100 different human AMPs are known to exhibit broad-spectrum antibacterial activity. Because of the increased frequency of resistance to conventional antibiotics there is an interest in developing AMPs as an alternative antibacterial therapy. Several cationic peptides that are derivatives of AMPs from the human innate immune system are currently in clinical development. There are also ongoing clinical studies aimed at modulating the expression of AMPs to boost the human innate immune response. In this review we discuss the potential problems associated with these therapeutic approaches. There is considerable experimental data describing mechanisms by which bacteria can develop resistance to AMPs. As for any type of drug resistance, the rate by which AMP resistance would emerge and spread in a population of bacteria in a natural setting will be determined by a complex interplay of several different factors, including the mutation supply rate, the fitness of the resistant mutant at different AMP concentrations, and the strength of the selective pressure. Several studies have already shown that AMP-resistant bacterial mutants display broad cross-resistance to a variety of AMPs with different structures and modes of action. Therefore, routine clinical administration of AMPs to treat bacterial infections may select for resistant bacterial pathogens capable of better evading the innate immune system. The ramifications of therapeutic levels of exposure on the development of AMP resistance and bacterial pathogenesis are not yet understood. This is something that needs to be carefully studied and monitored if AMPs are used in clinical settings.

  • 15.
    Andersson, Dan I
    et al.
    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.
    Roth, John R
    The origin of mutants under selection: Interactions of mutation, growth and selection2010In: EcoSal - Escherichia coli and Salmonella: cellular and molecular biology / [ed] Böck et al, Washington, DC: ASM Press , 2010Chapter in book (Refereed)
  • 16.
    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.

  • 17.
    Andersson, D.I
    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.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Antibiotikaresistens här för att stanna?1998In: Läkartidningen, ISSN 0023-7205, E-ISSN 1652-7518, Vol. 95, no 37, p. 3940-3944Article, review/survey (Other academic)
  • 18.
    Andersson, DI
    et al.
    Uppsala University.
    Hughes, Diarmaid
    Uppsala University. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Muller's Ratchet Impairs Fitness of Bacteria1995Other (Other academic)
  • 19.
    Arwidsson, Ola
    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.
    Evidence against reciprocal recombination as the basis for tuf gene conversion in Salmonella enterica serovar Typhimurium2004In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 338, no 3, p. 463-467Article in journal (Refereed)
    Abstract [en]

    The duplicate tuf genes on the Salmonella enterica serovar Typhimurium chromosome co-evolve by a RecA-, RecB-dependent gene conversion mechanism. Gene conversion is defined as a non-reciprocal transfer of genetic information. However, in a replicating bacterial chromosome there is a possibility that a reciprocal genetic exchange between different tuf genes sitting on sister chromosomes could result in "apparent" gene conversion. We asked whether the major mechanism of tuf gene conversion was classical or apparent. We devised a genetic selection that allowed us to isolate and examine both expected products from a reciprocal recombination event between the tuf genes. Using this selection we tested within individual cultures for a correlation in the frequency of jackpots as expected if recombination were reciprocal. We found no correlation, either in the frequency of each type of recombinant product, or in the DNA sequences of the products resulting from each recombination event. We conclude that the evidence argues in favor of a non-reciprocal gene conversion mechanism as the basis for tuf gene co-evolution.

  • 20.
    Banin, Ehud
    et al.
    Bar Ilan Univ, Fac Life Sci, IL-82900 Ramat Gan, Israel..
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kuipers, Oscar P.
    Univ Groningen, Mol Genet, NL-9747 AG Groningen, Netherlands..
    Bacterial pathogens, antibiotics and antibiotic resistance: Editorial2017In: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 41, no 3, p. 450-452, article id fux016Article in journal (Other academic)
  • 21.
    Bergman, Jessica
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hammarlöf, Disa L
    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.
    Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 2, p. e90486-Article in journal (Other academic)
    Abstract [en]

    Salmonella enterica grows extremely slowly when it depends on tufA499 (encoding the Gln125Arg mutant form of EF-Tu) to drive protein synthesis. We screened a plasmid library for multi-copy suppressors of the slow growth phenotype and identified spoT as a candidate. The spoT gene encodes a dual function enzyme with both ppGpp synthetase and hydrolase activities. When spoT was cloned behind an arabinose-inducible promoter the growth rate of the mutant strain increased in response to arabinose addition. We found that the slow-growing mutant strain had a relatively high basal level of ppGpp during exponential growth in rich medium. Overexpression of spoT significantly reduced this level of ppGpp suggesting that inappropriately high ppGpp levels might cause the slow growth rate associated with tufA499. We tested this hypothesis by inactivating relA (codes for RelA, a ribosome-associated ppGpp synthetase) in the mutant strain. This inactivation decreased the level of ppGpp in the mutant strain and increased its growth rate. Based on these data we propose that ribosomes depending on tufA499 for their supply of ternary complex (EF-Tu•GTP•aa-tRNA) experience amino acid starvation and that RelA on these starving ribosomes produces an excess of the alarmone ppGpp. This results in a suboptimal partitioning of transcription activity between genes important for fast growth in rich medium and genes important for growth in a poor medium. Accordingly, mutant bacteria growing in a rich medium act physiologically as though they were growing in a nutrient-poor environment. We propose that this generates a vicious circle and contributes to the extreme slow-growth phenotype associated with mutant EF-Tu. Reducing the level of ppGpp increases the growth rate of the mutant because it breaks this circle and reduces the wasteful misdirection of resources in the cell.

  • 22.
    Bergman, Jessica M
    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 Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, United States of America.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Acetate availability and utilization supports the growth of mutant sub-populations on aging bacterial colonies2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 10, p. e109255-Article in journal (Refereed)
    Abstract [en]

    When bacterial colonies age most cells enter a stationary phase, but sub-populations of mutant bacteria can continue to grow and accumulate. These sub-populations include bacteria with mutations in rpoB (RNA polymerase β-subunit) or rpoS (RNA polymerase stress-response sigma factor). Here we have identified acetate as a nutrient present in the aging colonies that is utilized by these mutant subpopulations to support their continued growth. Proteome analysis of aging colonies showed that several proteins involved in acetate conversion and utilization were upregulated during aging. Acetate is known to be excreted during the exponential growth phase but can be imported later during the transition to stationary phase and converted to acetyl-CoA. Acetyl-CoA is used in multiple processes, including feeding into the TCA cycle, generating ATP via the glyoxylate shunt, as a source of acetyl groups for protein modification, and to support fatty acid biosynthesis. We showed that deletion of acs (encodes acetyl-CoA synthetase; converts acetate into acetyl-CoA) significantly reduced the accumulation of rpoB and rpoS mutant subpopulations on aging colonies. Measurement of radioactive acetate uptake showed that the rate of conversion decreased in aging wild-type colonies, was maintained at a constant level in the rpoB mutant, and significantly increased in the aging rpoS mutant. Finally, we showed that the growth of subpopulations on aging colonies was greatly enhanced if the aging colony itself was unable to utilize acetate, leaving more acetate available for mutant subpopulations to use. Accordingly, the data show that the accumulation of subpopulations of rpoB and rpoS mutants on aging colonies is supported by the availability in the aging colony of acetate, and by the ability of the subpopulation cells to convert the acetate to acetyl-CoA.

  • 23.
    Bjorkman, J
    et al.
    Uppsala University.
    Hughes, Diarmaid
    Uppsala University. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Andersson, DI
    Uppsala University.
    Virulence of antibiotic-resistant Salmonella typhimurium1998In: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, ISSN 0027-8424, Vol. 95, no 7, p. 3949-3953Article in journal (Other academic)
    Abstract [en]

    We show that most Salmonella typhimurium mutants resistant to streptomycin, rifampicin, and nalidixic acid are avirulent in mice, Of seven resistant mutants examined, sis were avirulent and one was similar to the wild type In competition experiments in mi

  • 24.
    Björkman, J
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Nagaev, I
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Berg, O G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Molecular Evolution.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Andersson, Dan I
    Effects of environment on compensatory mutations to ameliorate costs of antibiotic resistance2000In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 287, no 5457, p. 1479-1482Article in journal (Refereed)
    Abstract [en]

    Most types of antibiotic resistance impose a biological cost on bacterial fitness. These costs can be compensated, usually without loss of resistance, by second-site mutations during the evolution of the resistant bacteria in an experimental host or in a laboratory medium. Different fitness-compensating mutations were selected depending on whether the bacteria evolved through serial passage in mice or in a laboratory medium. This difference in mutation spectra was caused by either a growth condition-specific formation or selection of the compensated mutants. These results suggest that bacterial evolution to reduce the costs of antibiotic resistance can take different trajectories within and outside a host.

  • 25.
    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.

  • 26.
    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.

  • 27.
    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.
    Huseby, Douglas L.
    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.
    Having your cake and eating it - Staphylococcus aureus small colony variants can evolve faster growth rate without losing their antibiotic resistance2017In: MICROBIAL CELL, ISSN 2311-2638, Vol. 4, no 8, p. 275-277Article in journal (Refereed)
    Abstract [en]

    Staphylococcus aureus can produce small colony variants (SCVs) during infections. These cause significant clinical problems because they are difficult to detect in standard microbiological screening and are associated with persistent infections. The major causes of the SCV phenotype are mutations that inhibit respiration by inactivation of genes of the menadione or hemin biosynthesis pathways. This reduces the production of ATP required to support fast growth. Importantly, it also decreases cross-membrane potential in SCVs, resulting in decreased uptake of cationic compounds, with reduced susceptibility to aminoglycoside antibiotics as a consequence. Because SCVs are slow-growing (mutations in men genes are associated with growth rates in rich medium similar to 30% of the wild-type growth rate) bacterial cultures are very susceptible to rapid takeover by faster-growing mutants (revertants or suppressors). In the case of reversion, the resulting fast growth is obviously associated with the loss of antibiotic resistance. However, direct reversion is relatively rare due to the very small genetic target size for such mutations. We explored the phenotypic consequences of SCVs evolving faster growth by routes other than direct reversion, and in particular whether any of those routes allowed for the maintenance of antibiotic resistance. In a recent paper (mBio 8: e00358-17) we demonstrated the existence of several different routes of SCV evolution to faster growth, one of which maintained the antibiotic resistance phenotype. This discovery suggests that SCVs might be more adaptable and problematic that previously thought. They are capable of surviving as a slow-growing persistent form, before evolving into a significantly faster-growing form without sacrificing their antibiotic resistance phenotype.

  • 28.
    Brandis, Gerrit
    et al.
    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.
    A large chromosomal inversion causes the formation of an unstable tufA/B hybrid operonManuscript (preprint) (Other academic)
  • 29.
    Brandis, Gerrit
    et al.
    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 protects the tuf genes from inactivation by mobile genetic elementsManuscript (preprint) (Other academic)
  • 30.
    Brandis, Gerrit
    et al.
    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.
    Genetic characterization of compensatory evolution in strains carrying rpoB Ser531Leu, the rifampicin resistance mutation most frequently found in clinical isolates2013In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, no 11, p. 2493-2497Article in journal (Refereed)
    Abstract [en]

    Objectives: The evolution of rifampicin resistance in Mycobacterium tuberculosis is a major threat to effective tuberculosis therapy. Much is known about the initial emergence of rifampicin resistance, but the further evolution of these resistant strains has only lately been subject to investigation. Although resistance can be caused by many different mutations in rpoB, among clinical M. tuberculosis isolates the mutation rpoB S531L is overwhelmingly the most frequently found. Clinical isolates with rpoB S531L frequently carry additional mutations in genes for RNA polymerase subunits, and it has been speculated that these are fitness-compensatory mutations, ameliorating the fitness cost of the primary resistance mutation. We tested this hypothesis using Salmonella as a model organism. Methods: We created the rpoB S531L mutation in Salmonella and then evolved independent lineages with selection for mutants with increased relative fitness. Relative fitness associated with putative compensatory mutations was measured after genetic reconstruction in isogenic strains. Results: Compensatory mutations were identified in genes coding for different subunits of RNA polymerase: rpoA, rpoB and rpoC. Genetic reconstructions demonstrated that each of these secondary mutations reduced the fitness cost of the rpoB S531L resistance mutation. Conclusions: The compensatory mutations identified in Salmonella cluster in similar locations to the additional mutations found in M. tuberculosis isolates. These new data strongly support the idea that many of the previously identified rpoA, rpoB and rpoC mutations in rifampicin-resistant M. tuberculosis (rpoB S531L) are indeed fitness-compensatory mutations.

  • 31.
    Brandis, Gerrit
    et al.
    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 Selective Advantage of Synonymous Codon Usage Bias in Salmonella2016In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 12, no 3, article id e1005926Article in journal (Refereed)
    Abstract [en]

    The genetic code in mRNA is redundant, with 61 sense codons translated into 20 different amino acids. Individual amino acids are encoded by up to six different codons but within codon families some are used more frequently than others. This phenomenon is referred to as synonymous codon usage bias. The genomes of free-living unicellular organisms such as bacteria have an extreme codon usage bias and the degree of bias differs between genes within the same genome. The strong positive correlation between codon usage bias and gene expression levels in many microorganisms is attributed to selection for translational efficiency. However, this putative selective advantage has never been measured in bacteria and theoretical estimates vary widely. By systematically exchanging optimal codons for synonymous codons in the tuf genes we quantified the selective advantage of biased codon usage in highly expressed genes to be in the range 0.2–4.2 x 10−4 per codon per generation. These data quantify for the first time the potential for selection on synonymous codon choice to drive genome-wide sequence evolution in bacteria, and in particular to optimize the sequences of highly expressed genes. This quantification may have predictive applications in the design of synthetic genes and for heterologous gene expression in biotechnology.

  • 32.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pietsch, Franziska
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Alemayehu, Rahel
    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.
    Comprehensive phenotypic characterization of rifampicin resistance mutations in Salmonella provides insight into the evolution of resistance in Mycobacterium tuberculosis2015In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 70, no 3, p. 680-685Article in journal (Refereed)
    Abstract [en]

    Objectives: Mutations in the beta-subunit of RNA polymerase (RNAP), encoded by rpoB, are responsible for rifampicin resistance (Rif(R)). Although many mutations in rpoB can reduce susceptibility, only a few are frequent amongst Rif(R) clinical Mycobacterium tuberculosis (MTB) isolates. It has been suggested that there is a negative correlation between the fitness costs of Rif(R) mutations and their respective clinical frequency, but so far comparable fitness cost measurements have only been conducted for a very limited number of Rif(R) mutations. We tested this hypothesis using Salmonella and Mycobacterium smegmatis as model organisms. Methods: We constructed 122 different Rif(R) mutations in Salmonella. MICs and relative fitness costs in the presence and absence of rifampicin were determined for each mutant, including for a smaller number of Rif(R) M. smegmatis strains. Results were compared with available mutation frequency data from clinical MTB isolates. Results: (i) Rif(R) mutations frequently found in MTB isolates have a fitness cost in Salmonella Typhimurium and M. smegmatis. (ii) Clinically frequent Rif(R) mutations have a high rifampicin MIC. (iii) There is a strong correlation between the magnitude of the fitness cost of a Rif(R) mutation in Salmonella Typhimurium or M. smegmatis and the frequency with which that mutation is associated with secondary (putative compensatory) mutations in RNAP of clinical MTB isolates. Conclusions: This suggests that the success of Rif(R) mutations in clinical MTB isolates may be dependent not only on a low initial fitness cost, but rather the results of three factors: (i) a high rifampicin MIC; (ii) a relatively low initial fitness cost; and (iii) the ability to additionally acquire compensatory mutations selected to further reduce fitness cost.

  • 33.
    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. 

  • 34.
    Cao, Sha
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandis, Gerrit
    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.
    Alternative Evolutionary Pathways for Drug-Resistant Small Colony Variant Mutants in Staphylococcus aureus2017In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 3, article id e00358-17Article in journal (Refereed)
    Abstract [en]

    Staphylococcus aureus is known to generate small colony variants (SCVs) that are resistant to aminoglycoside antibiotics and can cause persistent and recurrent infections. The SCV phenotype is unstable, and compensatory mutations lead to restored growth, usually with loss of resistance. However, the evolution of improved growth, by mechanisms that avoid loss of antibiotic resistance, is very poorly understood. By selection with serial passaging, we isolated and characterized different classes of extragenic suppressor mutations that compensate for the slow growth of small colony variants. Compensation occurs by two distinct bypass mechanisms: (i) translational suppression of the initial SCV mutation by mutant tRNAs, ribosomal protein S5, or release factor 2 and (ii) mutations that cause the constitutive activation of the SrrAB global transcriptional regulation system. Although compensation by translational suppression increases growth rate, it also reduces antibiotic susceptibility, thus restoring a pseudo-wild-type phenotype. In contrast, an evolutionary pathway that compensates for the SCV phenotype by activation of SrrAB increases growth rate without loss of antibiotic resistance. RNA sequence analysis revealed that mutations activating the SrrAB pathway cause upregulation of genes involved in peptide transport and in the fermentation pathways of pyruvate to generate ATP and NAD(+), thus explaining the increased growth. By increasing the growth rate of SCVs without the loss of aminoglycoside resistance, compensatory evolution via the SrrAB activation pathway represents a threat to effective antibiotic therapy of staphylococcal infections. IMPORTANCE Small colony variants (SCVs) of Staphylococcus aureus are a significant clinical problem, causing persistent and antibiotic-resistant infections. However, SCVs are unstable and can rapidly evolve growth-compensated mutants. Previous data suggested that growth compensation only occurred with the loss of antibiotic resistance. We have used selection with serial passaging to uncover four distinct pathways of growth compensation accessible to SCVs. Three of these paths (reversion, intragenic suppression, and translational suppression) increase growth at the expense of losing antibiotic resistance. The fourth path activates an alternative transcriptional program and allows the bacteria to produce the extra ATP required to support faster growth, without losing antibiotic resistance. The importance of this work is that it shows that drug-resistant SCVs can evolve faster growth without losing antibiotic resistance.

  • 35.
    De Rosa, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lu, Lu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Zamaratski, Edouard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Szałaj, Natalia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wadensten, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lenhammar, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gising, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Roos, Annette K.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandt, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Karlen, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Design, synthesis and in vitro biological evaluation of oligopeptides targeting E. coli type I signal peptidase (LepB)2017In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 25, no 3, p. 897-911Article in journal (Refereed)
    Abstract [en]

    Type I signal peptidases are potential targets for the development of new antibacterial agents. Here we report finding potent inhibitors of E. coli type I signal peptidase (LepB), by optimizing a previously reported hit compound, decanoyl-PTANA-CHO, through modifications at the N- and C-termini. Good improvements of inhibitory potency were obtained, with IC50s in the low nanomolar range. The best inhibitors also showed good antimicrobial activity, with MICs in the low μg/mL range for several bacterial species. The selection of resistant mutants provided strong support for LepB as the target of these compounds. The cytotoxicity and hemolytic profiles of these compounds are not optimal but the finding that minor structural changes cause the large effects on these properties suggests that there is potential for optimization in future studies.

  • 36.
    Ehrenberg, Måns
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Bilgin, Neş’e
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Dincbas, Vildan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Karimi, Reza
    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.
    Abdulkarim, Farhad
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    tRNA-ribosome interactions1995In: Biochemistry and Cell Biology, ISSN 0829-8211, E-ISSN 1208-6002, Vol. 73, no 11-12, p. 1049-1054Article, review/survey (Refereed)
    Abstract [en]

    Direct measurements of the rates of dissociation of dipeptidyl-tRNA from the ribosome show that hyperaccurate SmP and SmD ribosomes have unstable A-site binding of peptidyl-tRNA, while P-site binding is extremely stable in relation to the wild type. Error-prone Ram ribosomes, on the other hand, have stable A-site and unstable P-site binding of peptidyl-tRNA. At least for these mutant ribosomes, we conclude that stabilization of peptidyl-tRNA in one site destabilizes binding in the other. Elongation factor Tu (EF-Tu) undergoes a dramatic structural transition from its GDP-bound form to its active GTP-bound form, in which it binds aa-tRNA (aminoacyl-tRNA) in ternary complex. The effects of substitution mutations at three sites in domain I of EF-Tu, Gln124, Leu120, and Tyr160, all of which point into the domain I-domain III interface in both the GTP and GDP conformations of EF-Tu, were examined. Mutations at each position cause large reductions in aa-tRNA binding. An attractive possibility is that the mutations alter the domain I-domain III interface such that the switching of EF-Tu between different conformations is altered, decreasing the probability of aa-tRNA binding. We have previously found that two GTPs are hydrolyzed per peptide bond on EF-Tu, the implication being that two molecules of EF-Tu may interact on the ribosome to catalyze the binding of a single aa-tRNA to the A-site. More recently we found that ribosomes programmed with mRNA constructs other than poly(U), including the sequence AUGUUUACG, invariably use two GTPs per peptide bond in EF-Tu function. Other experiments measuring the protection of aa-tRNA from deacylation or from RNAse A attack show that protection requires two molecules of EF-Tu, suggesting an extended ternary complex. To remove remaining ambiguities in the interpretion of these experiments, we are making direct molecular weight determinations with neutron scattering and sedimentation-diffusion techniques.

  • 37.
    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.

  • 38.
    Hammarlöf, Disa L.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and 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.
    Temperature-sensitive mutants of RNase E in Salmonella enterica2011In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, no 23, p. 6639-6650Article in journal (Refereed)
    Abstract [en]

    RNase E has an important role in mRNA turnover and stable RNA processing although the reason for its essentiality is unknown. We isolated conditional mutants of RNase E to provide genetic tools to probe its essential function. In Salmonella enterica serovar Typhimurium an extreme slow-growth phenotype caused by mutant EF-Tu (Gln125Arg, tufA499) can be rescued by mutants of RNase E that have reduced activity. We exploited this phenotype to select mutations in RNase E and screened these for temperature sensitivity (ts) for growth. Four different ts mutations were identified, all in the N-terminal domain of RNase E: Gly66→Cys; Ile207→Ser; Ile207→Asn; Ala327→Pro. We also selected second-site mutations in RNase E that reversed temperature-sensitivity. The complete set of RNase E mutations (53 primary mutations including the ts mutations, and 23 double mutations) were analyzed for their possible effects on the structure and function of RNase E using the available 3-D structures. Most single mutations were predicted to destabilize the structure while second-site mutations that reversed the ts phenotype were predicted to restore stability to the structure. Three isogenic strain pairs carrying single or double mutations in RNase E (ts, and ts plus second-site mutation) were tested for their effects on the degradation, accumulation and processing of mRNA, rRNA and tRNA. The greatest defect was observed on rne mRNA autoregulation and this correlated with ability to rescue the tufA499-associated slow growth phenotype. This is consistent with the RNase E mutants being defective in initial binding or subsequent cleavage of an mRNA critical for fast growth.

  • 39.
    Hickman, Rachel A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Cars, Thomas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Malmberg, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Cars, Otto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Cell-wall-inhibiting antibiotic combinations with activity against multidrug-resistant Klebsiella pneumoniae and Escherichia coli2014In: Clinical Microbiology and Infection, ISSN 1198-743X, E-ISSN 1469-0691, Vol. 20, no 4, p. O267-O273Article in journal (Refereed)
    Abstract [en]

    The increasing prevalence of hospital and community-acquired infections caused by multidrug-resistant (MDR) bacterial pathogens is rapidly limiting the options for effective antibiotic therapy. Systematic studies on combinations of already available antibiotics that could provide an effective treatment against MDR bacteria are needed. We tested combinations of antibiotics that target one important physiological function (peptidoglycan synthesis) at several steps, and studied Enterobacteriaceae (Klebsiella pneumoniae and Escherichia coli) for which multidrug resistance associated with ESBL-producing plasmids has become a major problem. To measure the effectiveness of antibiotics alone and in combination, we used checkerboard assays, static antibiotic concentration time-kill assays, and an improved in-vitro kinetic model that simulates human pharmacokinetics of multiple simultaneously administered antibiotics. The target strains included an MDR K. pneumoniae isolate responsible for a recent major hospital outbreak. A double combination (fosfomycin and aztreonam) and a triple combination (fosfomycin, aztreonam and mecillinam) were both highly effective in reducing bacterial populations in all assays, including the in vitro kinetic model. These combinations were effective even though each of the MDR strains was resistant to aztreonam alone. Our results provide an initial validation of the potential usefulness of a combination of antibiotics targeting peptidoglycan synthesis in the treatment of MDR Gram-negative bacteria. We suggest that a combination of fosfomycin with aztreonam could become a useful treatment option for such infections and should be further studied.

  • 40.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Both genes for EF-Tu in Salmonella typhimurium are individually dispensible for growth1990In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 215, no 1, p. 41-51Article in journal (Refereed)
    Abstract [en]

    Each of the two genes encoding EF-Tu in Salmonella typhimurium has been inactivated using a mini-Mu MudJ insertion. Eleven independently isolated insertions are described, six in tufA and five in tufB. Transduction analysis shows that the inserted MudJ is 100% linked to the appropriate tuf gene. A mutant strain with electrophoretically distinguishable EF-TuA and EF-TuB was used to show, on two-dimensional gels, that the MudJ insertions result in the loss of the appropriate EF-Tu protein. Southern blotting, using cloned Escherichia coli tuf sequences as probes, shows that each MudJ insertion results in the physical breakage of the appropriate tuf gene. The degree of growth-rate impairment associated with each tuf inactivation is independent of which tuf gene is inactivated. The viability of S. typhimurium strains with either tuf gene inactive contrasts strongly with data suggesting that in the closely related bacterium E. coli, an active tufA gene is essential for growth. Finally the strains described here facilitate the analysis of phenotypes associated with individual mutant or wild-type Tus both in vivo and in vitro.

  • 41.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Co-evolution of the tuf genes links gene conversion with the generation of chromosomal inversions2000In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 297, no 2, p. 355-364Article in journal (Refereed)
    Abstract [en]

    The tufA and tufB genes in Salmonella typhimurium co-evolve by recombination and exchange of genetic material. A model is presented which predicts that co-evolution is achieved by gene conversions and chromosomal inversions. Analysis of recombinants reveals that conversion and inversion each occur with similar rates and each depends on RecBCD activity. The model predicts sequence structures for different classes of post-recombination tuf genes. Sequence analysis reveals the presence of each of these structures and classes, with a predicted bias in the absence of mismatch repair. An implication of these data is that co-evolution of gene families can be linked with the generation of chromosomal rearrangements.

  • 42.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Comparative genomics and the development of novel antimicrobials2008In: Comparative genomics: basic and applied research, CRC Press, Taylor and Francis Group, Boca Raton, FL, USA , 2008, p. 177-192Chapter in book (Refereed)
  • 43.
    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.

  • 44.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Evaluating genome dynamics: the constraints on rearrangements within bacterial genomes2000In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 1, no 6, p. 0006.1-0006.8Article, review/survey (Other academic)
    Abstract [en]

    Inversions and translocations distinguish the genomes of closely related bacterial species, but most of these rearrangements preserve the relationship between the rearranged fragments and the axis of chromosome replication. Within species, such rearrangements are found less frequently, except in the case of clinical isolates of human pathogens, where rearrangements are very frequent.

  • 45.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Exploiting genomics, genetics and chemistry to combat antibiotic resistance2003In: Nature reviews genetics, ISSN 1471-0056, E-ISSN 1471-0064, Vol. 4, no 6, p. 432-441Article, review/survey (Refereed)
    Abstract [en]

    To address the worsening problem of antibiotic-resistant bacteria there is an urgent need to develop new antibiotics. Comparative genomics and molecular genetics are being applied to produce lists of essential new targets for compound screening programmes. Combinatorial chemistry and structural biology are being applied to rapidly explore and optimize the interactions between lead compounds and their biological targets. Several compounds that have been identified from target-based screens are now in development, but technical and economic constraints might result in a trickle, rather than a flood, of new antibiotics onto the market in the near future.

  • 46.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Impact of homologous recombination and gene conversion on genomic organisation and stability1999In: Organization of the prokaryotic genome / [ed] Robert L. Charlebois, American Society for Microbiology, Washington, USA , 1999, p. 109-128Chapter in book (Other academic)
  • 47.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Selection and Evolution of Resistance to Antimicrobial Drugs2014In: IUBMB Life - A Journal of the International Union of Biochemistry and Molecular Biology, ISSN 1521-6543, E-ISSN 1521-6551, Vol. 66, no 8, p. 521-529Article, review/survey (Refereed)
    Abstract [en]

    The overuse and misuse of antibiotics over many years has selected a high frequency of resistance among medically important bacterial pathogens. The evolution of resistance is complex, frequently involving multiple genetic alterations that minimize biological fitness costs and/or increase the resistance level. Resistance is selected at very low drug concentrations, such as found widely distributed in the environment, and this selects for resistant mutants with a high fitness. Once resistance with high fitness is established in a community it is very difficult to reduce its frequency. Addressing the problem of resistance is essential if we are to ensure a future where we can continue to enjoy effective medical control of bacterial infections. This will require several actions including the discovery and development of novel antibiotics, the creation of a continuous pipeline of drug discovery, and the implementation of effective global antibiotic stewardship to reduce the misuse of antibiotics and their release into the environment.

  • 48.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Using the power of genetic suppressors to probe the essential functions of RNase E2016In: Current Genetics, ISSN 0172-8083, E-ISSN 1432-0983, Vol. 62, no 1, p. 53-57Article, review/survey (Refereed)
    Abstract [en]

    This review describes how, using the power of genetic suppressor analysis, mRNA turnover in bacteria was shown to be an essential function of RNase E. RNase E is an essential multifunctional enzyme in bacteria, involved in the processing of stable RNAs to their mature forms (rRNAs and tRNAs) and in the turnover of most mRNAs. Genetic suppressor analysis was successfully used to address whether mRNA turnover is one of the essential functions of RNase E. Conditional lethal mutations in rne were shown to be suppressible by three different classes of extragenic suppressors, including a class that caused overexpression of RelE. The only known function of RelE is the cleavage of mRNA in the ribosomal A-site. Suppression of the conditional lethal defect in rne by RelE overexpression provides strong genetic evidence that mRNA turnover is one of the essential functions of RNase E. Several hypotheses that could explain why mRNA turnover is essential are discussed. Suppressor analysis is an old-fashioned but very powerful approach that can be usefully applied to address a wide variety of important questions in biology and genetics. In this work suppressor analysis has revealed that mRNA turnover is an essential function of RNase E, a conclusion that raises a host of interesting questions for future research.

  • 49.
    Hughes, Diarmaid
    et al.
    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.
    Adventures with mutation and selection in beehive and cow country2010In: The Lure of Bacterial Genetics: a Tribute to John Roth / [ed] Maloy, S., Hughes, K.T. and Casadesus, J, Washington, DC: ASM Press , 2010, p. 245-253Chapter in book (Other academic)
  • 50.
    Hughes, Diarmaid
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Andersson, Dan I
    Antibiotic Development and Resistance2001Book (Refereed)
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