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Garmendia, Eva
Publications (4 of 4) Show all publications
Garmendia, E., Brandis, G. & Hughes, D. (2018). Transcriptional Regulation Buffers Gene Dosage Effects on a Highly Expressed Operon in Salmonella. mBio, 9(5), Article ID e01446-18.
Open this publication in new window or tab >>Transcriptional Regulation Buffers Gene Dosage Effects on a Highly Expressed Operon in Salmonella
2018 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 9, no 5, article id e01446-18Article in journal (Refereed) Published
Abstract [en]

Highly expressed genes are commonly located close to the origin of replication of bacterial chromosomes (OriC). This location skew is thought to reflect selective advantages associated with gene dosage effects during the replication cycle. The expression of constitutively expressed genes can vary up to fivefold based on chromosomal location, but it is not clear what level of variation would occur in naturally regulated operons. We tested the magnitude of the chromosome location effect using EF-Tu (tufA, tufB), an abundant protein whose cellular level correlates with, and limits, the maximum growth rate. We translocated the Salmonella tufB operon to four locations across the chromosome. The distance from OriC had only a small effect on growth rate, consistent with this operon having the natural ability to upregulate expression and compensate for reduced gene dosage. In contrast, when the total EF-Tu concentration was limiting for the growth rate (tufA deleted), we observed a strong gene dosage effect when tufB was located further from OriC. However, only a short period of experimental evolution was required before the bacteria adapted to this EF-Tu starvation situation by acquiring genetic changes that increased expression levels from the translocated tufB gene, restoring growth rates. Our findings demonstrate that, at least for the tufB operon, gene dosage is probably not the dominant force selecting for a chromosomal location close to OriC. We suggest that the colocation of highly expressed genes close to OriC might instead be selected because it enhances their coregulation during various growth states, with gene dosage being a secondary benefit. IMPORTANCE A feature of bacterial chromosomes is that highly expressed essential genes are usually located close to the origin of replication. Because bacteria have overlapping cycles of replication, genes located close to the origin will often be present in multiple copies, and this is thought to be of selective benefit where high levels of expression support high growth rate. However, the magnitude of this selective effect and whether other forces could be at play are poorly understood. To study this, we translocated a highly expressed essential operon, tufB, to different locations and measured growth fitness. We found that transcriptional regulation buffered the effects of translocation and that even under conditions where growth rate was reduced, genetic changes that increased the expression of tufB were easily and rapidly selected. We conclude, at least for tufB, that forces other than gene dosage may be significant in selecting for chromosomal location.

Place, publisher, year, edition, pages
AMER SOC MICROBIOLOGY, 2018
Keywords
chromosome organization, EF-Tu, location bias, tufB
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-369516 (URN)10.1128/mBio.01446-18 (DOI)000449472200015 ()30206172 (PubMedID)
Funder
Swedish Research Council, 2016-04449Swedish Research Council, 2017-03953Knut and Alice Wallenberg Foundation, 2009.0251Carl Tryggers foundation , CTS16: 194Carl Tryggers foundation , CTS17: 204
Available from: 2018-12-17 Created: 2018-12-17 Last updated: 2018-12-17Bibliographically approved
Garmendia, E. (2017). A Unified Multitude: Experimental Studies of Bacterial Chromosome Organization. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>A Unified Multitude: Experimental Studies of Bacterial Chromosome Organization
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Bacteria are many, old and varied; different bacterial species have been evolving for millions of years and show many disparate life-styles and types of metabolism. Nevertheless, some of the characteristics regarding how bacteria organize their chromosomes are relatively conserved, suggesting that they might be both ancient and important, and that selective pressures inhibit their modification. This thesis aims to study some of these characteristics experimentally, assessing how changes affect bacterial growth, and how, after changing conserved features, bacteria might evolve.

First, we experimentally tested what are the constraints on the horizontal transfer of a gene highly important for bacterial growth. Second, we investigated the significance of the location and orientation of a highly expressed and essential operon; and we experimentally evolved strains with suboptimal locations and orientations to assess how bacteria could adapt to these changes. Thirdly, we sought to understand the accessibility of different regions of the bacterial chromosome to engage in homologous recombination. And lastly, we constructed bacterial strains with chromosomal inversions to assess what effect the inversions had on growth rate, and how bacteria carrying costly inversions could evolve to reduce these costs.

The results provide evidence for different selective forces acting to conserve these chromosome organizational traits. Accordingly, we found that evolutionary distance, functional conservation, suboptimal expression and impaired network connectivity of a gene can affect the successful transfer of genes between bacterial species. We determined that relative location of an essential and highly expressed operon is critical for supporting fast growth rate, and that its location seems to be more important than its orientation. We also found that both the location, and relative orientation of separated duplicate sequences can affect recombination rates between these sequences in different regions of the chromosome. Finally, the data suggest that the importance of having the two arms of a circular bacterial chromosome approximately equal in size is a strong selective force acting against certain type of chromosomal inversions.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. p. 66
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1392
Keywords
bacterial evolution, chromosome organization and structure, chromosomal inversions, EF-Tu, horizontal gene transfer
National Category
Microbiology Genetics Evolutionary Biology
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-332471 (URN)978-91-513-0140-2 (ISBN)
Public defence
2017-12-15, room B42, Uppsala Biomedical Centre (BMC), Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2017-11-24 Created: 2017-10-29 Last updated: 2018-03-07
Kacar, B., Garmendia, E., Tuncbag, N., Andersson, D. I. & Hughes, D. (2017). Functional Constraints on Replacing an Essential Gene with Its Ancient and Modern Homologs. mBio, 8(4), Article ID e01276-17.
Open this publication in new window or tab >>Functional Constraints on Replacing an Essential Gene with Its Ancient and Modern Homologs
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2017 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 4, article id e01276-17Article in journal (Refereed) Published
Abstract [en]

Genes encoding proteins that carry out essential informational tasks in the cell, in particular where multiple interaction partners are involved, are less likely to be transferable to a foreign organism. Here, we investigated the constraints on transfer of a gene encoding a highly conserved informational protein, translation elongation factor Tu (EF-Tu), by systematically replacing the endogenous tufA gene in the Escherichia coli genome with its extant and ancestral homologs. The extant homologs represented tuf variants from both near and distant homologous organisms. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 billion years ago (bya). Our results demonstrate that all of the foreign tuf genes are transferable to the E. coli genome, provided that an additional copy of the EF-Tu gene, tufB, remains present in the E. coli genome. However, when the tufB gene was removed, only the variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth which demonstrates the limited functional interchangeability of E. coli tuf with its homologs. Relative bacterial fitness correlated with the evolutionary distance of the extant tuf homologs inserted into the E. coli genome. This reduced fitness was associated with reduced levels of EF-Tu and reduced rates of protein synthesis. Increasing the expression of tuf partially ameliorated these fitness costs. In summary, our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.IMPORTANCE Horizontal gene transfer (HGT) is a fundamental driving force in bacterial evolution. However, whether essential genes can be acquired by HGT and whether they can be acquired from distant organisms are very poorly understood. By systematically replacing tuf with ancestral homologs and homologs from distantly related organisms, we investigated the constraints on HGT of a highly conserved gene with multiple interaction partners. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 bya. Only variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth, demonstrating the limited functional interchangeability of E. coli tuf with its homologs. Our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.

Keywords
EF-Tu, ancient genes, horizontal gene transfer, proteobacteria, tuf
National Category
Microbiology
Research subject
Biology with specialization in Microbiology; Biology with specialization in Molecular Biology; Biology with specialization in Evolutionary Genetics
Identifiers
urn:nbn:se:uu:diva-330501 (URN)10.1128/mBio.01276-17 (DOI)000409384300045 ()28851849 (PubMedID)
Available from: 2017-10-02 Created: 2017-10-02 Last updated: 2017-12-19Bibliographically approved
Larsson Hammarlöf, D., Bergman, J. M., Garmendia, E. & Hughes, D. (2015). Turnover of mRNAs is one of the essential functions of RNase E. Molecular Microbiology, 98(1), 34-45
Open this publication in new window or tab >>Turnover of mRNAs is one of the essential functions of RNase E
2015 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 98, no 1, p. 34-45Article in journal (Refereed) Published
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.

National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-265838 (URN)10.1111/mmi.13100 (DOI)000362725800004 ()26094815 (PubMedID)
Funder
Swedish Research CouncilSwedish Foundation for Strategic Research Knut and Alice Wallenberg Foundation
Available from: 2015-11-03 Created: 2015-11-03 Last updated: 2017-12-01Bibliographically approved
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