A genome-wide screen for bacterial envelope biogenesis mutants identifies a novel factor involved in cell wall precursor metabolism.
Paradis-Bleau, C., Kritikos, G., Orlova, K., Typas, A. & Bernhardt, T.G.
PLoS Genet. 2014 Jan;10(1):e1004056. doi: 10.1371/journal.pgen.1004056. Epub 2014Jan 2.
The cell envelope of Gram-negative bacteria is a formidable barrier that is difficult for antimicrobial drugs to penetrate. Thus, the list of treatments effective against these organisms is small and with the rise of new resistance mechanisms is shrinking rapidly. New therapies to treat Gram-negative bacterial infections are therefore sorely needed. This goal will be greatly aided by a detailed mechanistic understanding of envelope assembly. Although excellent progress in the identification of essential envelope biogenesis systems has been made in recent years, many aspects of the process remain to be elucidated. We therefore developed a simple, quantitative, and high-throughput assay for mutants with envelope biogenesis defects and used it to screen an ordered single-gene deletion library of Escherichia coli. The screen was robust and correctly identified numerous mutants known to be involved in envelope assembly. Importantly, the screen also implicated 102 genes of unknown function as encoding factors that likely impact envelope biogenesis. As a proof of principle, one of these factors, ElyC (YcbC), was characterized further and shown to play a critical role in the metabolism of the essential lipid carrier used for the biogenesis of cell wall and other bacterial surface polysaccharides. Further analysis of the function of ElyC and other hits identified in our screen is likely to uncover a wealth of new information about the biogenesis of the Gram-negative envelope and the vulnerabilities in the system suitable for drug targeting. Moreover, the screening assay described here should be readily adaptable to other organisms to study the biogenesis of different envelope architectures.
Country-specific antibiotic use practices impact the human gut resistome.
Forslund, K., Sunagawa, S., Kultima, J.R., Mende, D.R., Arumugam, M., Typas, A. & Bork, P.
Genome Res. 2013 Jul;23(7):1163-9. doi: 10.1101/gr.155465.113. Epub 2013 Apr 8.
Despite increasing concerns over inappropriate use of antibiotics in medicine and food production, population-level resistance transfer into the human gut microbiota has not been demonstrated beyond individual case studies. To determine the "antibiotic resistance potential" for entire microbial communities, we employ metagenomic data and quantify the totality of known resistance genes in each community (its resistome) for 68 classes and subclasses of antibiotics. In 252 fecal metagenomes from three countries, we show that the most abundant resistance determinants are those for antibiotics also used in animals and for antibiotics that have been available longer. Resistance genes are also more abundant in samples from Spain, Italy, and France than from Denmark, the United States, or Japan. Where comparable country-level data on antibiotic use in both humans and animals are available, differences in these statistics match the observed resistance potential differences. The results are robust over time as the antibiotic resistance determinants of individuals persist in the human gut flora for at least a year.
High-throughput approaches to understanding gene function and mapping network architecture in bacteria.
Brochado, A.R. & Typas, A.
Curr Opin Microbiol. 2013 Apr;16(2):199-206. doi: 10.1016/j.mib.2013.01.008. Epub2013 Feb 9.
Advances in sequencing technology have provided an unprecedented view of bacterial diversity, along with a daunting number of novel genes. Within this new reality lies the challenge of developing large-scale approaches to assign function to the new genes and place them in pathways. Here, we highlight recent advances on this front, focusing on how high-throughput gene-gene, gene-drug and drug-drug interactions can yield functional and mechanistic inferences in bacteria.
Fe-S cluster biosynthesis controls uptake of aminoglycosides in a ROS-less death pathway.
Ezraty, B., Vergnes, A., Banzhaf, M., Duverger, Y., Huguenot, A., Brochado, A.R., Su, S.Y., Espinosa, L., Loiseau, L., Py, B., Typas, A. & Barras, F.
Science. 2013 Jun 28;340(6140):1583-7. doi: 10.1126/science.1238328.
All bactericidal antibiotics were recently proposed to kill by inducing reactive oxygen species (ROS) production, causing destabilization of iron-sulfur (Fe-S) clusters and generating Fenton chemistry. We find that the ROS response is dispensable upon treatment with bactericidal antibiotics. Furthermore, we demonstrate that Fe-S clusters are required for killing only by aminoglycosides. In contrast to cells, using the major Fe-S cluster biosynthesis machinery, ISC, cells using the alternative machinery, SUF, cannot efficiently mature respiratory complexes I and II, resulting in impendence of the proton motive force (PMF), which is required for bactericidal aminoglycoside uptake. Similarly, during iron limitation, cells become intrinsically resistant to aminoglycosides by switching from ISC to SUF and down-regulating both respiratory complexes. We conclude that Fe-S proteins promote aminoglycoside killing by enabling their uptake.
From the regulation of peptidoglycan synthesis to bacterial growth and morphology.
Typas, A., Banzhaf, M., Gross, C.A. & Vollmer, W.
Nat Rev Microbiol. 2012 Feb;10(2):123-36. doi: 10.1038/nrmicro2677.
How bacteria grow and divide while retaining a defined shape is a fundamental question in microbiology, but technological advances are now driving a new understanding of how the shape-maintaining bacterial peptidoglycan sacculus grows. In this Review, we highlight the relationship between peptidoglycan synthesis complexes and cytoskeletal elements, as well as recent evidence that peptidoglycan growth is regulated from outside the sacculus in Gram-negative bacteria. We also discuss how growth of the sacculus is sensitive to mechanical force and nutritional status, and describe the roles of peptidoglycan hydrolases in generating cell shape and of D-amino acids in sacculus remodelling.
Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo.
Oh, E., Becker, A.H., Sandikci, A., Huber, D., Chaba, R., Gloge, F., Nichols, R.J., Typas, A., Gross, C.A., Kramer, G., Weissman, J.S. & Bukau, B.
Cell. 2011 Dec 9;147(6):1295-308.
As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting, and folding factors. Here, we present a selective ribosome profiling strategy that enables global monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the Escherichia coli chaperone trigger factor (TF) reveal that, though TF can interact with many polypeptides, beta-barrel outer-membrane proteins are the most prominent substrates. Loss of TF leads to broad outer-membrane defects and premature, cotranslational protein translocation. Whereas in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after approximately 100 amino acids are translated. Moreover, excess TF interferes with cotranslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting, and folding factors. PAPERCLIP:
Escherichia coli sigma senses sequence and conformation of the promoter spacer region.
Singh, S.S., Typas, A., Hengge, R. & Grainger, D.C.
Nucleic Acids Res. 2011 Jul;39(12):5109-18. Epub 2011 Mar 11.
In bacteria, promoter identification by RNA polymerase is mediated by a dissociable sigma factor. The housekeeping sigma(70) factor of Escherichia coli recognizes two well characterized DNA sequence elements, known as the '-10' and '-35' hexamers. These elements are separated by 'spacer' DNA, the sequence of which is generally considered unimportant. Here, we use a combination of bioinformatics, genetics and biochemistry to show that sigma(70) can sense the sequence and conformation of the promoter spacer region. Our data illustrate how alterations in spacer region sequence can increase promoter activity. This stimulatory effect requires sigma(70) side chain R451, which is located in close proximity to the non-template strand at promoter position -18. Conversely, R451 is not required to mediate transcriptional stimulation by improvement of the -10 element. Mutation of sigma(70) residue R451, which is highly conserved, results in reduced growth rate, consistent with a central role in promoter recognition.
Phenotypic landscape of a bacterial cell.
Nichols, R.J., Sen, S., Choo, Y.J., Beltrao, P., Zietek, M., Chaba, R., Lee, S., Kazmierczak, K.M., Lee, K.J., Wong, A., Shales, M., Lovett, S., Winkler, M.E., Krogan, N.J., Typas, A. & Gross, C.A.
Cell. 2011 Jan 7;144(1):143-56. Epub 2010 Dec 23.
The explosion of sequence information in bacteria makes developing high-throughput, cost-effective approaches to matching genes with phenotypes imperative. Using E. coli as proof of principle, we show that combining large-scale chemical genomics with quantitative fitness measurements provides a high-quality data set rich in discovery. Probing growth profiles of a mutant library in hundreds of conditions in parallel yielded > 10,000 phenotypes that allowed us to study gene essentiality, discover leads for gene function and drug action, and understand higher-order organization of the bacterial chromosome. We highlight new information derived from the study, including insights into a gene involved in multiple antibiotic resistance and the synergy between a broadly used combinatory antibiotic therapy, trimethoprim and sulfonamides. This data set, publicly available at http://ecoliwiki.net/tools/chemgen/, is a valuable resource for both the microbiological and bioinformatic communities, as it provides high-confidence associations between hundreds of annotated and uncharacterized genes as well as inferences about the mode of action of several poorly understood drugs.
Regulation of peptidoglycan synthesis by outer-membrane proteins.
Typas, A., Banzhaf, M., van den Berg van Saparoea, B., Verheul, J., Biboy, J., Nichols, R.J., Zietek, M., Beilharz, K., Kannenberg, K., von Rechenberg, M., Breukink, E., den Blaauwen, T., Gross, C.A. & Vollmer, W.
Cell. 2010 Dec 23;143(7):1097-109.
Growth of the mesh-like peptidoglycan (PG) sacculus located between the bacterial inner and outer membranes (OM) is tightly regulated to ensure cellular integrity, maintain cell shape, and orchestrate division. Cytoskeletal elements direct placement and activity of PG synthases from inside the cell, but precise spatiotemporal control over this process is poorly understood. We demonstrate that PG synthases are also controlled from outside of the sacculus. Two OM lipoproteins, LpoA and LpoB, are essential for the function, respectively, of PBP1A and PBP1B, the major E. coli bifunctional PG synthases. Each Lpo protein binds specifically to its cognate PBP and stimulates its transpeptidase activity, thereby facilitating attachment of new PG to the sacculus. LpoB shows partial septal localization, and our data suggest that the LpoB-PBP1B complex contributes to OM constriction during cell division. LpoA/LpoB and their PBP-docking regions are restricted to gamma-proteobacteria, providing models for niche-specific regulation of sacculus growth.
High-throughput, quantitative analyses of genetic interactions in E. coli.
Typas, A., Nichols, R.J., Siegele, D.A., Shales, M., Collins, S.R., Lim, B., Braberg, H., Yamamoto, N., Takeuchi, R., Wanner, B.L., Mori, H., Weissman, J.S., Krogan, N.J. & Gross, C.A.
Nat Methods. 2008 Sep;5(9):781-7.
Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor-driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli.
Stationary phase reorganisation of the Escherichia coli transcription machinery by Crl protein, a fine-tuner of sigmas activity and levels.
Typas, A., Barembruch, C., Possling, A. & Hengge, R.
EMBO J. 2007 Mar 21;26(6):1569-78. Epub 2007 Mar 1.
Upon environmental changes, bacteria reschedule gene expression by directing alternative sigma factors to core RNA polymerase (RNAP). This sigma factor switch is achieved by regulating relative amounts of alternative sigmas and by decreasing the competitiveness of the dominant housekeeping sigma(70). Here we report that during stationary phase, the unorthodox Crl regulator supports a specific sigma factor, sigma(S) (RpoS), in its competition with sigma(70) for core RNAP by increasing the formation of sigma(S)-containing RNAP holoenzyme, Esigma(S). Consistently, Crl has a global regulatory effect in stationary phase gene expression exclusively through sigma(S), that is, on sigma(S)-dependent genes only. Not a specific promoter motif, but sigma(S) availability determines the ability of Crl to exert its function, rendering it of major importance at low sigma(S) levels. By promoting the formation of Esigma(S), Crl also affects partitioning of sigma(S) between RNAP core and the proteolytic sigma(S)-targeting factor RssB, thereby playing a dual role in fine-tuning sigma(S) proteolysis. In conclusion, Crl has a key role in reorganising the Escherichia coli transcriptional machinery and global gene expression during entry into stationary phase.
The molecular basis of selective promoter activation by the sigmaS subunit of RNA polymerase.
Typas, A., Becker, G. & Hengge, R.
Mol Microbiol. 2007 Mar;63(5):1296-306.
Different environmental stimuli cause bacteria to exchange the sigma subunit in the RNA polymerase (RNAP) and, thereby, tune their gene expression according to the newly emerging needs. Sigma factors are usually thought to recognize clearly distinguishable promoter DNA determinants, and thereby activate distinct gene sets, known as their regulons. In this review, we illustrate how the principle sigma factor in stationary phase and in stressful conditions in Escherichia coli, sigmaS (RpoS), can specifically target its large regulon in vivo, although it is known to recognize the same core promoter elements in vitro as the housekeeping sigma factor, sigma70 (RpoD). Variable combinations of cis-acting promoter features and trans-acting protein factors determine whether a promoter is recognized by RNAP containing sigmaS or sigma70, or by both holoenzymes. How these promoter features impose sigmaS selectivity is further discussed. Moreover, additional pathways allow sigmaS to compete more efficiently than sigma70 for limiting amounts of core RNAP (E) and thereby enhance EsigmaS formation and effectiveness. Finally, these topics are discussed in the context of sigma factor evolution and the benefits a cell gains from retaining competing and closely related sigma factors with overlapping sets of target genes.
The -35 sequence location and the Fis-sigma factor interface determine sigmas selectivity of the proP (P2) promoter in Escherichia coli.
Typas, A., Stella, S., Johnson, R.C. & Hengge, R.
Mol Microbiol. 2007 Feb;63(3):780-96.
The P2 promoter of proP, encoding a transporter for proline and glycine betaine in Escherichia coli, is a unique paradigm, where master regulators of different growth stages, Fis and sigma(S) (RpoS), collaborate to achieve promoter activation. It is also the only case described where Fis functions as class II transcriptional activator (centred at -41). Here we show that the degenerate -35 sequence, and the location of the Fis binding site, which forces a suboptimal 16 bp spacing between the -35 and -10 elements, allow only sigma(S) but not sigma(70) to function at proP (P2). Moreover, the interface between Fis and sigma(S) seems better suited to sigma(S), due to a single residue difference between sigma(S) and sigma(70). Nevertheless, Fis can activate RNA polymerase containing sigma(70) at a proP (P2) promoter variant, in which a typical sigma(70)-35 recognition sequence has been introduced at a 17 bp distance from the -10 hexamer. In summary, we elucidate the rules that govern sigma factor selectivity in the presence of a class II activator, provide new insight into transcriptional activation by Fis from this position, and clarify, why the proP (P2) promoter is precisely activated during a short time window of the growth cycle, when Fis and sigma(S) are both present.
The sigmaS subunit of RNA polymerase as a signal integrator and network master regulator in the general stress response in Escherichia coli.
Klauck, E., Typas, A. & Hengge, R.
Sci Prog. 2007;90(Pt 2-3):103-27.
The sigmaS (RpoS) subunit of RNA polymerase in Escherichia coli is a key master regulator which allows this bacterial model organism and important pathogen to adapt to and survive environmentally rough times. While hardly present in rapidly growing cells, sigmaS strongly accumulates in response to many different stress conditions, partly replaces the vegetative sigma subunit in RNA polymerase and thereby reprograms this enzyme to transcribe sigmaS-dependent genes (up to 10% of the E. coli genes). In this review, we summarize the extremely complex regulation of sigmaS itself and multiple signal input at the level of this master regulator, we describe the way in which sigmaS specifically recognizes "stress" promoters despite their similarity to vegetative promoters, and, while being far from comprehensive, we give a short overview of the far-reaching physiological impact of sigmaS. With sigmaS being a central and multiple signal integrator and master regulator of hundreds of genes organized in regulatory cascades and sub-networks or regulatory modules, this system also represents a key model system for analyzing complex cellular information processing and a starting point for understanding the complete regulatory network of an entire cell.
Role of the spacer between the -35 and -10 regions in sigmas promoter selectivity in Escherichia coli.
Typas, A. & Hengge, R.
Mol Microbiol. 2006 Feb;59(3):1037-51.
In vitro, the sigma(s) subunit of RNA polymerase (RNAP), RpoS, recognizes nearly identical -35 and -10 promoter consensus sequences as the vegetative sigma70. In vivo, promoter selectivity of RNAP holoenzyme containing either sigma(s) (Esigma(s)) or sigma70 (Esigma70) seems to be achieved by the differential ability of the two holoenzymes to tolerate deviations from the promoter consensus sequence. In this study, we suggest that many natural sigma(s)-dependent promoters possess a -35 element, a feature that has been considered as not conserved among sigma(s)-dependent promoters. These -35 hexamers are mostly non-optimally spaced from the -10 region, but nevertheless functional. A +/- 2 bp deviation from the optimal spacer length of 17 bp or the complete absence of a -35 consensus sequence decreases overall promoter activity, but at the same time favours Esigma(s) in its competition with Esigma70 for promoter recognition. On the other hand, the reduction of promoter activity due to shifting of the -35 element can be counterbalanced by an activity-stimulating feature such as A/T-richness of the spacer region without compromising Esigma(s) selectivity. Based on mutational analysis of sigma(s), we suggest a role of regions 2.5 and 4 of sigma(s) in sensing sub-optimally located -35 elements.
Differential ability of sigma(s) and sigma70 of Escherichia coli to utilize promoters containing half or full UP-element sites.
Typas, A. & Hengge, R.
Mol Microbiol. 2005 Jan;55(1):250-60.
The sigma(s) subunit of RNA polymerase (RNAP) is the master regulator of the general stress response in Escherichia coli. Nevertheless, the selectivity of promoter recognition by the housekeeping sigma70-containing and sigma5-containing RNAP holoenzymes (Esigma70 and Esigma(s) respectively) is not yet fully clarified, as they both recognize nearly identical -35 and -10 promoter consensus sequences. In this study, we show that in a subset of promoters, Esigma(s) favours the presence of a distal UP-element half-site, and at the same time is unable to take advantage of a proximal half-site or a full UP-element. This is reflected by the frequent occurrence of distal UP-element half-sites in natural sigma(s)-dependent promoters and the absence of proximal half-sites. Esigma70, however, exhibits the opposite preference. The presence of the -35 element is a prerequisite for this differential behaviour. In the absence of the -35 element, half or full UP-element sites play no role in sigma selectivity, but the distal subsite leads to an equivalent, if not greater, transcriptional stimulation than the proximal one for both sigma factors. Finally, experiments using single amino acid substitutions of sigma(s) indicate that the foundation for this preference lies in an inability of sigma(s) to interact with the a subunit C-terminal domain.