Stationary-Phase Regulation of RpoS Translation in Escherichia coli

ABSTRACT In enteric bacteria, adaptation to a number of different stresses is mediated by the RpoS protein, one of several sigma factors that collectively allow a tailored transcriptional response to environmental cues. Stress stimuli including low temperature, osmotic shock, nutrient limitation, and growth to stationary phase (SP) all result in a substantial increase in RpoS abundance and activity. The mechanism of regulation depends on the specific signal but may occur at the level of transcription, translation, protein activity, or targeted proteolysis. In both Escherichia coli and Salmonella enterica, SP induction of RpoS in rich medium is >30 fold and includes effects on both transcription and translation. Recently, we found that SP control of rpoS transcription in S. enterica involves repression of the major rpoS promoter during exponential phase by the global transcription factor Fis. Working primarily with E. coli, we now show that 24 nucleotides of the rpoS ribosome-binding site (RBS) are necessary and sufficient for a large part of the increase in rpoS translation as cells grow to SP. Genetic evidence points to an essential role for the leader nucleotides just upstream of the Shine-Dalgarno sequence but is conflicted on the question of whether sequence or structure is important. SP regulation of rpoS is conserved between E. coli and S. enterica. When combined with an fis mutation to block transcriptional effects, replacement of the rpoS RBS sequence by the lacZ RBS eliminates nearly all SP induction of RpoS.

Download Full-text

Role of rpoS in the regulation of glyoxalase III in Escherichia coli.

Acta Biochimica Polonica ◽

10.18388/abp.2004_3570 ◽

2004 ◽

Vol 51 (3) ◽

pp. 857-860 ◽

Cited By ~ 10

Author(s):

Ludmil Benov ◽

Fatima Sequeira ◽

Anees F Beema

Keyword(s):

Escherichia Coli ◽

Stationary Phase ◽

Enteric Bacteria ◽

Glyoxalase I ◽

Aldehyde Reductase ◽

E Coli ◽

Rpos Mutant ◽

Phosphorylated Sugars ◽

Glyoxalase Iii

Methylglyoxal is an endogenous electrophile produced in Escherichia coli by the enzyme methylglyoxal synthase to limit the accumulation of phosphorylated sugars. In enteric bacteria methylglyoxal is detoxified by the glutathione-dependent glyoxalase I/II system, by glyoxalase III, and by aldehyde reductase and alcohol dehydrogenase. Here we demonstrate that glyoxalase III is a stationary-phase enzyme. Its activity reached a maximum at the entry into the stationary phase and remained high for at least 20 h. An rpoS- mutant displayed normal glyoxalase I and II activities but was unable to induce glyoxalase III in stationary phase. It thus appears that glyoxalase III is regulated by rpoS and might be important for survival of non-growing E. coli cultures.

Download Full-text

Mannose-Binding Activity of Escherichia coli: a Determinant of Attachment and Ingestion of the Bacteria by Macrophages

Infection and Immunity ◽

10.1128/iai.29.2.417-424.1980 ◽

1980 ◽

Vol 29 (2) ◽

pp. 417-424

Author(s):

Zvi Bar-Shavit ◽

Rachel Goldman ◽

Itzhak Ofek ◽

Nathan Sharon ◽

David Mirelman

Keyword(s):

Escherichia Coli ◽

Stationary Phase ◽

Pathogenic Bacteria ◽

Binding Activity ◽

Exponential Phase ◽

Restrictive Temperature ◽

Filamentous Bacteria ◽

Phagocytic Cells ◽

E Coli ◽

Mannose Binding

Recently, it was suggested that a mannose-specific lectin on the bacterial cell surface is responsible for the recognition by phagocytic cells of certain nonopsonized Escherichia coli strains. In this study we assessed the interaction of two strains of E. coli at different phases of growth with a monolayer of mouse peritoneal macrophages and developed a direct method with [ 14 C]mannan to quantitate the bacterial mannose-binding activity. Normal-sized bacteria were obtained from logarithmic and stationary phases of growth. Nonseptated filamentous cells were formed by growing the organisms in the presence of cephalexin or at a restrictive temperature. Attachment to macrophages of all bacterial forms was inhibited by methyl α- d -mannoside and mannan but not by other sugars tested. The attachment of stationary phase and filamentous bacteria to macrophages, as well as their mannose-binding activity, was similar, whereas in the exponential-phase bacteria they were markedly reduced. The results show a linear relation between the two parameters ( R = 0.98, P < 0.001). The internalization of the filamentous cells attached to macrophages during 45 min of incubation was much less efficient (20%) compared to that of exponential-phase, stationary-phase, or antibody-coated filamentous bacteria (90%). The results indicate that the mannose-binding activity of E. coli determines the recognition of the organisms by phagocytes. They further suggest that administration of β-lactam antibiotics may impair elimination of certain pathogenic bacteria by inducing the formation of filaments which are inefficiently internalized by the host's phagocytic cells.

Download Full-text

Functional Analysis of Genes Comprising the Locus of Heat Resistance in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01400-17 ◽

2017 ◽

Vol 83 (20) ◽

Cited By ~ 10

Author(s):

Ryan Mercer ◽

Oanh Nguyen ◽

Qixing Ou ◽

Lynn McMullen ◽

Michael G. Gänzle

Keyword(s):

Escherichia Coli ◽

Heat Shock ◽

Heat Shock Proteins ◽

Heat Resistance ◽

Growth Phase ◽

Genomic Island ◽

Enteric Bacteria ◽

Content Type ◽

E Coli ◽

Shock Proteins

ABSTRACT The locus of heat resistance (LHR) is a 15- to 19-kb genomic island conferring exceptional heat resistance to organisms in the family Enterobacteriaceae, including pathogenic strains of Salmonella enterica and Escherichia coli. The complement of LHR-comprising genes that is necessary for heat resistance and the stress-induced or growth-phase-induced expression of LHR-comprising genes are unknown. This study determined the contribution of the seven LHR-comprising genes yfdX1 GI, yfdX2, hdeD GI, orf11, trx GI, kefB, and psiE GI by comparing the heat resistances of E. coli strains harboring plasmid-encoded derivatives of the different LHRs in these genes. (Genes carry a subscript “GI” [genomic island] if an ortholog of the same gene is present in genomes of E. coli.) LHR-encoded heat shock proteins sHSP20, ClpKGI, and sHSPGI are not sufficient for the heat resistance phenotype; YfdX1, YfdX2, and HdeD are necessary to complement the LHR heat shock proteins and to impart a high level of resistance. Deletion of trx GI, kefB, and psiE GI from plasmid-encoded copies of the LHR did not significantly affect heat resistance. The effect of the growth phase and the NaCl concentration on expression from the putative LHR promoter p2 was determined by quantitative reverse transcription-PCR and by a plasmid-encoded p2:GFP promoter fusion. The expression levels of exponential- and stationary-phase E. coli cells were not significantly different, but the addition of 1% NaCl significantly increased LHR expression. Remarkably, LHR expression in E. coli was dependent on a chromosomal copy of evgA. In conclusion, this study improved our understanding of the genes required for exceptional heat resistance in E. coli and factors that increase their expression in food. IMPORTANCE The locus of heat resistance (LHR) is a genomic island conferring exceptional heat resistance to several foodborne pathogens. The exceptional level of heat resistance provided by the LHR questions the control of pathogens by current food processing and preparation techniques. The function of LHR-comprising genes and their regulation, however, remain largely unknown. This study defines a core complement of LHR-encoded proteins that are necessary for heat resistance and demonstrates that regulation of the LHR in E. coli requires a chromosomal copy of the gene encoding EvgA. This study provides insight into the function of a transmissible genomic island that allows otherwise heat-sensitive enteric bacteria, including pathogens, to lead a thermoduric lifestyle and thus contributes to the detection and control of heat-resistant enteric bacteria in food.

Download Full-text

Multiplex Polymerase Chain Reaction Assay for Identification of Enterotoxigenic Escherichia Coli Strains

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870101300405 ◽

2001 ◽

Vol 13 (4) ◽

pp. 308-311 ◽

Cited By ~ 22

Author(s):

Jacek Osek

Keyword(s):

Escherichia Coli ◽

Polymerase Chain Reaction ◽

Multiplex Polymerase Chain Reaction ◽

Direct Determination ◽

Enteric Bacteria ◽

Enterotoxigenic Escherichia Coli ◽

Chain Reaction ◽

Gram Negative ◽

E Coli ◽

Polymerase Chain

A multiplex polymerase chain reaction (PCR) system was developed for identification of enterotoxigenic Escherichia coli (ETEC) strains and to differentiate them from other gram negative enteric bacteria. This test simultaneously amplifies heat-labile (LTI) and heat-stable (STI and STII) toxin sequences and the E. coli-specific universal stress protein ( uspA). The specificity of the method was validated by single PCR tests performed with the reference E. coli and non- E. coli strains and with bacteria isolated from pig feces. The multiplex PCR allowed the rapid and specific identification of enterotoxin-positive E. coli and may be used as a method for direct determination of ETEC and to differentiate them from other E. coli and gram-negative enteric isolates.

Download Full-text

The l-Isoaspartyl Protein Repair Methyltransferase Enhances Survival of Aging Escherichia coli Subjected to Secondary Environmental Stresses

Journal of Bacteriology ◽

10.1128/jb.180.10.2623-2629.1998 ◽

1998 ◽

Vol 180 (10) ◽

pp. 2623-2629 ◽

Cited By ~ 57

Author(s):

Jonathan E. Visick ◽

Hui Cai ◽

Steven Clarke

Keyword(s):

Escherichia Coli ◽

Stationary Phase ◽

Protein Denaturation ◽

Environmental Stresses ◽

Protein Repair ◽

Repeated Heating ◽

E Coli ◽

Competitive Disadvantage ◽

Biological World

ABSTRACT Like its homologs throughout the biological world, thel-isoaspartyl protein repair methyltransferase ofEscherichia coli, encoded by the pcm gene, can convert abnormal l-isoaspartyl residues in proteins (which form spontaneously from asparaginyl or aspartyl residues) to normal aspartyl residues. Mutations in pcm were reported to greatly reduce survival in stationary phase and when cells were subjected to heat or osmotic stresses (C. Li and S. Clarke, Proc. Natl. Acad. Sci. USA 89:9885–9889, 1992). However, we subsequently demonstrated that those strains had a secondary mutation inrpoS, which encodes a stationary-phase-specific sigma factor (J. E. Visick and S. Clarke, J. Bacteriol. 179:4158–4163, 1997). We now show that the rpoS mutation, resulting in a 90% decrease in HPII catalase activity, can account for the previously observed phenotypes. We further demonstrate that a new pcmmutant lacks these phenotypes. Interestingly, the newly constructedpcm mutant, when maintained in stationary phase for extended periods, is susceptible to environmental stresses, including exposure to methanol, oxygen radical generation by paraquat, high salt concentrations, and repeated heating to 42°C. The pcmmutation also results in a competitive disadvantage in stationary-phase cells. All of these phenotypes can be complemented by a functionalpcm gene integrated elsewhere in the chromosome. These data suggest that protein denaturation and isoaspartyl formation may act synergistically to the detriment of aging E. coli and that the repair methyltransferase can play a role in limiting the accumulation of the potentially disruptive isoaspartyl residues in vivo.

Download Full-text

The Escherichia coli K-12 NarL and NarP Proteins Insulate the nrf Promoter from the Effects of Integration Host Factor

Journal of Bacteriology ◽

10.1128/jb.00975-06 ◽

2006 ◽

Vol 188 (21) ◽

pp. 7449-7456 ◽

Cited By ~ 21

Author(s):

Douglas F. Browning ◽

David J. Lee ◽

Alan J. Wolfe ◽

Jeffrey A. Cole ◽

Stephen J. W. Busby

Keyword(s):

Escherichia Coli ◽

Response Regulator ◽

Regulatory Region ◽

Enteric Bacteria ◽

Integration Host Factor ◽

Host Factor ◽

Receptor Protein ◽

E Coli ◽

Serovar Typhimurium ◽

K 12

ABSTRACT The Escherichia coli K-12 nrf operon promoter can be activated fully by the FNR protein (regulator of fumarate and nitrate reduction) binding to a site centered at position −41.5. FNR-dependent transcription is suppressed by integration host factor (IHF) binding at position −54, and this suppression is counteracted by binding of the NarL or NarP response regulator at position −74.5. The E. coli acs gene is transcribed from a divergent promoter upstream from the nrf operon promoter. Transcription from the major acsP2 promoter is dependent on the cyclic AMP receptor protein and is modulated by IHF and Fis binding at multiple sites. We show that IHF binding to one of these sites, located at position −127 with respect to the nrf promoter, has a positive effect on nrf promoter activity. This activation is dependent on the face of the DNA helix, independent of IHF binding at other locations, and found only when NarL/NarP are not bound at position −74.5. Binding of NarL/NarP appears to insulate the nrf promoter from the effects of IHF. The acs-nrf regulatory region is conserved in other pathogenic E. coli strains and related enteric bacteria but differs in Salmonella enterica serovar Typhimurium.

Download Full-text

Quantitative profiling and dynamics of mRNA modifications in Escherichia coli

10.26434/chemrxiv-2021-0wc9g-v3 ◽

2021 ◽

Author(s):

Dimitar Plamenov Petrov ◽

Steffen Kaiser ◽

Stefanie Kaiser ◽

Kirsten Jung

Keyword(s):

Mass Spectrometry ◽

Escherichia Coli ◽

Stationary Phase ◽

Exponential Growth ◽

Gel Electrophoresis ◽

E Coli ◽

Physiological Processes ◽

Mrna Methylation ◽

Important Regulator ◽

Quantitative Profiling

mRNA methylation is an important regulator of many physiological processes in eukaryotes but has not been studied in depth in prokaryotes. In contrast to the large number of eukaryotic mRNA modifications that have been described, N6-methyladenosine (m6A) is the only modification of bacterial mRNA identified to date. Here, we used a gel electrophoresis-based RNA separation method and quantitatively analyzed the mRNA-specific modification profile of Escherichia coli using mass spectrometry. In addition to m6A, we provide evidence for the presence of 7-methylguanosine (m7G), and we found first hints for 5-methylcytidine (m5C), N6,N6-dimethyladenosine (m6,6A), 1-methylguanosine (m1G), 5-methyluridine (m5U), and pseudouridine (Ψ) in the mRNA of E. coli, which implies that E. coli has a complex mRNA modification pattern. Furthermore, we observed changes in the abundance of some mRNA modifications during the transition of E. coli from the exponential growth to the stationary phase as well as upon exposure to stress. This study reveals a previously underestimated level of regulation between transcription and translation in bacteria.

Download Full-text

Biophysical Properties of Escherichia coli Cytoplasm in Stationary Phase by Superresolution Fluorescence Microscopy

mBio ◽

10.1128/mbio.00143-20 ◽

2020 ◽

Vol 11 (3) ◽

Author(s):

Yanyu Zhu ◽

Mainak Mustafi ◽

James C. Weisshaar

Keyword(s):

Escherichia Coli ◽

Fluorescence Microscopy ◽

Rna Polymerase ◽

Stationary Phase ◽

Exponential Growth ◽

Mean Square Displacement ◽

Quiescent State ◽

Chromosomal Dna ◽

Content Type ◽

E Coli

ABSTRACT In nature, bacteria must survive long periods of nutrient deprivation while maintaining the ability to recover and grow when conditions improve. This quiescent state is called stationary phase. The biochemistry of Escherichia coli in stationary phase is reasonably well understood. Much less is known about the biophysical state of the cytoplasm. Earlier studies of harvested nucleoids concluded that the stationary-phase nucleoid is “compacted” or “supercompacted,” and there are suggestions that the cytoplasm is “glass-like.” Nevertheless, stationary-phase bacteria support active transcription and translation. Here, we present results of a quantitative superresolution fluorescence study comparing the spatial distributions and diffusive properties of key components of the transcription-translation machinery in intact E. coli cells that were either maintained in 2-day stationary phase or undergoing moderately fast exponential growth. Stationary-phase cells are shorter and exhibit strong heterogeneity in cell length, nucleoid volume, and biopolymer diffusive properties. As in exponential growth, the nucleoid and ribosomes are strongly segregated. The chromosomal DNA is locally more rigid in stationary phase. The population-weighted average of diffusion coefficients estimated from mean-square displacement plots is 2-fold higher in stationary phase for both RNA polymerase (RNAP) and ribosomal species. The average DNA density is roughly twice as high as that in cells undergoing slow exponential growth. The data indicate that the stationary-phase nucleoid is permeable to RNAP and suggest that it is permeable to ribosomal subunits. There appears to be no need to postulate migration of actively transcribed genes to the nucleoid periphery. IMPORTANCE Bacteria in nature usually lack sufficient nutrients to enable growth and replication. Such starved bacteria adapt into a quiescent state known as the stationary phase. The chromosomal DNA is protected against oxidative damage, and ribosomes are stored in a dimeric structure impervious to digestion. Stationary-phase bacteria can recover and grow quickly when better nutrient conditions arise. The biochemistry of stationary-phase E. coli is reasonably well understood. Here, we present results from a study of the biophysical state of starved E. coli. Superresolution fluorescence microscopy enables high-resolution location and tracking of a DNA locus and of single copies of RNA polymerase (the transcription machine) and ribosomes (the translation machine) in intact E. coli cells maintained in stationary phase. Evidently, the chromosomal DNA remains sufficiently permeable to enable transcription and translation to occur. This description contrasts with the usual picture of a rigid stationary-phase cytoplasm with highly condensed DNA.

Download Full-text

Genomewide Mutational Diversity in Escherichia coli Population Evolving in Prolonged Stationary Phase

mSphere ◽

10.1128/msphere.00059-17 ◽

2017 ◽

Vol 2 (3) ◽

Cited By ~ 13

Author(s):

Savita Chib ◽

Farhan Ali ◽

Aswin Sai Narain Seshasayee

Keyword(s):

Escherichia Coli ◽

Genetic Diversity ◽

Stationary Phase ◽

Phenotypic Diversity ◽

Model System ◽

Content Type ◽

Neutral Drift ◽

E Coli ◽

Evolution By Natural Selection ◽

Over Time

ABSTRACT Prolonged stationary phase in bacteria, contrary to its name, is highly dynamic, with extreme nutrient limitation as a predominant stress. Stationary-phase cultures adapt by rapidly selecting a mutation(s) that confers a growth advantage in stationary phase (GASP). The phenotypic diversity of starving E. coli populations has been studied in detail; however, only a few mutations that accumulate in prolonged stationary phase have been described. This study documented the spectrum of mutations appearing in Escherichia coli during 28 days of prolonged starvation. The genetic diversity of the population increases over time in stationary phase to an extent that cannot be explained by random, neutral drift. This suggests that prolonged stationary phase offers a great model system to study adaptive evolution by natural selection. Prolonged stationary phase is an approximation of natural environments presenting a range of stresses. Survival in prolonged stationary phase requires alternative metabolic pathways for survival. This study describes the repertoire of mutations accumulating in starving Escherichia coli populations in lysogeny broth. A wide range of mutations accumulates over the course of 1 month in stationary phase. Single nucleotide polymorphisms (SNPs) constitute 64% of all mutations. A majority of these mutations are nonsynonymous and are located at conserved loci. There is an increase in genetic diversity in the evolving populations over time. Computer simulations of evolution in stationary phase suggest that the maximum frequency of mutations observed in our experimental populations cannot be explained by neutral drift. Moreover, there is frequent genetic parallelism across populations, suggesting that these mutations are under positive selection. Finally, functional analysis of mutations suggests that regulatory mutations are frequent targets of selection. IMPORTANCE Prolonged stationary phase in bacteria, contrary to its name, is highly dynamic, with extreme nutrient limitation as a predominant stress. Stationary-phase cultures adapt by rapidly selecting a mutation(s) that confers a growth advantage in stationary phase (GASP). The phenotypic diversity of starving E. coli populations has been studied in detail; however, only a few mutations that accumulate in prolonged stationary phase have been described. This study documented the spectrum of mutations appearing in Escherichia coli during 28 days of prolonged starvation. The genetic diversity of the population increases over time in stationary phase to an extent that cannot be explained by random, neutral drift. This suggests that prolonged stationary phase offers a great model system to study adaptive evolution by natural selection.

Download Full-text

In Vivo Modulation of a DnaJ Homolog, CbpA, by CbpM

Journal of Bacteriology ◽

10.1128/jb.01757-06 ◽

2007 ◽

Vol 189 (9) ◽

pp. 3635-3638 ◽

Cited By ~ 14

Author(s):

Matthew R. Chenoweth ◽

Nancy Trun ◽

Sue Wickner

Keyword(s):

Escherichia Coli ◽

Cell Growth ◽

Stationary Phase ◽

Specific Inhibitor ◽

Vitro Activity ◽

In Vitro Activity ◽

E Coli ◽

Hsp70 Chaperone

ABSTRACT CbpA, an Escherichia coli DnaJ homolog, can function as a cochaperone for the DnaK/Hsp70 chaperone system, and its in vitro activity can be modulated by CbpM. We discovered that CbpM specifically inhibits the in vivo activity of CbpA, preventing it from functioning in cell growth and division. Furthermore, we have shown that CbpM interacts with CbpA in vivo during stationary phase, suggesting that the inhibition of activity is a result of the interaction. These results reveal that the activity of the E. coli DnaK system can be regulated in vivo by a specific inhibitor.

Download Full-text