The SecB Chaperone Is Bifunctional in Serratia marcescens: SecB Is Involved in the Sec Pathway and Required for HasA Secretion by the ABC Transporter

ABSTRACT HasA is the secreted hemophore of the heme acquisition system (Has) of Serratia marcescens. It is secreted by a specific ABC transporter apparatus composed of three proteins: HasD, an inner membrane ABC protein; HasE, another inner membrane protein; and HasF, a TolC homolog. Except for HasF, the structural genes of the Has system are encoded by an iron-regulated operon. In previous studies, this secretion system has been reconstituted in Escherichia coli, where it requires the presence of the SecB chaperone, the Sec pathway-dedicated chaperone. We cloned and inactivated the secB gene from S. marcescens. We show that S. marcescens SecB is 93% identical to E. coli SecB and complements the secretion defects of a secB mutant of E. coli for both the Sec and ABC pathways of HasA secretion. In S. marcescens, SecB inactivation affects translocation by the Sec pathway and abolishes HasA secretion. This demonstrates that S. marcescens SecB is the genuine chaperone for HasA secretion in S. marcescens. These results also demonstrate that S. marcescens SecB is bifunctional, as it is involved in two separate secretion pathways. We investigated the effects of secB point mutations in the reconstituted HasA secretion pathway by comparing the translocation of a Sec substrate in various mutants. Two different patterns of SecB residue effects were observed, suggesting that SecB functions may differ for the Sec and ABC pathways.

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Genetic analysis of the role of the conserved inner membrane protein CvpA in EHEC resistance to deoxycholate

Journal of Bacteriology ◽

10.1128/jb.00661-20 ◽

2020 ◽

Author(s):

Alyson R. Warr ◽

Rachel T. Giorgio ◽

Matthew K. Waldor

Keyword(s):

Stress Response ◽

Membrane Protein ◽

Bile Salt ◽

Cell Envelope ◽

Enteric Bacteria ◽

Enteric Pathogens ◽

Inner Membrane ◽

Bacterial Phyla ◽

E Coli ◽

Inner Membrane Protein

The function of cvpA, a bacterial gene predicted to encode an inner membrane protein, is largely unknown. Early studies in E. coli linked cvpA to Colicin V secretion and recent work revealed that it is required for robust intestinal colonization by diverse enteric pathogens. In enterohemorrhagic E. coli (EHEC), cvpA is required for resistance to the bile salt deoxycholate (DOC). Here, we carried out genome-scale transposon-insertion mutagenesis and spontaneous suppressor analysis to uncover cvpA’s genetic interactions and identify common pathways that rescue the sensitivity of a ΔcvpA EHEC mutant to DOC. These screens demonstrated that mutations predicted to activate the σE-mediated extracytoplasmic stress response bypass the ΔcvpA mutant’s susceptibility to DOC. Consistent with this idea, we found that deletions in rseA and msbB and direct overexpression of rpoE restored DOC resistance to the ΔcvpA mutant. Analysis of the distribution of CvpA homologs revealed that this inner membrane protein is conserved across diverse bacterial phyla, in both enteric and non-enteric bacteria that are not exposed to bile. Together, our findings suggest that CvpA plays a role in cell envelope homeostasis in response to DOC and similar stress stimuli in diverse bacterial species. IMPORTANCE Several enteric pathogens, including Enterohemorrhagic E. coli (EHEC), require CvpA to robustly colonize the intestine. This inner membrane protein is also important for secretion of a colicin and EHEC resistance to the bile salt deoxycholate (DOC), but its function is unknown. Genetic analyses carried out here showed that activation of the σE-mediated extracytoplasmic stress response restored the resistance of a cvpA mutant to DOC, suggesting that CvpA plays a role in cell envelope homeostasis. The conservation of CvpA across diverse bacterial phyla suggests that this membrane protein facilitates cell envelope homeostasis in response to varied cell envelope perturbations.

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Repression of the Inner Membrane Lipoprotein NlpA by Rns in Enterotoxigenic Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.01714-06 ◽

2006 ◽

Vol 189 (5) ◽

pp. 1627-1632 ◽

Cited By ~ 23

Author(s):

Maria D. Bodero ◽

M. Carolina Pilonieta ◽

George P. Munson

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Enterotoxigenic Escherichia Coli ◽

Start Codon ◽

Inner Membrane ◽

E Coli ◽

Inner Membrane Protein

ABSTRACT The expression of the inner membrane protein NlpA is repressed by the enterotoxigenic Escherichia coli (ETEC) virulence regulator Rns, a member of the AraC/XylS family. The Rns homologs CfaD from ETEC and AggR from enteroaggregative E. coli also repress expression of nlpA. In vitro DNase I and potassium permanganate footprinting revealed that Rns binds to a site overlapping the start codon of nlpA, preventing RNA polymerase from forming an open complex at nlpAp. A second Rns binding site between positions −152 and −195 relative to the nlpA transcription start site is not required for repression. NlpA is not essential for growth of E. coli under laboratory conditions, but it does contribute to the biogenesis of outer membrane vesicles. As outer membrane vesicles have been shown to contain ETEC heat-labile toxin, the repression of nlpA may be an indirect mechanism through which the virulence regulators Rns and CfaD limit the release of toxin.

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Tail-Anchored Inner Membrane Protein ElaB Increases Resistance to Stress While Reducing Persistence in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00057-17 ◽

2017 ◽

Vol 199 (9) ◽

Cited By ~ 11

Author(s):

Yunxue Guo ◽

Xiaoxiao Liu ◽

Baiyuan Li ◽

Jianyun Yao ◽

Thomas K. Wood ◽

...

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Membrane Proteins ◽

Membrane Protein ◽

Stationary Phase ◽

Transmembrane Domain ◽

Inner Membrane ◽

Content Type ◽

E Coli ◽

Inner Membrane Protein

ABSTRACT Host-associated bacteria, such as Escherichia coli, often encounter various host-related stresses, such as nutritional deprivation, oxidative stress, and temperature shifts. There is growing interest in searching for small endogenous proteins that mediate stress responses. Here, we characterized the small C-tail-anchored inner membrane protein ElaB in E. coli. ElaB belongs to a class of tail-anchored inner membrane proteins with a C-terminal transmembrane domain but lacking an N-terminal signal sequence for membrane targeting. Proteins from this family have been shown to play vital roles, such as in membrane trafficking and apoptosis, in eukaryotes; however, their role in prokaryotes is largely unexplored. Here, we found that the transcription of elaB is induced in the stationary phase in E. coli and stationary-phase sigma factor RpoS regulates elaB transcription by binding to the promoter of elaB. Moreover, ElaB protects cells against oxidative stress and heat shock stress. However, unlike membrane peptide toxins TisB and GhoT, ElaB does not lead to cell death, and the deletion of elaB greatly increases persister cell formation. Therefore, we demonstrate that disruption of C-tail-anchored inner membrane proteins can reduce stress resistance; it can also lead to deleterious effects, such as increased persistence, in E. coli. IMPORTANCE Escherichia coli synthesizes dozens of poorly understood small membrane proteins containing a predicted transmembrane domain. In this study, we characterized the function of the C-tail-anchored inner membrane protein ElaB in E. coli. ElaB increases resistance to oxidative stress and heat stress, while inactivation of ElaB leads to high persister cell formation. We also demonstrated that the transcription of elaB is under the direct regulation of stationary-phase sigma factor RpoS. Thus, our study reveals that small inner membrane proteins may have important cellular roles during the stress response.

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Growth Inhibition by External Potassium of Escherichia coli Lacking PtsN (EIIANtr) Is Caused by Potassium Limitation Mediated by YcgO

Journal of Bacteriology ◽

10.1128/jb.01029-15 ◽

2016 ◽

Vol 198 (13) ◽

pp. 1868-1882 ◽

Cited By ~ 5

Author(s):

Ravish Sharma ◽

Tomohiro Shimada ◽

Vinod K. Mishra ◽

Suchitra Upreti ◽

Abhijit A. Sardesai

Keyword(s):

Escherichia Coli ◽

Membrane Protein ◽

Phosphorylation Site ◽

Phosphotransferase System ◽

Inner Membrane ◽

Content Type ◽

E Coli ◽

External Potassium ◽

Inner Membrane Protein ◽

External K

ABSTRACTThe absence of PtsN, the terminal phosphoacceptor of the phosphotransferase system comprising PtsP-PtsO-PtsN, inEscherichia coliconfers a potassium-sensitive (Ks) phenotype as the external K+concentration ([K+]e) is increased above 5 mM. A growth-inhibitory increase in intracellular K+content, resulting from hyperactivated Trk-mediated K+uptake, is thought to cause this Ks. We provide evidence that the Ksof the ΔptsNmutant is associated with K+limitation. Accordingly, the moderate Ksdisplayed by the ΔptsNmutant was exacerbated in the absence of the Trk and Kup K+uptake transporters and was associated with reduced cellular K+content. Conversely, overproduction of multiple K+uptake proteins suppressed the Ks. Expression of PtsN variants bearing the H73A, H73D, and H73E substitutions of the phosphorylation site histidine of PtsN complemented the Ks. Absence of the predicted inner membrane protein YcgO (also called CvrA) suppressed the Ks, which was correlated with elevated cellular K+content in the ΔptsNmutant, but the ΔptsNmutation did not alter YcgO levels. Heterologous overexpression ofycgOalso led to Ksthat was associated with reduced cellular K+content, exacerbated by the absence of Trk and Kup and alleviated by overproduction of Kup. Our findings are compatible with a model that postulates that Ksin the ΔptsNmutant occurs due to K+limitation resulting from activation of K+efflux mediated by YcgO, which may be additionally stimulated by [K+]e, implicating a role for PtsN (possibly its dephosphorylated form) as an inhibitor of YcgO activity.IMPORTANCEThis study examines the physiological link between the phosphotransferase system comprising PtsP-PtsO-PtsN and K+ion metabolism inE. coli. Studies on the physiological defect that renders anE. colimutant lacking PtsN to be growth inhibited by external K+indicate that growth impairment results from cellular K+limitation that is mediated by YcgO, a predicted inner membrane protein. Additional observations suggest that dephospho-PtsN may inhibit and external K+may stimulate K+limitation mediated by YcgO. It is speculated that YcgO-mediated K+limitation may be an output of a response to certain stresses, which by modulating the phosphotransfer capacity of the PtsP-PtsO-PtsN phosphorelay leads to growth cessation and stress tolerance.

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Genetic analyses link the conserved inner membrane protein CvpA to the σE extracytoplasmic stress response

10.1101/2020.09.25.314492 ◽

2020 ◽

Author(s):

Alyson R. Warr ◽

Rachel T. Giorgio ◽

Matthew K. Waldor

Keyword(s):

Stress Response ◽

Membrane Protein ◽

Bile Salt ◽

Enteric Pathogens ◽

Inner Membrane ◽

Genetic Analyses ◽

Bacterial Phyla ◽

E Coli ◽

Stress Response Pathway ◽

Inner Membrane Protein

AbstractThe function of cvpA, a bacterial gene predicted to encode an inner membrane protein, is largely unknown. Early studies in E. coli linked cvpA to Colicin V secretion and recent work revealed that it is required for robust intestinal colonization by diverse enteric pathogens. In enterohemorrhagic E. coli (EHEC), cvpA is required for resistance to the bile salt deoxycholate (DOC). Here, we carried out genome-scale transposon-insertion mutagenesis and spontaneous suppressor analysis to uncover cvpA’s genetic interactions and identify common pathways that rescue the sensitivity of a ΔcvpA EHEC mutant to DOC. Collectively, these screens led to the hypothesis that the ΔcvpA mutant is impaired in its capacity to activate the σE-mediated stress response. This idea was supported by showing that mutations that activate σE, either indirectly or through its direct overexpression, can restore the ΔcvpA mutant’s resistance to DOC. Analysis of the distribution of CvpA homologs revealed that this inner membrane protein is conserved across bacterial phyla, in both enteric and non-enteric bacteria that are not exposed to bile. Together, our findings suggest that CvpA may function in triggering activation of the σE stress response pathway in response to DOC as well as additional stimuli.ImportanceSeveral enteric pathogens, including Enterohemorrhagic E. coli (EHEC), require cvpA to robustly colonize the intestine. This inner membrane is also important for secretion of a colicin and EHEC resistance to the bile salt deoxycholate, but its function is unknown. Genetic analyses carried out here suggest that cvpA is required to trigger the σE stress response pathway in response to deoxycholate. Since CvpA is conserved across diverse bacterial phyla, we propose that this inner membrane protein is important for activation of this stress response pathway in response to diverse perturbations of the cell envelope.

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The Escherichia coli Yej Transporter Is Required for the Uptake of Translation Inhibitor Microcin C

Journal of Bacteriology ◽

10.1128/jb.01028-07 ◽

2007 ◽

Vol 189 (22) ◽

pp. 8361-8365 ◽

Cited By ~ 68

Author(s):

Maria Novikova ◽

Anastasia Metlitskaya ◽

Kirill Datsenko ◽

Teymur Kazakov ◽

Alexey Kazakov ◽

...

Keyword(s):

Escherichia Coli ◽

Abc Transporter ◽

Trna Synthetase ◽

Single Locus ◽

Membrane Transporter ◽

Inner Membrane ◽

Site Specific ◽

E Coli ◽

Translation Inhibitor ◽

Transposon Insertions

ABSTRACT Microcin C (McC), a peptide-nucleotide antibiotic, targets aspartyl-tRNA synthetase. By analyzing a random transposon library, we identified Escherichia coli mutants resistant to McC. Transposon insertions were localized to a single locus, yejABEF, which encodes components of a putative inner membrane ABC transporter. Analysis of site-specific mutants established that all four components of the transporter are required for McC sensitivity. Since aspartyl-tRNA synthetase in yej mutant extracts was fully sensitive to McC, we conclude that yej mutations interfere with McC uptake and that YejABEF is the only inner membrane transporter responsible for McC uptake in E. coli. Other substrates of YejABEF remain to be identified.

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The Escherichia coli Inner Membrane Protein YhiM is Necessary for Efficient Attachment of Bacteriophage T4

Fine Focus ◽

10.33043/ff.4.1.103-114 ◽

2018 ◽

Vol 4 (1) ◽

pp. 103-114

Author(s):

M.A. Evans ◽

P.T. Spieth ◽

R.L. Sparks-Thissen

Keyword(s):

Escherichia Coli ◽

Membrane Protein ◽

Bacteriophage T4 ◽

Inner Membrane ◽

E Coli ◽

Obligate Intracellular ◽

Intracellular Parasites ◽

Acid Shock ◽

Inner Membrane Protein

Bacteriophages are obligate intracellular parasites, but many of the cellular proteins involved in replication have not been identified. We have tested the role of the inner membrane protein YhiM in bacteriophage replication. YhiM is a conserved (21) membrane protein in Escherichia coli (E. coli) thought to be localized to the cytoplasmic membrane that is necessary for cell survival under conditions of cell stress, including acid shock, low osmolarity and high temperature. We show here that YhiM is necessary for replication of the bacteriophage T4. It also plays a modest role in the replication of T1, T3, and T5 but it does not play a role in the replication of ΦX174. Our data indicated that no replication of T4 occurs in cells missing YhiM. This block in infection is due to a block in attachment of the virus to the cell surface.

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