A method to map changes in bacterial surface composition induced by regulatory RNAs in Escherichia coli and Staphylococcus aureus

Hammann, Philippe; Parmentier, Delphine; Cerciat, Marie; Reimegård, Johan; Helfer, Anne‐Catherine; Boisset, Sandrine; Guillier, Maude; Vandenesch, F.; Wagner, E. Gerhart H.; Romby, Pascale; Fechter, Pierre

doi:10.1016/j.biochi.2014.07.011

Cited by 8 publications

(4 citation statements)

References 15 publications

(13 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As rfaH is required for efficient LPS biosynthesis (48)(49)(50)(51), it is likely that rfaH mutants have OM permeability unrelated to the pathway affected by RpoS. YdgH is predicted to be a periplasmic protein, and its levels have been suggested to be downregulated by MicA, an sRNA that increases upon entry to stationary phase but is transcriptionally regulated by sigma E, the envelope stress sigma factor (52)(53)(54)(55). Thus, YdgH may play a role in stationary-phase envelope permeability while not being directly regulated by RpoS.…”

Section: Resultsmentioning

confidence: 99%

Novel RpoS-Dependent Mechanisms Strengthen the Envelope Permeability Barrier during Stationary Phase

2017

View full text Add to dashboard Cite

Gram-negative bacteria have effective methods of excluding toxic compounds, including a largely impermeable outer membrane (OM) and a range of efflux pumps. Furthermore, when cells become nutrient limited, RpoS enacts a global expression change providing cross-protection against many stresses. Here, we utilized sensitivity to an anionic detergent (sodium dodecyl sulfate [SDS]) to probe changes occurring to the cell's permeability barrier during nutrient limitation. Escherichia coli is resistant to SDS whether cells are actively growing, carbon limited, or nitrogen limited. In actively growing cells, this resistance depends on the AcrAB-TolC efflux pump; however, this pump is not necessary for protection under either carbon-limiting or nitrogen-limiting conditions, suggesting an alternative mechanism(s) of SDS resistance. In carbon-limited cells, RpoS-dependent pathways lessen the permeability of the OM, preventing the necessity for efflux. In nitrogen-limited but not carbon-limited cells, the loss of rpoS can be completely compensated for by the AcrAB-TolC efflux pump. We suggest that this difference simply reflects the fact that nitrogen-limited cells have access to a metabolizable energy (carbon) source that can efficiently power the efflux pump. Using a transposon mutant pool sequencing (Tn-Seq) approach, we identified three genes, sanA, dacA, and yhdP, that are necessary for RpoS-dependent SDS resistance in carbon-limited stationary phase. Using genetic analysis, we determined that these genes are involved in two different envelope-strengthening pathways. These genes have not previously been implicated in stationary-phase stress responses. A third novel RpoS-dependent pathway appears to strengthen the cell's permeability barrier in nitrogen-limited cells. Thus, though cells remain phenotypically SDS resistant, SDS resistance mechanisms differ significantly between growth states. IMPORTANCE Gram-negative bacteria are intrinsically resistant to detergents and many antibiotics due to synergistic activities of a strong outer membrane (OM) permeability barrier and efflux pumps that capture and expel toxic molecules eluding the barrier. When the bacteria are depleted of an essential nutrient, a program of gene expression providing cross-protection against many stresses is induced. Whether this program alters the OM to further strengthen the barrier is unknown. Here, we identify novel pathways dependent on the master regulator of stationary phase that further strengthen the OM permeability barrier during nutrient limitation, circumventing the need for efflux pumps. Decreased permeability of nutrient-limited cells to toxic compounds has important implications for designing new antibiotics capable of targeting Gram-negative bacteria that may be in a growth-limited state.KEYWORDS Escherichia coli, RpoS, efflux pumps, outer membrane, permeability, stationary phase

show abstract

Section: Resultsmentioning

confidence: 99%

Novel RpoS-Dependent Mechanisms Strengthen the Envelope Permeability Barrier during Stationary Phase

2017

View full text Add to dashboard Cite

show abstract

“…Proteomics assesses sRNA expression-dependent changes in the abundance of total protein or cellular subfractions. Differential proteomic analysis by a DiGE approach (e.g., Hammann et al, 2014;Mann et al, 2012;Udekwu et al, 2005), label-free quantification of proteins by spectral count LCeMSeMS (Romilly et al, 2014), or stable isotope labeling by amino acids in cell culture (SILAC; (Ong et al, 2002)) map the sRNA effect on the proteome. New developments that help target identification on a global scale involve ribosome profiling (Guo, Updegrove, et al, 2014;Ingolia, 2014) and the use of protein cofactors as baits to purify regulatory complexes.…”

Section: Experimental Strategiesmentioning

confidence: 99%

Small RNAs in Bacteria and Archaea

Wagner

Romby

2015

Advances in Genetics

Self Cite

456

245

View full text Add to dashboard Cite

“…One possibility is that in the periplasm, YdgH plays a role in the maturation of outer membrane-bound biomolecules. In support of this speculation, overexpression of micA, a small RNA that is a primary effector of the σ E envelope stress response, leads to upregulation of ydgH (71). Alternatively, its absence may lead to envelope stress, leading to activation of σ E .…”

Section: Discussionmentioning

confidence: 93%

A genome-scale antibiotic screen inSerratia marcescensidentifies YdgH as a conserved modifier of cephalosporin and detergent susceptibility

Lazarus

Warr

Westervelt

et al. 2021

Preprint

View full text Add to dashboard Cite

Serratia marcescens, a member of the order Enterobacterales, is adept at colonizing healthcare environments and an important cause of invasive infections. Antibiotic resistance is a daunting problem in S. marcescens because in addition to plasmid-mediated mechanisms, most isolates have considerable intrinsic resistance to multiple antibiotic classes. To discover endogenous modifiers of antibiotic susceptibility in S. marcescens, a high-density transposon insertion library was subjected to sub-minimal inhibitory concentrations of two cephalosporins, cefoxitin and cefepime, as well as the fluoroquinolone ciprofloxacin. Comparisons of transposon insertion abundance before and after antibiotic exposure identified hundreds of potential modifiers of susceptibility to these agents. Using single gene deletions, we validated several candidate modifiers of cefoxitin susceptibility and chose ydgH, a gene of unknown function, for further characterization. In addition to cefoxitin, deletion of ydgH in S. marcescens resulted in decreased susceptibility to multiple 3rd generation cephalosporins, and in contrast, to increased susceptibility to both cationic and anionic detergents. YdgH is highly conserved throughout the Enterobacterales, and we observed similar phenotypes in Escherichia coli O157:H7 and Enterobacter cloacae mutants. YdgH is predicted to localize to the periplasm and we speculate that it may be involved there in cell envelope homeostasis. Collectively, our findings provide insight into chromosomal mediators of antibiotic resistance in S. marcescens and will serve as a resource for further investigations of this important pathogen.

show abstract

A method to map changes in bacterial surface composition induced by regulatory RNAs in Escherichia coli and Staphylococcus aureus

Cited by 8 publications

References 15 publications

Novel RpoS-Dependent Mechanisms Strengthen the Envelope Permeability Barrier during Stationary Phase

Novel RpoS-Dependent Mechanisms Strengthen the Envelope Permeability Barrier during Stationary Phase

Small RNAs in Bacteria and Archaea

A genome-scale antibiotic screen inSerratia marcescensidentifies YdgH as a conserved modifier of cephalosporin and detergent susceptibility

Contact Info

Product

Resources

About