RpoS-independent evolution reveals the importance of attenuated cAMPCRP regulation in high hydrostatic pressure resistance acquisition in E. coli

High hydrostatic pressure (HHP) processing is an attractive non-thermal alternative to food pasteurization. Nevertheless, the large inter- and intra-species variations in HHP resistance among foodborne pathogens and the ease by which they can acquire extreme resistance are an issue of increasing concern. Since RpoS activity has been considered as a central determinant in the HHP resistance of E. coli and its pathovars, this study probed for the potential of an E. coli MG1655 ΔrpoS mutant to acquire HHP resistance by directed evolution. Despite the higher initial HHP sensitivity of the ΔrpoS mutant compared to the wild-type strain, evolved lineages of the former readily managed to restore or even succeed wild-type levels of resistance. A number of these ΔrpoS derivatives were affected in cAMP/CRP regulation, and this could be causally related to their HHP resistance. Subsequent inspection revealed that some of previously isolated HHP-resistant mutants derived from the wild-type strain also incurred a causal decrease in cAMP/CRP regulation. cAMP/CRP attenuated HHP-resistant mutants also exhibited higher resistance to fosfomycin, a preferred treatment for STEC infections. As such, this study reveals attenuation of cAMP/CRP regulation as a relevant and RpoS-independent evolutionary route towards HHP resistance in E. coli that coincides with fosfomycin resistance

The expression of virulence increases outer-membrane permeability and sensitivity to envelope stress in Salmonella Typhimurium

Environmental cues modulate the expression of virulence in bacterial pathogens. However, while cues that upregulate virulence are often intuitive and mechanistically well understood, this is less so for cues that downregulate virulence. In this study, we noticed that upregulation of the HilD virulence regulon in Salmonella Typhimurium ( S .Tm) sensitized cells to membrane stress mediated by cholate, Tris/EDTA or heat. Further monitoring of membrane status and stress resistance of S .Tm cells in relation to virulence expression, revealed that co-expressed virulence factors embedded in the envelope (including the Type Three Secretion System 1 and the flagella) increased permeability, and stress sensitivity of the membrane. Importantly, pretreating the bacteria by sublethal stress inhibited virulence expression and restored stress resistance. As such, these results demonstrate a trade-off between virulence and stress resistance, which explains the downregulation of virulence expression in response to harsh environments in S .Tm

Metabolite profiling and peptidoglycan analysis of transient cell wall-deficient bacteria in a new Escherichia coli model system

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd. Many bacteria are able to assume a transient cell wall-deficient (or L-form) state under favourable osmotic conditions. Cell wall stress such as exposure to β-lactam antibiotics can enforce the transition to and maintenance of this state. L-forms actively proliferate and can return to the walled state upon removal of the inducing agent. We have adopted Escherichia coli as a model system for the controlled transition to and reversion from the L-form state, and have studied these dynamics with genetics, cell biology and 'omics' technologies. As such, a transposon mutagenesis screen underscored the requirement for the Rcs phosphorelay and colanic acid synthesis, while proteomics show only little differences between rods and L-forms. In contrast, metabolome comparison reveals the high abundance of lysophospholipids and phospholipids with unsaturated or cyclopropanized fatty acids in E.coliL-forms. This increase of membrane lipids associated with increased membrane fluidity may facilitate proliferation through bud formation. Visualization of the residual peptidoglycan with a fluorescently labelled peptidoglycan binding protein indicates de novo cell wall synthesis and a role for septal peptidoglycan synthesis during bud constriction. The DD-carboxypeptidases PBP5 and PBP6 are threefold and fourfold upregulated in L-forms, indicating a specific role for regulation of crosslinking during L-form proliferation.Peer Reviewe

RpoS-independent evolution reveals the importance of attenuated cAMP/CRP regulation in high hydrostatic pressure resistance acquisition in E. coli

High hydrostatic pressure (HHP) processing is an attractive non-thermal alternative to food pasteurization. Nevertheless, the large inter- and intra-species variations in HHP resistance among foodborne pathogens and the ease by which they can acquire extreme resistance are an issue of increasing concern. Since RpoS activity has been considered as a central determinant in the HHP resistance of E. coli and its pathovars, this study probed for the potential of an E. coli MG1655 ΔrpoS mutant to acquire HHP resistance by directed evolution. Despite the higher initial HHP sensitivity of the ΔrpoS mutant compared to the wild-type strain, evolved lineages of the former readily managed to restore or even succeed wild-type levels of resistance. A number of these ΔrpoS derivatives were affected in cAMP/CRP regulation, and this could be causally related to their HHP resistance. Subsequent inspection revealed that some of previously isolated HHP-resistant mutants derived from the wild-type strain also incurred a causal decrease in cAMP/CRP regulation. cAMP/CRP attenuated HHP-resistant mutants also exhibited higher resistance to fosfomycin, a preferred treatment for STEC infections. As such, this study reveals attenuation of cAMP/CRP regulation as a relevant and RpoS-independent evolutionary route towards HHP resistance in E. coli that coincides with fosfomycin resistance.status: publishe

Impact of Protein Aggregates on Sporulation and Germination of Bacillus subtilis.

In order to improve our general understanding of protein aggregate (PA) management and impact in bacteria, different model systems and processes need to be investigated. As such, we developed an inducible synthetic PA model system to investigate PA dynamics in the Gram-positive model organism Bacillus subtilis. This confirmed previous observations that PA segregation in this organism seems to follow the Escherichia coli paradigm of nucleoid occlusion governing polar localization and asymmetric segregation during vegetative growth. However, our findings also revealed that PAs can readily persist throughout the entire sporulation process after encapsulation in the forespore during sporulation. Moreover, no deleterious effects of PA presence on sporulation, germination and spore survival against heat or UV stress could be observed. Our findings therefore indicate that the sporulation process is remarkably robust against perturbations by PAs and misfolded proteins

Impact of Protein Aggregates on Sporulation and Germination of Bacillus subtilis

Peer reviewed: TrueAcknowledgements: We thank Matthew Cabeen for his input on the genetic engineering of B. subtilis.Funder: UKRI Sensor CDT studentshipIn order to improve our general understanding of protein aggregate (PA) management and impact in bacteria, different model systems and processes need to be investigated. As such, we developed an inducible synthetic PA model system to investigate PA dynamics in the Gram-positive model organism Bacillus subtilis. This confirmed previous observations that PA segregation in this organism seems to follow the Escherichia coli paradigm of nucleoid occlusion governing polar localization and asymmetric segregation during vegetative growth. However, our findings also revealed that PAs can readily persist throughout the entire sporulation process after encapsulation in the forespore during sporulation. Moreover, no deleterious effects of PA presence on sporulation, germination and spore survival against heat or UV stress could be observed. Our findings therefore indicate that the sporulation process is remarkably robust against perturbations by PAs and misfolded proteins

Impact of Protein Aggregates on Sporulation and Germination of <i>Bacillus subtilis</i>

In order to improve our general understanding of protein aggregate (PA) management and impact in bacteria, different model systems and processes need to be investigated. As such, we developed an inducible synthetic PA model system to investigate PA dynamics in the Gram-positive model organism Bacillus subtilis. This confirmed previous observations that PA segregation in this organism seems to follow the Escherichia coli paradigm of nucleoid occlusion governing polar localization and asymmetric segregation during vegetative growth. However, our findings also revealed that PAs can readily persist throughout the entire sporulation process after encapsulation in the forespore during sporulation. Moreover, no deleterious effects of PA presence on sporulation, germination and spore survival against heat or UV stress could be observed. Our findings therefore indicate that the sporulation process is remarkably robust against perturbations by PAs and misfolded proteins

Novel cryptic prophage operon modulates stress resistance in Escherichia coli O157:H7

Prokaryotic genome plasticity has paved the way for an explosive diversification within bacterial species. Part of this plasticity originates from horizontal gene transfer that takes place within the bacterial kingdom with bacterial phage genes being amongst the most common exchanged fragments. These phage genetic elements can constitute up to one fifth of the complete prokaryotic genetic blueprint but its influence on the hosts physiology remains ill defined. Escherichia coli O157:H7 is a notorious foodborne pathogen potentiated by the large amount of horizontally transferred genomic material which differentiates it from harmless gut-inhabiting relatives such as E. coli K-12. Upon screening a transposon knock-out library of E. coli O157:H7 in search of high pressure (HP) resistant mutants, we came across a cryptic prophage encoded operon (hpsP-hpsQ) uniquely found in this strain that can modulate the stress resilience of the bacterial host. Disrupting the first part of this viral operon leads to a significant increase in resistance to HP as well as cross-resistance to heat with the underlying mechanism still being elusive. Upon overexpression HpsP not only proved to be a genotoxic protein with (in vitro and in vivo) DNA binding capacity, but was also able to quench the potent toxic effect of HpsQ, the second operon member. Moreover, HpsP functionality can be blocked by complementing with its own N-terminal ATPase domain thus abolishing its genotoxic DNA binding and starting host resilience modulation.status: publishe

Metabolite profiling and peptidoglycan analysis of transient cell wall-deficient bacteria in a new Escherichia coli model system

Many bacteria are able to assume a transient cell wall-deficient (or L-form) state under favourable osmotic conditions. Cell wall stress such as exposure to -lactam antibiotics can enforce the transition to and maintenance of this state. L-forms actively proliferate and can return to the walled state upon removal of the inducing agent. We have adopted Escherichia coli as a model system for the controlled transition to and reversion from the L-form state, and have studied these dynamics with genetics, cell biology and omics' technologies. As such, a transposon mutagenesis screen underscored the requirement for the Rcs phosphorelay and colanic acid synthesis, while proteomics show only little differences between rods and L-forms. In contrast, metabolome comparison reveals the high abundance of lysophospholipids and phospholipids with unsaturated or cyclopropanized fatty acids in E.coliL-forms. This increase of membrane lipids associated with increased membrane fluidity may facilitate proliferation through bud formation. Visualization of the residual peptidoglycan with a fluorescently labelled peptidoglycan binding protein indicates de novo cell wall synthesis and a role for septal peptidoglycan synthesis during bud constriction. The DD-carboxypeptidases PBP5 and PBP6 are threefold and fourfold upregulated in L-forms, indicating a specific role for regulation of crosslinking during L-form proliferation

The expression of virulence genes increases membrane permeability and sensitivity to envelope stress in Salmonella Typhimurium.

Virulence gene expression can represent a substantial fitness cost to pathogenic bacteria. In the model entero-pathogen Salmonella Typhimurium (S.Tm), such cost favors emergence of attenuated variants during infections that harbor mutations in transcriptional activators of virulence genes (e.g., hilD and hilC). Therefore, understanding the cost of virulence and how it relates to virulence regulation could allow the identification and modulation of ecological factors to drive the evolution of S.Tm toward attenuation. In this study, investigations of membrane status and stress resistance demonstrate that the wild-type (WT) expression level of virulence factors embedded in the envelope increases membrane permeability and sensitizes S.Tm to membrane stress. This is independent from a previously described growth defect associated with virulence gene expression in S.Tm. Pretreating the bacteria with sublethal stress inhibited virulence expression and increased stress resistance. This trade-off between virulence and stress resistance could explain the repression of virulence expression in response to harsh environments in S.Tm. Moreover, we show that virulence-associated stress sensitivity is a burden during infection in mice, contributing to the inherent instability of S.Tm virulence. As most bacterial pathogens critically rely on deploying virulence factors in their membrane, our findings could have a broad impact toward the development of antivirulence strategies