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Mycobacterium tuberculosis possesses a dynamic cell envelope, which consists of a peptidoglycan layer, a mycolic acid layer, and an arabinogalactan polysaccharide. This envelope possesses a highly complex and unique structure representing a barrier that protects and assists the growth of M. tuberculosis and allows its adaptation to the host. It regulates the immune response of the host cells, causing their damage. Therefore, the cell envelope of M. tuberculosis is an attractive target for vaccine and drug development. The emergence of multidrug-resistant as well as extensively drug resistant tuberculosis and co-infection with HIV prevented an effective control of this disease. Thus, the discovery and development of new drugs is a major keystone for TB treatment and control. This review mainly summarizes the development of drug enzymes involved in the biosynthesis of the cell wall in M. tuberculosis, and other potential drug targets in this pathway, to provide more effective strategies for the development of new drugs.

The Tol-Pal system spans the cell envelope of Gram-negative bacteria, transducing the potential energy of the proton motive force (PMF) into dissociation of the TolB-Pal complex at the outer membrane (OM), freeing the lipoprotein Pal to bind the cell wall. The primary physiological role of Tol-Pal is to maintain OM integrity during cell division through accumulation of Pal molecules at division septa. How the protein complex couples the PMF at the inner membrane into work at the OM is unknown. The effectiveness of this trans-envelope energy transduction system is underscored by the fact that bacteriocins and bacteriophages co-opt Tol-Pal as part of their import/infection mechanisms. Mechanistic understanding of this process has been hindered by a lack of structural data for the inner membrane TolQ-TolR stator, of its complexes with peptidoglycan (PG) and TolA, and of how these elements combined power events at the OM. Recent studies on the homologous stators of Ton and Mot provide a starting point for understanding how Tol-Pal works. Here, we combine ab initio protein modeling with previous structural data on sub-complexes of Tol-Pal as well as mutagenesis, crosslinking, co-conservation analysis and functional data. Through this composite pooling of in silico , in vitro , and in vivo data, we propose a mechanism for force generation in which PMF-driven rotary motion within the stator drives conformational transitions within a long TolA helical hairpin domain, enabling it to reach the TolB-Pal complex at the OM. [ABSTRACT FROM AUTHOR]

The Tol-Pal system spans the cell envelope of Gram-negative bacteria, transducing the potential energy of the proton motive force (PMF) into dissociation of the TolB-Pal complex at the outer membrane (OM), freeing the lipoprotein Pal to bind the cell wall. The primary physiological role of Tol-Pal is to maintain OM integrity during cell division through accumulation of Pal molecules at division septa. How the protein complex couples the PMF at the inner membrane into work at the OM is unknown. The effectiveness of this trans-envelope energy transduction system is underscored by the fact that bacteriocins and bacteriophages co-opt Tol-Pal as part of their import/infection mechanisms. Mechanistic understanding of this process has been hindered by a lack of structural data for the inner membrane TolQ-TolR stator, of its complexes with peptidoglycan (PG) and TolA, and of how these elements combined power events at the OM. Recent studies on the homologous stators of Ton and Mot provide a starting point for understanding how Tol-Pal works. Here, we combine ab initio protein modeling with previous structural data on sub-complexes of Tol-Pal as well as mutagenesis, crosslinking, co-conservation analysis and functional data. Through this composite pooling of in silico, in vitro, and in vivo data, we propose a mechanism for force generation in which PMF-driven rotary motion within the stator drives conformational transitions within a long TolA helical hairpin domain, enabling it to reach the TolB-Pal complex at the OM.

Listeria monocytogenes continues to be a food safety challenge owing to its stress tolerance and virulence traits. Several listeriosis outbreaks have been linked to the consumption of contaminated ready-to-eat food products. Numerous interventions, including nisin application, are presently employed to mitigate against L. monocytogenes risk in food products. In response, L. monocytogenes deploys several defense mechanisms, reducing nisin efficacy, that are not yet fully understood. Cold shock proteins (Csps) are small, highly conserved nucleic acid-binding proteins involved in several gene regulatory processes to mediate various stress responses in bacteria. L. monocytogenes possesses three csp gene paralogs; cspA , cspB , and cspD. Using a panel of single, double, and triple csp gene deletion mutants, the role of Csps in L. monocytogenes nisin tolerance was examined, demonstrating their importance in nisin stress responses of this bacterium. Without csp genes, a L. monocytogenes Δ cspABD mutant displayed severely compromised growth under nisin stress. Characterizing single (Δ cspA , Δ cspB , and Δ cspD) and double (Δ cspBD , Δ cspAD , and Δ cspAB) csp gene deletion mutants revealed a hierarchy (cspD > cspB > cspA) of importance in csp gene contributions toward the L. monocytogenes nisin tolerance phenotype. Individual eliminations of either cspA or cspB improved the nisin stress tolerance phenotype, suggesting that their expression has a curbing effect on the expression of nisin resistance functions through CspD. Gene expression analysis revealed that Csp deficiency altered the expression of DltA, MprF, and penicillin-binding protein-encoding genes. Furthermore, the Δ cspABD mutation induced an overall more electronegative cell surface, enhancing sensitivity to nisin and other cationic antimicrobials as well as the quaternary ammonium compound disinfectant benzalkonium chloride. These observations demonstrate that the molecular functions of Csps regulate systems important for enabling the constitution and maintenance of an optimal composed cell envelope that protects against cell-envelope-targeting stressors, including nisin. Overall, our data show an important contribution of Csps for L. monocytogenes stress protection in food environments where antimicrobial peptides are used. Such knowledge can be harnessed in the development of better L. monocytogenes control strategies. Furthermore, the potential that Csps have in inducing cross-protection must be considered when combining hurdle techniques or using them in a series. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes continues to be a food safety challenge owing to its stress tolerance and virulence traits. Several listeriosis outbreaks have been linked to the consumption of contaminated ready-to-eat food products. Numerous interventions, including nisin application, are presently employed to mitigate against L. monocytogenes risk in food products. In response, L. monocytogenes deploys several defense mechanisms, reducing nisin efficacy, that are not yet fully understood. Cold shock proteins (Csps) are small, highly conserved nucleic acid-binding proteins involved in several gene regulatory processes to mediate various stress responses in bacteria. L. monocytogenes possesses three csp gene paralogs; cspA, cspB, and cspD. Using a panel of single, double, and triple csp gene deletion mutants, the role of Csps in L. monocytogenes nisin tolerance was examined, demonstrating their importance in nisin stress responses of this bacterium. Without csp genes, a L. monocytogenes ΔcspABD mutant displayed severely compromised growth under nisin stress. Characterizing single (ΔcspA, ΔcspB, and ΔcspD) and double (ΔcspBD, ΔcspAD, and ΔcspAB) csp gene deletion mutants revealed a hierarchy (cspD > cspB > cspA) of importance in csp gene contributions toward the L. monocytogenes nisin tolerance phenotype. Individual eliminations of either cspA or cspB improved the nisin stress tolerance phenotype, suggesting that their expression has a curbing effect on the expression of nisin resistance functions through CspD. Gene expression analysis revealed that Csp deficiency altered the expression of DltA, MprF, and penicillin-binding protein-encoding genes. Furthermore, the ΔcspABD mutation induced an overall more electronegative cell surface, enhancing sensitivity to nisin and other cationic antimicrobials as well as the quaternary ammonium compound disinfectant benzalkonium chloride. These observations demonstrate that the molecular functions of Csps regulate systems important for enabling the constitution and maintenance of an optimal composed cell envelope that protects against cell-envelope-targeting stressors, including nisin. Overall, our data show an important contribution of Csps for L. monocytogenes stress protection in food environments where antimicrobial peptides are used. Such knowledge can be harnessed in the development of better L. monocytogenes control strategies. Furthermore, the potential that Csps have in inducing cross-protection must be considered when combining hurdle techniques or using them in a series.

Colistin represents a last-line treatment option for infections caused by multidrug resistant Gram-negative pathogens, including Pseudomonas aeruginosa. Colistin resistance generally involves the modification of the lipid A moiety of lipopolysaccharide (LPS) with positively charged molecules, namely phosphoethanolamine (PEtN) or 4-amino-4-deoxy-L-arabinose (Ara4N), that reduce colistin affinity for its target. Several lines of evidence highlighted lipid A aminoarabinosylation as the primary colistin resistance mechanism in P. aeruginosa , while the contribution of phosphoethanolamination remains elusive. PEtN modification can be due to either endogenous (chromosomally encoded) PEtN transferase(s) (e.g., EptA in P. aeruginosa) or plasmid borne MCR enzymes, commonly found in enterobacteria. By individually cloning eptA and mcr-1 into a plasmid for inducible gene expression, we demonstrated that MCR-1 and EptA have comparable PEtN transferase activity in P. aeruginosa and confer colistin resistance levels similar to those provided by lipid A aminoarabinosylation. Notably, EptA, but not MCR-1, negatively affects P. aeruginosa growth and, to a lesser extent, cell envelope integrity when expressed at high levels. Mutagenesis experiments revealed that PEtN transferase activity does not account for the noxious effects of EptA overexpression, that instead requires a C-terminal tail unique to P. aeruginosa EptA, whose function remains unknown. Overall, this study shows that both endogenous and exogenous PEtN transferases can promote colistin resistance in P. aeruginosa , and that PEtN and MCR-1 mediated resistance has no impact on growth and cell envelope homeostasis, suggesting that there may be no fitness barriers to the spread of mcr-1 in P. aeruginosa. [ABSTRACT FROM AUTHOR]

Colistin represents a last-line treatment option for infections caused by multidrug resistant Gram-negative pathogens, including Pseudomonas aeruginosa. Colistin resistance generally involves the modification of the lipid A moiety of lipopolysaccharide (LPS) with positively charged molecules, namely phosphoethanolamine (PEtN) or 4-amino-4-deoxy-L-arabinose (Ara4N), that reduce colistin affinity for its target. Several lines of evidence highlighted lipid A aminoarabinosylation as the primary colistin resistance mechanism in P. aeruginosa, while the contribution of phosphoethanolamination remains elusive. PEtN modification can be due to either endogenous (chromosomally encoded) PEtN transferase(s) (e.g., EptA in P. aeruginosa) or plasmid borne MCR enzymes, commonly found in enterobacteria. By individually cloning eptA and mcr-1 into a plasmid for inducible gene expression, we demonstrated that MCR-1 and EptA have comparable PEtN transferase activity in P. aeruginosa and confer colistin resistance levels similar to those provided by lipid A aminoarabinosylation. Notably, EptA, but not MCR-1, negatively affects P. aeruginosa growth and, to a lesser extent, cell envelope integrity when expressed at high levels. Mutagenesis experiments revealed that PEtN transferase activity does not account for the noxious effects of EptA overexpression, that instead requires a C-terminal tail unique to P. aeruginosa EptA, whose function remains unknown. Overall, this study shows that both endogenous and exogenous PEtN transferases can promote colistin resistance in P. aeruginosa, and that PEtN and MCR-1 mediated resistance has no impact on growth and cell envelope homeostasis, suggesting that there may be no fitness barriers to the spread of mcr-1 in P. aeruginosa.

The brucellae are facultative intracellular bacteria with a cell envelope rich in phosphatidylcholine (PC). PC is abundant in eukaryotes but rare in prokaryotes, and it has been proposed that Brucella uses PC to mimic eukaryotic-like features and avoid innate immune responses in the host. Two PC synthesis pathways are known in prokaryotes: the PmtA-catalyzed trimethylation of phosphatidylethanolamine and the direct linkage of choline to CDP-diacylglycerol catalyzed by the PC synthase Pcs. Previous studies have reported that B. abortus and B. melitensis possess non-functional PmtAs and that PC is synthesized exclusively via Pcs in these strains. A putative choline transporter ChoXWV has also been linked to PC synthesis in B. abortus. Here, we report that Pcs and Pmt pathways are active in B. suis biovar 2 and that a bioinformatics analysis of Brucella genomes suggests that PmtA is only inactivated in B. abortus and B. melitensis strains. We also show that ChoXWV is active in B. suis biovar 2 and conserved in all brucellae except B. canis and B. inopinata. Unexpectedly, the experimentally verified ChoXWV dysfunction in B. canis did not abrogate PC synthesis in a PmtA-deficient mutant, which suggests the presence of an unknown mechanism for obtaining choline for the Pcs pathway in Brucella. We also found that ChoXWV dysfunction did not cause attenuation in B. suis biovar 2. The results of these studies are discussed with respect to the proposed role of PC in Brucella virulence and how differential use of the Pmt and Pcs pathways may influence the interactions of these bacteria with their mammalian hosts.

The brucellae are facultative intracellular bacteria with a cell envelope rich in phosphatidylcholine (PC). PC is abundant in eukaryotes but rare in prokaryotes, and it has been proposed that Brucella uses PC to mimic eukaryotic-like features and avoid innate immune responses in the host. Two PC synthesis pathways are known in prokaryotes: the PmtA-catalyzed trimethylation of phosphatidylethanolamine and the direct linkage of choline to CDP-diacylglycerol catalyzed by the PC synthase Pcs. Previous studies have reported that B. abortus and B. melitensis possess non-functional PmtAs and that PC is synthesized exclusively via Pcs in these strains. A putative choline transporter ChoXWV has also been linked to PC synthesis in B. abortus. Here, we report that Pcs and Pmt pathways are active in B. suis biovar 2 and that a bioinformatics analysis of Brucella genomes suggests that PmtA is only inactivated in B. abortus and B. melitensis strains. We also show that ChoXWV is active in B. suis biovar 2 and conserved in all brucellae except B. canis and B. inopinata. Unexpectedly, the experimentally verified ChoXWV dysfunction in B. canis did not abrogate PC synthesis in a PmtA-deficient mutant, which suggests the presence of an unknown mechanism for obtaining choline for the Pcs pathway in Brucella. We also found that ChoXWV dysfunction did not cause attenuation in B. suis biovar 2. The results of these studies are discussed with respect to the proposed role of PC in Brucella virulence and how differential use of the Pmt and Pcs pathways may influence the interactions of these bacteria with their mammalian hosts. [ABSTRACT FROM AUTHOR]

S-layers are self-assembled crystalline 2D lattices enclosing the cell envelopes of several bacteria and archaea. Despite their abundance, the landscape of S-layer structure and function remains a land of wonder. By virtue of their location, bacterial S-layers have been hypothesized to add structural stability to the cell envelope. In addition, S-layers are implicated in mediating cell-environment and cell-host interactions playing a key role in adhesion, cell growth, and division. Significant strides in the understanding of these bacterial cell envelope components were made possible by recent studies that have provided structural and functional insights on the critical S-layer and S-layer-associated proteins (SLPs and SLAPs), highlighting their roles in pathogenicity and their potential as therapeutic or vaccine targets. In this mini-review, we revisit the sequence-structure-function relationships of S-layers, SLPs, and SLAPs in Gram-positive pathogens, focusing on the best-studied classes, Bacilli (Bacillus anthracis) and Clostridia (Clostridioides difficile). We delineate the domains and their architectures in archetypal S-layer proteins across Gram-positive genera and reconcile them with experimental findings. Similarly, we highlight a few key “flavors” of SLPs displayed by Gram-positive pathogens to assemble and support the bacterial S-layers. Together, these findings indicate that S-layers are excellent candidates for translational research (developing diagnostics, antibacterial therapeutics, and vaccines) since they display the three crucial characteristics: accessible location at the cell surface, abundance, and unique lineage-specific signatures.

S-layers are self-assembled crystalline 2D lattices enclosing the cell envelopes of several bacteria and archaea. Despite their abundance, the landscape of S-layer structure and function remains a land of wonder. By virtue of their location, bacterial S-layers have been hypothesized to add structural stability to the cell envelope. In addition, S-layers are implicated in mediating cell-environment and cell-host interactions playing a key role in adhesion, cell growth, and division. Significant strides in the understanding of these bacterial cell envelope components were made possible by recent studies that have provided structural and functional insights on the critical S-layer and S-layer-associated proteins (SLPs and SLAPs), highlighting their roles in pathogenicity and their potential as therapeutic or vaccine targets. In this mini-review, we revisit the sequence-structure-function relationships of S-layers, SLPs, and SLAPs in Gram-positive pathogens, focusing on the best-studied classes, Bacilli (Bacillus anthracis) and Clostridia (Clostridioides difficile). We delineate the domains and their architectures in archetypal S-layer proteins across Gram-positive genera and reconcile them with experimental findings. Similarly, we highlight a few key "flavors" of SLPs displayed by Gram-positive pathogens to assemble and support the bacterial S-layers. Together, these findings indicate that S-layers are excellent candidates for translational research (developing diagnostics, antibacterial therapeutics, and vaccines) since they display the three crucial characteristics: accessible location at the cell surface, abundance, and unique lineage-specific signatures. [ABSTRACT FROM AUTHOR]

Cell envelope proteins from Mycobacterium avium subspecies paratuberculosis (MAP) that are antigenically distinct from closely related mycobacterial species are potentially useful for Johne's Disease (JD) diagnosis. We evaluated the potential of ELISAs, based on six antigenically distinct recombinant MAP cell envelope proteins (SdhA, FadE25_2, FadE3_2, Mkl, DesA2, and hypothetical protein MAP1233) as well as an extract of MAP total cell envelope proteins, to detect antibodies against MAP in the sera of infected cattle. The sensitivity (Se) and specificity (Sp) of an ELISA based on MAP total cell envelope proteins, when analyzing 153 bovine serum samples, was 75 and 96%, respectively. Analysis of the same samples, using a commercial serum ELISA resulted in a Se of 56% and Sp of 99%. Results of ELISA analysis using plates coated with recombinant cell envelope proteins ranged from a highest Se of 94% and a lowest Sp of 79% for Sdh A to a lowest Se of 67% and a highest Sp of 95% for hypothetical protein MAP1233. Using polyclonal antibodies to MAP total cell envelope proteins, immunohistochemical analysis of intestinal and lymph node tissues from JD-positive cattle detected MAP organisms whereas antibodies to recombinant proteins did not. Finally, polyclonal antibodies to MAP total cell envelope protein and to recombinant SdhA, FadE25_2, and DesA2 proteins immunomagnetically separated MAP microorganisms spiked in PBS. These results suggest that antigenically distinct MAP cell envelope proteins and antibodies to these proteins may have potential to detect MAP infection in dairy cattle.

The cell biology of Chloroflexota is poorly studied. We applied cryo-focused ion beam milling and cryo-electron tomography to study the ultrastructural organization of thermophilic Roseiflexus castenholzii and Chloroflexus aggregans , and mesophilic " Ca. Viridilinea mediisalina." These species represent the three main lineages within a group of multicellular filamentous anoxygenic phototrophic Chloroflexota bacteria belonging to the Chloroflexales order. We found surprising structural complexity in the Chloroflexales. As with filamentous cyanobacteria, cells of C. aggregans and " Ca. Viridilinea mediisalina" share the outer membrane-like layers of their intricate multilayer cell envelope. Additionally, cells of R. castenholzii and " Ca. Viridilinea mediisalina" are connected by septal channels that resemble cyanobacterial septal junctions. All three strains possess long pili anchored close to cell-to-cell junctions, a morphological feature comparable to that observed in cyanobacteria. The cytoplasm of the Chloroflexales bacteria is crowded with intracellular organelles such as different types of storage granules, membrane vesicles, chlorosomes, gas vesicles, chemoreceptor-like arrays, and cytoplasmic filaments. We observed a higher level of complexity in the mesophilic strain compared to the thermophilic strains with regards to the composition of intracellular bodies and the organization of the cell envelope. The ultrastructural details that we describe in these Chloroflexales bacteria will motivate further cell biological studies, given that the function and evolution of the many discovered morphological traits remain enigmatic in this diverse and widespread bacterial group. [ABSTRACT FROM AUTHOR]

It is the enormous adaptive capacity of microorganisms, which is key to their competitive success in nature, but also challenges antibiotic treatment of human diseases. To deal with a diverse set of stresses, bacteria are able to reprogram gene expression using a wide variety of transcription factors. Here, we focused on the MarR-type regulator MalR conserved in the Corynebacterineae, including the prominent pathogens Corynebacterium diphtheriae and Mycobacterium tuberculosis. In several corynebacterial species, the malR gene forms an operon with a gene encoding a universal stress protein (uspA). Chromatin affinity purification and sequencing (ChAP-Seq) analysis revealed that MalR binds more than 60 target promoters in the C. glutamicum genome as well as in the large cryptic prophage CGP3. Overproduction of MalR caused severe growth defects and an elongated cell morphology. ChAP-Seq data combined with a global transcriptome analysis of the malR overexpression strain emphasized a central role of MalR in cell envelope remodeling in response to environmental stresses. For example, prominent MalR targets are involved in peptidoglycan biosynthesis and synthesis of branched-chain fatty acids. Phenotypic microarrays suggested an altered sensitivity of a ΔmalR mutant toward several β-lactam antibiotics. Furthermore, we revealed MalR as a repressor of several prophage genes, suggesting that MalR may be involved in the control of stress-responsive induction of the large CGP3 element. In conclusion, our results emphasize MalR as a regulator involved in stress-responsive remodeling of the cell envelope of C. glutamicum and suggest a link between cell envelope stress and the control of phage gene expression.

Aquatic environments are reservoirs of the human pathogen Vibrio cholerae O1, which causes the acute diarrheal disease cholera. Upon low temperature or limited nutrient availability, the cells enter a viable but non-culturable (VBNC) state. Characteristic of this state are an altered morphology, low metabolic activity, and lack of growth under standard laboratory conditions. Here, for the first time, the cellular ultrastructure of V. cholerae VBNC cells raised in natural waters was investigated using electron cryo-tomography. This was complemented by a comparison of the proteomes and the peptidoglycan composition of V. cholerae from LB overnight cultures and VBNC cells. The extensive remodeling of the VBNC cells was most obvious in the passive dehiscence of the cell envelope, resulting in improper embedment of flagella and pili. Only minor changes of the peptidoglycan and osmoregulated periplasmic glucans were observed. Active changes in VBNC cells included the production of cluster I chemosensory arrays and change of abundance of cluster II array proteins. Components involved in iron acquisition and storage, peptide import and arginine biosynthesis were overrepresented in VBNC cells, while enzymes of the central carbon metabolism were found at lower levels. Finally, several pathogenicity factors of V. cholerae were less abundant in the VBNC state, potentially limiting their infectious potential. This study gives unprecedented insight into the physiology of VBNC cells and the drastically altered presence of their metabolic and structural proteins.

Bacteriocins of Gram-negative bacteria are typically multi-domain proteins that target and kill bacteria of the same or closely related species. There is increasing interest in protein bacteriocin import; from a fundamental perspective to understand how folded proteins are imported into bacteria and from an applications perspective as species-specific antibiotics to combat multidrug resistant bacteria. In order to translocate across the cell envelope and cause cell death, protein bacteriocins hijack nutrient uptake pathways. Their import is energized by parasitizing intermembrane protein complexes coupled to the proton motive force, which delivers a toxic domain into the cell. A plethora of genetic, structural, biochemical, and biophysical methods have been applied to find cell envelope components involved in bacteriocin import since their discovery almost a century ago. Here, we review the various approaches that now exist for investigating how protein bacteriocins translocate into Gram-negative bacteria and highlight areas of research that will need methodological innovations to fully understand this process. We also highlight recent studies demonstrating how bacteriocins can be used to probe organization and architecture of the Gram-negative cell envelope itself.

Cable bacteria are long, multicellular micro-organisms that are capable of transporting electrons from cell to cell along the longitudinal axis of their centimeter-long filaments. The conductive structures that mediate this long-distance electron transport are thought to be located in the cell envelope. Therefore, this study examines in detail the architecture of the cell envelope of cable bacterium filaments by combining different sample preparation methods (chemical fixation, resin-embedding, and cryo-fixation) with a portfolio of imaging techniques (scanning electron microscopy, transmission electron microscopy and tomography, focused ion beam scanning electron microscopy, and atomic force microscopy). We systematically imaged intact filaments with varying diameters. In addition, we investigated the periplasmic fiber sheath that remains after the cytoplasm and membranes were removed by chemical extraction. Based on these investigations, we present a quantitative structural model of a cable bacterium. Cable bacteria build their cell envelope by a parallel concatenation of ridge compartments that have a standard size. Larger diameter filaments simply incorporate more parallel ridge compartments. Each ridge compartment contains a ~50 nm diameter fiber in the periplasmic space. These fibers are continuous across cell-to-cell junctions, which display a conspicuous cartwheel structure that is likely made by invaginations of the outer cell membrane around the periplasmic fibers. The continuity of the periplasmic fibers across cells makes them a prime candidate for the sought-after electron conducting structure in cable bacteria.

It is the enormous adaptive capacity of microorganisms, which is key to their competitive success in nature, but also challenges antibiotic treatment of human diseases. To deal with a diverse set of stresses, bacteria are able to reprogram gene expression using a wide variety of transcription factors. Here, we focused on the MarR-type regulator MalR conserved in the Corynebacterineae , including the prominent pathogens Corynebacterium diphtheriae and Mycobacterium tuberculosis. In several corynebacterial species, the malR gene forms an operon with a gene encoding a universal stress protein (uspA). Chromatin affinity purification and sequencing (ChAP-Seq) analysis revealed that MalR binds more than 60 target promoters in the C. glutamicum genome as well as in the large cryptic prophage CGP3. Overproduction of MalR caused severe growth defects and an elongated cell morphology. ChAP-Seq data combined with a global transcriptome analysis of the malR overexpression strain emphasized a central role of MalR in cell envelope remodeling in response to environmental stresses. For example, prominent MalR targets are involved in peptidoglycan biosynthesis and synthesis of branched-chain fatty acids. Phenotypic microarrays suggested an altered sensitivity of a Δ malR mutant toward several β-lactam antibiotics. Furthermore, we revealed MalR as a repressor of several prophage genes, suggesting that MalR may be involved in the control of stress-responsive induction of the large CGP3 element. In conclusion, our results emphasize MalR as a regulator involved in stress-responsive remodeling of the cell envelope of C. glutamicum and suggest a link between cell envelope stress and the control of phage gene expression. [ABSTRACT FROM AUTHOR]

Aquatic environments are reservoirs of the human pathogen Vibrio cholerae O1, which causes the acute diarrheal disease cholera. Upon low temperature or limited nutrient availability, the cells enter a viable but non-culturable (VBNC) state. Characteristic of this state are an altered morphology, low metabolic activity, and lack of growth under standard laboratory conditions. Here, for the first time, the cellular ultrastructure of V. cholerae VBNC cells raised in natural waters was investigated using electron cryo-tomography. This was complemented by a comparison of the proteomes and the peptidoglycan composition of V. cholerae from LB overnight cultures and VBNC cells. The extensive remodeling of the VBNC cells was most obvious in the passive dehiscence of the cell envelope, resulting in improper embedment of flagella and pili. Only minor changes of the peptidoglycan and osmoregulated periplasmic glucans were observed. Active changes in VBNC cells included the production of cluster I chemosensory arrays and change of abundance of cluster II array proteins. Components involved in iron acquisition and storage, peptide import and arginine biosynthesis were overrepresented in VBNC cells, while enzymes of the central carbon metabolism were found at lower levels. Finally, several pathogenicity factors of V. cholerae were less abundant in the VBNC state, potentially limiting their infectious potential. This study gives unprecedented insight into the physiology of VBNC cells and the drastically altered presence of their metabolic and structural proteins. [ABSTRACT FROM AUTHOR]

Bacteriocins of Gram-negative bacteria are typically multi-domain proteins that target and kill bacteria of the same or closely related species. There is increasing interest in protein bacteriocin import; from a fundamental perspective to understand how folded proteins are imported into bacteria and from an applications perspective as species-specific antibiotics to combat multidrug resistant bacteria. In order to translocate across the cell envelope and cause cell death, protein bacteriocins hijack nutrient uptake pathways. Their import is energized by parasitizing intermembrane protein complexes coupled to the proton motive force, which delivers a toxic domain into the cell. A plethora of genetic, structural, biochemical, and biophysical methods have been applied to find cell envelope components involved in bacteriocin import since their discovery almost a century ago. Here, we review the various approaches that now exist for investigating how protein bacteriocins translocate into Gram-negative bacteria and highlight areas of research that will need methodological innovations to fully understand this process. We also highlight recent studies demonstrating how bacteriocins can be used to probe organization and architecture of the Gram-negative cell envelope itself. [ABSTRACT FROM AUTHOR]

Lipid-modified cupredoxin azurin (Laz) is involved in electron transport in Neisseria and proposed to act as an electron donor to the surface-displayed nitrite reductase AniA. We identified Laz in Neisseria gonorrhoeae cell envelopes and naturally elaborated membrane vesicles in proteomic investigations focused on discovering new vaccine and therapeutic targets for this increasingly difficult to treat pathogen. Its surface exposure in N. meningitidis suggested Laz could be a vaccine candidate for N. gonorrhoeae. Here we characterized the localization, expression, and role of Laz within the gonococcal cell envelope and challenged the hypothesis that Laz and AniA interact. While we demonstrate that Laz indeed shows some good features of a vaccine antigen, such as stable expression, high conservation, and ability to elicit antibodies that cross-react with a diverse panel of Neisseria, it is not a surface-displayed lipoprotein in the gonococcus. This discovery eliminates Laz as a gonorrhea vaccine candidate, further highlighting the necessity of examining homologous protein localization between closely related species. Absence of Laz slightly altered cell envelope integrity but was not associated with growth defects in vitro, including during anoxia, implicating the presence of other electron pathways to AniA. To further dissect the implied AniA-Laz interaction, we utilized biolayer interferometry and optimized and executed chemical cross-linking coupled with immunoblotting to covalently link interacting protein partners in living gonococci. This method, applied for the first time in N. gonorrhoeae research to interrogate protein complexes, was validated by the appearance of the trimer form of AniA, as well as by increased formation of the β-barrel assembly machinery complex, in the presence of cross-linker. We conclude that Laz is not an electron donor to AniA based on their distinct subcellular localization, discordant expression during infection of the female mouse lower genital tract, and lack of interaction in vivo and in vitro.

Mycobacteria are well known for their taxonomic diversity, their impact on global health, and for their atypical cell wall and envelope. In addition to a cytoplasmic membrane and a peptidoglycan layer, the cell envelope of members of the order Corynebacteriales, which include Mycobacterium tuberculosis, also have an arabinogalactan layer connecting the peptidoglycan to an outer membrane, the so-called “mycomembrane.” This unusual cell envelope composition of mycobacteria is of prime importance for several physiological processes such as protection from external stresses and for virulence. Although there have been recent breakthroughs in the elucidation of the composition and organization of this cell envelope, its evolutionary origin remains a mystery. In this perspectives article, the characteristics of the cell envelope of mycobacteria with respect to other actinobacteria will be dissected through a molecular evolution framework in order to provide a panoramic view of the evolutionary pathways that appear to be at the origin of this unique cell envelope. In combination with a robust molecular phylogeny, we have assembled a gene matrix based on the presence or absence of key determinants of cell envelope biogenesis in the Actinobacteria phylum. We present several evolutionary scenarios regarding the origin of the mycomembrane. In light of the data presented here, we also propose a novel alternative hypothesis whereby the stepwise acquisition of core enzymatic functions may have allowed the sequential remodeling of the external cell membrane during the evolution of Actinobacteria and has led to the unique mycomembrane of slow-growing mycobacteria as we know it today.

The brucellae are facultative intracellular bacteria that cause a worldwide extended zoonosis. One of the pathogenicity mechanisms of these bacteria is their ability to avoid rapid recognition by innate immunity because of a reduction of the pathogen-associated molecular pattern (PAMP) of the lipopolysaccharide (LPS), free-lipids, and other envelope molecules. We investigated the Brucella homologs of lptA, lpxE, and lpxO, three genes that in some pathogens encode enzymes that mask the LPS PAMP by upsetting the core-lipid A charge/hydrophobic balance. Brucella lptA, which encodes a putative ethanolamine transferase, carries a frame-shift in B. abortus but not in other Brucella spp. and phylogenetic neighbors like the opportunistic pathogen Ochrobactrum anthropi. Consistent with the genomic evidence, a B. melitensis lptA mutant lacked lipid A-linked ethanolamine and displayed increased sensitivity to polymyxin B (a surrogate of innate immunity bactericidal peptides), while B. abortus carrying B. melitensis lptA displayed increased resistance. Brucella lpxE encodes a putative phosphatase acting on lipid A or on a free-lipid that is highly conserved in all brucellae and O. anthropi. Although we found no evidence of lipid A dephosphorylation, a B. abortus lpxE mutant showed increased polymyxin B sensitivity, suggesting the existence of a hitherto unidentified free-lipid involved in bactericidal peptide resistance. Gene lpxO putatively encoding an acyl hydroxylase carries a frame-shift in all brucellae except B. microti and is intact in O. anthropi. Free-lipid analysis revealed that lpxO corresponded to olsC, the gene coding for the ornithine lipid (OL) acyl hydroxylase active in O. anthropi and B. microti, while B. abortus carrying the olsC of O. anthropi and B. microti synthesized hydroxylated OLs. Interestingly, mutants in lptA, lpxE, or olsC were not attenuated in dendritic cells or mice. This lack of an obvious effect on virulence together with the presence of the intact homolog genes in O. anthropi and B. microti but not in other brucellae suggests that LptA, LpxE, or OL β-hydroxylase do not significantly alter the PAMP properties of Brucella LPS and free-lipids and are therefore not positively selected during the adaptation to intracellular life.

Introduction: Mycobacteria have several unique cellular characteristics, such as multiple cell envelope layers, elongation at cell poles, asymmetric cell division, and accumulation of intracytoplasmic lipid inclusions, which contributes to their survival under stress conditions. However, the understanding of these characteristics in clinical Mycobacterium tuberculosis (M. tuberculosis) isolates and under host stress is limited. We previously reported the influence of host stress on the cell length distribution in a large set of clinical M. tuberculosis isolates (n = 158). Here, we investigate the influence of host stress on the cellular ultrastructure of few clinical M. tuberculosis isolates (n = 8) from that study. The purpose of this study is to further understand the influence of host stress on the cellular adaptations of clinical M. tuberculosis isolates.Methods: We selected few M. tuberculosis isolates (n = 8) for analyzing the cellular ultrastructure ex vivo in sputum and under in vitro stress conditions by transmission electron microscopy. The cellular adaptations of M. tuberculosis in sputum were correlated with the ultrastructure of antibiotic sensitive and resistant isolates in liquid culture, under oxidative stress, iron deficiency, and exposure to isoniazid.Results: In sputum, M. tuberculosis accumulated intracytoplasmic lipid inclusions. In liquid culture, clinical M. tuberculosis revealed isolate to isolate variation in the extent of intracytoplasmic lipid inclusions, which were absent in the laboratory strain H37Rv. Oxidative stress, iron deficiency, and exposure to isoniazid increased the accumulation of lipid inclusions and decreased the thickness of the cell envelope electron transparent layer in M. tuberculosis cells. Furthermore, intracytoplasmic compartments were observed in iron deficient cells.Conclusion: Our ultrastructural analysis has revealed significant influence of host stress on the cellular adaptations in clinical M. tuberculosis isolates. These adaptations may contribute to the survival of M. tuberculosis under host and antibiotic stress conditions. Variation in the cellular adaptations among clinical M. tuberculosis isolates may correlate with their ability to persist in tuberculosis patients during antibiotic treatment. These observations indicate the need for further analyzing these cellular adaptations in a large set of clinical M. tuberculosis isolates. This will help to determine the significance of these cellular adaptations in the tuberculosis treatment.

In contrast to bacteria, all archaea possess cell walls lacking peptidoglycan and a number of different cell envelope components have also been described. A paracrystalline protein surface layer, commonly referred to as S-layer, is present in nearly all archaea described to date. S-layers are composed of only one or two proteins and form different lattice structures. In this review, we summarize current understanding of archaeal S-layer proteins, discussing topics such as structure, lattice type distribution among archaeal phyla and glycosylation. The hexagonal lattice type is dominant within the phylum Euryarchaeota, while in the Crenarchaeota this feature is mainly associated with specific orders. S-layers exclusive to the Crenarchaeota have also been described, which are composed of two proteins. Information regarding S-layers in the remaining archaeal phyla is limited, mainly due to organism description through only culture-independent methods. Despite the numerous applied studies using bacterial S-layers, few reports have employed archaea as a study model. As such, archaeal S-layers represent an area for exploration in both basic and applied research.

Lipid-modified cupredoxin azurin (Laz) is involved in electron transport in Neisseria and proposed to act as an electron donor to the surface-displayed nitrite reductase AniA. We identified Laz in Neisseria gonorrhoeae cell envelopes and naturally elaborated membrane vesicles in proteomic investigations focused on discovering new vaccine and therapeutic targets for this increasingly difficult to treat pathogen. Its surface exposure in N. meningitidis suggested Laz could be a vaccine candidate for N. gonorrhoeae. Here we characterized the localization, expression, and role of Laz within the gonococcal cell envelope and challenged the hypothesis that Laz and AniA interact. While we demonstrate that Laz indeed shows some good features of a vaccine antigen, such as stable expression, high conservation, and ability to elicit antibodies that cross-react with a diverse panel of Neisseria , it is not a surface-displayed lipoprotein in the gonococcus. This discovery eliminates Laz as a gonorrhea vaccine candidate, further highlighting the necessity of examining homologous protein localization between closely related species. Absence of Laz slightly altered cell envelope integrity but was not associated with growth defects in vitro , including during anoxia, implicating the presence of other electron pathways to AniA. To further dissect the implied AniA-Laz interaction, we utilized biolayer interferometry and optimized and executed chemical cross-linking coupled with immunoblotting to covalently link interacting protein partners in living gonococci. This method, applied for the first time in N. gonorrhoeae research to interrogate protein complexes, was validated by the appearance of the trimer form of AniA, as well as by increased formation of the β-barrel assembly machinery complex, in the presence of cross-linker. We conclude that Laz is not an electron donor to AniA based on their distinct subcellular localization, discordant expression during infection of the female mouse lower genital tract, and lack of interaction in vivo and in vitro. [ABSTRACT FROM AUTHOR]

Mycobacteria are well known for their taxonomic diversity, their impact on global health, and for their atypical cell wall and envelope. In addition to a cytoplasmic membrane and a peptidoglycan layer, the cell envelope of members of the order Corynebacteriales , which include Mycobacterium tuberculosis , also have an arabinogalactan layer connecting the peptidoglycan to an outer membrane, the so-called "mycomembrane." This unusual cell envelope composition of mycobacteria is of prime importance for several physiological processes such as protection from external stresses and for virulence. Although there have been recent breakthroughs in the elucidation of the composition and organization of this cell envelope, its evolutionary origin remains a mystery. In this perspectives article, the characteristics of the cell envelope of mycobacteria with respect to other actinobacteria will be dissected through a molecular evolution framework in order to provide a panoramic view of the evolutionary pathways that appear to be at the origin of this unique cell envelope. In combination with a robust molecular phylogeny, we have assembled a gene matrix based on the presence or absence of key determinants of cell envelope biogenesis in the Actinobacteria phylum. We present several evolutionary scenarios regarding the origin of the mycomembrane. In light of the data presented here, we also propose a novel alternative hypothesis whereby the stepwise acquisition of core enzymatic functions may have allowed the sequential remodeling of the external cell membrane during the evolution of Actinobacteria and has led to the unique mycomembrane of slow-growing mycobacteria as we know it today. [ABSTRACT FROM AUTHOR]

Introduction: Mycobacteria have several unique cellular characteristics, such as multiple cell envelope layers, elongation at cell poles, asymmetric cell division, and accumulation of intracytoplasmic lipid inclusions, which contributes to their survival under stress conditions. However, the understanding of these characteristics in clinical Mycobacterium tuberculosis (M. tuberculosis) isolates and under host stress is limited. We previously reported the influence of host stress on the cell length distribution in a large set of clinical M. tuberculosis isolates (n = 158). Here, we investigate the influence of host stress on the cellular ultrastructure of few clinical M. tuberculosis isolates (n = 8) from that study. The purpose of this study is to further understand the influence of host stress on the cellular adaptations of clinical M. tuberculosis isolates. Methods: We selected few M. tuberculosis isolates (n = 8) for analyzing the cellular ultrastructure ex vivo in sputum and under in vitro stress conditions by transmission electron microscopy. The cellular adaptations of M. tuberculosis in sputum were correlated with the ultrastructure of antibiotic sensitive and resistant isolates in liquid culture, under oxidative stress, iron deficiency, and exposure to isoniazid. Results: In sputum, M. tuberculosis accumulated intracytoplasmic lipid inclusions. In liquid culture, clinical M. tuberculosis revealed isolate to isolate variation in the extent of intracytoplasmic lipid inclusions, which were absent in the laboratory strain H37Rv. Oxidative stress, iron deficiency, and exposure to isoniazid increased the accumulation of lipid inclusions and decreased the thickness of the cell envelope electron transparent layer in M. tuberculosis cells. Furthermore, intracytoplasmic compartments were observed in iron deficient cells. Conclusion: Our ultrastructural analysis has revealed significant influence of host stress on the cellular adaptations in clinical M. tuberculosis isolates. These adaptations may contribute to the survival of M. tuberculosis under host and antibiotic stress conditions. Variation in the cellular adaptations among clinical M. tuberculosis isolates may correlate with their ability to persist in tuberculosis patients during antibiotic treatment. These observations indicate the need for further analyzing these cellular adaptations in a large set of clinical M. tuberculosis isolates. This will help to determine the significance of these cellular adaptations in the tuberculosis treatment. [ABSTRACT FROM AUTHOR]

The brucellae are facultative intracellular bacteria that cause a worldwide extended zoonosis. One of the pathogenicity mechanisms of these bacteria is their ability to avoid rapid recognition by innate immunity because of a reduction of the pathogenassociated molecular pattern (PAMP) of the lipopolysaccharide (LPS), free-lipids, and other envelope molecules. We investigated the Brucella homologs of lptA, lpxE, and lpxO, three genes that in some pathogens encode enzymes that mask the LPS PAMP by upsetting the core-lipid A charge/hydrophobic balance. Brucella lptA, which encodes a putative ethanolamine transferase, carries a frame-shift in B. abortus but not in other Brucella spp. and phylogenetic neighbors like the opportunistic pathogen Ochrobactrum anthropi. Consistent with the genomic evidence, a B. melitensis lptA mutant lacked lipid A-linked ethanolamine and displayed increased sensitivity to polymyxin B (a surrogate of innate immunity bactericidal peptides), while B. abortus carrying B. melitensis lptA displayed increased resistance. Brucella lpxE encodes a putative phosphatase acting on lipid A or on a free-lipid that is highly conserved in all brucellae and O. anthropi. Although we found no evidence of lipid A dephosphorylation, a B. abortus lpxE mutant showed increased polymyxin B sensitivity, suggesting the existence of a hitherto unidentified free-lipid involved in bactericidal peptide resistance. Gene lpxO putatively encoding an acyl hydroxylase carries a frame-shift in all brucellae except B. microti and is intact in O. anthropi. Free-lipid analysis revealed that lpxO corresponded to olsC, the gene coding for the ornithine lipid (OL) acyl hydroxylase active in O. anthropi and B. microti, while B. abortus carrying the olsC of O. anthropi and B. microti synthesized hydroxylated OLs. Interestingly, mutants in lptA, lpxE, or olsCwere not attenuated in dendritic cells or mice. This lack of an obvious effect on virulence together with the presence of the intact homolog genes in O. anthropi and B. microti but not in other brucellae suggests that LptA, LpxE, or OL β-hydroxylase do not significantly alter the PAMP properties of Brucella LPS and free-lipids and are therefore not positively selected during the adaptation to intracellular life. [ABSTRACT FROM AUTHOR]

Bacteria will accompany humans in our exploration of space, making it of importance to study their adaptation to the microgravity environment. To investigate potential phenotypic changes for bacteria grown in space, Escherichia coli was cultured onboard the International Space Station with matched controls on Earth. Samples were challenged with different concentrations of gentamicin sulfate to study the role of drug concentration on the dependent variables in the space environment. Analyses included assessments of final cell count, cell size, cell envelope thickness, cell ultrastructure, and culture morphology. A 13-fold increase in final cell count was observed in space with respect to the ground controls and the space flight cells were able to grow in the presence of normally inhibitory levels of gentamicin sulfate. Contrast light microscopy and focused ion beam/scanning electron microscopy showed that, on average, cells in space were 37% of the volume of their matched controls, which may alter the rate of molecule–cell interactions in a diffusion-limited mass transport regime as is expected to occur in microgravity. TEM imagery showed an increase in cell envelope thickness of between 25 and 43% in space with respect to the Earth control group. Outer membrane vesicles were observed on the spaceflight samples, but not on the Earth cultures. While E. coli suspension cultures on Earth were homogenously distributed throughout the liquid medium, in space they tended to form a cluster, leaving the surrounding medium visibly clear of cells. This cell aggregation behavior may be associated with enhanced biofilm formation observed in other spaceflight experiments.

Bacteria will accompany humans in our exploration of space, making it of importance to study their adaptation to the microgravity environment. To investigate potential phenotypic changes for bacteria grown in space, Escherichia coli was cultured onboard the International Space Station with matched controls on Earth. Samples were challenged with different concentrations of gentamicin sulfate to study the role of drug concentration on the dependent variables in the space environment. Analyses included assessments of final cell count, cell size, cell envelope thickness, cell ultrastructure, and culture morphology. A 13-fold increase in final cell count was observed in space with respect to the ground controls and the space flight cells were able to grow in the presence of normally inhibitory levels of gentamicin sulfate. Contrast light microscopy and focused ion beam/scanning electron microscopy showed that, on average, cells in space were 37% of the volume of their matched controls, which may alter the rate of molecule-cell interactions in a diffusion-limited mass transport regime as is expected to occur in microgravity. TEM imagery showed an increase in cell envelope thickness of between 25 and 43% in space with respect to the Earth control group. Outer membrane vesicles were observed on the spaceflight samples, but not on the Earth cultures. While E. coli suspension cultures on Earth were homogenously distributed throughout the liquid medium, in space they tended to form a cluster, leaving the surrounding medium visibly clear of cells. This cell aggregation behavior may be associated with enhanced biofilm formation observed in other spaceflight experiments. [ABSTRACT FROM AUTHOR]

Keywords: cell cycle; nucleoid; divisome; cell division; cell envelope; cell shape; chromosome replication Cell cycle, nucleoid, divisome, cell division, cell envelope, cell shape, chromosome replication. [Extracted from the article]

The function of membrane proteases range from general house-keeping to regulation of cellular processes. Although the biological role of these enzymes in archaea is poorly understood, some of them are implicated in the biogenesis of the archaeal cell envelope and surface structures. The membrane-bound ATP-dependent Lon protease is essential for cell viability and affects membrane carotenoid content in Haloferax volcanii. At least two different proteases are needed in this archaeon to accomplish the posttranslational modifications of the S-layer glycoprotein. The rhomboid protease RhoII is involved in the N-glycosylation of the S-layer protein with a sulfoquinovose-containing oligosaccharide while archaeosortase ArtA mediates the proteolytic processing coupled-lipid modification of this glycoprotein facilitating its attachment to the archaeal cell surface. Interestingly, two different signal peptidase I homologs exist in H. volcanii, Sec11a and Sec11b, which likely play distinct physiological roles. Type IV prepilin peptidase PibD processes flagellin/pilin precursors, being essential for the biogenesis and function of the archaellum and other cell surface structures in H. volcanii.

Listeria monocytogenes is a Gram-positive pathogen responsible for the manifestation of human listeriosis, an opportunistic foodborne disease with an associated high mortality rate. The key to the pathogenesis of listeriosis is the capacity of this bacterium to trigger its internalization by non-phagocytic cells and to survive and even replicate within phagocytes. The arsenal of virulence proteins deployed by L. monocytogenes to successfully promote the invasion and infection of host cells has been progressively unveiled over the past decades. A large majority of them are located at the cell envelope, which provides an interface for the establishment of close interactions between these bacterial factors and their host targets. Along the multistep pathways carrying these virulence proteins from the inner side of the cytoplasmic membrane to their cell envelope destination, a multiplicity of auxiliary proteins must act on the immature polypeptides to ensure that they not only maturate into fully functional effectors but also are placed or guided to their correct position in the bacterial surface. As the major scaffold for surface proteins, the cell wall and its metabolism are critical elements in listerial virulence. Conversely, the crucial physical support and protection provided by this structure make it an ideal target for the host immune system. Therefore, mechanisms involving fine modifications of cell envelope components are activated by L. monocytogenes to render it less recognizable by the innate immunity sensors or more resistant to the activity of antimicrobial effectors. This review provides a state-of-the-art compilation of the mechanisms used by L. monocytogenes to organize its surface for virulence, with special focus on those proteins that work behind the frontline, either supporting virulence effectors or ensuring the survival of the bacterium within its host.

The function of membrane proteases range from general house-keeping to regulation of cellular processes. Although the biological role of these enzymes in archaea is poorly understood, some of them are implicated in the biogenesis of the archaeal cell envelope and surface structures. The membrane-bound ATP-dependent Lon protease is essential for cell viability and affects membrane carotenoid content in Haloferax volcanii. At least two different proteases are needed in this archaeon to accomplish the posttranslational modifications of the S-layer glycoprotein. The rhomboid protease RhoII is involved in the N-glycosylation of the S-layer protein with a sulfoquinovose-containing oligosaccharide while archaeosortase ArtA mediates the proteolytic processing coupled-lipid modification of this glycoprotein facilitating its attachment to the archaeal cell surface. Interestingly, two different signal peptidase I homologs exist in H. volcanii, Sec11a and Sec11b, which likely play distinct physiological roles. Type IV prepilin peptidase PibD processes flagellin/pilin precursors, being essential for the biogenesis and function of the archaellum and other cell surface structures in H. volcanii. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a Gram-positive pathogen responsible for the manifestation of human listeriosis, an opportunistic foodborne disease with an associated high mortality rate. The key to the pathogenesis of listeriosis is the capacity of this bacterium to trigger its internalization by non-phagocytic cells and to survive and even replicate within phagocytes. The arsenal of virulence proteins deployed by L. monocytogenes to successfully promote the invasion and infection of host cells has been progressively unveiled over the past decades. A large majority of them is located at the cell envelope, which provides an interface for the establishment of close interactions between these bacterial factors and their host targets. Along the multistep pathways carrying these virulence proteins from the inner side of the cytoplasmic membrane to their cell envelope destination, a multiplicity of auxiliary proteins must act on the immature polypeptides to ensure that they not only maturate into fully functional effectors but also are placed or guided to their correct position in the bacterial surface. As the major scaffold for surface proteins, the cell wall and its metabolism are critical elements in listerial virulence. Conversely, the crucial physical support and protection provided by this structure make it an ideal target for the host immune system. Therefore, mechanisms involving fine modifications of cell envelope components are activated by L. monocytogenes to render it less recognizable by the innate immunity sensors or more resistant to the activity of antimicrobial effectors. This review provides a state-of-the-art compilation of the mechanisms used by L. monocytogenes to organize its surface for virulence, with special focus on those proteins that work "behind the frontline", either supporting virulence effectors or ensuring the survival of the bacterium within its host. [ABSTRACT FROM AUTHOR]