Membrane proteome of Acinetobacter radioresistens S13 during aromatic exposure

Study of the bacterial membrane proteome, though in its early stages, is a field of growing interest in the search for information about nutrient transport and processing. We tested different strategies and chemical compounds to extract proteins from the membranes (inner and outer) of Acinetobacter radioresistens S13, a Gram-negative bacterium selected for its ability to degrade aromatics. A. radioresistens S13 was monitored under different growth substrate conditions, using acetate, benzoate or phenol as sole carbon source. Two-dimensional gel electrophoresis map analysis of membrane extracts from benzoate- and phenol-grown cells reveals differences versus controls (acetate-grown cultures). Primarily, a different pattern of spots was observed and, in particular, some proteins were only expressed in the presence of aromatic substrate. Among these, we detected a Na(+)/H(+) antiporter, whose function is likely to be regulation of intracellular pH, and an ABC type sugar transport system, probably involved in capsular polysaccharide translocation. We also identified other proteins, detectable in acetate-grown but over-expressed in aromatic-grown cells. These include: (1) an outer membrane protein ascribable to an OmpA-like protein, recently described in the literature as "alasan", a bioemulsifying agent involved in solubilizing and enhancing bioavailability of hydrocarbons; (2) a trimeric porin of the PhoE family also belonging to the outer membrane and involved in facilitating the transport of anions (especially phosphate); and (3) two glycosyl transferases probably involved in capsules and/or lipopolysaccharide biosynthesis. Study of the bacterial membrane proteome helps to elucidate the role of the membrane as modulable site enabling communication between internal and external environments.     

ZraP is a periplasmic molecular chaperone and a repressor of the zinc-responsive two-component regulator ZraSR

The bacterial envelope is the interface with the surrounding environment and is consequently subjected to a barrage of noxious agents including a range of compounds with antimicrobial activity. The ESR (envelope stress response) pathways of enteric bacteria are critical for maintenance of the envelope against these antimicrobial agents. In the present study, we demonstrate that the periplasmic protein ZraP contributes to envelope homoeostasis and assign both chaperone and regulatory function to ZraP from Salmonella Typhimurium. The ZraP chaperone mechanism is catalytic and independent of ATP; the chaperone activity is dependent on the presence of zinc, which is shown to be responsible for the stabilization of an oligomeric ZraP complex. Furthermore, ZraP can act to repress the two-component regulatory system ZraSR, which itself is responsive to zinc concentrations. Through structural homology, ZraP is a member of the bacterial CpxP family of periplasmic proteins, which also consists of CpxP and Spy. We demonstrate environmental co-expression of the CpxP family and identify an important role for these proteins in Salmonella's defence against the cationic antimicrobial peptide polymyxin B.     

Intraspecific differences in bacterial responses to modelled reduced gravity

AIMS: Bacteria are important residents of water systems, including those of space stations which feature specific environmental conditions, such as lowered effects of gravity. The purpose of this study was to compare responses with modelled reduced gravity of space station, water system bacterial isolates with other isolates of the same species. METHODS AND RESULTS: Bacterial isolates, Stenotrophomonas paucimobilis and Acinetobacter radioresistens, originally recovered from the water supply aboard the International Space Station (ISS) were grown in nutrient broth under modelled reduced gravity. Their growth was compared with type strains S. paucimobilis ATCC 10829 and A. radioresistens ATCC 49000. Acinetobacter radioresistens ATCC 49000 and the two ISS isolates showed similar growth profiles under modelled reduced gravity compared with normal gravity, whereas S. paucimobilis ATCC 10829 was negatively affected by modelled reduced gravity. CONCLUSIONS: These results suggest that microgravity might have selected for bacteria that were able to thrive under this unusual condition. These responses, coupled with impacts of other features (such as radiation resistance and ability to persist under very oligotrophic conditions), may contribute to the success of these water system bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Water quality is a significant factor in many environments including the ISS. Efforts to remove microbial contaminants are likely to be complicated by the features of these bacteria which allow them to persist under the extreme conditions of the systems.     

Allosteric activation of a bacterial stress sensor

In Gram-negative bacteria, envelope stress signals such as unfolded outer membrane proteins (OMP) activate the periplasmic protease DegS. This protease then triggers a cellular pathway to alleviate the stress. Now Sohn et al. (2007) show conclusively that inhibition of DegS is relieved allosterically by binding of the C-terminal sequences in unfolded OMPs to the PDZ domain of DegS.     

Recognition of β-strand motifs by RseB is required for σ(E) activity in Escherichia coli

Gram-negative bacteria react to misfolded proteins in the envelope through a myriad of different stress response pathways. This cohort of pathways allows the bacteria to specifically respond to different types of damage, and many of these have been discovered to have key roles in the virulence of bacterial pathogens. Misfolded outer membrane proteins (OMPs) are typically recognized by the σ(E) pathway, a highly conserved envelope stress response pathway. We examined the features of misfolded OMPs with respect to their ability to generate envelope stress responses. We determined that the secondary structure, particularly the potential to form β strands, is critical to inducing the σ(E) response in an RseB-dependent manner. The sequence of the potential β-strand motif modulates the strength of the σ(E) response generated by the constructs. By understanding the details of how such stress response pathways are activated, we can gain a greater understanding of how bacteria survive in harsh environments.     

Expression of glutathione S-transferase and peptide methionine sulphoxide reductase in Ochrobactrum anthropi is correlated to the production of reactive oxygen species caused by aromatic substrates

Peptide methionine sulphoxide reductase (MsrA) and glutathione S-transferases (GSTs) are considered as detoxification enzymes. In the xenobiotics-degrading bacterium Ochrobactrum anthropi the two enzymes are co-induced by toxic concentrations of aromatic substrates such as phenol and 4-chlorophenol. In aerobic organisms, degradation of aromatic substrates by mono- and dioxygenases leads to a generation of oxidative stress that causes the occurrence of reactive oxygen species (ROS). A capillary electrophoretic method, using the intracellular conversion of dihydrorhodamine-123 into rhodamine-123, was developed to measure the content of ROS in the bacteria. The presence of toxic concentrations of the aromatic substrate 4-chlorophenol, an inducer of GST and MsrA, leads to a significant increase in the production of ROS. These results strongly suggest that GST and MsrA enzymes are part of the bacterial defence mechanism against particular oxidative stress conditions. As oxidative stress is known to be present predominantly close to the cytoplasmic membrane, we investigated the subcellular distribution of both MsrA and GST enzymes in this bacterium grown in the presence of 4-chlorophenol. By Western blotting, MsrA and GST was assayed in the cytoplasm as well as in the periplasm. Moreover, immunolocalisation by colloidal gold immunoelectron microscopy identified the two proteins associated with the cell envelope.     

Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response

Conditions that impair protein folding in the Gram-negative bacterial envelope cause stress. The destabilizing effects of stress in this compartment are recognized and countered by a number of signal transduction mechanisms. Data presented here reveal another facet of the complex bacterial stress response, release of outer membrane vesicles. Native vesicles are composed of outer membrane and periplasmic material, and they are released from the bacterial surface without loss of membrane integrity. Here we demonstrate that the quantity of vesicle release correlates directly with the level of protein accumulation in the cell envelope. Accumulation of material occurs under stress, and is exacerbated upon impairment of the normal housekeeping and stress-responsive mechanisms of the cell. Mutations that cause increased vesiculation enhance bacterial survival upon challenge with stressing agents or accumulation of toxic misfolded proteins. Preferential packaging of a misfolded protein mimic into vesicles for removal indicates that the vesiculation process can act to selectively eliminate unwanted material. Our results demonstrate that production of bacterial outer membrane vesicles is a fully independent, general envelope stress response. In addition to identifying a novel mechanism for alleviating stress, this work provides physiological relevance for vesicle production as a protective mechanism.     

A synergistic role for two predicted inner membrane proteins of Escherichia coli in cell envelope integrity

The bacterial cytoplasmic membrane is a principal site of protein translocation, lipid and peptidoglycan biogenesis, signal transduction, transporters and energy generating components of the respiratory chain. Although 25–30% of bacterial proteomes consist of membrane proteins, a comprehensive understanding of their influence on fundamental cellular processes is incomplete. Here, we show that YciB and DcrB, two small cytoplasmic membrane proteins of previously unknown functions, play an essential synergistic role in maintaining cell envelope integrity of Escherichia coli. Lack of both YciB and DcrB results in pleiotropic cell defects including increased levels of lipopolysaccharide, membrane vesiculation, dynamic shrinking and extension of the cytoplasmic membrane accompanied by lysis and cell death. The stalling of an abundant outer membrane lipoprotein, Lpp, at the periplasmic face of the inner membrane leads to lethal inner membrane–peptidoglycan linkages. Additionally, the periplasmic chaperone Skp contributes to yciB dcrB mutant cell death by possibly mistargeting stalled porins into the inner membrane. Consistent with the idea of a compromised envelope in the yciB dcrB mutant, multiple envelope stress response systems are induced, with Cpx signal transduction being required for growth. Taken together, our results suggest a fundamental role for YciB and DcrB in cell envelope biogenesis.     

A third envelope stress signal transduction pathway in Escherichia coli

Escherichia coli uses overlapping envelope stress responses to adapt to insults to the bacterial envelope that cause protein misfolding. The sigmaE and Cpx envelope stress responses are activated by both common and distinct envelope stresses and respond by increasing the expression of the periplasmic protease DegP as well as target genes unique to each response. The sigmaE pathway is involved in outer membrane protein (OMP) folding quality control whereas the Cpx pathway plays an important role in the assembly of at least one pilus. Previously, we identified the spy gene as a new Cpx regulon member of unknown function. Interestingly, induction of spy expression by severe envelope stresses such as spheroplasting is only partially dependent on an intact Cpx signalling pathway, unlike other Cpx-regulated genes. Here we show that the BaeS sensor kinase and BaeR response regulator also control expression of spy in response to envelope stress. BaeS and BaeR do not affect expression of other known Cpx-regulated genes, however, baeR cpxR double mutants show increased sensitivity to envelope stresses relative to either single mutant alone. We propose that the Bae signal transduction pathway controls a third envelope stress response in E. coli that induces expression of a distinct set of adaptive genes.     

Regulation of the Escherichia coli sigma-dependent envelope stress response

The Escherichia colisigma(E)-dependent stress response pathway controls the expression of genes encoding periplasmic folding catalysts, proteases, biosynthesis enzymes for lipid A (a component of lipopolysaccharide or LPS) and other proteins known or predicted to function in or produce components of the envelope. When E. coli is subjected to heat or other stresses that generate unfolded envelope proteins, sigma(E) activity is induced. Four key players in this signal transduction pathway have been identified: RseA, an inner membrane sigma(E) antisigma factor; RseB, a periplasmic protein that binds to the periplasmic face of RseA; and the DegS and YaeL proteases. The major point of regulation, the interaction between sigma(E) and RseA, is primarily controlled by the stability of RseA. Envelope stress promotes RseA degradation, which occurs by a proteolytic cascade initiated by DegS. There is evidence that one sigma(E)-inducing stress (OmpC overexpression) directly activates DegS to cleave RseA. Secondarily, envelope stress may relieve RseB-mediated enhancement of RseA activity. Additional levels of control upon sigma(E) activity may become evident upon further study of this stress response pathway.     

A dual function for SecA in the assembly of single spanning membrane proteins in Escherichia coli

The assembly of bacterial membrane proteins with large periplasmic loops is an intrinsically complex process because the SecY translocon has to coordinate the signal recognition particle-dependent targeting and integration of transmembrane domains with the SecA-dependent translocation of the periplasmic loop. The current model suggests that the ATP hydrolysis by SecA is required only if periplasmic loops larger than 30 amino acids have to be translocated. In agreement with this model, our data demonstrate that the signal recognition particle- and SecA-dependent multiple spanning membrane protein YidC becomes SecA-independent if the large periplasmic loop connecting transmembrane domains 1 and 2 is reduced to less than 30 amino acids. Strikingly, however, we were unable to render single spanning membrane proteins SecA-independent by reducing the length of their periplasmic loops. For these proteins, the complete assembly was always SecA-dependent even if the periplasmic loop was reduced to 13 amino acids. If, however, the 13-amino acid-long periplasmic loop was fused to a downstream transmembrane domain, SecA was no longer required for complete translocation. Although these data support the current model on the SecA dependence of multiple spanning membrane proteins, they indicate a novel function of SecA for the assembly of single spanning membrane proteins. This could suggest that single and multiple spanning membrane proteins are processed differently by the bacterial SecY translocon.     

Cellular Architecture of Treponema pallidum: Novel Flagellum, Periplasmic Cone, and Cell Envelope as Revealed by Cryo Electron Tomography

High-resolution cryo electron tomography (cryo-ET) was utilized to visualize Treponema pallidum, the causative agent of syphilis, at the molecular level. Three-dimensional (3D) reconstructions from 304 infectious organisms revealed unprecedented cellular structures of this unusual member of the spirochetal family. High-resolution cryo-ET reconstructions provided detailed structures of the cell envelope, which is significantly different from that of Gram-negative bacteria. The 4-nm lipid bilayer of both outer membrane and cytoplasmic membrane resolved in 3D reconstructions, providing an important marker for interpreting membrane-associated structures. Abundant lipoproteins cover the outer leaflet of the cytoplasmic membrane, in contrast to the rare outer membrane proteins visible by scanning probe microscopy. High-resolution cryo-ET images also provided the first observation of T. pallidum chemoreceptor arrays, as well as structural details of the periplasmically located cone-shaped structure at both ends of the bacterium. Furthermore, 3D subvolume averages of periplasmic flagellar motors and flagellar filaments from living organisms revealed the novel flagellar architectures that may facilitate their rotation within the confining periplasmic space. Our findings provide the most detailed structural understanding of periplasmic flagella and the surrounding cell envelope, which enable this enigmatic bacterium to efficiently penetrate tissue and to escape host immune responses.     

细菌菌影作为新型疫苗和递送载体的研究进展

细菌菌影（bacterial ghost,BG）是革兰阴性菌在噬菌体PhiX174的裂解基因E的作用下形成不含核酸、核糖体等胞质内容物的细菌空壳。这种细菌空壳保留了与天然细菌一样的完整外膜结构,且不具有活菌样的致病作用,可作为疫苗无需佐剂就能诱导机体产生体液免疫应答和细胞免疫应答。菌影内部及外膜上可装载DNA、抗原和药物等异源物质,易被机体免疫细胞识别捕获,使其成为一种新型的生物递送载体。另外,菌影具有制备简单,易于保存等优点。细菌菌影在疾病预防和治疗方面具有广阔的应用前景。     

Hfq reduces envelope stress by controlling expression of envelope‐localized proteins and protein complexes in enteropathogenic Escherichia coli

Gram‐negative bacteria possess several envelope stress responses that detect and respond to damage to this critical cellular compartment. The σ^E envelope stress response senses the misfolding of outer membrane proteins (OMPs), while the Cpx two‐component system is believed to detect the misfolding of periplasmic and inner membrane proteins. Recent studies in several Gram‐negative organisms found that deletion of hfq, encoding a small RNA chaperone protein, activates the σ^E envelope stress response. In this study, we assessed the effects of deleting hfq upon activity of the σ^E and Cpx responses in non‐pathogenic and enteropathogenic (EPEC) strains of Escherichia coli. We found that the σ^E response was activated in Δhfq mutants of all E. coli strains tested, resulting from the misregulation of OMPs. The Cpx response was activated by loss of hfq in EPEC, but not in E. coli K‐12. Cpx pathway activation resulted in part from overexpression of the bundle‐forming pilus (BFP) in EPEC Δhfq. We found that Hfq repressed expression of the BFP via PerA, a master regulator of virulence in EPEC. This study shows that Hfq has a more extensive role in regulating the expression of envelope proteins and horizontally acquired virulence genes in E. coli than previously recognized.     

The catechol 1,2 dioxygenase system of Acinetobacter radioresistens: isoenzymes, inductors and gene localisation

Two different isozymes (Iso A and Iso B) of catechol 1,2 dioxygenase (C1,2O) were isolated from cultures of A. radioresistens grown in two different media, containing phenol and benzoate respectively. In the phenol medium the bacteria expressed about 90% of Iso A, whereas in the benzoate medium the Iso A/Iso B ratio was 40:60. The two proteins have different molecular masses, isoelectric points and N-terminal sequences that are not consistent with simple post-translational modifications. Furthermore, their behaviour differs at high temperatures (42 degrees C-47 degrees C) and at moderately acidic pH (pH 6.0): Iso A proved to be the more stable under conditions of environmental stress. Hybridisation analysis with an A. calcoaceticus catA-derived probe revealed that A. radioresistens C1,2O proteins are encoded by two chromosomally located genes. Bidimensional electrophoresis (2DE) maps of crude extracts of cells grown in different carbon sources (phenol, benzoate and acetate) clearly demonstrated a differential induction pattern for the two proteins. The hypothesis of a double set of genes, one for benzoate catabolism and the other for phenol catabolism, is discussed, and analogies are drawn with other known C1,2Os.     

Inhibition of mammalian cathepsins byPlesiomonas shigelloides

To study molecular mechanisms underlying self-defense of the bacterial pathogen Plesiomonas shigelloides against host inflammatory and immune responses, we evaluated its interactions with mammalian papain-like cathepsins that are essential for host immunity. When grown under anaerobic, but not aerobic, conditions, P. shigelloides was shown to bind and inhibit papain, a model representative of the papain family of cysteine proteinases. This points to mammalian cathepsins as likely physiological targets of a novel cysteine-proteinase inhibitor expressed on bacterial cell surface. Both papain and mammalian cathepsins L and B were inhibited by periplasmic extracts of aerobically and anaerobically grown bacteria, the inhibitory activity being higher in the latter. Inhibition by both intact cells and periplasmic samples was rapid and efficient. The results suggest a possible defensive role of bacterial inhibitors of cathepsins during invasion of a mammalian host. The bacteria thus may modulate host protective responses through inhibiting cathepsins involved in antigen processing and presentation.     

Transfer of phospholipid and protein into the envelope of gram-negative bacteria by liposome fusion

A liposome-bacterial fusion system was developed in order to introduce preformed terminal complement complexes, C5b-9, into the outer membrane of Gram-negative bacteria. Liposomes were prepared from a total phospholipid extract of Salmonella minnesota Re595. Fusion between liposomes and Salmonella sp. or Escherichia coli 17 was dependent on time, temperature, pH, and Ca2+ and PO4- concentration. Only Salmonella sp. with attenuated LPS core regions were able to fuse efficiently with liposomes. It was demonstrated that fusion of liposomes with S. minnesota Re595 or E. coli 17 under optimum conditions resulted in (i) quantitative transfer of the self-quenching fluorescent membrane probe octadecyl rhodamine B chloride from the liposomal bilayer to the bacterial envelope, (ii) transfer of radiolabeled liposomal phospholipid to the bacterial outer membrane and its subsequent translocation to the cytoplasmic membrane, demonstrated by isolation of the bacterial membranes following fusion, and (iii) delivery of liposome-entrapped horseradish peroxidase (HRP) to the periplasmic space, confirmed by a chemiluminescent assay. Following fusion of liposomes incorporating C5b-9 complexes with S. minnesota Re595 or E. coli 17, immunological analysis of the isolated membranes revealed C5b-9 complexes located exclusively in the outer membrane.     

Catabolic pathways and cellular responses of Pseudomonas putida P8 during growth on benzoate with a proteomics approach

The catabolic pathways and cellular responses of Pseudomonas putida P8 during growth on benzoate were studied through proteomics approach. Two-dimensional gel electrophoresis (2-DE) gel profiles of P. putida cells grown on 100 and 800 mg/L benzoate were quantitatively compared using threshold criteria and statistical tools. Protein spots of interest were identified through database searching based on peptide mass fingerprints (PMFs) obtained using matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Eight catabolic enzymes involved in both the ortho-cleavage (CatB, PcaI, and PcaF) and the meta-cleavage (DmpC, DmpD, DmpE, DmpF, and DmpG) pathways for benzoate biodegradation were identified in P. putida grown on 800 mg/L of benzoate while no meta-cleavage pathway enzymes were observed in the 2-DE gel profiles of P. putida grown on 100 mg/L of benzoate. The activation of both the ortho- and the meta-cleavage pathways in P. putida P8 grown on high benzoate concentration was confirmed directly at the protein level. In addition, another 28 differentially expressed proteins were also identified, including proteins involved in (i) detoxification and stress response (AhpC, ATPase-like ATP-binding region, putative DNA-binding stress protein, SodB and catalase/peroxidase HPI); (ii) carbohydrate, amino acid/protein and energy metabolism (isocitrate dehydrogenase, SucC, SucD, AcnB, GabD, ArcA, ArgI, Efp and periplasmic binding proteins of several ABC-transporters); and (iii) cell envelope and cell division (bacterial surface antigen family protein and MinD). Based on the data obtained, physiological changes of P. putida in response to growth on benzoate at different concentrations were discussed.