Cj0596 is a periplasmic peptidyl prolyl cis-trans isomerase involved in Campylobacter jejuni motility, invasion, and colonization

Background  

Campylobacter jejuni is a gastrointestinal pathogen of humans, but part of the normal flora of poultry, and therefore grows well at the respective body temperatures of 37°C and 42°C. Proteomic studies on temperature regulation in C. jejuni strain 81–176 revealed the upregulation at 37°C of Cj0596, a predicted periplasmic chaperone that is similar to proteins involved in outer membrane protein folding and virulence in other bacteria.     

Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens: implications for the formation of a T-DNA transport structure.

Plant cell transformation by Agrobacterium tumefaciens involves the transfer of a single-stranded DNA-protein complex (T-complex) from the bacterium to the plant cell. One of the least understood and important aspects of this process is how the T-complex exits the bacterium. The eleven virB gene products have been proposed to specify the DNA export channel on the basis of their predicted hydrophobicity. To determine the cellular localization of the VirB proteins, two different cell fractionation methods were employed to separate inner and outer membranes. Seven VirB-specific antibodies were used on Western blots (immunoblots) to detect the proteins in the inner and outer membranes and soluble (containing cytoplasm and periplasm) fractions. VirB5 was in both the inner membrane and cytoplasm. Six of the VirB proteins were detected in the membrane fractions only. Three of these, VirB8, VirB9, and VirB10, were present in both inner and outer membrane fractions regardless of the fractionation method used. Three additional VirB proteins, VirB1, VirB4, and VirB11, were found mainly in the inner membrane fraction by one method and were found in both inner and outer membrane fractions by a second method. These results confirm the membrane localization of seven VirB proteins and strengthen the hypothesis that VirB proteins are involved in the formation of a T-DNA export channel or gate. That most of the VirB proteins analyzed are found in both inner and outer membrane fractions suggest that they form a complex pore structure that spans both membranes, and their relative amounts in the two membrane fractions reflect their differential sensitivity to the experimental conditions.     

Characterization of an OmpA-like outer membrane protein of the acidophilic iron-oxidizing bacterium, <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

An OmpA family protein (FopA) previously reported as one of the major outer membrane proteins of an acidophilic iron-oxidizing bacterium Acidithiobacillus ferrooxidans was characterized with emphasis on the modification by heat and the interaction with peptidoglycan. A 30-kDa band corresponding to the FopA protein was detected in outer membrane proteins extracted at 75°C or heated to 100°C for 10 min prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the band was not detected in outer membrane proteins extracted at ≤40°C and without boiling prior to electrophoresis. By Western blot analysis using the polyclonal antibody against the recombinant FopA, FopA was detected as bands with apparent molecular masses of 30 and 90 kDa, suggesting that FopA existed as an oligomeric form in the outer membrane of A. ferrooxidans. Although the fopA gene with a sequence encoding the signal peptide was successfully expressed in the outer membrane of Escherichia coli, the recombinant FopA existed as a monomer in the outer membrane of E. coli. FopA was detected in peptidoglycan-associated proteins from A. ferrooxidans. The recombinant FopA also showed the peptidoglycan-binding activity.     

Ultrastructure of Pollen Grains from Forced and Unforced Shrubs of Common Lilac

Forcing makes possible to induce plant flowering independently of the season. In lilac, high temperature is the factor that breaks deep dormancy. The deepest dormancy occurs between the end of October and the end of December. Depending on the depth of dormancy, the starting temperatures required for forcing are 37°C in November, 31°C in December, and 25°C in January–March. Under natural conditions, the temperature inducing the inflorescence bud breaking is 6°C, whereas 9°C and 13°C or more allow inflorescence elongation and flowering, respectively (Kronenberg, 1994). In the present work, the effect of high temperature at the beginning of the forcing cycle on the structure of developing pollen grains of common lilac was investigated. Pollen grains from the outdoor-grown (control) shrubs showed no signs of degeneration. They were spherical, three-colpate to colporoidate, and bicellular, and contained large numbers of lipid bodies. High temperatures at the early forcing cycle (November) resulted in the degeneration of most microspores. The first signs of degeneration (cytoplasm plasmolysis) were observed at the tetrad stage and in mature anthers; the microspores consisted only of the outer and inner sporopolenin layers.     

Proteomic analysis of cell envelope from Staphylococcus xylosus C2a, a coagulase-negative staphylococcus

Staphylococcus xylosus is a saprophytic bacterium commonly found on skin of mammals but also used for its organoleptic properties in manufacturing of fermented meat products. This bacterium is able to form biofilms and to colonize biotic or abiotic surfaces, processes which are mediated, to a certain extent, by cell-envelope proteins. Thus, the present investigation aimed at evaluating and adapting different existing methods for cell-envelope subproteome analyses of the strain S. xylosus C2a. The protocol selected consisted initially of a lysostaphin treatment producing protoplasts and giving a fraction I enriched in cell wall proteins. A second fraction enriched in membrane proteins was then efficiently recovered by a procedure involving delipidation with a mixture of tributyl phosphate, methanol, and acetone and solubilization with a buffer containing ASB14. Proteins were separated using two-dimensional gel electrophoresis (2-DE) and identified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A total of 168 protein spots was identified corresponding to 90 distinct proteins. To categorize and analyze these proteomic data, a rational bioinformatic approach was carried out on proteins identified within cell envelope of S. xylosus C2a. Thirty-four proteins were predicted as membrane-associated with 91% present, as expected, within fraction II enriched in membrane proteins: 24 proteins were predicted as membranal, 3 as lipoproteins, and 7 as components of membrane protein complex. Eighteen out of 25 (72%) proteins predicted as secreted were indeed identified in fraction I enriched in cell wall proteins: 6 proteins were predicted as secreted via Sec translocon, and the remaining 19 proteins were predicted as secreted via unknown secretion system. Eighty-one percent (25/31) of proteins predicted as cytoplasmic were found in fraction II: 8 were clearly predicted as interacting temporarily with membrane components. By coupling conventional 2-DE and bioinformatic analysis, the approach developed allows fractionating, resolving, and analyzing a significant and important set of cell envelope proteins from a coagulase-negative staphylococcus, that is, S. xylosus C2a.     

Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism

Grapes (Vitis vinifera) are a valuable fruit crop and wine production is a major industry. Global warming and expanded range of cultivation will expose grapes to more temperature stresses in future. Our study investigated protein level responses to abiotic stresses, with particular reference to proteomic changes induced by the impact of four different temperature stress regimes, including both hot and cold temperatures, on cultured grape cells. Cabernet Sauvignon cell suspension cultures grown at 26°C were subjected to 14 h of exposure to 34 and 42°C for heat stress, and 18 and 10°C for cold stress. Cells from the five temperatures were harvested in biological triplicates and label‐free quantitative shotgun proteomic analysis was performed. A total of 2042 non‐redundant proteins were identified from the five temperature points. Fifty‐five proteins were only detected in extreme heat stress conditions (42°C) and 53 proteins were only detected at extreme cold stress conditions (10°C). Gene Ontology (GO) annotations of differentially expressed proteins provided insights into the metabolic pathways that are involved in temperature stress in grape cells. Sugar metabolism displayed switching between alternative and classical pathways during temperature stresses. Additionally, nine proteins involved in the phenylpropanoid pathway were greatly increased in abundance at extreme cold stress, and were thus found to be cold‐responsive proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD000977 ( http://proteomecentral.proteomexchange.org/dataset/PXD000977 ).     

Influence of Substrates on the Surface Characteristics and Membrane Proteome of Fibrobacter succinogenes S85

Although Fibrobacter succinogenes S85 is one of the most proficient cellulose degrading bacteria among all mesophilic organisms in the rumen of herbivores, the molecular mechanism behind cellulose degradation by this bacterium is not fully elucidated. Previous studies have indicated that cell surface proteins might play a role in adhesion to and subsequent degradation of cellulose in this bacterium. It has also been suggested that cellulose degradation machinery on the surface may be selectively expressed in response to the presence of cellulose. Based on the genome sequence, several models of cellulose degradation have been suggested. The aim of this study is to evaluate the role of the cell envelope proteins in adhesion to cellulose and to gain a better understanding of the subsequent cellulose degradation mechanism in this bacterium. Comparative analysis of the surface (exposed outer membrane) chemistry of the cells grown in glucose, acid-swollen cellulose and microcrystalline cellulose using physico-chemical characterisation techniques such as electrophoretic mobility analysis, microbial adhesion to hydrocarbons assay and Fourier transform infra-red spectroscopy, suggest that adhesion to cellulose is a consequence of an increase in protein display and a concomitant reduction in the cell surface polysaccharides in the presence of cellulose. In order to gain further understanding of the molecular mechanism of cellulose degradation in this bacterium, the cell envelope-associated proteins were enriched using affinity purification and identified by tandem mass spectrometry. In total, 185 cell envelope-associated proteins were confidently identified. Of these, 25 proteins are predicted to be involved in cellulose adhesion and degradation, and 43 proteins are involved in solute transport and energy generation. Our results supports the model that cellulose degradation in F. succinogenes occurs at the outer membrane with active transport of cellodextrins across for further metabolism of cellodextrins to glucose in the periplasmic space and inner cytoplasmic membrane.     

Membrane Vesicles: A Common Feature in the Extracellular Matter of Cold-Adapted Antarctic Bacteria

Many Gram-negative, cold-adapted bacteria from the Antarctic environment produce large amounts of extracellular matter, which has potential biotechnology applications. We examined the ultrastructure of extracellular matter from five Antarctic bacteria (Shewanella livingstonensis NF22^T, Shewanella vesiculosa M7^T, Pseudoalteromonas sp. M4.2, Psychrobacter fozii NF23^T, and Marinobacter guineae M3B^T) by transmission electron microscopy after high-pressure freezing and freeze substitution. All analyzed extracellular matter appeared as a netlike mesh composed of a capsular polymer around cells and large numbers of membrane vesicles (MVs), which have not yet been described for members of the genera Psychrobacter and Marinobacter. MVs showed the typical characteristics described for these structures, and seemed to be surrounded by the same capsular polymer as that found around the cells. The analysis of MV proteins from Antarctic strains by SDS-PAGE showed different banding profiles in MVs compared to the outer membrane, suggesting some kind of protein sorting during membrane vesicle formation. For the psychrotolerant bacterium, S. livingstonensis NF22^T, the growth temperature seemed to influence the amount and morphology of MVs. In an initial attempt to elucidate the functions of MVs for this psychrotolerant bacterium, we conducted a proteomic analysis on membrane vesicles from S. livingstonensis NF22^T obtained at 4 and 18°C. At both temperatures, MVs were highly enriched in outer membrane proteins and periplasmic proteins related to nutrient processing and transport in Gram-negative bacteria suggesting that MVs could be related with nutrient sensing and bacterial survival. Differences were observed in the expression of some proteins depending on incubation temperature but further studies will be necessary to define their roles and implications in the survival of bacteria in the extreme Antarctic environment.     

Bacterial proteins with cleaved or uncleaved signal peptides of the general secretory pathway

Correct protein compartmentalization is a key step for molecular function and cell viability, and this is especially true for membrane and externalized proteins of bacteria. Recent proteomic reports of Bacillus subtilis have shown that many proteins with Sec-like signal peptides and absence of a transmembrane helix domain are still observed in membrane-enriched fractions, but further evidence about signal peptide cleavage or soluble protein contamination is still needed. Here we report a proteomic screening of identified peptides in culture filtrate, membrane fraction and whole cell lysate of Mycobacterium tuberculosis. We were able to detect peptide sequencing evidence that shows that the predicted signal peptide was kept uncleaved for several types of proteins such as mammalian cell entry (Mce) proteins and PE or PE-PGRS proteins. Label-free quantitation of all proteins identified in each fraction showed that the majority of these proteins with uncleaved signal peptides are, indeed, enriched in the Triton X-114 lipid phase. Some of these proteins are likely to be located in the inner membrane while others may be outer membrane proteins.     

Expression of CspA and GST by an Antarctic psychrophilic bacterium Colwellia sp. NJ341 at near-freezing temperature

Summary A psychrotrophic bacterium Colwellia sp. NJ341 from Antarctic sea ice could grow at −5 and 22 °C, and the extent of cellular protein content and growth were greater at low temperatures (0–10 °C) than at higher temperatures. SDS-PAGE analysis demonstrated the presence of a 7 kDa cold-shock protein. The further result of two-dimensional electrophoresis (2-DE) showed that two proteins a and c were newly synthesized at near-freezing temperatures. With matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) analysis, proteins a and c were identified as glutathione S-transferase (GST) and cold-shock protein A (CspA), respectively, which were involved in cold-adaptation at near-freezing temperature in an Antarctic psychrophilic bacterium Colwellia sp. NJ341.     

Proteomics of life at low temperatures: trigger factor is the primary chaperone in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

The proteomes expressed at 4°C and 18°C by the psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis have been compared using two‐dimensional differential in‐gel electrophoresis, showing that translation, protein folding, membrane integrity and anti‐oxidant activities are upregulated at 4°C. This proteomic analysis revealed that the trigger factor is the main upregulated protein at low temperature. The trigger factor is the first molecular chaperone interacting with virtually all newly synthesized polypeptides on the ribosome and also possesses a peptidyl‐prolyl cis‐trans isomerase activity. This suggests that protein folding at low temperatures is a rate‐limiting step for bacterial growth in cold environments. It is proposed that the psychrophilic trigger factor rescues the chaperone function as both DnaK and GroEL (the major bacterial chaperones but also heat‐shock proteins) are downregulated at 4°C. The recombinant psychrophilic trigger factor is a monomer that displays unusually low conformational stability with a Tm value of 33°C, suggesting that the essential chaperone function requires considerable flexibility and dynamics to compensate for the reduction of molecular motions at freezing temperatures. Its chaperone activity is strongly temperature‐dependent and requires near‐zero temperature to stably bind a model‐unfolded polypeptide.     

Global analysis of the membrane subproteome of Pseudomonas aeruginosa using liquid chromatography-tandem mass spectrometry

Pseudomonas aeruginosa is one of the most significant opportunistic bacterial pathogens in humans causing infections and premature death in patients with cystic fibrosis, AIDS, severe burns, organ transplants, or cancer. Liquid chromatography coupled online with tandem mass spectrometry was used for the large-scale proteomic analysis of the P. aeruginosa membrane subproteome. Concomitantly, an affinity labeling technique, using iodoacetyl-PEO biotin to tag cysteinyl-containing proteins, permitted the enrichment and detection of lower abundance membrane proteins. The application of these approaches resulted in the identification of 786 proteins. A total of 333 proteins (42%) had a minimum of one transmembrane domain (ranging from 1 to14) and 195 proteins were classified as hydrophobic based on their positive GRAVY values (ranging from 0.01 to 1.32). Key integral inner and outer membrane proteins involved in adaptation and antibiotic resistance were conclusively identified, including the detection of 53% of all predicted opr-type porins (outer integral membrane proteins) and all the components of the mexA-mexB-oprM transmembrane protein complex. This work represents one of the most comprehensive proteomic analyses of the membrane subproteome of P. aeruginosa and for prokaryotes in general.     

Band 3 tyr-phosphorylation in normal and glucose-6-phospate dehydrogenase-deficient human erythrocytes

Artificial transformation of Escherichia coli with plasmid DNA in presence of CaCl₂ is a widely used technique in recombinant DNA technology. However, exact mechanism of DNA transfer across cell membranes is largely obscure. In this study, measurements of both steady state and time-resolved anisotropies of fluorescent dye trimethyl ammonium diphenyl hexatriene (TMA-DPH), bound to cellular outer membrane, indicated heat-pulse (0°C→42°C) step of the standard transformation procedure had lowered considerably outer membrane fluidity of cells. The decrease in fluidity was caused by release of lipids from cell surface to extra-cellular medium. A subsequent cold-shock (42°C→0°C) to the cells raised the fluidity further to its original value and this was caused by release of membrane proteins to extra-cellular medium. When the cycle of heat-pulse and cold-shock steps was repeated, more release of lipids and proteins respectively had taken place, which ultimately enhanced transformation efficiency gradually up to third cycle. Study of competent cell surface by atomic force microscope showed release of lipids had formed pores on cell surface. Moreover, the heat-pulse step almost depolarized cellular inner membrane. In this communication, we propose heat-pulse step had two important roles on DNA entry: (a) Release of lipids and consequent formation of pores on cell surface, which helped DNA to cross outer membrane barrier, and (b) lowering of membrane potential, which facilitated DNA to cross inner membrane of E. coli.     

Brij detergents reveal new aspects of membrane microdomain in erythrocytes

Membrane microdomains enriched in cholesterol, sphingolipids (rafts), and specific proteins are involved in important physiological functions. However their structure, size and stability are still controversial. Given that detergent-resistant membranes (DRMs) are in the liquid-ordered state and are rich in raft-like components, they might correspond to rafts at least to some extent. Here we monitor the lateral order of biological membranes by characterizing DRMs from erythrocytes obtained with Brij-98, Brij-58, and TX-100 at 4?°C and 37?°C. All DRMs were enriched in cholesterol and contained the raft markers flotillin-2 and stomatin. However, sphingomyelin (SM) was only found to be enriched in TX-100-DRMs – a detergent that preferentially solubilizes the membrane inner leaflet – while Band 3 was present solely in Brij-DRMs. Electron paramagnetic resonance spectra showed that the acyl chain packing of Brij-DRMs was lower than TX-100-DRMs, providing evidence of their diverse lipid composition. Fatty acid analysis revealed that the SM fraction of the DRMs was enriched in lignoceric acid, which should specifically contribute to the resistance of SM to detergents. These results indicate that lipids from the outer leaflet, particularly SM, are essential for the formation of the liquid-ordered phase of DRMs. At last, the differential solubilization process induced by Brij-98 and TX-100 was monitored using giant unilamellar vesicles. This study suggests that Brij and TX-100-DRMs reflect different degrees of lateral order of the membrane microdomains. Additionally, Brij DRMs are composed by both inner and outer leaflet components, making them more physiologically relevant than TX-100-DRMs to the studies of membrane rafts.     

Proteomics of Colwellia psychrerythraea at subzero temperatures – a life with limited movement,flexible membranes and vital DNA repair

The mechanisms that allow psychrophilic bacteria to remain metabolically active at subzero temperatures result from form and function of their proteins. We present first proteomic evidence of physiological changes of the marine psychrophile Colwellia psychrerythraea 34H (Cp34H) after exposure to subzero temperatures (?1, and ?10°C in ice) through 8 weeks. Protein abundance was compared between different treatments to understand the effects of temperature and time, independently and jointly, within cells transitioning to, and being maintained in ice. Parallel [3H]‐leucine and [3H]–thymidine incubations indicated active protein and DNA synthesis to ?10°C. Mass spectrometry‐based proteomics identified 1763 proteins across four experimental treatments. Proteins involved in osmolyte regulation and polymer secretion were found constitutively present across all treatments, suggesting that they are required for metabolic success below 0°C. Differentially abundant protein groups indicated a reallocation of resources from DNA binding to DNA repair and from motility to chemo‐taxis and sensing. Changes to iron and nitrogen metabolism, cellular membrane structures, and protein synthesis and folding were also revealed. By elucidating vital strategies during life in ice, this study provides novel insight into the extensive molecular adaptations that occur in cold‐adapted marine organisms to sustain cellular function in their habitat.     

Analysis of Proteins Expressed by an Abiotic Stress Tolerant Pseudomonas putida (NBAII-RPF9) Isolate Under Saline and High Temperature Conditions

Pseudomonas putida (NBAII-RPF9) was identified as an abiotic stress tolerant bacterium capable of growing at 45 °C as well as in 1 M NaCl. The proteins expressed by this bacterium when subjected to these two stresses were analyzed by 2D gel and MALDI-TOF/MS. Two parameters viz., heat/saline shock (20 min at 45 °C/1 M solid NaCl added at mid log phase and incubated for 1 h) and heat/saline tolerance (24 h growth at 45 °C/in 1 M NaCl) were studied. Under heat shock 13 upregulated proteins and 1 downregulated protein were identified and under tolerance 6 upregulated proteins were identified. GroES and GroEL proteins were expressed under both tolerance and shock. Under saline shock 11 upregulated proteins were identified whereas under saline tolerance 6 upregulated proteins were identified and all these proteins had pI between 3 and 10 with molecular weights ranging from 14.3 to 97 kDa. Aspartate carbamoyltransferase was common under both the saline conditions studied. The analysis revealed involvement of heat stress responsive molecular chaperones and membrane proteins during heat stress. During salt stress, proteins involved in metabolic processes were found to be upregulated to favor growth and adaptation of the bacterium. Heat shock chaperones viz., DnaK and DnaJ were expressed under both saline and heat stress. This is the first report of protein profile obtained from a single bacterium under saline and heat stress and the studies reveal the complex mechanisms adapted by the organism to survive under high temperature or saline conditions.     

Proteomic characterization of the outer membrane vesicle of the halophilic marine bacterium <Emphasis Type="Italic">Novosphingobium pentaromativorans</Emphasis> US6-1

Novosphingobium pentaromativorans US6-1 is a Gram-negative halophilic marine bacterium able to utilize several polycyclic aromatic hydrocarbons such as phenanthrene, pyrene, and benzo[a]pyrene. In this study, using transmission electron microscopy, we confirmed that N. pentaromativorans US6-1 produces outer membrane vesicles (OMVs). N. pentaromativorans OMVs (hereafter OMV_Novo) are spherical in shape, and the average diameter of OMV_Novo is 25–70 nm. Proteomic analysis revealed that outer membrane proteins and periplasmic proteins of N. pentaromativorans are the major protein components of OMV_Novo. Comparative proteomic analysis with the membrane-associated protein fraction and correlation analysis demonstrated that the outer membrane proteins of OMV_Novo originated from the membrane- associated protein fraction. To the best of our knowledge, this study is the first to characterize OMV purified from halophilic marine bacteria.