Systematic analysis of native membrane protein complexes in Escherichia coli

The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.     

A Small Heat Shock Protein Enables Escherichia coli To Grow at a Lethal Temperature of 50°C Conceivably by Maintaining Cell Envelope Integrity

It is essential for organisms to adapt to fluctuating growth temperatures. Escherichia coli, a model bacterium commonly used in research and industry, has been reported to grow at a temperature lower than 46.5°C. Here we report that the heterologous expression of the 17-kDa small heat shock protein from the nematode Caenorhabditis elegans, CeHSP17, enables E. coli cells to grow at 50°C, which is their highest growth temperature ever reported. Strikingly, CeHSP17 also rescues the thermal lethality of an E. coli mutant deficient in degP, which encodes a protein quality control factor localized in the periplasmic space. Mechanistically, we show that CeHSP17 is partially localized in the periplasmic space and associated with the inner membrane of E. coli, and it helps to maintain the cell envelope integrity of the E. coli cells at the lethal temperatures. Together, our data indicate that maintaining the cell envelope integrity is crucial for the E. coli cells to grow at high temperatures and also shed new light on the development of thermophilic bacteria for industrial application.     

Comparison of Cellulolytic Activities in Clostridium thermocellum and Three Thermophilic, Cellulolytic Anaerobes

Avicelase, carboxymethyl cellulase (CMCase), and β-glucosidase activities have been compared between Clostridium thermocellum and three extremely thermophilic, cellulolytic anaerobes, isolates TP8, TP11, and KT8. The three isolates were all small, gram-negative staining, oval-ended rods which occurred singly and, at exponential phase, in long chains. They were nonflagellated and no spores were visible. The KT8 and TP11 isolates caused clumping of the cellulose during growth. In all four organisms the CMCase activity paralleled cell growth; however, in C. thermocellum and TP8 the avicelase activity did not increase until early stationary phase. Total CMCase activity in C. thermocellum was significantly higher than in the three isolates; however, avicelase activities were much more comparable among the four organisms. C. thermocellum produced higher levels of ethanol, and all four organisms produced similar concentrations of acetate. The amounts of free and bound CMCase and avicelase activities were investigated. In C. thermocellum and TP8 most of the CMCase and avicelase activities were bound to the cellulose in the medium. In contrast, most of the CMCase activity in TP11 and KT8 was free in the culture supernatant; a significant percentage of avicelase activity was also free. The TP8 isolate was also grown on a defined medium with urea as sole nitrogen source and cellulose serving as the carbon source. Under these conditions the pattern of enzyme production was the same as that in the enriched medium, although the level of that production was considerably reduced.     

Preliminary characterization of some Streptomyces species isolated from a composting process and their antimicrobial potential

The aim of this study was to screen Streptomycetes isolates with antimicrobial and antiviral activity, in a search for new metabolites. The isolates were obtained from a composting process, and identified based on morphological characteristics and molecular biological methods. The antimicrobial activity was determined using the double-layer agar method against 53 test organisms (bacteria, yeasts, and filamentous fungi). All isolates were grown in submerged culture, in mineral salts-starch-casein (SC) broth and ISP2 media, and the filtrate cultures were used in the assays for antibacterial and antiviral activity. Bovine Herpes virus (BoHV-I) was used for the antiviral activity. The morphological and molecular characteristics confirmed that all 25 isolates belonged to the genus Streptomyces. In the assay for antimicrobial activity, 80% of the Streptomyces isolates were able to inhibit at least one of the test organisms. Of these, 80% were active against bacteria and 45% against fungi. Eight of the isolates showed a broad spectrum of inhibitory activity; of these, the isolate Streptomyces spp. 1S was able to inhibit 46 of the test organisms, and, most importantly, the 16 Gram-negative strains were inhibited. Of the 25 isolates, 44.4% of the isolates were able to grow and produce bioactive metabolites when grown in submerged culture. Four extracts showed a cytopathic effect in 10 CCID₅₀ MDBK cell, even though no viricidal effect was observed. The results obtained with these isolates indicated good biotechnological potential of these Streptomyces strains.     

Characterization of Root Surface and Endorhizosphere Pseudomonads in Relation to Their Colonization of Roots

An extensive colonization of the endorhizosphere by fluorescent pseudomonads was observed in tomato plants grown on artificial substrates. These studies reveal that a significantly higher percentage of pseudomonads obtained from the endorhizosphere (30%) reduced plant growth than those obtained from the root surface (4%). Lipopolysaccharide patterns, cell envelope protein patterns, and other biochemical characteristics indicated that Pseudomonas isolates obtained from the endorhizosphere are distinct from Pseudomonas isolates obtained from the root surface. Isolates from the endorhizosphere especially were able to recolonize the endorhizosphere of both sterile and nonsterile tomato roots. The ability of the endorhizosphere isolates to colonize the endorhizosphere significantly correlated with their agglutination by tomato root agglutinin but did not correlate with chemotaxis to seed exudates of tomato. No correlation between colonization of the endorhizosphere and agglutination by root agglutinin could be demonstrated for the root surface isolates. We propose that agglutination of specific Pseudomonas strains by root agglutinin is of importance in the initial phase of adherence of bacteria to the root surface.     

Characterization of Clinical and Environmental Mycobacterium avium Spp. Isolates and Their Interaction with Human Macrophages

Members of the Mycobacterium avium complex (MAC) are naturally occurring bacteria in the environment. A link has been suggested between M. avium strains in drinking water and clinical isolates from infected individuals. There is a need to develop new screening methodologies that can identify specific virulence properties of M. avium isolates found in water that predict a level of risk to exposed individuals. In this work we have characterized 15 clinical and environmental M. avium spp. isolates provided by the US Environmental Protection Agency (EPA) to improve our understanding of the key processes involved in the binding, uptake and survival of these isolates in primary human macrophages. M. avium serovar 8 was predominant among the isolates studied. Different amounts and exposure of mannose-capped lipoarabinomannan (ManLAM) and glycopeptidolipids (GPLs), both major mycobacterial virulence factors, were found among the isolates studied. Reference clinical isolate 104 serovar 1 and clinical isolates 11 and 14 serovar 8 showed an increased association with macrophages. Serum opsonization increased the cell association and survival at 2 h post infection for all isolates. However, only the clinical isolates 104 and 3 among those tested showed an increased growth in primary human macrophages. The other isolates varied in their survival in these cells. Thus we conclude that the amounts of cell envelope ManLAM and GPL, as well as GPL serovar specificity are not the only important bacterial factors for dictating the early interactions of M. avium with human macrophages.     

Agar Pitting Gram-negative Rods in the Subgingival Flora of Dogs

The phenotypic characteristics as well as the prevalence and quantity (colony forming units/mL/sample) of 39 bacterial isolates of agar pitting Gram-negative rods, from two diseased and one healthy subgingival site from each of 16 pet dogs with naturally occurring periodontitis, were investigated. Phenotypic features were determined with use of standard biochemical methods, by enzymatic profiling with the API ZYM system, and by cellular fatty acid profiling. The organisms detected were motile, catalase-negative Campylobacter sp., present in 69% of the dogs and in 29% of the subgingival samples (69/29), motile, catalase-positive Campylobacter sp. (63/29), Eikenella corrodens (25/10), organisms closely resembling E. corrodens but nitrate-negative and unable to grow in air, designated E. corrodens -like (19/8), Bilophila wadsworthia (6/2), and non-motile Campylobacter sp. (6/2). The most frequent organisms were the motile Campylobacter isolates constituting 72% of all isolates. No statistically significant differences were detected between the diseased and healthy subgingival sites, with regard to the prevalence of any of these groups of organisms. Furthermore, the bacterial isolates were detected in almost equal numbers in the diseased and healthy sample sites. Hence, no association between dog periodontitis and the agar pitting Gram-negative rods was established. The phenotypic data also suggest, that the organisms that were only presumptively identified in the present study (Campylobacter sp., E. corrodens -like), may not have been previously described.     

Characterization and identification of actinomycetes isolated from ‘fired plots’ under shifting cultivation in northeast Himalaya, India

A total of 35 actinomycetes was isolated from soil samples collected after fire operations at agricultural sites under shifting cultivation in northeast India. More than one-half of these isolates were observed in viable but nonculturable (VBNC) state. Five isolates were always seen embedded with slimy bacteria during subculture; 11 morphologically distinct and cultivable isolates were subjected to characterization and identification. The isolates developed circular to irregular colonies of between 3 and 6 mm on tryptone yeast extract agar plates at 28 °C following 7 days of incubation. The isolates could survive at temperatures between 4 and 50 °C (optimum 28 °C), and pH 5–11 (optimum 8). The isolates varied in cell morphology, utilization of carbon sources, sensitivity to antibiotics, and salt tolerance. Based on 16S rRNA sequencing, the isolates revealed maximum similarity to the genus Streptomyces (9), and to Kitasatospora and Nocardia (1 each). Several isolates were found to be positive for production of lytic (chitinase and glucanase) and industrially important (amylase, lipase, and protease) enzymes. The occurrence of actinomycetes in VBNC state and embedded with bacteria was attributed to coping mechanisms associated with these organisms under stress (high temperature) conditions. The cultivable cultures extend the opportunity for further investigations on ecological resilience during fire operations.     

TraG Encoded by the pIP501 Type IV Secretion System Is a Two-Domain Peptidoglycan-Degrading Enzyme Essential for Conjugative Transfer

pIP501 is a conjugative broad-host-range plasmid frequently present in nosocomial Enterococcus faecalis and Enterococcus faecium isolates. We focus here on the functional analysis of the type IV secretion gene traG, which was found to be essential for pIP501 conjugative transfer between Gram-positive bacteria. The TraG protein, which localizes to the cell envelope of E. faecalis harboring pIP501, was expressed and purified without its N-terminal transmembrane helix (TraGΔTMH) and shown to possess peptidoglycan-degrading activity. TraGΔTMH was inhibited by specific lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A. Analysis of the TraG sequence suggested the presence of two domains which both could contribute to the observed cell wall-degrading activity: an N-terminal soluble lytic transglycosylase domain (SLT) and a C-terminal cysteine-, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The protein domains were expressed separately, and both degraded peptidoglycan. A change of the conserved glutamate residue in the putative catalytic center of the SLT domain (E87) to glycine resulted in almost complete inactivity, which is consistent with this part of TraG being a predicted lytic transglycosylase. Based on our findings, we propose that TraG locally opens the peptidoglycan to facilitate insertion of the Gram-positive bacterial type IV secretion machinery into the cell envelope.     

Insights into the Function of YciM,a Heat Shock Membrane Protein Required To Maintain Envelope Integrity in Escherichia coli

The cell envelope of Gram-negative bacteria is an essential organelle that is important for cell shape and protection from toxic compounds. Proteins involved in envelope biogenesis are therefore attractive targets for the design of new antibacterial agents. In a search for new envelope assembly factors, we screened a collection of Escherichia coli deletion mutants for sensitivity to detergents and hydrophobic antibiotics, a phenotype indicative of defects in the cell envelope. Strains lacking yciM were among the most sensitive strains of the mutant collection. Further characterization of yciM mutants revealed that they display a thermosensitive growth defect on low-osmolarity medium and that they have a significantly altered cell morphology. At elevated temperatures, yciM mutants form bulges containing cytoplasmic material and subsequently lyse. We also discovered that yciM genetically interacts with envC, a gene encoding a regulator of the activity of peptidoglycan amidases. Altogether, these results indicate that YciM is required for envelope integrity. Biochemical characterization of the protein showed that YciM is anchored to the inner membrane via its N terminus, the rest of the protein being exposed to the cytoplasm. Two CXXC motifs are present at the C terminus of YciM and serve to coordinate a redox-sensitive iron center of the rubredoxin type. Both the N-terminal membrane anchor and the C-terminal iron center of YciM are important for function.     

A Genome-Wide Screen for Bacterial Envelope Biogenesis Mutants Identifies a Novel Factor Involved in Cell Wall Precursor Metabolism

The cell envelope of Gram-negative bacteria is a formidable barrier that is difficult for antimicrobial drugs to penetrate. Thus, the list of treatments effective against these organisms is small and with the rise of new resistance mechanisms is shrinking rapidly. New therapies to treat Gram-negative bacterial infections are therefore sorely needed. This goal will be greatly aided by a detailed mechanistic understanding of envelope assembly. Although excellent progress in the identification of essential envelope biogenesis systems has been made in recent years, many aspects of the process remain to be elucidated. We therefore developed a simple, quantitative, and high-throughput assay for mutants with envelope biogenesis defects and used it to screen an ordered single-gene deletion library of Escherichia coli. The screen was robust and correctly identified numerous mutants known to be involved in envelope assembly. Importantly, the screen also implicated 102 genes of unknown function as encoding factors that likely impact envelope biogenesis. As a proof of principle, one of these factors, ElyC (YcbC), was characterized further and shown to play a critical role in the metabolism of the essential lipid carrier used for the biogenesis of cell wall and other bacterial surface polysaccharides. Further analysis of the function of ElyC and other hits identified in our screen is likely to uncover a wealth of new information about the biogenesis of the Gram-negative envelope and the vulnerabilities in the system suitable for drug targeting. Moreover, the screening assay described here should be readily adaptable to other organisms to study the biogenesis of different envelope architectures.     

Characterization of Vibrio fluvialis-Like Strains Implicated in Limp Lobster Disease

Studies were undertaken to characterize and determine the pathogenic mechanisms involved in a newly described systemic disease in Homarus americanus (American lobster) caused by a Vibrio fluvialis-like microorganism. Nineteen isolates were obtained from eight of nine lobsters sampled. Biochemically, the isolates resembled V. fluvialis, and the isolates grew optimally at 20°C; none could grow at temperatures above 23°C. The type strain (1AMA) displayed a thermal reduction time (D value) of 5.77 min at 37°C. All of the isolates required at least 1% NaCl for growth. Collectively, the data suggest that these isolates may embody a new biotype. Pulsed-field gel electrophoresis (PFGE) analysis of the isolates revealed five closely related subgroups. Some isolates produced a sheep hemagglutinin that was neither an outer membrane protein nor a metalloprotease. Several isolates possessed capsules. The isolates were highly susceptible to a variety of antibiotics tested. However, six isolates were resistant to erythromycin. Seventeen isolates harbored plasmids. Lobster challenge studies revealed that the 50% lethal dose of a plasmid-positive strain was 100-fold lower than that of a plasmid-negative strain, suggesting that the plasmid may enhance the pathogenicity of these microorganisms in lobsters. Microorganisms that were recovered from experimentally infected lobsters exhibited biochemical and PFGE profiles that were indistinguishable from those of the challenge strain. Tissue affinity studies demonstrated that the challenge microorganisms accumulated in heart and midgut tissues as well as in the hemolymph. Culture supernatants and polymyxin B lysates of the strains caused elongation of CHO cells in tissue culture, suggesting the presence of a hitherto unknown enterotoxin. Both plasmid-positive and plasmid-negative strains caused significant dose-related intestinal fluid accumulations in suckling mice. Absence of viable organisms in the intestinal contents of mice suggests that these microorganisms cause diarrhea in mice by intoxication rather than by an infectious process. Further, these results support the thermal reduction data at 37°C and suggest that the mechanism(s) that led to fluid accumulation in mice differs from the disease process observed in lobsters by requiring neither the persistence of viable microorganisms nor the presence of plasmids. In summary, results of lobster studies satisfy Koch's postulates at the organismal and molecular levels; the findings support the hypothesis that these V. fluvialis-like organisms were responsible for the originally described systemic disease, which is now called limp lobster disease.     

Membrane proteins of Rhodopseudomonas spheroides III. Isolation,purification, and characterization of cell envelope proteins

Cell envelopes (cell wall and cell membrane) from aerobically grown cells of Rhodopseudomonas spheroides were isolated and purified by a combination of differential centrifugation and centrifugation through 40% sucrose. Cell envelope protein from aerobically grown cells was resolved by dodecyl sulphate-polyacrylamide gel electrophoresis. Biochemical characterization of selected envelope membrane proteins demonstrated heterogeneity between different protein species. Amino acid analyses of individual proteins revealed between 50–60 mole% nonpolar residues.Envelope membranes derived from anaerobically grown cells were also isolated and purified by a combination of differential centrifugation, column chromatography on Sepharose 2B, and centrifugation in 40% sucrose. The dodecyl sulphate-polyacrylamide gel patterns of anaerobic and aerobic envelope membrane proteins were very similar and the results suggest a common protein structure.     

The region of the envelope gene of human immunodeficiency virus type 1 responsible for determination of cell tropism.

Different isolates of human immunodeficiency virus type 1 (HIV-1) vary in the cell tropisms they display, i.e., the range of cell types in which they are able to establish a productive infection. Here, we report on the phenotypes of recombinants between two molecularly cloned strains of HIV-1. Our results prove that the envelope glycoprotein gp120 is solely responsible for the difference in cell tropism between the two parental isolates and that no other genes or sequences are involved in determining the cell tropism of these strains. The region of the envelope involved in the determination of cell tropism includes sequences which encode the V3 loop of gp120. Control of cell tropism by this region of the virus env gene is a general phenomenon which applies to many different HIV-1 isolates.     

Psychrophilic endophytic fungi with biological activity inhabit Cupressaceae plant family

Psychrophilic microorganisms are cold-adapted organisms that have an optimum growth temperature below 15 °C, and often below 5 °C. Endophytic microorganisms live inside healthy plants and biosynthesize an array of secondary metabolites which confer major ecological benefits to their host. We provide information, for the first time, on an endophytic association between bioactive psychrophilic fungi and trees in Cupressaceae plant family living in temperate to cold, semi-arid habitats. We have recovered psychrophilic endophytic fungi (PEF) from healthy foliar tissues of Cupressus arizonica, Cupressus sempervirens and Thuja orientalis (Cupressaceae, Coniferales). In total, 23 such fungi were found out of 110 endophytic fungal isolates. They were identified as ascomycetous fungi, more specifically Phoma herbarum, Phoma sp. and Dothideomycetes spp., all from Dothideomycetes. The optimal growth temperature for all these 23 fungal isolates was 4 °C, and the PEF isolates were able to biosynthesize secondary metabolite at this temperature. Extracted metabolites from PEF showed significant antiproliferative/cytotoxic, antifungal and antibacterial effects against phytopathogenic fungi and bacteria. Of special interest was their antibacterial activity against the ice-nucleation active bacterium Pseudomonas syringae. Accordingly, we suggest that evergreen Cupressaceae plants may benefit from their psychrophilic endophytic fungi during cold stress. Whether such endosymbionts confer any ecological and evolutionary benefits to their host plants remains to be investigated in vivo.     

Human Plasma and the Antibacterial Effect of Peritoneal Dialysis Solutions

The influence of human plasma on the antibacterial effect of solutions for peritoneal dialysis was studied. The solutions contained 43 mEq per litre of either acetate or lactate as the source of base. Enough pooled human plasma was added to half of each solution to give a total concentration of a gramme of protein per litre. The numbers of viable organisms from 15 clinical isolates each of Staphylococcus aureus, Escherichia coli, and Pseudomonas species were counted before and after incubation in the four solutions. Numbers of viable Staph. aureus and E. coli diminished consistently after incubation in all four solutions, but the greatest decreases occurred in the acetate solution which contained no plasma. Plasma abolished the greater antibacterial effect of acetate on these organisms. Differences between numbers of viable Pseudomonas sp. after incubation in the four solutions were not significant. The diffusion of substances from plasma into dialysis fluids during peritoneal dialysis, therefore, may abolish the greater antibacterial effect of solutions made with acetate rather than lactate.     

The phylogenetic and ecological context of cultured and whole genome-sequenced planktonic bacteria from the coastal NW Mediterranean Sea

Microbial isolates are useful models for physiological and ecological studies and can also be used to reassemble genomes from metagenomic analyses. However, the phylogenetic diversity that can be found among cultured marine bacteria may vary significantly depending on the isolation. Therefore, this study describes a set of 136 bacterial isolates obtained by traditional isolation techniques from the Blanes Bay Microbial Observatory, of which seven strains have had the whole genome sequenced. The complete set was compared to a series of environmental sequences obtained by culture-independent techniques (60 DGGE sequences and 303 clone library sequences) previously obtained by molecular methods. In this way, each isolate was placed in both its “ecological” (time of year, nutrient limitation, chlorophyll and temperature values) context or setting, and its “phylogenetic” landscape (i.e. similar organisms that were found by culture-independent techniques, when they were relevant, and when they appeared). Nearly all isolates belonged to the Gammaproteobacteria, Alphaproteobacteria, or the Bacteroidetes (70, 40 and 20 isolates, respectively). Rarefaction analyses showed similar diversity patterns for sequences from isolates and molecular approaches, except for Alphaproteobacteria where cultivation retrieved a higher diversity per unit effort. Approximately 30% of the environmental clones and isolates formed microdiversity clusters constrained at 99% 16S rRNA gene sequence identity, but the pattern was different in Bacteroidetes (less microdiversity) than in the other main groups. Seventeen cases (12.5%) of nearly complete (98–100%) rRNA sequence identity between isolates and environmental sequences were found: nine in the Alphaproteobacteria, five in the Gammaproteobacteria, and three in the Bacteroidetes, indicating that cultivation could be used to obtain at least some organisms representative of the various taxa detected by molecular methods. Collectively, these results illustrated the largely unexplored potential of culturing on standard media for complementing the study of microbial diversity by culture-independent techniques and for obtaining phylogenetically distinct model organisms from natural seawater.     

Identification of Eubacteria Isolated from Leaf Cavities of Four Species of the N-Fixing Azolla Fern as Arthrobacter Conn and Dimmick

The nutritional and physiological characteristics of 15 isolates from four species of the Azolla fern were determined. Although some minor variation existed in levels of urease activity, ability to utilize xylose, and formation of N₂ gas from NO₃, all 15 isolates were rather similar and believed to represent a single species. These eubacteria exhibited aminopeptidase activity and became viscous when treated with KOH, similar to gram-negative organisms; however, the absence of lipopolysaccharide and 2-keto-3-deoxyoctonate in cell walls indicated that they are truly gram-positive organisms. They are unusual because peptidoglycan could not be detected during most of their growth cycle. The presence of lysine as the major diamino acid in cell wall hydrolysates, the inability to hydrolyze cellulose, and the distinctive developmental pattern with rods and “V” forms present during log phase, becoming progressively shorter until cocci dominated during stationary and death phases, indicated that these organisms belong to the genus Arthrobacter Conn and Dimmick. With the exception of the inability to hydrolyze gelatin, their characteristics are consistent with those of the type species, Arthrobacter globiformis Conn and Dimmick.