Proteomic analysis of Proteus mirabilis outer membrane proteins reveals differential expression in vivo vs. in vitro conditions

Proteus mirabilis is an opportunistic pathogen that frequently causes complicated urinary tract infections. Among a wide spectrum of potential virulence factors, outer membrane proteins (OMPs) are critical for bacterial interactions and survival in different environments. In this work, we used a proteomic approach to assess P. mirabilis in vivo OMPs expression compared to in vitro, including iron replete and iron-restricted conditions. Three putative iron receptors, IreA, PMI0842, and PMI2596, were detected both in bacterium grown in vivo and in vitro under iron-restricted conditions. A prophage gene product, PMI1721, was detected only on in vivo growing bacterium, suggesting a potential role yet to be disclosed on the surface of P. mirabilis. Plasminogen, a host protein, was co-purified with OMPs of in vivo grown bacteria, which is in accordance with previous observations and suggests that plasminogen bound to P. mirabilis surface may be associated to virulence as seen in other bacterial pathogens. Western blots using sera of experimentally challenged mice showed that iron-regulated proteins are expressed and highly immunogenic during infection. This work confirms observations made by others for P. mirabilis and reveals details not yet described, suggesting new aspects of the bacterium pathogenesis that remain unknown.     

Characterization of a new murein-associated lipoprotein in the outer membrane of Proteus mirabilis

A murein-associated outer membrane protein from Proteus mirabilis has been isolated. Since the protein carries ester- as well as amide-linked fatty acids it can be classified as a second outer membrane lipoprotein. An apparent molecular weight of 15,000 for this protein was determined from amino acid analysis and sodium dodecylsulfate/polyacrylamide gel electrophoresis. The amino acid composition, however, does not show similarities with the amino acid composition of the lipoprotein covalently linked to murein, which has a molecular weight of 7,300 as described previously in Proteus mirabilis.Abbreviation SDS sodium dodecylsulfate     

Heat effect on the structure and activity of the recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrococcus horikoshii

Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290+/-8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90 degrees C, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 degrees C was 4 h). The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. K(m) values of the recombinant enzyme are comparable for both substrates: 0.2 mM for L-glutamate and 0.53 mM for 2-oxoglutarate. The enzyme was activated by heating at 80 degrees C for 1 h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.     

Expression and immunogenicity analysis of two iron-regulated outer membrane proteins of Vibrio parahaemolyticus

Genes of two iron-regulated outer membrane proteins of Vibrio parahaemolyticus zj2003, a pathogenic strain isolated from large yellow croaker （Pseudosciaena crocea）, psuA and pvuA, were cloned and expressed as N-terminal His6-tagged proteins in Escherichia coli BL21（DE3）. The recombinant fusion proteins were purified with nickel chelate affinity chromatography. To analyze the immunogenicity of the proteins, groups of large yellow croaker were immunized with the purified recombinant psuA, pvuA or both, by intraperitoneal injection. Antibody response was assessed by enzyme-linked immunosorbent assay. Titers to the recombinant proteins increased from log2 3.25 to log29.80, 4-8 weeks following immunization. The relative percent survival of the groups vaccinated with psuA, pvuA, or a combination of the two, reached 50%, 62.5% and 75%, respectively. Western blot analysis was carried out with the serum from unvaccinated survival fish after infection. Both recombinant proteins were detected, indicating that these two proteins of V. parahaemolyticus zj2003 were immunogenic and could produce synergistic effects during in vivo infection, and they might be considered as important components for developing an aquaculture vaccine against this pathogen.     

Histochemical and biochemical urease localization in the periplasm and outer membrane of two Proteus mirabilis strains

Proteus mirabilis, a gram-negative bacillus, is often implicated in the formation of infectious kidney stones. As ureolytic activity of this organism is thought to play a major role in its pathogenesis, we adapted our recently described urease localization technique to visualize urease activity in vivo. Urease activity was ultrastructurally localized in two clinically isolated P. mirabilis strains by precipitating the enzymatic reaction product (ammonia) with sodium tetraphenylboron. Subsequent silver staining of the cells revealed urease activity to be predominantly associated with the periplasm and outer membranes of each strain. Biochemical measurements of urease activity in P. mirabilis cell fractions correlated well with histochemical observations in that the majority of urease activity was associated with the periplasm. Membrane-bound urease activity of these strains was associated mainly with the peptidoglycan in the detergent-insoluble (outer membrane) fraction.     

Nitrogen-limited behaviour of micro-organisms growing in the presence of large concentrations of ammonium ions

Cells of Klebsiella pneumoniae NCTC 418 grown at low culture pH values (4.5-5) in a glucose-limited chemostat culture contained elevated levels of glutamate synthase (EC 2.6.1.53). This can be taken as an indication that these cells show the physiology of nitrogen-limited cells, in spite of the fact that high concentrations (about 80 mM) of ammonium ions were present in the culture extracellular fluids. This phenomenon can be explained by the rapid diffusion of ammonia (NH3) through the cell membrane, leading to very low cytoplasmic ammonium (NH4+) and NH3 levels in cells that possess an almost neutral cytoplasmic pH value, but are growing at low culture pH values.     

Regulation of the expression of ferredoxin-glutamate synthase in barley

We have investigated the regulation of ferredoxin–glutamate synthase (Fd-GOGAT) in leaves of barley (Hordeum vulgare L. cv. Maris Mink) at the mRNA, protein and enzyme activity levels. Studies of the changes in Fd-GOGAT during plant development showed that the activity in shoots increases rapidly after germination to reach a maximum (on a fresh-weight basis) at day 10 and then declines markedly to less than 50% of the maximal activity by day 30, this decline being correlated with an equivalent loss of Fd-GOGAT protein. Growing the plants in darkness reduced the maximum activity attained in the shoots, but did not affect the overall pattern of the changes or their timing. The activity of Fd-GOGAT increased two- to three-fold within 48 h when etiolated leaves were exposed to light, and Northern blots indicated that the induction occurred at the mRNA level. However, whilst a carbon source could at least partially substitute for light in the induction of nitrate reductase activity, no induction of Fd-GOGAT activity was seen when etiolated leaves were treated with either sucrose or glucose. Interestingly, the levels of Fd-GOGAT mRNA and activity remained high up to a period of 16 h or 72 h darkness, respectively. Compared with plants grown in N-free medium, light-grown plants supplied with nitrate had almost two-fold higher Fd-GOGAT activities and increased Fd-GOGAT mRNA levels, but nitrate had no effect on the abundance of the enzyme or its mRNA in etiolated plants, indicating that light is required for nitrate induction of barley Fd-GOGAT. Received: 23 April 1997 / Accepted: 28 May 1997     

Ammonium assimilation in Proteus vulgaris,Bacillus pasteurii,and Sporosarcina ureae

No active uptake of ammonium was detected in Proteus vulgaris, Bacillus pasteurii, and Sporosarcina ureae, which indicates that these bacteria depend on the passive diffusion of ammonia across the cell membrane. In P. vulgaris the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway and glutamate dehydrogenase (GDH) were present, and these enzymes exhibited high affinities for ammonium. In B. pasteurii and S. ureae, however, no GS activity was detected, and GOGAT activity was only present in S. ureae. GDH enzymes were present in these two organisms, but showed only low affinity for ammonium, with apparent K _m-values of 55.2 mM in B. pasteurii and 36.7 mM in S. ureae, repectively. These observations explain why P. vulgaris is able to grow at neutral pH and low ammonium concentration (2 mM), while B. pasteurii and S. ureae require high ammonium concentration (40 mM) and alkaline pH for growth.Non-standard abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - GT glutamyl transferase - MA methylammonium - NB nutrient broth - YE yeast extract - NA nocotinic acid     

Association of iron-regulated outer membrane proteins of Neisseria meningitidis with the RmpM (class 4) protein

The RmpM (class 4) protein of Neisseria meningitidis has previously been shown to be associated with the outer membrane porins. In the present study, we demonstrate that this protein forms complexes with the lactoferrin receptor LbpA, the transferrin receptor TbpA and the siderophore receptor FrpB as well. This complexation apparently resulted in a stabilization of oligomeric forms of these iron-regulated proteins. In vitro experiments further revealed a reduced ability to acquire iron from human lactoferrin in the rmpM mutant. Furthermore, all TonB-dependent receptors investigated here appeared to exist as oligomers (probably dimers), suggesting that this is a general feature of this class of proteins.     

Changes in the organization of the outer membrane of Proteus mirabilis during swarming: freeze-fracture structure and membrane fluidity analysis.

Freeze-fracture studies of short, nonswarming Proteus mirabilis revealed the characteristic gram-negative profile of fractured inner membrane with densely packed particles and sectioned outer membrane with little or no fracture plane. Long swarming cells, however, fractured easily along both the inner membrane and a second membrane, probably the outer membrane. The inner membrane had a typical profile, whereas the outer membrane had fewer but more prominent particles. Isolation and purification of the inner and outer membranes of the short and long bacteria and examination of them with electron paramagnetic resonance measurements after spinlabeling supported the above observations. The outer membrane of swarmer cells allowed higher mobility of the spin label than did the outer membrane of the nonswarming short cells, which showed a typical rigid profile. These results suggest that regions of lipid bilayer appear in the outer membrane during swarmer formation. Previous observation of the behavior and biochemistry of P. mirabilis during swarming are discussed in light of these results.     

The effect of various culture conditions on the levels of ammonia assimilatory enzymes of Corynebacterium callunae

Corynebacterium callunae (NCIB 10338) grows faster on glutamate than ammonia when used as sole nitrogen sources. The levels of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (GOGAT; EC 1.4.1.13) of C. callunae were found to be influenced by the nitrogen source. Accordingly, the levels of GS and GOGAT activities were decreased markedly under conditions of ammonia excess and increased under low nitrogen conditions. In contrast, glutamate dehydrogenase (GDH; EC 1.4.1.4) activities were not significantly affected by the type or the concentration of the nitrogen source supplied. The carbon source in the growth medium could also affect GDH, GS and GOGAT levels. Of the carbon sources tested in the presence of 2 mM or 10 mM ammonium chloride as the nitrogen source pyruvate, acetate, fumarate and malate caused a decrease in the levels of all three enzymes as compared with glucose. GDH, GS and GOGAT levels were slightly influenced by aeration. Also, the enzyme levels varied with the growth phase. Methionine sulfoximine, an analogue of glutamine, markedly inhibited both the growth of C. callunae cells and the transferase activity of GS. The apparent K _m values of GDH for ammonia and glutamate were 17.2 mM and 69.1 mM, respectively. In the NADPH-dependent reaction of GOGAT, the apparent K _m values were 0.1 mM for -ketoglutarate and 0.22 mM for glutamine.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase     

Reconstitution of model membranes from phospholipid and outer membrane proteins of Proteus mirabilis. Role of proteins in the formation of hydrophilic pores and protection of membranes against detergents.

Outer membrane proteins extracted from isolated cell walls of Proteus mirabilis were able to combine the cell wall phospholipids in a model membrane system. The presence of outer membrane proteins in vesicular model membranes mediated the release of previously entrapped [14C]sucrose while [3H]inulin was retained. Incorporation of lipopolysaccharide from the same cell walls was not required for the formation of such selectively permeable membranes. Three major outer membrane proteins of apparent molecular weights 39000, 36000 and 17000 were isolated using acetic acid and sodium deoxycholate solution as solvents and avoiding the strongly denaturing sodium dodecyl sulfate. The isolated proteins were assayed for their ability to form hydrophilic pores in reconstituted membranes. The trypsin-sensitive 39000-Mr protein and the peptidoglycan-associated 36000-Mr protein were equally effective in this function whereas the 17000-Mr protein mediated little penetration of low molecular weight solute. The 39000-Mr and 36000-Mr proteins also protected reconstituted membrane vesicles from disruption by detergent while 17000-Mr protein was ineffective in this regard.     

Glucose fermentation to acetate and alanine in resting cell suspensions of Pyrococcus furiosus: Proposal of a novel glycolytic pathway based on 13C labelling data and enzyme activities

Abstract Suspensions of maltose-grown cells of the hyperthermophilic archaeon Pyrococcus furiosus , when incubated at 90°C with 35 mM [1-¹³C]glucose or [3-¹³C]glucose, consumed glucose at a rate of about 10 nmol min⁻¹ (mg protein)⁻¹. Acetate (10 mM), alanine (3 mM), CO₂ and H₂ were the fermentation products. The ¹³C-labelling pattern in alamine and acetate were analyzed. With [1-¹³C]glucose the methyl group of both alanine and acetate was labelled; with [3-¹³C]glucose only the carboxyl group of alanine was labelled whereas acetate was unlabelled. Extracts of maltose-grown cells contained glucose isomerase (12.8 U mg⁻¹, 100°C), ketohexokinase (0.23 U mg⁻¹, 100°C), and fructose 1-phosphate aldolase (0.06 U mg^{−1, 100°C). Enzymes catalyzing the formation of fructose 1,6-bisphosphate from fructose 1-phosphate or fructose 6-phosphate could not be detected. As publihed previously by our group and other authors P. furiosus also contains enzymes of glyceraldehyde conversion to 2-phosphoglycerate according to a non-phosphorylated Entner-Doudoroff pathway, of dihydroxyacetone phosphate conversion to 2-phosphoglycerate according to the Embden-Meyerhof pathway, and of 2-phosphoglycerate conversion - via pyruvate - to acetate and alanine. Based on the enzyme activities in P. furiosus , the following pathway for glucose degradation to alanine and acetate in cell suspensions is proposed which can explain the [13}C]glucose labelling data: glucose→ fructose → fructose 1- phosphate → dihydroxyacetone phosphate + glyceraldehyde and further conversion of both trioses to alanine and acetate via pyruvate.     

The enzymes of the ammonia assimilation in Pseudomonas aeruginosa

Glutamine synthetase from Pseudomonas aeruginosa is regulated by repression/derepression of enzyme synthesis and by adenylylation/deadenylylation control. High levels of deadenylylated biosynthetically active glutamine synthetase were observed in cultures growing with limiting amounts of nitrogen while synthesis of the enzyme was repressed and that present was adenylylated in cultures with excess nitrogen.NADP-and NAD-dependent glutamate dehydrogenase could be separated by column chromatography and showed molecular weights of 110,000 and 220,000, respectively. Synthesis of the NADP-dependent glutamate dehydrogenase is repressed under nitrogen limitation and by growth on glutamate. In contrast, NAD-dependent glutamate dehydrogenase is derepressed by glutamate. Glutamate synthase is repressed by glutamate but not by excess nitrogen.     

Siderophore-mediated iron transport correlates with the presence of specific iron-regulated proteins in the outer membrane of Rhizobium meliloti.

A universal chemical assay used to detect the production of siderophores in a range of Rhizobium strains showed that production is strain specific. Iron nutrition bioassays carried out on Rhizobium meliloti strains to determine cross-utilization of their siderophores showed that R. meliloti 2011, 220-5, and 220-3 could each use the siderophores produced by the other two but not the siderophore produced by R. meliloti DM4 (and vice versa). Mutants of R. meliloti 2011 and 220-5 defective in siderophore production were isolated by Tn5-mob mutagenesis. The Tn5-mob-containing EcoRI fragment of mutant R. meliloti 220-5-1 was cloned into pUC19. By using this fragment as a probe, the presence of a homologous region was observed in R. meliloti 2011 and 220-3 but not in R. meliloti DM4. A complementing cosmid from a gene bank of R. meliloti 2011 was identified by using the same probe. Introduction of this cosmid into R. meliloti 102F34, a strain not producing a siderophore, resulted in the ability of this strain to produce a siderophore and also in the ability to utilize the siderophores produced by R. meliloti 2011, 220-5, and 220-3 but not the siderophore produced by R. meliloti DM4. A comparative analysis of the outer membrane proteins prepared from iron-deficient cultures of R. meliloti 102F34 and 102F34 harboring the cosmid revealed the presence, in the latter, of a low-iron-induced outer membrane protein corresponding to a low-iron-induced protein in R. meliloti 2011, 220-5, and 220-3. This protein is not present in R. meliloti DM4. The results suggest that R. meliloti 2011, 220-5, and 220-3 produce siderophores that are identical or sufficiently similar in structure to be transported by the membrane transport system of each strain while also indicating that utilization of a particular siderophore is correlated with the presence of specific outer membrane proteins.     

Intracytoplasmic membrane vesiculation in light-harvesting mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus

Abstract In Chlamydomonas reinhardtii there are three glutamate dehydrogenase isozymes which can use both NADH and NADPH as cofactors and respond differently to different nitrogen sources and several stress conditions. From data of induction of isozymes in different metabolic situations, we propose a possible physiological role for each of them in algal carbon and nitrogen metabolism.     

Some properties of glutamate dehydrogenase,glutamine synthetase and glutamate synthase from Corynebacterium callunae

Characteristics of the three major ammonia assimilatory enzymes, glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT) in Corynebacterium callunae (NCIB 10338) were examined. The GDH of C. callunae specifically required NADPH and NADP⁺ as coenzymes in the amination and deamination reactions, respectively. This enzyme showed a marked specificity for -ketoglutarate and glutamate as substrates. The optimum pH was 7.2 for NADPH-GDH activity (amination) and 9.0 for NADP⁺-GDH activity (deamination). The results showed that NADPH-GDH and NADP⁺-GDH activities were controlled primarily by product inhibition and that the feedback effectors alanine and valine played a minor role in the control of NADPH-GDH activity. The transferase activity of GS was dependent on Mn⁺² while the biosynthetic activity of the enzyme was dependent on Mg²⁺ as essential activators. The pH optima for transferase and biosynthetic activities were 8.0 and 7.0, respectively. In the transfer reaction, the K _m values were 15.2 mM for glutamine, 1.46 mM for hydroxylamine, 3.5×10^-3 mM for ADP and 1.03 mM for arsenate. Feedback inhibition by alanine, glycine and serine was also found to play an important role in controlling GS activity. In addition, the enzyme activity was sensitive to ATP. The transferase activity of the enzyme was responsive to ionic strength as well as the specific monovalent cation present. GOGAT of C. callunae utilized either NADPH or NADH as coenzymes, although the latter was less effective. The enzyme specifically required -ketoglutarate and glutamine as substrates. In cells grown in a medium with glutamate as the nitrogen source, the optimum pH was 7.6 for NADPH-GOGAT activity and 6.8 for NADH-GOGAT activity. Findings showed that NADPH-GOGAT and NADH-GOGAT activities were controlled by product inhibition caused by NADP⁺ and NAD⁺, respectively, and that ATP also had an important role in the control of NADPH-GOGAT activity. Both activities of GOGAT were found to be inhibited by azaserine.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase     

The role of outer membrane proteins in peptide uptake by Escherichia coli

Abstract The influence of various outer membrane proteins on peptide penetration through the outer membrane in Escherichia coli was assessed by determining peptide transport kinetics in wild type and outer membrane protein-deficient strains. Peptide uptake was measured in whole cells by using a fluorescamine-based assay to monitor continuously the removal of peptides from the medium. Transport data were collected and processed using a microcomputer to give overall K _m and V _max values for peptide transport in each strain. In the mutants, K _m values were changed more markedly then V _max values reflecting an alteration in diffusion through the envelope. This approach shows that porins are involved in facilitating peptide penetration and that the OmpF channel appears to be more important than either OmpC or PhoE proteins. The loss of OmpA protein also decreases outer membrane permeability towards peptides, although whether this protein forms pores itself or exists more to maintain the functional integrity of other proteins is not known.     

Identification and antibody-therapeutic targeting of chloramphenicol-resistant outer membrane proteins in Escherichia coli

Bacterial resistance to an antibiotic may result from survival in a suddenly strong antibiotic or in sub-minimum inhibitory concentration of the drug. Their shared proteins responsible for the resistance should be potential targets for designing new drugs to inhibit the growth of the antibiotic-resistant bacteria. In the current study, comparative proteomic methodologies were used for identification of sharedly altered outer membrane proteins (OM proteins) that are responsible for chloramphenical (CAP)-resistant Escherichia coli and for survival in medium with suddenly strong CAP treatment. Six differential OM proteins and another protein with unknown location were determined to be sharedly CAP-resistant-related proteins with the use of 2-DE/MS, Western blotting and gene mutant methods, in which TolC, OmpT, OmpC, and OmpW were critically altered proteins and potential targets for designing of the new drugs. Furthermore, a novel method of specific antibody combating bacterial growth was developed on these OM proteins. Only anti-TolC showed a very significant inhibition on bacterial growth in medium with CAP when antisera to TolC, OmpC, OmpT, and OmpW were separately utilized. The growth of CAP-resistant E. coli and its original strain was completely inhibited when they bound with anti-TolC and survived in 1/8 MIC of CAP. This observed result is basically the same to the finding that DeltatolC was survived in the same concentration of the antibiotic. Our study demonstrates that the enhancement of expression of antibody target with antibiotic could be very effective approach compared to using a drug alone, which highlights a potential way for treatment of infection by antibiotic-resistant bacteria.