We report the cloning of the gene encoding the 32-kDa lipoprotein, designated LipL32, the most prominent protein in the leptospiral protein profile. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment to design an oligonucleotide probe. A Lambda-Zap II library containing EcoRI fragments of Leptospira kirschneri DNA was screened, and a 5.0-kb DNA fragment which contained the entire structural lipL32 gene was identified. Several lines of evidence indicate that LipL32 is lipid modified in a manner similar to that of other procaryotic lipoproteins. The deduced amino acid sequence of LipL32 would encode a 272-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by a lipoprotein signal peptidase cleavage site. LipL32 is intrinsically labeled during incubation of L. kirschneri in media containing
New vaccine strategies are needed for prevention of leptospirosis, a widespread human and veterinary disease caused by invasive spirochetes belonging to the genus Leptospira. We have examined the immunoprotective capacity of the leptospiral porin OmpL1 and the leptospiral outer membrane lipoprotein LipL41 in the Golden Syrian hamster model of leptospirosis. Specialized expression plasmids were developed to facilitate expression of leptospiral proteins inEscherichia coli as the membrane-associated proteins OmpL1-M and LipL41-M. Although OmpL1-M expression is highly toxic inE. coli, this was accomplished by using plasmid pMMB66-OmpL1, which has undetectable background expression without induction. LipL41-M expression and processing were enhanced by altering its lipoprotein signal peptidase cleavage site to mimic that of the murein lipoprotein. Active immunization of hamsters with E. coli membrane fractions containing a combination of OmpL1-M and LipL41-M was found to provide significant protection against homologous challenge with Leptospira kirschneri serovar grippotyphosa. At 28 days after intraperitoneal inoculation, survival in animals vaccinated with both proteins was 71% (95% confidence interval [CI], 53 to 89%), compared with only 25% (95% CI, 8 to 42%) in the control group (P < 0.001). On the basis of serological, histological, and microbiological assays, no evidence of infection was found in the vaccinated survivors. The protective effects of immunization with OmpL1-M and LipL41-M were synergistic, since significant levels of protection were not observed in animals immunized with either OmpL1-M or LipL41-M alone. In contrast to immunization with the membrane-associated forms of leptospiral proteins, hamsters immunized with His6-OmpL1 and His6-LipL41 fusion proteins, either alone or in combination, were not protected. These data indicate that the manner in which OmpL1 and LipL41 associates with membranes is an important determinant of immunoprotection.
The sucABCD genes of Escherichia coli encode subunits for two enzymes of the tricarboxylic acid (TCA) cycle, ␣-ketoglutarate dehydrogenase (sucAB) and succinyl coenzyme A synthetase (sucCD). To examine how these genes are expressed in response to changes in oxygen and carbon availability, a set of sucA-lacZ, sucC-lacZ, sdhCDAB-sucA-lacZ, and sdhC-lacZ fusions were constructed and analyzed in vivo. While the expression of a sucA-lacZ fusion was low under all cell growth conditions tested, the expression of the sucA gene from the upstream sdhC promoter was considerably higher and varied by up to 14-fold depending on the carbon substrate used. Expression of the sdhCDAB-sucA-lacZ fusion varied by fourfold in response to oxygen. In contrast, no expression was seen from a sucC-lacZ reporter fusion, indicating that no promoter immediately precedes the sucCD genes. Taken together, these findings demonstrate that the oxygen and carbon control of sucABCD gene expression occurs by transcriptional regulation of the upstream sdhC promoter. The weaker sucA promoter provides an additional low constitutive level of sucABCD gene expression to supplement transcription from the sdhC promoter. The negative control of sucABCD gene expression seen under anaerobic conditions, like that for the sdhCDAB genes, is provided by the arcA and fnr gene products. These findings establish that the differential expression of eight genes for three of the TCA cycle enzymes in E. coli is controlled from one regulatory element.The two tricarboxylic acid (TCA) cycle enzymes in Escherichia coli, ␣-ketoglutarate dehydrogenase and succinyl coenzyme A synthetase (succinyl-CoA synthetase), are encoded by thesucAB and sucCD genes, respectively (4). ␣-Ketoglutarate dehydrogenase catalyzes the oxidative decarboxylation of ␣-ketoglutarate to generate succinyl-CoA and carbon dioxide, along with the production of NADH plus H ϩ (20). SuccinylCoA synthetase catalyzes the interconversion of succinyl-CoA and succinate, and this interconversion is accompanied by the production or hydrolysis of GTP (10, 15). Both TCA cycle enzymes participate in the cyclic flow of carbon from acetylCoA to carbon dioxide during aerobic cell growth conditions (4). This process provides reducing equivalents in the form of NADH for subsequent use in electron transport-linked phosphorylation reactions. Succinyl-CoA synthetase also participates in the noncyclic or branched pathway operative during anaerobic cell growth conditions to provide carbon intermediates for cell biosynthetic reactions (10, 15). The results of enzyme assays and two-dimensional protein gel electrophoresis studies indicate that the cellular synthesis of ␣-ketoglutarate dehydrogenase is suppressed by anaerobiosis and glucose and is induced by acetate or other oxidized carbon sources (1,5,24).The sucABCD genes are located at 16.7 min in the E. coli chromosome near the genes for two other TCA cycle enzymes, succinate dehydrogenase (sdhCDAB) and citrate synthase (gltA) (Fig. 1) (25). Little information is available concer...
Profiles of the proteolytic activities found in Bacteroides gingivalis culture supernatants, outer membranes, vesicles, and cell extracts were analyzed in sodium dodecyl sulfate-polyacrylamide gels containing covalently bound bovine serum albumin. A total of eight distinct bands of proteolytic activity could be detected. Four of these were found in the culture supernatant (P1, P2, P3, and P4). The outer membranes, vesicles, and the cell extract each contained seven major proteolytic bands (P1, P3, P4, P5, P6, P7, and P8). No activity was found in the membrane-free extract, suggesting that the proteases were associated with the cell envelope. With the exception of P7 and P8, all the proteolytic bands were dependent on reducing agents for activity. The eight proteolytic bands were distributed in an identical manner in all four strains of B. gingivalis studied. The effects of protease inhibitors, pH, and heat were determined. SulThydryl group reagents and N-a-p-tosyl-L-lysine chloromethyl ketone reduced proteolytic activity. The optimum pH was found to be between 7 and 8. A 30-min preincubation at 50°C inactivated the P6, P7, and P8 proteolytic bands. All proteolytic activity was lost after the samples were heated at 75°C for 30 min.
Isocitrate dehydrogenase, the icd gene product, has been studied extensively regarding the regulation of enzymatic activity and its relationship to the metabolic flux between the tricarboxylic acid cycle and the glyoxylate bypass. In this study, the transcriptional regulation of icd gene expression was monitored by using an icd-lacZ gene fusion and shown to vary over a 15-fold range in response to changes in oxygen and carbon availability. Anaerobic cell growth resulted in fivefold-lower icd-lacZ expression than during aerobic growth. This negative control is mediated by the arcA and fnr gene products. When different carbon compounds were used for cell growth, icd-lacZ expression varied threefold. The results of continuous cell culture studies indicated that this control may be due to variations in cell growth rate rather than to catabolite repression. DNase I footprinting at the icd promoter revealed a 42-bp ArcA-phosphate-protected region that overlaps the start site of icd transcription. Phosphorylation of ArcA considerably enhanced its binding to DNA, while ArcA-phosphate exhibited an apparent dissociation value of approximately 0.1 M. Based on these studies, ArcA appears to function as a classical repressor of transcription by binding at a site overlapping the icd promoter during anaerobic cell growth conditions.The tricarboxylic acid (TCA) cycle enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the conversion of isocitrate to ␣-ketoglutarate, with concomitant production of NADH and carbon dioxide (3, 15). In Escherichia coli, ICDH activity is regulated by posttranslational modification involving a phosphorylation of serine 113 within the homodimeric protein (16,17,34,36). This reaction is catalyzed by the AceK protein, isocitrate dehydrogenase kinase/phosphatase, to inactivate ICDH under conditions when the cell is grown on acetate or fatty acids. AceK also serves as a protein phosphatase that restores ICDH activity under alternative cell growth conditions when glucose or its saccharide precursors are present. The modulation of ICDH enzyme activity in the cell aids in maintaining the optimal amounts of TCA cycle intermediates, since this enzyme is at the branch point for carbon flow to the glyoxylate bypass pathway (3,14). In the competing reactions, isocitrate lyase converts part of the isocitrate pool to glyoxylate and succinate, while malate synthase then combines glyoxylate with acetyl coenzyme A to form malate. Thus, when E. coli is grown on acetate or its direct precursors, the glyoxylate bypass reactions makes it possible for the cell to generate four-carbon compounds needed for biosynthetic reactions while also balancing its needs for energy via TCA cycle-derived NADH and FADH.Little is known about the control of isocitrate dehydrogenase (icd) gene expression in E. coli under different cell growth conditions. Early studies by Gray et al. demonstrated that ICDH enzyme activity varied over a 10-fold range depending on the availability of oxygen and the composition of the cell growth mediu...
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