- Volume 140, Issue 2, 1994
Volume 140, Issue 2, 1994
- Genetics And Molecular Biology
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Stability and function of the signal peptide of the pCloDF13-derived bacteriocin release protein
Summary: The pCloDF13-derived bacteriocin release protein (BRP) is synthesized as a prelipoprotein with a signal peptide which remains stable after processing. This signal peptide accumulates in the cytoplasmic membrane and is, together with the mature BRP, required for efficient release of cloacin DF13. We investigated the structural requirements for stability of the BRP signal peptide by constructing hybrid signal peptides consisting of parts of the BRP and Lpp signal peptides. Signal peptide stability was investigated by pulse-labelling and pulse-chase experiments. To study the functioning of the BRP signal peptide, the hybrid constructs were tested for their ability to promote BRP-mediated cloacin DF13-release and their ability to affect the viability of the host cells. The results obtained suggest that the N-terminal part of the BRP signal peptide together with the C-terminal alanine residue are important for stability. When expressed as a separate entity, all mutant signal peptides that contain a part of the BRP signal peptide are capable of affecting cell viability. The results indicated a possible correlation between stability of the BRP signal peptide and cloacin DF13-release.
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Molecular characterization of a 40 kDa OmpC-like porin from Serratia marcescens
More LessSummary: An oligonucleotide that encodes the N-terminal portion of a 41 kDa porin of Serratia marcescens was used to probe S. marcescens UOC-51 genomic DNA. An 11 kb EcoRI fragment which hybridized with the oligonucleotide was subcloned into Escherichia coli, examined for expression, and sequenced. The product expressed by the cloned gene was 40 kDa. The nucleotide sequence has an ORF of 1.13 kb. When the deduced amino acid sequence was aligned and compared to other enterobacterial porins the cloned S. marcescens porin most closely resembled E. coli OmpC. Although we did not detect osmoregulation or thermoregulation of any porins in S. marcescens UOC-51, sequences analogous to the E. coli osmoregulator OmpR-binding regions are seen upstream to the cloned gene. We examined the regulation of the S. marcescens porin in E. coli and found that its expression increased in a high salt environment. A micF gene, whose transcriptional product functions to inhibit synthesis of OmpF by hybridizing with the ompF transcript, was also seen upstream of the S. marcescens ompC. An alignment with the E. coli micF gene revealed that the functional region of the S. marcescens micF gene is conserved. Based on the results obtained we have determined that S. marcescens UOC-51 produces a 40 kDa porin similar to the E. coli OmpC porin.
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- Pathogenicity And Medical Microbiology
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Phagocytosis by pig alveolar macrophages of Actinobacillus pleuropneumoniae serotype 2 mutant strains defective in haemolysin II (Apxll) and pleurotoxin (ApxIII)
More LessSummaryThe ability of pig alveolar macrophages to phagocytose Actinobacillus pleuropneumoniae HK 361, which produces both haemolysin II (Apxlll) and pleurotoxin (Apxlll), has been studied. Macrophages incubated with HK 361 in the presence of normal pig serum were rapidly killed. Incubation of the macrophages with a haemolysin-deficient mutant (HK 361 e), which possesses only cytotoxic activity (Apxlll), also caused gross damage to the macrophages. A mutant (HK 361 h) which produces neither Apxll nor Apxlll in its culture supernatant allowed longer survival of the macrophages than did either the parent strain or mutant e when incubated with normal pig serum. Prolonged incubation with mutant h resulted in an increase in the number of damaged macrophages, but not to the same extent as with either HK 361 or mutant e. The number of mutant h cells phagocytosed in the presence of normal pig serum was low. The addition of either hyperimmune rabbit serum, raised against whole formalin-treated HK 361 cells, or convalescent pig serum from a pig recovering from a serotype 3 infection, which contained antibody against both Apxll and Apxlll, did not increase the survival of macrophages incubated with either HK 361 or mutant e. However, incubation of mutant h with convalescent pig serum did result in damage-free macrophages. This serum, which possessed neutralizing capabilities against the toxic activities of Apxll and Apxlll, enhanced the number of mutant h cells phagocytosed compared to the numbers phagocytosed in normal pig serum. Killed bacteria were rapidly phagocytosed and did not damage macrophages. The number of phagocytosed killed bacteria appeared to be similar to that seen with live mutant h cells when incubated in the presence of convalescent pig serum. The presence of a toxic activity associated with the haemolysin-and cytotoxin-negative mutant may represent an additional cell-associated toxin which is not present in its culture supernatant. It appears therefore, that in the absence of extracellular Apxll and Apxlll and in the presence of convalescent pig serum A. pleuropneumoniae is readily phagocytosed.
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A new assay for the invasive adenylate cyclase toxin of Bordetella pertussis based on its morphological effects on the fibronectinstimulated spreading of BHK21 cells
SummaryWhen baby hamster kidney (BHK) cells are allowed to spread on fibronectincoated substrata in the absence of serum and the presence of agents which elevate intracellular 3’:5’-cyclic AMP (cAMP) levels they adopt an abnormal, stellated morphology. To determine whether the invasive adenylate cyclase (AC) toxin of Bordetella pertussis induced the same response, cell extracts were prepared from several B. pertussis strains. They were characterized for AC toxin production by enzymic assay and by immunobiotting with an AC-toxin-specific monoclonal antibody. Extracts of strains producing AC toxin induced elevated levels of intracellular cAMP in BHK cells and promoted a stellation response during cell spreading. Extracts prepared from strains defective in AC toxin production showed no effect. Using image analysis to quantify the morphological change, we have demonstrated that the effect of AC toxin on cell spreading is dose dependent. This technique is a rapid and sensitive assay for the invasive AC toxin.
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Isolation and characterization of Bordetella parapertussis-like bacteria from ovine lungs
More LessSummaryBacteria resembling two Bordetella species were isolated from both normal and pneumonic ovine lungs using a selective charcoal agar. Twenty-eight of the 33 isolates showed similarities to stock NCTC B. parapertussis strains in their SDS-PAGE gel protein profiles, in their biochemical reactions and in causing browning on tyrosine agar. Five isolates behaved similarly to stock B. bronchiseptica strains, in being actively motile, in giving identical positive reactions in three out of four biochemical tests and in causing no colour change in tyrosine agar. Multilocus enzyme electrophoresis separated the isolates into two electrophoretic types distinguishable from those of stock B. parapertussis and stock B. bronchiseptica strains.
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- Physiology And Growth
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Organization and regulation of the mitochondrial oxidative pathway in Mucor rouxii
More LessSummary: Mitochondria from spores, anaerobic yeasts and aerobic mycelium of Mucor rouxii were isolated and the components of the respiratory pathways were analysed. Our data suggest the presence in these mitochondria of three different NADH dehydrogenases, three b- and two c-type cytochromes, plus three different terminal oxidases: cytochrome c oxidase, an incompletely characterized cytochrome which, by its spectral characteristics, behaves as a cytochrome o, and a salicylhydroxamic-acid-sensitive oxidase. The composition of the respiratory chain, as well as the relative proportions of cytochromes and oxidases in the mitochondria, were distinct at each developmental stage. Our data suggest that the development of the respiratory pathways during differentiation is mainly regulated by the oxygen tension. Besides this role, oxygen also influences the relative number of mitochondria in the cells and their ultrastructural organization.
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Anaerobic degradation of pimelate by newly isolated denitrifying bacteria
More LessSummary: A C7 dicarboxylic (pimelic) acid derivative is postulated as an intermediate in anaerobic degradation of benzoate. Four strains of Gram-negative, nitrate-reducing bacteria capable of growth with both pimelate and benzoate as sole carbon and energy source were isolated. The metabolism of strain LP-1, which was enriched from activated sludge with pimelate as substrate, was studied in detail. This strain grew only with oxygen or with oxidized nitrogen compounds as electron acceptor. In the presence of nitrate, a wide range of substrates excluding C1 compounds was degraded. The new isolate was catalase- and oxidase-positive, and had one single polar flagellum. Strain LP-1 was tentatively classified within the family Pseudomonadaceae. The catabolism of pimelate and benzoate was studied in cell-free extracts of strain LP-1. Both acids were activated with coenzyme A in a Mg2+- and ATP-dependent reaction. The corresponding acyl-CoA synthetases were specifically induced by the respective growth substrate. Pimelate was also activated by CoA transfer from succinyl-CoA. Pimelyl-CoA was oxidized by cell-free extracts in the presence of potassium ferricyanide. Degradation to glutaryl-CoA and acetyl-CoA proceeded by a sequence of β-oxidation-like reactions. Glutaryl-CoA dehydrogenase and glutaconyl-CoA decarboxylase activities were expressed in cells grown with pimelate or benzoate, indicating the specific involvement of these enzyme activities in anaerobic degradation of these two acids. Enzyme activities responsible for further degradation of the resulting crotonyl-CoA to acetyl-CoA via classical β-oxidation were also detected.
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