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Volume 144,
Issue 9,
1998
Volume 144, Issue 9, 1998
- Pathogenicity And Medical Microbiology
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Airway hyper-responsiveness to neurokinin A and bradykinin following Mycoplasma pneumoniae infection associated with reduced epithelial neutral endopeptidase
More LessTo determine whether mycoplasma infection produces airway hyper-responsiveness to tachykinins and bradykinin and, if so, to elucidate the role of neutral endopeptidase (NEP), isolated hamster tracheal segments were studied under isometric conditions in vitro. Nasal inoculation with Mycoplasma pneumoniae potentiated contractile responses to neurokinin A and bradykinin, causing a leftward shift of the dose-response curves to a lower concentration by 1 log unit for each agonist, whereas there was no response with acetylcholine. Pretreatment of tissues with the NEP inhibitor phosphoramidon augmented neurokinin A- and bradykinin-induced contractions in saline-treated control tissues, but did not further potentiate the responsiveness in M. pneumoniae-infected tissues. NEP activity in the tracheal epithelium, but not in epithelium-denuded tissues, was decreased in infected animals. These results suggest that M. pneumoniae infection causes airway bronchoconstrictor hyper-responsiveness to neurokinin A and bradykinin and that this effect may be associated with an inhibition of epithelial NEP activity.
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Hydrolysis of the soft amphiphilic antimicrobial agent tetradecyl betainate is retarded after binding to and killing Salmonella typhimurium
More LessHydrolysis of tetradecyl betainate (B14), a fast-acting microbicidal amphiphilic quaternary ammonium compound (QAC) being an ester of betaine and tetradecanol, occurred after binding to Salmonella typhimurium, resulting in release of the water-soluble betaine portion and retention of the lipophilic tetradecanol. The rate of the hydrolysis was significant but retarded in comparison to B14 in solution. As in free solution, the hydrolysis of substance bound to S. typhimurium was increased in an alkaline environment and by heat. At pH 6.0 and 20 ° the hydrolysis of bound ester was about 10% after 180 min, whereas at pH 9.0 and 50 ° it was about 50% after 60 min. These results are consistent with a model where amphiphilic QACs are inserted into the bacterial outer membrane (OM) with the quaternary ammonium head group facing outwards and the lipophilic portion, including the ester bond, being in the membrane lipid environment enough for retarding the hydrolysis. However, calculation of the mean concentration of B14 in the bacteria at MBC99 (minimum bactericidal concentration required to kill 99% of cells) showed a 7000-8000 times greater concentration than in the medium. At this concentration, when most B14 is considered to be bound to the OM, the available surface area for each molecule was only 2 å2. This is only 6-7% of that required for close packing of the quaternary ammonium head group (30 å2), indicating that a three-dimensional, presumably continuous arrangement was formed. Since B14 is hydrolysed after its binding to bacteria with microbicidal effect, it may be used under conditions where stable QACs might be harmful to the close or the common environment.
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Cloning of the haemocin locus of Haemophilus influenzae type b and assessment of the role of haemocin in virulence
More LessThe bacteriocin haemocin (HMC) is produced by most type b strains of Haemophilus influenzae, including strains determined to be genetically diverse, and is toxic to virtually all non-type b strains of H. influenzae, both encapsulated and non-encapsulated. Examination of the deduced amino acid sequences of several genes upstream of the previously identified HMC immunity gene (hmcl) revealed several features common to class II bacteriocins of certain Gram-positive bacteria. Mutagenesis of the open reading frame immediately upstream of hmcl resulted in a loss of the HMC production phenotype. When an HMC-producing strain of H. influenzae and the HMC-deficient isogenic mutant were compared for invasion in the infant-rat model, the HMC-producing strain was found to invade significantly earlier; however, a significantly higher number of rats infected with the isogenic mutant became bacteraemic as compared with those infected with the HMC-producing parent.
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- Physiology And Growth
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Nitrogen-starvation-induced chlorosis in Synechococcus PCC 7942: adaptation to long-term survival
More LessWhen deprived of essential nutrients, the non-diazotrophic cyanobacterium Synechococcus sp. strain PCC 7942 undergoes a proteolytic degradation of the phycobiliproteins, its major light-harvesting pigments. This process is known as chlorosis. This paper presents evidence that the degradation of phycobiliproteins is part of an acclimation process in which growing cells differentiate into non-pigmented cells able to endure long periods of starvation. The time course of degradation processes differs for various photosynthetic pigments, for photosystem I and photosystem II activities and is strongly influenced by the illumination and by the experimental conditions of nutrient deprivation. Under standard experimental conditions of combined nitrogen deprivation, three phases of the differentiation process can be defined. The first phase corresponds to the well-known phycobiliprotein degradation, in phase 2 the cells lose chlorophyll a prior to entering phase 3, the fully differentiated state, in which the cells are still able to regenerate pigmentation after the addition of nitrate to the culture. An analysis of the protein synthesis patterns by two-dimensional gel electrophoresis during nitrogen starvation indicates extensive differential gene expression, suggesting the operation of tight regulatory mechanisms.
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Bacillus subtilis genes for the utilization of sulfur from aliphatic sulfonates
More LessA 5 kb region upstream of katA at 82°G on the Bacillus subtilis chromosome contains five ORFs organized in an operon-like structure. Based on sequence similarity, three of the ORFs are likely to encode an ABC transport system (ssuBAC) and another to encode a monooxygenase (ssuD). The deduced amino acid sequence of the last ORF (ygaN) shows no similarity to any known protein. B. subtilis can utilize a range of aliphatic sulfonates such as alkanesulfonates, taurine, isethionate and sulfoacetate as a source of sulfur, but not when ssuA and ssuC are disrupted by insertion of a neomycin-resistance gene. Utilization of aliphatic sulfonates was not affected in a strain lacking 3′-phosphoadenosine 5′-phosphosulfate (PAPS) sulfotransferase, indicating that sulfate is not an intermediate in the assimilation of sulfonate-sulfur. Sulfate or cysteine prevented expression of β-galactosidase from a transcriptional ssuD::IacZ fusion. It is proposed that ssuBACD encode a system for ATP-dependent transport of alkanesulfonates and an oxygenase required for their desulfonation.
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F- phenocopies: characterization of expression of the F transfer region in stationary phase
More LessThe phenomenon of ‘F- phenocopies’ in which F+ cells become transfer-deficient in stationary phase seems contradictory to the proposed role for F transfer in adaptive mutation during stationary phase induced by nutrient limitation. The expression of a range of transfer genes at the transcriptional and translational level in stationary phase has been characterized as well as the degree of nicking at the origin of transfer, oriT. Transfer efficiency rapidly decreased in mid-exponential phase, coincident with a decrease in traM transcripts. Approximately 2 h later, the transcript for traA, encoding F-pilin, also decreased to undetectable levels. The levels of TraA (pilin), TraD, TraJ and TraT remained fairly constant well into stationary phase while the levels of TraM and Tral decreased to undetectable levels in early stationary phase. A null mutation in the gene for the alternative s factor, rpoS, did not affect mating efficiency or transcript levels but did increase the stability of TraM and Tral in stationary phase. Nicking at oriT was detected at maximal levels in early stationary phase and at low levels in late stationary phase. The results suggest that the F-pilus transfer apparatus is maintained in the cell envelope after transcription of the transfer region from the main promoter, Py, has ceased with down-regulation of traM transcription being the first step detected in this process. The presence of a low level of nicking at oriT in stationary phase is consistent with a role for F in promoting adaptive mutation.
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The vanadate-tolerant yeast Hansenula polymorpha undergoes cellular reorganization during growth in, and recovery from, the presence of vanadate
More LessWhen present at intracellular concentrations above micromolar, vanadate becomes toxic to most organisms. However, the yeast Hansenula polymorpha is able to grow on vanadate concentrations in the millimolar range, showing at the same time modifications in cellular ultrastructure and polyphosphate metabolism. Here, the development of the ultrastructural changes, and of vacuolar and secretory activities, during exponential growth on vanadate and upon a return to vanadate-free conditions was investigated. External invertase secretion was inhibited by vanadate, as shown by a decrease in external invertase activity, an intracellular accumulation of small vesicles and a cytoplasmic accumulation of internal invertase. An aberrant appearance of the cell wall and defects in cellular surface growth, possibly linked to defects in secretion, were also observed. However, inhibition of the secretory pathway was not complete since the activity of another secreted enzyme, exoglucanase, increased in the presence of vanadate. Growth on vanadate was also accompanied by an enhancement of vacuolar proteolysis, as indicated by an increase in carboxypeptidase Y activity. However, these modifications were all reversible upon return to vanadate-free conditions, with the normalization process being complex and involving new and dramatic ultrastructural changes and activation of an autophagic mechanism. This mechanism is involved in the elimination/resorption of the observed vanadate-induced aberrant cell structures and/or sites involved in vanadate accumulation, a necessary prerequisite for restoration of conventional ultrastructure and metabolic functions.
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Glutamine biosynthesis and the utilization of succinate and glutamine by Rhizobium etli and Sinorhizobium meliloti
More LessSinorhizobium meliloti 1021 and Rhizobium etli CE3 turn over nitrogen and carbon from glutamine to ammonium and CO2, respectively. Some of the ammonium released is assimilated back into glutamine, indicating that a glutamine cycle similar to that in Neurospora operates in Rhizobium. In addition, a previously unrecognized metabolic pathway in Rhizobium was discovered - namely, conversion of glutamine-carbon to γ-hydroxybutyric acid and β-hydroxybutyric acid. Additionally, some of the 2-oxoglutarate derived from glutamine catabolism in Rhizobium is converted to succinate in glutamine-containing medium. Both S. meliloti 1021 and R. etli CE3 oxidize succinate preferentially over glutamine when provided with both carbon sources. In contrast to Sinorhizobium meliloti 1021 and Rhizobium etli CE3, an S. meliloti double mutant that lacks both glutamine synthetase (GS) I and II preferentially oxidizes glutamine over succinate when supplied with both substrates. GSII activity is induced in wild-type S. meliloti 1021 and R. etli CE3 grown in succinate-glutamine medium, and this enzyme participates in the cycling of glutamine-carbon and -nitrogen. On the other hand, GSII activity is repressed in both micro-organisms when glutamine is the only carbon source. These findings show that, in medium containing both glutamine and succinate, glutamine synthesis helps drive the utilization of succinate. When glutamine is in excess as an energy-providing substrate its synthesis is restricted, allowing for more effective utilization of glutamine as an energy source.
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- Plant-Microbe Interactions
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Bacterial chemotactic motility is important for the initiation of wheat root colonization by Azospirillum brasilense
More LessBacteria of the genus Azospirillum are able to colonize plant roots. Using the glucuronidase (GUS) reporter system, various Azospirillum mutants, including mutants affected in chemotactic motility or extracellular polysaccharide biosynthesis, were investigated for their capacity to initiate wheat root colonization at the root hair zones. Only non-flagellated mutants and a generally non-chemotactic mutant exhibited a strongly reduced colonization ability as compared to the wild-type. No role of the Azospirillum calcofluor-binding polysaccharide in primary wheat root colonization could be observed. This is the first report demonstrating directly, by using different motility mutants, the requirement of bacterial motility in the establishment of the Azospirillum-plant root association.
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Mutants of Rhizobium tropici strain CIAT899 that do not induce chlorosis in plants
More LessType B strains of Rhizobium tropici induce severe foliar chlorosis when applied at planting to seeds of symbiotic host and non-host dicotyledonous plants. A Tn5-induced mutant, designated CT4812, of R. tropici strain CIAT899 that was unable to induce chlorosis was isolated. Cloning and sequencing of the DNA flanking the transposon in CT4812 revealed that the Tn5 insertion is located in a gene similar to glnD, which encodes uridylyltransferase/uridylyl-removing enzyme in enteric bacteria. Two marker-exchange mutants with insertions in glnD also failed to induce chlorosis in bean (Phaseolus vulgaris) plants. The 5′-most insertion in glnD (in mutant strain ME330) abolished the ability of R. tropici to utilize nitrate as a sole carbon source, whereas a mutation in glnD further downstream (in mutant strain ME245) did not have an obvious effect on nitrate utilization. A gene similar to the Salmonella typhimurium virulence gene mviN overlaps the 3′ end of the R. tropici glnD homologue. A mutation in mviN had no effect on the ability of CIAT899 to induce chlorosis in bean plants. Therefore the glnD homologue, but not mviN, appears to be required for induction of chlorosis in plants by R. tropici strain CIAT899. A high nitrogen:carbon ratio in the rhizosphere of bean plants also prevented R. tropici from inducing chlorosis in bean plants. Mutations in either the glnD homologue or mviN had no significant effect on root nodule formation or acetylene reduction activity. A mutation in mviN eliminated motility in R. tropici. The sequence data, the inability of the glnD mutant to utilize nitrate, and the role of the R. tropici glnD gene in chlorosis induction in plants,a process that is nitrogen regulated, suggest that glnD plays a role in nitrogen sensing in R. tropici as its homologues do in other organisms.
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A stomatin-like protein encoded by the slp gene of Rhizobium etli is required for nodulation competitiveness on the common bean
More LessRhizobium etli strain TAL182 is a competitive strain for effective nodulation of beans. From this strain, a novel gene was isolated, slp, which is 669 bp in size and required for nodulation competition on the common bean. The slp knockout mutant of TAL182 is defective in nodulation competition, shows reduced growth in the presence of 200 mM NaCl, KCl or LiCl and is complemented by the cloned slp gene. The deduced amino acid sequence of slp shows 66-72% similarity to stomatin proteins of Homo sapiens, Mus musculus and Caenorhabditis elegans. Expression of slp in Escherichia coli from a T7 promoter shows a 26 kDa protein which cross-reacts with human-stomatin-specific polyclonal antibody. Like the human stomatin protein, the slp-deduced protein, Slp, is very hydrophilic except for a single hydrophobic membrane-spanning domain. Among various bean-nodulating rhizobia, slp is present in R. etli, Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici type A strains but is absent in R. tropici type B strains. It is also absent in Bradyrhizobium and several other Rhizobium spp.
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- Systematics And Evolution
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Shigella and enteroinvasive Escherichia coli strains are derived from distinct ancestral strains of E. coli
More LessThe differentiation between Shigella subspecies, and the phylogenetic position of Shigella clones within Escherichia coli clones was determined by analysis of restriction fragment length polymorphisms of rDNA (ribotyping). Seventy-five Shigella strains belonging to the four subspecies and 13 enteroinvasive E. coli (EIEC) strains were compared with the 72 E. coli strains of the ECOR collection, which have been classified into four phylogenetic groups (A, B1, B2 and D). Seventeen Shigella dysenteriae ribotypes, 12 Shigella flexneri ribotypes, 23 Shigella boydii ribotypes, 12 Shigella sonnei ribotypes and 13 EIEC ribotypes were identified following digestion with HindIII and EcoRI. Correspondence analysis of the data showed that S. boydii serotype 13 strains were distantly related to the other Shigella strains, and that S. sonnei and S. flexneri were distinct from S. boydii and S. dysenteriae. The ribotypes of Shigella and ECOR strains were indistinguishable, and S. sonnei, S. flexneri and most S. dysenteriae strains were closely related to phylogenetic group D, whereas S. dysenteriae serotype 1 strains belonged to phylogenetic group B1, and S. boydii strains were evenly distributed between the two groups. The Shigella strains were distantly related to group B2, which contains E. coli strains frequently implicated in extra-intestinal infections in humans. In contrast, the 13 EIEC strains were more widely distributed between phylogenetic groups B1, A and B2. Thus, there was no primordial Shigella species and Shigella and EIEC strains are derived from different ancestral strains.
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