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Archives of Microbiology (v.191, #1)
Effects on haemolytic activity of single proline substitutions in the Bordetella pertussis CyaA pore-forming fragment by Busaba Powthongchin; Chanan Angsuthanasombat (pp. 1-9).
The recombinant Bordetella pertussis CyaA pore-forming (CyaA-PF) fragment was previously shown to be expressed separately in Escherichia coli as a soluble precursor that can be in vivo palmitoylated to exert haemolytic activity. In this study, PCR-based mutagenesis was employed to investigate the contributions to haemolysis of five predicted helices within the N-terminal hydrophobic region of the CyaA-PF fragment. Single proline substitutions were made for alanine near the centre of each predicted helix as a means of disrupting local secondary structure. All mutant proteins were over-expressed in E. coli as a 126-kDa soluble protein at levels comparable to the wild-type. Marked reductions in haemolytic activity against sheep erythrocytes of mutants, A510P, A538P, A583P and A687P pertaining to the putative helices 1500–522, 2529–550, 3571–593 and 5678–698, respectively, were observed. However, a slight decrease in haemolytic activity was found for the proline replacement in the predicted helix 4602–627 (A616P). MALDI–TOF–MS and LC–MS–MS analyses verified the palmitoylation at Lys983 of all five mutants as identical to that of the CyaA-PF wild-type protein, indicating that toxin modification via this acylation was not affected by the mutations. Altogether, these results suggest that structural integrity of the predicted helices 1, 2, 3 and 5, but not helix 4, is important for haemolytic activity, particularly for the putative transmembrane helices 2 and 3 that might conceivably be involved in pore formation of the CyaA-PF fragment.
Keywords: Adenylate cyclase-haemolysin; Bordetella pertussis ; Haemolytic activity; Pore-forming toxin; Palmitoylation; Transmembrane helices
Mutation in the cobO gene generates auxotrophy for cobalamin and methionine and impairs the symbiotic properties of Sinorhizobium fredii HH103 with soybean and other legumes by Carlos Medina; Juan Carlos Crespo-Rivas; Javier Moreno; María Rosario Espuny; María Teresa Cubo (pp. 11-21).
We report here the isolation of a methionine and cobalamin mutant strain (SVQ336) of Sinorhizobium fredii HH103 obtained by Tn5-lacZ mutagenesis. Sequence analysis showed that the transposon was inserted into a gene homologous to cobO. This gene codes for a cobalamin adenosyltransferase which is involved in the biosynthesis of vitamin B12. Another HH103 cobO mutant (strain SVQ524), was constructed by the insertion of Ω interposon. Both cobO mutants required the addition of methionine because cobalamin acts as a cofactor of the enzyme MetH, which catalyses the last step of the methionine biosynthesis. Mutant SVQ524 failed to nodulate on Vigna radiate but was able to nodulate on Glycine max cvs. Williams and Peking and Cajanus cajan, although the total number of nodules formed was highly reduced in comparison with that of plants inoculated with the wild-type strain HH103. The roots of these plants did not seem to secrete enough cobalamin and/or methionine to support growth of cobalamin/methionine auxotrophs in the rhizosphere. In all cases, the phenotype of SVQ524 was nearly overcome by the addition of methionine or cobalamin to the plant growth media or by the presence of a copy of the cobO gene in cosmid pMUS756.
Keywords: Sinorhizobium fredii ; Cobalamin; Methionine; Root exudation; Nodulation
The involvement of sortase A in high virulence of STSS-causing Streptococcus suis serotype 2 by Changjun Wang; Ming Li; Youjun Feng; Feng Zheng; Yaqing Dong; Xiuzhen Pan; Gong Cheng; Ruiping Dong; Dan Hu; Xiaodan Feng; Junchao Ge; Di Liu; Jing Wang; Min Cao; Fuquan Hu; Jiaqi Tang (pp. 23-33).
Sortase A (SrtA), originally identified as a transpeptidase in Staphylococcus aureus, plays key roles in full virulence of pathogenic bacteria. In silico genome-wide search suggested a srtA homologue from 05ZYH33, a Chinese human isolate of streptococcal toxic shock syndrome (STSS)-causing Streptococcus suis serotype 2 (S. suis 2, SS2). An isogenic srtA mutant (ΔsrtA) of 05ZYH33 strain was obtained by homologous recombination. Immunofluorescence analysis revealed that two known virulence-associated surface proteins featuring Leu-Pro-X-Thr-Gly motif, muramidase-released protein and surface antigen one, were absent in the ΔsrtA. Piglet infection experiments showed that deletion of srtA attenuated the full virulence of 05ZYH33 strain, and impaired its colonizing potential in specific organs. Furthermore, the ΔsrtA displayed significant reduction in adherence to human cells (Hep-2 and human umbilical vein endothelial cells). Collectively, we concluded that SrtA was involved in the virulence manifestation of STSS-causing SS2.
Keywords: Streptococcus suis serotype 2; Sortase A; LPXTG motif; Pathogenicity
Stress tolerance, genetic analysis and symbiotic properties of root-nodulating bacteria isolated from Mediterranean leguminous shrubs in Central Spain by Beatriz Ruiz-Díez; Susana Fajardo; Miguel Angel Puertas-Mejía; María del Rosario de Felipe; Mercedes Fernández-Pascual (pp. 35-46).
Nine root-nodulating bacterial isolates were obtained from the leguminous shrubs Spartium junceum, Adenocarpus hispanicus, Cytisus purgans, Cytisus laburnuum, Retama sphaerocarpa and Colutea arborescens in areas of Central Spain. A poliphasic approach analyzing phenotypic, symbiotic and genetic properties was used to study their diversity and characterize them in relation to Mediterranean conditions. Stress tolerance assays revealed marked variations in salinity, extreme pH and cadmium tolerance compared with reference strains, with the majority showing salinity, alkalinity and Cd tolerance and three of them growing at acid pH. Variation within the 16S rRNA gene was examined by amplified 16S rDNA restriction analysis (ARDRA) and direct sequencing to show genetic diversity. Phylogeny confirmed the close relationship of four isolates with Bradyrhizobium canariense, three with Phylobacterium myrsinacearum, one with Rhizobium rhizogenes and another with Mesorhizobium huakuii. The cross inoculation tests revealed wide spectra of nodulation. This is the first report of P. myrsinacearum being able to nodulate these leguminous shrubs, and also the first time reported the association between B.canariense, R. rhizogenes and M. huakuii and C. laburnuum, C. purgans and C. arborescens, respectively. These results suggested that native rhizobia could be suitable candidates as biofertilizers and/or inoculants of leguminous shrubs with restoration or revegetation purposes in Mediterranean areas.
Keywords: Leguminous shrubs; Rhizobia; Salinity; pH; Cd; Nodulation; 16S ribosomal gene; Genotypic characterization; Mediterranean areas
p38 MAPK as a signal transduction component of heavy metals stress in Euglena gracilis by Daniel Rios-Barrera; Alicia Vega-Segura; Valerie Thibert; Jose S. Rodríguez-Zavala; M. Eugenia Torres-Marquez (pp. 47-54).
Living organisms are subject to stress, and among these stressors, heavy metals exposure triggers accumulation of sulfur metabolites. Among these metabolites, glutathione and phytochelatins are found in several organisms, such as Euglena gracilis. Pre-exposing E. gracilis to low concentrations of Hg2+ generates a population with resistance to even 0.2 mM Cd2+, and this resistance relies partly on phytochelatins. p38 MAPK is stimulated by stress and is involved in apoptotic as well as survival mechanisms. In this study, we explored its participation in heavy metal-induced stress and its possible role in sulfur metabolite accumulation. We found that about 51% of the E. gracilis pretreated with Hg2+ becomes resistant to Cd2+ and proliferates despite the presence of this metal. The accumulation of the sulfur metabolites γ-glu-cys, glutathione and phytochelatin 2 displayed cyclic patterns that were disturbed by a challenge with Cd2+. We observed a p38 MAPK-like activity that was stimulated by acute or chronic heavy metal exposure, and its inhibition by SB203580 slightly diminished the accumulation of sulfur compounds. p38 MAPK inhibition also affected basal levels of glutathione in either pretreated or control cells. Thus, it appears that p38 MAPK mediates redox stress component of the signal pathway induced by heavy metals.
Keywords: p38 MAPK; Phytochelatins; GSH; Cadmium; Heavy metals; Euglena gracilis ; Protist; Cyclic metabolite pattern
Effect of tungsten and molybdenum on growth of a syntrophic coculture of Syntrophobacter fumaroxidans and Methanospirillum hungatei by Caroline M. Plugge; Bo Jiang; Frank A. M. de Bok; Chingling Tsai; Alfons J. M. Stams (pp. 55-61).
The effect of tungsten (W) and molybdenum (Mo) on the growth of Syntrophobacter fumaroxidans and Methanospirillum hungatei was studied in syntrophic cultures and the pure cultures of both the organisms. Cells that were grown syntropically were separated by Percoll density centrifugation. Measurement of hydrogenase and formate dehydrogenase levels in cell extracts of syntrophically grown cells correlated with the methane formation rates in the co-cultures. The effect of W and Mo on the activity of formate dehydrogenase was considerable in both the organisms, whereas hydrogenase activity remained relatively constant. Depletion of tungsten and/or molybdenum, however, did not affect the growth of the pure culture of S. fumaroxidans on propionate plus fumarate significantly, although the specific activities of hydrogenase and especially formate dehydrogenase were influenced by the absence of Mo and W. This indicates that the organism has a low W or Mo requirement under these conditions. Growth of M. hungatei on either formate or H2/CO2 required tungsten, and molybdenum could replace tungsten to some extent. Our results suggest a more prominent role for H2 as electron carrier in the syntrophic conversion of propionate, when the essential trace metals W and Mo for the functioning of formate dehydrogenase are depleted.
Keywords: Formate dehydrogenase; Hydrogenase; Interspecies hydrogen and formate transfer; Propionate; Tungsten; Molybdenum; Syntrophy
Differentiated pellicle organization and lipopeptide production in standing culture of Bacillus subtilis strains by Marlène Chollet-Imbert; Frédérique Gancel; Christian Slomianny; Philippe Jacques (pp. 63-71).
Pellicle formation and lipopeptide production was analysed in standing cultures of different Bacillus subtilis strains producing two or three families of lipopeptides. Despite its ability to produce surfactin, B. Subtilis ATCC 6633 was unable to form stable pellicle at air–water interface. For the ATTC 21332 and ATCC 9943 strains, it was shown for the first time that the lipopeptides were also produced in standing cultures at productivities similar or lower than those obtained when the culture medium is agitated. A differentiated behaviour was observed between these strains in repetitive batch cultures. B. subtilis 9943 formed a wrinkled, thinner and more resistant pellicle than B. subtilis 21332. The structure of the pellicle determined by electron microscopy observations showed that cells of B. subtilis 9943 formed microcolonies whereas those of B. subtilis 21332 rapidly died. Under these conditions, surfactin production by strain 21332 decreased after 2 days whereas it remained stable for B. subtilis 9943 during the 6 days of the cultures. These data indicate that cells of B. subtilis strains growing in pellicle can produce lipopeptides differently depending on their cellular organisation.
Keywords: Bacillus subtilis ; Pellicle; Lipopeptides
Factors accelerating pyrimidine production in Deinococcus radiophilus by Don McPhail; Man-Kim Cheung; Judith Brown; Margaret Shepherdson (pp. 73-82).
In studying the pyrimidine synthesising pathway in Deinococcus radiophilus two instances of anomalous behaviour were observed. One was the strikingly different results obtained for two types of assay for carbamoyl phosphate synthetase. Both depend on the fixation of 14C from the substrate bicarbonate to give radioactive products. In the coupled assay the carbamoyl phosphate product of the enzyme is converted to carbamoyl aspartate in the presence of aspartate and aspartate transcarbamoylase. In the direct assay aspartate is omitted from the reaction mixture and the carbamoyl phosphate is converted to urea. It was found that the radioactive counts in the direct assay were about 5% of those measured in the coupled assay. The second anomaly was that omission of glutamine from both assay mixtures had no significant effect on the fixation of radioactive carbon. These results suggested that aspartate amino-N could be the source of nitrogen for glutamine synthesis by a substrate-channelled pathway which delivered glutamine to carbamoyl phosphate synthetase, and that externally added glutamine could not access its binding site on the enzyme.
Keywords: Molecular evolution; Substrate-channelling; Pyrimidine synthesis
Acanthamoeba castellanii an environmental host for Shigella dysenteriae and Shigella sonnei by Amir Saeed; Hadi Abd; Benjamin Edvinsson; Gunnar Sandström (pp. 83-88).
The interaction between Shigella dysenteriae or Shigella sonnei and Acanthamoeba castellanii was studied by viable counts, gentamicin assay and electron microscopy. The result showed that Shigella dysenteriae or Shigella sonnei grew and survived in the presence of amoebae for more than 3 weeks. Gentamicin assay showed that the Shigella were viable inside the Acanthamoeba castellanii which was confirmed by electron microscopy that showed the Shigella localized in the cytoplasm of the Acanthamoeba castellanii. In conclusion, the relationship between Shigella dysenteriae and Shigella sonnei with Acanthamoeba castellanii is symbiotic, and accordingly free-living amoebae may serve as a transmission reservoir for Shigella in water.
Keywords: A. castellanii ; Shigella ; Environmental reservoir
Unusual production of glutathione in Actinobacteria by Todd Johnson; Gerald L. Newton; Robert C. Fahey; Mamta Rawat (pp. 89-93).
Most Actinobacteria produce mycothiol as the major thiol. In addition to mycothiol Rhodococcus AD45 generates a substantial level of glutathione possibly using genes acquired in a lateral transfer. Instead of mycothiol, Rubrobacter radiotolerans and Rubrobacter xylanophilus produce glutathione, whose synthesis appears to involve enzymes substantially different from those in other organisms.
Keywords: Mycothiol; Actinomycetes; Glutathione; Rubrobacter ; Rhodococcus