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Archives of Microbiology (v.181, #2)
Genomics of the ccoNOQP-encoded cbb 3 oxidase complex in bacteria by Céline Cosseau; Jacques Batut (pp. 89-96).
Many bacteria adapt to microoxic conditions by synthesizing a particular cytochrome c oxidase (cbb 3) complex with a high affinity for O2, encoded by the ccoNOQP operon. A survey of genome databases indicates that ccoNOQP sequences are widespread in all sub-branches of Proteobacteria but otherwise are found only in bacteria of the CFB group (Cytophaga, Flexibacter, Bacteroides). Our analysis of available genome sequences suggests four major strategies of regulating ccoNOQP expression in response to O2. The most widespread strategy involves direct regulation by the O2-responsive protein Fnr. The second strategy involves an O2-insensitive paralogue of Fnr, FixK, whose expression is regulated by the O2-responding FixLJ two-component system. A third strategy of mixed regulation operates in bacteria carrying both fnr and fixLJ-fixK genes. Another, not yet identified, strategy is likely to operate in the ε-Proteobacteria Helicobacter pylori and Campylobacter jejuni which lack fnr and fixLJ-fixK genes. The FixLJ strategy appears specific for the α-subclass of Proteobacteria but is not restricted to rhizobia in which it was originally discovered.
Keywords: cbb 3 terminal oxidase; ccoNOQP ; fnr ; fixLJ-fixK ; O2 regulation
An attachment tip and pili-like structures in insect- and plant-pathogenic spiroplasmas of the class Mollicutes by El-Desouky Ammar; Dave Fulton; Xiaodong Bai; Tea Meulia; Saskia A. Hogenhout (pp. 97-105).
Ultrastructural studies using scanning electron microscopy (SEM), negative-staining transmission electron microscopy (TEM), and thin-sectioning TEM on four species of Spiroplasma, in vitro and/or in vivo, indicated that their helices commonly possess one tapered end (tip structure) and one blunt or round end. These tip structures appeared morphologically different from the rest of the helix, exhibiting an electron-dense conical or rod-shaped core. In thin sections of the midgut of the leafhopper Dalbulus elimatus, the tip structures of Spiroplasma kunkelii in the midgut lumen were mostly aligned between microvilli, perpendicular to the apical plasma membrane of epithelial cells. These tip structures appeared frequently attached or closely apposed to the plasma membrane, in which cup-shaped invaginations close to the tips were observed. Pleomorphic forms of spiroplasma, enclosed in membranous vesicles, were found in the cytoplasm of the midgut epithelial cells. These findings suggest that the tip structure may be involved in the orientation and attachment of spiroplasma helices in relation to their host cells, and thus may be functionally comparable to the “attachment organelle” of mycoplasmas. Additionally, pili-like structures were observed by negative-staining TEM on the surface of Spiroplasma melliferum, and in thin sections of S. kunkelii infecting the leafhopper vector Dalbulus gelbus.
Keywords: Spiroplasma kunkelii ; Spiroplasma citri ; Spiroplasma melliferum ; Spiroplasma floricola ; Dalbulus elimatus ; Dalbulus gelbus ; Mycoplasma ; Corn stunt spiroplasma; Leafhopper vector; Ultrastructure
Purification of saliva agglutinin of Streptococcus intermedius and its association with bacterial aggregation and adherence by Taihei Yamaguchi (pp. 106-111).
Streptococcus intermedius strain 1208-1 cells were aggregated in the presence of saliva. The saliva agglutinin was purified by centrifugation, filtration, and gel filtration. SDS-PAGE analyses indicated that the purified agglutinin consisted of two high-molecular-mass proteins. Aggregation was dependent on calcium over pH 5.5, with 1 mM being the most effective concentration. Boiling inactivated purified agglutinin. S. intermedius strain 3 and Streptococcus mutans strain 1 were aggregated in the purified agglutinin. After adsorption with strain 1208-1 cells, the saliva sample did not exhibit any aggregation activity, and the agglutinin bands were no longer visible by SDS-PAGE. Adherence analyses demonstrated that the purified agglutinin immobilized on the surfaces of polystyrene wells, actinomyces cells, and apatite beads accounted for the binding of streptococcus cells. Agglutinin also effectively inhibited adherence to apatite beads coated with native saliva.
Keywords: Streptococcus intermedius ; Saliva aggregation; Saliva agglutinin; Adherence; Actinomyces naeslundii.
Mutation analysis of the different tfd genes for degradation of chloroaromatic compounds in Ralstonia eutropha JMP134 by Caroline Laemmli; Christoph Werlen; Jan Roelof van der Meer (pp. 112-121).
Ralstonia eutropha JMP134 possesses two sets of similar genes for degradation of chloroaromatic compounds, tfdCDEFB (in short: tfd I cluster) and tfdD II C II E II F II B II (tfd II cluster). The significance of two sets of tfd genes for the organism has long been elusive. Here, each of the tfd genes in the two clusters on the original plasmid pJP4 was replaced by double recombination with a gene fragment in which a kanamycin resistance gene was inserted into the respective tfd gene’s reading frame. The insertion mutants were all tested for growth on 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), and 3-chlorobenzoate (3-CBA). None of the tfdD II C II E II F II B II genes appeared to be essential for growth on 2,4-D or on 3-CBA. Mutations in tfdC, tfdD and tfdF also did not abolish but only retarded growth on 2,4-D, indicating that they were redundant to some extent as well. Of all tfd genes tested, only tfdE and tfdB were absolutely essential, and interruption of those two reading frames abolished growth on 2,4-D, 3-CBA (tfdE only), and MCPA completely. Interestingly, strains with insertion mutations in the tfd I cluster and those in tfdD II , tfdC II , tfdE II and tfdB II were severely effected in their growth on MCPA, compared to the wild-type. This indicated that not only the tfd I cluster but also the tfd II cluster has an essential function for R. eutropha during growth on MCPA. In contrast, insertion mutation of tfdD II resulted in better growth of R. eutropha JMP134 on 3-CBA, which is most likely due to the prevention of toxic metabolite production in the absence of TfdDII activity.
Keywords: Ralstonia eutropha JMP134; tfd genes; Insertion mutagenesis; 2,4-Dichlorophenoxyacetic acid; 2-Methyl-4-chlorophenoxyacetic acid; 3-Chlorobenzoate
Molecular characteristics and transcription of the gene encoding a multifunctional alcohol dehydrogenase in relation to the deactivation of pyruvate formate-lyase in the ruminal bacterium Streptococcus bovis by Narito Asanuma; Takahiro Yoshii; Tsuneo Hino (pp. 122-128).
To clarify the deactivation mechanism of pyruvate formate-lyase (PFL) and its role in the regulation of fermentation in Streptococcus bovis, the molecular properties and genetic expression of multifunctional alcohol dehydrogenase (ADHE) were investigated. S. bovis was found to have ADHE, which was deduced to consist of 872 amino acids with a molecular mass of 97.4 kDa. The ADHE was shown to harbor three enzyme activities: (1) alcohol dehydrogenase, (2) coenzyme-A-linked acetaldehyde dehydrogenase that catalyzes the conversion of acetyl-CoA to ethanol, and (3) PFL deactivase. Similar to Escherichia coli ADHE, S. bovis ADHE required Fe2+ for its activity. The gene encoding ADHE (adhE) was shown to be monocistronic. The level of adhE mRNA changed in parallel with the mRNA levels of the genes encoding PFL (pfl) and PFL-activating enzyme (act) as the growth conditions changed, although these genes are independently transcribed. Synthesis of ADHE, PFL-activating enzyme, and PFL appears to be regulated concomitantly. Overexpression of ADHE did not cause a change in the formate-to-lactate ratio. It is conceivable that ADHE is not significantly involved in the reversible inactivation of active PFL under anoxic conditions. Partition of the flow from pyruvate appears to be mainly regulated by the activities of lactate dehydrogenase and PFL.
Keywords: adhE ; Alcohol dehydrogenase; Pyruvate formate-lyase deactivase; Ruminal bacterium; Streptococcus bovis
Ethiopian soils harbor natural populations of rhizobia that form symbioses with common bean (Phaseolus vulgaris L.) by Desta Beyene; Serawit Kassa; Franklin Ampy; Amha Asseffa; Tadesse Gebremedhin; Peter van Berkum (pp. 129-136).
The diversity and taxonomic relationships of 83 bean-nodulating rhizobia indigenous to Ethiopian soils were characterized by PCR-RFLP of the internally transcribed spacer (ITS) region between the 16S and 23S rRNA genes, 16S rRNA gene sequence analysis, multilocus enzyme electrophoresis (MLEE), and amplified fragment-length polymorphism. The isolates fell into 13 distinct genotypes according to PCR-RFLP analysis of the ITS region. Based on MLEE, the majority of these genotypes (70%) was genetically related to the type strain of Rhizobium leguminosarum. However, from analysis of their 16S rRNA genes, the majority was placed with Rhizobium etli. Transfer and recombination of the 16S rRNA gene from presumptively introduced R. etli to local R. leguminosarum is a possible theory to explain these contrasting results. However, it seems unlikely that bean rhizobia originating from the Americas (or Europe) extensively colonized soils of Ethiopia because Rhizobium tropici, Rhizobium gallicum, and Rhizobium giardinii were not detected and only a single ineffective isolate of R. etli that originated from a remote location was identified. Therefore, Ethiopian R. leguminosarum may have acquired the determinants for nodulation of bean from a low number of introduced bean-nodulating rhizobia that either are poor competitors for nodulation of bean or that failed to survive in the Ethiopian environment. Furthermore, it may be concluded from the genetic data presented here that the evidence for separating R. leguminosarum and R. etli into two separate species is inconclusive.
Keywords: Rhizobium ; Phylogeny; Ethiopia; 16S rRNA; Nitrogen fixation; Phaseolus vulgaris ; Symbiosis
Escherichia coli RNase E and RNase G cleave a Bacillus subtilis transcript at the same site in a structure-dependent manner by Gustav Hambraeus; Blanka Rutberg (pp. 137-143).
The decay of Bacillus subtilis aprE leader-lacZ mRNA was examined in Escherichia coli wild-type and in mutants deficient in RNase E, RNase G, or both. Two versions of the mRNA were studied: the wild-type mRNA, which has a stem-loop at the 5′ end, and a mutant mRNA, with a single-stranded 5′ end. The half-life of both transcripts was determined by RNase E, the half-life of the mutant transcript being one-third of that of the wild-type transcript. RNase G cleaved both transcripts at a site within an AU-rich sequence in the stem-loop region, but cleavage was much more efficient when the stem-loop was destabilized. RNase E cleaved at the same site, but less efficiently and only in the mutant transcript.
Keywords: mRNA degradation; RNase E; RNase G
The effect of FITA mutations on the symbiotic properties of Sinorhizobium fredii varies in a chromosomal-background-dependent manner by José María Vinardell; Francisco Javier López-Baena; Angeles Hidalgo; Francisco Javier Ollero; Ramón Bellogín; María del Rosario Espuny; Francisco Temprano; Francisco Romero; Hari B. Krishnan; Steven G. Pueppke; José Enrique Ruiz-Sainz (pp. 144-154).
nodD1 of Sinorhizobium fredii HH103, which is identical to that of S. fredii USDA257 and USDA191, repressed its own expression. Spontaneous flavonoid-independent transcription activation (FITA) mutants of S. fredii HH103 M (=HH103 RifR pSym::Tn5-Mob) showing constitutive expression of nod genes were isolated. No differences were found among soybean cultivar Williams plants inoculated with FITA mutants SVQ250 or SVQ253 or with the parental strain HH103M. Soybean plants inoculated with mutant SVQ255 formed more nodules, and those inoculated with mutant SVQ251 had symptoms of nitrogen starvation. Sequence analyses showed that all of the FITA mutants carried a point mutation in their nodD1 coding region. Mutants SVQ251 and SVQ253 carried the same mutation, but only the former was symbiotically impaired, which indicated the presence of an additional mutation elsewhere in the genome of mutant SVQ251. Mutants SVQ251 and SVQ255 were outcompeted by the parental strain for nodulation of soybean cultivar Williams. The symbiotic plasmids of mutants SVQ251 and SVQ255 (pSym251 and pSym255, respectively) and that (pSymHH103M) of the parental strain were transferred to pSym-cured derivatives of S. fredii USDA192 and USDA193 (USDA192C and USDA193C, respectively). Soybean responses to inoculation with S. fredii USDA192C and USDA193C transconjugants carrying pSym251 and pSymHH103M were not significantly different, whereas more nodules were formed after inoculation with transconjugants carrying pSym255. Only transconjugant USDA192C(pSym255) produced a significant increase in soybean dry weight.
Keywords: Sinorhizobium fredii ; nodD ; FITA mutations; Soybean; Nodulation
Substrate specificities and electron paramagnetic resonance properties of benzylsuccinate synthases in anaerobic toluene and m-xylene metabolism by Knut Verfürth; Antonio J. Pierik; Christina Leutwein; Susanne Zorn; Johann Heider (pp. 155-162).
The anaerobic degradation pathways of toluene and m-xylene are initiated by addition of a fumarate cosubstrate to the methyl group of the hydrocarbon, yielding (R)-benzylsuccinate and (3-methylbenzyl)succinate, respectively, as first intermediates. These reactions are catalyzed by a novel glycyl-radical enzyme, (R)-benzylsuccinate synthase. Substrate specificities of benzylsuccinate synthases were analyzed in Azoarcus sp. strain T and Thauera aromatica strain K172. The enzyme of Azoarcus sp. strain T converts toluene, but also all xylene and cresol isomers, to the corresponding succinate adducts, whereas the enzyme of T. aromatica is active with toluene and all cresols, but not with any xylene isomer. This corresponds to the capabilities of Azoarcus sp. strain T to grow on either toluene or m-xylene, and of T. aromatica to grow on toluene as sole hydrocarbon substrate. Thus, differences in the substrate spectra of the respective benzylsuccinate synthases of the two strains contribute to utilization of different aromatic hydrocarbons, although growth on different substrates also depends on additional determinants. We also provide direct evidence by electron paramagnetic resonance (EPR) spectroscopy that glycyl radical enzymes corresponding to substrate-induced benzylsuccinate synthases are specifically detectable in anoxically prepared extracts of toluene- or m-xylene-grown cells. The presence of the EPR signals and the determined amount of the radical are consistent with the respective benzylsuccinate synthase activities. The properties of the EPR signals are highly similar to those of the prototype glycyl radical enzyme pyruvate formate lyase, but differ slightly from previously reported parameters for partially purified benzylsuccinate synthase.
Keywords: Anaerobic metabolism; Benzylsuccinate synthase; Glycyl radical enzyme; Electron paramagnetic spectroscopy; Substrate specificity; Toluene; Xylenes; Cresols; Thauera ; Azoarcus
Utilization of aminoaromatic acids by a methanogenic enrichment culture and by a novel Citrobacter freundii strain by Olga Savelieva; Irina Kotova; Wim Roelofsen; Alfons J. M. Stams; Alexander Netrusov (pp. 163-170).
Following incubation of mesophilic methanogenic floccular sludge from a lab-scale upflow anaerobic sludge bed reactor used to treat cattle manure wastewater, a stable 5-aminosalicylate-degrading enrichment culture was obtained. Subsequently, a Citrobacter freundii strain, WA1, was isolated from the 5-aminosalicylate-degrading methanogenic consortium. The methanogenic enrichment culture degraded 5-aminosalicylate completely to CH4, CO2 and NH4 +, while C. freundii strain WA1 reduced 5-aminosalicylate with simultaneous deamination to 2-hydroxybenzyl alcohol during anaerobic growth with electron donors such as pyruvate, glucose or serine. When grown on pyruvate, C. freundii WA1 converted 3-aminobenzoate to benzyl alcohol and also reduced benzaldehyde to benzyl alcohol. Pyruvate was fermented to acetate, CO2, H2 and small amounts of lactate, succinate and formate. Less lactate (30%) was produced from pyruvate when C. freundii WA1 grew with 5-aminosalicylate as co-substrate.
Keywords: Anaerobiosis; Aminosalicylate; Aminobenzoate; Biodegradation; Fermentation; Citrobacter