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Archives of Microbiology (v.177, #4)
No Title by Nardia J. Baxter; Robert P. Hirt; Levente Bodrossy; Kornel L. Kovacs; Martin T. Embley; James I. Prosser; Colin J. Murrell (pp. 279-289).
The genes encoding the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Methylococcus capsulatus (Bath) were localised to an 8.3-kb EcoRI fragment of the genome. Genes encoding the large subunit (cbbL), small subunit (cbbS) and putative regulatory gene (cbbQ) were shown to be located on one cluster. Surprisingly, cbbO, a second putative regulatory gene, was not located in the remaining 1.2-kb downstream (3′) of cbbQ. However, probing of the M. capsulatus (Bath) genome with cbbO from Nitrosomonas europaea demonstrated that a cbbO homologue was contained within a separate 3.0-kb EcoRI fragment. Instead of a cbbR ORF being located upstream (5′) of cbbL, there was a moxR-like ORF that was transcribed in the opposite direction to cbbL. There were three additional ORFs within the large 8.3-kb EcoRI fragment: a pyrE-like ORF, an rnr-like ORF and an incomplete ORF with no sequence similarity to any known protein. Phylogenetic analysis of cbbL from M. capsulatus (Bath) placed it within clade A of the green-type Form 1 Rubisco. cbbL was expressed in M. capsulatus (Bath) when grown with methane as a sole carbon and energy source under both copper-replete and copper-limited conditions. M. capsulatus (Bath) was capable of autotrophic growth on solid medium but not in liquid medium. Preliminarily investigations suggested that other methanotrophs may also be capable of autotrophic growth. Rubisco genes were also identified, by PCR, in Methylococcus-like strains and Methylocaldum species; however, no Rubisco genes were found in Methylomicrobium album BG8, Methylomonas methanica S1, Methylomonas rubra, Methylosinus trichosporium OB3b or Methylocystis parvus OBBP.
Keywords: Methylococcus Ribulose bisphosphate carboxylase/oxygenase Methanotroph
No Title by Annita Toffanin; Esther Cadahia; Juan Imperial; Tomás Ruiz-Argüeso; Jose Palacios (pp. 290-298).
Moderate levels of urease activity (ca. 300 mU mg–1) were detected in Rhizobium leguminosarum bv. viciae UPM791 vegetative cells. This activity did not require urea for induction and was partially repressed by the addition of ammonium into the medium. Lower levels of urease activity (ca. 100 mU mg–1) were detected also in pea bacteroids. A DNA region of ca. 9 kb containing the urease structural genes (ureA, ureB and ureC), accessory genes (ureD, ureE, ureF, and ureG), and five additional ORFs (orf83, orf135, orf207, orf223, and orf287) encoding proteins of unknown function was sequenced. Three of these ORFs (orf83, orf135 and orf207) have a homologous counterpart in a gene cluster from Sinorhizobium meliloti, reported to be involved in urease and hydrogenase activities. R. leguminosarum mutant strains carrying Tn5 insertions within this region exhibited a urease-negative phenotype, but induced wild-type levels of hydrogenase and nitrogenase activities in bacteroids. orf287 encodes a potential transmembrane protein with a C-terminal GGDEF domain. A mutant affected in orf287 exhibited normal levels of urease activity in culture cells. Experiments aimed at cross-complementing Ni-binding proteins required for urease and hydrogenase synthesis (UreE and HypB, respectively) indicated that these two proteins are not functionally interchangeable in R. leguminosarum.
Keywords: Urease Rhizobium Nickel enzyme Hydrogenase
No Title by Meike Goenrich; Jan Bursy; Eva Hübner; Dietmar Linder; Arnold C. Schwartz; Julia A. Vorholt (pp. 299-303).
Recently, it has been shown that heterotrophic methylotrophic Proteobacteria contain tetrahydrofolate (H4F)- and tetrahydromethanopterin (H4MPT)-dependent enzymes. Here we report on the purification of two methylene tetrahydropterin dehydrogenases from the methylotroph Hyphomicrobium zavarzinii ZV580. Both dehydrogenases are composed of one type of subunit of 31 kDa. One of the dehydrogenases is NAD(P)-dependent and specific for methylene H4MPT (specific activity: 680 U/mg). Its N-terminal amino acid sequence showed sequence identity to NAD(P)-dependent methylene H4MPT dehydrogenase MtdB from Methylobacterium extorquens AM1. The second dehydrogenase is specific for NADP and methylene H4F (specific activity: 180 U/mg) and also exhibits methenyl H4F cyclohydrolase activity. Via N-terminal amino acid sequencing this dehydrogenase was identified as belonging to the classical bifunctional methylene H4F dehydrogenases/cyclohydrolases (FolD) found in many bacteria and eukarya. Apparently, the occurrence of methylene tetrahydrofolate and methylene tetrahydromethanopterin dehydrogenases is not uniform among different methylotrophic α-Proteobacteria. For example, FolD was not found in M. extorquens AM1, and the NADP-dependent methylene H4MPT dehydrogenase MtdA was present in the bacterium that also shows H4F activity.
Keywords: C1-metabolism Methylotrophic bacteria Methanogenic archaea Tetrahydromethanopterin Tetrahydrofolate
No Title by Agustín Aranda; Amparo Querol; Marcel.li del Olmo (pp. 304-312).
In the production of sherry wines, the process of biological aging is essential for the development of their organoleptic properties. This process involves velum formation by "flor" yeasts. Several of these yeast strains have been isolated and characterized with regard to their genetic, physiological and metabolic properties. In this work, we studied their resistance to cold-, osmotic-, oxidative-, ethanol- and acetaldehyde-stress, and found, in most cases, a correlation between resistance to acetaldehyde stress and ethanol stress and isolation from "soleras." Moreover, gene expression analysis revealed induction of the heat shock protein (HSP) genes HSP12, HSP82, and especially HSP26 and HSP104, under acetaldehyde stress in most of the strains. In strain C, there was a clear correlation between resistance to ethanol and acetaldehyde, the high induction of HSP genes by these compounds and its presence as the predominant strain in most levels of several soleras.
Keywords: Sherry wines Stress resistance Acetaldehyde Ethanol Gene expression HSP12 HSP26 HSP82 HSP104
No Title by Sibylle Kaspar; Michael Bott (pp. 313-321).
For the CitA-CitB (DpiB-DpiA) two-component signal transduction system from Escherichia coli, three diverse functions have been reported: induction of the citrate fermentation genes citCDEFXGT, repression of the regulator gene appY, and destabilization of the inheritance of iteron-containing plasmids such as pSC101. This poses the question of the principal biological role of this system. Here it is shown that the periplasmic domain of the E. coli sensor kinase CitA functions as a high-affinity citrate receptor. Two CitA derivatives were purified by affinity chromatography and subjected to binding studies using isothermal titration calorimetry (ITC). One of them, termed CitA215MBP, comprised the N-terminal part of CitA (amino acid residues 1–215), including the two transmembrane helices, and was fused to the amino terminus of the E. coli maltose-binding protein lacking its signal peptide. The second CitA derivative, designated CitAPEc, encompassed only the periplasmic domain (amino acid residues 38–177). CitA215MBP bound citrate at 25 °C with a K d of 0.3 µM and a binding stoichiometry of up to 0.9 in 50 mM sodium phosphate buffer, pH 7. Binding was driven by the enthalpy change (ΔH of –95.7 kJ mol–1), whereas the entropy change was not favorable for binding (TΔS of –58.6 kJ mol–1). ITC experiments with CitAPEc yielded similar K d values for citrate (0.15–1.0 µM). Besides citrate, also isocitrate (K d ~15 µM) and tricarballylate (K d ~50 µM), but not malate were bound by CitAPEc. The results favor the assumption that the primary biological function of the CitA-CitB system is the regulation of the citrate fermentation genes.
Keywords: Citrate fermentation Citrate receptor Escherichia coli Isothermal titration calorimetry Ligand binding Response regulator Sensor kinase Two-component system
No Title by Jesse G. Dillon; Cecelia M. Tatsumi; Patrick G. Tandingan; Richard W. Castenholz (pp. 322-331).
The UV-screening pigment scytonemin is found in many species of ensheathed cyanobacteria. Past work has shown that the pigment is synthesized in response to exposure to UV-A irradiance. This study investigated the effect of other correlated stress factors including heat, osmotic and oxidative stress on the synthesis of scytonemin in a clonal cyanobacterial isolate (Chroococcidiopsis sp.) from an epilithic desert crust. Stress experiments were carried out both in conjunction with UV-A irradiance and in isolation. Increases in both temperature and photooxidative conditions in conjunction with UV-A caused a synergistic increase in the rate of scytonemin production. In contrast, increased salt concentration under UV-A irradiance inhibited scytonemin synthesis. However, unlike the responses to temperature and oxidative stress, cells synthesized low levels of scytonemin under osmotic stress in the absence of scytonemin-inducing irradiance. These results suggest that scytonemin induction may be regulated as a part of a complex stress response pathway in which multiple environmental signals affect its synthesis.
Keywords: Cyanobacteria Scytonemin UV Osmotic stress Temperature stress Oxidative stress Desert crusts
No Title by Margaret M. Wallace; David W. Miller; Shirley Raps (pp. 332-338).
The characterization of pMa025, a plasmid isolated from the unicellular, toxin-producing cyanobacterium Microcystis aeruginosa UV025, is described. A recombinant plasmid, pMaL [pMa025-pBluescript II SK(–)] was constructed for mapping, sequencing, and development of shuttle vectors capable of transforming both Escherichia coli and M. aeruginosa. pMa025 is 8,018 bp in length and has a G+C content of 62.3 mol%. Nineteen presumptive ORFs, ORF A – ORF S were identified using ATG or GTG as initiation codons. Fifteen different ORFs, ORF a – ORF o were identified using TGA as a degenerate codon for tryptophan. GTG was the start codon in two-thirds of the putative ORFs when TGA was the termination codon. GTG was the start codon in one-third of the putative ORFs when TGA was used as a codon for tryptophan. The deduced amino acid sequence from ORF j (3,114 bp) was significantly similar to that of a putative plasmid replication protein, RepA, from plasmid pUH24 of Synecho coccus sp. strain PCC7942. M. aeruginosa UV027 and E. coli were transformed to carbenicillin resistance with pMaL-D7, a 6.4-kb hybrid plasmid (3.46 kb pMa025, 2.95 kb pBluescript II) generated from the nested deletion strategy. pMaL-D7 will be used as a shuttle vector.
Keywords: Microcystis aeruginosa Plasmid Nucleotide sequence Shuttle vector
No Title by Takuro Nunoura; Saki Akihara; Ken Takai; Yoshihiko Sako (pp. 339-344).
A novel, extremely thermophilic bacterium was isolated from a shallow marine hydrothermal vent at depth of 22 m in Tachibana Bay, Nagasaki Prefecture, Japan. Cells were gram-negative, non-spore-forming, motile rods. Growth was observed between 52 and 78 °C (optimum 70 °C), pH 5 and 8 (optimum pH 7) and 0–4.5% NaCl (optimum 1.0%). The isolate was a strictly aerobic heterotroph utilizing yeast extract and trypticase peptone. The G+C content of the genomic DNA is 69 mol%. Analysis of 16S rDNA sequences indicated that strain Ts1a is closely related to Thermaerobacter marianensis. The differences in physiology and DNA-DNA similarity between strain Ts1a and T. marianensis showed that strain Ts1a represents a new species of Thermaerobacter. The type strain of T. nagasakiensis is strain Ts1a (=JCM11223, DSM 14512).
Keywords: Thermaerobacter nagasakiensis sp. nov. Obligate aerobe Extreme thermophile Marine bacterium
No Title by David Tropel; Christine Meyer; Jean Armengaud; Yves Jouanneau (pp. 345-351).
In the chlorobenzene degrader Pseudomonas putida GJ31, chlorocatechol is formed as an intermediate and cleaved by a meta-cleavage extradiol chlorocatechol dioxygenase, which has previously been shown to be exceptionally resistant to inactivation by substituted catechols. The gene encoding this dioxygenase (cbzE) is preceded by a gene (cbzT) potentially encoding a ferredoxin, the function of which was studied. The cbzT gene product was overproduced in Escherichia coli and purified in recombinant form. Two homologous proteins, CdoT and AtdS, encoded by genes identified in strains degrading nitrobenzene and aniline, respectively, were also purified and characterized. All three proteins showed spectroscopic properties typical for [2Fe-2S] ferredoxins. The chlorocatechol dioxygenase from strain GJ31 (CbzE) was fully inactivated when 4-methylcatechol was used as substrate. Inactivated CbzE could be rapidly reactivated in vitro in the presence of purified CbzT and a source of reductant. It is inferred that the ability of strain GJ31 to metabolize both chlorobenzene and toluene might depend on the regeneration of the chlorocatechol dioxygenase activity mediated by CbzT. Three CbzT-like ferredoxins, including AtdS, were found to be competent in the reactivation of CbzE, whereas XylT, a protein known to mediate reactivation of the catechol dioxygenase from P. putida mt2 (XylE), was ineffective. Accordingly, CbzT formed a covalent complex with CbzE when cross-linked with a carbodiimide, whereas XylT did not. In the reverse situation, CbzT was found to reactivate XylE as efficiently as XylT and formed an heterologous covalent complex with this enzyme upon cross-linking. We conclude that CbzT, CdoT and AtdS are isofunctional ferredoxins that appear to be involved in the reactivation of their cognate catechol dioxygenases. Based on primary structure comparisons, residues of the ferredoxins possibly involved in the molecular interaction with catechol dioxygenases were identified and their significance is discussed.
Keywords: [2Fe-2S] ferredoxin Catechol dioxygenase Methylcatechol Enzyme reactivation Cross-linking
No Title by José V. Cañizares; Claudia Pallotti; Isabel Saínz-Pardo; María Iranzo; Salvador Mormeneo (pp. 352-357).
Saccharomyces cerevisiae presents two alternative vegetative forms of growth, switching between yeast forms to pseudohyphal forms depending on the specific environmental conditions. To identify genes involved in cell wall morphogenesis, a haploid S. cerevisiae monomorphic mutant, W27, which exhibits pseudohyphal growth in the absence of the normal external signals that induce the formation of filamentous forms, was characterized. S. cerevisiae W27 did not demonstrate agar-invasive growth, a characteristic of most filamentous strains. The mutant wall had no obvious alterations with respect to mannan and glucan content, but had three times more chitin than the parental strain. This produced an increase in the amount of proteins linked covalently to chitin. The same protein species, however, were released from the cell walls of the mutant and the parental strain. The W27 mutation was complemented with a genomic library and the SRD2/ECM23 gene was identified as the complementing ORF. Transformation of S. cerevisiae W27 with the Ycplac33 vector carrying the SRD2 gene produced the original phenotype. These results suggest that the SRD2 gene acts as a negative regulator of pseudohyphal growth.
Keywords: Wall morphogenesis Pseudohyphal growth Agar-invasive growth Polysaccharides distribution SRD2 gene Saccharomyces cerevisiae