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Archives of Microbiology (v.177, #2)
No Title by Yuri A. Trotsenko; Valentina N. Khmelenina (pp. 123-131).
This review summarizes recent findings on the biology of obligate methanotrophic bacteria living in various extreme environments. By using molecular ecology techniques, it has become clear that obligate methanotrophs are ubiquitous in nature and well adapted to high or low temperature, pH and salinity. The isolation and characterization of pure cultures has led to the discovery of several new genera and species of extremophilic/tolerant methanotrophs. Their major physiological role is participation in the methane cycle and supplying C1 intermediates and various metabolites to other members of microbial communities in extreme ecosystems. To survive under extreme conditions, methanotrophs have developed diverse structure-function adaptive mechanisms including cell-surface layer formation, changes in cellular phospholipid composition and de novo synthesis of organic osmolytes such as ectoine, 5-oxoproline and sucrose. However, despite the above advances, basic knowledge of other stress protectants, as well as bioenergetic and genetic aspects of methanotroph adaptation, is still lacking. This information is necessary for better understanding the molecular mechanisms underlying the versatility of methanotrophs and for the development of novel biotechnological processes.
Keywords: Extreme environments Obligate methanotrophs Ecophysiology Adaptive mechanisms Taxonomy Phylogeny
No Title by Kerstin Hermuth; Birgitta Leuthner; Johann Heider (pp. 132-138).
The first step in anaerobic toluene degradation is the addition of a fumarate cosubstrate to the methyl group of toluene, as catalyzed by the glycyl radical enzyme benzylsuccinate synthase. The bssDCAB genes code for the subunits of benzylsuccinate synthase (BssA, B and C) and an additional enzyme implicated in activating the enzyme by introducing the glycyl radical (BssD). Quantitation of the amounts of benzylsuccinate synthase and activating enzyme showed that both proteins are only synthesized in toluene-grown cells, and that the activating enzyme is present in about 14-fold lower amounts. Two mRNA species of the bss gene cluster were identified, one beginning in front of bssD, and a second in front of bssC. Only the first mRNA 5′-end correlates with a toluene-induced promoter, which is similar to that preceding the bbs operon coding for the further enzymes of toluene catabolism of the same strain. The second mapped 5′-end appears to be generated by endonucleolytic processing. The mRNA segment containing the bssD gene is very short-lived, while that containing the bssCAB genes is more stable. The RNA stability data are consistent with the observed amounts of encoded gene products. Furthermore, the previously known bssDCAB genes are apparently cotranscribed with a fifth gene (bssE) whose product may function as a putative ATP-dependent chaperone for assembly and/or activation of benzylsuccinate synthase.
Keywords: Anaerobic toluene catabolism Benzylsuccinate synthase Promoter Operon structure
No Title by Jeanette M. Norton; Javier J. Alzerreca; Yuichi Suwa; Martin G. Klotz (pp. 139-149).
Autotrophic ammonia-oxidizing bacteria use the essential enzyme ammonia monooxygenase (AMO) to transform ammonia to hydroxylamine. The amo operon consists of at least three genes, amoC, amoA, and amoB; amoA encodes the subunit containing the putative enzyme active site. The use of the amo genes as functional markers for ammonia-oxidizing bacteria in environmental applications requires knowledge of the diversity of the amo operon on several levels: (1) the copy number of the operon in the genome, (2) the arrangement of the three genes in an individual operon, and (3) the primary sequence of the individual genes. We present a database of amo gene sequences for pure cultures of ammonia-oxidizing bacteria representing both the β- and the γ-subdivision of Proteobacteria in the following genera: Nitrosospira (6 strains), Nitrosomonas (5 strains) and Nitrosococcus (2 strains). The amo operon was found in multiple (2–3) nearly identical copies in the β-subdivision representatives but in single copies in the γ-subdivision ammonia oxidizers. The analysis of the deduced amino acid sequence revealed strong conservation for all three Amo peptides in both primary and secondary structures. For the amoA gene within the β-subdivision, nucleotide identity values are approximately 85% within the Nitrosomonas or the Nitrosospira groups, but approximately 75% when comparing between these groups. Conserved regions in amoA and amoC were identified and used as primer sites for PCR amplification of amo genes from pure cultures, enrichments and the soil environment. The intergenic region between amoC and amoA is variable in length and may be used to profile the community of ammonia-oxidizing bacteria in environmental samples. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00203-001-0369-z.
Keywords: Ammonia monooxygenase Nitrosospira Nitrosomonas amoA Nitrification PCR Nucleotide sequence Bacterial diversity
No Title by Kantcho L. Lahtchev; Vika D. Semenova; Ilia I. Tolstorukov; Ida van der Klei; Marten Veenhuis (pp. 150-158).
Genetically defined strains of the yeast Hansenula polymorpha were constructed from a clone of H. polymorpha CBS4732 with very low mating and sporulation abilities. Mating, spore viability, and the percentage of four-spore-containing asci were increased to a level at which tetrad analysis was possible. Auxotrophic mutations in 30 genes were isolated and used to construct strains with multiple markers for mapping studies, transformation with plasmid DNA, and mutant screening. Various other types of mutants were isolated and characterized, among them mutants that displayed an altered morphology, methanol-utilization deficient mutants and strains impaired in the biosynthesis of alcohol oxidase and catalase. Also, the mutability of H. polymorpha CBS4732 vs H. polymorpha NCYC495 was compared. The data revealed clear differences in frequencies of appearance and mutational spectra of some mutants isolated. Many of the mutants isolated had good mating abilities, and diploids resulting from their crossing displayed high sporulation frequencies and high spore viability. Most of the markers used revealed normal Mendelian segregation during meiosis.The frequency of tetratype spore formation was lower than in Saccharomyces cerevisiae suggesting a lower frequency of recombination during the second meiotic division. The properties of genetically defined strains of H. polymorpha CBS4732 as well as their advantages for genetics and molecular studies are discussed.
Keywords: Hansenula polymorpha Methylotrophic yeast Genetic analysis Mutant isolation
No Title by Antje Porthun; Michael Bernhard; Bärbel Friedrich (pp. 159-166).
The actinomycete Rhodococcus opacus MR11 harbors a bidirectional NAD-reducing [NiFe] hydrogenase (SH). This cytoplasmic enzyme is composed of two heterodimeric modules which catalyze distinct enzymatic activities. The hydrogenase moiety mediates H2:benzyl viologen oxidoreductase activity and the FMN-containing diaphorase module displays NADH:benzyl viologen oxidoreductase activity. The SH of Rh. opacus resembles [NiFe] hydrogenases present in strains of the proteobacterium Ralstonia eutropha and in species of cyanobacteria. Heterologous expression of active [NiFe] hydrogenases failed in most cases due to protein-assisted maturation processes implicated in the assembly of the NiFe bimetallic site. This study reports on the construction of a recombinant plasmid harboring the four SH subunit genes hoxFUYH and the associated endopeptidase gene hoxW from Rh. opacus under the regime of the SH promoter from R. eutropha H16. The resulting recombinant plasmid restored lithoautotrophic growth in a R. eutropha mutant impaired in H2-oxidizing ability. The SH of Rh. opacus was functionally active in R. eutropha and displayed the typical features described for its natural host. It readily dissociated in vitro into two active subforms. Dissociation was accompanied by the loss of the H2-dependent NAD-reducing activity, which was partially reconstituted by addition of 5 mM MgSO4 and 0.5 mM NiCl2. Activity and stability of the SH from Rh. opacus were enhanced almost three-fold by co-overexpression of the SH-associated metal insertion genes hypA2B2F2 of R. eutropha. Under optimal conditions the heterologously expressed Rh. opacus SH catalyzed NAD-reduction at a specific activity of 1.7 units per mg protein, which is approximately 30% of the yield obtained for the R. eutropha SH. The results indicate that, despite an enormous phylogenetic distance of the two bacterial species, their SH proteins are highly related.
Keywords: Rhodococcus opacus Ralstonia eutropha Heterologous gene expression Bidirectional NAD-reducing [NiFe] hydrogenase
No Title by Tímea Hanczár; Robert Csáki; Levente Bodrossy; Colin J. Murrell; Kornél L. Kovács (pp. 167-172).
Methylococcus capsulatus (Bath) was shown to contain two distinct hydrogenases, a soluble hydrogenase and a membrane-bound hydrogenase. This is the first report of a membrane-bound hydrogenase in methanotrophs. Both enzymes were expressed apparently constitutively under normal growth conditions. The soluble hydrogenase was capable of reducing NAD+ with molecular hydrogen. The activities of both soluble and particulate methane monooxygenases could be driven by molecular hydrogen. This confirmed that molecular hydrogen could be used as a source of reducing power for methane oxidation. Hydrogen-driven methane monooxygenase activities tolerated elevated temperatures and moderate oxygen concentrations. The significance of these findings for biotechnological applications of methanotrophs is discussed.
Keywords: Metalloenzyme [NiFe]hydrogenase Methane monooxygenase Methanotrophic bacteria Thermotolerance Methyloccoccus capsulatus (Bath) NAD+-reducing hydrogenase Hydrogen metabolism Methane metabolism
No Title by Elena Borodina; Donovan P. Kelly; Peter Schumann; Frederick A. Rainey; Naomi L. Ward-Rainey; Ann P. Wood (pp. 173-183).
Novel methylotrophic Arthrobacter and Hyphomicrobium species are described. Constitutive membrane-associated dimethylsulfone- and dimethylsulfoxide-reductases were found in Arthrobacter methylotrophus strain TGA and Hyphomicrobium sulfonivorans strain S1. Enzyme activities increased during growth with dimethylsulfone or dimethylsulfoxide, respectively, and different ratios of activity with different growth substrates indicated that they are separate enzymes. SDS-PAGE showed some membrane-associated polypeptides to be enhanced during growth with dimethylsulfone (54 kDa in H. sulfonivorans, 21–24 kDa, 54 kDa and 80 kDa in A. methylotrophus). Western blotting with anti-dimethylsulfoxide-reductase antibody showed cross-reaction with 54- and 21-kDa polypeptides in A. methylotrophus. All strains contained rhodanese and sulfur oxygenase after growth with dimethylsulfone. Sulfite was oxidized in the Arthrobacter species by APS reductase and sulfite dehydrogenase. H. sulfonivorans oxidized sulfite with APS reductase, which is unusual for an α-proteobacterium. The Arthrobacter species were distinguished from each other and from other Arthrobacter and Micrococcus species by 16S rRNA gene sequence analysis. The menaquinone and fatty acid profiles of the Arthrobacter species were similar. Their peptidoglycan structures were L-Lys–L-Ser–L-Thr–L-Ala for A. sulfonivorans and L-Lys–L-Ala2–4 for A. methylotrophus. H. sulfonivorans exhibited gross morphology typical for Hyphomicrobium, but possessed helically twisted prosthecae. 16S rRNA gene sequence analysis showed it to be distinct from all the other Hyphomicrobium, Filomicrobium and Pedomicrobium species sequenced to date. Formal descriptions of the new species are given.
Keywords: Arthrobacter sulfonivorans Arthrobacter methylotrophus Hyphomicrobium sulfonivorans Dimethylsulfone Reductases Regulation Taxonomy Sulfur oxidation
No Title by Bong-Hui Kim; Karin Wengerter; Roland Benz (pp. 184-191).
In this communication it is demonstrated that the cell wall of the gram-positive bacterium Micromonospora purpurea contains a cell wall channel for the passage of hydrophilic solutes. The channel-forming protein was identified in sucrose step-density-gradient fractions of the cell envelope and in whole cell extracts using either organic solvent or detergent and the lipid bilayer technique. The fractions of the sucrose step-density centrifugation were assayed for NADH-oxidase activity and for the formation of ion-permeable channels in lipid bilayers. The highest NADH-oxidase activity and the highest channel-forming ability were found in different fractions. The cell wall fraction was identified by the presence of meso-diaminopimelic acid and contained an ion-permeable channel with the extremely high single-channel conductance of about 14 nS in 1 M KCl. The channel-forming unit was purified to homogeneity by FPLC on a HiTrap-Q column. It was identified as a heat- and SDS-resistant 200-kDa band on SDS-PAGE and formed the same general diffusion pores in lipid bilayer membranes as those formed by detergent extracts of the cell wall fraction of the sucrose step-density centrifugation. The channels were slightly selective for potassium ions over chloride, possibly caused by an excess of negative charges in or near the channel.
Keywords: Channel formation Cell wall Mycolic acid Micromonospora purpurea Lipid bilayer membrane Permeability barrier
No Title by J. Seah; J. Kwang (pp. 192-196).
The Escherichia coli H serogroup is determined by flagellin, which has both H-type-specific and cross-reactive epitopes. The cross-reactive epitopes are responsible for the cross-reaction found in agglutination. To identify the specific epitope in H33 flagellin, the H33 flagellin gene was sequenced and the encoded central variable region (CVR) was determined. Four overlapping fragments of the CVR were prepared and their specificity was verified using different H-type antisera. Short fragments carrying potential H-type-specific determinants were selected, and monoclonal antibodies (MAbs) against these fragments were prepared. A murine MAb of subtype IgG1 showing specificity to H33 flagellin was produced. The epitope of the MAb was mapped to amino acid residues 250–260.
Keywords: Escherichia coli Epitope Flagellin FliC Epitope mapping Serotype
No Title by Henriette Stavri; Terry J. Beveridge; Dianne Moyles; Abed Athamna; Ronald J. Doyle (pp. 197-199).
Helcococcus kunzii is a gram-positive, catalase-negative opportunist. The organism has been isolated from the lower extremities and breast masses of several patients. A clinical isolate of Helcococcus kunzii was shown to possess a hemagglutinin-lectin with a specificity for N-acetylglucosamine and lactose, two structurally unrelated carbohydrates. The lectin is sensitive to protease, heat and mutanolysin. Electron microscopy failed to reveal fimbriae or fibrillae, suggesting that the lectin is associated with peptidoglycan or the cytoplasmic membrane. It is likely that the lectin is involved in adhesion and colonization of H. kunzii.
Keywords: Helcococcus Lectin Hemagglutinin Adhesin