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Archives of Microbiology (v.171, #6)
Biochemical and phylogenetic analyses of psychrophilic isolates belonging to the Arthrobacter subgroup and description of Arthrobacter psychrolactophilus, sp. nov. by Jennifer Loveland-Curtze; Peter P. Sheridan; Kevin R. Gutshall; J. E. Brenchley (pp. 355-363).
During our work on psychrophilic microorganisms we obtained a large collection of new isolates. In order to identify six of these, we examined their growth properties, cell wall compositions, and their 16S rRNA gene sequences. The results showed that all of the isolates are gram-positive, aerobic, contain lysine in their cell walls, and belong to the high mol% G+C Arthrobacter subgroup. Phylogenetic analysis of the 16S rRNA genes grouped five isolates obtained from a small geographical region into a monophyletic clade. Isolate B7 had a 16S rRNA sequence that was 94.3% similar to that of Arthrobacter polychromogenes and 94.4% similar to that of Arthrobacter oxydans. Primary characteristics that distinguish isolate B7 from the Arthrobacter type strain (Arthrobacter globiformis) and A. polychromogenes include lack of growth at 37°C, growth at 0–5°C, the ability to use lactose as a sole carbon source, and the absence of blue pigments. Because of these differences, isolate B7 was chosen as a type strain representing a new Arthrobacter species, Arthrobacter psychrolactophilus. The sixth isolate, LV7, differed from the other five because it did not have the rod/ coccus morphological cycle and was most closely related to Arthrobacter agilis.
Keywords: Key wordsArthrobacter psychrolactophilus; Psychrophilic; Cell wall; Rod/coccus morphology; 16S rRNA phylogeny
Heterologous expression of soluble methane monooxygenase genes in methanotrophs containing only particulate methane monooxygenase by John S. Lloyd; Paolo De Marco; Howard Dalton; J. C. Murrell (pp. 364-370).
The methanotrophs Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b contain particulate methane monooxygenase (pMMO) and soluble methane monooxygenase (sMMO) genes. Other methanotrophs such as Methylomicrobium album BG8 and Methylocystis parvus OBBP contain only pMMO genes. Although molecular genetic techniques are poorly developed in methanotrophs, sMMO genes were expressed in methanotrophs normally containing only pMMO genes. This was achieved by conjugation using broad-host-range plasmids containing the native promoter and sMMO genes from Mc. capsulatus (Bath) and Ms. trichosporium OB3b. sMMO genes derived from Ms. trichosporium OB3b were expressed in an active form in Mcy. parvus OBBP and in Mm. album BG8. Therefore, all of the genes required for active sMMO synthesis were contained on the broad-host-range plasmids and were expressed in the heterologous hosts. Constitutive synthesis of pMMO was observed in Mm. album BG8 when grown at high and low copper-to-biomass ratios, while transcription of the recombinant sMMO genes was only observed under growth conditions of low copper-to-biomass ratios. Therefore, the regulatory protein(s) for sMMO synthesis was also present on the plasmid used, or the heterologous host contained a regulatory system for sMMO. Expression of sMMO genes in methanotrophs containing only pMMO will assist further investigations on the expression and regulation of MMO genes in methanotrophs.
Keywords: Key words Heterologous expression; Methane; oxidation; Methane monooxygenase; Methanotroph; Regulation
Metabolic state of Zymomonas mobilis in glucose-, fructose-, and xylose-fed continuous cultures as analysed by 13C- and 31P-NMR spectroscopy by A. A. De Graaf; Katharina Striegel; Rolf M. Wittig; Birgit Laufer; Günter Schmitz; Wolfgang Wiechert; Georg A. Sprenger; Hermann Sahm (pp. 371-385).
The reasons for the well-known significantly different behaviour of the anaerobic, gram-negative, ethanologenic bacterium Zymomonas mobilis during growth on fructose (i.e. decreased growth and ethanol yields, increased by-product formation) as compared to that on its second natural substrate, glucose, have remained unexplained. A xylose-fermenting recombinant strain of Z. mobilis that was recently constructed in our laboratory also unexpectedly displayed an increased formation of by-products and a strongly reduced growth rate as compared to the parent strain. Therefore, a comprehensive study employing recently developed NMR-based methods for the in vivo analysis of intracellular phosphorylated pool sizes and metabolic fluxes was undertaken to enable a global characterization of the intracellular metabolic state of Z. mobilis during growth on 13C-labelled glucose, fructose and xylose in defined continuous cultures. The 13C-NMR flux analysis indicated that ribose 5-phosphate is synthesized via the nonoxidative pentose phosphate pathway in Z. mobilis, and it identified a metabolic bottleneck in the recombinant xylose-fermenting Z. mobilis strain at the level of heterologous xylulokinase. The 31P-NMR analyses revealed a global alteration of the levels of intracellular phosphorylated metabolites during growth on fructose as compared to that on glucose. The results suggest that this is primarily caused by an elevated concentration of intracellular fructose 6-phosphate.
Keywords: Key wordsZymomonas mobilis; Metabolic flux; analysis; Sugar phosphates; Glucose; Fructose; Xylose; 13C-NMR; In vivo 31P-NMR; Rate-limiting step
The acuH gene of Aspergillus nidulans, required for growth on acetate and long-chain fatty acids, encodes a putative homologue of the mammalian carnitine/acylcarnitine carrier by J. Ramón De Lucas; A. I. Domínguez; Susana Valenciano; Geoffrey Turner; F. Laborda (pp. 386-396).
The Aspergillus nidulans acuH gene, required for growth on acetate and long-chain fatty acids, was cloned by complementation of the acuH13 mutation. Northern blotting analysis showed that transcription of the acuH gene occurs in acetate-grown mycelium and at higher levels in oleate-grown mycelium, but not during growth on glucose minimal medium. The acuH gene encodes a protein of 326 amino acids that belongs to the mitochondrial carrier family. The ACUH protein contains three related segments of approximately 100 amino acids in length, each segment comprising two hydrophobic domains that are probably folded into two transmembrane α-helices linked by an extensive polar region. Sequence comparisons suggest that the acuH gene of A. nidulans encodes the homologue of the carnitine/acylcarnitine carrier of rat and man. The uncharacterised proteins YOR100C of Saccharomyces cerevisiae, COLT of Drosophila melanogaster, and DIF-1 of Caenorhabditis elegans also seem to be homologues of ACUH. In addition to the motifs present in all members of the mitochondrial carrier family, we propose the highly conserved motif R(A,S)(V,F)PANAA(T,C)F within the sixth hydrophobic domain of these proteins as the characteristic feature of the carnitine carrier subfamily. The proposed function of the ACUH protein is the transport of acetylcarnitine molecules from the cytosol to the mitochondrial matrix, a process required during growth on acetate or on long-chain fatty acids.
Keywords: Key wordsAspergillus nidulans; acuH; Carnitine; carrier; Acetate; Fatty acids; Acetyl-CoA transport; YOR100C; dif-1; colt
Lapstatin, a new aminopeptidase inhibitor produced by Streptomyces rimosus, inhibits autogenous aminopeptidases by B. Repič Lampret; Jurka Kidrič; Bogdan Kralj; Ljubinka Vitale; Miroslav Pokorny; Metka Renko (pp. 397-404).
Lapstatin, a low-molecular-weight aminopeptidase inhibitor, was purified to homogeneity from Streptomyces rimosus culture filtrates. The purification procedure included extraction with methanol, followed by chromatography on Dowex 50WX4, AG50WX4, and HPLC RP C18 columns. By amino acid analysis, mass spectrometry, and NMR spectroscopy, the structure of lapstatin was shown to be 3-amino-2-hydroxy-4-methylpentanoylvaline. Lapstatin inhibited the extracellular leucine aminopeptidases from Streptomyces rimosus, Streptomyces griseus, and Aeromonas proteolytica with an IC50 in the range of 0.3–2.4 μM. IC50 values for other enzymes tested were at least tenfold higher. Leucine aminopeptidase from Streptomyces griseus was inhibited in a competitive manner, with an inhibition constant of 5 × 10–7 M. Lapstatin is the first low-molecular-weight compound isolated from streptomycetes shown to inhibit an autogenous aminopeptidase.
Keywords: Key words Extracellular peptide inhibitor; Leucine aminopeptidase; Streptomyces rimosus
Selective enrichment and characterization of Roseospirillum parvum, gen. nov. and sp. nov., a new purple nonsulfur bacterium with unusual light absorption properties by Jens Glaeser; J. Overmann (pp. 405-416).
A new type of phototrophic purple bacterium, strain 930I, was isolated from a microbial mat covering intertidal sandy sediments of Great Sippewissett Salt Marsh (Woods Hole, Mass., USA). The bacterium could only be enriched at a wavelength of 932 (± 10) nm. Cells were vibrioid- to spirilloid-shaped and motile by means of bipolar monotrichous flagellation. The intracytoplasmic membranes were of the lamellar type. Photosynthetic pigments comprised bacteriochlorophyll a and the carotenoids spirilloxanthin and lycopenal. The isolated strain exhibited an unusual, long-wavelength absorption maximum at 911 nm. Sulfide or thiosulfate served as electron donor for anoxygenic phototrophic growth. During growth on sulfide, elemental sulfur globules formed outside the cells. Elemental sulfur could not be further oxidized to sulfate. In the presence of sulfide plus bicarbonate, fructose, acetate, propionate, butyrate, valerate, 2-oxoglutarate, pyruvate, lactate, malate, succinate, fumarate, malonate, casamino acids, yeast extract, L(+)-alanine, and L(+)-glutamate were assimilated. Sulfide, thiosulfate, or elemental sulfur served as a reduced sulfur source for photosynthetic growth. Maximum growth rates were obtained at pH 7.9, 30 °C, 50 μmol quanta m–2 s–1 of daylight fluorescent tubes, and a salinity of 1–2% NaCl. The strain grew microaerophilically in the dark at a partial pressure of 1 kPa O2. The DNA base composition was 71.2 mol% G + C. Sequence comparison of 16S rRNA genes indicated that the isolate is a member of the α-Proteobacteria and is most closely related to Rhodobium orientis at a similarity level of 93.5%. Because of the large phylogenetic distance to known phototrophic species of the α-Proteobacteria and of its unique absorption spectrum, strain 930I is described as a new genus and species, Roseospirillum parvum gen. nov. and sp. nov.
Keywords: Key words Purple nonsulfur bacteria; Light-harvesting complex; Infrared radiation; Photosynthetic energy transfer; Anoxygenic photosynthesis; Phototaxis; Microbial mats
Morpholine-induced formation of l-alanine dehydrogenase activity in Mycobacterium strain HE5 by Grit Schuffenhauer; Thomas Schräder; J. R. Andreesen (pp. 417-423).
An NAD-dependent, morpholine-stimulated l-alanine dehydrogenase activity was detected in crude extracts from morpholine-, pyrrolidine-, and piperidine-grown cells of Mycobacterium strain HE5. Addition of morpholine to the assay mixture resulted in an up to 4.6-fold increase of l-alanine dehydrogenase activity when l-alanine was supplied at suboptimal concentration. l-Alanine dehydrogenase was purified to near homogeneity using a four-step purification procedure. The native enzyme had a molecular mass of 160 kDa and contained one type of subunit with a molecular mass of 41 kDa, indicating a tetrameric structure. The sequence of 30 N-terminal amino acids was determined and showed a similarity of up to 81% to that of various alanine dehydrogenases. The pH optimum for the oxidative deamination of l-alanine, the only amino acid converted by the enzyme, was determined to be pH 10.1, and apparent K m values for l-alanine and NAD were 1.0 and 0.2 mM, respectively. K m values of 0.6, 0.02, and 72 mM for pyruvate, NADH, and NH4 +, respectively, were estimated at pH 8.7 for the reductive amination reaction.
Keywords: Key words Alanine dehydrogenase; Ammonia; assimilation; Mycobacterium; Morpholine degradation
Generation and initial characterization of Pseudomonas stutzeri KC mutants with impaired ability to degrade carbon tetrachloride by Lycely Del C. Sepúlveda-Torres; Narayanan Rajendran; Michael J. Dybas; Craig S. Criddle (pp. 424-429).
Under iron-limiting conditions, Pseudomonas stutzeri KC secretes a small but as yet unidentified factor that transforms carbon tetrachloride (CT) to CO2 and nonvolatile products when activated by reduction at cell membranes. Pseudomonas fluorescens and other cell types activate the factor. Triparental mating was used to generate kanamycin-resistant lux::Tn5 recombinants of strain KC. Recombinants were streaked onto the surface of agar medium plugs in microtiter plates and were then screened for carbon tetrachloride degradation by exposing the plates to gaseous 14C-carbon tetrachloride. CT+ recombinants generated nonvolatile 14C-labeled products, but four CT– recombinants did not generate significant nonvolatile 14C-labeled products and had lost the ability to degrade carbon tetrachloride. When colonies of P. fluorescens were grown next to colonies of CT+ recombinants and were exposed to gaseous 14C-carbon tetrachloride, 14C-labeled products accumulated around the P. fluorescens colonies, indicating that the factor secreted by CT+ colonies had diffused through the agar and become activated. When P. fluorescens was grown next to CT– colonies, little carbon tetrachloride transformation was observed, indicating a lack of active factor. Expression of lux reporter genes in three of the CT– mutants was regulated by added iron and was induced under the same iron-limiting conditions that induce carbon tetrachloride transformation in the wild-type.
Keywords: Key words Transposon mutagenesis; Carbon; tetrachloride; Biotransformation; Biodegradation; Luciferase; Pseudomonas stutzeri KC; Reporter genes; Mutants
Linear alkanesulfonates as carbon and energy sources for gram-positive and gram-negative bacteria by Wolfram Reichenbecher; J. Colin Murrell (pp. 430-438).
Several bacteria from soil and rainwater samples were enriched and isolated with propanesulfonate or butanesulfonate as sole carbon and energy source. Most of the strains isolated utilized nonsubstituted alkanesulfonates with a chain length of C3–C6 and the substituted sulfonates taurine and isethionate as carbon and energy source. A gram-positive isolate, P40, and a gram-negative isolate, P53, were characterized in more detail. Phylogenetic analysis grouped strain P40 within group IV of the genus Rhodococcus and showed a close relationship with Rhodococcus opacus. After phylogenetic and physiological analyses, strain P53 was identified as Comamonas acidovorans. Both bacteria also utilized a wide range of sulfonates as sulfur source. Strain P40, but not strain P53, released sulfite into the medium during dissimilation of sulfonated compounds. Cell-free extracts of strain P53 exhibited high sulfite oxidase activity [2.34 U (mg protein)–1] when assayed with ferricyanide, but not with cytochrome c. Experiments with whole-cell suspensions of both strains showed that the ability to dissimilate 1-propanesulfonate was specifically induced during growth on this substrate and was not present in cells grown on propanol, isethionate or taurine. Whole-cell suspensions of both strains accumulated acetone when oxidizing the non-growth substrate 2-propanesulfonate. Strain P40 cells also accumulated sulfite under these conditions. Stoichiometric measurements with 2-propanesulfonate as substrate in oxygen electrode experiments indicate that the nonsubstituted alkanesulfonates were degraded by a monooxygenase. When strain P53 grew with nonsubstituted alkanesulfonates as carbon and energy source, cells expressed high amounts of yellow pigments, supporting the proposition that an oxygenase containing iron sulfur centres or flavins was involved in their degradation.
Keywords: Key wordsComamonas acidovorans; Rhodococcus; Alkanesulfonates; Monooxygenases; Sulfonates
Analysis of the expression and function of the σB-dependent general stress regulon of Bacillus subtilis during slow growth by T. Schweder; Angela Kolyschkow; Uwe Völker; Michael Hecker (pp. 439-443).
Glucose-limited continuous cultures were used to analyze σB activity at decreasing growth rates. Expression of the σB-dependent genes gsiB and ctc started to increase at a growth rate of 0.2 h–1, and both genes were induced approximately fivefold at a growth rate of 0.1 h–1 as compared to expression at the maximal growth rate. However, maximal σB activity was only reached when the growth stopped as a result of the exhaustion of the carbon and energy source glucose. During glucose-limited growth, increased expression of the general stress regulon at growth rates below 0.2 h–1 did not provide wild-type cells with a growth advantage over sigB mutants. Instead, expression of the stress regulon seems to constitute a significant burden during glucose-limited growth, resulting in a selective growth advantage of the sigB mutant as compared to the wild-type at a growth rate of 0.08 h–1.
Keywords: Key wordsBacillus subtilis; sigB; Chemostat; Glucose limitation; Slow growth; Survival