Competition between the facultatively chemolithotrophic Thiobacillus A2, an obligately chemolithotrophic Thiobacillus and a heterotrophic spirillum for inorganic and organic substrates

Competition in a chemostat between the versatile Thiobacillus A2 and the specialized T. neapolitanus for thiosulfate as the sole growth-limiting substrate, led to dominance of the specialized over the versatile organism, at dilution rates 0.025 h^-1. Increasing concentrations of acetate or glycollate in the thiosulfate medium caused increased relative numbers of T. A2 in steady states at D=0.07 h^-1. Eventually, with 10–12 mmol of organic substrate per litre, complete dominance of T. A2 over T. neapolitanus occurred.Mixed cultures of T. A2 and a specialized spirillumshaped heterotroph, competing for acetate as sole growth-limiting substrate resulted in complete dominance of the heterotroph at dilution rates of 0.07 and 0.15 h^-1. In this case increasing concentrations of thiosulfate in the acetate medium, up to 10 mM, eventually led to the elimination of the heterotroph.These results have been interpreted as evidence that T. A2 was growing mixotrophically. As the concentration of the second substrate was raised, the number of T. A2 cells increased and as a result T. A2 consumed an increasing portion of the common substrate.In mixed chemostat cultures containing all three organisms, T. A2 could maintain itself with all tested ratios of acetate and thiosulfate in the inflowing medium. The heterotroph was excluded from the culture below a relatively low acetate to thiosulfate ratio, whilst above a relatively high acetate to thiosulfate ratio T. neapolitanus was completely eliminated.These results were discussed in relation to the ecological niche of Thiobacillus A2-type organisms.     

Purification and some properties of ubiquinol oxidase from obligately chemolithotrophic iron-oxidizing bacterium, Thiobacillus ferrooxidans NASF-1

Ubiquinol-oxidizing activity was detected in an acidophilic chemolithotrophic iron-oxidizing bacterium, T. ferrooxidans. The ubiquinol oxidase was purified 79-fold from plasma membranes of T. ferrooxidans NASF-1 cells. The purified oxidase is composed of two polypeptides with apparent molecular masses of 32,600 and 50,100 Da, as measured by gel electrophoresis in the presence of sodium dodecyl sulfate. The absorption spectrum of the reduced enzyme at room temperature showed big peaks at 530 and 563, and a small broad peak at 635 nm, indicating the involvement of cytochromes b and d. Characteristic peaks of cytochromes a and c were not observed in the spectrum at around 600 and 550 nm, respectively. This enzyme combined with CO, and its CO-reduced minus reduced difference spectrum showed peaks at 409 nm and 563 nm and a trough at 431 nm. These results indicated that the oxidase contained cytochrome b, but the involvement of cytochrome d was not clear. The enzyme catalyzed the oxidations of ubiquinol-2 and reduced N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride. The ubiquinol oxidase activity was activated by the addition of albumin and lecithin to the reaction mixture and inhibited by the respiratory inhibitors KCN, HQNO, NaN3, and antimycin A1, although the enzyme was relatively resistant to KCN, and the divalent cation, Zn2+, compared with ubiquinol oxidases of E. coli.     

Isolation and characterization of Thiobacillus hydrothermalis sp. nov., a mesophilic obligately chemolithotrophic bacterium isolated from a deep-sea hydrothermal vent in Fiji Basin

A novel obligately chemolithotrophic Thiobacillus species isolated from a deep-sea hydrothermal vent is described. This organism grows lithoautotrophically on thiosulphate, tetrathionate, sulphide and sulphur which are oxidized to sulphate. The isolate is slightly halophilic and markedly halotolerant, showing optimum growth at pH 7.5 and at 35°C. The G+C content of the DNA is 67.1 mol%. The 16S rRNA sequence is distinct from any other Thiobacilli sequences. Phylogenetic analysis shows the organism to be a representative of the -group of proteobacteria and a specific relative of Thiobacillus neapolitanus. The ubiquinone is ubiquinone-8. These characters distinguish the isolate from any other Thiobacillus or Thiomicrospira species previously reported and is a new species described as Thiobacillus hydrothermalis. The type strain is isolate R3, DSM7121.     

An obligately endosymbiotic mycorrhizal fungus itself harbors obligately intracellular bacteria.

Arbuscular-mycorrhizal fungi are obligate endosymbionts that colonize the roots of almost 80% of land plants. This paper describes the employment of a combined morphological and molecular approach to demonstrate that the cytoplasm of the arbuscular-mycorrhizal fungus Gigaspora margarita harbors a further bacterial endosymbiont. Intracytoplasmic bacterium-like organisms (BLOs) were detected ultrastructurally in its spores and germinating and symbiotic mycelia. Morphological observations with a fluorescent stain revealed about 250,000 live bacteria inside each spore. The sequence for the small-subunit rRNA gene obtained for the BLOs from the spores was compared with those for representatives of the eubacterial lineages. Molecular phylogenetic analysis unambiguously showed that the endosymbiont of G. margarita was an rRNA group II pseudomanad (genus Burkholderia). PCR assays with specifically designed oligonucleotides were used to check that the sequence came from the BLOs. Successful amplification was obtained when templates from both the spores and the symbiotic mycelia were used. A band of the expected length was also obtained from spores of a Scutellospora sp. No bands were given by the negative controls. These findings indicate that mycorrhizal systems can include plant, fungal, and bacterial cells.     

Genetic transformation in Methylobacterium organophilum.

Several mutants have been isolated from the facultative methylotroph, Methylobacterium organophilum, using either N-methyl-N'-nitro-N-nitrosoguanidine or ultraviolet light as mutagens. One of these isolates, a glutamate auxotroph lacking isocitrate dehydrogenase, has been transformed to prototrophy, using wild-type DNA, at a frequency of 0-5%. Competence and DNA uptake occur only in cultures which are near the end of exponential growth, and maximal transformation requires a DNA concentration of 100 mug ml-1.     

Genetic transformation in Rhizobium trifolii.

Markers controlling the synthesis of amino acids and organic bases as well as streptomycin resistance and sensitivity to acriflavine were transformed in Rhizobium trifolii. The results indicate that the str marker was transformed independently of leu, his, ade and trp markers. Co-transformation of leu and utra markers ranged from 3 to 7%, whereas that of thi and acr was 10%.     

Carbon monoxide-dependent chemolithotrophic growth of Clostridium thermoautotrophicum.

The acetogen Clostridium thermoautotrophicum was cultivated under CO-dependent chemolithotrophic conditions. CO-dependent growth profiles and energetics indicated that supplemental CO2 was fundamental to efficient growth at the expense of CO. Overall product stoichiometry approximated 6.5CO----CH3CO2H + 3.5CO2 + 0.6 cell C + 0.5 unrecovered C. Initial CO/CO2 ratios of 2 to 4 yielded optimal doubling times and cell yields. Maximal YCO values approximated 2.5 g of cell dry weight per mol of CO consumed; YH2 was considerably lower than YCO. Cross-transfer growth experiments and protein profiles indicated differential expression of genes between CO and methanol cultures.     

Genetic transformation of yeast

Genetic transformation was first described by Griffith in 1928 and has since been demonstrated in a variety of organisms, including many species of fungi. This review focuses on the history and technology of the transformation of Saccharomyces cerevisiae. The application of protocols developed for S. cerevisiae to other important yeast species is discussed. The protocols for transformation by spheroplasting, LiAc/ssDNA/PEG, and electroporation are compared, and possible mechanisms for transformation are discussed.     

Genetic transformation of Pseudomonas phaseolicola by R-factor DNA.

Summary The bacterium Pseudomonas phaseolicola was successfully transformed, for the first time, with R-factors RSF1010 and pBR322 DNA by a calciumshock and heat-pulse technique. Frequency of transformation for RSF1010 ranged from 0.8×10^-7 to 3.1×10^-6 and was ca. 0.4×10^-8 for pBR322.     

Genetic transformation of Rhodopseudomonas sphaeroides by plasmid DNA.

A broad-host-range cloning vector, pUI81, was constructed in vitro from plasmids RSF1010 and pSL25 (a pBR322 derivative) and used to assay for transformation in Rhodopseudomonas sphaeroides. Washing cells with 500 mM Tris was an effective means of inducing competence for DNA uptake. Transformation frequencies as high as 10(-5) (transformants per viable cell) have been achieved by incubating Tris-treated cells with plasmid DNA, 100 mM CaCl2, and 20% polyethylene glycol 6000. Maximum frequencies were obtained when recipient cells were spread onto selective media after a 6.5-h outgrowth period in antibiotic-free medium. The structure (open circular versus closed, covalent circular), size, and concentration of plasmid DNA all significantly affected the transformation frequency. Four different plasmids, all small and suitable as cloning vectors, have been introduced by transformation into several different R. sphaeroides strains. Recombinant DNA carried on small, nonconjugative plasmids with broad host ranges can now be directly transferred to R. sphaeroides by this method.