Identification of a gene for a rubrerythrin/nigerythrin-like protein in Spirillum volutans by using amino acid sequence data from mass spectrometry and NH2-terminal sequencing.

A hydrogen peroxide-resistant mutant of the catalase-negative microaerophile, Spirillum volutans, constitutively expresses a 21.5 kDa protein that is undetectable and non-inducible in the wild-type cells. Part of the gene that encodes the protein was cloned using amino acid sequence data obtained by both mass spectrometry and NH2-terminal sequencing. The deduced 158 amino acid polypeptide shows high relatedness to rubrerythrin and nigerythrin previously described in the anaerobes Clostridium perfringens and Desulfovibrio vulgaris. The protein also shows high similarity to putative rubrerythrin proteins found in the anaerobic archeons Archaeoglobus fulgidus, Methanococcus jannaschii and Methanobacterium thermoautotrophicum. This is the first report of this type of protein in an organism that must respire with oxygen. It seems likely that the novel combination of methodologies used in this study could be applied to the rapid cloning of other genes in bacteria for which no genomic library yet exists.

Toxicity of organomercury compounds: bioassay results as a basis for risk assessment.

A former mercury plant, where mercury salts and organomercurials for pesticide use were produced, caused soil contamination in high concentrations. Typical organomercurial products included ethylmercury, phenylmercury, methoxyethylmercury and ethoxyethylmercury compounds. Risk assessment of these sites must be carried out before any major clean-up processes can be planned. A sensitive speciation technique for the various organomercury species in environmental matrices is a prerequisite for toxicity investigations. In this connection, a high-performance liquid chromatography-atomic fluorescence spectrometry (HPLC-AFS) technique has been developed to differentiate between and determine the presence of eight organomercury compounds in environmental samples. Using this technique, methylmercury, ethylmercury and phenylmercury and some unknown organomercury species were found in soil samples collected from the sites of an old mercury products producing plant. With regard to risk assessment, it is necessary to assess the toxicity of the organomercurials. As different microbial metabolic pathways react differently to mercury and its compounds, batteries of bioassays are, therefore, useful to evaluate the toxicity of pollutants. To describe the toxicity and genotoxicity of MeHg+, MeOEtHg+, EtHg+, EtOEtHg+ and PhHg+, p-tolymercury chloride, nitromersol and Hg2+ six bioassays were used: resazurin reduction method, Spirillum volutans test, nematode toxicity assay Panagrellus redivivus, Toxi-Chromotest and SOS-Chromotest. A ranking of the toxicity of the organomercurial is shown. The SOS-Chromotest indicated genotoxicity for 5-7 organomercurials.

Bacteremia with Anaerobiospirillum succiniciproducens.

Anaerobiospirillum succiniciproducens is a motile, spiral anaerobic bacterium with bipolar tufts of flagella. Reports of clinical illness due to A. succiniciproducens are rare. In a retrospective review of anaerobic isolates referred to the Centers for Disease Control (CDC) from January 1, 1975, through January 31, 1986, isolates of A. succiniciproducens from the blood of 21 patients were identified. A single patient whose blood isolate had not been received at CDC was included in the review. These 22 patients were from 15 states. Their mean age was 58.6 years. Underlying disorders included alcoholism, atherosclerosis, malignancy, surgery, diabetes mellitus, and dental caries. Clinical features included gastrointestinal tract signs and symptoms in 17 (77%) of 22, fever greater than 38 degrees C in seven (37%) of 19, and leukocytosis of more than 10,000 cells/mm3 in 11 (58%) of 19. Although 16 patients received antimicrobial therapy, its effect on outcome was unclear. A. succiniciproducens was reported to have contributed to the deaths of seven patients. Disorders predisposing patients to anaerobic infections may put them at increased risk for A. succiniciproducens bacteremia. The presence of antecedent gastrointestinal tract signs and symptoms suggests that the gastrointestinal tract might be the primary portal of entry.

Resistencia a antibioticos en cepas de Azospirillum sp / Antibiotic resistance of Azospirillum sp. strains

Se determinaron los niveles de resistencia a nueve antibióticos de 32 cepas de Azospirillum sp. Las cepas fueron sensible a tetraciclina, kanamicina, cloramfenicol y ácido nalidíxico, y resistentes a altos niveles de ampicilina y carbenicilina. Para la mayoría de las cepas se pudo determinar un antibiotipo por medio del cual podrían ser identificadas

Resistencia a antibioticos en cepas de Azospirillum sp / Antibiotic resistance of Azospirillum sp. strains

Se determinaron los niveles de resistencia a nueve antibióticos de 32 cepas de Azospirillum sp. Las cepas fueron sensible a tetraciclina, kanamicina, cloramfenicol y ácido nalidíxico, y resistentes a altos niveles de ampicilina y carbenicilina. Para la mayoría de las cepas se pudo determinar un antibiotipo por medio del cual podrían ser identificadas (AU)

Determination of the spiral conformation of Aquaspirillum spp. by scanning electron microscopy of elongated cells induced by cephalexin treatment.

The effect of the beta-lactam antibiotic cephalexin on the spiral conformation of cells of Aquaspirillum spp. was examined by scanning electron microscopy. A. itersonii and A. peregrinum, which are known to have a left-handed spiral shape, elongated and still showed left-handed spirals in medium containing cephalexin. The spiral conformation of the elongated cells is therefore considered to represent the natural condition. The spiral conformations of A. metamorphum and A. psychrophilum grown in ordinary cultures were difficult to determine because they have short cells without a complete spiral. After cephalexin treatment, the cells of these species elongated and displayed spiral forms, right-handed in A. metamorphum and left-handed in A. psychrophilum. This elongation method may be useful for checking and determination of the spiral handedness of short spiral or curved bacteria such as vibrios.

Deoxyribonucleic acid-mediated transformation of Spirillum lipoferum.

The methodology for deoxyribonucleic acid-mediated transformation of Spirillum lipoferum to resistance to various antimicrobial agents is reported.

Effect of oxygen and nitrate on nitrogen fixation and denitrification by Azospirillum brasilense grown in continuous culture.

Azospirillum brasilense was grown continuously at various levels of dissolved oxygen (O2) in a nitrogen-free medium containing malates as the carbon source. Steady-state cultures were established only at O2 concentrations less than 0.0150 atm (1 atm = 101.325 Pa) and rates of acetylene reduction (N2 fixation) and efficiencies of N2 fixation were maximal between 0.0050-0.0075 atm dissolved O2. These cultures appeared to be O2- or N2-limited. There was no evidence of a respiratory protective mechanism in this organism. Anaerobic denitrifying steady-state cultures were established with nitrate (NO3-) as the sole nitrogen source with no detectable N2 fixation. N2 fixation, but no denitrification, was observed when NO3- was decreased to 10 microgram N per millilitre at 0.003 atm dissolved O2. In samples removed from the culture vessel, either activity could be induced with a lag of approximately 120 min by incubation under appropriate conditions.

Nitrate and nitrite reductase negative mutants of N2-fixing Azospirillum spp.

Chlorate resistant spontaneous mutants of Azospirillum spp. (syn. Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants from A. brasilense and 13 from A. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr-). Most of the mutants were also nitrite reductase negative (nir-), only 3 remaining nir+. Two mutants from nr+ nir+ parent strains lost only nir and became like the nr+ nir- parent strain of A. brasilense. No parent strain or nr+ mutant showed any nitrogenase activity with 10 mM NO3-. In all nr- mutants, nitrogenase was unaffected by 10 mM NO3-. Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir-. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr- nir-) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.

Dependence of the superficial layers of Spirillum putridiconchylium on Ca2+ or Sr2+.

Chelating agents disrupted the superficial layers on Spirillum putridiconchylium and adsorption of cationized ferritin indicated that both upper and lower surfaces of superficial layer fragments, as well as the outer membrane surface, possessed areas which were negatively charged. Growth of the bacterium in 1% casamino acids (vitamin free) resulted in cells which were devoid of the superficial layers, and negative staining of these cells revealed in amorphous precipitate together with a vesicular outer membrane component extruding from their surfaces into the medium. Addition of either 1 mM Ca2+ or 1 mM Sr2+ to the growth medium produced the typical regularly structured cell surface, whereas addition of equal concentrations of Li+, Na+, K+, Mg2+, Ba2+, Mn2+, Fe3+, or three polyamines produced the structureless surface.

Nitrate reductase and soluble cytochrome c in Spirillum itersonii.

The addition of nitrate to cultures of Spirillum itersonii incubated under low aeration produced a diauxic growth pattern in which the second exponential phase was preceded by the appearance of nitrite in the medium. The organism also grew anaerobically in the presence of nitrate. Nitrate reductase activity could be demonstrated in cell-free extracts by use of reduced methyl viologen as the electron donor. The enzyme was located in the supernatant fraction after centrifugation of extracts for 2 hr at 40,000 x g, and it sedimented as a single peak when centrifuged in a sucrose gradient. Nitrate reductase activity was found in cells grown with low aeration without nitrate, but was increased about twofold by addition of nitrate. Enzyme activity was negligible in cells grown with high aeration. The proportion of soluble cytochrome c was increased two- to threefold in cells grown with nitrate. The specific activities of nitrate reductase and soluble cytochrome c rose when nitrate or nitrite was added to cell suspensions incubated with low aeration; nitrite was more effective than nitrate during the early stages of incubation. A nitrate reductase-negative mutant synthesized increased amounts of soluble cytochrome c in response to nitrate or to nitrite in the cell suspension system. It is concluded that enhanced synthesis of soluble cytochrome c does not require the presence of a functional nitrate reductase.