Respiration-Dependent Proton and Sodium Pumps in a Psychrophilic Bacterium, Vibrio sp. Strain ABE-1

Respiration-dependent proton and sodium flows in a psychrophilicbacterium, Vibrio sp. strain ABE-1, were examined. At alkalinepH, this bacterium grew without being affected by a proton conductor,carbonylcyanide m-chlorophenylhydrazone (CCCP). O₂-pulse intoanaerobic cell suspensions prepared with Na^$-free buffers inducedtransient alkalization in the presence of CCCP and acidificationat pH 8.5 and 6.5, respectively. However, using cells preparedwith Na^$-containing buffer, the transient pH changes of thecell suspension could be simultanously detected at both pHs.Several inhibitory experiments suggested that the acidificationand alkalization should be attributed to a respiration-dependentprimary H^$ pump and Na^$ pump, respectively, and that the latterwas similar to that first reported in a marine bacterium, Vibrioalginolyticus. This Na^$ pump may have supported the CCCP-resistantgrowth at alkaline pH. The H^$ and Na^$ pumps operated very actively at low temperatures,such as 5?C, and should markedly help sustain bacterial growthat low temperatures. (Received May 30, 1987; Accepted November 13, 1987)     

Cloning, Nucleotide Sequences and Differential Expression of the nifH and nifH-Like (frxC) Genes from the Filamentous Nitrogen-Fixing Cyanobacterium Plectonema boryanum

The frxC gene, found in liverwort chloroplast DNA, encodes aprotein of unknown function. The deduced amino acid sequenceof the protein shows significant homology to that of ni-trogenaseFe-protein encoded by the nifH gene. We have cloned the frxCand nifH genes from the nitrogen-fixing cyanobacterium Plectonemaboryanum, using frxC- and nifH-specific probes, and have determinedtheir nucleotide sequences. The amino acid sequence deducedfrom the frxC gene of P. boryanum exhibits 83% homology to thatof the protein encoded by the/rxCgene from liverwort, whereasit exhibits only 34% homology to that encoded by the nifH genefrom the same organism, namely, P. boryanum. Northern blot analysisshowed that the frxC gene was transcribed more actively undernitrogenase-repressed conditions than under nitrogenase-inducedconditions, suggesting that the FrxC protein has a functiondistinct from nitrogen fixation. These results, together withthe phylogenetic relationship between the nifH and frxC genes,indicate that the frxC and nifH genes are derived from a commonancestral gene but have evolved independently to encode proteinswith different functions. (Received April 27, 1991; Accepted August 12, 1991)     

Analysis of bacterial populations in a grassland soil according to rates of development on solid media

Abstract The process of colony formation by bacteria from grassland soil sampled in April, July and September was simulated by a colony-forming curve (CFC). The CFC was a super-imposition of several component curves (cCFC) given theoretically by the first order reaction (FOR) model [3,6]. The pattern of FOR model curves was not influenced by the time of sampling and four cCFCs were always recognized during an incubation period of 160 h. It was considered that the CFC describes an inherent property of the bacterial population of the field. Bacterial isolates were obtained from colonies produced in each of four cCFCs on agar plates. Isolates corresponding to one cCFC were classified as one group. The bacterial isolates were characterized by morphological and physiological tests and subsequently clustered. Few oligotrophic bacteria were obtained among bacteria which produced visible colonies within 63 h of incubation time. On the other hand, approx. 50% of bacteria which produced v colonies after 63 h were oligotrophic bacteria. The time required for the appearance of the first colony, t _r of the FOR model, was very similar in the isolates belonging to one group. A close linear relationship was observed between t _r value and doubling time of isolates.     

Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus and structural comparison with other NAD(P)+-dependent dehydrogenases

The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was found to carry an about 9 kilobase pair plasmid, which contained 4.6 kilobase pairs of B. stearothermophilus DNA. The nucleotide sequence including the 1287 base pair coding region of the leucine dehydrogenase gene was determined by the dideoxy chain termination method. The translated amino acid sequence was confirmed by automated Edman degradation of several peptide fragments produced from the purified enzyme by trypsin digestion. The polypeptide contained 429 amino acid residues corresponding to the subunit (Mr 49,000) of the hexameric enzyme. Comparison of the amino acid sequence of leucine dehydrogenase with those of other pyridine nucleotide dependent oxidoreductases registered in a protein data bank revealed significant sequence similarity, particularly between leucine and glutamate dehydrogenases, in the regions containing the coenzyme binding domain and certain specific residues with catalytic importance.     

Increase in peripheral large granular lymphocytes in postpartum autoimmune thyroiditis

In order to elucidate the mechanism of postpartum aggravation of autoimmune thyroid disease (AITD), we serially examined the change in the proportion of peripheral large granular lymphocytes (LGL), which have activities of NK, K and/or cytotoxic T cells, in their postpartum period. Within 6 months postpartum, the percentage of LGL increased transiently in patients with AITD who remained euthyroid, or developed destructive thyrotoxicosis and/or hypothyroidism due to thyroiditis and even in normal controls. These changes in the LGL percentage were more obvious in the patients who had marked postpartum thyroid dysfunction. In contrast, we did not find a definite increase in the LGL percentage within 6 months postpartum in patients with Graves' disease who relapsed into Graves' thyrotoxicosis. These deta suggest that the increase in LGL in the postpartum period may be related to the induction of postpartum destructive thyrotoxicosis and/or hypothyroidism in AITD.     

Diaminopropionate ammonia-lyase from Salmonella typhimurium. Purification and characterization of the crystalline enzyme, and sequence determination of the pyridoxal 5'-phosphate binding peptide

We have found a wide occurrence of alpha,beta-diaminopropionate ammonia-lyase in bacteria and actinomycetes. Considerable amounts of this enzyme were found in Salmonella typhimurium. The enzyme was purified and crystallized from S. typhimurium (IFO 12529). The relative molecular mass of the native enzyme, estimated by the ultracentrifugal equilibrium method, is 89,000 Da, and the enzyme consists of two subunits identical in molecular mass. The enzyme exhibits absorption maxima at 278 and 413 nm and contains 2 mol of pyridoxal 5'-phosphate(pyridoxal-P)/mol of enzyme. The enzyme catalyzes the alpha,beta-elimination reaction of both L- and D-alpha,beta-diaminopropionate, the most suitable substrates, to form pyruvate and ammonia. The L- and D-isomers of serine were also degraded, though slowly. After the internal Schiff base with pyridoxal-P had been reduced with sodium borohydride, followed by trypsin or lysyl endopeptidase digestion of the enzyme, we determined the sequence of about 20 amino acid residues around the lysine residue which binds pyridoxal-P. No homology was found in either the amino acid sequence of the pyridoxal-P binding peptide or the amino-terminal amino acid sequence between the enzyme and other pyridoxal-P-dependent enzymes.     

Thermostable alanine racemase from Bacillus stearothermophilus: molecular cloning of the gene, enzyme purification, and characterization

The alanine racemase (EC 5.1.1.1) gene of a thermophilic bacterium, Bacillus stearothermophilus, was cloned and expressed in Escherichia coli C600 with vector plasmid pICR301, which was constructed from pBR322 and the L-alanine dehydrogenase gene derived from B. stearothermophilus. A coupled assay method with L-alanine dehydrogenase and tetrazolium salts was used to detect visually the alanine racemase activity in the clones. Alanine racemase overproduced in a clone carrying the plasmid pICR4, 12 kilobases of DNA, was purified from cell extracts about 340-fold to homogeneity by five steps including heat treatment. The overproduced enzyme was confirmed to originate from B. stearothermophilus by an immunochemical cross-reaction with the enzyme of B. stearothermophilus. The purified enzyme has a molecular weight of about 78 000 and consists of two identical subunits of Mr of 39 000. At the optimum temperature (50 degrees C), the enzyme has a specific activity of 1800 units/mg (Vmax, D- to L-alanine). Resolution and reconstitution experiments together with the absorption spectrum of the enzyme clearly indicate that alanine racemase of B. stearothermophilus is a pyridoxal 5'-phosphate enzyme.     

Morphology,physics, chemistry and biology of Lake Rara in West Nepal

A survey of oligotrophic Lake Rara, the biggest lake in Nepal, was carried out from 1982 till 1984. Mean depth is 100 m, and maximum depth is 167 m. The surface area covers 9.8 km², and the lake contains 0.98 km³ volume of water. Transparency was about 16 m, photoquantum yield decreased exponentially with depth below 5 m, and the extinction coefficient was 8.3 × 10⁻². The concentration of Chl.-a was in the range of 0.06–0.46 mg m⁻³, and total nitrogen was 18–30 μg 1⁻¹. The whole water column was well oxygenated. Primary productivity was extremely low. It has more than 30 inflowing brooks and one outlet. The water quality of the brooks changes drastically with their location. The pH, electrical conductivity, and EDTA hardness in the waters from a landslide area were high. In the waters from a rich pine forest they were extremely low. The zooplankton consisted of two species of protozoa, five species of rotifers, two species of Cladocera, and two species of Copepoda. The zooplankton density range was 6200–16200 individuals m⁻³. The minimum was on November 11th, 1983 and the maximum on August 19th, 1983.     

Isolation and characterization of cDNA for chicken muscle adenylate kinase

A cDNA clone for muscle adenylate kinase was isolated from a cDNA library of chick skeletal muscle poly(A)+ RNA, and the DNA sequence was determined. The cDNA insert had 854 nucleotides, which consisted of the 5'-untranslated sequence of 57 nucleotides, the sequence of 582 nucleotides coding for 194 amino acids, and the 3'-untranslated sequence of 163 nucleotides and the poly(A) tail of 52 nucleotides. The amino acid sequence predicted from the nucleotide sequence was highly homologous with the reported sequences of human, calf, porcine, and rabbit muscle adenylate kinases. RNA blot analysis of poly(A)+ RNA from various chicken tissues revealed a single species of mRNA of approximately 850 nucleotides and its tissue-specific distribution. The induction of muscle adenylate kinase mRNA synthesis during the chick embryogenesis was also demonstrated by the blot analysis. Southern blot analysis indicated a single gene for muscle adenylate kinase in the chicken genome.