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Researchers report the first known identification of nitrogen-fixing bacteria in pulp and paper mill primary clarifiers. About half of the culturable cells contained nitrogenase and 90% of these cells were identified as Klebsiella strains.

Results reveal that mutations affecting the degradation pathway of poly-3-hydroxybutyrate occur on the chromosome and the megaplasmid of the bacterium. Data on the chromosomal loci are presented.

Mutations that mediated the ability of Pck-deficient Rhizobium meliloti mutants to utilize succinate and tricarboxylic acid (TCA) intermediates were analyzed to characterize the biosynthesis of phosphoenolpyruvate (PEP). The activity of pyruvate orthophosphate dikinase (PPDK) in Rhizobium (Sinorhizobium) meliloti was enhanced by pckA suppressor mutations. Gluconeogenesis from malate in the pckA suppressor strain was also mediated by the combined activities of malic enzyme and PPDK.

Diphosphopyridine-nucleotide-dependent malic enzyme (DME) of Rhizobium meliloti is needed for symbiotic nitrogen fixation since it forms a pathway in which malate is converted to acetyl-CoA in bacteroids. This enzyme shows maximal activity with NAD+ as cofactor but has reduced activity when NADP+ is used. Acetyl-CoA strongly inhibited DME activity but not TME activity. The dme gene was expressed in free-living cells and in nitrogen-fixing bacteroids while tme gene was repressed in bacteroids.

The tme gene of Rhizobium meliloti which encodes the triphosphopyridine nucleotide (NADP+)-dependent malic enzyme was cloned and localized to a 3.7-kb region. It was discovered that (NADP+)-dependent malic enzyme activity was not needed for N2 fixation, as tme mutants induced N2-fixing root nodules on alfalfa. Also, the apparent (NADP+)-dependent malic enzyme activity observed in wild-type activity appeared to be caused by the utilization of NADP+ by (NAD+)-dependent malic enzyme.

Tn5 insertion and subcloning of the phosphoenolpyruvate carboxykinase (pckA) gene of Rhizobium meliloti helps map and analyze the DNA sequence and expression of this gene. A 536-amino-acid protein is encoded by pckA, which is expressed in enhanced levels in cells with succinate or arabinose as carbon sources. Primer extension analysis of this gene's promoter suggests that it belongs to the sigma70 class of promoters.

Research has been conducted on replicated river biofilms cultivated in rotating annular reactors. The effect of seasonal variations and nutrient inputs on biofilms' genetic compositions and physiological activities has been investigated via the use of physiological and molecular biology techniques.

DEAE-cellulose chromatography of extracts of free-living Rhizobium meliloti cells revealed separate NAD+-dependent and NADP+-dependent malic enzyme activities. The NAD+ malic enzyme exhibited more activity with NAD+ as cofactor, but also showed some activity with NADP+. The NADP+ malic enzyme only showed activity when NADP+ was supplied as cofactor. Three independent transposon-induced mutants of R. meliloti which lacked NAD+ malic enzyme activity (dme-) but retained NADP+ malic enzyme activity were isolated. In an otherwise wild-type background, the dine mutations did not alter the carbon utilization phenotype; however, nodules induced by these mutants failed to fix N2. Structurally, these nodules appeared to develop like wild-type nodules up to the stage where N2-fixation would normally begin. These results support the proposal that NAD+ malic enzyme, together with pyruvate dehydrogenase, functions in the generation of acetyl-CoA required for TCA cycle function in N2-fixing bacteroids which metabolize C4-dicarboxylic acids supplied by the plant. [ABSTRACT FROM AUTHOR]

The goal of this study was to initiate investigation of the genetics of bacterial poly-3-hydroxybutyrate (PHB) metabolism at the community level. We constructed metagenome libraries from activated sludge and soil microbial communities in the broad-host-range IncP cosmid pRK7813. Several unique clones were isolated from these libraries by functional heterologous complementation of a Sinorhizobium meliloti bdhA mutant, which is unable to grow on the PHB cycle intermediate D-3-hydroxybutyrate due to absence of the enzyme D-3-hydroxybutyrate dehydrogenase activity. Clones that conferred D-3-hydroxybutyrate utilization on Escherichia coli were also isolated. Although many of the S. meliloti bdhA mutant complementing clones restored D-3-hydroxybutyrate dehydrogenase activity to the mutant host, for some of the clones this activity was not detectable. This was also the case for almost all of the clones isolated in the E. coli selection. Further analysis was carried out on clones isolated in the S. meliloti complementation. Transposon mutagenesis to locate the complementing genes, followed by DNA sequence analysis of three of the genes, revealed coding sequences that were broadly divergent but lay within the diversity of known short-chain dehydrogenase/reductase encoding genes. In some cases, the amino acid sequence identity between pairs of deduced BdhA proteins was <35%, a level at which detection by nucleic acid hybridization based methods would probably not be successful. [ABSTRACT FROM AUTHOR]