Fifteen mutants of Sinorhizobium (Rhizobium ) meliloti unable to utilize acetate as a sale carbon source (Ace-) were characterized in this study. Merodiploid complementation tests showed that nine of these mutations were in loci distinct from previously described gluconeogenic loci. The chromosomal locations of the mutations were determined, and complementing clones were isolated from the cosmid library of S. meliloti genomic DNA. The mutants were placed into four groups (I--IV) based on genetic linkage in phage co-transduction. None of the mutations were in glyoxylate shunt enzyme-encoding genes. Nucleotide sequence analysis of ace mutants from Groups III and IV showed mutations in genes encoding acetyl-CoA synthetase ( acsB) and anaerobic coproporphyrinogen III oxidase (hemN ) respectively. Cell extracts of the hemN mutant exhibited double the isocitrate lyase levels of the wild type. The acsB mutant lacked acetyl-CoA synthetase activity and had an interesting growth phenotype; it was able to grow on low concentrations of acetate only. (Abstract shortened by UMI.)

This research followed acetate carbon (C) uptake, metabolism, and fate through a typical modern Kraft pulp and paper mill AS system. Freshly collected mill biomass (AS floc suspensions) was placed under conditions representing four key phases of AS biotreatment: (1) initial acetate uptake by aerated starved AS; (2) ongoing acetate uptake; (3) aerobic metabolism of acetate-loaded AS in acetate-stripped effluent; and (4) anaerobic, settled biomass metabolism. Conditions mimicked the mill system as closely as possible. Acetate carbon uptake kinetics and conversion to CO2, growth products, PHB, and secreted metabolites in each of the four phases were measured. The role of PHB synthesis in the initial stripping of acetate from mill effluent and the PHB production potential of this mill AS were also investigated. / Results showed that acetate was rapidly taken up by high-affinity systems in the AS. During the initial exposure of mill-starved AS, acetate greatly stimulated AS-O2 uptake, and was quickly converted to PHB and CO 2. Upon depletion of available effluent acetate, as occurs in the downstream sections of the aeration tank, O2-uptake rates decreased and the acetate-C stored in AS-PHB was slowly released as CO2, and partly used for growth. Under secondary clarifier-like anaerobic conditions, the AS released virtually no CO2. However, substantial amounts of PHB were used for growth under anaerobic conditions and a small proportion of the original acetate C exited the cells as organic acids.

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Many bacteria accumulate carbon stores as poly-3-hydroxybutyrate (PHB) when growth is limited but carbon availability is not. This stored carbon can then be utilized during conditions of limited carbon availability. The net PHB accumulation in the cell is dependent on the balance between PHB synthesis and degradation. Sinorhizobium meliloti accumulates PHB in the free-living stage but not in the symbiotic stage. The physiological role of the PHB cycle in S. meliloti is unknown. As a first step to understand the genetics of PHB degradation, transposon-generated mutants that were not able to use PHB degradation intermediates, such as 3-hydroxybutyrate and acetoacetate, as a sole carbon source, were isolated. Genetic mapping revealed that there were at least three chromosomal loci involved in acetoacetate metabolism. Identification of these three loci determined that in S. meliloti: (1) acetoacetyl-CoA synthetase (AcsA), encoded by acsA2 gene, rather than the enzyme acetoacetate:succinyl-CoA transferase, is the enzyme that catalyzes activation of acetoacetate to acetoacetyl-CoA; (2) PHB synthase, encoded by phbC, is required for acetoacetate utilization; (3) a putative transporter protein encoding gene, aau-3, may also be involved in acetoacetate metabolism. acsA2 and aau-3 were 78% linked in co-transduction, while phbC was mapped to somewhere else on the chromosome. Biochemical analysis revealed that acsA2::Tn5 mutants lacked AcsA activity but not acetoacetate:succinyl-CoA transferase activity, while phbC::Tn5 maintained similar level of AcsA activity as wild type in vitro. PHB was absent in the phbC mutant. / One transposon-generated mutant, age-1, showed enhanced growth rate on acetoacetate medium. Genetic mapping and transductional analysis indicated that the location of the mutation in age-1 is tightly linked to acsA2. Fine mapping with PCR and DNA sequence techniques showed that Tn5 in age-1 was located at 132 by upstream of the putative translation start site of acsA2. Gene expression analysis indicated that age-1 insertion results in elevated transcription of acsA2. Thus enhanced growth rate on acetoacetate was due to the increased gene expression. acsA2 transcription was induced by acetoacetate and 3-hydroxybutyrate, and repressed by glucose and acetate. / All mutants formed root nodules that fixed nitrogen with varying decrease of impairment. Acetoacetate metabolism and the PHB degradation are not essential for symbiosis.