Browsing by Author "Reid, Shez"

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    Regulation and characterization of the nitrogen assimilatory gene cluster in Clostridium acetobutylicum P262
    (2000) Stutz, Helen; Reid, Shez
    The solventogenic Clostridium acetobutylicum strain P262 was used for the commercial production of solvents in South Africa from 1945 to 1983 (Jones and Woods, 1986a). Our laboratory has focused on understanding two: fundamental aspects of its physiology, namely nitrogen metabolism and electron transport pathways. The long-term goal is the potential genetic modification of nitrogen utilization and/or electron distribution in the cell, to manipulate fermentation patterns for improved growth rate and solvent yields. The main aims of this research project were to: extend the sequence analysis of the glnA locus encoding the glutamine synthetase (GS) enzyme (Janssen et al., 1990; Fierro-Monti et al., 1992); to identify and characterize the locus encoding the structural genes for the glutamate synthase (GOGAT) enzyme; and to detennine the mechanisms and growth conditions which regulate the activity of these two key enzymes of nitrogen assimilation. In addition, we were interested in characterizing clone pMET13Cl, which was isolated by a selection system developed to clone genes involved in electron transport from C. acetobutylicum (Santangelo et al., 1991). Sequence analyses revealed that the region downstream of glnA and the putative regulatory gene, glnR (Reid and Woods, 1995), encoded the structural genes for the large (a) and small (J) subunits of GOGAT, respectively. This is the first report in which the genes encoding GS and GOGAT are genetically linked. The a subunit was designated gltA, and the downstream f3 subllll.it was designated gltB. All the likely cofactor and substrate binding sites identified in GOGAT enzymes (Vanoni and Curti, 1999) were conserved in the deduced gltA and gltB polypeptides. The identity of the gltA and gltB genes were functionally confirmed by complementation studies involving their co-expression from separate constructs in an E. coli glutamate auxotroph (strain: Mx:3004), which restored the ability of this mutant to grow with ammonia as the sole source of nitrogen. Physiological studies on the germination, growth and differentiation patterns of C. acetobutylicum P262 were assessed in relation to different nitrogen conditions. Significantly, organic nitrogen (casamino acids) was the preferred source of nitrogen, and not ammonia as ll previously asswned. These studies led to the development of nitrogen limiting conditions (0.025% casamino acids + 0.15% glutamine) and nitrogen rich conditions (0.2% casamino acids) used for regulatory studies. GOG AT activity was optimised. It appeared to be sensitive to oxygen and specific for the co-enzyme NADH. Both GS and GOGAT activities were regulated by the nitrogen source in a similar way: induced in the nitrogen limiting conditions and repressed in the nitrogen rich conditions. Northern blot analyses, in conjunction with the enzyme activity profiles and feedback inhibition studies, indicated that GS and GOGAT activities were regulated primarily at the level of transcription. Furthermore, glnA. andglnR, and gltA and gltB are each transcribed as an operon under nitrogen limiting conditions. No assimilatory GDH activity could be detected. The implications of these results, as well as sequence features identified, are discussed in context with a proposed model for the regulation of GS and GOGAT activity in C. acetobutylicum P262. Analysis of plasmid pMET13Cl identified a gene whose predicted -46 kDa product was associated with an electron transport function. The deduced amino acid sequence was not typical of electron transport proteins, but rather shared striking homology to bacterial GOGAT p subunit polypeptides. This p subunit-like gene was thus designated gltX. We were, however, unable to relate it to GOGAT activity or nitrogen metabolism. Rather, it appears to belong to a novel family of FAD-dependent NAD(P)H oxidoreductases suggested by Vanoni and Curti (1999) and supported by an analysis of the evolutionary relationships of the GOGAT subunits/domains from various sources.
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    Studies on Clostridium acetobutylicum P262 : sporulation induction and analysis of an oligosporogenous, solvent-deficient mutant
    (1994) Babb, Brendan Lloyd; Reid, Shez; Woods, Dave
    The ability of various fermentation products to induce sporulation was in order to design a sporulation induction medium for Clostridium acetobutylicum P262. Of acetic acid, butyric acetone and butanol, was found to be most effective at induction. Induction was more efficient at low pH values under certain conditions. The heat resistance of mature spores was determined and the optlmal temperature for spore quantification was shown to be 75·C. acetobutylicum mutants m5 06 were by transposon mutagenesis using the conjugative transposon Tn925::Tn917, not transposon Tn925 as previously thought [Babb. B.L. 1990. B.Sc. (Honours) thesis, University of Cape Town. South Africa]. The spore development and the fermentation profiles of mutants were in batch over a period of 60h. Mutant m5 was shown to be oligosporogenous with majority of cells blocked at sporulation stage H. It was deficient in acetone and butanol production. Mutant 06 proceeded to sporulation stage VII and produced acetone and butanol at levels similar to that of the wild type strain. Mutants 06 appeared to contain two respectively from Southern hybridization experiments using a probe for left transposon junction. However, when a probe to right transposon junction was used, the chromosomal deoxyribonucleic acid (DNA) of mutant m5 was shown to contain approximately eight junction sites. The cause for the anomalous hybridization pattern was non-specific restriction enzyme activity nor a result of independent transposition of transposon Tn917.
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    Studies on the regulation of nitrogen metabolism in clostridium acetobutylicum NCP 262 and clostridium beijerinckii NCIMB 8052
    (1999) Quixley, Keith Windsor McRostie; Reid, Shez
    In order to produce Clostridium acetobutylicum strains with improved solventogenic capabilities through genetic manipulation, a thorough understanding of the mechanisms governing solvent production is required. Nitrogen metabolism has been shown to be important in the regulation of sporulation and solventogenesis, and the enzyme glutamine synthetase, encoded by the gene ginA, is central to this aspect of metabolism. This work represents a continuation of studies on the glutamine synthetase enzyme of C. acetobutylicum NCP 262. Previous work identified a gene, glnR, which is thought to encode an anti terminator protein involved in the regulation of ginA transcription. An attempt was made to demonstrate a regulatory role for glnR in a system using the cloned C. acetobutylicum NCP 262 ginA with and without glnR in the E. coli glnAntrBC mutant YMCll. Although the enzyme was efficiently expressed and fully functional, no evidence could be found of any influence of glnR on GS activity in the heterologous host. In an attempt to locate other genes involved in ginA regulation the region upstream of ginA in C. acetobutylicum NCP 262 was sequenced. Although no aceesory regulatory genes were found, an incomplete open reading frame encoding a putative aspartokinase or bifunctional aspartokinase/homoserine dehydrogenase was identified. As attempts to reconstitute the C. acetobutylicum NCP 262 ginA regulatory system in E. coli were unsuccessful, it was necessary to find an alternative to heterologous host. C. beijerinckii NCIMB 8052, which is closely related to C. acetobutylicum NCP 262, was evaluated as a model organism for the study of nitrogen metabolism in the latter. It was found that organic nitrogen in the form of cas amino acids was preferred over ammonium, and that GS activity was induced under conditions of nitrogen limitation, and repressed in cultures grOWL in the presence of high concentration of cas amino acids. Southern hybridisation experiments identified homologues of ginA, glnR and gltA in C. beijerinckii NCIMB 8052, and a clone was isolated from a partial gene library of this organism which complemented glutamine auxotrophy in E. coli YMCll. The clone was sequenced and found to carry two complete and one incomplete ORF's which shared a high degree of nucleotide sequence similarity with ginA, ginR and gltA genes of C. acetobutylicum NCP 262 (87.8%, 86.5.% and 86.8% respectively). In addition, the relative arrangement of the genes was similar. Primer extension experiments identified four transcriptional start sites, two of which corresponded approximately to those previously identified in C. acetobutylicum NCP 262. Transcription from all four was found to increase under conditions of nitrogen limitation. Leaders of three of the transcripts were found to be capable of forming identical terminator structures upstream of the start codon of ginA, however potential antiterminator structures differed for each transcript. A reporter system was developed for use in C. beijerinckii NCIMB 8052, based on the eglA gene cloned from C. acetobutylicum NCP 262, cloned into the Bacillus/Clostridium shuttle vector pFNKl. When eglA was under the control of its own promoter, the endoglucanase activity was expressed and efficiently secreted into the culture medium by C. beijerinckii NCIMB 8052. The pH and temperature optima of the enzyme activity were found to be between 5.0 and 6.0 and 45°C respectively. These parameters differed from those previously reported for an endoglucanase activity in C. acetobutylicum NCP 262. Expression of eglA appeared to be repressed by cellobiose and fructose, but not by glucose, sucrose or galactose. A promoterless eglA was placed under the control of ginA and scr promoters. Very little eglA expression was observed for the scr promoter-eglA in any of the above-mentioned carbon sources, indicating either a disruption of normal regulation in the cell, or the absence of essential regions on the cloned promoter fragment. The ginA promotereglA fusion produced large amounts of endoglucanase activity under conditions of nitrogen excess and limitation, indicating the absence of a negative regulatory factor. GS activity in the same cultures was regulated normally, indicating that the loss of regulation was most likely to be due to the absence of an essential factor on the fusion construct. It was shown, nevertheless, that efficient expression of the reporter could be obtained under the control of a heterologous promoter. Potential problems with the reporter system were discussed and suggestions were made for improvement ofthe system. v Transcription from all four was found to increase under conditions of nitrogen limitation. Leaders of three of the transcripts were found to be capable of forming identical terminator structures upstream of the start codon of ginA, however potential antiterminator structures differed for each transcript. A reporter system was developed for use in C. beijerinckii NCIMB 8052, based on the eglA gene cloned from C. acetobutylicum NCP 262, cloned into the Bacillus/Clostridium shuttle vector pFNKl. When eglA was under the control of its own promoter, the endoglucanase activity was expressed and efficiently secreted into the culture medium by C. beijerinckii NCIMB 8052. The pH and temperature optima of the enzyme activity were found to be between 5.0 and 6.0 and 45°C respectively. These parameters differed from those previously reported for an endoglucanase activity in C. acetobutylicum NCP 262. Expression of eglA appeared to be repressed by cellobiose and fructose, but not by glucose, sucrose or galactose. A promoterless eglA was placed under the control of ginA and scr promoters. Very little eglA expression was observed for the scr promoter-eglA in any of the above-mentioned carbon sources, indicating either a disruption of normal regulation in the cell, or the absence of essential regions on the cloned promoter fragment. The glnA promotereglA fusion produced large amounts of endoglucanase activity under conditions of nitrogen excess and limitation, indicating the absence of a negative regulatory factor. GS activity in the same cultures was regulated normally, indicating that the loss of regulation was most likely to be due to the absence of an essential factor on the fusion construct. It was shown, nevertheless, that efficient expression of the reporter could be obtained under the control of a heterologous promoter. Potential problems with the reporter system were discussed and suggestions were made for improvement of the system.