Regulation of the ammonia assimilatory enzymes in Salmonella typhimurium.

The regulation of glutamate dehydrogenase (EC 1.4.1.4), glutamine synthetase (EC 6.3.1.2), and glutamate synthase (EC 2.6.1.53) was examined for cultures of Salmonella typhimurium grown with various nitrogen and amino acid sources. In contrast to the regulatory pattern observed in Klebsiella aerogenes, the glutamate dehydrogenase levels of S. typhimurium do not decrease when glutamine synthetase is derepressed during growth with limiting ammonia. Thus, it appears that the S. typhimurium glutamine synthetase does not regulate the synthesis of glutamate dehydrogenase as reported for K. aerogenes. The glutamate dehydrogenase activity does increase, however, during growth of a glutamate auxotroph with glutamate as a limiting amino acid source. The regulation of glutamate synthase levels is complex with the enzyme activity decreasing during growth with glutamate as a nitrogen source, and during growth of auxotrophs with either glutamine or glutamate as limiting amino acids.     

Regulation of urease and ammonia assimilatory enzymes in Selenomonas ruminantium.

Urease and glutamine synthetase activities in Selenomonas ruminantium strain D were highest in cells grown in ammonia-limited, linear-growth cultures or when certain compounds other than ammonia served as the nitrogen source and limited the growth rate in batch cultures. Glutamate dehydrogenase activity was highest during glucose (energy)-limited growth or when ammonia was not growth limiting. A positive correlation (R = 0.96) between glutamine synthetase and urease activities was observed for a variety of growth conditions, and both enzyme activities were simultaneously repressed when excess ammonia was added to ammonia-limited, linear-growth cultures. The glutamate analog methionine sulfoximine (MSX), inhibited glutamine synthetase activity in vitro, but glutamate dehydrogenase, glutamate synthase, and urease activities were not affected. The addition of MSX (0.1 to 100 mM) to cultures growing with 20 mM ammonia resulted in growth rate inhibition that was dependent upon the concentration of MSX and was overcome by glutamine addition. Urease activity in MSX-inhibited cultures was increased significantly, suggesting that ammonia was not the direct repressor of urease activity. In ammonia-limited, linear-growth cultures, MSX addition resulted in growth inhibition, a decrease in GS activity, and an increase in urease activity. These results are discussed with respect to the importance of glutamine synthetase and glutamate dehydrogenase for ammonia assimilation under different growth conditions and the relationship of these enzymes to urease.     

Oxygen regulation in Salmonella typhimurium

Regulation by oxygen of the peptidase T (pepT) locus of Salmonella typhimurium was studied by measuring beta-galactosidase levels in strains containing a pepT::Mu d1(Apr lac) operon fusion. beta-Galactosidase was induced in anaerobic cultures and late-exponential and stationary-phase aerated cultures. Peptidase T activity also was induced under these growth conditions. pepT+ but not pepT strains will utilize as amino acid sources the tripeptides Leu-Leu-Leu and Leu-Gly-Gly only when grown anaerobically. Mutations at two loci, oxrA and oxrB (oxygen regulation) prevent induction of the pepT locus. The oxrA locus is homologous to the fnr locus of Escherichia coli. We have isolated 12 independent Mu d1 insertions (oxd::Mu d1, oxygen dependent) that show induction of beta-galactosidase in anaerobic cultures and stationary-phase aerated cultures. These insertions fall into nine classes based on map location. All of the oxd::Mu d1 insertions are regulated by oxrA and oxrB and therefore define a global regulon that responds to oxygen limitation.     

Molecular studies of an fi+ plasmid from strains of Salmonella typhimurium

Summary Plasmid DNA has been isolated from five fi ⁺ strains of Salmonella typhimurium of independent origin, including type 36 and LT2. The mean contour length of the plasmids was between 27.3 and 29.3 m. A variant line of S. typhimurium type 36 which was fi ^- yeilded no plasmid DNA. These results support the hypothesis that the fi ⁺ property of S. typhimurium is coded by a plasmid. In S. typhimurium 36 this plasmid, designated MP10₃₆, also appears to code for restriction of non-donor-specific phages. Molecular studies indicate that superinfection of S. typhimurium 36 with the kanamycin resistance determinant K, which results in loss of the fi ⁺ property, is correlated with loss of MP10₃₆. Reassociation experiments demonstrate a high degree of homology between the DNA of all five S. typhimurium plasmids, and between MP10₃₆ and K. MP10₃₆ has some homology with F and F-like R factors, but not with plasmids of other compatibility groups. A recombinant between an ampicillin resistance determinant and MP10₃₆ is autotransferable at low frequency. The significance of these findings is discussed.     

The effect of various culture conditions on the levels of ammonia assimilatory enzymes of Corynebacterium callunae

Corynebacterium callunae (NCIB 10338) grows faster on glutamate than ammonia when used as sole nitrogen sources. The levels of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (GOGAT; EC 1.4.1.13) of C. callunae were found to be influenced by the nitrogen source. Accordingly, the levels of GS and GOGAT activities were decreased markedly under conditions of ammonia excess and increased under low nitrogen conditions. In contrast, glutamate dehydrogenase (GDH; EC 1.4.1.4) activities were not significantly affected by the type or the concentration of the nitrogen source supplied. The carbon source in the growth medium could also affect GDH, GS and GOGAT levels. Of the carbon sources tested in the presence of 2 mM or 10 mM ammonium chloride as the nitrogen source pyruvate, acetate, fumarate and malate caused a decrease in the levels of all three enzymes as compared with glucose. GDH, GS and GOGAT levels were slightly influenced by aeration. Also, the enzyme levels varied with the growth phase. Methionine sulfoximine, an analogue of glutamine, markedly inhibited both the growth of C. callunae cells and the transferase activity of GS. The apparent K _m values of GDH for ammonia and glutamate were 17.2 mM and 69.1 mM, respectively. In the NADPH-dependent reaction of GOGAT, the apparent K _m values were 0.1 mM for -ketoglutarate and 0.22 mM for glutamine.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase     

Complex typing of Salmonella typhimurium hospital strains

A salmonellosis outbreak, caused by S. typhimurium, was investigated with the use of some microbiological and molecular-biological methods of typing. This investigation revealed that the outbreak was caused by the "outbreak" electrotype of the multi-resistant variant of the infective agent, found to have several plasmidovars. The possibilities and limitations of typing by sensitivity to antibiotics and plasmid DNA profile were shown. These methods of intraspecific typing were regarded as methods making it possible to establish the heterogeneity of S. typhimurium with the use of intraclonal markers.     

Global regulation by CsrA in Salmonella typhimurium

CsrA is a regulator of invasion genes in Salmonella enterica serovar Typhimurium. To investigate the wider role of CsrA in gene regulation, we compared the expression of Salmonella genes in a csrA mutant with those in the wild type using a DNA microarray. As expected, we found that expression of Salmonella pathogenicity island 1 (SPI-1) invasion genes was greatly reduced in the csrA mutant, as were genes outside the island that encode proteins translocated into eukaryotic cells by the SPI-1 type III secretion apparatus. The flagellar synthesis operons, flg and fli, were also poorly expressed, and the csrA mutant was aflagellate and non-motile. The genes of two metabolic pathways likely to be used by Salmonella in the intestinal milieu also showed reduced expression: the pdu operon for utilization of 1,2-propanediol and the eut operon for ethanolamine catabolism. Reduced expression of reporter fusions in these two operons confirmed the microarray data. Moreover, csrA was found to regulate co-ordinately the cob operon for synthesis of vitamin B12, required for the metabolism of either 1,2-propanediol or ethanolamine. Additionally, the csrA mutant poorly expressed the genes of the mal operon, required for transport and use of maltose and maltodextrins, and had reduced amounts of maltoporin, normally a dominant protein of the outer membrane. These results show that csrA controls a number of gene classes in addition to those required for invasion, some of them unique to Salmonella, and suggests a co-ordinated bacterial response to conditions that exist at the site of bacterial invasion, the intestinal tract of a host animal.     

The regulation of ammonia assimilating enzymes in Lemma minor

Summary Lemna minor has the potential to assimilate ammonia via either the glutamine or glutamate pathways. A 3-4 fold variation in the level of ferredoxindependent glutamate synthase may occur, when plants are grown on different nitrogen sources, but these changes show no simple relationship to changes in the endogenous pool of glutamate. High activities of glutamate synthase and glutamine synthetase at low ammonia availability suggests that these two enzymes function in the assimilation of low ammonia concentrations. Increasing ammonia availability leads to a reduction in level of glutamate synthase and glutamine synthetase and an increase in the level of glutamate dehydrogenase. Glutamine synthetase and glutamate dehydrogenase are subject to concurrent regulation, with glutamine rather than ammonia, exerting negative control on glutamine synthetase and positive control on glutamate dehydrogenase. The changes in the ratio of these two enzymes in response to the internal pool of glutamine could regulate the direction of the flow of ammonia into amino acids via the two alternative routes of assimilation.Abbreviations GS Glutamine synthetase - GDH Glutamate dehydrogenase - GOGAT Glutamate synthase     

Relationship of ammonia excretion,dinitrogen fixation and ammonia assimilatory enzymes to encystation in a continuous culture ofAzospirillum brasilense

Continuous culture studies have been carried out on a strain ofA. brasilense to study ammonia excretion, dinitrogen fixation and ammonia assimilatory enzymes (glutamate-ammonia ligase and glutamate synthase (NADPH)) in encysted conditions. High glutamate synthase (NADPH) and low ammonia excretion was observed at the time of induction of cyst formation. Low and oscillating nitrogenase activity was observed throughout the experiment.