Gamma-Glutamylpolyamine Synthetase GlnA3 Is Involved in the First Step of Polyamine Degradation Pathway in Streptomyces coelicolor M145.

Streptomyces coelicolor M145 was shown to be able to grow in the presence of high concentrations of polyamines, such as putrescine, cadaverine, spermidine, or spermine, as a sole nitrogen source. However, hardly anything is known about polyamine utilization and its regulation in streptomycetes. In this study, we demonstrated that only one of the three proteins annotated as glutamine synthetase-like protein, GlnA3 (SCO6962), was involved in the catabolism of polyamines. Transcriptional analysis revealed that the expression of glnA3 was strongly induced by exogenous polyamines and repressed in the presence of ammonium. The ΔglnA3 mutant was shown to be unable to grow on defined Evans agar supplemented with putrescine, cadaverine, spermidine, and spermine as sole nitrogen source. HPLC analysis demonstrated that the ΔglnA3 mutant accumulated polyamines intracellularly, but was unable to degrade them. In a rich complex medium supplemented with a mixture of the four different polyamines, the ΔglnA3 mutant grew poorly showing abnormal mycelium morphology and decreased life span in comparison to the parental strain. These observations indicated that the accumulation of polyamines was toxic for the cell. An in silico analysis of the GlnA3 protein model suggested that it might act as a gamma-glutamylpolyamine synthetase catalyzing the first step of polyamine degradation. GlnA3-catalyzed glutamylation of putrescine was confirmed in an enzymatic in vitro assay and the GlnA3 reaction product, gamma-glutamylputrescine, was detected by HPLC/ESI-MS. In this work, the first step of polyamine utilization in S. coelicolor has been elucidated and the putative polyamine utilization pathway has been deduced based on the sequence similarity and transcriptional analysis of homologous genes expressed in the presence of polyamines.

Regulation of aerobic and anaerobic D-malate metabolism of Escherichia coli by the LysR-type regulator DmlR (YeaT).

Escherichia coli K-12 is able to grow under aerobic conditions on D-malate using DctA for D-malate uptake and the D-malate dehydrogenase DmlA (formerly YeaU) for converting D-malate to pyruvate. Induction of dmlA encoding DmlA required an intact dmlR (formerly yeaT) gene, which encodes DmlR, a LysR-type transcriptional regulator. Induction of dmlA by DmlR required the presence of D-malate or L- or meso-tartrate, but only D-malate supported aerobic growth. The regulator of general C(4)-dicarboxylate metabolism (DcuS-DcuR two-component system) had some effect on dmlA expression. The anaerobic L-tartrate regulator TtdR or the oxygen sensors ArcB-ArcA and FNR did not have a major effect on dmlA expression. DmlR has a high level of sequence identity (49%) with TtdR, the L- and meso-tartrate-specific regulator of L-tartrate fermentation in E. coli. dmlA was also expressed at high levels under anaerobic conditions, and the bacteria had D-malate dehydrogenase activity. These bacteria, however, were not able to grow on D-malate since the anaerobic pathway for D-malate degradation has a predicted yield of < or = 0 ATP/mol D-malate. Slow anaerobic growth on D-malate was observed when glycerol was also provided as an electron donor, and D-malate was used in fumarate respiration. The expression of dmlR is subject to negative autoregulation. The network for regulation and coordination of the central and peripheral pathways for C(4)-dicarboxylate metabolism by the regulators DcuS-DcuR, DmlR, and TtdR is discussed.

Regulation of tartrate metabolism by TtdR and relation to the DcuS-DcuR-regulated C4-dicarboxylate metabolism of Escherichia coli.

Escherichia coli catabolizes L-tartrate under anaerobic conditions to oxaloacetate by the use of L-tartrate/succinate antiporter TtdT and L-tartrate dehydratase TtdAB. Subsequently, L-malate is channelled into fumarate respiration and degraded to succinate by the use of fumarase FumB and fumarate reductase FrdABCD. The genes encoding the latter pathway (dcuB, fumB and frdABCD) are transcriptionally activated by the DcuS-DcuR two-component system. Expression of the L-tartrate-specific ttdABT operon encoding TtdAB and TtdT was stimulated by the LysR-type gene regulator TtdR in the presence of L- and meso-tartrate, and repressed by O(2) and nitrate. Anaerobic expression required a functional fnr gene, and nitrate repression depended on NarL and NarP. Expression of ttdR, encoding TtdR, was repressed by O(2), nitrate and glucose, and positively regulated by TtdR and DcuS. Purified TtdR specifically bound to the ttdR-ttdA promoter region. TtdR was also required for full expression of the DcuS-DcuR-dependent dcuB gene in the presence of tartrate. Overall, expression of the ttdABT genes is subject to L-/meso-tartrate-dependent induction, and to aerobic and nitrate repression. The control is exerted directly at ttdA and in addition indirectly by regulating TtdR levels. TtdR recognizes a subgroup (L- and meso-tartrate) of the stimuli perceived by the sensor DcuS, which responds to all C(4)-dicarboxylates; both systems apparently communicate by mutual regulation of the regulatory genes.