Background: Members of the genus Rhodococcus are frequently found in soil and other natural environments and are highly resistant to stresses common in those environments. The accumulation of storage compounds permits cells to survive and metabolically adapt during fluctuating environmental conditions. The purpose of this study was to perform a genome-wide bioinformatic analysis of key genes encoding metabolism of diverse storage compounds by Rhodococcus jostii RHA1 and to examine its ability to synthesize and accumulate triacylglycerols (TAG), wax esters, polyhydroxyalkanoates (PHA), glycogen and polyphosphate (PolyP). Results: We identified in the RHA1 genome: 14 genes encoding putative wax ester synthase/acyl-CoA: diacylglycerol acyltransferase enzymes (WS/DGATs) likely involved in TAG and wax esters biosynthesis; a total of 54 genes coding for putative lipase/esterase enzymes possibly involved in TAG and wax ester degradation; 3 sets of genes encoding PHA synthases and PHA depolymerases; 6 genes encoding key enzymes for glycogen metabolism, one gene coding for a putative polyphosphate kinase and 3 putative exopolyphosphatase genes. Where possible, key amino acid residues in the above proteins (generally in active sites, effectors binding sites or substrate binding sites) were identified in order to support gene identification. RHA1 cells grown under N-limiting conditions, accumulated TAG as the main storage compounds plus wax esters, PHA (with 3-hydroxybutyrate and 3-hydroxyvalerate monomers), glycogen and PolyP. Rhodococcus members were previously known to accumulate TAG, wax esters, PHAs and polyP, but this is the first report of glycogen accumulation in this genus.
The two-component signal transduction system (TCS) BarA/UvrY activates transcription of CsrB and CsrC noncoding RNAs, which act by sequestering the RNA-binding global regulatory protein CsrA. Here, we show that the metabolic end products formate and acetate provide a physiological stimulus for this TCS and thus link posttranscriptional regulation by the Csr system to the metabolic state of the cell.
The oleaginous bacterium Rhodococus opacus strain PD630 serves as a model organism to investigate the metabolism of storage triacylglycerols (TAGs) in bacteria. The key enzyme catalysing the last step of TAG biosynthesis in bacteria is a promiscuous acyltransferase (Atf), exhibiting acyl-CoA acyltransferase activity to both diacylglycerols (DGAT activity) and fatty alcohols (wax ester synthase, WS activity). An 800 bp PCR product was obtained from chromosomal DNA of strain PD630 by using degenerate primers designed from conserved stretches of Atf proteins of Acinetobacter baylyi strain ADP1 and Mycobacterium smegmatis mc 2 155. The atf fragment was used as a probe on a strain PD630 gene library, resulting in the identification of a 3948 bp chromosomal DNA fragment containing the complete atf1 gene. An atf1 disruption mutant of strain PD630 exhibited a TAG-leaky phenotype and accumulated up to 50 % less fatty acids than the wild-type, with significantly reduced oleic acid content when cultivated in the presence of gluconate or oleic acid. Whereas DGAT activity was drastically reduced in comparison to the wild-type, WS activity remained almost unchanged in the mutant.RT-PCR analysis of gluconate-grown cells of strain PD630 showed that there is expression of atf1 under conditions of TAG synthesis. To identify additional Atfs in strain PD630, PCR employing non-degenerate primers deduced from Rhodococcus jostii RHA1 sequence data was used. This yielded nine additional atf-homologous genes exhibiting 88-99 % sequence identity to the corresponding strain RHA1 enzymes. Besides Atf1 only Atf2 exhibited high DGAT and/or WS activity when heterologously expressed in Escherichia coli.
The hybrid sensor kinase BarA and its cognate response regulator UvrY, members of the two-component signal transduction family, activate transcription of CsrB and CsrC noncoding RNAs. These two small RNAs act by sequestering the RNA binding protein CsrA, which posttranscriptionally regulates translation and/or stability of its target mRNAs. Here, we provide evidence that CsrA positively affects, although indirectly, uvrY expression, at both the transcriptional and translational levels. We also demonstrate that CsrA is required for properly switching BarA from its phosphatase to its kinase activity. Thus, the existence of a feedback loop mechanism that involves the Csr and BarA/UvrY global regulatory systems is exposed. The BarA/UvrY two-component signal transduction system (TCS) of Escherichia coli consists of the membrane-bound sensor kinase BarA and its cognate response regulator UvrY (1). BarA, which belongs to the subfamily of tripartite sensor kinases (2, 3), senses and responds to the presence of formate and acetate but also to that of other short-chain fatty acids (4). Phosphorylated BarA catalyzes the transphosphorylation of UvrY (1), a typical response regulator of the FixJ family (1, 5), which in turn activates expression of the noncoding RNAs of the carbon storage regulation (Csr) system, CsrB and CsrC. These small regulatory RNAs possess repeated sequence elements that allow them to interact with multiple copies of the RNA binding protein CsrA and thereby prevent its regulatory interaction with its mRNA targets (6).CsrA is a small, dimeric RNA binding protein that coordinates gene expression by positively or negatively regulating the translation, stability, and/or elongation of its target transcripts (7,8). CsrA directly interacts with the 5= untranslated leaders of target mRNAs at sites characterized by a GGA sequence that is often located within the loop of a short stem-loop structure (9-11). In this way, CsrA activates exponential-phase processes while it represses several stationary-phase functions (12). CsrA is widely distributed among eubacteria (13) and regulates expression of genes for virulence factors (14, 15), quorum sensing (16, 17), motility (18,19), carbon metabolism (20, 21), biofilm formation (22, 23), cyclic di-GMP synthesis (24), and peptide uptake (10).Curiously, activation of csrB transcription, which depends directly on UvrY-P, does not take place in a csrA mutant strain (25). Therefore, it has been suggested that CsrA has a positive effect on the BarA/UvrY TCS. In this study, we confirmed and extended these results by examining the effects of CsrA on either the expression or the activity of BarA and UvrY. Our results demonstrate that CsrA, apparently indirectly, is required for proper uvrY expression and also for activation of the BarA kinase activity. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. The strains and plasmids used in this work are listed in csrB-lacZ) construction, the barA gene was deleted by homologous recombination using the lambda Red ...
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