The essential role of nitrogen limitation in expression of xplA and degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in Gordonia sp. strain KTR9.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a widely used explosive and a major soil and groundwater contaminant. Organisms such as Gordonia sp. KTR9, capable of degrading RDX and using it as an N source, may prove useful for bioremediation of contaminated sites. XplA is a cytochrome P450 monooxygenase responsible for RDX degradation. Expression of xplA in KTR9 was not induced by RDX but was strongly induced (50-fold) during N-limited growth. When glnR, encoding a regulatory protein affecting N assimilation in diverse Actinobacteria, was deleted from KTR9, the bacterium lost the ability to use nitrate, nitrite, and RDX as N sources. Deletion of glnR also abolished the inhibition of xplA expression by nitrite. Our results confirm the essential role of GlnR in regulating assimilation of nitrite, but there was no evidence for a direct role of GlnR in regulating XplA expression. Rather, the general availability of nitrogen repressed XplA expression. We conclude that the inability of the glnR mutant to use RDX as an N source was due to its inability to assimilate nitrite, an intermediate in the assimilation of nitrogen from RDX. Regulation of XplA does not seem adaptive for KTR9, but it is important for RDX bioremediation with KTR9 or similar bacteria.

A novel GlnR target gene, nnaR, is involved in nitrate/nitrite assimilation in Streptomyces coelicolor.

GlnR is the global transcriptional regulator of nitrogen assimilation in Streptomyces coelicolor. Under nitrogen starvation, GlnR controls the transcription of at least nine genes associated with nitrogen metabolism. In this study, we identified a new GlnR target gene, SCO2958, named nnaR (nitrate/nitrite assimilation regulator). In silico analysis of NnaR revealed the presence of two distinct domains: an N-terminal uroporphyrinogen-III synthase (HemD)-like enzymatic domain and a C-terminal DNA binding domain. Complementation experiments with a haemin auxotroph Escherichia coli ΔhemD mutant strain revealed that NnaR has no HemD activity. Physiological studies of an S. coelicolor nnaR : : Tn5062 mutant showed that NnaR is involved in regulating nitrite reduction. By electrophoretic mobility shift assays the functionality of the NnaR DNA binding domain was confirmed, and it was found that NnaR binds in front of the genes narK (putative nitrate extrusion protein), nirB (nitrite reductase), nirA (putative nitrite/sulphite reductase) and nasA (putative nitrate reductase), which are associated with nitrate/nitrite assimilation. Furthermore, a cooperative binding of NnaR together with GlnR to the nirB promoter was observed, suggesting that NnaR may act as a GlnR co-activator.

Proteomic analysis of the GlnR-mediated response to nitrogen limitation in Streptomyces coelicolor M145.

GlnR is the global regulator of nitrogen assimilation in Streptomyces coelicolor M145 and other actinobacteria. Two-dimensional polyacrylamide gel electrophoresis analyses were performed to identify new GlnR target genes by proteomic comparison of wild-type S. coelicolor M145 and a ΔglnR mutant. Fifty proteins were found to be differentially regulated between S. coelicolor M145 and the ΔglnR mutant. These spots were identified by nanoHPLC-ESI-MS/MS and classified according to their cellular role. Most of the identified proteins are involved in amino acid biosynthesis and in carbon metabolism, demonstrating that the role of GlnR is not restricted to nitrogen metabolism. Thus, GlnR is supposed to play an important role in the global metabolic control of S. coelicolor M145.

The dynamic architecture of the metabolic switch in Streptomyces coelicolor.

BACKGROUND: During the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples. RESULTS: Surprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis. CONCLUSIONS: Our study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological setting.

Tuber-specific cphA expression to enhance cyanophycin production in potatoes.

The production of biodegradable polymers that can be used to substitute petrochemical compounds in commercial products in transgenic plants is an important challenge for plant biotechnology. Nevertheless, it is often accompanied by reduced plant fitness. To decrease the phenotypic abnormalities of the sprout and to increase polymer production, we restricted cyanophycin accumulation to the potato tubers by using the cyanophycin synthetase gene (cphA(Te)) from Thermosynechococcus elongatus BP-1, which is under the control of the tuber-specific class 1 promoter (B33). Tuber-specific cytosolic (pB33-cphA(Te)) as well as tuber-specific plastidic (pB33-PsbY-cphA(Te)) expression resulted in significant polymer accumulation solely in the tubers. In plants transformed with pB33-cphA(Te), both cyanophycin synthetase and cyanophycin were detected in the cytoplasm leading to an increase up to 2.3% cyanophycin of dry weight and resulting in small and deformed tubers. In B33-PsbY-cphA(Te) tubers, cyanophycin synthetase and cyanophycin were exclusively found in amyloplasts leading to a cyanophycin accumulation up to 7.5% of dry weight. These tubers were normal in size, some clones showed reduced tuber yield and sometimes exhibited brown sunken staining starting at tubers navel. During a storage period over of 32 weeks of one selected clone, the cyanophycin content was stable in B33-PsbY-cphA(Te) tubers but the stress symptoms increased. However, all tubers were able to germinate. Nitrogen fertilization in the greenhouse led not to an increased cyanophycin yield, slightly reduced protein content, decreased starch content, and changes in the amounts of bound and free arginine and aspartate, as compared with control tubers were observed.

The roles of the nitrate reductase NarGHJI, the nitrite reductase NirBD and the response regulator GlnR in nitrate assimilation of Mycobacterium tuberculosis.

Mycobacterium tuberculosis can utilize various nutrients including nitrate as a source of nitrogen. Assimilation of nitrate requires the reduction of nitrate via nitrite to ammonium, which is then incorporated into metabolic pathways. This study was undertaken to define the molecular mechanism of nitrate assimilation in M. tuberculosis. Homologues to a narGHJI-encoded nitrate reductase and a nirBD-encoded nitrite reductase have been found on the chromosome of M. tuberculosis. Previous studies have implied a role for NarGHJI in nitrate respiration rather than nitrate assimilation. Here, we show that a narG mutant of M. tuberculosis failed to grow on nitrate. A nirB mutant of M. tuberculosis failed to grow on both nitrate and nitrite. Mutant strains of Mycobacterium smegmatis mc(2)155 that are unable to grow on nitrate were isolated. The mutants were rescued by screening a cosmid library from M. tuberculosis, and a gene with homology to the response regulator gene glnR of Streptomyces coelicolor was identified. A DeltaglnR mutant of M. tuberculosis was generated, which also failed to grow on nitrate, but regained its ability to utilize nitrate when nirBD was expressed from a plasmid, suggesting a role of GlnR in regulating nirBD expression. A specific binding site for GlnR within the nirB promoter was identified and confirmed by electrophoretic mobility shift assay using purified recombinant GlnR. Semiquantitative reverse transcription PCR, as well as microarray analysis, demonstrated upregulation of nirBD expression in response to GlnR under nitrogen-limiting conditions. In summary, we conclude that NarGHJI and NirBD of M. tuberculosis mediate the assimilatory reduction of nitrate and nitrite, respectively, and that GlnR acts as a transcriptional activator of nirBD.

The Streptomyces coelicolor GlnR regulon: identification of new GlnR targets and evidence for a central role of GlnR in nitrogen metabolism in actinomycetes.

Streptomyces coelicolor GlnR is a global regulator that controls genes involved in nitrogen metabolism. By genomic screening 10 new GlnR targets were identified, including enzymes for ammonium assimilation (glnII, gdhA), nitrite reduction (nirB), urea cleavage (ureA) and a number of biochemically uncharacterized proteins (SCO0255, SCO0888, SCO2195, SCO2400, SCO2404, SCO7155). For the GlnR regulon, a GlnR binding site which comprises the sequence gTnAc-n(6)-GaAAc-n(6)-GtnAC-n(6)-GAAAc-n(6) has been found. Reverse transcription analysis of S. coelicolor and the S. coelicolor glnR mutant revealed that GlnR activates or represses the expression of its target genes. Furthermore, glnR expression itself was shown to be nitrogen-dependent. Physiological studies of S. coelicolor and the S. coelicolor glnR mutant with ammonium and nitrate as the sole nitrogen source revealed that GlnR is not only involved in ammonium assimilation but also in ammonium supply. blast analysis demonstrated that GlnR-homologous proteins are present in different actinomycetes containing the glnA gene with the conserved GlnR binding site. By DNA binding studies, it was furthermore demonstrated that S. coelicolor GlnR is able to interact with these glnA upstream regions. We therefore suggest that GlnR-mediated regulation is not restricted to Streptomyces but constitutes a regulon conserved in many actinomycetes.

Conjugative DNA transfer in Streptomyces: SpdB2 involved in the intramycelial spreading of plasmid pSVH1 is an oligomeric integral membrane protein that binds to dsDNA.

In the current model of conjugal plasmid transfer in mycelium-forming streptomycetes, plasmid transfer by the FtsK-like TraB protein is followed by the subsequent spreading of the newly transferred plasmid within the neighbouring mycelial compartments. Several plasmid-encoded Spd proteins are involved in the plasmid spreading by an unknown mechanism. spdB2 of the conjugative pSVH1 plasmid of Streptomyces venezuelae was heterologously expressed in Escherichia coli and Streptomyces lividans, with a C-terminal His-tag-encoding sequence. Induction of spdB2-His expression affected viability in both species. The integral membrane protein SpdB2-His was eluted from the membrane fraction of S. lividans with Triton X-100, and purified as a soluble protein by Ni-NTA affinity chromatography. Cross-linking experiments with glutaraldehyde showed that SpdB2-His formed oligomers. SpdB2-His had a nonspecific DNA-binding activity: while all types of dsDNA were bound, single-stranded M13-DNA was not recognized. The spd genes of the spdB3-spd79-spdB2 operon of pSVH1 were simultaneously expressed in E. coli with different affinity tags. While expression of StrepII-SpdB3 was not detected, Spd79-flag and SpdB2-His were localized in the membrane fraction of E. coli. In the absence of SpdB2, most of the Spd79-flag protein was found in the cytoplasmic fraction, indicating that SpdB2 affects localization of Spd79. Pulldown assays with StrepII-TraB protein of pSVH1 demonstrated that TraB interacted with SpdB2, suggesting that the septal DNA translocator TraB is also involved in intramycelial plasmid spreading.

Nitrogen metabolism in Streptomyces coelicolor: transcriptional and post-translational regulation.

Glutamine synthetases (GS) are key enzymes of nitrogen metabolism. Most bacteria contain only one type of GS enzyme encoded by glnA. Streptomyces coelicolor, the model organism for Gram-positive streptomycetes, however is characterized by two functional GS (glnA, glnII) involved in nitrogen assimilation. In addition, three GS-like genes were identified which do not exhibit GS enzyme activity. The control of nitrogen assimilation and metabolism is mediated by transcriptional and post-translational regulation systems. The OmpR-like regulators GlnR and GlnRII are involved in transcriptional control of important nitrogen metabolism genes (glnA, glnII, amtB, glnK, glnD). Although GlnR and GlnRII share identical binding regions, their physiological impact is different. GSI activity is modulated post-translationally by the adenylyltransferase GlnE in response to the nitrogen concentration whereas no post-translational modifications of GSII are known. The PII/GlnD system also responds to changes in nitrogen conditions. The adenylyltransferase GlnD, which resembles the uridylyltransferase of Enterobacteriaceae, modifies PII under low-nitrogen conditions. Furthermore, PII is processed at its N-terminus in response to an ammonium shock. Apparently the function of the PII protein of S. coelicolor is different from that of the PII proteins of Enterobacteriaceae.

Unique conjugation mechanism in mycelial streptomycetes: a DNA-binding ATPase translocates unprocessed plasmid DNA at the hyphal tip.

A single plasmid-encoded protein, the septal DNA translocator TraB, is sufficient to promote conjugal plasmid transfer in mycelial streptomycetes. To analyse the molecular mechanism of conjugation the closely related TraB proteins from plasmids pSG5 of Streptomyces ghanaensis and pSVH1 of Streptomyces venezuelae were characterized. TraB of pSG5 was expressed as a fusion protein with eGFP and found to be localized at the hyphal tips of Streptomyces lividans by fluorescence microscopy, which strongly indicates that conjugation takes place at the tips of the mating mycelium. The TraB protein of pSVH1 was heterologously expressed in S. lividans with an N-terminal strep-tagII and purified as a soluble protein to near homogeneity. The purified protein was shown to hydrolyse ATP and to bind to a 50 bp non-coding pSVH1 sequence containing a 14 bp direct repeat. The protein-DNA complex was too large to enter an agarose gel, indicating that multimers of TraB were bound to the DNA. Denaturation of the protein-DNA complex released unprocessed plasmid DNA demonstrating that the TraB protein does not possess nicking activity. Our experimental data provide evidence that conjugal DNA transfer in streptomycetes is mediated by the septal DNA translocator TraB, an plasmid-encoded ATPase that interacts non-covalently with DNA and translocates an unprocessed double-stranded DNA molecule at the hyphal tip into the recipient.

Modular architecture of the conjugative plasmid pSVH1 from Streptomyces venezuelae.

The conjugative rolling circle replication (RCR) type plasmid pSVH1 from the chloramphenicol producer Streptomyces venezuelae was characterized by DNA sequence analysis and insertion/deletion analysis. Nucleotide sequence of the 12,652 bp pSVH1 revealed 11 open reading frames with high coding probability for which putative functions could be assigned. Beside the replication initiator gene rep for RCR, pSVH1 contained only genes involved in conjugative transfer. The transfer gene traB encoding the septal DNA translocator TraB is regulated by the GntR-type transcriptional regulator TraR. Six spd genes involved in intra-mycelial plasmid spreading are organized in two operons, consisting of two and three translationally coupled genes. Subcloning experiments demonstrated that the transfer gene traB represents a kill function and localized the pSVH1 minimal replicon consisting of rep and the dso origin to a 2072-bp fragment. Plasmid pSVH1 showed a modular architecture. Its replication region resembled that of the Streptomyces natalensis plasmid pSNA1, while the transfer and spread regions involved in conjugative plasmid transfer were highly similar to the corresponding regions of the Streptomyces ghanaensis plasmid pSG5.