Post-translational Serine/Threonine Phosphorylation and Lysine Acetylation: A Novel Regulatory Aspect of the Global Nitrogen Response Regulator GlnR in S. coelicolor M145.

Soil-dwelling Streptomyces bacteria such as S.coelicolor have to constantly adapt to the nitrogen (N) availability in their habitat. Thus, strict transcriptional and post-translational control of the N-assimilation is fundamental for survival of this species. GlnR is a global response regulator that controls transcription of the genes related to the N-assimilation in S. coelicolor and other members of the Actinomycetales. GlnR represents an atypical orphan response regulator that is not activated by the phosphorylation of the conserved aspartate residue (Asp 50). We have applied transcriptional analysis, LC-MS/MS analysis and electrophoretic mobility shift assays (EMSAs) to understand the regulation of GlnR in S. coelicolor M145. The expression of glnR and GlnR-target genes was revisited under four different N-defined conditions and a complex N-rich condition. Although, the expression of selected GlnR-target genes was strongly responsive to changing N-concentrations, the glnR expression itself was independent of the N-availability. Using LC-MS/MSanalysis we demonstrated that GlnR was post-translationally modified. The post-translational modifications of GlnR comprise phosphorylation of the serine/threonine residues and acetylation of lysine residues. In the complex N-rich medium GlnR was phosphorylated on six serine/threonine residues and acetylated on one lysine residue. Under defined N-excess conditions only two phosphorylated residues were detected whereas under defined N-limiting conditions no phosphorylation was observed. GlnR phosphorylation is thus clearly correlated with N-rich conditions. Furthermore, GlnR was acetylated on four lysine residues independently of the N-concentration in the defined media and on only one lysine residue in the complex N-rich medium. Using EMSAs we demonstrated that phosphorylation inhibited the binding of GlnR to its targets genes, whereas acetylation had little influence on the formation of GlnR-DNA complex. This study clearly demonstrated that GlnR DNA-binding affinity is modulated by post-translational modifications in response to changing N-conditions in order to elicit a proper transcriptional response to the latter.

Quantitative proteomics of the human skin secretome reveal a reduction in immune defense mediators in ectodermal dysplasia patients.

In healthy human skin host defense molecules such as antimicrobial peptides (AMPs) contribute to skin immune homeostasis. In patients with the congenital disease ectodermal dysplasia (ED) skin integrity is disturbed and as a result patients have recurrent skin infections. The disease is characterized by developmental abnormalities of ectodermal derivatives and absent or reduced sweating. We hypothesized that ED patients have a reduced skin immune defense because of the reduced ability to sweat. Therefore, we performed a label-free quantitative proteome analysis of wash solution of human skin from ED patients or healthy individuals. A clear-cut difference between both cohorts could be observed in cellular processes related to immunity and host defense. In line with the extensive underrepresentation of proteins of the immune system, dermcidin, a sweat-derived AMP, was reduced in its abundance in the skin secretome of ED patients. In contrast, proteins involved in metabolic/catabolic and biosynthetic processes were enriched in the skin secretome of ED patients. In summary, our proteome profiling provides insights into the actual situation of healthy versus diseased skin. The systematic reduction in immune system and defense-related proteins may contribute to the high susceptibility of ED patients to skin infections and altered skin colonization.

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 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.

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.