The Streptomyces NrdR transcriptional regulator is a Zn ribbon/ATP cone protein that binds to the promoter regions of class Ia and class II ribonucleotide reductase operons. (73/441)

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomyces spp. contain genes coding for two RNRs, either of which is sufficient for vegetative growth. The class Ia RNR is encoded by the nrdAB genes, and the class II RNR is encoded by nrdJ, which is coexpressed with nrdR. We previously showed that the Streptomyces coelicolor nrdR gene encodes a protein, NrdR, which represses transcription of both sets of RNR genes. NrdR is a member of a highly conserved family of proteins that is confined exclusively to prokaryotes. In this report, we describe a physical and biochemical characterization of the S. coelicolor NrdR protein and show that it is a zinc-ATP/dATP-containing protein that binds to the promoter regions of both Streptomyces RNR operons. The NrdR N terminus contains a zinc ribbon motif that is necessary for binding to the upstream regulatory region of both RNR operons. The latter contains two 16-bp direct repeat sequences, termed NrdR boxes, which are located proximal to, or overlap with, the promoter regions. These experiments support the view that NrdR controls the transcription of RNR genes by binding to the NrdR box sequences. We also show that the central NrdR ATP cone domain binds ATP and dATP and that mutations that abolish ATP/dATP binding significantly reduce DNA binding, suggesting that the ATP cone domain may allosterically regulate NrdR binding. We conclude that NrdR is a widely conserved regulator of RNR genes, binding to specific sequence elements in the promoter region and thereby modulating transcription.  (+info)

RNA:protein ratio of the unicellular organism as a characteristic of phosphorous and nitrogen stoichiometry and of the cellular requirement of ribosomes for protein synthesis. (74/441)

BACKGROUND: Mean phosphorous:nitrogen (P:N) ratios and relationships of P:N ratios with the growth rate of organisms indicate a surprising similarity among and within microbial species, plants, and insect herbivores. To reveal the cellular mechanisms underling this similarity, the macromolecular composition of seven microorganisms and the effect of specific growth rate (SGR) on RNA:protein ratio, the number of ribosomes, and peptide elongation rate (PER) were analyzed under different conditions of exponential growth. RESULTS: It was found that P:N ratios calculated from RNA and protein contents in these particular organisms were in the same range as the mean ratios reported for diverse organisms and had similar positive relationships with growth rate, consistent with the growth-rate hypothesis. The efficiency of protein synthesis in microorganisms is estimated as the number of active ribosomes required for the incorporation of one amino acid into the synthesized protein. This parameter is calculated as the SGR:PER ratio. Experimental and theoretical evidence indicated that the requirement of ribosomes for protein synthesis is proportional to the RNA:protein ratio. The constant of proportionality had the same values for all organisms, and was derived mechanistically from the characteristics of the protein-synthesis machinery of the cell (the number of nucleotides per ribosome, the average masses of nucleotides and amino acids, the fraction of ribosomal RNA in the total RNA, and the fraction of active ribosomes). Impairment of the growth conditions decreased the RNA:protein ratio and increased the overall efficiency of protein synthesis in the microorganisms. CONCLUSION: Our results suggest that the decrease in RNA:protein and estimated P:N ratios with decrease in the growth rate of the microorganism is a consequence of an increased overall efficiency of protein synthesis in the cell resulting from activation of the general stress response and increased transcription of cellular maintenance genes at the expense of growth related genes. The strong link between P:N stoichiometry, RNA:protein ratio, ribosomal requirement for protein synthesis, and growth rate of microorganisms indicated by the study could be used to characterize the N and P economy of complex ecosystems such as soils and the oceans.  (+info)

Evolution of the terminal regions of the Streptomyces linear chromosome. (75/441)

Comparative analysis of the Streptomyces chromosome sequences, between Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces ambofaciens ATCC23877 (whose partial sequence is released in this study), revealed a highly compartmentalized genetic organization of their genome. Indeed, despite the presence of specific genomic islands, the central part of the chromosome appears highly syntenic. In contrast, the chromosome of each species exhibits large species-specific terminal regions (from 753 to 1,393 kb), even when considering closely related species (S. ambofaciens and S. coelicolor). Interestingly, the size of the central conserved region between species decreases as the phylogenetic distance between them increases, whereas the specific terminal fraction reciprocally increases in size. Between highly syntenic central regions and species-specific chromosomal parts, there is a notable degeneration of synteny due to frequent insertions/deletions. This reveals a massive and constant genomic flux (from lateral gene transfer and DNA rearrangements) affecting the terminal contingency regions. We speculate that a gradient of recombination rate (i.e., insertion/deletion events) toward the extremities is the force driving the exclusion of essential genes from the terminal regions (i.e., chromosome compartmentalization) and generating a fast gene turnover for strong adaptation capabilities.  (+info)

The CarA/LitR-family transcriptional regulator: Its possible role as a photosensor and wide distribution in non-phototrophic bacteria. (76/441)

LitR is a MerR-family regulator that controls light-induced carotenogenesis in Streptomyces coelicolor. The introduction of litR conferred photo-dependent transcription activities on Streptomyces griseus, which does not perform photo-responsive carotenogenesis. LitR discovers homologs encoded at illumination-related loci of various non-phototrophic bacterial genomes. Probably, LitR acts not only as a transcriptional regulator but also a photosensor.  (+info)

Critical residues and novel effects of overexpression of the Streptomyces coelicolor developmental protein BldB: evidence for a critical interacting partner. (77/441)

The bldB gene of Streptomyces coelicolor encodes the best-characterized member of a family of small proteins that have low isoelectric points but that lack any previously characterized sequence motifs. BldB is dimeric and is required for the efficient production of antibiotics and spore-forming cells, called aerial hyphae, by growing colonies. The mechanism of action of BldB and its relatives is unknown. Here, we have explored amino acids in BldB that either are highly conserved or have been implicated in function genetically. We show that five amino acids are important for its function at physiological expression levels. Mutations in three of these amino acids gave rise to proteins that were either monomeric or unstable in vivo, while two others are not. We find that overexpression of bldB in S. coelicolor blocks sporulation prior to sporulation-specific septation but permits the formation of aerial hyphae. Vegetative septation was apparently normal in both the bldB null mutant and the bldB overexpression strain. To our surprise, overexpression of the dimerization-competent but functionally defective alleles caused a dramatic acceleration of sporulation. Our results suggest that BldB makes at least one important contact with another subcellular constituent and that a loss or alteration of this interaction impairs the phenotypic properties of the organism.  (+info)

A recA null mutation may be generated in Streptomyces coelicolor. (78/441)

The recombinase RecA plays a crucial role in homologous recombination and the SOS response in bacteria. Although recA mutants usually are defective in homologous recombination and grow poorly, they nevertheless can be isolated in almost all bacteria. Previously, considerable difficulties were experienced by several laboratories in generating recA null mutations in Streptomyces, and the only recA null mutants isolated (from Streptomyces lividans) appeared to be accompanied by a suppressing mutation. Using gene replacement mediated by Escherichia coli-Streptomyces conjugation, we generated recA null mutations in a series of Streptomyces coelicolor A3(2) strains. These recA mutants were very sensitive to mitomycin C but only moderately sensitive to UV irradiation, and the UV survival curves showed wide shoulders, reflecting the presence of a recA-independent repair pathway. The mutants segregated minute colonies with low viability during growth and produced more anucleate spores than the wild type. Some crosses between pairs of recA null mutants generated no detectable recombinants, showing for the first time that conjugal recombination in S. coelicolor is recA mediated, but other mutants retained the ability to undergo recombination. The nature of this novel recombination activity is unknown.  (+info)

Mapping essential domains of Mycobacterium smegmatis WhmD: insights into WhiB structure and function. (79/441)

A growing body of evidence suggests that the WhiB-like proteins exclusive to the GC-rich actinomycete genera play significant roles in pathogenesis and cell division. Each of these proteins contains four invariant cysteine residues and a conserved helix-turn-helix motif. whmD, the Mycobacterium smegmatis homologue of Streptomyces coelicolor whiB, is essential in M. smegmatis, and the conditionally complemented mutant M. smegmatis 628-53 undergoes filamentation under nonpermissive conditions. To identify residues critical to WhmD function, we developed a cotransformation-based assay to screen for alleles that complement the filamentation phenotype of M. smegmatis 628-53 following inducer withdrawal. Mycobacterium tuberculosis whiB2 and S. coelicolor whiB complemented the defect in M. smegmatis 628-53, indicating that these genes are true functional orthologues of whmD. Deletion analysis suggested that the N-terminal 67 and C-terminal 12 amino acid residues are dispensable for activity. Site-directed mutagenesis indicated that three of the four conserved cysteine residues (C90, C93, and C99) and a conserved aspartate (D71) are essential. Mutations in a predicted loop glycine (G111) and an unstructured leucine (L116) were poorly tolerated. The region essential for WhmD activity encompasses 6 of the 10 residues conserved in all seven M. tuberculosis WhiBs, as well as in most members of the WhiB family identified thus far. WhmD structure was found to be sensitive to the presence of a reducing agent, suggesting that the cysteine residues are involved in coordinating a metal ion. Iron-specific staining strongly suggested that WhmD contains a bound iron atom. With this information, we have now begun to comprehend the functional significance of the conserved sequence and structural elements in this novel family of proteins.  (+info)

Evolution of new function in the GTP cyclohydrolase II proteins of Streptomyces coelicolor. (80/441)

The genome sequence of Streptomyces coelicolor contains three open reading frames (sco1441, sco2687, and sco6655) that encode proteins with significant (>40%) amino acid identity to GTP cyclohydrolase II (GCH II), which catalyzes the committed step in the biosynthesis of riboflavin. The physiological significance of the redundancy of these proteins in S. coelicolor is not known. However, the gene contexts of the three proteins are different, suggesting that they may serve alternate biological niches. Each of the three proteins was overexpressed in Escherichia coli and characterized to determine if their functions are biologically overlapping. As purified, each protein contains 1 molar equiv of zinc/mol of protein and utilizes guanosine 5'-triphosphate (GTP) as substrate. Two of these proteins (SCO 1441 and SCO 2687) produce the canonical product of GCH II, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (APy). Remarkably, however, one of the three proteins (SCO 6655) converts GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (FAPy), as shown by UV-visible spectrophotometry, mass spectrometry, and NMR. This activity has been reported for a GTP cyclohydrolase III protein from Methanocaldococcus jannaschii [Graham, D. E., Xu, H., and White, R. H. (2002) Biochemistry 41, 15074-15084], which has no amino acid sequence homology to SCO 6655. Comparison of the sequences of these proteins and mapping onto the structure of the E. coli GCH II protein [Ren, J., Kotaka, M., Lockyer, M., Lamb, H. K., Hawkins, A. R., and Stammers, D. K. (2005) J. Biol. Chem. 280, 36912-36919] allowed identification of a switch residue, Met120, which appears to be responsible for the altered fate of GTP observed with SCO 6655; a Tyr is found in the analogous position of all proteins that have been shown to catalyze the conversion of GTP to APy. The Met120Tyr variant of SCO 6655 acquires the ability to catalyze the conversion of GTP to APy, suggesting a role for Tyr120 in the late phase of the reaction. Our data are consistent with duplication of GCH II in S. coelicolor promoting evolution of a new function. The physiological role(s) of the gene clusters that house GCH II homologues will be discussed.  (+info)