Genes essential for morphological development and antibiotic production in Streptomyces coelicolor are targets of BldD during vegetative growth.

BldD is a transcriptional regulator essential for morphological development and antibiotic production in Streptomyces coelicolor. Here we identify the BldD regulon by means of chromatin immunoprecipitation-microarray analysis (ChIP-chip). The BldD regulon encompasses ~167 transcriptional units, of which more than 20 are known to play important roles in development (e.g. bldA, bldC, bldH/adpA, bldM, bldN, ssgA, ssgB, ftsZ, whiB, whiG, smeA-ssfA) and/or secondary metabolism (e.g. nsdA, cvn9, bldA, bldC, leuA). Strikingly, 42 BldD target genes (~25% of the regulon) encode regulatory proteins, stressing the central, pleiotropic role of BldD. Almost all BldD binding sites identified by ChIP-chip are present in the promoters of the target genes. An exception is the tRNA gene bldA, where BldD binds within the region encoding the primary transcript, immediately downstream of the position corresponding to the processed, mature 3 end of the tRNA. Through gene overexpression, we identified a novel BldD target gene (cdgA) that influences differentiation and antibiotic production. cdgA encodes a GGDEF domain protein, implicating c-di-GMP in the regulation of Streptomyces development. Sequence analysis of the upstream regions of the complete regulon identified a 15 bp inverted repeat that functions as a high-affinity binding site for BldD, as was shown by electrophoretic mobility shift assays and DNase I footprinting analysis. High-scoring copies of the BldD binding site were found at relevant positions in the genomes of other bacteria containing a BldD homologue, suggesting the role of BldD is conserved in sporulating actinomycetes.

BldG and SCO3548 interact antagonistically to control key developmental processes in Streptomyces coelicolor.

The similarity of BldG and the downstream coexpressed protein SCO3548 to anti-anti-sigma and anti-sigma factors, respectively, together with the phenotype of a bldG mutant, suggests that BldG and SCO3548 interact as part of a regulatory system to control both antibiotic production and morphological differentiation in Streptomyces coelicolor. A combination of bacterial two-hybrid, affinity purification, and far-Western analyses demonstrated that there was self-interaction of both BldG and SCO3548, as well as a direct interaction between the two proteins. Furthermore, a genetic complementation experiment demonstrated that SCO3548 antagonizes the function of BldG, similar to other anti-anti-sigma/anti-sigma factor pairs. It is therefore proposed that BldG and SCO3548 form a partner-switching pair that regulates the function of one or more sigma factors in S. coelicolor. The conservation of bldG and sco3548 in other streptomycetes demonstrates that this system is likely a key regulatory switch controlling developmental processes throughout the genus Streptomyces.

Antimicrobial activity in the egg wax of the African cattle tick Amblyomma hebraeum (Acari: Ixodidae).

Eggs of the tick Amblyomma hebraeum Koch (Acari: Ixodidae) inhibited the growth of Escherichia coli and Serratia marcescens (Gram-negative bacteria) in solid culture, but not the growth of Staphylococcus epidermidis, and only marginally the growth of Bacillus subtilis (Gram-positive bacteria). When egg wax was extracted with chloroform/methanol (2:1), the extract contained antibacterial activity, but the denuded eggs did not. When assayed against bacteria in liquid culture, the aqueous phase inhibited the growth of S. epidermidis. However, the activity against E. coli was lost during extraction. The antimicrobial component of the aqueous phase was heat stable (100 degrees C for 10 min), resistant to proteinase K (15 min at 55 degrees C) and to pronase (30 min at 37 degrees C). The antibacterial activity in the aqueous phase increased the permeability of the cell membrane of susceptible bacterial cells within 30 min. However, lysis of the cells was detected by optical density measurements (OD(600 nm)) only after 1.5 h. The most evident cytological changes observed by transmission electron microscopy were a thickening of the cell wall and the appearance of numerous electron lucent areas within the cytoplasm of treated bacteria. Gené's organ, the egg-waxing organ in ticks, grew enormously during the first 16 days post-engorgement, and gained antimicrobial activity by day 10 (when oviposition began). This suggests that Gené's organ is the major source of the antibacterial substance in the egg wax. The vitellogenic hormone in A. hebraeum, 20-hydroxyecdysone, when injected into recently engorged females, did not stimulate growth of Gené's organ or precocious secretion of antimicrobial activity.

Expression of ccaR, encoding the positive activator of cephamycin C and clavulanic acid production in Streptomyces clavuligerus, is dependent on bldG.

In Streptomyces coelicolor, bldG encodes a putative anti-anti-sigma factor that regulates both aerial hypha formation and antibiotic production, and a downstream transcriptionally linked open reading frame (orf3) encodes a putative anti-sigma factor protein. A cloned DNA fragment from Streptomyces clavuligerus contained an open reading frame that encoded a protein showing 92% identity to the S. coelicolor BldG protein and 91% identity to the BldG ortholog in Streptomyces avermitilis. Sequencing of the region downstream of bldG in S. clavuligerus revealed the presence of an open reading frame encoding a protein showing 72 and 69% identity to the ORF3 proteins in S. coelicolor and S. avermitilis, respectively. Northern analysis indicated that, as in S. coelicolor, the S. clavuligerus bldG gene is expressed as both a monocistronic and a polycistronic transcript, the latter including the downstream orf3 gene. High-resolution S1 nuclease mapping of S. clavuligerus bldG transcripts revealed the presence of three bldG-specific promoters, and analysis of expression of a bldGp-egfp reporter indicated that the bldG promoter is active at various stages of development and in both substrate and aerial hyphae. A bldG null mutant was defective in both morphological differentiation and in the production of secondary metabolites, such as cephamycin C, clavulanic acid, and the 5S clavams. This inability to produce cephamycin C and clavulanic acid was due to the absence of the CcaR transcriptional regulator, which controls the expression of biosynthetic genes for both secondary metabolites as well as the expression of a second regulator of clavulanic acid biosynthesis, ClaR. This makes bldG the first regulatory protein identified in S. clavuligerus that functions upstream of CcaR and ClaR in a regulatory cascade to control secondary metabolite production.

BldD from Streptomyces coelicolor is a non-essential global regulator that binds its own promoter as a dimer.

We have shown that the bldD gene of Streptomyces coelicolor, while required for antibiotic production and morphological differentiation, is not essential for viability. We have also demonstrated that BldD forms a higher order complex both in solution and when bound to target DNA. Purified BldD exists in three forms in solution, as a tetramer, dimer and monomer, but only in the dimeric form when bound to its own promoter/operator.

The putative anti-anti-sigma factor BldG is post-translationally modified by phosphorylation in Streptomyces coelicolor.

The Streptomyces coelicolor bldG gene encodes a protein showing similarity to the SpoIIAA and RsbV anti-anti-sigma factors of Bacillus subtilis. Purified maltose binding protein-BldG could be phosphorylated in vitro by wild-type S. coelicolor crude extract, and both the phosphorylated and unphosphorylated forms of BldG could be detected in vivo using isoelectric focusing. ATP was shown to serve as the phosphoryl group donor, and phosphorylation of BldG was abolished when the putative phosphorylation site was changed from a serine to an alanine residue. A bldG mutant strain expressing the non-phosphorylatable BldG protein was unable to undergo morphological differentiation or produce antibiotics even after prolonged incubation, suggesting that phosphorylation of BldG is necessary for proper development in S. coelicolor.

Iron-regulated phenylalanyl-tRNA synthetase activity in Azotobacter vinelandii.

Azotobacter vinelandii strain UA22 was produced by pTn5luxAB mutagenesis, such that the promoterless luxAB genes were transcribed in an iron-repressible manner. Tn5luxAB was localized to a fragment of chromosomal DNA encoding the thrS, infC, rpmI, rplT, pheS and pheT genes, with Tn5 inserted in the 3'-end of pheS. The isolation of this mutation in an essential gene was possible because of polyploidy in Azotobacter, such that strain UA22 carried both wild-type and mutant alleles of pheS. Phenylalanyl-tRNA synthetase activity and PHES::luxAB reporter activity was partially repressed under iron-sufficient conditions and fully derepressed under iron-limited conditions. The ferric uptake regulator (Fur) bound to a DNA sequence immediately upstream of luxAB, within the pheS gene, but PHES::luxAB reporter activity was not affected by phenylalanine availability. This suggests there is novel regulation of pheST in A. vinelandii by iron availability.

The positive activator of cephamycin C and clavulanic acid production in Streptomyces clavuligerus is mistranslated in a bldA mutant.

In Streptomyces coelicolor bldA encodes the principal leucyl tRNA for translation of UUA codons and controls pigmented antibiotic production by the presence of TTA codons in the genes encoding the pathway-specific activators of actinorhodin and undecylprodigiosin biosynthesis. In Streptomyces clavuligerus the gene encoding the pathway-specific activator of both cephamycin C and clavulanic acid production, ccaR, also contains a TTA codon and was expected to exhibit bldA-dependence. A cloned S. clavuligerus DNA fragment containing a sequence showing 91% identity to the S. coelicolor bldA-encoded tRNA was able to restore antibiotic production and sporulation to bldA mutants of S. coelicolor and the closely related Streptomyces lividans. A null mutation of the bldA gene in S. clavuligerus resulted in the expected sporulation defective phenotype, but unexpectedly had no effect on antibiotic production. Transcript analysis showed no difference in the levels of ccaR transcripts in the wild-type and bldA mutant strains, ruling out any effect of elevated levels of the ccaR mRNA. Furthermore, when compared to the wild-type strain, the bldA mutant showed no differences in the levels of CcaR, suggesting that the single TTA codon in ccaR is mistranslated efficiently. The role of codon context in bldA dependence is discussed.

Study of the bldG locus suggests that an anti-anti-sigma factor and an anti-sigma factor may be involved in Streptomyces coelicolor antibiotic production and sporulation.

A cloned 2.5 kb DNA fragment that can restore antibiotic production and sporulation to a bldG mutant encodes a 113 aa protein showing similarity to a family of anti-anti-sigma factors from Bacillus and Staphylococcus; and the deduced product of a closely spaced downstream ORF, designated ORF3, shows similarity to cognate anti-sigma factors. The homologues in Bacillus regulate the activity of sporulation- and stress-response-specific sigma factors. However, there is no sigma factor gene near bldG and ORF3. bldG is transcribed both as a monocistronic and a polycistronic mRNA, the latter including the downstream ORF3 gene. The two transcripts were present at all time points during growth and both were upregulated when aerial mycelium and pigmented antibiotics were seen. At all time points, the monocistronic bldG transcript was two- to threefold more abundant than the polycistronic transcript. Mapping of the mRNA 5' ends indicated that bldG transcription is initiated from two transcription start sites located 82 and 123 bp upstream of the bldG translation start. A constructed bldG null mutant had the same phenotype as previously isolated bldG point mutations, some of which were shown to have potentially significant base changes within bldG. When compared to the wild-type strain, the null mutant showed no differences in the levels of transcription from the two bldG promoters. These results suggest that bldG is not involved in autoregulation.

The bldA-encoded tRNA is poorly expressed in the bldI mutant of Streptomyces coelicolor A3(2).

The bldA gene, encoding a leucyl tRNA recognizing the UUA codon, is expressed at significantly lower levels in the bldI mutant, Streptomyces coelicolor J703, than in the parent S. coelicolor A3(2). Expression of a TTA-containing reporter gene was reduced in the bldI mutant, as was the mature, 87 nucleotide, form of the bldA-encoded tRNA. This reduced level of the tRNA was also seen when the bldA gene was introduced on a high-copy-number plasmid into the bldI mutant, suggesting that maximal bldA expression may require a bldI-dependent promoter.