Transcriptional switch on of ssgA by A-factor, which is essential for spore septum formation in Streptomyces griseus.
ABSTRACT: A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) triggers morphological development and secondary metabolism in Streptomyces griseus. A transcriptional activator (AdpA) in the A-factor regulatory cascade switches on a number of genes required for both processes. AdBS11 was identified in a library of the DNA fragments that are bound by AdpA and mapped upstream of ssgA, which is essential for septum formation in aerial hyphae. Gel mobility shift assays and DNase I footprinting revealed three AdpA-binding sites at nucleotide positions about -235 (site 1), -110 (site 2), and +60 (site 3) with respect to the transcriptional start point, p1, of ssgA. ssgA had two transcriptional start points, one starting at 124 nucleotides (p1) and the other starting at 79 nucleotides (p2) upstream of the start codon of ssgA. Of the three binding sites, only sites 1 and 2 were required for transcriptional activation of p1 and p2 by AdpA. The transcriptional switch on of ssgA required the extracytoplasmic function sigma factor, sigma(AdsA), in addition to AdpA. However, it was unlikely that sigma(AdsA) recognized the two ssgA promoters, since their -35 and -10 sequences were not similar to the promoter sequence motifs recognized by sigma(BldN), a sigma(AdsA) homologue of Streptomyces coelicolor A3(2). An ssgA disruptant formed aerial hyphae, but did not form spores, irrespective of the carbon source of the medium, which indicated that ssgA is a member of the whi genes. Transcriptional analysis of ssfR, located just upstream of ssgA and encoding an IclR-type transcriptional regulator, suggested that no read-through from ssfR into ssgA occurred, and ssgA was transcribed in the absence of ssfR. ssgA was thus found to be controlled by AdpA and not by SsfR to a detectable extent. SsfR appeared to regulate spore septum formation independently of SsgA or through interaction with SsgA in some unknown way, because an ssfR disruptant also showed a whi phenotype.
Project description:A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) at an extremely low concentration triggers streptomycin production and aerial mycelium formation in Streptomyces griseus. A-factor induces the expression of an A-factor-dependent transcriptional activator, AdpA, essential for both morphological and physiological differentiation by binding to the A-factor receptor protein ArpA, which has bound and repressed the adpA promoter, and dissociating it from the promoter. Nine DNA fragments that were specifically recognized and bound by histidine-tagged AdpA were isolated by cycles of a gel mobility shift-PCR method. One of them was located in front of a gene encoding an extracytoplasmic function sigma factor belonging to a subgroup of the primary sigma(70) family. The cloned gene was named AdpA-dependent sigma factor gene (adsA), and the gene product was named sigma(AdsA). Transcription of adsA depended on A-factor and AdpA, since adsA was transcribed at a very low and constant level in an A-factor-deficient mutant strain or in an adpA-disrupted strain. Consistent with this, transcription of adsA was greatly enhanced at or near the timing of aerial hyphae formation, as determined by low-resolution S1 nuclease mapping. High-resolution S1 mapping determined the transcriptional start point 82 nucleotides upstream of the translational start codon. DNase I footprinting showed that AdpA bound both strands symmetrically between the transcriptional start point and the translational start codon; AdpA protected the antisense strand from positions +7 to +41 with respect to the transcriptional start point and the sense strand from positions +12 to +46. A weak palindrome was found in the AdpA-binding site. The unusual position bound by AdpA as a transcriptional activator, in relation to the promoter, suggested the presence of a mechanism by which AdpA activates transcription of adsA in some unknown way. Disruption of the chromosomal adsA gene resulted in loss of aerial hyphae formation but not streptomycin or yellow pigment production, indicating that sigma(AdsA) is involved only in morphological development and not in secondary metabolic function. The presence of a single copy in each of the Streptomyces species examined by Southern hybridization suggests a common role in morphogenesis in this genus.
Project description:SsgA-like proteins are a family of actinomycete-specific regulatory proteins that control cell division and spore maturation in streptomycetes. SsgA and SsgB together activate sporulation-specific cell division by controlling the localization of FtsZ. Here we report the identification of novel regulators that control the transcription of the ssgA-like genes. Transcriptional regulators controlling ssg gene expression were identified using a DNA-affinity capture assay. Supporting transcriptional and DNA binding studies showed that the ssgA activator gene ssgR is controlled by the TetR-family regulator AtrA, while the ?-butyrolactone-responsive AdpA (SCO2792) and SlbR (SCO0608) and the metabolic regulator Rok7B7 (SCO6008) were identified as candidate regulators for the cell division genes ssgA, ssgB and ssgG. Transcription of the cell division gene ssgB depended on the sporulation genes whiA and whiH, while ssgR, ssgA and ssgD were transcribed independently of the whi genes. Our work sheds new light on the mechanisms by which sporulation-specific cell division is controlled in Streptomyces.
Project description:The role of ssgA in cell division and development of streptomycetes was analyzed. An ssgA null mutant of Streptomyces coelicolor produced aerial hyphae but failed to sporulate, and ssgA can therefore be regarded as a novel whi gene. In addition to the morphological changes, antibiotic production was also disturbed, with strongly reduced actinorhodin production. These defects could be complemented by plasmid-borne ssgA. In the wild-type strain, transcription of ssgA was induced by nutritional shift-down and was shown to be linked to that of the upstream-located gene ssgR, which belongs to the family of iclR-type transcriptional regulator genes. Analysis of mycelium harvested from liquid-grown cultures by transmission electron microscopy showed that septum formation had strongly increased in ssgA-overexpressing strains in comparison to wild-type S. coelicolor and that spore-like compartments were produced at high frequency. Furthermore, the hyphae were significantly wider and contained irregular and often extremely thick septa. These data underline the important role for ssgA in Streptomyces cell division.
Project description:AdpA is a key transcriptional activator in the A-factor regulatory cascade in Streptomyces griseus, activating a number of genes required for secondary metabolism and morphological differentiation. Of the five chymotrypsin-type serine protease genes, sprA, sprB, and sprD were transcribed in response to AdpA, showing that these protease genes are members of the AdpA regulon. These proteases were predicted to play the same physiological role, since these protease genes were transcribed in a similar time course during growth and the matured enzymes showed high end-to-end similarity to one another. AdpA bound two sites upstream of the sprA promoter approximately at positions -375 and -50 with respect to the transcriptional start point of sprA. Mutational analysis of the AdpA-binding sites showed that both AdpA-binding sites were essential for transcriptional activation. AdpA bound a single site at position -50 in front of the sprB promoter and greatly enhanced the transcription of sprB. The AdpA-binding site at position -40 was essential for transcription of sprD, although there was an additional AdpA-binding site at position -180. Most chymotrypsin activity excreted by S. griseus was attributed to SprA and SprB, because mutant deltasprAB, having a deletion in both sprA and sprB, lost almost all chymotrypsin activity, as did mutant deltaadpA. Even the double mutant deltasprAB and triple mutant deltasprABD grew normally and developed aerial hyphae and spores over the same time course as the wild-type strain.
Project description:The pleiotropic transcriptional regulator AdpA positively controls morphological differentiation and regulates secondary metabolism in most Streptomyces species. Streptomyces xiamenensis 318 has a linear chromosome 5.96?Mb in size. How AdpA affects secondary metabolism and morphological differentiation in such a naturally minimized genomic background is unknown. Here, we demonstrated that AdpA Sx , an AdpA orthologue in S. xiamenensis, negatively regulates cell growth and sporulation and bidirectionally regulates the biosynthesis of xiamenmycin and polycyclic tetramate macrolactams (PTMs) in S. xiamenensis 318. Overexpression of the adpASx gene in S. xiamenensis 318 had negative effects on morphological differentiation and resulted in reduced transcription of putative ssgA, ftsZ, ftsH, amfC, whiB, wblA1, wblA2, wblE, and a gene encoding sporulation-associated protein (sxim_29740), whereas the transcription of putative bldD and bldA genes was upregulated. Overexpression of adpASx led to significantly enhanced production of xiamenmycin but had detrimental effects on the production of PTMs. As expected, the transcriptional level of the xim gene cluster was upregulated, whereas the PTM gene cluster was downregulated. Moreover, AdpA Sx negatively regulated the transcription of its own gene. Electrophoretic mobility shift assays revealed that AdpA Sx can bind the promoter regions of structural genes of both the xim and PTM gene clusters as well as to the promoter regions of genes potentially involved in the cell growth and differentiation of S. xiamenensis 318. We report that an AdpA homologue has negative effects on morphological differentiation in S. xiamenensis 318, a finding confirmed when AdpA Sx was introduced into the heterologous host Streptomyces lividans TK24.IMPORTANCE AdpA is a key regulator of secondary metabolism and morphological differentiation in Streptomyces species. However, AdpA had not been reported to negatively regulate morphological differentiation. Here, we characterized the regulatory role of AdpA Sx in Streptomyces xiamenensis 318, which has a naturally streamlined genome. In this strain, AdpA Sx negatively regulated cell growth and morphological differentiation by directly controlling genes associated with these functions. AdpA Sx also bidirectionally controlled the biosynthesis of xiamenmycin and PTMs by directly regulating their gene clusters rather than through other regulators. Our findings provide additional evidence for the versatility of AdpA in regulating morphological differentiation and secondary metabolism in Streptomyces.
Project description:The rpoH regulatory region of different members of the enteric bacteria family was sequenced or downloaded from GenBank and compared. In addition, the transcriptional start sites of rpoH of Yersinia frederiksenii and Proteus mirabilis, two distant members of this family, were determined. Sequences similar to the sigma(70) promoters P1, P4 and P5, to the sigma(E) promoter P3 and to boxes DnaA1, DnaA2, cAMP receptor protein (CRP) boxes CRP1, CRP2 and box CytR present in Escherichia coli K12, were identified in sequences of closely related bacteria such as: E.coli, Shigella flexneri, Salmonella enterica serovar Typhimurium, Citrobacter freundii, Enterobacter cloacae and Klebsiella pneumoniae. In more distant bacteria, Y.frederiksenii and P.mirabilis, the rpoH regulatory region has a distal P1-like sigma(70) promoter and two proximal promoters: a heat-induced sigma(E)-like promoter and a sigma(70) promoter. Sequences similar to the regulatory boxes were not identified in these bacteria. This study suggests that the general pattern of transcription of the rpoH gene in enteric bacteria includes a distal sigma(70) promoter, >200 nt upstream of the initiation codon, and two proximal promoters: a heat-induced sigma(E)-like promoter and a sigma(70) promoter. A second proximal sigma(70) promoter under catabolite-regulation is probably present only in bacteria closely related to E.coli.
Project description:AdpA in the A-factor regulatory cascade in Streptomyces griseus activates a number of genes required for secondary metabolism and morphological differentiation, forming an AdpA regulon. The Streptomyces subtilisin inhibitor (SSI) gene, sgiA, in S. griseus was transcribed in response to AdpA, showing that sgiA is a member of the AdpA regulon. AdpA bound a single site upstream of the sgiA promoter at approximately position -70 with respect to its transcriptional start point. Mutational analysis of the AdpA-binding site showed that the AdpA-binding site was essential for transcriptional activation. Mutants in which sgiA was disrupted had higher trypsin, chymotrypsin, metalloendopeptidase, and total protease activities than the wild-type strain, which showed that SgiA modulated the activities of these extracellularly produced proteases. Because a number of genes encoding chymotrypsins, trypsins, and metalloendopeptidases, most of which are SSI-sensitive proteases, are also under the control of AdpA, the A-factor regulatory cascade was thought to play a crucial role in modulating the extracellular protease activities by triggering simultaneous production of the proteases and their inhibitor at a specific timing during growth. Mutants in which sgiA was disrupted grew normally and formed aerial hyphae and spores with the same time course as the wild-type strain. However, exogenous addition of purified SgiA to substrate mycelium grown on agar medium resulted in a delay in aerial mycelium formation, indicating that SgiA is involved in aerial hypha formation in conjunction with proteases.
Project description:In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) switches on aerial mycelium formation and secondary metabolite biosynthesis. An A-factor-dependent transcriptional activator, AdpA, activates multiple genes required for morphological development and secondary metabolism in a programmed manner. A region upstream of a zinc-containing metalloendopeptidase gene (sgmA) was found among the DNA fragments that had been isolated as AdpA-binding sites. The primary product of sgmA consisted of N-terminal pre, N-terminal pro, mature, and C-terminal pro regions. sgmA was transcribed in an AdpA-dependent manner, and its transcription was markedly enhanced at the timing of aerial mycelium formation. AdpA bound two sites in the region upstream of the sgmA promoter; one was at about nucleotide position -60 (A site) with respect to the transcriptional start point of sgmA, and the other was at about position -260 (B site), as determined by DNase I footprinting. Transcriptional analysis with mutated promoters showed that the A site was essential for the switching on of sgmA transcription and that the B site was necessary for the marked enhancement of transcription at the timing of aerial mycelium formation. Disruption of the chromosomal sgmA gene resulted in a delay in aerial hypha formation by half a day. SgmA is therefore suggested to be associated with the programmed morphological development of Streptomyces, in which this peptidase, perhaps together with other hydrolytic enzymes, plays a role in the degradation of proteins in substrate hyphae for reuse in aerial hypha formation.
Project description:The Bacillus subtilis spoIIIA locus encodes eight proteins, SpoIIIAA to SpoIIIAH, which are expressed in the mother cell during endospore formation and which are essential for the activation of sigma(G) in the forespore. Complementation studies indicated that this locus may be transcribed from two promoters, one promoter upstream from the first gene and possibly a second unidentified promoter within the locus. Fragments of the spoIIIA locus were expressed at an ectopic site to complement the sporulation-defective phenotype of a spoIIIAH deletion, and we determined that complementation required a fragment of DNA that extended into spoIIIAF. To confirm that there was a promoter located in spoIIIAF, we constructed transcriptional fusions to lacZ and found strong sporulation-induced promoter activity. Primer extension assays were used to determine the transcription start site, and point mutations introduced into the -10 and -35 regions of the promoter reduced its activity. This promoter is transcribed by sigma(E)-RNA polymerase and is repressed by SpoIIID. Therefore, we concluded that the spoIIIA locus is transcribed from two promoters, one at the start of the locus (P1(spoIIIA)) and the other within the locus (P2(spoIIIA)). Based on Campbell integrations and reverse transcription-PCR analysis of the P2(spoIIIA) region, we determined that P2(spoIIIA) is sufficient for transcription of spoIIIAG and spoIIIAH. Inactivation of P2(spoIIIA) blocked spore formation, indicating that P2(spoIIIA) is essential for expression of spoIIIAG and spoIIIAH. The P2(spoIIIA) activity is twice the P1(spoIIIA) activity; therefore, larger amounts of SpoIIIAG and SpoIIIAH than of proteins encoded at the upstream end of the locus may be required.
Project description:The alternative sigma factor sigma(B) of Staphylococcus aureus controls the expression of a variety of genes, including virulence determinants and global regulators. Genetic manipulations and transcriptional start point (TSP) analyses showed that the sigB operon is transcribed from at least two differentially controlled promoters: a putative sigma(A)-dependent promoter, termed sigB(p1), giving rise to a 3.6-kb transcript covering sa2059-sa2058-rsbU-rsbV-rsbW-sigB, and a sigma(B)-dependent promoter, sigB(p3), initiating a 1.6-kb transcript covering rsbV-rsbW-sigB. TSP and promoter-reporter gene fusion experiments indicated that a third promoter, tentatively termed sigB(p2) and proposed to lead to a 2.5-kb transcript, including rsbU-rsbV-rsbW-sigB, might govern the expression of the sigB operon. Environmental stresses, such as heat shock and salt stress, induced a rapid response within minutes from promoters sigB(p1) and sigB(p3). In vitro, the sigB(p1) promoter was active in the early growth stages, while the sigB(p2) and sigB(p3) promoters produced transcripts throughout the growth cycle, with sigB(p3) peaking around the transition state between exponential growth and stationary phase. The amount of sigB transcripts, however, did not reflect the concentration of sigma(B) measured in cell extracts, which remained constant over the entire growth cycle. In a guinea pig cage model of infection, sigB transcripts were as abundant 2 and 8 days postinoculation as values found in vitro, demonstrating that sigB is indeed transcribed during the course of infection. Physical interactions between staphylococcal RsbU-RsbV, RsbV-RsbW, and RsbW-sigma(B) were inferred from a yeast (Saccharomyces cerevisiae) two-hybrid approach, indicating the presence of a partner-switching mechanism in the sigma(B) activation cascade similar to that of Bacillus subtilis. The finding that overexpression of RsbU was sufficient to trigger an immediate and strong activation of sigma(B), however, signals a relevant difference in the regulation of sigma(B) activation between B. subtilis and S. aureus in the cascade upstream of RsbU.