Structure and function of the arginine repressor-operator complex from Bacillus subtilis.

In many bacteria, the concentration of L-arginine is controlled by a transcriptional regulator, the arginine repressor. In Bacillus subtilis this transcription factor is called AhrC and has roles in both the repression and activation of the genes involved in arginine metabolism. It interacts with 18 bp ARG boxes in the promoters of arginine biosynthetic and catabolic operons. AhrC is a hexamer and each subunit has two domains. The C-terminal domains form the core, mediating inter-subunit interactions and L-arginine binding, while the N-terminal domains contain a winged helix-turn-helix DNA-binding motif and are arranged around the periphery. Upon binding of the co-repressor L-arginine there is a approximately 15 degrees relative rotation between core C-terminal trimers. Here, we report the X-ray crystal structure of a dimer of the N-terminal domains of AhrC (NAhrC) in complex with an 18 bp DNA ARG box operator, refined to 2.85 A resolution. Comparison of the N-terminal domains within this complex with those of the free domain reveals that the flexible beta-wings of the DNA-binding motif in the free domain form a stable dimer interface in the protein-DNA complex, favouring correct orientation of the recognition helices. These are then positioned to insert into adjacent turns of the major groove of the ARG box, whilst the wings contact the minor groove. There are extensive contacts between the protein and the DNA phosphodiester backbone, as well as a number of direct hydrogen bonds between conserved amino acid side chains and bases. Combining this structure with other crystal structures of other AhrC components, we have constructed a model of the repression complex of AhrC at the B. subtilis biosynthetic argC operator and, along with transcriptome data, analysed the origins of sequence specificity and arginine activation.

A high-resolution structure of the DNA-binding domain of AhrC, the arginine repressor/activator protein from Bacillus subtilis.

In Bacillus subtilis the concentration of L-arginine is controlled by the transcriptional regulator AhrC, which interacts with 18 bp DNA operator sites called ARG boxes in the promoters of arginine biosynthetic and catabolic operons. AhrC is a 100 kDa homohexamer, with each subunit having two domains. The C-terminal domains form the core, mediating intersubunit interactions and binding of the co-repressor L-arginine, whilst the N-terminal domains contain a winged helix-turn-helix DNA-binding motif and are arranged around the periphery. The N-terminal domain of AhrC has been expressed, purified and characterized and it has been shown that the fragment still binds DNA operators as a recombinant monomer. The DNA-binding domain has also been crystallized and the crystal structure refined to 1.0 A resolution is presented.

Structure of the C-terminal effector-binding domain of AhrC bound to its corepressor L-arginine.

The arginine repressor/activator protein (AhrC) from Bacillus subtilis belongs to a large family of multifunctional transcription factors that are involved in the regulation of bacterial arginine metabolism. AhrC interacts with operator sites in the promoters of arginine biosynthetic and catabolic operons, acting as a transcriptional repressor at biosynthetic sites and an activator of transcription at catabolic sites. AhrC is a hexamer of identical subunits, each having two domains. The C-terminal domains form the core of the protein and are involved in oligomerization and L-arginine binding. The N-terminal domains lie on the outside of the compact core and play a role in binding to 18 bp DNA operators called ARG boxes. The C-terminal domain of AhrC has been expressed, purified and characterized, and also crystallized as a hexamer with the bound corepressor L-arginine. Here, the crystal structure refined to 1.95 A is presented.

Streptomycin production by Streptomyces griseus can be modulated by a mechanism not associated with change in the adpA component of the A-factor cascade.

In Streptomyces coelicolor, AtrA is an activator of transcription of the actinorhodin cluster-situated regulator gene actII-ORF4. In previous work, we showed that S. coelicolor AtrA binds in vitro to the promoter of S. griseus strR, the streptomycin cluster-situated regulator. We show here that S. griseus carries a single close homologue of atrA and that expression of S. coelicolor AtrA in S. griseus causes a DNA binding-dependent reduction in streptomycin production and in the mRNA levels of strR and genes of streptomycin biosynthesis. However, there is no effect on the level of the mRNA of adpA, which is the only transcription factor that has so far been characterised for strR. The adpA gene is directly regulated by ArpA, the receptor protein for the gamma-butyrolactone signalling molecule A-factor. Therefore, to our knowledge, our results provide the first in vivo evidence that A-factor-ArpA-AdpA-StrR regulatory cascade represents only part of the full complexity of regulation of streptomycin biosynthesis in S. griseus. The potential biotechnological application of our findings is discussed.

Transcriptional activation of the pathway-specific regulator of the actinorhodin biosynthetic genes in Streptomyces coelicolor.

The Streptomyces produce a plethora of secondary metabolites including antibiotics and undergo a complex developmental cycle. As a means of establishing the pathways that regulate secondary metabolite production by this important bacterial genus, the model species Streptomyces coelicolor and its relatives have been the subject of several genetic screens. However, despite the identification and characterization of numerous genes that affect antibiotic production, there is still no overall understanding of the network that integrates the various environmental and growth signals to bring about changes in the expression of biosynthetic genes. To establish new links, we are taking a biochemical approach to identify transcription factors that regulate antibiotic production in S. coelicolor. Here we describe the identification and characterization of a transcription factor, designated AtrA, that regulates transcription of actII-ORF4, the pathway-specific activator of the actinorhodin biosynthetic gene cluster in S. coelicolor. Disruption of the corresponding atrA gene, which is not associated with any antibiotic gene cluster, reduced the production of actinorhodin, but had no detectable effect on the production of undecylprodigiosin or the calcium-dependent antibiotic. These results indicate that atrA has specificity with regard to the biosynthetic genes it influences. An orthologue of atrA is present in the genome of Streptomyces avermitilis, the only other streptomycete for which there is a publicly available complete sequence. We also show that S. coelicolor AtrA can bind in vitro to the promoter of strR, a transcriptional activator unrelated to actII-ORF4 that is the final regulator of streptomycin production in Streptomyces griseus. These findings provide further evidence that the path leading to the expression of pathway-specific activators of antibiotic biosynthesis genes in disparate Streptomyces may share evolutionarily conserved components in at least some cases, even though the final activators are not related, and suggests that the regulation of streptomycin production, which serves an important paradigm, may be more complex than represented by current models.

Collection and characterisation of bacterial membrane proteins.

A general strategy for the amplified expression in Escherichia coli of membrane transport and receptor proteins from other bacteria is described. As an illustration we report the cloning of the putative alpha-ketoglutarate membrane transport gene from the genome of Helicobacter pylori, overexpression of the protein tagged with RGS(His)6 at the C-terminus, and its purification in mg quantities. The retention of structural and functional integrity was verified by circular dichroism spectroscopy and reconstitution of transport activity. This strategy for overexpression and purification is extended to additional membrane proteins from H. pylori and from other bacteria.

Benzothiazole degradation by Rhodococcus pyridinovorans strain PA: evidence of a catechol 1,2-dioxygenase activity.

The pathway for biodegradation of benzothiazole (BT) and 2-hydroxybenzothiazole (OBT) by Rhodococcus pyridinovorans strain PA was studied in detail. The kinetics of biodegradation were monitored by in situ (1)H nuclear magnetic resonance (NMR) in parallel with reversed-phase high-performance liquid chromatography (HPLC). Successive oxidations from BT to OBT and then from OBT to dihydroxybenzothiazole were observed. Further insight was obtained by using a mutant strain with impaired ability to grow on BT and OBT. The precise structure of another intermediate was determined by in situ two-dimensional (1)H-(13)C NMR and HPLC-electrospray ionization mass spectrometry; this intermediate was found to be a ring-opening product (a diacid structure). Detection of this metabolite, together with the results obtained by (1)H and (19)F NMR when cells were incubated with 3-fluorocatechol, demonstrated that a catechol 1,2-dioxygenase is involved in a pathway for biodegradation of BTs in this Rhodococcus strain. Our results show that catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities may both be involved in the biodegradation of BTs depending on the culture conditions.

End-product control of expression of branched-chain amino acid biosynthesis genes in Streptomyces coelicolor A3(2): paradoxical relationships between DNA sequence and regulatory phenotype.

The branched-chain protein amino acids isoleucine, valine and leucine can provide precursors for synthesis of complex polyketide secondary metabolites in streptomycetes; therefore the regulation of their own synthesis is of interest. DNA sequences upstream of ilvBNC, ilvD, leuA, leuB, ilvE and leuCD in Streptomyces coelicolor A3(2) have been obtained in this laboratory or as part of the S. coelicolor genome sequencing project. Upstream of ilvB and leuA, typical features of classical attenuator systems can be discerned, in particular hypothetical short ORFs with runs of Ile/Val/Leu and Leu codons, respectively. No such features are apparent upstream of other genes or gene clusters present. All five upstream regions were fused to xylE (encoding catechol dioxygenase, CO) as a reporter gene in the SCP2*-based low-copy-number vector pIJ2839. All wild-type regions showed strong depression of CO activity in the presence of all three branched-chain amino acids whether or not the attenuation features were present. By site-directed mutagenesis, the Ile/Val/Leu and Leu triplets in the putative attenuator peptides for ilvB and leuA were replaced by ones for other amino acids. In the case of ilvB, this had no effect at all; for leuA, the wild-type regulatory phenotype persisted in at least some experiments. It was concluded that (i) an unknown regulatory mechanism must be operating in the ilv/leu system of S. coelicolor A3(2) in place of classical attenuation; and (ii) it is unsafe to infer the functioning of a regulatory mechanism from sequence homologies alone.