Colin P. Smith

Structure, Biosynthetic Origin, and Engineered Biosynthesis of Calcium-Dependent Antibiotics from Streptomyces coelicolor

The calcium-dependent antibiotic (CDA), from Streptomyces coelicolor, is an acidic lipopeptide comprising an N-terminal 2,3-epoxyhexanoyl fatty acid side chain and several nonproteinogenic amino acid residues. S. coelicolor grown on solid media was shown to produce several previously uncharacterized peptides with C-terminal Z-dehydrotryptophan residues. The CDA biosynthetic gene cluster contains open reading frames encoding nonribosomal peptide synthetases, fatty acid synthases, and enzymes involved in precursor supply and tailoring of the nascent peptide. On the basis of protein sequence similarity and chemical reasoning, the biosynthesis of CDA is rationalized. Deletion of SCO3229 (hmaS), a putative 4-hydroxymandelic acid synthase-encoding gene, abolishes CDA production. The exogenous supply of 4-hydroxymandelate, 4-hydroxyphenylglyoxylate, or 4-hydroxyphenylglycine re-establishes CDA production by the DeltahmaS mutant. Feeding analogs of these precursors to the mutant resulted in the directed biosynthesis of novel lipopeptides with modified arylglycine residues.

Substrate induction and catabolite repression of the Streptomyces coelicoior glycerol operon are mediated through the GyIR protein

The pathway for glycerol catabolism In Streptomyces coelicolor is determined by the gylCABX operon, which is transcribed from two closely spaced glycerol‐inducible, glucose‐repressible promoters. Glucose (or catabolite) repression of gyl is known to be exerted by a general catabolite repression system In which the soluble glucose kinase plays a central role. The gylR gene is contained in a separate glycerol‐inducible, weakly glucose‐repressible transcription unit immediately upstream from the gyl operon. The role of gylR in the regulation of gyl transcription was assessed by introducing specific null mutations into the chromosomal gylR gene. Direct quantification of gyl transcripts from the gylR null mutants grown on different carbon sources demonstrated that GylR is the repressor of the gylCABX operon and also revealed that GylR functions as a negative autoregulator. Moreover, the transcriptional analysis revealed that the gylR null mutants were relieved of glucose repression of both gylCABX and gylR. We conclude that both substrate induction and catabolite repression of gyl are mediated through the GylR protein. This is the first direct evidence that catabolite repression In Streptomyces Is not exerted at the transcriptional level by a general ‘catabolite repressor protein’. Models for catabolite repression are discussed.

THE DNAK OPERON OF STREPTOMYCES-COELICOLOR ENCODES A NOVEL HEAT-SHOCK PROTEIN WHICH BINDS TO THE PROMOTER REGION OF THE OPERON

Transcriptional studies have demonstrated that the dnaK gene of Streptomyces coelicolor A3(2) is contained within a 4.3 kb operon. The operon is transcribed from a single (transiently) heat‐inducible promoter, dnaKp, that resembles the typical vegetative (σ70‐recognized) eubacterial consensus promoter sequence. dnaK transcription was found to be heat‐inducible at all stages of development in surface‐grown cultures. In addition, at the normal growth temperature of 30°C, dnaK transcript levels were shown to vary at different stages of development, being more abundant in young germinating cultures and in mycelium undergoing sporogenesis. The nucleotide sequence of the dnaK operon has been completed, revealing the gene organization 5′‐dnaK‐grpE‐dnaJ‐orfX. orfX represents a novel heat‐shock gene. Its predicted product displays high similarity to the GlnR repressor proteins of Bacillus spp. and to the MerR family of eubacterial transcriptional regulators. The S. coelicolor OrfX protein has been over‐produced in Escherichia coli, and DNA‐binding experiments indicate that it interacts specifically with the dnaKp region, binding to three partially related inverted repeat sequences; they are centred at −75, −49 and +4, respectively, relative to the transcription start site of the operon. These results suggest that OrfX plays a direct role in the regulation of the dnaK operon.

The HspR regulon of Streptomyces coelicolor: a role for the DnaK chaperone as a transcriptional co‐repressor†

The dnaK operon of Streptomyces coelicolor encodes the DnaK chaperone machine and HspR, the transcriptional repressor of the operon; HspR confers repression by binding to several inverted repeat sequences in the promoter region, dnaKp. Here, we demonstrate that HspR specifically requires the presence of DnaK protein to retard a dnaKp fragment in gel‐shift assays. This requirement is independent of the co‐chaperones, DnaJ and GrpE, and it is ATP independent. Furthermore the retarded protein–DNA complex can be ‘supershifted’ by anti‐DnaK monoclonal antibody, demonstrating that DnaK forms an integral component of the complex. It was shown in DNase I footprinting experiments that refolding and specific binding of HspR to its DNA target does not require DnaK. We conclude that the formation of the stable DnaK–HspR–DNA ternary complex does not depend on the chaperoning activity of DnaK. In affinity chromatography experiments using whole‐cell extracts, DnaK was shown to co‐purify with HspR, providing additional evidence that the two proteins interact in vivo; it was not possible to purify HspR away from DnaK in any experiments unless a powerful denaturant was used. The level of heat shock induction of chromosomal DnaK could be partially suppressed by expressing dnaK extrachromosomally from a heterologous promoter. In addition, it is shown that DnaK confers enhanced HspR‐mediated repression of transcription in vitro. Taken together, these results suggest that DnaK functions as a transcriptional co‐repressor by binding to HspR at its operator sites. In this model, the DnaK–HspR system would represent a novel example of feedback regulation of gene expression by a molecular chaperone, in which DnaK directly activates a repressor, rather than inactivates an activator (as is the case in the DnaK–σ32 and Hsp70–HSF systems of other organisms).

Sequence and transcriptional analysis of the nourseothricin acetyltransferase-encoding gene nat1 from Streptomyces noursei

We have determined the nucleotide (nt) sequence of nat1, a gene encoding nourseothricin (Nc) acetyltransferase (AT) from Streptomyces noursei, and its transcriptional start point (tsp). The nt sequence upstream from the coding region is completely different from that of the stat gene (encoding streptothricin AT) from Streptomyces lavendulae [S. Horinouchi, K. Furuya, M. Nishiyama, H. Suzuki and T. Beppu, J. Bacteriol. 169 (1987) 1929-1937], even though the nt sequences of the two genes and the deduced amino acid (aa) sequences of the two enzymes show a high degree of similarity. Another stat gene, derived from a Gram-negative plasmid, showed only deduced aa similarity, but not nt sequence similarity, to the above two. A database search for related aa sequences did not reveal any clear-cut homologies to other types of protein. A multiple aa sequence alignment of several ATs is presented.

Physical identification of a chromosomal locus encoding biosynthetic genes for the lipopeptide calcium-dependent antibiotic (CDA) of Streptomyces coelicolor A3(2)

Putative peptide-synthetase-encoding DNA fragments were isolated from the Streptomyces coelicolor A3(2) chromosome using a PCR-based approach and mapped to a single approximately 35 kb segment. In integrative transformation experiments, DNA fragments from this region disrupted production of the calcium-dependent antibiotic (CDA) and had sequences characteristic of non-ribosomal peptide synthetases, thus proving that the cda locus had been cloned.

A ‘Gram-negative-type’ DNA polymerase III is essential for replication of the linear chromosome of Streptomyces coelicolor A3(2)

The Streptomyces coelicolor dnaE gene, encoding the catalytic α‐subunit of DNA polymerase III (pol III) was isolated by genetic complementation of a temperature‐sensitive DNA replication mutant, S. coelicolor ts‐38. The deduced protein sequence (1179 residues) is highly similar to the Escherichia coli‐type pol III α‐subunit, rather than to the PolC‐type α‐subunit that is known to be essential for replication in the ‘low Gu2003+u2003C’ Gram‐positive bacteria such as Bacillus subtilisThe dnaE gene is able to restore replication to a ‘slow stop’ mutant (ts‐38) and a ‘fast stop’ mutant (ts‐114); the dnaE gene of ts‐38 carries a single amino acid substitution (Glu‐802 to Lys), and the mutation in ts‐114 has been mapped between codons 697 and 1062 of dnaE. Mutant ts‐38 is considered to be defective in assembly of the multisubunit pol III holoenzyme and, hence, in initiation of replication, whereas ts‐114 is defective in chain elongation. This study provides the first evidence that a DnaE‐type pol III is essential for replication in a Gram‐positive bacterium. In addition, the complementation studies suggest that the C‐terminal 117 residues are not essential for DnaE function in S. coelicolor. When integrated at a distant site on the chromosome, a fragment containing the 3′ half of dnaE (codons 697–1179) is capable of rescuing ts‐38 (but not ts‐114) at the restrictive temperature; it was demonstrated that homogenotization was responsible for this phenomenon.

Streptomyces coelicolor A3(2): from genome sequence to function

Abstract The huge genome (8.7 Mb) and developmental complexity of Streptomyces coelicolor A3(2) present significant challenges for functional genomics. Nevertheless, effective microarrays of PCR-generated gene fragments have been generated in two laboratories, and significant information is accruing from proteome analysis. Genomewide transposon mutagenesis and highly efficient PCR-targeted gene disruption are making genetic intervention straightforward.

Investigations of Expression and Protein Sequence of the Aminoacetyltransferase Gene Nat1 from Streptomyces Noursei

Nourseothricin (Nc) belongs to the group of streptothricin (St) antibiotics that includes grisin and racemomycin, which exhibit antibacterial, antifungal and antiviral activity (Bocker and Bergter, 1986). Although they are not used therapeutically they give remarkable effects as fodder additives in animal husbandry (Bocker and Bergter, 1986). Resistance against the streptothricins is mediated via acetylation (Keeratipibul, 1983, Tschaepe et al., 1984, Haupt et al., 1986). From NMR studies Kobayashi et al. (1987) found that in Streptomyces lavendulae this involves a monoacetylation of the beta-amino-group of the beta-lysine moiety of the streptothricin. The gene (stat) for this enzymatic activity was cloned and sequenced and its transcription start shown to be near the translational start (Horinouchi et al., 1987). In addition, a transposon-specified St-acetyltransferase (SAT) was identified in enterobacteria isolated from animals treated with Nc (Tschaepe et al., 1984). The nucleotide sequence of this gene was determined by Hein et al. (1989). From Streptomyces noursei a gene (nat)1 conferring resistanc to Nc via acetylation was cloned as plasmid pNAT1 and analyzed (Krugel et al., 1988).