Janet White

Transcriptional regulation of the redD transcriptional activator gene accounts for growth-phase-dependent production of the antibiotic undecylprodigiosin in Streptomyces coelicolor A3(2)

Transcription of redD, the activator gene required for production of the red‐pigmented antibiotic undecylprodigiosin by Streptomyces coelicolor A3(2), showed a dramatic increase during the transition from exponential to stationary phase. The increase in redD expression was followed by transcription of redX, a biosynthetic structural gene, and the appearance of the antibiotic in the mycelium, and coincided with the intracellular appearance of ppGpp. However, ppGpp production elicited either by nutritional shift‐down of, or addition of serine hydroxamate to, exponentially growing cultures had no stimulatory effect on redD transcription. The presence of redD on a multicopy plasmid resulted in elevated levels of the redD transcript and production of redX and undecylprodigiosin during exponential growth; the normal growth‐phase‐dependent production of undecylprodigiosin appeared to be mediated entirely through the redD promoter, which shows limited similarity to the consensus sequence for the major class of eubacterial promoters.

The mRNA for the 23S rRNA methylase encoded by the ermE gene of Saccharopolyspora erythraea is translated in the absence of a conventional ribosome‐binding site

Transcriptional analysis of the ermE gene of Saccharopolyspora erythraea, which confers resistance to erythromycin by N6‐dimethylation of 23S rRNA and which is expressed from two promoters, ermEp1 and ermEp2, revealed a complex regulatory region in which transcription is initiated in a divergent and overlapping manner. Two promoters (eryC1p1 and eryC1p2) were identified for the divergently transcribed erythromycin biosynthetic gene eryC1, which plays a role in the formation of desosamine or its attachment to the macrolide ring. Transcription from eryC1p2 starts at the same position as that of ermEp1, but on the opposite strand of the DNA helix, suggesting co‐ordinate regulation of genes for erythromycin production and resistance. ErmEp1 initiates transcription at, and one nucleotide before, the ermE translational start codon. Site‐directed and deletion mutagenesis, combined with immunochemical analysis, demonstrated that the ermEp1 transcript is translated in the absence of a conventional ribosome‐binding site to give rise to the full‐length 23S rRNA methylase. Deletion of the ‐35 region of ermEp1 reduced, but did not abolish, promoter activity, reminiscent of the‘extended ‐10’class of bacterial promoters which, like ermEp1, possess TGN motifs immediately upstream of their 10 regions and which initiate transcription seven nucleotides downstream of the ‐10 region.

Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacl-galR family of regulatory genes

malR of Streptomyces coelicolor A3(2) encodes a homologue of the LacI/GalR family of repressor proteins, and is divergently transcribed from the malEFG gene cluster, which encodes components of an ATP‐dependent transport system that is required for maltose utilization. Transcription of malE was induced by maltose and repressed by glucose. Disruption or deletion of malR resulted in constitutive, glucose‐insensitive malE transcription at a level markedly above that observed in the parental malR+ strain, and overproduction of MalR prevented growth on maltose as carbon source. Consequently, MalR plays a crucial role in both substrate induction and glucose repression of maltose utilization. malR is expressed from a single promoter with transcription initiating at the first G of the predicted GTG translation start codon.

A response-regulator-like activator of antibiotic synthesis from Streptomyces coelicolor A3(2) with an amino-terminal domain that lacks a phosphorylation pocket.

In Streptomyces coelicolor A3(2), bldA mutants that lack the tRNA for the rare leucine codon UUA fail to make the red undecylprodigiosin antibiotic complex. To find out why, red-pigmented while bald (Pwb) derivatives of a bldA mutant were isolated. Using a cloning strategy that allowed for (and demonstrated) dominance of the mutations, they were localized to the red gene cluster. By using insert-mediated integration of a phi C31 phage-based vector, one of the Pwb mutations was more precisely located between red structural genes to a segment of approximately 1 kb about 4 kb from the known pathway-specific regulatory gene redD. The segment contained most of an ORF (redZ) encoding a protein (RedZ) with end-to-end similarity to response regulators of diverse function from a variety of bacteria. Remarkably, in RedZ hydrophobic residues replace nearly all of the charged residues that usually make up the phosphorylation pocket present in typical response regulators, including the aspartic acid residue that is normally phosphorylated by a cognate sensory protein kinase. A single TTA codon in redZ provided a potential explanation for the bldA-dependence of undecylprodigiosin synthesis. This codon was unchanged in three Pwb mutants, but further analysis of one of the mutants revealed a potential up-promoter mutation. It seems possible that a combination of low-level natural translation of the UUA codon by a charged non-cognate tRNA, coupled with increased transcription of redZ in the Pwb mutant allows the accumulation of a threshold level of the RedD protein.

Cloning, characterization and disruption of a (p)ppGpp synthetase gene (relA) of Streptomyces coelicolor A3(2)

An internal segment of the (p)ppGpp synthetase gene, relA, of Streptomyces coelicolor A3(2) was amplified from genomic DNA using the polymerase chain reaction and used as a hybridization probe to isolate the complete gene from a cosmid library. relA lies downstream of a gene (apt) that apparently encodes adenine phosphoribosyltransferase and is transcribed from two promoters, relAp1 and relAp2, and by transcriptional readthrough from apt. While the level of relAp2 transcripts remained relatively constant, relAp1 activity apparently peaked during transition phase, following a decline in readthrough transcription from apt. Disruption of relA using an att− derivative of the temperate phage φC31 abolished ppGpp synthesis on amino acid depletion. When grown on agar, the disruptants grew more slowly than a control lysogen made with an att+ phage vector and gave smaller colonies that sporulated normally. The relA mutation had no consistent or marked effect on actinorhodin production in either liquid‐ or agar‐grown cultures, indicating that elevated levels of (p)ppGpp are not essential for triggering the onset of antibiotic production.

The malEFG gene cluster of Streptomyces coelicolor A3(2): characterization, disruption and transcriptional analysis

Abstract The malEFG gene cluster of the Gram-positive mycelial actinomycete Streptomyces coelicolor A3(2) was cloned and sequenced. MalEFG show only limited similarity to homologues involved in maltose and maltodextrin transport in other bacteria. Disruption of malE prevented the utilization of maltose as carbon source. Transcription of malE was induced by maltose and repressed by glucose.

The use of pNJ5000 as an intermediate vector for the genetic manipulation of Agrobacterium Ti-plasmids

The use of broad-host-range plasmids derived from RP4 as intermediate vectors for the transfer of narrow-host-range recombinant plasmids from Escherichia coli to Agrobacterium tumefaciens as a preliminary to marker exchange is described. Recombinant plasmids having a ColE1 type origin were linked to the RP4 derivative. Cointegrate formation appeared to take place by RecA-independent, homologous recombination within a short piece of DNA derived from the beta-lactamase gene of Tn1/Tn3 carried by both vector components, so that it never disrupted the recombinant portion of the construction. pNJ5000 provides an unstable intermediate vector for use in marker exchange experiments, while its stable relative pNJ1020 provides a carrier for use in binary vector systems.

The obligate aerobe Streptomyces coelicolor A3(2) synthesizes three active respiratory nitrate reductases

Streptomyces coelicolor A3(2) synthesizes three membrane-associated respiratory nitrate reductases (Nars). During aerobic growth in liquid medium the bacterium was able to reduce 50 mM nitrate stoichiometrically to nitrite. Construction and analysis of a mutant in which all three narGHJI operons were deleted showed that it failed to reduce nitrate. Deletion of the gene encoding MoaA, which catalyses the first step in molybdenum cofactor biosynthesis, also prevented nitrate reduction, consistent with the Nars being molybdoenzymes. In contrast to the triple narGHJI mutant, the moaA mutant was also unable to use nitrate as sole nitrogen source, which indicates that the assimilatory nitrate reductases in S. coelicolor are also molybdenum-dependent. Analysis of S. coelicolor growth on solid medium demonstrated that Nar activity is present in both spores and mycelium (hypha). Development of a survival assay with the nitrate analogue chlorate revealed that wild-type S. coelicolor spores and mycelium were sensitive to chlorate after anaerobic incubation, independent of the presence of nitrate, while both the moaA and triple nar mutants were chlorate-resistant. Complementation of the triple nar mutant with the individual narGHJI operons delivered on cosmids revealed that each operon encoded an enzyme that was synthesized and active in nitrate or chlorate reduction. The data obtained from these studies allow a tentative assignment of Nar1 activity to spores, Nar2 to spores and mycelium, and Nar3 exclusively to mycelium.

Transcriptional regulation of the four promoters of the agarase gene (dagA) of Streptomyces coelicolor A3(2)

The agarase gene (dagA) of Streptomyces coelicolor A3(2) is transcribed from four promoters that are recognized by at least three, and probably four, different RNA polymerase holoenzymes, each containing a different sigma factor. S1 nuclease protection studies revealed that transcription from all four promoters is induced by the products of agar hydrolysis and strongly repressed by glucose. Mutants deficient in glucose kinase activity were defective in glucose repression of all four promoters. Mutants were isolated or identified in which transcription from all four promoters had become inducer-independent (i.e. constitutive), establishing the existence of a repressor gene for dagA that does not appear to be located within 9 kb of the structural gene. The cloned dagA gene was also constitutively expressed in the closely related strain Streptomyces lividans, which does not normally make agarase and which presumably lacks the repressor gene. Glucose was still able to repress dagA transcription even under conditions of constitutive expression, suggesting that glucose kinase does not mediate its effect via inducer exclusion. Relative differences in the use of the four promoters were not detected during different stages of growth of surface-grown cultures, although dagA transcription appeared to peak during the production of aerial mycelium.

Nitrate respiration in the actinomycete Streptomyces coelicolor.

Streptomyces coelicolor is an obligate aerobic, filamentous soil-dwelling bacterium. Remarkably, the genome of S. coelicolor has three copies of the narGHJI operon that encodes respiratory nitrate reductase. This review summarizes our current views on the requirements for multiple nitrate reductases in S. coelicolor.