- Volume 145, Issue 9, 1999

Four adjacent genes (sipW, sipX, sipY and sipZ) encoding different type I signal peptidases, were isolated on a 7860 bp DNA fragment from Streptomyces lividans TK21. Three of the sip genes constitute an operon and the fourth is the first gene of another operon encompassing three additional, unrelated genes. A DNA fragment containing the four sip genes complemented an Escherichia coli type I signal peptidase mutant when cloned in a multicopy plasmid. Clustering of four different type I signal peptidase genes seems, so far, to be a unique feature of Streptomyces.

To identify negative regulatory genes for cellular differentiation in Streptomyces griseus, DNA fragments repressing the normal developmental processes were cloned on a high-copy-number plasmid. One of these DNA fragments markedly repressed aerial mycelium and spore formation on solid media containing glucose or galactose, but not on media containing maltose or mannitol. The fragment contained three complete ORFs; precise subcloning revealed that a 249 bp fragment located in the promoter region between ORF1 and ORF3 was sufficient for repression. Quantification of the promoter activities by using a thermostable malate dehydrogenase gene as a reporter showed that the promoter for ORF3 (PORF3) maintained high activity in mycelia grown in the presence of glucose but lost activity rapidly in maltose medium. PORF3 activity increased markedly when the promoter sequence was introduced on a high-copy-number plasmid. The results suggested that carbon-source-dependent deactivation of PORF3 mediated by a transcriptional repressor may initiate differentiation in S. griseus.

amfC plays a regulatory role in aerial mycelium formation in Streptomyces griseus and is distributed widely among Streptomyces species. Disruption of the chromosomal amfC gene in Streptomyces coelicolor A3(2) severely reduced formation of aerial hyphae, indicating that amfC is important in morphological development. In addition, the disruption caused S. coelicolor A3(2) M130 to produce much less actinorhodin, and to produce undecylprodigiosin at a later stage of growth, indicating that amfC also regulates secondary metabolism. S1 nuclease mapping showed that transcription of actII-ORF4, the pathway-specific transcriptional activator in the act gene cluster, was greatly reduced in the amfC disruptants. The defect in secondary metabolite formation was suppressed or overcome by a mutation in sre-1. Consequently, an amfC-disrupted strain derived from S. coelicolor A3(2) M145, an actinorhodin-overproducing strain due to the sre-1 mutation, still produced a large amount of actinorhodin. Extra copies of amfC in strains M130 and M145 did not change spore-chain morphology or secondary metabolite formation. However, the spores in these strains remained white even after prolonged incubation. Since only spore pigmentation was affected, all known whi genes, except whiE, responsible for the polyketide spore pigment formation, were assumed to function normally. S1 nuclease mapping showed that transcription of whiEP1, one of the promoters in the whiE locus, was reduced in S. coelicolor A3(2) containing extra copies of amfC. Introducing amfC into several other Streptomyces species, such as Streptomyces lividans, Streptomyces lavendulae and Streptomyces lipmanii, also abolished spore pigment formation. An increase in the amount of AmfC appeared to disturb the maturation of spores.

In Streptomyces coelicolor A3(2), a protein serine/threonine kinase (AfsK) and its target protein (AfsR) control secondary metabolism. AfsK and AfsR homologues (AfsK-g and AfsR-g) from Streptomyces griseus showed high end-to-end similarity in amino acid sequence with the respective S. coelicolor A3(2) proteins, as determined by cloning and nucleotide sequencing. AfsK-g and a fusion protein between AfsK-g and thioredoxin (TRX–AfsK-g) produced in high yield as inclusion bodies in Escherichia coli were solubilized with urea, purified by column chromatography and then refolded to an active form by dialysis to gradually remove the urea. AfsR-g was also fused to glutathione S-transferase (GST–AfsR-g); the fusion product in the soluble fraction in E. coli was purified. Incubation of AfsK-g or TRX–AfsK-g in the presence of [γ-32P]ATP yielded autophosphorylated products containing phosphoserine and phosphothreonine residues. In addition, TRX–AfsK-g phosphorylated serine and threonine residues of GST–AfsR-g in the presence of [γ-32P]ATP. Disruption of chromosomal afsK-g had no effect on A-factor or streptomycin production, irrespective of the culture conditions. The afsK-g disruptants did not form aerial mycelium or spores on media containing glucose at concentrations higher than 1%, but did form spores on mannitol- and glycerol-containing media; this suggests that afsK-g is essential for morphogenesis in the presence of glucose. Introduction of afsK-g restored aerial mycelium formation in the disruptants. The phenotype of afsR-g disruptants was similar to that of afsK-g disruptants; introduction of afsR-g restored the defect in aerial mycelium formation on glucose-containing medium. Thus the AfsK/AfsR system in S. griseus is conditionally needed for morphological differentiation, whereas in S. coelicolor A3(2) it is conditionally involved in secondary metabolism.

The tsf genes from Streptomyces coelicolor A3(2) and Streptomyces ramocissimus, encoding the guanine-nucleotide exchange factor EF-Ts, were cloned and sequenced. Streptomycetes have multiple and highly divergent EF-Tu species, with EF-Tu1 and EF-Tu3 showing only about 65% amino acid sequence identity, and yet these can apparently interact with a single EF-Ts species. tsf lies in an operon with rpsB, which encodes ribosomal protein S2. The amino acid sequence of S2 from S. coelicolor differs from most other bacterial S2 homologues in having a C-terminal extension of 70 aa residues with a highly repetitive organization, the function of which is unknown. Transcription analysis of the rpsB–tsf operon of S. coelicolor by promoter probing, nuclease S1 mapping and Northern blotting revealed that the genes give rise to a bicistronic transcript from a single promoter upstream of rpsB. An attenuator was identified in the rpsB–tsf intergenic region; it results in an approximately 2:1 ratio of rpsB vs tsf transcripts. Although tuf1, encoding the major EF-Tu, is located in the rpsL ribosomal protein operon, an additional promoter in the fus–tuf1 intergenic region leads to a significant excess of EF-Tu over ribosomes. Most amino acid residues known from the Escherichia coli crystal structure of the EF-Tu·EF-Ts complex to be directly involved in interaction between the two elongation factors are conserved between E. coli and Streptomyces. However, whenever interaction residues in the EF-Tu moiety show divergence among Streptomyces EF-Tu1, EF-Tu2 and EF-Tu3, the single Streptomyces EF-Ts exhibits compensatory substitutions of the corresponding residues. These apparently enable productive interaction to occur with all three EF-Tus.

In a transposition mutant of Streptomyces lividans TK24, the usually glucose-repressible expression of a heterologous α-amylase gene (aml) became resistant to glucose repression. The transposon had inserted into an ORF called sblA which encodes a 274 aa product sharing significant sequence similarities with various phosphatases that act on small phosphorylated substrates. sblA was transcribed as a monocistronic mRNA and its transcription was enhanced at the transition phase. Because its transcriptional and putative translational start points coincide, sblA is likely to be translated in the absence of a conventional RBS. The sblA-disrupted mutant is characterized by early growth arrest in glucose-grown cultures and by partial relief of glucose repression of aml expression.

An internal adenylyltransferase gene (glnE) fragment from Streptomyces coelicolor was amplified using heterologous PCR primers derived from consensus motifs. The sequence had significant similarity to bacterial glnE genes, and included a motif typical of the C-terminal adenylyltransferase domain of GlnE. glnE from S. coelicolor lies on the AseI-C fragment of the chromosome and is localized near glnA (encoding glutamine synthetase I, GSI) and glnII (encoding GSII). To analyse the function of GlnE in S. coelicolor, glnE (S. coelicolor E4) and glnA (S. coelicolor HT107) gene replacement mutants were constructed. The GSI activity of the glnE mutant was not down-regulated after an ammonium shock. However, the GSI activity of the wild-type cells decreased to 60% of the original activity. The glnA mutant is not glutamine auxotrophic, but in the γ-glutamyltransferase assay no GSI activity was detected in unshifted and shifted HT107 cells. By snake venom phosphodiesterase treatment the GSI activity in the wild-type can be reconstituted, whereas no alteration is observed in the E4 mutant. Additionally, the loss of short-term GSI regulation in the E4 mutant was accompanied by an increased glutamine:glutamate ratio.

The cloning, using a PCR approach, of genes from both Streptomyces coelicolor and Streptomyces avermitilis encoding an acyl-CoA dehydrogenase (AcdH), putatively involved in the catabolism of branched-chain amino acids, is reported. The deduced amino acid sequences of both genes have a high similarity to prokaryotic and eukaryotic short-chain acyl-CoA dehydrogenases. When the S. coelicolor and S. avermitilis acyl-CoA dehydrogenase genes (acdH) were expressed in Escherichia coli, each of the AcdH flavoproteins was able to oxidize the branched-chain acyl-CoA derivatives isobutyryl-CoA, isovaleryl-CoA and cyclohexylcarbonyl-CoA, as well as the short straight-chain acyl-CoAs n-butyryl-CoA and n-valeryl-CoA in vitro. NMR spectral data confirmed that the oxidized product of isobutyryl-CoA is methacrylyl-CoA, which is the expected product at the acyl-CoA dehydrogenase step in the catabolism of valine in streptomycetes. Disruption of the S. avermitilis acdH produced a mutant unable to grow on solid minimal medium containing valine, isoleucine or leucine as sole carbon sources. Feeding studies with 13C triple-labelled isobutyrate revealed a significant decrease in the incorporation of label into the methylmalonyl-CoA-derived positions of avermectin in the acdH mutant. In contrast the mutation did not affect incorporation into the malonyl-CoA-derived positions of avermectin. These results are consistent with the acdH gene encoding an acyl-CoA dehydrogenase with a broad substrate specificity that has a role in the catabolism of branched-chain amino acids in S. coelicolor and S. avermitilis.

Modular polyketide synthases (PKSs) are a large family of multifunctional enzymes responsible for the biosynthesis of numerous bacterial natural products such as erythromycin and rifamycin. Advanced genetic analysis of these remarkable systems is often seriously hampered by the large size (>40 kb) of PKS gene clusters, and, notwithstanding their considerable fundamental and biotechnological significance, by the lack of suitable methods for engineering non-selectable modifications in chromosomally encoded PKS genes. The development of a facile host–vector strategy for genetic engineering of the rifamycin PKS in the producing organism, Amycolatopsis mediterranei S699, is described here. The genes encoding all 10 modules of the rifamycin PKS were replaced with a hygromycin-resistance marker gene. In a similar construction, only the first six modules of the PKS were replaced. The deletion hosts retained the ability to synthesize the primer unit 3-amino-5-hydroxybenzoic acid (AHBA), as judged by co-synthesis experiments with a mutant strain lacking AHBA synthase activity. Suicide plasmids carrying a short fragment from the 5′ flanking end of the engineered deletion, an apramycin-resistance marker gene, and suitably engineered PKS genes could be introduced via electroporation into the deletion hosts, resulting in the integration of PKS genes and biosynthesis of a reporter polyketide in quantities comparable to those produced by the wild-type organism. Since this strategy for engineering recombinant PKSs in A. mediterranei requires only a selectable single crossover and eliminates the need for tedious non-selectable double-crossover experiments, it makes rifamycin PKS an attractive target for extensive genetic manipulation.

The absA locus in Streptomyces coelicolor A3(2) was identified because mutations in it uncoupled sporulation from antibiotic synthesis: absA mutants failed to produce any of the four antibiotics characteristic of S. coelicolor. These mutants are now shown to contain point mutations in the absA1 gene which encodes the histidine kinase sensor-transmitter protein of a two-component signalling system. The absA1 non-antibiotic-producing mutants, which are unpigmented, spontaneously acquire pigmented colony sectors. Genetic analysis established that the pigmented sectors contain second-site suppressive mutations, sab (for suppressor of ab s). Phenotypic characterization showed that sab strains produce all four antibiotics; some overproduce antibiotics and are designated Pha, for precocious hyperproduction of antibiotics. A set of sab mutations responsible for suppression was localized by plasmid-mediated and protoplast fusion mapping techniques to the vicinity of the absA locus. DNA cloned from this region was used to construct phage that could transduce sab mutations. Sequence analysis of sab strains defined sab mutations in both the absA1 gene and the absA2 gene; the latter encodes the two-component system’s response regulator.

Streptomyces ambofaciens produces the macrolide antibiotic spiramycin, an inhibitor of protein synthesis, and possesses multiple resistance mechanisms to the produced antibiotic. Several resistance determinants have been isolated from S. ambofaciens and studies with one of them, srmA, which hybridized with ermE (the erythromycin-resistance gene from Saccharopolyspora erythraea), are detailed here. The nucleotide sequence of srmA was determined and the mechanism by which its product confers resistance was characterized. The SrmA protein is a methyltransferase which introduces a single methyl group into A-2058 (Escherichia coli numbering scheme) in the large rRNA, thereby conferring an MLS (macrolide–lincosamide–streptogramin type B) type I resistance phenotype. A mutant of S. ambofaciens in which srmA was inactivated was viable and still produced spiramycin, indicating that srmA is dispensable, at least in the presence of the other resistance determinants.

A region of the Streptomyces coelicolor A3(2) chromosome was identified and cloned by using as a probe the lipase gene from Streptomyces exfoliatus M11. The cloned region consisted of 6286 bp, and carried a complete lipase gene, lipA, as well as a gene encoding a transcriptional activator (lipR). The S. coelicolor A3(2) lipA gene encodes a functional extracellular lipase 82% identical to the S. exfoliatus M11 lipase; the partially purified S. coelicolor enzyme showed a preference for substrates of short to medium chain length. Transcription of lipA was completely dependent on the presence of lipR, and occurred from a single promoter similar to the lipA promoters of S. exfoliatus M11 and Streptomyces albus G. These three Streptomyces lipA promoters have well-conserved −10 and −35 regions, as well as additional conserved sequences upstream of the −35 region, which could function as targets for transcriptional activation by the cognate LipR regulators. The Streptomyces LipR activators are related to other bacterial regulators of a similar size, constituting a previously unidentified family of proteins that includes MalT, AcoK, AlkS, AfsR, five mycobacterial proteins of unknown function and some Streptomyces regulators in antibiotic synthesis clusters. A lipase-deficient strain of S. coelicolor was constructed and found to be slightly affected in production of the polyketide antibiotic actinorhodin.

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.

In Streptomyces albus, Hsp18, a protein belonging to the family of small heat-shock proteins, can be detected only at high temperature. Disruption of orfY, located upstream and in the opposite orientation to hsp18, resulted in an elevated level of hsp18 mRNA at low temperature. Genetic and biochemical experiments indicated that the product of orfY, now called RheA (Repressor of h sp eighteen), directly represses hsp18. In Escherichia coli, an hsp18′–bgaB transcriptional fusion was repressed in a strain expressing S. albus RheA. DNA-binding experiments with crude extracts of E. coli overproducing RheA indicated that RheA interacts specifically with the hsp18 promoter. Transcription analysis of rheA in the S. albus wild-type and in rheA mutant strains suggested that RheA represses transcription not only of hsp18 but also of rheA itself.

The genetics of non-oxidative decarboxylation of aromatic acids are poorly understood in both prokaryotes and eukaryotes. Although such reactions have been observed in numerous micro-organisms acting on a variety of substrates, the genes encoding enzymes responsible for these processes have not, to our knowledge, been reported in the literature. Here, the isolation of a streptomycete from soil (Streptomyces sp. D7) which efficiently converts 4-hydroxy-3-methoxybenzoic acid (vanillic acid) to 2-methoxyphenol (guaiacol) is described. Protein two-dimensional gel analysis revealed that several proteins were synthesized in response to vanillic acid. One of these was characterized by partial amino-terminal sequencing, leading to the cloning of a gene cluster from a genomic DNA lambda phage library, consisting of three ORFs, vdcB (602 bp), vdcC (1424 bp) and vdcD (239 bp). Protein sequence comparisons suggest that the product of vdcB (201 aa) is similar to phenylacrylate decarboxylase of yeast; the putative products of vdcC (475 aa) and vdcD (80 aa) are similar to hypothetical proteins of unknown function from various micro-organisms, and are found in a similar cluster in Bacillus subtilis. Northern blot analysis revealed the synthesis of a 2·5 kb mRNA transcript in vanillic-acid-induced cells, suggesting that the cluster is under the control of a single inducible promoter. Expression of the entire vdc gene cluster in Streptomyces lividans 1326 as a heterologous host resulted in that strain acquiring the ability to decarboxylate vanillic acid to guaiacol non-oxidatively. Both Streptomyces sp. strain D7 and recombinant S. lividans 1326 expressing the vdc gene cluster do not, however, decarboxylate structurally similar aromatic acids, suggesting that the system is specific for vanillic acid. This catabolic system may be useful as a component for pathway engineering research focused towards the production of valuable chemicals from forestry and agricultural by-products.

Quantitative experiments on the interaction of Salmonella choleraesuis and Salmonella dublin with porcine and bovine intestinal epithelia yielded no evidence to suggest that host restriction of S. choleraesuis and S. dublin for pigs and calves respectively could be explained in terms of the patterns of intestinal invasion observed in ligated ileal loops in vivo, at 3 h after challenge. No evidence was found to support the idea that Peyer’s patches, or specifically M cells, are the major route of entry for these serotypes in vivo. Three hours after loop inoculation, each serotype was recovered in comparable numbers from either absorptive or Peyer’s patch mucosae present in the same ileal loop, indicating that both types of tissue are involved in the early stages of the enteropathogenic process induced by both serotypes. More detailed transmission electron microscopic (TEM) analyses of follicle-associated epithelia (FAE) challenged with S. choleraesuis showed that in the same region of FAE, organisms invaded both M cells and enterocytes directly; comparable detailed TEM studies with S. dublin could not be carried out because of the tissue-destructive properties of this serotype. S. dublin was clearly more histotoxic than S. choleraesuis as had previously been found in rabbits: this difference is almost certainly due to a tissue-damaging toxin which is neither host nor gut-tissue specific. The tissue-destructive potential of S. dublin has profound implications for the measurement of and the assignment of significance to the invasiveness of S. dublin. S. dublin was nearly always seen entering gut cells in micro-colonies whereas S. choleraesuis entered mainly as single organisms or small groups of two or three.

Each of 314 strains of Pseudomonas aeruginosa recovered from 87 French cystic fibrosis (CF) patients was typed by multilocus enzyme electrophoresis to investigate the genetic diversity, the relatedness and the molecular epidemiology of strains isolated from cases of chronic pulmonary colonization. Comparison of allele profiles at 18 enzyme loci identified 17 electrophoretic types (ETs). Of the 314 isolates, 290 (92%) were either ET1 (n=127) or ET2 (n=163), which differed only at the shikimate dehydrogenase (SKD) locus. The mean genetic diversity (H) was 0·138. These results suggest that there is cross-colonization between patients and/or that two predominant groups of strains are able to colonize French CF patients. Sequential isolates collected from 18 patients during a period of 12–28 months were analysed to assess genomic variability and its relationship to clinical outcome. Six patients were colonized by a stable strain. For the others, double infections or changes in colonization over time were observed. No relationships were detected between the clinical outcome and the persistence of stable isolates, the emergence of transient superinfecting variants, the presence of multiple ETs or the shift of ET during the monitoring.