Fiona Flett

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.

PIGMENTED ANTIBIOTIC PRODUCTION BY STREPTOMYCES COELICOLOR A3(2) : KINETICS AND THE INFLUENCE OF NUTRIENTS

SUMMARY: The production of the pigments actinorhodin and undecylprodigiosin by Streptomyces coelicolor A3(2) was examined in a chemically defined medium which permits dispersed growth of the organism. The physiological controls on the production of the two pigments were markedly disparate. Actinorhodin production occurred mainly in the stationary phase of batch cultures grown with glucose and sodium nitrate as the principal carbon and nitrogen sources. In the same batch cultures, undecylprodigiosin accumulated during the exponential growth phase. The production of both pigments was sensitive to the levels of ammonium and phosphate in the medium. Actinorhodin production was exquisitely sensitive to ammonium concentration, and was completely inhibited by as little as 1 mM-ammonium chloride, whereas more than 50 mM-ammonium chloride was required to prevent undecylprodigiosin production. A similar, but less extreme effect was seen with phosphate: actinorhodin production was completely inhibited by 24 mM-phosphate, whereas undecylprodigiosin was still formed at this high phosphate concentration. The effects of ammonium inhibition of pigmented antibiotic production were relieved by reducing the concentration of phosphate in the medium, but changing the ammonium concentration had no effect on phosphate inhibition. Thus the regulation of pigment production by these two nutrients is interrelated, with phosphate control being epistatic to that of ammonium. The results implicate a phosphorylated intermediate as a major regulator of secondary metabolite synthesis by S. coelicolor.

The Streptomyces lividans 66 chromosome contains a 1 MB deletogenic region flanked by two amplifiable regions.

Genetic instability in Streptomyces species often involves large deletions sometimes accompanied by DNA amplification. Two such systems in Streptomyces lividans 66 involve the production of mutants sensitive to chloramphenicol and the production of mutants resistant to the galactose analogue 2-deoxygalactose, respectively. Overlapping cosmids were isolated that span the ca. 1 Mb region between the two amplifiable regions. The structure of the region was confirmed by restriction mapping using the rarely cutting enzymes AseI, BfrI and DraI and pulsed-field gel electrophoresis. The region contains a non-clonable gap flanked by inverted repeats; the structure is consistent with the presence of a physical gap, i.e. a linear chromosome.

Growth rate control of protein and nucleic acid content in Streptomyces coelicolor A3(2) and Escherichia coli B/r

Escherichia coli possesses regulatory mechanisms that coordinate cell growth with the synthesis of essential macromolecules (protein, RNA and DNA). While fundamental differences have been identified in the growth habit and chromosome structure of E. coli and Streptomyces, little is known about these regulatory mechanisms in filamentous bacteria. This paper reports on the relationship between the macromolecule content of S. coelicolor A3(2) and its specific growth rate. The protein, RNA and DNA contents (g per 100 g biomass) of S. coelicolor A3(2) grown in steady-state continuous culture over a range of specific growth rates (0.025-0.3 h-1) were 31-45, 10-22 and 3.5-4.5% (w/w), respectively. This composition is qualitatively similar to that of other microorganisms. Changes in the macromolecular content of S. coelicolor A3(2) and E. coli B/r with specific growth rate appear to be essentially similar. However, the data indicate that the RNA content of S. coelicolor A3(2), grown under the conditions used, exceeds that of E. coli grown at the same specific growth rate. The data also suggest that overlapping rounds of replication are not a feature of DNA synthesis in S. coelicolor A3(2). This may be a function of the organisms low maximum specific growth rate. Alternatively, it may be a consequence of regulatory mechanisms which act to inhibit the initiation of DNA synthesis in a linear chromosome which is already undergoing replication.

DNA deletions in spontaneous chloramphenicol-sensitive mutants of Streptomyces coelicolor A3(2) and Streptomyces lividans 66

SummaryA mutant of Streptomyces coelicolor A3(2) highly resistant to chloramphenicol was selected. It had amplified some chromosomal DNA fragments to a copy number of 20–50. Some of the amplified fragments were cloned and used as hybridisation probes to investigate the spontaneous chloramphenicol-sensitive mutants which occur at high frequency in this species and the closely related species Streptomyces lividans 66. These investigations demonstrated that chloramphenicol sensitivity in both species is associated with large deletions that are at least 40 kb in length.

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 G + C’ 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.

DNA amplification and genetic instability in Streptomyces.

Genetic instability is very common in Streptomyces species, but only affects specific genes in any one strain. It sometimes occurs at high frequency spontaneously, but may be stimulated by treatments such as UV irradiation or intercalating agents. Deletion of genes occurs and may be accompanied by DNA amplifications. It is unlikely that there is plasmid involvement in most cases. Little is yet known about the molecular mechanisms of deletion and DNA amplification. Genetic instability can be a problem during commercial antibiotic production. DNA amplification of cloned genes is potentially useful for achieving both stability and high gene dosage.

Intraspecific and Intergeneric Mobilization of Non-conjugative Resistance Plasmids by a 24-5 Megadalton Conjugative Plasmid of Neisseria gonorrhoeae

pLE2451, a 24.5 megadalton conjugative plasmid from Neisseria gonorrhoeae, was capable of efficiently mobilizing gonococcal beta-lactamase plasmids between gonococci and from gonococci to Haemophilus influenzae and restriction-deficient Escherichia coli. Donor strains of N. gonorrhoeae carrying pLE2451 were also found to be capable of mobilizing a variety of non-conjugative plasmids originally derived from enteric bacteria or Haemophilus species when such plasmids were resident in E. coli. Nevertheless, pLE2451 was not detected physically in E. coli or H. influenzae transconjugants. This suggests that the plasmid is unstable in these hosts but survives transiently to provide transfer functions for mobilization. The proficiency of pLE2451 in promoting intraspecific and intergeneric mobilization was not paralleled by pUB701, pRI234 and pFR16017, a series of conjugative plasmids derived originally from Haemophilus species. These plasmids were incapable of mobilizing even Haemophilus beta-lactamase plasmids, such as RSF0885, between Haemophilus species.

Isolation and characterization of temperature-sensitive mutants of Streptomyces coelicolor A3(2) blocked in macromolecular synthesis

A collection of temperature-sensitive mutants of Streptomyces coelicolor A3(2) was isolated. The majority of the mutants showed an osmotically remedial phenotype. Mutants defective in macromolecular synthesis were identified and characterized further. Four mutants were found in which DNA replication was defective, but which continued to synthesize RNA and protein at the restrictive temperature (39 degrees C). The kinetics of cessation of DNA synthesis allowed a tentative identification of slow (initiation) and fast (elongation) stop dna mutants. The inhibition of DNA replication in the four mutants was found to be reversible on returning to the permissive temperature (30 degrees C), but only after a delay of about 2 h. Three other mutants were identified which showed not only cessation of DNA replication at the restrictive temperature, but also defects in other macromolecular synthesis events.

Analysis of Large Deletions and Characterization of the Deletion Endpoints Associated with an Amplifiable DNA Region in Streptomyces Lividans

The phenomenon, that Streptomyces species can lose spontaneously certain phenotypes at frequenzies between 10–3 and 10–1 has been recognized since at least 1913 (Beijerinck 1913). This genetic instability is very common in many Streptomyces species and can affect a variety of genes; however, only specific genes are affected in any one strain (reviewed by Cullum et al., 1986; Hutter and Eckhardt, 1988). Frequently antibiotic producing strains, including some of commercial importance, are subject to genetic instability: they lose the ability to produce antibiotics (e.g. tetracyclines), i.e. they “degenerate”. As plasmid-curing agents such as acriflavine and ethidium bromide or UV-irradiation increased the frequency of mutation drastically, several authors suggested that the loss of a plasmid caused the loss of antibiotic production and they concluded that genes (or regulatory genes) for antibiotic production are coded on plasmids.