Functional Analysis Of NsdA Gene And The Bioinformatics Of TTA Codon In Strptomyces Coelicolor

SCTA1 is a cosmid with an insert of chromosomal DNA from Streptomyces coelicolorA3(2). Former study has shown that its insertion into the chromosome of S. coelicolorstrains, causing a duplication of a ca. 40-kb segment, delayed actinorhodin antibioticproduction and sporulation, implying that it carded a gene negatively affecting theseprocesses. Subcloning of the SCTA1 insert DNA resulted in identification of an openreading frame SCO5582 as nsdA, a gene negatively affecting Streptomyces differentiation.In this study, disruption of the chromosomal nsdA caused overproduction of actinorhodin.This was correlated with premature expression of a pathway-specific regulatory gene(actâ…¡-orf4), implying that nsdA in the wild type strain indirectly repressed expression of theactinorhodin biosynthesis cluster, nsdA expression was up-regulated upon aerial myceliuminitiation, and was strongest in the aerial mycelium. NsdA has a conserved SXR site. Asite-directed mutation (S458A) in this site in NsdA abolished its function. However, theS458 site was not found to be phosphorylated in Streptomyces. Blast searching showed thateight NsdA analogues are present in S. coelicolor genome. Disruption of an nsdA-like geneSC04114 had no obvious phenotypic effects on S. coelicolor. The nsdA orthologueSAV2652 in S. avermitilis could complement the S. coelicolor nsdA null mutant phenotype.The rarest codon in the high G+C genome of Streptomyces coelicolor is TTA,corresponding in mRNA to the UUA codon that is recognised by a developmentallyimportant tRNA encoded by the bldA gene. There are 145 TTA-containing genes in thechromosome of S. coelicolor. Only 42 of these are represented in the genome ofStreptomyces avermitilis, among which only 12 have a TTA codon in both species. The TTAcodon is less represented in highly-expressed genes, housekeeping genes and orthologousgenes, and is more represented in functional-unknown, extrachromosomal or weaklyexpressed genes. In our laboratory, 21 TTA-containing chromosomal genes in S. coelicolorwere disrupted, including 12 of the 42 genes that are common to both S. avermitiUis and S.coelicolor. None of the mutant strains showed any obvious phenotypic differences from thewild-type strain under tested conditions. Possible reasons for this, and the role andevolution of the observed distribution of TTA codons among Streptomyces genes werediscussed.