Functional evidence that the principal DNA replication origin of the Streptomyces coelicolor chromosome is close to the dnaA-gyrB region.

The degree of overrepresentation of selected chromosomal genes in rapidly growing cultures of Streptomyces coelicolor was assessed by quantitative DNA hybridization analysis. The results are consistent with the hypothesis that the principal origin of replication is close to the dnaA-gyrB region, in the center of the linear chromosome, and that replication proceeds bidirectionally.     

Polyhydroxyalkanoate accumulation in Streptomyces coelicolor affected by SCO7613 gene region

Polyhydroxyalkanoate (PHA) is stored as an important carbon and energy source in bacterial cells. For biomedical applications, gram-positive bacteria can be better sources of PHAs, since they lack outer membrane lipopolysaccharide. Although gram-positive Streptomyces coelicolor A3(2) has been indicated as a high potential PHA producer, pha C gene that encodes the key enzyme PHA synthase in the metabolic pathway is not determined in its genome. BLAST search results of the GenBank database argued that SCO7613 could specify a putative polyhydroxyalkanoate synthase (PhaC) responsible for PHA biosynthesis. Deduced amino acid sequence of SCO7613 showed the presence of conserved lipase box like sequence, ⁵⁵⁵GASAG⁵⁵⁹, in which serine residue was present as the active nucleophile. Present study describes deletion of putative S. coelicolor pha C gene via PCR dependent method. We showed that SCO7613 is not an essential gene in S. coelicolor and its deletion affected PHA accumulation negatively although it is not ceased. Transcomplementation abolished the mutant phenotype, demonstrating that the decrease in PHA resulted from the deletion of SCO7613.     

Glutamate synthesis in Streptomyces coelicolor.

Both glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) are involved in glutamate synthesis in Streptomyces coelicolor. The highest levels of GDH were seen in extracts of cells grown with high levels of ammonium as the nitrogen source. GOGAT activity was reduced two- to threefold in extracts of cells grown with good sources of glutamate. S. coelicolor mutants deficient in GOGAT (Glt-) required glutamate for growth with L-alanine, asparagine, arginine, or histidine as the nitrogen source but grew like wild-type cells when ammonium, glutamine, or aspartate was the nitrogen source. The glt mutations were tightly linked to hisA1. Mutants deficient in both GOGAT and GDH (Gdh-) required glutamate for growth in all media. The gdh-5 mutation was mapped to the left region of the S. coelicolor chromosomal map, between proA1 and uraA1.     

Analysis of gene expression in operons of Streptomyces coelicolor

Background  

Recent studies have shown that microarray-derived gene-expression data are useful for operon prediction. However, it is apparent that genes within an operon do not conform to the simple notion that they have equal levels of expression.     

Cytological evidence for association of the ends of the linear chromosome in Streptomyces coelicolor

The chromosome of the filamentous bacterium Streptomyces coelicolor is linear, but the genetic map is circular. We present cytological evidence based on the use of fluorescence in situ hybridization showing that the ends of the chromosome frequently colocalize, in agreement with the idea that the ends are held together, effectively forming a circular chromosome. These observations provide a possible explanation for how a linear bacterial chromosome can exhibit a circular genetic map.     

Integration of SCP1, a giant linear plasmid, into the Streptomyces coelicolor chromosome.

SCP1, coding for the methylenomycin biosynthetic genes in Streptomyces coelicolor, is a giant linear plasmid of 350 kb. Extensive physical characterization revealed that SCP1 has unusually long terminal inverted repeats (TIR) of about 80 kb on both ends and an insertion sequence, IS466, at the end of the right TIR (TIR-R), and the 5'-ends are attached to a terminal protein. In the NF strain S. coelicolor 2612, SCP1 is integrated into the chromosome at the 9-o'clock position. Analysis of the two junctions between the SCP1 DNA and the chromosomal DNA revealed that the left junction had an almost intact left terminus of SCP1, while the right junction was composed of IS466, completely deleting TIR-R. Based on these results, we presented a possible formation mechanism of the NF strain, which is characterized by integration of SCP1 into the chromosome via an interaction of the target site and the combined ends of the racket-frame structure of SCP1 followed by deletion of TIR-R. We also hypothesized that this type of integration of a giant linear plasmid might be involved in the origin and distribution of the chromosomal antibiotic biosynthetic gene clusters in microorganisms.     

Identification of DNA amplifications near the center of the Streptomyces coelicolor M145 chromosome

Linear streptomycete chromosomes frequently undergo spontaneous gross DNA rearrangements at the terminal regions. Large DNA deletions of the chromosome ends are in many cases associated with tandemly reiterated DNA amplifications, found at the border of the deletable areas. In contrast to previous reports, we have discovered amplifications near the center of the Streptomyces coelicolor M145 chromosome. The detected amplified units of DNA are 19.9 kb and 16 kb in length and exist in copy numbers of 30 and 40, respectively. Both amplifications were located in the same region and share at least 3.6 kb.     

Streptomyces coelicolor A3(2) lacks a genomic island present in the chromosome of Streptomyces lividans 66

Streptomyces lividans ZX1 has become a preferred host for DNA cloning in Streptomyces species over its progenitor, the wild-type strain 66 (stock number 1326 from the John Innes Center collection), especially when stable DNA is crucial for in vitro electrophoresis, because DNA from strain 66 contains a novel modification that makes it sensitive to oxidative double-strand cleavage during electrophoresis. Detailed analysis of this modification-deficient mutant (ZX1) revealed that it has several additional phenotypic traits associated with a chromosomal deletion of ca. 90 kb, which was cloned and mapped by using a cosmid library. Comparative sequence analysis of two clones containing the left and right deletion ends originating from strain 66 and one clone with the deletion and fused sequence cloned from strain ZX1 revealed a perfect 15-bp direct repeat, which may have mediated deletion and fusion to yield strain ZX1 by site-specific recombination. Analysis of AseI linking clones in the deleted region in relation to the published AseI map of strain ZX1 yielded a complete AseI map for the S. lividans 66 genome, on which the relative positions of a cloned phage phiHAU3 resistance (phiHAU3r) gene and the dnd gene cluster were precisely localized. Comparison of S. lividans ZX1 and its progenitor 66, as well as the sequenced genome of its close relative, Streptomyces coelicolor M145, reveals that the ca. 90-kb deletion in strain ZX1 may have originated from an insertion from an unknown source.     

The phosphotransferase system of Streptomyces coelicolor.

We have investigated the crr gene of Streptomyces coelicolor that encodes a homologue of enzyme IIAGlucose of Escherichia coli, which, as a component of the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) plays a key role in carbon regulation by triggering glucose transport, carbon catabolite repression, and inducer exclusion. As in E. coli, the crr gene of S. coelicolor is genetically associated with the ptsI gene that encodes the general phosphotransferase enzyme I. The gene product IIACrr was overproduced, purified, and polyclonal antibodies were obtained. Western blot analysis revealed that IIACrr is expressed in vivo. The functionality of IIACrr was demonstrated by phosphoenolpyruvate-dependent phosphorylation via enzyme I and the histidine-containing phosphoryl carrier protein HPr. Phosphorylation was abolished when His72, which corresponds to the catalytic histidine of E. coli IIAGlucose, was mutated. The capacity of IIACrr to operate in sugar transport was shown by complementation of the E. coli glucose-PTS. The striking functional resemblance between IIACrr and IIAGlucose was further demonstrated by its ability to confer inducer exclusion of maltose to E. coli. A specific interaction of IIACrr with the maltose permease subunit MalK from Salmonella typhimurium was uncovered by surface plasmon resonance. These data suggest that this IIAGlucose-like protein may be involved in carbon metabolism in S. coelicolor.     

Conserved regulatory elements in the promoter region of the N-CAM gene.

Genomic clones containing 5'-flanking sequences, the first exon, and the entire first intron from the chicken N-CAM gene were characterized by restriction mapping and DNA sequencing. A > 600-bp segment that includes the first exon is very G + C-rich and contains a large proportion of CpG dinucleotides, suggesting that it represents a CpG island. SP-1 and AP-1 consensus elements are present, but no TATA- or CCAAT-like elements were found within 300 bp upstream of the first exon. Comparison of the chicken promoter region sequence with similar regions of the human, rat, and mouse N-CAM genes revealed that some potential regulatory elements including a "purine box" seen in mouse and rat N-CAM genes, one of two homeodomain binding regions seen in mammalian N-CAM genes, and several potential SP-1 sites are not conserved within this region. In contrast, high CpG content, a homeodomain binding sequence, an SP-1 element, an octomer element, and an AP-1 element are conserved in all four genes. The first intron of the chicken gene is 38 kb, substantially smaller than the corresponding intron from mammalian N-CAM genes. Together with previous studies, this work completes the cloning of the chicken N-CAM gene, which contains at least 26 exons distributed over 85 kb.     

Amplifying sequences in the Streptomyces coelicolor A3 (2) gene

The unstable feature of ristomycin resistance in S. coelicolor A3 (2) was studied. It was shown that the frequency of ristomycin-resistant derivatives was high in both chloramphenicol sensitive mutants and their resistant revertants. The 15- and 20-kb DNA sequences capable of amplifying were detected in the chloramphenicol resistant revertants. In the genomes of the studied strains they were represented by 50 and 40 copies, respectively.     

A combined genetic and physical map of the Streptomyces coelicolor A3(2) chromosome.

The restriction enzymes AseI (ATTAAT), DraI (TTTAAA), and SspI (AATATT) cut the Streptomyces coelicolor A3(2) chromosome into 17, 8, and 25 fragments separable by pulsed-field gel electrophoresis (PFGE). The sums of their lengths indicated that the chromosome consists of about 8 Mb of DNA, some 75% more than that of Escherichia coli K-12. A physical map of the chromosome was constructed for AseI and DraI, using single and double digests, linking clones, cross-hybridization of restriction fragments, and locations of genetically mapped genes, insertion sequences, prophages, and the integrated SCP1 and SLP1 plasmids on the physical map. The physical map was aligned with the previously established genetic map, revealing that the two long opposite quadrants of the genetic map that are almost devoid of markers (the silent regions at 3 o'clock and 9 o'clock) are indeed physically long rather than being hot spots for genetic exchange. They must therefore contain long stretches of DNA different in function from the remainder of the genome. Consistent with this conclusion are the locations of significant deletions in both of the silent regions. Of these, a 40-kb deletion in the 9 o'clock region accompanied or followed integration of the SCP1 linear plasmid to produce the NF fertility state. PFGE analysis of Streptomyces lividans 66, a close relative of S. coelicolor A3(2), was hampered by the previously described susceptibility of its DNA to degradation during electrophoresis. However, ZX7, a mutant derivative of S. lividans lacking the DNA modification responsible for this degradation, yielded good PFGE preparations. Not more than 7 of the 17 S. coelicolor AseI fragments could be shared by the S. lividans strain.