SUMMARY: A restriction map of ø A7 DNA (46·7 kb) was established for nine endonucleases (Bc/I, ClaI, EcoRI, EcoRV, HpaI, PvuI, SacII, SphI and XbaI) which cut the phage genome up to 11 times. There were no sites for BamHI, Bg/II, HindIII, PstI, PvuII, SacI or Sa/I. ø A7 DNA, circularized through its cohesive ends, could integrate into the genome of several Streptomyces hosts, to form stable lysogens. Integration occurred by recombination between unique attachment sites on the phage (attP) and the host (attB) genomes. The attP site has been located on the ø A7 restriction map. Deletion mutants of ø A7 DNA were obtained by selecting for pyrophosphate- or EDTA-resistant clones. The deletions occurred either near the left-hand end of the conventional restriction map, or about 18 kb from the right-hand end, close to, but not affecting the unique SacII site. Together, the deletions defined at least 7·9 of DNA (16·9% of the phage genome) non-essential for plaque formation. ø A7 DNA was introduced into S. lividans protoplasts by liposome-assisted transfection. Since the phage does not adsorb to intact cells of this strain, and therefore does not form plaques, an overlay of S. antibioticus spores was used to detect the infectious progeny released by the protoplasts. Using this technique, ø A7 could be introduced into S. antibioticus with an efficiency of about 6 × 106 p.f.u. per μg DNA (equivalent to 3 × 10-4 p.f.u. per DNA molecule).
SUMMARY: The bialaphos resistance gene, bar, was used as a selectable marker to isolate the bialaphos production genes (bap) from the Streptomyces viridochromogenes genome. The S. viridochromogenes bar gene was cloned on overlapping restriction fragments using pIJ680 and pIJ702 in the bialaphos-sensitive host, S. lividans. Although the restriction endonuclease cleavage map of these fragments was not similar to the bap cluster of S. hygroscopicus, the presence and location of bar and four other bap genes as well as a gene required for the transcriptional activation of the cluster (brpA) was demonstrated by heterologous cloning experiments using a series of previously characterized bialaphos-nonproducing S. hygroscopicus mutants. Since recombination-deficient mutants of streptomycetes have not been isolated, restored function provided by cloned homologous DNA results from both recombination (marker rescue) and complementation in trans. In contrast to our previously reported homologous cloning experiments where we were able to define the position of mutant alleles by recombination, in these heterologous cloning experiments we observed little if any recombination between plasmid-cloned genes and the chromosome. As a result, this approach allowed us to define the location and orientation of functional genes using a genetic complementation test. The organization of the clustered S. viridochromogenes bap genes was indistinguishable from the corresponding S. hygroscopicus mutant alleles. The fact that the S. viridochromogenes transcriptional regulatory gene, brpA, functioned in S. hygroscopicus implied that some transcriptional regulatory signals may also be interchangeable. In these two Streptomyces species, which have considerable nucleotide sequence divergence, the complex biochemical and genetic organization of the bialaphos biosynthetic pathway is conserved.
SUMMARY: The coding characteristics of four plasmids expressing a protein (BCP) which comigrates with bacterioferritin were examined and the nucleotide sequence of a common 1985 bp segment from the 53 min region of the Escherkhia coli linkage map was determined. Three open reading-frames (orf1, orf2 and orf3) were detected, and orf2 (bcp, 156 amino acid codons) appeared to encode the bacterioferritin comigratory protein, BCP. The translation product of orf3 (205 amino acid codons) resembled the iron-sulphur protein component (DMS B subunit) of the anaerobic dimethylsulphoxide reductase complex of E. coli.
SUMMARY: The lacZ gene from Streptococcus thermophilus A054, a commercial yogurt strain, was cloned on a 7·2 kb PstI fragment in Escherichia coli and compared with the previously cloned lacZ gene from S. thermophilus ATCC 19258. Using the dideoxy chain termination method, the DNA sequences of both lacZ structural genes were determined and found to be 3071 bp in length. When the two sequences were more closely analysed, 21 nucleotide differences were detected, of which only nine resulted in amino acid changes in the proteins, the remainder occurring in wobble positions of the respective codons. Only three bases separated the termination codon for the lacS gene from the initiation codon for lacZ, suggesting that the lactose utilization genes are organized as an operon. The amino acid sequence of the β-galactosidase, derived from the DNA sequence, corresponds to a protein with a molecular mass of 116860 Da. Comparison of the S. thermophilus amino acid sequences with those from Lactobacillus bulgaricus, E. coli and Klebsiella pneumoniae showed 48, 35 and 32·5% identity respectively. Although little sequence homology was observed at the DNA level, many regions conserved in the amino acid squence were identified when the β-galactosidase proteins from S. thermophilus, E. coli and L. bulgaricus were compared.
SUMMARY: Myxococcus xanthus mutants defective in myxospore germination have been isolated both by a selective and by a non-selective method after UV or Tn5-lac-induced mutagenesis. The ability of these mutants to germinate in germinant solutions other than those used for their isolation has been tested. Six of seven mutants isolated behaved as germination-defective in all germinants. Germination of the seventh mutant was conditional on the germinant used, being normal in Casamino acids but defective in a Casitone-based medium. Genetic analysis of the four mutant strains carrying Tn5-lac insertions revealed that the transposon had disrupted a different locus in each mutant, so that the four mutants defined four unlinked loci involved in the germination process (gerA, gerB, gerC, gerD). Strain MR307 was studied in more detail. Cloning of the gene affected in this mutant, gerC, and construction of merodiploids revealed that the wild-type allele is dominant over the mutated one. In vitro construction of lacZ fusions allowed study of gerC expression throughout the M. xanthus life cycle, revealing that the gene affected by insertion at ΩMR307 is developmentally regulated.