The sre gene (ORF469) of the R4 phage encodes a protein similar to the resolvase-DNA invertase family proteins. Insertional gene disruption of sre prevented a lysogen from entering the lytic cycle, implying that Sre protein is a site-specific recombinase needed for excision of the R4 prophage genome (M. Matsuura, T. Noguchi, T. Aida, M. Asayama, H. Takahashi, and M. Shirai, J. Gen. Appl. Microbiol. 41:53-61, 1995). To determine whether this sre gene is also necessary for the integration reaction, we studied its function by integration plasmid analysis. When deletions, frameshifts, and site-directed mutations that caused an amino acid substitution of Ser-17 for Ala were introduced into the sre structural gene, transformation efficiency of Streptomyces parvulus 2297 with these plasmid DNAs was severely reduced. However, an adenine insertion just before the possible initiation codon of the sre gene did not significantly decrease the efficiency. These data suggest that the Sre protein is a site-specific recombinase responsible for integration of the R4 phage genome.Streptomyces strains are important organisms because they produce a variety of useful secondary metabolites, for example, antibiotics, antitumor reagents, and immunosuppressors (3). R4 is an actinophage that has been studied in depth from the viewpoint of its practical application (12-17). To improve the genetic engineering of Streptomyces strains, site-specific recombination of this R4 phage has been studied (11, 21). Our data suggested that the ORF469 gene (sre) (DDBJ accession no. D38173), which was found adjacent to the attP recombination site of the R4 phage genome, is essential for excision of the prophage genome and probably encodes a site-specific recombinase responsible for the excisive recombination (11). The protein encoded by ORF469 falls not into the integrase family but into the resolvase-DNA invertase family and is highly similar to Tn2501 resolvase and SpoIVCA of Bacillus subtilis (11). The ORF469 protein and SpoIVCA have additional C-terminal halves consisting of about 300 amino acids that typical resolvases do not have, and this kind of recombinase has been also found in Anabaena spp. (XisF) (2,20). In this study, we showed that the protein encoded by the ORF469 gene (sre) is a site-specific recombinase responsible for integration of the R4 phage genome into the Streptomyces parvulus 2297 chromosome.Construction of integration plasmids. To introduce a thiostrepton resistance gene (tsr) (6) as a selection marker into an integration plasmid, a 1.1-kb BclI fragment from pIJ702 containing the tsr gene was inserted by blunt-end ligation into the SmaI site of pAT95 (the SmaI site was in the multicloning site of the pUC18 vector), which contains a 13-kb BclI fragment carrying the ORF469 gene and the attP recombination site from the R4 phage genome (11), to generate pAT96L (data not shown). A 5.6-kb EcoRI-ScaI fragment carrying tsr, ORF469 (sre), and attP was isolated from pAT96L and inserted between the SmaI and EcoRI sites of pUC118 to create ...

Solid media on a base of B-12 or CB medium with agarose or agarose of low melting temperature were developed for the cultivation of Microcystis species. The media with 0.4% gel showed the highest number of CFU, and increasing the gel concentration resulted in a reduction of the number of CFU. There was no difference in the numbers of CFU between pour and spread plates made of the solid media. By using the solid media, 31 clones of Microcystis species were isolated from natural blooms in Lake Kasumigaura, and 5 axenic strains (1 of M. wesenbergii and 4 of M. aeruginosa) were established from the clones.

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We performed molecular characterization of the RpoD1 protein encoded by the rpoD1 gene isolated from a cyanobacterium, Microcystis aeruginosa K-81. The deduced amino acid sequence (416 aa, 48,871 Da) of RpoD1 exhibited extensive similarity to those of proteins of the eubacterial RpoD family (Escherichia coli sigma 70 homologs). We overproduced and purified RpoD1 (54 kDa) from E. coli. Biological and biochemical analyses suggested that RpoD1 has a function homologous to that of E. coli sigma 70 as follows: (i) the RpoD1 protein complemented an rpoD mutant of E. coli strain YN543 (rpoD285) and (ii) the heterologous RNA polymerase holoenzyme reconstituted from the E. coli core enzyme and recombinant RpoD1 was specifically transcribed from E. coli promoters. Furthermore, Western blot analysis with antiserum against Synechococcus sp. strain PCC 7942 RpoD1 (a principal sigma factor of the sigma 70 type) indicated that M. aeruginosa K-81 RpoD1 (sigma A1) is the principal sigma factor, which is a major component of the sigma subunit on exponential cell growth.

Microcystis strains (2 toxic and 18 nontoxic to mice) were isolated from toxic waterblooms that had been collected from Lake Kasumigaura, Ibaraki Prefecture, Japan, in August 1985. Thirteen of the strains (2 toxic and 11 nontoxic) were Microcystis aeruginosa, 2 (nontoxic) were Microcystis wesenbergii, and the other 5 were difficult to identify. Six (1 toxic and 4 nontoxic M. aeruginosa and 1 M. wesenbergit) of these 20 strains were established as axenic cultures. A toxic and axenic strain of M. aeruginosa, K-139, was used to study the relationship between growth conditions and toxicity. Cells in early-to-mid-log phase showed the highest toxicity (50% lethal dose, 7.5 mg of cells per kg of mouse), and maximum toxicity was not affected by growth temperatures between 22 and 30°C. Purification and characterization of the toxins from K-139 cells were also conducted, and at least two toxins were detected. One of the toxins (molecular mass, 980 daltons) has not been reported previously. The main target of the toxin in mice was the liver. Marked congestion and necrosis in the parenchymal cells around the central veins of the liver were observed microscopically in specimens that had been prepared from the mice with acute toxicity after injection with the toxin.