Expression analysis of the spi gene in the pock-forming plasmid pSA1.1 from Streptomyces azureus and localization of its product during differentiation

The sporulation inhibitory gene spi in the pock-forming conjugative plasmid pSA1.1 of Streptomyces azureus was introduced into cells via a high or low copy number vector to examine the effect of gene dosage on the growth of Streptomyces lividans TK24 as a host. In transformants carrying a high spi copy number, nutrient mycelial growth was inhibited, as was morphological differentiation from substrate mycelium to aerial mycelium on solid media. The degree of inhibition depended on the spi gene dosage, but the presence of pSA1.1 imp genes, which encode negative repressor proteins for spi, relieved the inhibition. Confocal images of Spi tagged with enhanced green fluorescent protein in cells on solid media revealed that spi expression was initiated at the time of elongation of substrate mycelium, that its expression increased dramatically at septation in aerial hyphae, and that the expression was maximal during prespore formation. Expression of spi covered the whole of the hyphae, and the level of expression at the tip of the hyphae during prespore formation was about sixfold greater than during substrate mycelial growth and threefold greater than during aerial mycelial growth. Thus, localized expression of spi at particular times may inhibit sporulation until triggering imp expression to repress its inhibitory effects.     

Screening of compounds stimulating spore formation and mycelial growth of pock-forming plasmid-carrying strains in Streptomyces azureus

Summary Sporulation-stimulating compounds were screened by using mutant PK100a of Streptomyces azureus ATCC 14921, in which spore formation was markedly inhibited by the pock-forming plasmid pSA1.1. On agar media, cysteine, bacitracin, glutathione and -NAD induced the formation of coloured spores or spore mass of strain PK100a. These compounds also stimulated the spore formation of the wild-type and its plasmid-cured (good spore-forming) strains and the growth of aerial mycelia of these three strains. This screening method appears to be an effective method for the screening of substances that stimulate spore formation and mycelial growth of streptomycetes or that overcome the inhibitory effect of pock-forming plasmids. Correspondence to: S. Ogata     

Specific Lysogenicity in Streptomyces azureus

Slope (or plate) cultures of thiostrepton-producing Streptomyces azureus (ATCC 14921) often showed spontaneously developing plaques. Plaques increased in number during serial subcultures. The production of aerial mycelia and sporulating aerial hyphae was interrupted by the overlapping plaques, whereas the growth of substrate mycelia continued in the plaques. These abnormal (eroded) cultures were easily restored to their normal conditions once they were passed through liquid cultures under shaking conditions. A few phage particles were found in the plaques, together with some headless tails and numerous tail tips which formed a hexagonal crystal or a large crystal mass when viewed in an electron microscope. No lytic phenomenon and no phage production were found in the liquid cultures, although all mycelia and spores harbored phage-producing abilities. It was also found that the propagation of phages was successful in solid culture, but not in liquid culture. The whole phage was named SAt2, which belongs to group B of Bradley's morphological classification. From these results, it is considered that S. azureus is lysogenic with temperate phage SAt2, of which virulent mutants are able to infect the aerial mycelia and sporulating hyphae of their lysogenic host.     

Detection of the single-stranded DNA of Streptomyces plasmid pSA1.1 and a binding histone-like protein

Abstract Streptomyces plasmid pSA1.1 accumulated single-stranded DNA as replication intermediates in S. lividans ; therefore, this plasmid was considered to replicate by a rolling-circle mechanism. A DNA-binding protein (pI > 9.7 and about 10 kDa) was purified on a denatured DNA-Cellulose column, then on a native DNA-Cellulose column. The N-terminal amino acid sequence of this protein has a high homology with bacterial histone-like proteins. In the gel retardation assay, this protein bound with the single-stranded DNA of pSA1.1. We propose that this protein may participate in the replication of pSA1.1.     

A temperate phage of Streptomyces azureus

A new phage, SAt1, was isolated from soil on Streptomyces azureus ATCC 14921. This phage was able to lysogenize S. azureus. The percentage of lysogenic responses was ca. 10%. Electron microscopic observation showed that this phage belonged to group B of Bradley's morphological classification. The molecular mass of SAt1 DNA was ca. 24 megadaltons. The guanine-plus-cytosine content and the density of SAt1 DNA were ca. 71% and 1.724 g/cm3, respectively. A cleavage map of SAt1 DNA was constructed with restriction endonucleases BanIII, BglII, HindIII, and XbaI. Furthermore, some other characteristics of this phage were investigated.     

Gene encoding a replication initiator protein and replication origin of conjugative plasmid pSA1.1 of Streptomyces cyaneus ATCC 14921

pSA1.1 is a 9.1-kb multicopy plasmid originally isolated from Streptomyces cyaneus (formerly S. azureus) ATCC 14921. This plasmid accumulates single-stranded DNA in S. lividans and is therefore considered to replicate by a rolling-circle replication. In the present work, the rep gene encoding the replication initiator protein and the replication origin ori of pSA1.1 were determined. The rep and ori are located on separate regions. The Rep protein of pSA1.1 belongs to superfamily I which includes A proteins of phages. Nucleotide sequence of the surrounding putative nicking site of pSA1.1 shows good agreement with those of the pC194 group plasmids and phages. The direction of replication was also determined.     

Purine Auxotrophic Mutants with Altered Spore Color in Streptomyces azureus. ATCC 14921

A purine auxotroph with pale purple spores and a derivative with yellowish orange spores were obtained from the wild-type strain of Streptomyces azureus ATCC 14921, which has bluish green spores. The changed color or pigmentation in the mutants was limited to the spores. They accumulated AIR (5′-phosphoribosyl-5-aminoimidazole) due to the lack of AIR carboxylase activity.     

Isolation and characterization of pock-forming plasmids for Streptomyces griseus from soil actinomycetes

Thirty independent actinomycetes strains carrying plasmids were isolated from soil. These plasmids were purified as cccDNA by CsCl-EtBr equilibrium density-gradient centrifugation. Plasmids that induce "pocks", namely formation of circular zones of sporulation-inhibition, were selected by protoplast transformation of streptomycin-producing strain, Streptomyces griseus ATCC10137. Six pock-forming plasmids, pOA7, pOA11, pOA15, pOA23, pOA29 and pOA30, were obtained, and their cleavage maps, transformation frequencies, and copy numbers, as well as their stability, are described.     

Efficient plasmid transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts.

A procedure for efficient transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts with plasmid DNA was developed. Transformation frequencies with S. fradiae protoplasts were strongly influenced by the temperatures for cell growth, protoplast formation, and protoplast regeneration. Transformation frequencies for both species were also influenced by the culture age before protoplast formation, the source and concentration of polyethylene glycol, the transformation-inducing agent, the concentration of protoplasts used in the transformation procedure, and the number of protoplasts added to regeneration plates. Transformation frequencies were substantially higher for both species when calf thymus DNA and protamine sulfate were added to the transformation mix. With S. fradiae, transformation frequencies were much lower with plasmid DNA prepared from other species than with the same plasmids prepared from S. fradiae, suggesting that S. fradiae expresses restriction and modification. With the modified transformation procedures using DNA prepared from homologous hosts, S. ambofaciens and S. fradiae are now transformed routinely at frequencies of 10(6) to 10(7) transformants per micrograms of plasmid DNA.     

Multicopy plasmid stability in Escherichia coli requires host-encoded functions that lead to plasmid site-specific recombination

Summary The heritable stability of the multicopy plasmid ColE1 and its natural relatives, requires the presence in the plasmid of a site (cer in ColE1) that acts as a substrate for site-specific recombination, thereby maintaining plasmids in the monomeric state. Multimerization, promoted by homologous recombination, leads to plasmid loss. Here we show that the Escherichia coli chromosome encodes at least two unlinked functions that act on cer and its analogous sites, to promote stabilizing site-specific recombination. One of these functions is encoded by a gene residing on a cosmid that also contains the argI and pyrB genes, mapping it to the 96–97 min region of the E. coli map.     

Characterization of a natural cointegrate of the pock-forming plasmids pSK1 and pSK2 of Streptomyces kasugaensis MB273

Strains carrying only one species of pock-forming plasmid, designated as pSK3, were isolated from two different derivative strains of Streptomyces kasugaensis MB273 which contained three species of plasmids, pSK1, pSK2 and pSK3. Single and double digestion of pSK3 with seven restriction endonucleases yielded fragments identical with those of pSK3 and assignable to those obtained from pSK1 and pSK2. In particular, digestion with BglII alone or in combination with other restriction endonucleases afforded the same size fragments as those of pSK1 and pSK2. Strains containing pSK3 induced pocks on lawns of strains carrying pSK1 or pSK2 and resisted pock formation by the latter strains. Therefore, it was concluded that pSK3 was a pSK3 derivative with elevated pock-forming ability and represented a composite plasmid consisting of two elements, pSK1 and pSK2, without any loss of their plasmid functions. Deletion derivative plasmids constructed from the BglII fragments of pSK3 provided evidence supporting the above conclusion. Pock formation by a pSK3-containing strain against strains carrying pSK1, or pSK2 or no plasmid accompanied the transfer of pSK3 from the former to the latter. Segregation of pSK1 and pSK2 from pSK3 was observed in mycelium from pocks caused by pSK3-containing strains and on subculture of pSK3-containing strains.     

Characterization of a mutant of thiostrepton-producing Streptomyces azureus ATCC 14921 and its plasmids

A mutant, strain PK10, of Streptomyces azureus ATCC 14921 and its two plasmids were characterized and compared with another mutant, PK 100, and its plasmid. One PK 10 plasmid of 8.8 kb was identical to a pock-forming plasmid, pSA1.1, of PK100. The other olasmid which was found only in PK10 nd named pSA1.2 (size, 7.6 kb), was a non-pock forming derivative of pSA1.1 with deletions in two different regions (about 1.2 kb and 30 b long). The pcok-forming ability of strain PK10 on a plasmid-free strain was lower than that of strain PK100 which contained only pSA1.1. Strain PK10 had fewer copies of pSA1.1 than strain PK100, and had normal spore formation and thiostrepton production, which were depressed in the strain PK100. The pSA1.1 from both PK10 and PK100 amplified to 20 to 30 copies in the transformants and inhibited theri spore formation and thiostrepton production. Thus, the function of pSA1.1 appeared to be depressed by pSA1.2.     

Effects of UV dose on formation of spontaneously developing pocks in Streptomyces azureus ATCC14921

Spontaneously developing pocks (S pocks) of Streptomyces azureus ATCC14921 were formed by the both functions of conjugative plasmid pSA1 and lysogenic phage SAt2. The formation was affected by the dose of UV irradiation. The mean pock diameter in cultures treated with UV light at 0, 7.1, 14.2 and 21.3 x 10(2) microW. erg/cm, respectively, were 1.3, 0.4, 2.2, and 0.5 mm. The dose affected conjugative plasmid pSA1 related to pock formation. There was UV damage of autonomous pSA1 replicon and UV induction of the chromosomal integrated sequence. Increases and decreases in the amount of autonomous pSA1 replicon corresponded to increases and decreases, respectively, in the diameter of the pocks. Both pSA1 and SAt2 syntheses were developed in the large pocks (1.3 and 2.2 mm), but only SAt2 synthesis was developed in the pinhole pocks (0.4 and 0.5 mm).     

Mobilization function of the pBHR1 plasmid, a derivative of the broad-host-range plasmid pBBR1

The pBHR1 plasmid is a derivative of the small (2.6-kb), mobilizable broad-host-range plasmid pBBR1, which was isolated from the gram-negative bacterium Bordetella bronchiseptica (R. Antoine and C. Locht, Mol. Microbiol. 6:1785-1799, 1992). Plasmid pBBR1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable transposons from gram-positive bacteria. We show that the Mob protein specifically recognizes a 52-bp sequence which contains, in addition to the transfer origin, the promoter of the mob gene. We demonstrate that this gene is autoregulated. The binding of the Mob protein to the 52-bp sequence could thus allow the formation of a protein-DNA complex with a double function: relaxosome formation and mob gene regulation. We show that the Mob protein is a relaxase, and we located the nic site position in vitro. After sequence alignment, the position of the nic site of pBBR1 corresponds with those of the nick sites of the Bacteroides mobilizable transposon Tn4555 and the streptococcal plasmid pMV158. The oriT of the latter is characteristic of a family of mobilizable plasmids that are found in gram-positive bacteria and that replicate by the rolling-circle mechanism. Plasmid pBBR1 thus appears to be a new member of this group, even though it resides in gram-negative bacteria and does not replicate via a rolling-circle mechanism. In addition, we identified two amino acids of the Mob protein necessary for its activity, and we discuss their involvement in the mobilization mechanism.     

Characterization of the incompatibility region of Streptomyces plasmid pSL1

The incompatibility region of the Streptomyces plasmid pSL1 was identified as a 240-bp segment, though some other function from the vector plasmid was also necessary. A 540-bp fragment including the 240-bp region was enough for full expression of incompatibility. Inserted mutation analysis led to a more detailed location of the region essential for replication.     

Physical characterization of SCP1, a giant linear plasmid from Streptomyces coelicolor.

SCP1, coding for the methylenomycin biosynthesis genes in Streptomyces coelicolor, was shown to be a giant linear plasmid of 350 kb with a copy number of about four by analysis with pulsed-field gel electrophoresis. A detailed physical map of SCP1 was constructed by extensive digestion with six restriction endonucleases, by DNA hybridization experiments, and finally by cloning experiments. SCP1 has unusually long terminal inverted repeats of 80 kb on both ends and an insertion sequence at the end of the right terminal inverted repeat. Analysis by pulsed-field gel electrophoresis in agarose containing sodium dodecyl sulfate revealed that a protein is bound to the terminal 4.1-kb SpeI fragments derived from both ends of SCP1. Treatment with lambda exonuclease or exonuclease III and SpeI digestion also indicated that the 5' ends of SCP1 are attached to a protein.     

Two distinct conjugal transfer systems on Streptomyces plasmid pZL1

The mechanism of conjugal transfer of plasmids in Gram-negative and unicellular Gram-positive bacteria is commonly via a type IV secretion system （T4SS） [1]. The genes encoding the T4S proteins are usually arranged in a single operon or a few operons. In Gram-negative Agrobacterium tumefaciens, the T4SS is encoded by the virB and virD operons （11 and 5 genes, respectively） [2]. Streptomyces are multicellular mycelial Gram-positive bac- teria that form unicellular spores. There are fundamental dif- ferences in the mechanisms of conjugal transfer between Streptomyces plasmids and those of Gram-negative and uni- cellular Gram-positive bacteria. Conjugal transfer of Streptomyces plasmids requires a tra gene encoding an FtsK/SpolIIE-family DNA translocator and a few adjacent genes [3]. No bacterial T4SS has previously been found on Streptomyces conjugative plasmids. We reported here the co- existence of both a T4SS-like and an FtsK/SpolIIE-family DNA translocator on a 128-kb Streptomyces plasmid, pZL 1.     

Identification and properties of a novel clt locus in the Streptomyces phaeochromogenes plasmid pJV1.

A novel clt locus required for efficient transfer of the Streptomyces phaeochromogenes plasmid pJV1 was identified and mapped. The clt region was functional in both orientations, and its absence caused a severe reduction in plasmid transfer. Chromosome mobilization, on the other hand, was not affected by absence of the clt locus. The clt region showed structural, but not sequence, similarity to transfer origins of gram-negative plasmids.