Nucleotide sequence and characterization of the cryptic Bacillus thuringiensis plasmid pGI3 reveal a new family of rolling circle replicons.

The complete nucleotide sequence of plasmid pGI3 from Bacillus thuringiensis subsp. thuringiensis H1.1. was obtained. Although this 11,365-bp molecule contained at least 11 putative open reading frames (ORFs), extensive database searches did not reveal any homologous sequences with the exception of ORF6, which displayed similarity to the largest ORF of pSTK1, a 1,883-bp cryptic plasmid isolated from Bacillus stearothermophilus. Deletion analysis to determine the pGI3 minimal replicon revealed that ORF6 is the rep gene. Replication occurred via a single-stranded DNA (ssDNA) intermediate, as demonstrated by S1 treatment and Southern hybridization in nondenaturating conditions. Interestingly, however, no homology was found between the pGI3 (ORF6) and pSTK1 (ORF3) rep genes and those from other single-stranded DNA plasmids, nor was there any DNA similarity to the double-strand origins of replication characterized so far, indicating that pGI3 and pSTK1 form another, new family of ssDNA plasmids. PCR analysis revealed that the pGI3 rep gene is largely distributed among B. thuringiensis strains but can also be found in B. cereus and B. mycoides strains, albeit at a lower frequency. Finally, segregation experiments performed with B. subtilis and B. thuringiensis showed that the pGI3 derivatives, including the minimal replicon, were segregationally stable at temperatures suitable for B. thuringiensis growth (<43 degrees C).     

苏云金芽胞杆菌拟步行甲亚种YBT-1765中稳定质粒pBMB175的克隆和功能分析

从苏云金芽胞杆菌拟步行甲亚种YBT_1765中克隆得到一个大小约15.2kb的质粒pBMB175,构建了该质粒的限制性图谱,通过功能验证,将其最小的复制区定位在一个1151bp的片段上。分析了包含有这个复制区的一个大小为4152bp的核苷酸序列,该片段包含有3个编码框(ORF1、OFR2和ORF3)。氨基酸序列同源性比较发现,ORF1(767AA)与UvrD_旋促酶、重组酶RecD和RecB家族具有20%～30%的相似性;ORF2(149AA)没有发现与任何已知序列具有同源性;ORF3(83AA)与pGI3中一个未知功能的蛋白(ORF7)具有34%的相似性。通过缺失及序列比较分析推测ORF2可能编码一种新的复制蛋白。因此pBMB175的复制类型可能属于一类新的复制家族。利用最小复制区构建的重组质粒在无抗生素选择压力下可稳定遗传40多代,具备构建稳定遗传质粒载体的潜力。     

Sequence analysis and characterization of pOM1, a small cryptic plasmid from Butyrivibrio fibrisolvens, and its use in construction of a new family of cloning vectors for Butyrivibrios.

As a preliminary step in the development of vector systems, we have isolated and begun to characterize small, cryptic plasmids from several strains of the rumen bacterium Butyrivibrio fibrisolvens. We present here the complete nucleotide sequence of Butyrivibrio plasmid pOM1, which was isolated from B. fibrisolvens Bu49. While it is very similar in size to the previously characterized Butyrivibrio plasmids pRJF1 and pRJF2, pOM1 exhibits a restriction pattern which is quite distinct. Analysis of sequence data reveals that pOM1 contains only two open reading frames of significant length (ORF1 and ORF2), both of which are required for self-replication and maintenance. The protein encoded in ORF1 shows homologies with Pre (plasmid recombination enzyme) proteins encoded in plasmids from gram-positive organisms such as Staphylococcus aureus, Streptococcus agalactiae, Lactobacillus plantarum, and Bacillus thuringiensis. The putative translation product of ORF2, on the other hand, resembles Rep (replication) proteins of a different group of gram-positive plasmids, for which the Staphylococcus plasmid pSN2 is a prototype. Unlike the other characterized-Butyrivibrio plasmids, pOM1 appears to replicate via a rolling-circle mechanism. Experimental evidence showing the presence of a single-stranded replication intermediate consistent with this mechanism is presented. pOM1 has been used in the construction of a new Escherichia coli-B. fibrisolvens shuttle vector, pSMerm1, which has been successfully used to introduce a cloned gene into B. fibrisolvens harboring the pRJF1 plasmid.     

Nucleotide sequence analysis of pRS1, a cryptic plasmid from Oenococcus oeni

A new cryptic plasmid, pRS1, from an Oenococcus oeni strain isolated from Spanish wines is reported. Nucleotide sequence analysis (2523 bp) revealed the presence of three major open reading frames (ORFs) whose nucleotide sequence and encoded proteins exhibit high homology with those of pOg32, a previously described plasmid of O. oeni. Common features in other plasmids from O. oeni (i.e., pLo13 and pOg32) have been found in pRS1. They have three major ORFs in the same strand; the putative encoded proteins by two of these ORFs exhibit homology with the replication (Rep) and the recombination (Pre) proteins, respectively, of the pT181 plasmid family and related gram-positive bacteria plasmids; these plasmids contain the DNA sequences required for plasmid replication by the rolling circle mechanism and for recombination (i.e., double-strand origin, DSO; single-strand origin, SSO; recombination-specific sites, RSA and RSB); and finally, all these plasmids have a third ORF of unknown function. These features suggest that pRS1 could constitute together with pLo13 and pOg32 a family of small cryptic plasmids of O. oeni.     

Self-transfer and mobilisation capabilities of the pXO2-like plasmid pBT9727 from Bacillus thuringiensis subsp. konkukian 97-27

Recent characterisations of plasmids related to the anthrax virulence plasmids pXO1 and pXO2 in clinical isolates of Bacillus cereus and Bacillus thuringiensis have contributed to the emerging picture of a virulence-associated plasmid pool in the B. cereus sensu lato group. The family of pXO2-like plasmids includes the conjugative plasmid pAW63 from the biopesticide strain B. thuringiensis subsp. kurstaki HD73 and the heretofore cryptic plasmid pBT9727 from the clinical strain B. thuringiensis subsp. konkukian 97-27. Comparative sequence analysis of these three plasmids suggested that they were derived from an ancestral conjugative plasmid, with pAW63 retaining its self-transfer capabilities, and pXO2 having lost them through genetic drift. Such properties had not been investigated in pBT9727, but sequence homologies led us to predict that it may possess self-transfer capabilities. Here, we report that pBT9727 is indeed conjugative, and is able to promote its own transfer as well as that of small mobilisable plasmids.     

Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids.

The replication origins of three large Bacillus thuringiensis plasmids, derived from B. thuringiensis HD263 subsp. kurstaki, have been cloned in Escherichia coli and sequenced. The replication origins, designated ori 43, ori 44, and ori 60, were isolated from plasmids of 43, 44, and 60 MDa, respectively. Each cloned replication origin exhibits incompatibility with the resident B. thuringiensis plasmid from which it was derived. Recombinant plasmids containing the three replication origins varied in their ability to transform strains of B. thuringiensis, Bacillus megaterium, and Bacillus subtilis. Analysis of the derived nucleotide and amino acid sequences indicates that the replication origins are nonhomologous, implying independent derivations. No significant homology was found to published sequences of replication origins derived from the single-stranded DNA plasmids of gram-positive bacteria, and shuttle vectors containing the three replication origins do not appear to generate single-stranded DNA intermediates in B. thuringiensis. The replication origin regions of the large plasmids are each characterized by a single open reading frame whose product is essential for replication in B. thuringiensis. The putative replication protein of ori 60 exhibits partial homology to the RepA protein of the Bacillus stearothermophilus plasmid pTB19. The putative replication protein of ori 43 exhibits weak but extensive homology to the replication proteins of several streptococcal plasmids, including the open reading frame E replication protein of the conjugative plasmid pAM beta 1. The nucleotide sequence of ori 44 and the amino acid sequence of its putative replication protein appear to be nonhomologous to other published replication origin sequences.     

Replication Mechanism and Sequence Analysis of the Replicon of pAW63, a Conjugative Plasmid from Bacillus thuringiensis

A 5.8-kb fragment of the large conjugative plasmid pAW63 from Bacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMbeta1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMbeta1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMbeta1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the Enterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repC and prgO, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti.     

The smr gene resides on a novel plasmid pSP187 identified in a Staphylococcus pasteuri isolate recovered from unpasteurized milk

This work describes a novel plasmid pSP187 (5550 bp) carrying the small multidrug resistance determinant smr encoding resistance to quaternary ammonium compounds (QACs). pSP187 was identified in a Staphylococcus pasteuri isolate recovered from bulk milk in a dairy cattle herd in Norway. Sequence analysis revealed 6 putative ORFs in addition to the smr gene within a cassette with identical genetic organization to that found in the pSK41-like Staphylococcus aureus plasmid pTZ22. A protein homology search suggested the gene product of ORF7 to be a putative replication initiation protein, while ORF2 was predicted to encode a protein homologous to members of FtsK/SpoIIIE cell division-DNA segregation protein families. Sequence similarities to some initiator proteins of rolling circle replicons (RCR) indicated that pSP187 uses a RCR mode of replication, supported by the detection of intermediate ssDNA using S1 nuclease treatment and hybridization analysis. Interestingly, a 30-bp sequence found upstream from ORF7 showed high similarity to other dyad symmetry motifs proposed as putative double-strand origins of replication in the plasmids pGI3 (Bacillus thuringiensis), pSTK1 (Bacillus stearothermophilus), and pER1-2 (Streptococcus thermophilus). In conclusion: The novel smr-containing plasmid pSP187 is the first member of RCR group VI to be identified in a Staphylococcus sp.     

The patchwork nature of rolling-circle plasmids: comparison of six plasmids from two distinct Bacillus thuringiensis serotypes

Bacillus thuringiensis, the entomopathogenic bacteria from the Bacillus cereus group, harbors numerous extrachromosomal molecules whose sizes vary from 2 to more than 200kb. Apart from the genes coding for the biopesticide delta-endotoxins located on large plasmids, little information has been obtained on these plasmids and their contribution to the biology of their host. In this paper, we embarked on a detailed comparison of six small rolling-circle replicating (RCR) plasmids originating from two major B. thuringiensis strains. The complete nucleotide sequences of plasmid pGI1, pGI2, pGI3, pTX14-1, pTX14-2, and pTX14-3 have been obtained and compared. Replication functions, comprising, for each plasmid, the gene encoding the Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified and analyzed. Two new families, or homology groups, of RCR plasmids originated from the studies of these plasmids (Group VI based on pGI3 and Group VII based on pTX14-3). On five of the six plasmids, loci involved in conjugative mobilization (Mob-genes and origin of transfer (oriT)) were identified. Plasmids pTX14-1, pTX14-2, and pTX14-3 each harbor an ORF encoding a polypeptide containing a central domain with repetitive elements similar to eukaryotic collagen (Gly-X-Y triplets). These genes were termed bcol for Bacillus-collagen-like genes.     

Distribution of 2-kb miniplasmid pBMB2062 from Bacillus thuringiensis kurstaki YBT-1520 strain in Bacillus species

A multi-copy and small plasmid pBMB2062 from Bacillus thuringiensis kurstaki YBT-1520 strain was cloned and characterized and its distribution was analyzed using dot-blot analysis with the ORF1 fragment as a probe. Bacillus species of 84 serotypes were evaluated. The pBMB2062 plasmid was found to be present in commercial B. thuringiensis kurstaki (H3abc) and aizawai (H7) insecticides of various serotypes, and one Bacillus cereus UW85 strain (produced Zwittermicin fungicide and Cry toxin synergist). The sequences of 7 pBMB2062-like plasmids from randomly selected Bacillus species (positive signal in the dot-blot analysis) were highly conserved. Two open reading frames (ORFs), ORF1 and ORF2, were present in this plasmid. ORF1 was found to be necessary for plasmid replication, whereas ORF2 did not play a role in replication or stability. Based on its sequence homology, ORF2 was a putative solitary antitoxin. Furthermore, the copy number of the replicon of pBMB2062 was higher than those of ori1030 and ori44 based on the thermogenic data, and ori2062 could drive the stable replication of a recombinant plasmid (11 kb total size) in B. thuringiensis.     

Cloning and partial characterization of three small cryptic plasmids from Bacillus thuringiensis

The strain H1.1 of Bacillus thuringiensis var. thuringiensis harbors three small cryptic plasmids: pGI1, pGI2, and pGI3 (8.2, 9.2, and 10.6 kb, respectively). Two of these plasmids (i.e., pGI2 and pGI3) were successfully cloned in their entirety into the vector pBR322, whereas only overlapping DNA fragments covering pGI1 were obtained in Escherichia coli. A curing-hybridization technique was used to obtain isolates of B. thuringiensis missing one or another small cryptic plasmid. These derivatives were examined for any change in a phenotypic trait, but no specific function could be assigned to one of these plasmids. Hybridization and restriction mapping data revealed that the transposon Tn4430 accounts for 45% of the pGI2 plasmid DNA.     

Complete nucleotide sequence and characterization of pSNA1 from pimaricin-producing Streptomyces natalensis that replicates by a rolling circle mechanism

A cryptic plasmid, pSNA1, has been identified in the pimaricin-producing Streptomyces natalensis strain ATCC 27448. pSNA1 has been mapped with restriction endonucleases and its complete nucleotide sequence was determined. The circular DNA molecule is 9367 bp in length and has a 71.3% G+C content. Its estimated copy number is 30. Analysis of the sequence and codon preferences indicated that pSNA1 contains seven open reading frames [encoding peptides larger than 90 amino acid (aa) residues], ORF 1 to ORF 7, located on both strands of pSNA1. ORF 3 codes for a protein (476 aa) that shows high sequence similarity to replication-associated proteins in Streptomyces plasmids known to replicate via the rolling circle mechanism. Accumulation of single-strand intermediates further indicates that pSNA1 replicates via the rolling circle replication model. ORF 1 encodes a polypeptide of 246 aa that shares homology with KorA proteins encoded by other streptomycete plasmids. ORF 4 (SpdA) codes for a protein (161 aa) possibly involved in intramycelial plasmid transfer. Protein encoded by ORF 2 (309 aa) shares homology with a Streptomyces protein (SpdB2) also involved in plasmid spreading.     

Intraspecies plasmid transformation of Bacillus thuringiensis

Bacillus thuringiensis strains 1009, 3004, 3029 of the natural isolates of var. dendrolimus harbouring two large plasmids were transformed by a mix of small plasmids DNA. One of transforming plasmids carried a gene for tetracyclinresistance. The natural isolates of var. dendrolimus strain 2002 and var. berliner strain 1035 were used as donors for plasmid DNA transformation. Three variants of plasmid spectra registered in tetracycliresistant transformants are discussed.     

Comparison of the in vivo and in vitro activity of the delta-endotoxin of Bacillus thuringiensis var. morrisoni (HD-12) and two of its constituent proteins after cloning and expression in Escherichia coli

The insecticidal crystal delta-endotoxin of Bacillus thuringiensis var. morrisoni HD-12 contains at least five polypeptides in the range 126-140 kDa. Immune blotting revealed that individual proteins in this complex share homology with a range of other B. thuringiensis delta-endotoxins. In vivo the native HD-12 crystal killed a lepidopteran larva (Pieris brassicae) and a dipteran larva (Anopheles gambiae), but not the related dipteran Aedes aegypti. In vitro the solubilized activated crystal lysed Choristoneura fumiferana cells (lepidopteran) and dipteran cells derived from Anopheles gambiae and Culex quinquefasciatus but not those from Aedes aegypti. An intragenic probe derived from a B. thuringiensis var. sotto lepidoptera-specific delta-endotoxin gene hybridized with one of six plasmids extracted from HD-12. When cloned into pUC18 two HindIII fragments from this plasmid (pEG1 and pEG2) were shown to encode polypeptides cross-reacting with HD-12 antiserum. Escherichia coli lysates containing pEG2 were toxic in vivo to lepidoptera and diptera larvae and in vitro to a broader range of insect cell lines than the native crystal. E. coli cells containing pEG3, a subclone derived from pEG1, synthesised large amounts of a 140-kDa protein in the cytoplasm as inclusion bodies. The cytotoxicity of the protein encoded by pEG3 was restricted to C. fumiferana and A. gambiae cell lines.     

Identification of self-transmissible plasmids in four Bacillus thuringiensis subspecies.

The transfer of plasmids by mating from four Bacillus thuringiensis subspecies to Bacillus anthracis and Bacillus cereus recipients was monitored by selecting transcipients which acquired plasmid pBC16 (Tcr). Transcipients also inherited a specific large plasmid from each B. thuringiensis donor at a high frequency along with a random array of smaller plasmids. The large plasmids (ca. 50 to 120 megadaltons), pXO13, pXO14, pXO15, and pXO16, originating from B. thuringiensis subsp. morrisoni, B. thuringiensis subsp. toumanoffi, B. thuringiensis subsp. alesti, and B. thuringiensis subsp. israelensis, respectively, were demonstrated to be responsible for plasmid mobilization. Transcipients containing any of the above plasmids had donor capability, while B. thuringiensis strains cured of each of them were not fertile, indicating that the plasmids confer conjugation functions. Confirmation that pXO13, pXO14, and pXO16 were self-transmissible was obtained by the isolation of fertile B. anthracis and B. cereus transcipients that contained only pBC16 and one of these plasmids. pXO14 was efficient in mobilizing the toxin and capsule plasmids, pXO1 and pXO2, respectively, from B. anthracis transcipients to plasmid-cured B. anthracis or B. cereus recipients. DNA-DNA hybridization experiments suggested that DNA homology exists among pXO13, pXO14, and the B. thuringiensis subsp. thuringiensis conjugative plasmids pXO11 and pXO12. Matings performed between strains which each contained the same conjugative plasmid demonstrated reduced efficiency of pBC16 transfer. However, in many instances when donor and recipient strains contained different conjugative plasmids, the efficiency of pBC16 transfer appeared to be enhanced.     

Amino acid sequence and entomocidal activity of the P2 crystal protein. An insect toxin from Bacillus thuringiensis var. kurstaki

The gene encoding the 66-kDa entomocidal protein (P2 protein or mosquito factor) from Bacillus thuringiensis var. kurstaki has been isolated by the use of a 62-mer oligonucleotide probe that encoded 21 amino acids of the P2 protein NH2 terminus. The DNA sequence of the gene, designated cryBI, was unique from the published sequences of other B. thuringiensis genes. However, the amino acid sequence of the P2 protein, as deduced from the DNA sequence of the cryBI gene, was found to contain a sequence of 100 amino acids having 37% homology to a group of B. thuringiensis entomocidal proteins, the P1 proteins. Late stationary phase Bacillus megaterium cells harboring the cloned B. thuringiensis cryBI gene contained large aggregates of the P2 protein, and the cells were highly toxic to both lepidopteran and dipteran larvae. In contrast, Escherichia coli cells harboring the cloned cryBI gene contained very low levels of the P2 protein. DNA blot hybridization experiments showed that certain B. thuringiensis strains contained at least one cryBI-related DNA sequence in addition to the cryBI gene itself.     

Transfer of chromosomal genes and plasmids in Bacillus thuringiensis.

A low frequency of chromosomal gene transfer from Bacillus thuringiensis to Bacillus cereus was detected by cell mating, with a tryptophan marker being the most frequently transferred gene among four that were tested. The process was resistant to DNase and was not mediated by cell filtrates. Among several B. thuringiensis subspecies tested, transfer was best with a derivative of B. thuringiensis subsp. kurstaki HD1, which lost several plasmids. All of the B. cereus recombinants contained at least one plasmid from the donor B. thuringiensis; frequently, it was a plasmid that encoded a protoxin gene. In matings with B. thuringiensis subsp. kurstaki HD1, a 29-megadalton plasmid that contained a ca. 2.5-kilobase region of homology with the chromosome was always transferred. No detectable transfer of chromosomal genes was found in B. thuringiensis subsp. kurstaki HD1 strains lacking this plasmid, suggesting that there may be chromosome mobilization.