Detection and characterization of naturally occurring plasmids in Bacillus licheniformis

Twenty-two Bacillus licheniformis strains, freshly isolated from pasture-land, were studied for the presence of plasmid DNA. Among these strains, 14 were shown to harbor one or more plasmids of different size. Southern-hybridization experiments showed a high homology between all plasmids investigated and a 2.2-kb PvuII/HindIII fragment of pBL1, a B. licheniformis plasmid previously isolated. Three fragments of pBL1, including the 2.2-kb PvuII/HindIII region, were cloned into pJH101 vector. The resulting chimeras were able to transform Bacillus subtilis. The fragment with high homology probably contains the region with the replicative functions of plasmids from B. licheniformis species.     

Complete Sequence of Bacillus subtilis Plasmid p1414 and Comparison with Seven Other Plasmid Types Found in Russian Soil Isolates of Bacillus subtilis

We determined the complete sequence of a cryptic 7949-bp plasmid isolated from naturally occurring Bacillus subtilis found in Russian soil from Moscow. We found 15 putative open reading frames (ORFs), all of which were preceded by a ribosome binding site. One encodes the gene (rep) which should be essential for vegetative rolling circle replication (RCR). The putative double-stranded origin as well as a palT1-like single-stranded origin was also identified. The predicted product of another ORF showed similarity to a moblization protein while a third showed similarity to a ubiquitous family of small proteins whose members have so far been associated with stress response. We used fragments with these latter ORFs to probe representatives of seven other groups of cryptic RCR plasmids from geographically related B. subtilis isolates. All plasmids carried the mob function, suggesting a common ancestor for the rep/mob region but the putative hsp was present only on some of the plasmids. This suggests that the putative hsp gene is not an essential plasmid component and may therefore be present as a phenotypic marker-perhaps providing response to stress. This adds weight to the growing evidence that these small Bacillus plasmids may not be cryptic but may provide an adaptive advantage for the host in its natural environment.     

Experimental basis for a stable plasmid, pLS30, to shuttle between Bacillus subtilis species by conjugational transfer

The use of Bacillus subtilis 168 as the initial host for molecular cloning and subsequent delivery of the engineered DNA to other Bacillus hosts appears attractive, and would lead to an efficient DNA manipulation system. However, methods of delivery to other Bacillus species are limited due to their inability to develop natural competence. An alternative, unexplored conjugational transfer method drew our attention and a B. subtilis native plasmid, pLS30, isolated from B. subtilis (natto) strain IAM1168 was characterized for this aim. The nucleotide sequence (6,610 bp) contained the mob gene and its recognition sequence, oriT, that features pLS30 as a mobile plasmid between Bacillus species on conjugational transfer. Plasmid pLS3001, a chimera with a pBR322-based plasmid prepared in Escherichia coli to confer an antibiotic resistance marker, showed apparent mobilizing activity in the pLS20-mediated conjugational transfer system recently established. The rep gene and associated palT1-like sequence common to all other pLS plasmids previously sequenced indicated that pLS30 is a typical rolling circle replicating (RCR) type plasmid. Due to the significant stability of pLS30 in IAM1168, application of a mobile plasmid would allow quick propagation to Bacillus species.     

Homology among nearly all plasmids infecting three Bacillus species.

We have surveyed naturally occurring plasmids in strains of Bacillus subtilis and the closely related species B. mojavensis and B. licheniformis. Previous studies have failed to find host-benefitting functions for plasmids of these species, suggesting that these plasmids are nonmutualistic. Only one type of plasmid was found in each plasmid-bearing strain, suggesting that most of the plasmids infecting these Bacillus species are in the same incompatibility group. A sample of 18 plasmids from these species ranged in size from 6.9 to 16 kb, with all but 6 plasmids falling into three size groups. These groups differed in the sizes of their host ranges and geographical ranges. All but 1 of the 18 plasmids from these three host species are homologous with one another. The cryptic plasmids from these three species are far less diverse than are plasmids (from other species) that are known to benefit their bacterial hosts. The low-level diversity among these cryptic plasmids is consistent with the hypothesis that host-benefitting adaptations play an important role in fostering the coexistence of plasmid populations, but other explanations for the low-level plasmid diversity are possible. Comparison of the phylogenies of the plasmids with those of their hosts suggests that Bacillus plasmids are horizontally transferred in nature at a low rate similar to that found for the colicin plasmids of Escherichia coli.     

Nucleotide sequence and analysis of pBL1, a bacteriocin-producing plasmid from Lactococcus lactis IPLA 972

The complete sequence of the 10.9-kbp bacteriocinogenic plasmid pBL1 from Lactococcus lactis subsp. lactis IPLA 972 has been determined. Thirteen ORFs were encountered, of which 5 were incomplete. pBL1 proved to be a narrow-host-range plasmid which replicates neither in Bacilus subtilis nor in Lactobacillus spp. The structural organization of the pBL1 replication region was highly similar to other well-known theta-replicating plasmids of lactococci, at both the untranslated (the replication origin) and the translated (repB and orfX) sequences. As in other plasmids, the product of orfX was not necessary for plasmid replication. However, it was shown to be involved in plasmid stability. Three genes organized in an operon-like structure encompassed, most likely, the bacteriocin-encoding region. Upstream of the origin of replication a nicking site (oriT) was found. This oriT sequence proved to be functional by mobilization of plasmids wearing it. One complete and several partial IS elements were identified on pBL1.     

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).     

Small rolling circle plasmids in Bacillus subtilis and related species: organization, distribution, and their possible role in host physiology

Bacillus subtilis and related species (Bacillus licheniformis, Bacillus pumilus, Bacillus amyloliquefaciens, and Bacillus mojavensis) represent a group of bacteria largely studied and widely employed by industry. Small rolling circle replicating plasmids of this group of bacteria have been intensively studied as they represent a convenient model for genetic research and for the construction of molecular tools for the genetic modification of their hosts. Through the computational analysis of the available plasmid sequences to date, the first part of this review focuses on the main stages that the present model for rolling circle replication involves, citing the research data which helped to elucidate the mechanism by which these molecules replicate. Analysis of the distribution and phylogeny of the small RC plasmids inside the Bacillus genus is then considered, emphasizing the low level of diversity observed among these plasmids through the in silico analysis of their organization and the sequence divergence of their replication module. Finally, the parasitic vs. mutualistic nature of small rolling circle plasmids is briefly discussed.     

Molecular analysis of the replication region of the conjugative Streptococcus agalactiae plasmid pIP501 in Bacillus subtilis. Comparison with plasmids pAM beta 1 and pSM19035.

The large conjugative plasmid pIP501 was originally isolated from Streptococcus agalactiae. To study the molecular basis of pIP501 replication we determined the nucleotide sequence of a 2.2 kb DNA segment which is essential and sufficient for autonomous replication of pIP501 derived plasmids, in Bacillus subtilis cells. This region can be divided into two functionally discrete segments: a 496 bp region (oriR) that acts as an origin of replication, and a 1488 bp segment coding for an essential replication protein (RepR). The RepR protein, which has a molecular mass of 57.4 kDa, could complement in trans a thermosensitive replicon bearing the pIP501 origin. Chimeric Rep proteins and replicons were obtained by domain swapping between rep genes of closely related streptococcal plasmids belonging to the inc18 group (pIP501, pAM beta 1 and pSM19035). The chimeras were functional in B. subtilis.     

Identification and molecular genetic analysis of replication functions of the bacteriocinogenic plasmid pIP404 from Clostridium perfringens

The replication functions of the bacteriocinogenic plasmid pIP404, from Clostridium perfringens, were localized to a 2.8-kb EcoRI-EcoRV fragment by cloning into a vector deficient for replication in Bacillus subtilis. This fragment contains two genes, cop and rep, which encode proteins and an 800-bp noncoding segment of complex structure consisting of multiple tandemly repeated sequences. The Cop protein is involved in copy number control, whereas the rep gene product is essential for plasmid replication. By deletion analysis the minimal origin of replication was defined as the rep gene plus most of the repeated sequences. A powerful promoter producing a 150-nucleotide RNA molecule, RNA1, that could act as an anti-sense RNA to the rep gene was detected in the "origin-like" region. In contrast to most other small plasmids of gram-positive bacteria, pIP404, and its derivatives, does not appear to replicate via a single stranded intermediate in either C. perfringens or B. subtilis.     

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.     

Cloning of the genes for penicillinase, penP and penI, of Bacillus licheniformis in some vector plasmids and their expression in Escherichia coli, Bacillus subtilis, and Bacillus licheniformis.

By using plasmid pMB9, penicillinase genes (penP and penI) from both the wild-type and constitutive strains of Bacillus licheniformis 9945A were cloned in EScherichia coli. When a low-copy-number plasmid was used, both wild-type and constitutive penicillinase genes could be transferred into Bacillus subtilis. However, when a high-copy-number plasmid was used, only the genes of the wild type could be transferred. These recombinant plasmids in B. subtilis could all be transferred by the protoplast transformation procedure into B. licheniformis. Transformants of E. coli were resistant to ampicillin (20 micrograms/ml) in spite of the low penicillinase activities (7 U/mg of cells). However, transformants of B. subtilis and B. licheniformis were sensitive to ampicillin (20 micrograms/ml) even in high penicillinase activities (more than 10,000 U/mg of cells). The secretion of penicillinase was rarely observed in E. coli. In contrast, penicillinases secreted from transformants of B. subtilis and B. licheniformis were around 30 and 60% of the total activities, respectively. We took advantage of the plasmids to permit the construction of hetero- and mero-polyploid structures in host cells, and we discuss a regulatory mechanism of penicillinase synthesis in B. licheniformis.     

Molecular cloning in Bacillus subtilis of a Bacillus licheniformis gene encoding a thermostable alpha amylase

A resident-plasmid cloning system developed for Bacillus subtilis has been used to isolate recombinant plasmids carrying DNA from Bacillus licheniformis which confer alpha-amylase activity on alpha-amylase-negative mutants of B. subtilis. These plasmids contain a 3550-bp insert at the EcoRI site of the plasmid pBD64. Subcloning various lengths of the B. licheniformis DNA has localised the gene to a 2550-bp BclI fragment. We present evidence that the cloned fragment codes for a B. licheniformis heat-stable alpha-amylase with a temperature optimum of 93 degrees C. The foreign gene is expressed efficiently in B. subtilis and is stably maintained.     

Bacillus sporulation gene spo0H codes for sigma 30 (sigma H).

The DNA sequences of the spo0H genes from Bacillus licheniformis and B. subtilis are described, and the predicted open reading frames code for proteins of 26,097 and 25,447 daltons, respectively. The two spo0H gene products are 91% identical to one another and about 25% identical to most of the procaryotic sigma factors. The predicted proteins have a conserved 14-amino-acid sequence at their amino terminal end, typical of sigma factors. Antibodies raised against the spo0H gene product of B. licheniformis specifically react with RNA polymerase sigma factor protein, sigma 30, purified from B. subtilis. We conclude that the spo0H genes of B. licheniformis and B. subtilis code for sigma 30, now known as sigma H.     

地衣芽胞杆菌dif序列的功能鉴定

地衣芽胞杆菌是重要的工业菌株,如何为其建立一种有效的基因删除技术是对该菌株进行遗传改良的基础。枯草芽胞杆菌difB8序列已经被成功用于枯草芽胞杆菌多基因的删除。在分析地衣芽胞杆菌基因组序列并获得与枯草芽胞杆菌difB8以。序列十分相似的一段序列difBLi的基础上,构建了在庆大霉素抗性基因两侧具有difBLi的重组质粒pMD19-difGm和pHY-XI’：：difGm,通过电击转化法将质粒pHY-XI’：：difGm导入B．1icheniformis ATCC14580中,筛选获得了具有庆大霉素抗性的转化子。在转化子的传代过程中,重组质粒的庆大霉素抗性基因在体内Xer／dif／f位点特异性重组系统的介导下通过其侧翼的dif位点进行同源重组而被准确删除。确证了地衣芽胞杆菌中dif序列的功能,为地衣芽胞杆菌基因组中多基因的删除提供了一种新的实验途径。     

Complete sequence of three plasmids from Bacillus thuringiensis INTA-FR7-4 environmental isolate and comparison with related plasmids from the Bacillus cereus group

Bacillus thuringiensis is an insect pathogen used worldwide as a bioinsecticide. It belongs to the Bacillus cereus sensu lato group as well as Bacillus anthracis and B. cereus. Plasmids from this group of organisms have been implicated in pathogenicity as they carry the genes responsible for different types of diseases that affect mammals and insects. Some plasmids, like pAW63 and pBT9727, encode a functional conjugation machinery allowing them to be transferred to a recipient cell. They also share extensive homology with the non-functional conjugation apparatus of pXO2 from B. anthracis. In this study we report the complete sequence of three plasmids from an environmental B. thuringiensis isolate from Argentina, obtained by a shotgun sequencing method. We obtained the complete nucleotide sequence of plasmids pFR12 (12 095 bp), pFR12.5 (12 459 bp) and pFR55 (55 712 bp) from B. thuringiensis INTA-FR7-4. pFR12 and pFR12.5 were classified as cryptic as they do not code for any obvious functions besides replication and mobilization. Both small plasmids were classified as RCR plasmids due to similarities with the replicases they encode. Plasmid pFR55 showed a structural organization similar to that observed for plasmids pAW63, pBT9727 and pXO2. pFR55 also shares a tra region with these plasmids, containing genes related to T4SS and conjugation. A comparison between pFR55 and conjugative plasmids led to the postulation that pFR55 is a conjugative plasmid. Genes related to replication functions in pFR55 are different to those described for plasmids with known complete sequences. pFR55 is the first completely sequenced plasmid with a replication machinery related to that of ori44. The analysis of the complete sequence of plasmids from an environmental isolate of B. thuringiensis permitted the identification of a near complete conjugation apparatus in pFR55, resembling those of plasmids pAW63, pBT9727 and pXO2. The availability of this sequence is a step forward in the study of the molecular basis of the conjugative process in Gram positive bacteria, particularly due to the similarity with known conjugation systems. It is also a contribution to the expansion of the non-pathogenic B. cereus plasmid gene pool.     

Sequence analysis of two cryptic plasmids from Bifidobacterium longum DJO10A and construction of a shuttle cloning vector

Bifidobacterium longum DJO10A is a recent human isolate with probiotic characteristics and contains two plasmids, designated pDOJH10L and pDOJH10S. The complete sequences of both these plasmids have now been determined and consist of two circular DNA molecules of 10,073 and 3,661 bp, with G+C contents of 62.2% and 66.2%, respectively. Plasmid pDOJH10L is a cointegrate plasmid consisting of DNA regions exhibiting very high sequence identity to two other B. longum plasmids, pNAC2 (98%) and pKJ50 (96%), together with another region. Interestingly, the rolling circular replication (RCR) regions of both the pNAC2- and pKJ50-like plasmids were disrupted during the recombination event leading to a further recombination event to acquire a functional replicon. This consists of a new fused rep gene and an RCR-type ori consisting of a conserved DnaA box in an AT-rich region followed by four contiguous repeated sequences consistent with an iteron structure and an inverted repeat. The smaller pDOJH10S had no sequence similarity to any other characterized plasmid from bifidobacteria. In addition, it did not contain any features consistent with RCR, which is the replication mechanism proposed for all the bifidobacteria plasmids characterized to date. It did exhibit sequence similarity with several theta replication-related replication proteins from other gram-positive, high-G+C bacteria, with the closest match from a Rhodococcus rhodochrous plasmid, suggesting a theta mechanism of replication. S1 nuclease analysis of both plasmids in B. longum DJO10A revealed single-strand DNA intermediates for pDOJH10L, which is consistent for RCR, but none were detected for pDOJH10S. As the G+C content of pDOJH10S is similar to that of Rhodococcus rhodochrous (67%) and significantly higher than that of B. longum (60.1%), it may have been acquired through horizontal gene transfer from a Rhodococcus species, as both genera are members of the Actinomycetes and are intestinal inhabitants. An Escherichia coli-B. longum shuttle cloning vector was constructed from pDOJH10S and the E. coli ori region of p15A, a lacZ gene with a multiple cloning site of pUC18, and a chloramphenicol resistance gene (CAT) of pCI372 and was transformed successfully into E. coli and B. longum. It could not be introduced into lactic acid bacteria (Lactococcus and Lactobacillus), showing it was not very promiscuous. It was stably maintained in B. longum in the absence of antibiotic pressure for 92 generations, which is consistent with the segregational stability of theta-replicating plasmids in gram-positive bacteria. This is the first cloning vector for bifidobacteria that does not utilize RCR and should be useful for the stable introduction of heterologous genes into these dominant inhabitants of the large intestine.     

Isolation and characteristics of a plasmid from the thermotolerant strain of Bacillus licheniformis

A 8.3 kb cryptic plasmid was isolated from the thermotolerant strain of Bacillus licheniformis 28KA and designated pLT83. The replicative (rep) region was localized on the plasmid map. The pLT83 plasmid labelled in vitro with an antibiotic resistance determinant is able to replicate in B. subtilis cells. The pLT83 plasmid replicates stably in B. licheniformis strain at higher temperatures (37-60 degrees C) than in B. subtilis cells (37-50 degrees C). The plasmid and its derivatives may be used as vectors for gene cloning in B. subtilis and B. licheniformis cells.