Characterization of plasmid pSY3 in <Emphasis Type="Italic">Sphingobium chungbukense</Emphasis> DJ77

This study determined the complete nucleotide sequence of the plasmid pSY3 from Sphingobium chungbukense DJ77. It was 35,735 bp long with a G+C content of 61.9%. Forty open reading frames (ORFs) were found. We predicted these ORFs would encode proteins associated with plasmid replication, conjugative transfer, transposition of genes, plasmid stability/partition, hypothetical protein, and some other functions. Genes for biodegradation were not found. No other plasmid homologous to pSY3 in the overall nucleotide sequence or gene organization could be found in the NCBI database.     

Is cell motility a plasmid-encoded function in the cyanobacterium Synechocystis 6803?

Abstract The unicellular facultatively heterotrophic cyanobacterium Synechocystis 6803 harbors 4 plasmids, pSY1–4, of 2.5, 5.2, 50 and 100 kb, respectively. We observed that the loss of the pSY2 plasmid and that of cell motility occurs at a high frequency. However, we showed that there is no direct correlation between these two phenomena, and that cell motility does not require a pSY2-encoded function. In addition, restriction analysis and hybridization experiments showed that pSY1 and pSY2 share no homologous DNA sequences.     

Analysis of the 7.6-kb cryptic plasmid pNC500 from <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> B-276 and construction of <Emphasis Type="Italic">Rhodococcus</Emphasis>–<Emphasis Type="Italic">E. coli</Emphasis> shuttle vector

Four circular cryptic plasmids were detected from propene-degrading Rhodococcus rhodochrous (formerly Nocardia corallina) B-276 and the smallest 7.6-kb plasmid, named pNC500 was used to construct Rhodococcus–E. coli shuttle vector, pNC5403. Sequence analysis of pNC500 revealed that the plasmid contains eight potential ORFs, namely 1 through 8. The deduced amino acid sequences for ORFs 3, 4, 6, and 7 show homology with those of Rep A, Rep B, DNA methyl-transferase (M.XamI), and restriction nuclease (R.XamI), respectively. The region responsible for replication in the potent oil-desulfurization bacterium, Rhodococcus opacus T09 was determined as 3.7 kb-XbaI/BalI fragment which contains ORFs 3 and 4, while no transformants were obtained when ORF 4 was partially deleted, suggesting that both are required for its replication. Alignment of the predicted amino acid sequences revealed that ORFs 3 and 4 were DNA binding protein and DNA primase, respectively. A compatibility test with pAL5000-related plasmid vector, pRHK1, which contains pRC4, revealed that pNC5403 was compatible with pRHK1 suggesting that each replication origin would be different. ORFs 3 and 4 containing a pNC5403 derivative, pN5DXB, was stably maintained for over 80 generations in the absence of antibiotic selective conditions.     

Characterization of the transfer-related <Emphasis Type="Italic">tra</Emphasis> region of the conjugative plasmid p19 from a <Emphasis Type="Italic">Bacillus subtilis</Emphasis> soil strain

The nucleotide sequences of three DNA fragments (total size 30574 bp) of the plasmid p19 from the Bacillus subtilis 19 soil strain have been determined. Thirty open reading frames (ORFs) have been identified in these fragments. oriT of the plasmid has also been identified. As shown by the search for homologs of hypothetical protein products of these ORFs in databases, such homology exists for 18 ORFs. The protein products of nine ORFs can be assumed to have specific functions. Several ORFs were inactivated via insertional mutagenesis, and the conjugation capacity of the mutant plasmids was estimated. According to the data on homology of protein products and the results of ORF inactivation, regions of a total size of about 20 kb from the DNA fragments sequenced by us were inferred to belong to the tra region of p19. As follows from the analysis of the identified ORFs of the p19 tra region, it differs from the earlier described tra regions of other plasmids, irrespective of a certain similarity with the corresponding regions of plasmids of gram-positive bacteria from the genera Bacillus, Clostridium, and Listeria.     

Characterisation of actI-homologous DNA encoding polyketide synthase genes from the monensin producer Streptomyces cinnamonensis

Summary Cloned DNA encoding polyketide synthase (PKS) genes from one Streptomyces species was previously shown to serve as a useful hybridisation probe for the isolation of other PKS gene clusters from the same or different species. In this work, the actI and actIII genes, encoding components of the actinorhodin PKS of Streptomyces coelicolor, were used to identify and clone a region of homologous DNA from the monensin-producing organism S. cinnamonensis. A 4799 by fragment containing the S. cinnamonensis act-homologous DNA was sequenced. Five open reading frames (ORFs 1–5) were identified on one strand of this DNA. The five ORFs show high sequence similarities to ORFs that were previously identified in the granaticin, actinorhodin, tetracenomycin and whiE PKS gene clusters. This allowed the assignment of the following putative functions to these five ORFS : a heterodimeric -ketoacyl synthase (ORF1 and ORF2), an acyl carrier protein (ORF3), a -ketoacyl reductase (ORF5), and a bifunctional cyclase/dehydrase (ORF4). The ORFs are encoded in the order ORFl-ORF2-ORF3-ORF5-ORF4, and ORFs-1 and -2 show evidence for translational coupling. This act-homologous region therefore appears to encode a PKS gene cluster. A gene disruption experiment using the vector pGM 160, and other evidence, suggests that this cluster is not essential for monensin biosynthesis but rather is involved in the biosynthesis of a cryptic aromatic polyketide in S. cinnamonensis. An efficient plasmid transformation system for S. cinnamonensis has been established, using the multicopy plasmids pWOR120 and pWOR125.     

A novel cryptic plasmid pBMB175 from <Emphasis Type="Italic">Bacillus thuringiensis </Emphasis>subsp. <Emphasis Type="Italic">tenebrionis </Emphasis>YBT-1765

A new cryptic plasmid pBMB175 from Bacillus thuringiensis subsp. tenebrionis YBT-1765 was isolated and characterized. Sequence analysis showed that pBMB175 (14,841 bp and 31% GC content) contained at least eighteen putative open reading frames (ORFs), among which nine ORFs displayed the homology with the hypothetical proteins in rolling-circle replication plasmid pGI3. Deletion analysis revealed that the pBMB175 minireplicon located in a novel 1,151 bp fragment. This fragment contains ORF7 coding sequence, which encodes a protein (Rep175, 149 amino acids [aa]) indispensable for plasmid replication. Rep175 has no significant homology with known function proteins. Furthermore, a putative double-strand origin (dso), having no DNA similarity with characterized dso of other replicon so far, was identified in this minireplicon fragment. These features showed that pBMB175 could be placed into a new plasmid family.     

Complete nucleotide sequence of pGS18, a 62.8-kb plasmid from <Emphasis Type="Italic">Geobacillus stearothermophilus</Emphasis> strain 18

The complete nucleotide sequence (62.8 kb) of pGS18, the largest sequenced plasmid to date from the species Geobacillus stearothermophilus, was determined. Computational analysis of sequence data revealed 65 putative open reading frames (ORFs); 38 were carried on one strand and 27 were carried on the other. These ORFs comprised 84.1% of the pGS18 sequence. Twenty-five ORFs (38.4%) were assigned to putative functions; four ORFs (6.2%) were annotated as pseudogenes. The amino acid sequences obtained from 29 ORFs (44.6%) had the highest similarity to hypothetical proteins of the other microorganisms, and seven (10.8%) had no significant similarity to any genes present in the current open databases. Plasmid replication region, strongly resembling that of the theta-type replicon, and genes encoding three different plasmid maintenance systems were identified, and a putative discontinuous transfer region was localized. In addition, we also found several mobile genetic elements and genes, responsible for DNA repair, distributed along the whole sequence of pGS18. The alignment of pGS18 with two other large indigenous plasmids of the genus Geobacillus highlighted the presence of well-conserved segments and has provided a framework that can be exploited to formulate hypotheses concerning the molecular evolution of these three plasmids.     

DnaG-dependent priming signals can substitute for the two essential DNA initiation signals in oriV of the broad host-range plasmid RSF1010.

Broad host-range plasmid RSF1010 contains in the oriV region two DNA initiation signals, ssiA(RSF1010) and ssiB(RSF1010), which are essential for plasmid replication. Each of ssiA and ssiB could be substituted functionally by either of the two G4-type (DnaG-dependent) priming signals, the oric of bacteriophage G4 and an ssi signal from plasmid pSY343 (an R1 plasmid derivative). Functions of the chimeric oriVs of RSF1010 thus constructed were dependent on the RSF1010-specific replication proteins, RepA, RepB' and RepC. When both of ssiA and ssiB were replaced by the G4-type ssi signals, functions of the chimeric oriVs were no longer dependent on RepB' (RSF1010-specific DNA primase). The replication activities of the chimeric oriVs of RSF1010 were not influenced markedly by the type of heterologous priming signals they contained. It is conceivable that DNA replication of RSF1010 does not need the priming mechanism for lagging strand synthesis and proceeds by the strand displacement mechanism.     

Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a

The complete nucleotide sequence and organization of the large virulence plasmid pCP301 (termed by us) of Shigella flexneri 2a strain 301 were determined and analyzed. The result showed that the entire DNA sequence of pCP301 is composed of 221618 bp which form a circular plasmid. Sequence analysis identified 272 open reading frames (ORFs), among which, 194 correspond to the proteins described previously, 61 have low identity (<60%) to known proteins and the rest 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include the genes associated with bacterial virulence, the genes associated with regulation and the genes relating to plasmid maintenance, stability and DNA metabolism. Insertion sequence (IS) elements are 68 kb in length and account for 30 percent of complete sequence of the plasmid which indicates that gene multiple rearrangements of the pCP301 have taken place in Shigella flexneri evolution history. The research result is helpful for interpreting the pathogenesis of Shigella, as well as the genetics and evolution of the plasmid.     

Nucleotide sequence of diatom plasmids: identification of open reading frames with similarity to site-specific recombinases

We have determined the nucleotide sequence of two small circular DNA plasmids, pCf1 and pCf2 [22], from the marine diatom Cylindrotheca fusiformis. pCf1 is 4273 bp, and pCf2 is 4079 bp in size. In each plasmid, all of the major open reading frames (ORFs) are encoded on the same DNA strand. Two ORFs are similar, comparing the two plasmids. ORF218 (pCf1) and ORF217 (pCf2) share 80% amino acid identity and ORF482 (pCf1) and ORF484 (pCf2) share 54% amino acid identity. ORF218/217 shows significant similarity (28–31% amino acid identity) to the Tn3 class of resolvases. Resolvases are most commonly found in bacterial transposons. However, two other features found in the Tn3 class of transposon are missing in the plasmids; an ORF encoding a transposase and terminal inverted repeat sequences. This, and data mapping the portions of the plasmids that hybridize to genomic chloroplast DNA, suggest that the plasmids do not contain active transposons. By analogy with the R46 plasmid from Enterobacter [5, 6], another potential role for the resolvases encoded by pCf1 and pCf2 is the conversion of multimeric forms of the plasmid to monomers. The similarity of ORF218/217 to resolvases documents the first identification of a potential coding function in an algal plasmid.     

Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of <Emphasis Type="Italic">Shigella flexneri</Emphasis> 2a

The complete nucleotide sequence and organization of the large virulence plasmid pCP301 (termed by us) of Shigella flexneri 2a strain 301 were determined and analyzed. The result showed that the entire DNA sequence of pCP301 is composed of 221618 bp which form a circular plasmid. Sequence analysis identified 272 open reading frames (ORFs), among which, 194 correspond to the proteins described previously, 61 have low identity (<60%) to known proteins and the rest 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include the genes associated with bacterial virulence, the genes associated with regulation and the genes relating to plasmid maintenance, stability and DNA metabolism. Insertion sequence (IS) elements are 68 kb in length and account for 30 percent of complete sequence of the plasmid which indicates that gene multiple rearrangements of the pCP301 have taken place in Shigella flexneri evolution history. The research result is helpful for interpreting the pathogenesis of Shigella, as well as the genetics and evolution of the plasmid.     

Characterization of a 24-kb plasmid pCGR2 newly isolated from Corynebacterium glutamicum

A 24-kb plasmid with 21 open reading frames (ORFs) was newly isolated from Corynebacterium glutamicum ATCC 14997 and named pCGR2. Three of its ORFs were indispensable for stable autonomous replication of pCGR2 in C. glutamicum: in the absence of selective pressure, deletion derivatives of pCGR2 containing the three ORFs showed stability in C. glutamicum for over 50 generations. The first of these ORFs encoded replicase repA whose gene product revealed high amino acid sequence similarity to corresponding gene products of C. glutamicum pCG1-family plasmids in general, and to that of pTET3 plasmid repA in particular. The other two ORFs were located upstream of repA and exhibited high sequence similarity to pTET3 parA and parB, respectively. Interestingly, plasmids based on the pCGR2 were compatible not only with those based on different family plasmids (pBL1, pCASE1) but also with those based on pCG1-family plasmid. Plasmids comprising pCGR2 repA showed a copy number of four in C. glutamicum. The number increased to 240 upon introduction of a mutation within the repA origin of the putative promoter for counter-transcribed RNA. This 60-fold increase in copy number should immensely contribute towards enhanced expression of desired genes in C. glutamicum.     

Structural analysis of the plasmid pTA1 isolated from the thermoacidophilic archaeon Thermoplasma acidophilum

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at pH1.8 and 56°C and has no cell wall. Plasmid pTA1 was found in some strains of the species. We sequenced plasmid pTA1 and analyzed the open reading frames (ORFs). pTA1 was found to be a circular DNA molecule of 15,723 bp. Eighteen ORFs were found; none of the gene products except ORF1 had sequence similarity to known proteins. ORF1 showed similarity to Cdc6, which is involved in genome-replication initiation in Eukarya and Archaea. T. acidophilum has two Cdc6 homologues in the genome. The homologue found in pTA1 is most similar to Tvo3, one of the three Cdc6 homologues found in the genome of Thermoplasma volcanium, among all of the Cdc6 family proteins. The phylogenetic analysis suggested that plasmid pTA1 is possibly originated from the chromosomal DNA of Thermoplasma.     

Genetic profile of pNOB8 from Sulfolobus: the first conjugative plasmid from an archaeon

The complete nucleotide sequence of the archaeal conjugative plasmid, pNOB8, from the Sulfolobus isolate NOB8-H2, was determined. The plasmid is 41 229 bp in size and contains about 50 ORFs. Several direct sequence repeats are present, the largest of which is a perfect 85-bp repeat and a site of intraplasmid recombination in foreign Sulfolobus hosts. This recombination event produces a major deletion variant, pNOB8-33, which is not stably maintained. Less than 20% of the ORFs could be assigned putative functions after extensive database searches. Tandem ORFs 315 and 470, within the deleted 8-kb region, show significant sequence similarity to the protein superfamilies of ParA (whole protein) and ParB (N-terminal half), respectively, that are important for plasmid and chromosome partitioning in bacteria. A putative cis-acting element is also present that exhibits six 24-mer repeats containing palindromic sequences which are separated by 39 or 42 bp. By analogy with bacterial systems, this element may confer plasmid incompatibility and define a group of incompatible plasmids in Archaea. Although several ORFs can form putative trans-membrane or membrane-binding segments, only two ORFs show significant sequence similarity to bacterial conjugative proteins. ORF630b aligns with the TrbE protein superfamily, which contributes to mating pair formation in Bacteria, while ORF1025 aligns with the TraG protein superfamily. We infer that the conjugative mechanism for Sulfolobus differs considerably from known bacterial mechanisms. Finally, two transposases were detected; ORF413 is flanked by an imperfect 32-bp inverted repeat with a 5-bp direct repeat at the ends, and ORF406 is very similar in sequence to an insertion element identified in the Sulfolobus solfataricus P2 genome. Received: March 10, 1998 / Accepted: May 2, 1998     

Development of a novel plasmid as a shuttle vector for heterologous gene expression in Mycoplasma yeatsii

A circular plasmid, pMyBK1, was detected in Mycoplasma yeatsii strain GIH(T). Analysis of the sequence of the 3432-bp replicon identified two predicted open reading frames (ORFs), one with sequence similarity to multiple plasmid mobilization proteins and one that matches only to hypothetical ORFs encoded by integrated chromosomal elements in the sequenced genomes of two Mycoplasma species. Shuttle vectors were constructed in Escherichia coli which could be introduced into M. yeatsii at high efficiency (10(4)-10(5) per μg DNA) by electroporation. Independent deletion analysis of the two ORFs disclosed that whereas mob was dispensable, orf2 was necessary for plasmid replication or maintenance. The absence of plasmid-encoded database matches for ORF2 indicates that pMyBK1 represents a novel plasmid family. One shuttle vector was used to demonstrate heterologous expression of the Mycoplasma fermentans malp gene and was stable during multiple passages. The host-plasmid system described has potential application for genetic manipulation in a genus for which few replicative vectors are available.     

Transformation of Neisseria gonorrhoeae: physical requirements of the transforming DNA

The 1600-bp (base pair) fragment encoding a portion of the nalidixic acid resistant DNA gyrase, subunit B, was characterized to determine what parameters effect transformation in the gonococcus. When this DNA (pSY2) was isolated from Escherichia coli, it was able to transform a variety of gonococcal strains to resistance to nalidixic acid via DNA-mediated transformation, irrespective of their restriction-modification phenotype. Nalidixic acid resistant transformants contained no plasmid DNA sequences that corresponded to the vector, as measured by plasmid screening procedures and colony hybridization techniques. Supercoiled and linear DNA transformed the gonococcus at the same efficiency. DNA fragments as small as 615 bp were able to transform the gonococcus. The presence of a 10-bp uptake sequence enhanced a DNA fragment's ability to transform the gonococcus by four orders of magnitude. When the fragment encoding the nalidixic acid resistant DNA gyrase was subcloned into M13mp18, both the replicative form and the single-stranded form of the phage were able to transform the gonococcus to nalidixic acid resistance.     

A runaway-replication plasmid pSY343 contains two ssi signals

Taking advantage of the plaque morphology method, we detected two single-stranded initiation (ssi) signals in the plasmid pSY343; one was in the 170-nucleotide (nt) EcoRV-ThaI segment (170P), and the other was in the 93-nt DraI-FnuDII segment (93F), which were designated as ssiA and ssiB, respectively. We cloned the two ssi signals in the filamentous phage vectors M13 delta lac184 and flR199. A conserved 7-nt consensus sequence involved in the n' recognition site for priming DNA initiation on single-stranded (ss) DNA templates (A. Van der Ende, R. Teerstra, H. Van der Avoort, and P.J. Weisbeek, 1983, Nucleic Acids Res. 11, 4957-4975) was found, three copies in 170P and one in 93F. These two ssi signals contain possible stem and loop structures. The 170P overlapped partly with the origin (ori) region of pSY343 and the 93F was away from the ori region. Growth of chimera phages such as M13 delta lac184/ssiA and M13 delta lac184/ssiB was 38- and 71-fold greater, respectively, than that of M13 delta lac184, 8 h after phage infection. The conversion efficiency in vivo of ss to replicative form (RF) DNA of these chimera phages carrying ssiA and ssiB was 1.9- and 2.2-fold greater, respectively, than that of M13 delta lac184, 50 min after infection.     

Structural features and expression analysis of a linear mitochondrial plasmid in rapeseed (Brassica napus L.)

A linear plasmid molecule about 11 kb in length is present in the mitochondria of some varieties of rapeseed (Brassica napus L.). This plasmid can be inherited from the male parent, through the pollen, as well as by the usual maternal route, although the main mitochondrial genome is maternally inherited in rapeseed. We determined the complete nucleotide sequence of this plasmid DNA and clarified its genetic organization. The length of the linear plasmid is 11,640 bp. At the termini of the plasmid molecule are inverted repeats of 327 bp. The GC content of the plasmid DNA is 30.9%; thus, the plasmid is quite AT-rich compared to the main mitochondrial genome in higher plants. The plasmid has six ORFs, two of which encode a phage-type DNA polymerase and a phage-type RNA polymerase, respectively. RT-PCR analyses revealed that all six ORFs are transcribed, and all four ORFs on the minus strand are probably cotranscribed from a single promoter located in the terminal inverted repeat. We also show here that at least three of the six ORFs are translated into proteins in rapeseed mitochondria, and expressed at relatively high levels in flowers, as shown by Western analysis. These results suggest that this linear DNA molecule is able to replicate as an autonomous replicon and to express the genes it carries in rapeseed mitochondria.