Futile attempts to differentiate provide molecular evidence for individual differences within a population of cells during cellular reprogramming

A cryptic plasmid from Arthrobacter rhombi PRH1, designated as pPRH, was sequenced and characterized. It was 5000 bp in length with a G+C content of 66 mol%. The plasmid pPRH was predicted to encode six putative open reading frames (ORFs), in which ORF2 and ORF3 formed the minimal replicon of plasmid pPRH and shared 55-61% and 60-69% homology, respectively, with the RepA and RepB proteins of reported rhodococcal plasmids. Sequence analysis revealed a typical ColE2-type ori located 45 bp upstream of the gene repA. Sequence and phylogenetic analysis led to the conclusion that pPRH is a representative of a novel group of pAL5000 subfamily of ColE2 family plasmids. Three shuttle vectors pRMU824, pRMU824Km and pRMU824Tc, encoding chloramphenicol resistance, were constructed. The latter two harboured additional antibiotic resistance genes kan and tet, respectively. All vectors successfully replicated in Escherichia coli, Arthrobacter and Rhodococcus spp. The vector pRMU824Km was employed for functional screening of 2-hydroxypyridine catabolism encoding genes from Arthrobacter sp. PY22. Sequence analysis of the cloned 6-kb DNA fragment revealed eight putative ORFs, among which hpyB gene encoded a putative monooxygenase.     

pS86, A New Theta-Replicating Plasmid from Enterococcus faecalis

The complete nucleotide sequence of the small (5149 bp) and cryptic plasmid pS86 from Enterococcus faecalis ssp. faecalis S-86 has been determined. Sequence analysis revealed six putative open reading frames (ORFs) encoding polypeptides of 28.3, 11.5, 8.4, 65.1, 7.3, and 11.96 kDa each. Based on sequence similarity, two cassettes have been identified in pS86: ORF1 codes for the replication initiation protein (Rep); ORF4 codes for a putative mobilization protein that shows similarities to Mob/Pre proteins from plasmids of Gram-positive bacteria. No function could be assigned to the other putative ORFs found. According to our results, pS86 plasmid could use a theta-mode of replication, similar to the recently described theta-type replicons from pUCL287 (Tetragenococcus halophila) and pLA1 or pLA105 (Lactobacillus acidophilus) plasmids. Received: 24 November 1999 / Accepted: 26 April 2000     

Characterization of a cryptic plasmid pM4 from Lactobacillus plantarum M4

A cryptic plasmid from Lactobacillus plantarum M4 isolated from fresh milk, designated as pM4, was sequenced and characterized. It was 3320 bp in length with a G+C content of 38.73 mol%. The plasmid pM4 was predicted to encode three putative ORFs, in which ORF1 shared 99% and 98% homology, respectively, with the Rep proteins of reported plasmids pWCFS101 and pF8801, members of the rolling circle replication (RCR) pC194 family. Sequence analysis revealed a typical pC194 family double strand origin (dso) and a putative single strand origin (sso) located upstream of the rep gene. Mung bean nuclease analysis and Southern hybridization confirmed the presence of single-stranded DNA (ssDNA) intermediates, suggesting that pM4 belongs to the RCR pC194 family. Accumulation of ssDNA in rifampicin-treated strains implied that the host-encoded RNA polymerase was involved in the conversion of ssDNA to double-stranded DNA. Furthermore, the relative copy number of pM4 was estimated to be about 25 in each cell by real-time PCR. The new RCR plasmid would be valuable in constructing cloning vectors for application in the food industry.     

Isolation of a low-molecular-weight, multicopy plasmid, pNHK101, from Thermus sp. TK10 and its use as an expression vector for T. thermophilus HB27

We isolated a small multicopy cryptic plasmid, pNHK101, from Thermus sp. TK10 for use as a replicon of a Thermus expression vector. The nucleotide sequence of pNHK101 revealed that this plasmid was 1564bp long, with a total G+C content of 66.8%, which was in agreement with that of Thermus genomic DNA. The sequence did not show any significant similarities to any other plasmids; also, the amino acid sequences of four putative open reading frames, found in the plasmid, did not show strong similarities to those in the databases, except the ORF1, which had very slight similarities to several replication proteins of plasmids from other bacteria. pNHK101 was able to replicate in Thermus thermophilus HB27 with copy number about 80, and was stably maintained at 60 degrees C, but became unstable at 70 degrees C. Based on pNHK101, we constructed a plasmid vector, pKMH052, containing the highly thermostable kanamycin resistance gene as a selective marker. The copy number of pKMH052 decreased to about one-fourth of that of pNHK101, but stability at 60 degrees C did not alter under non-selective conditions. pKMH052 was compatible with pTT8, and interestingly, the presence of pTT8 in the same cells improved the stability of pKMH052 at 70 degrees C. Cloning of the crtB gene of T. thermophilus HB27 encoding phytoene synthase into pKMH052, and introduction into T. thermophilus cells resulted in a 2.8-fold production of carotenoids, indicating the potential use of this plasmid for overexpression of genes from thermophiles and hyperthermophiles.     

Molecular characterization of a cryptic plasmid from the psychrotrophic antarctic bacterium Pseudoalteromonas sp. 643A

We report the identification and nucleotide sequence analysis of pKW1, a plasmid of the psychrotrophic bacterium Pseudoalteromonas sp. 643A isolated from the stomach of Antarctic krill Euphasia superba. pKW1 consists of 4583 bp, has a G+C content of 43% and seven putative open reading frames (ORFs). The deduced amino acid sequence from ORF-1 shared significant similarity with the plasmid replicase protein of Psychrobacter cryohalolentis, strain K5. The DNA region immediately downstream of the ORF-1 showed some homology with the Rep-binding sequence of the theta-replicating ColE2-type plasmids. The ORF-3 amino acid sequence revealed amino acid sequence homology with the mobilization protein of Psychrobacter sp. PRwf-1 and Moraxella catarrhalis, with identities of 28% and 25%, respectively. The ORF-4 showed 46% amino acid sequence homology with the putative relaxase/mobilization nuclease MobA of Hafnia alvei and 44% homology with the putative mobilization protein A of Pasterulla multocida. The copy number of pKW1 in Pseudoalteromonas sp. 643A was estimated of 15 copies per chromosome.     

Molecular characterization of a cryptic plasmid from Campylobacter jejuni

Campylobacter jejuni is a leading bacterial cause of human enterocolitis. Molecular genetic characterization of this pathogen has been hampered by the lack of genetic tools that are functional in this organism. Cloning vectors commonly used in other organisms usually do not replicate within C. jejuni. To develop a system for functional analysis of C. jejuni genes, a small plasmid (pCJ01) identified in a poultry isolate of C. jejuni was sequenced and characterized in this study. By using inverse PCR, the full sequence of pCJ01 was amplified and subsequently determined. Results indicate that pCJ01 is a circular molecule of 3212 bp, with a G + C content of 33.5%. A typical plasmid replication origin with iteron sequences is identified upstream of the DNA sequences encoding replication initiation proteins. Four open reading frames (ORFs) are present in pCJ01. ORF1 and ORF2 share high homology with the putative RepA and RepB proteins, respectively, of known C. coli plasmids. ORF3 and ORF4, of unknown function, do not exhibit homology with any sequences deposited in the GenBank database. Hydropathy analysis predicts that ORF3 and ORF4 contain multiple stretches of hydrophobic amino acids, suggesting that they may encode transmembrane proteins. Since pCJ01 is a small plasmid and can be readily prepared from C. jejuni, it may be modified for use in molecular characterization of C. jejuni virulence genes.     

Intergeneric transfer of the Enterococcus faecalis plasmid pIP501 to Escherichia coli and Streptomyces lividans and sequence analysis of its tra region

The nucleotide sequence of the transfer (tra) region of the multiresistance broad-host-range Inc18 plasmid pIP501 was completed. The 8629-bp DNA sequence encodes 10 open reading frames (orf), 9 of them are possibly involved in pIP501 conjugative transfer. The putative pIP501 tra gene products show highest similarity to the respective ORFs of the conjugative Enterococcus faecalis plasmids pRE25 and pAMbeta1, and the Streptococcus pyogenes plasmid pSM19035, respectively. ORF7 and ORF10 encode putative homologues of type IV secretion systems involved in transport of effector molecules from pathogens to host cells and in conjugative plasmid transfer in Gram-negative (G-) bacteria. pIP501 mobilized non-selftransmissible plasmids such as pMV158 between different E. faecalis strains and from E. faecalis to Bacillus subtilis. Evidence for the very broad-host-range of pIP501 was obtained by intergeneric conjugative transfer of pIP501 to a multicellular Gram-positive (G+) bacterium, Streptomyces lividans, and to G- Escherichia coli. We proved for the first time pIP501 replication, expression of its antibiotic resistance genes as well as functionality of the pIP501 tra genes in S. lividans and E. coli.     

Sequence analysis of a cryptic plasmid pCJ419 from Campylobacter jejuni and construction of an Escherichia coli-Campylobacter shuttle vector

A small cryptic plasmid, pCJ419, was identified in a human clinical isolate of Campylobacter jejuni, cloned and sequenced. pCJ419 is a circular molecule of 4013 bp with a G+C content of 27.1%. The products of four open reading frames (ORFs) share significant sequence similarity with putative proteins from known C. jejuni and Campylobacter coli plasmids. ORF-1 encodes a putative mobilisation protein (Mob). ORF-2 and ORF-3 encode proteins that have high identity to putative RepA and RepB proteins, respectively, of known C. jejuni and C. coli plasmids. ORF-4 encodes a protein that has high identity to a hypothetical protein of unknown function, Cjp32, previously described in a pVir plasmid of C. jejuni. Tandem repeating 22-bp sequences typical of a plasmid replication origin (ori) were identified upstream of the DNA sequences encoding putative replication initiation proteins. An Escherichia coli-Campylobacter shuttle cloning vector, pGU0202, was constructed using plasmid pMW2 that harbours a Campylobacter-derived kanamycin resistance gene [aph(3')-III]. The sequences encoding pCJ419 mob, RepA and RepB proteins were inserted upstream of aph(3')-III resulting in a stable construct of 6174 bp that was used to transform both E. coli and Campylobacter.     

Sequence analysis of two cryptic plasmids from an agricultural isolate of Campylobacter coli

As part of a study identifying plasmids in Campylobacter, we isolated and sequenced two novel cryptic plasmids from an agricultural isolate of Campylobacter coli. The larger of the two plasmids, p3384, is 3316 bp in length and has a G+C content of 31.18%. A typical origin of replication consisting of five iterons was observed directly upstream of the first of three putative ORFs. The smaller plasmid, p3386, is 2426 bp in length and has a G+C content of 26.22%. Of the three putative ORFs detected on p3386, one shared homology with a putative protein from Campylobacter upsaliensis. The unique sequence of p3386 makes it attractive for further study concerning the evolutionary relationship of this plasmid to other Campylobacter plasmids, and to other Campylobacter isolates.     

Analysis of the replication region of a mycobacterial plasmid, pMSC262.

We determined the nucleotide sequence of a DNA fragment which contains the replication region of pMSC262, a Mycobacterium scrofulaceum plasmid used to construct the Mycobacterium-Escherichia coli shuttle vector. The complete sequence of the fragment contained 2,504 bp with an overall G+C content of 69.8%. By deletion analysis, we found that the minimum length required for plasmid replication in M. bovis BCG was about 1.6 kb. Within this region, several open reading frames (ORFs) and a putative replication origin (ori) were identified by computer analysis. One of the ORFs, ORF2, which encodes a putative 28.9-kDa basic protein with characteristics of DNA-binding proteins, appeared to be involved in replication of the plasmid in BCG. By separation of ORF2 and the putative ori region, it was revealed that the relative locations of ORF2 and the putative ori region are likely important for replication in BCG. No DNA or amino acid homologies were found between this replication region and that of pAL5000, another mycobacterial plasmid used for vector plasmid construction. In addition, we found that this replicon did not lead to replication in E. coli and was compatible in BCG with pAL5000-derived vector plasmid pYUB75 (R. G. Barletta, D. D. Kim, S. B. Snapper, B. R. Bloom, and W. R. Jacobs, J., J. Gen. Microbiol. 138:23-30, 1992).     

Cryptic plasmid pSKU146 from the wall-less plant pathogen Spiroplasma kunkelii encodes an adhesin and components of a type IV translocation-related conjugation system

A cryptic plasmid of the wall-less plant pathogenic mollicute, Spiroplasma kunkelii CR2-3X, was cloned and its sequence analyzed. The 14,615 bp plasmid, designated pSKU146, has a nucleotide content of 28 mol% G + C, and contains 18 potential protein-coding regions (open reading frames, ORFs), of which six encode proteins that exhibit similarity to virulence-associated proteins involved in cell-to-cell adhesion or conjugal DNA transfer. One ORF encodes a 96 kDa protein, SkARP1, that is highly similar to SARP1 adhesin involved in attachment of Spiroplasma citri to insect vector gut membrane. Five ORFs encode proteins similar to TraE and Mob in walled bacteria, and to ORFs found in the integrative, conjugative element (ICEF) of Mycoplasma fermentans, respectively. Presence of domains similar to proteins of the Type IV secretion system in pathogenic bacteria suggests that spiroplasma possesses a related translocation system. Plasmid pSKU146 also contains two identical oriT regions each containing a nick sequence characteristic of the IncP conjugative plasmid family, as well as a 58 bp palindromic sequence, palSK1. Features in pSKU146 suggest that the plasmid functions as a mobile genetic element in conjugative transmission of spiroplasma pathogenicity-related genes.     

Bioinformatic and partial functional analysis of pEspA and pEspB, two plasmids from Exiguobacterium arabatum sp. nov. RFL1109

The complete nucleotide sequences of two plasmids from Exiguobacterium arabatum sp. nov. RFL1109, pEspA (4563bp) and pEspB (38,945bp), have been determined. Five ORFs were identified in the pEspA plasmid, and putative functions were assigned to two of them. Using deletion mapping approach, the Rep-independent replication region of pEspA, which functions in Bacillus subtilis, was localized within a 0.6kb DNA region. Analysis of the pEspB sequence revealed 42 ORFs. From these, function of two genes encoding enzymes of the Lsp1109I restriction-modification system was confirmed experimentally, while putative functions of another 18 ORFs were suggested based on comparative analysis. Three functional regions have been proposed for the pEspB plasmid: the putative conjugative transfer region, the region involved in plasmid replication and maintenance, and the region responsible for transposition of the IS21 family-like transposable elements.     

Isolation and characterization of two novel plasmids pCYM01 and pCYM02 of Cylindrospermum stagnale

Cyanobacteria play a vital role in supplying nitrogen into the soil and aquatic ecosystem. It has an extra chromosomal DNA, whose role is not yet defined well. Isolation and characterization of extra chromosomal DNA in cyanobacteria might help to understand its survival mechanism. Cylindrospermum stagnale isolated (and deposited in NRMCF 3001) from soil showed presence of four plasmids namely pCYLM01, pCYLM02, pCYLM03, and pCYLM04. The following plasmids pCYLM01 and pCYLM02 were subjected to restriction digestion using HindIII restriction enzyme and cloned into pBlueScriptSK(-) vector. The sequence of pCYLM01 contained 4 potential open reading frames (ORFs) that have amino acids in the range of 59–299. Among them, ORF1 shows high sequence homology to the bacterial replication initiator family protein as evident from BLASTP analysis. The analysis of 4359 bp plasmid pCYLM02 sequence revealed 7 ORFs which are longer than 50 amino acids in length. The ORF2 of pCYLM02 has 243 amino acids and is represented in the plasmid sequence from 3045 to 3776 bp. The ORF3 of pCYLM02 corresponds to the plasmid sequence from 2323 to 2976 and codes for a putative protein of 217 amino acids long. A number of small ORFs below 50 bp were also found in the sequence analysis.     

Sequence analysis and characterization of plasmid pSFD10 from Salmonella choleraesuis

The nucleotide sequence of a small plasmid, designated pSFD10, is isolated from the vaccine strain Salmonella choleraesuis C500 in China, has been determined. This plasmid is 4091 bp long with a total G+C content of 51.4%, which is in the range of Salmonella genomic DNA. Analysis of the complete nucleotide sequence reveals that pSFD10 has a high degree of similarity to ColE1-type plasmid, having the possible cer and rom genes, and a putative mobilization origin of ColE1-type. Plasmid pSFD10 possesses six main open reading frames (ORFs), five of which have a very high degree of amino acid identity to ColE1-type plasmid gene products involved in mobilization and copy number control. The other ORF (ORF6) encodes a putative protein, which has 49% homology to the invasion plasmid antigen J protein (IpaJ) secreted by the type III secretion apparatus of Shigella flexneri. In addition, pSFD10 belongs to a different incompatibility group than ColE1-type and pMB1-type to which it is related. Plasmid pSFD10 can be mobilized by the plasmid RP4 in E. coli.     

Complete DNA sequence and analysis of two cryptic plasmids isolated from Lactobacillus plantarum

The complete nucleotide sequence of two cryptic plasmids isolated from Lactobacillus plantarum strain AS1.2986 has been determined. The smaller plasmid, designated pLP2000, encodes a 37.0kDa Rep protein and has a 17bp sequence repeated 10 times. Sequence analysis of the larger plasmid, designated pLP9000, revealed nine putative open reading frames (ORFs). Based on sequence similarity, ORF1 codes for a putative magnesium transporter protein that shows similarities to CorA from plasmid pCIS3 (Lactococcus lactis). None of the nine ORFs shows similarity to any known Rep protein. Southern blot analysis indicates these two plasmids both replicate via a rolling circle (RC) mechanism.     

Sequence analysis and characterization of sulfonamide resistance plasmid pRF-1 from Salmonella enterica serovar Choleraesuis

The nucleotide sequence of a small plasmid, designated pRF-1, isolated from Salmonella enterica serovar Choleraesuis, was determined. We identified seven open reading frames (ORFs) encoded by 6066 nucleotides with a total G + C content of 53.6%. Analysis of the complete nucleotide sequence revealed a replicon of pRF-1 to have high similarity to the p15A origin of replication, with a possible cer-like region. ORF1, which is composed of 816 nucleotides, shows a high degree of similarity to dihydropteroate synthetase encoded by the sulII gene from plasmids in several enteropathogenic bacteria, which functions as the sulfonamide resistance determinant. In fact, Salmonella and Escherichia coli strains carrying pRF-1 were found to show strong resistance to sulfathiazole, suggesting that orf1 is a functional gene. Four of seven ORFs were found to encode putative proteins of unknown function.