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.     

Analysis of two marine metagenomes reveals the diversity of plasmids in oceanic environments

Plasmid diversity is still poorly understood in pelagic marine environments. Metagenomic approaches have the potential to reveal the genetic diversity of microbes actually present in an environment and the contribution of mobile genetic elements such as plasmids. By searching metagenomic datasets from flow cytometry-sorted coastal California seawater samples dominated by cyanobacteria (SynMeta) and from the Global Ocean Survey (GOS) putative marine plasmid sequences were identified as well as their possible hosts in the same samples. Based on conserved plasmid replication protein sequences predicted from the SynMeta metagenomes, PCR primers were designed for amplification of one plasmid family and used to confirm that metagenomic contigs of this family were derived from plasmids. These results suggest that the majority of plasmids in SynMeta metagenomes were small and cryptic, encoding mostly their own replication proteins. In contrast, probable plasmid sequences identified in the GOS dataset showed more complexity, consistent with a much more diverse microbial population, and included genes involved in plasmid transfer, mobilization, stability and partitioning. Phylogenetic trees were constructed based on common replication protein functional domains and, even within one replication domain family, substantial diversity was found within and between different samples. However, some replication protein domain families appear to be rare in the marine environment.     

Characterization of a region of plasmid pBL1 of Brevibacterium lactofermentum involved in replication via the rolling circle model.

The minimal region for autonomous replication of pBL1, a 4.5-kb cryptic plasmid of Brevibacterium lactofermentum ATCC 13869 that has been used to construct a variety of corynebacterium vectors, was shown to be contained on a 1.8-kb HindII-SphI DNA fragment. This region contains two open reading frames (ORFs) (ORF1 and ORF5) which are essential for pBL1 replication in B. lactofermentum. Accumulation of single-strand intermediates in some of the constructions indicates that plasmid pBL1 replicates via the rolling circle replication model; its plus strand and minus strand were identified by hybridization with two synthetic oligonucleotide probes complementary to each pBL1 strand. ORF1 seems to encode the Rep protein and showed partial homology with sequences for Rep proteins from Streptomyces plasmids which replicate via rolling circle replication such as pIJ101, pSB24, and pJV1.     

Identification and characterization of an active plasmid partition mechanism for the novel Lactococcus lactis plasmid pCI2000

The replication region of the lactococcal plasmid pCI2000 was subcloned and analyzed. The nucleotide sequence of one 5.6-kb EcoRI fragment which was capable of supporting replication when cloned on a replication probe vector revealed the presence of seven putative open reading frames (ORFs). One ORF exhibited significant homology to several replication proteins from plasmids considered to replicate via a theta mode. Deletion analysis showed that this ORF, designated repA, is indeed required for replication. The results also suggest that the origin of replication is located outside repA. Upstream and divergently transcribed from repA, an ORF that showed significant (48 to 64%) homology to a number of proteins that are required for faithful segregation of chromosomal or plasmid DNA of gram-negative bacteria was identified. Gene interruption and transcomplementation experiments showed that this ORF, designated parA, is required for stable inheritance of pCI2000 and is active in trans. This is the first example of such a partitioning mechanism for plasmids in gram-positive bacteria.     

Characterization of the cryptic plasmid pBGR1 from Bartonella grahamii and construction of a versatile Escherichia coli-Bartonella spp. shuttle cloning vector

We report herein the isolation and molecular characterization of pBGR1, the first native plasmid isolated from the genus Bartonella. Cloning and sequencing revealed a 2725-base pair (bp) cryptic plasmid comprising two open reading frames of considerable length, which were designated rep and mob. The regions containing rep and mob are separated by 140-bp inverted repeat sequences and display a difference in G + C content from one another. A 1435-bp SacI-BclI fragment containing the rep gene is sufficient to mediate replication in the species Bartonella henselae and Bartonella tribocorum, while this replicon does not appear to be functional in Escherichia coli. The Rep protein of 190 amino acids (aa) shares homology to putative replication proteins of cryptic plasmids of Gram-negative origin, which form a subgroup of the rolling-circle replication proteins of the pSN2 plasmid superfamily of Gram-positive bacteria. The Mob protein of 333 aa is related to mobilization proteins of several cryptic plasmids and is associated with a conserved recombination site A. The tra functions of RP4 can mobilize pBGR1 derivatives in a mob-dependent manner. Mobilizable pBGR1-based E. coli-Bartonella spp. shuttle vectors were constructed and were shown to be maintained in B. tribocorum during in vivo passage in a rat model in the absence of antibiotic selection. The small size and stability of these shuttle cloning vectors should render them particularly valuable for genetic studies in Bartonella spp.     

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.     

Sequence analysis of the cryptic plasmid pMG101 from Rhodopseudomonas palustris and construction of stable cloning vectors

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of alpha-proteobacteria.     

Identification of a Campylobacter jejuni-secreted protein required for maximal invasion of host cells

The food-borne pathogen Campylobacter jejuni is dependent on a functional flagellum for motility and the export of virulence proteins that promote maximal host cell invasion. Both the flagellar and non-flagellar proteins exported via the flagellar type III secretion system contain a sequence within the amino-terminus that directs their export from the bacterial cell. Accordingly, we developed a genetic screen to identify C. jejuni genes that encode a type III secretion amino-terminal sequence that utilizes the flagellar type III secretion system of Yersinia enterocolitica and a phospholipase reporter ( yplA ). We screened a library of 321 C. jejuni genes and identified proteins with putative type III secretion amino-terminal sequences. One gene identified by the screen was Cj1242. We generated a mutation in Cj1242 , and performed growth rate, motility, secretion and INT 407 cell adherence and internalization assays. The C. jejuni Cj1242 mutant was not altered in growth rate or motility when compared with the wild-type strain, but displayed an altered secretion profile and a reduction in host cell internalization. Based on the phenotype of the C. jejuni Cj1242 mutant, we designated the protein Campylobacter invasion antigen C (CiaC). Collectively, our findings indicate that CiaC is a potentially important virulence factor.     

Characterization of a novel plasmid from extremely halophilic Archaea: nucleotide sequence and function analysis

We determined the complete nucleotide sequence of the 16341 bp plasmid pHH205 of the extremely halophilic archaeon Halobacterium salinarum J7. The plasmid has a G+C content of 61.1%. A number of direct and inverted repeat sequences were found in pHH205, while no insertion sequences were found. Thirty-eight large open reading frames (ORFs) were identified in both strands, and most of them had no significant similarities to known proteins. A putative protein encoded by ORF31 showed 20-41% homology to some hypothetical proteins, which are annotated in several archaeal genome databases as predicted nucleic acid-binding proteins containing PIN domain. Sequence analysis using the GC skew procedure predicted a possible origin of replication. A 4.8 kb PvuII-SnaBI fragment containing both this region and ORF31 was shown to be able to restore replicate of pWL102, a replicon-deficient plasmid in Haloferax volcanii and in H. salinarum R1. Several methods failed to completely cure H. salinarum J7 of pHH205, suggesting that the plasmid probably played an important role in the growth and metabolism of the host. Our work describes a novel haloarchaeal replicon, which may be useful in the construction of cloning and shuttle vectors.     

Sequence analysis and characterization of plasmids from Streptococcus thermophilus

The nucleotide sequences of eight plasmids isolated from seven Streptococcus thermophilus strains have been determined. Plasmids pSt04, pER1-1, and pJ34 are related and replicate via a rolling circle mechanism. Plasmid pJ34 encodes for a replication initiation protein (RepA) and a small polypeptide with unknown function. Plasmids pSt04 and pER1-1 carry in addition to repA genes coding for small heat shock proteins (sHsp). Expression of these proteins is induced at elevated temperatures or low pH and increases the thermo- and acid resistance. Plasmids pER1-2 and pSt22-2 show identical sequences with five putative open reading frames (ORFs). The gene products of ORF1 and ORF4 reveal some similarities to transposon encoded proteins of Bacillus subtilis and Tn916. ORF1 of plasmid pSt106 encodes a protein similar to resolvases of different Gram-positive bacteria. Integrity of ORF2 and 3, encoding a putative DNA primase and a replication protein, is essential for replication. ORF1 to 3 of plasmid pSt08, which are organized in a tricistronic operon, encode a RepA protein, an adenosine-specific methyltransferase, and a type II restriction endonuclease. Another type II restriction-modification (R/M) system is encoded on plasmid pSt0 which is highly similar to those encoded on lactococcal plasmid pHW393 and B. subtilis plasmid pXH13. Plasmid-free derivatives of strains St0 and St08 show increased phage sensitivity, indicating that in the wild-type strains the R/M systems are functionally expressed. Recombinant plasmids based on the replicons of plasmids pSt04, pJ34, pSt106, pSt08, and pSt0, are able to replicate in Lactococcus lactis and B. subtilis, respectively, whereas constructs carrying pER1-2 only replicate in S. thermophilus.     

Analysis of the sequence of a new cryptic plasmid, pRJF2, from a rumen bacterium of the genus Butyrivibrio: Comparison with other Butyrivibrio plasmids and application in the development of a cloning vector

Abstract A small cryptic plasmid, pRJF2, from Butyrivibrio fibrisolvens strain OB157 was isolated and sequenced. The plasmid is similar in organisation to the previously sequenced Butyrivibrio plasmid, pRJF1, with two open reading frames, ORF1 and ORF2, flanking a region tentatively identified as the replication origin, and a region of unknown function defined by terminal 79 bp invert repeats. The sequences of ORF1, ORF2, and the presumptive replication origin are highly conserved. The sequence between the 79 bp invert repeats is not, and is therefore presumed to be of lesser functional significance, although the 5' and 3' termini are still highly conserved. The functional importance for plasmid replication of these regions was tested by constructing potential shuttle vectors, each lacking one or more of the regions of interest. When the region between the invert repeats was deleted and replaced by the erythromycin resistance gene from pAM β1 together with pUC18, to produce the 7.9 kb chimaeric plasmid pYK4, the construct was successfully transformed into E. coli and B. fibrisolvens by electroporation, and was stably maintained in both hosts. Both ORF1 and ORF2 were required for successful transformation of B. fibrisolvens .     

pVC, a small cryptic plasmid from the environmental isolate of Vibrio cholerae MP-1

A marine bacterium was isolated from Mai Po Nature Reserve of Hong Kong and identified as Vibrio cholerae MP-1. It contains a small plasmid designated as pVC of 3.8 kb. Four open reading frames (ORFs) are identified on the plasmid, but none of them shows homology to any known protein. Database search indicated that a 440 bp fragment is 96% identical to a fragment found in a small plasmid of another V. cholerae. Further experiments demonstrated that a 2.3 kb EcoRI fragment containing the complete ORF1, partial ORF4 and their intergenic region could self-replicate. Additional analyses revealed that sequence upstream of ORF1 showed the features characteristic of theta type replicons. Protein encoded by ORF1 has two characteristic motifs existed in most replication initiator proteins (Rep): the leucine zipper (LZ) motif located at the N-terminal region and the alpha helix-turn-alpha helix motif (HTH) located at the C-terminal end. The results suggest that pVC replicates via the theta type mechanism and is likely a novel type of theta replicon.     

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.     

Molecular Characterization of Extrachromosomal DNA Accompanying Primula Red Phytoplasma

The complete nucleotide sequence of an extrachromosomal element found in primula red isolate of ‘Candidatus Phytoplasma asteris’ (16SrI‐B subgroup) was determined. The plasmid, named pPrR, is 4378 bp in length and has 75% A+T content that is similar to that of the phytoplasma genome. It encodes six putative open reading frames (ORF) longer than 100 amino acids and two smaller ones. The structural organization of the rep gene is similar to that found in plasmids which replicate via rolling circle mechanism. Furthermore, it has homology to both the plasmid pLS1 family and helicase domains of replication‐associated proteins (Rap) of eukaryotic viruses and geminiviruses. The ORF arrangement and genes sequences are most similar to the pPARG1 plasmid from ‘Rehmannia glutinosa’ phytoplasma.     

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。