Plasmid diversity in neisseriae

Horizontal gene transfer constitutes an important force in prokaryotic genome evolution, and it is well-known that plasmids are vehicles for DNA transfer. Chromosomal DNA is frequently exchanged between pathogenic and commensal neisseriae, but relatively little is known about plasmid diversity and prevalence among these nasopharyngeal inhabitants. We investigated the plasmid contents of 18 Neisseria lactamica isolates and 20 nasopharyngeal Neisseria meningitidis isolates. Of 18 N. lactamica strains, 9 harbored one or more plasmids, whereas only one N. meningitidis isolate contained a plasmid. Twelve plasmids were completely sequenced, while five plasmid sequences from the public databases were also included in the analyses. On the basis of nucleic acid sequences, mobilization, and replicase protein alignments, we distinguish six different plasmid groups (I to VI). Three plasmids from N. lactamica appeared to be highly similar on the nucleotide level to the meningococcal plasmids pJS-A (>99%) and pJS-B (>75%). The genetic organizations of two plasmids show a striking resemblance with that of the recently identified meningococcal disease-associated (MDA) phage, while four putative proteins encoded by these plasmids show 25% to 39% protein identity to those encoded by the MDA phage. The putative promoter of the gene encoding the replicase on these plasmids contains a polycytidine tract, suggesting that replication is subjected to phase variation. In conclusion, extensive plasmid diversity is encountered among commensal neisseriae. Members of three plasmid groups are found in both pathogenic and commensal neisseriae, indicating plasmid exchange between these species. Resemblance between plasmids and MDA phage may be indicative of dissemination of phage-related sequences among pathogenic and commensal neisseriae.     

Complete DNA sequence of Yersinia enterocolitica serotype 0:8 low-calcium-response plasmid reveals a new virulence plasmid-associated replicon

The complete nucleotide sequence and organization of the Yersinia enterocolitica serotype 0:8 low-calcium-response (LCR) plasmid, pYVe8081, were determined. The 67,720-bp plasmid encoded all the genes known to be part of the LCR stimulon except for ylpA. Eight of 13 intact open reading frames of unknown function identified in pYVe8081 had homologues in Yersinia pestis plasmid pCD1 or in Y. enterocolitica serotype 0:9 plasmid pYVe227. A region of approximately 17 kbp showed no DNA identity to pCD1 or pYVe227 and contained six potential new genes, a possible new replicon, and two intact insertion sequence (IS) elements. One intact IS element, ISYen1, was a new IS belonging to the IS256 family. Several vestigial IS elements appeared different from the IS distribution seen in the other LCR plasmids. The RepA proteins encoded by Y. enterocolitica serotype 0:8 pYVeWA and pYVe8081 were identical. The putative pYVe8081 replicon showed significant homology to the IncL/M replicon of pMU407.1 but was only distantly related to the replicons of pCD1 and pYVe227. In contrast, the putative partitioning genes of pYVe8081 showed 97% DNA identity to the spy/sopABC loci of pCD1 and pYVe227. Sequence analysis suggests that Yersinia LCR plasmids are from a common ancestor but that Y. enterocolitica serotype 0:8 plasmid replicons may have evolved independently via cointegrate formation following a transposition event. The change in replicon structure is predicted to change the incompatibility properties of Y. enterocolitica serotype 0:8 plasmids from those of Y. enterocolitica serotype 0:9 and Y. pestis LCR plasmids.     

Molecular characterization of a trans-acting, positive effector (ipaR) of invasion plasmid antigen synthesis in Shigella flexneri serotype 5

A trans-acting, positive effector of invasion plasmid antigen (Ipa) synthesis has been identified and mapped on the pWR100 invasion plasmid of Shigella flexneri serotype 5 (strain M90T-W). Recombinant plasmids carrying this regulatory gene, designated ipaR, were found to restore full virulence to a non-invasive ipaR::Tn5 insertion mutant [M90T-W(pHS1042)] that had lost the ability to synthesize four Ipa antigens (IpaA, 70 kDa; IpaB, 62 kDa; IpaC, 42 kDa; and IpaD, 37 kDa). Genetic mapping of the ipaR gene positioned the locus on a 2.6 kb PstI-AccI fragment contained within a larger 8.0 kb EcoRI molecule that also encoded IpaD, IpaA, and two small proteins (27 kDa and 28 kDa). The trans regulatory effect of the ipaR product on ipaB, ipaC, ipaD, and ipaA expression was demonstrated by transforming compatible ipaBC, ipaDA, ipaR and ipaDAR plasmid recombinants, in various combinations, into M90T-A3, an isogenic invasion plasmid mutant of M90T-W that contained a deletion of the pWR100 ipaBCDA and ipaR loci; such transformants produced wild type levels of the IpaB, IpaC, IpaD and IpaA antigens only in the presence of IpaR+ plasmids. DNA sequence analysis of the ipaR region established that the intiation codon for ipaR is 459 bp from the 3'-end of the ipaA gene and that ipaR encodes a 309 amino acid residue protein. An interesting feature of the IpaR polypeptide was its strong sequence homology with the bacteriophage P1 partition protein ParB, consisting of a 42.8% amino acid identity over a 278 residue section of the aligned proteins.     

Herpesvirus ICP18.5 and DNA-binding protein genes are conserved in equine herpesvirus-1

The genome of equine herpesvirus-1 (EHV-1) contained three open reading frames (ORFs) in a 3.9 kbpBamHI-SmaI fragment at 0.38–0.41 map units in the long unique region. The most 5′ ORF encoded the carboxy terminus of a protein with 45–55 percent amino acid homology to the DNA-binding proteins (ICP8-DBP) of four other alphaherpesviruses. The middle ORF translated to a polypeptide of 775 residues with 43–55% homology to the ICP18.5 proteins. The most 3′ ORF encoded the EHV-1 glycoprotein B (gB) gene. Three mRNAs of 4.3, 4.4–4.8, and 3.5–3.9 kb (corresponding to the three sequenced ORFs) were all transcribed from the same strand. The gene order of this group was conserved in all herpesviruses examined.     

Co-infection by two distinct totivirus-like double-stranded RNA elements in Chalara elegans (Thielaviopsis basicola)

A full-length cDNA clone was developed from a 5.3 kb double-stranded (ds) RNA element present in strain CKP of the plant pathogenic fungus Chalara elegans. The complete nucleotide sequence was 5310 bp in length and sequence analysis revealed that it contained three large putative open reading frames (ORFs). ORF1 was initiated at nucleotide position 329 and encoded a putative coat protein, which shared some homology (35-45% amino acid identity) to other dsRNAs in the family Totiviridae. Both ORF2 and ORF3 were initiated at nucleotide positions 2619 and 4071, respectively, and encoded a putative RNA-dependent RNA polymerase (RdRp). Sequence comparison using deduced amino acid sequences of both ORF2 and ORF3 revealed that all RdRp conserved motifs shared highest homology (41% identity) to that of SsRNA1 of Totiviridae. This dsRNA in C. elegans was designated Chalara elegans RNA Virus 1 (CeRV1). During the development of the full-length cDNA clone of CeRV1, several partial cDNA clones from an additional dsRNA fragment in strain CKP were obtained, which when aligned with each other, produced one linear fragment which was 2336 bp long. Northern blot and sequence analysis of this second clone showed it differed in sequence composition from CeRV1. This dsRNA in C. elegans was designated Chalara elegans RNA Virus 2 (CeRV2). Sequence analysis of CeRV2 showed it contained all conserved motifs and shared some homology (45% amino acid identity) to RdRp regions of Totiviridae. The nucleotide and amino acid sequences of the conserved motifs of the RdRp regions between CeRV1 and CeRV2 showed an identity of 56% and 50%, respectively. These findings suggest that co-infection of two distinct totivirus-like dsRNAs (CeRV1 and CeRV2) in C. elegans, a first report in this fungus. Transmission electron microscopy of strain CKP of C. elegans revealed the presence of putative virus-like particles in the cytoplasm, which were similar both in shape and size to viruses in the Totiviridae.     

The coat protein genes of squash mosaic virus: cloning,sequence analysis,and expression in tobacco protoplasts

Summary Complementary DNA of the middle-component RNA of the melon strain of squash mosaic comovirus (SqMV) was cloned. Clones containing the coat protein genes were identified by hybridization with a degenerate oligonucleotide synthesized according to the amino acid sequence of a purified peptide fragment of the SqMV large coat protein. A clone containing of 2.5 kbp cDNA insert of SqMV M-RNA was sequenced. The total insert sequence of 2510 bp included a 2373 bp open reading frame (ORF) (encoding 791 amino acids), a 123 bp 3-untranslated region, and a poly(A) region. This ORF is capable of encoding both the 42 and 22 k SqMV coat proteins. Direct N-terminal sequence analysis of the 22 k coat protein revealed its presence at the 3 end of this ORF and the position of the proteolytic cleavage site (Q/S) used to separate the large and small coat proteins from each other. A putative location of the N-terminal proteolytic cleavage site of the 42 k coat protein (Q/N) was predicted by comparisons with the corresponding coat proteins of cowpea mosaic virus, red clover mottle virus, and bean-pod mottle virus. Although the available nucleotide sequences of these viruses revealed little similarity, their encoded coat proteins shared about 47% identity. The identity of the encoded 42 k and 22 k peptides was confirmed by engineering the respective gene regions for expression followed by transfer into tobacco protoplasts using the polyethylene glycol method. Both SqMV coat proteins were expressed in vivo as determined by their reactivity to SqMV coat protein specific antibodies.     

Nucleotide sequence of plasmid pA387 of Amycolatopsis benzoatilytica and construction of a conjugative shuttle vector

The complete nucleotide sequence of plasmid pA387 of Amycolatopsis benzoatilytica DSM 43387 was determined. Sequence analysis revealed that pA387 is 30,157 bp long and has a G+C content of 71.74%. To obtain a minimal transferable replicon capable of self-replication, a 2,176 bp fragment of pA387 was cloned, and we demonstrated that this fragment is sufficient for autonomous replication. The replication region of pA387 exhibited no significant homology to any known replication proteins available in databases. Putative maintenance and transfer functions were identified on pA387. The predicted products of open reading frames, ORF 2 and ORF 12, resembled the plasmid stabilizing proteins, a DNA resolvase and a ParA protein, respectively. The putative translational products of ORF 15 and ORF 16 showed similarity to known bacterial conjugation proteins, TraG and TraA, respectively. A conjugative Escherichia coli -Amycolatopsis shuttle-cloning vector was constructed by using the pA387 replicon and designated pSETRL1. Shuttle vector pSETRL1 successfully transformed Amycolatopsis mediterranei DSM 40773 and Amycolatopsis orientalis NBRC 12806 by conjugation and electroporation, and is likely to be a useful vector in Amycolatopsis research.     

New Calicivirus isolated from walrus

The nucleotide sequence and genome organization of a new member of Caliciviridae was determined. Cell culture inoculated with fecal matter from walrus was used to recover fragments of a new virus by Suppression Subtractive Hybridization (SSH). The isolate was identified as a member of the Vesivirus genus of Caliciviridae and designated the name Walrus Calicivirus (WCV). Sets of PCR primers spanning the entire putative genome were designed using known sequences of other vesiviruses. The assembled genome was 8289 nucleotides (nt) long and shared no more than 87% identity with sequences of the other members of the genus Vesivirus. The largest open reading frame (ORF1) between positions 4-5646 encoded a polyprotein. ORF2, found at position 5652-7778, encoded a putative capsid protein. ORF3 overlapped ORF2 and encoded a small basic protein. Comparative analysis of multiple caliciviral capsid proteins was performed to propose a uniform capsid structural organization for this viral family.     

Expression of the human papillomavirus type 6b L2 open reading frame in Escherichia coli: L2-beta-galactosidase fusion proteins and their antigenic properties

Human papillomavirus (HPV) type 6b genome contains two large open reading frames (ORFs), designated L1 and L2, in a putative late region. These ORFs are expected to code for viral structural proteins. To examine antigenic properties of a L2 gene product, we constructed two plasmids which contain N-terminal (L2-N) and internal (L2-I) regions of the HPV6b L2 ORF and then each region was expressed in Escherichia coli as a fusion protein with E. coli beta-galactosidase (beta-Gal). Both L2-N/beta-Gal fusion proteins reacted with anti-beta-Gal antibody, but did not react with the antibody prepared against bovine papillomavirus type 1 (BPV1), in contrast with a high reactivity of HPV6b L1-beta-Gal fusion protein with the anti-BPV1 antibody. Antibody raised against the L2-I/beta-Gal protein in a rabbit reacted with viral antigens in the nuclei of cells in superficial epithelium of the condyloma acuminatum tissue, but did not react with the antigens in the bovine papilloma tissue. This antibody recognized a protein from condyloma acuminata which migrates to the position of mol wt 70K-76K on an electrophoresed SDS-polyacrylamide gel. These results suggested that the L2 ORF of HPV6b codes for a capsid protein which is less cross-reactive than the L1 antigen with anti-BPV1 antibody.     

Sequence and genomic organization of a rabbit hemorrhagic disease virus isolated from a wild rabbit

Rabbit hemorrhagic disease virus (RHDV) is a member of the caliciviridae family. The nucleotidic sequence of a full-length cDNA of one RHDV isolate (RHDV-SD) is reported. The genome is 7437 bases long and includes two ORFs, ORF1 (7034 b) and ORF2 (353 b), coding for the polyprotein and the Vp12, respectively. The coding sequence for the second structural protein (the capsid protein, Vp60) is located at the 3 end of ORF1. Comparison of RHDV-SD with the German RHDV isolate revealed 470 nucleotide substitutions (96% homology). The deduced amino acid sequences of the two isolates are closely related (98% identity), and no hypervariable region could be identified either in the structural or nonstructural proteins.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number Z29514.     

Infectivity and complete nucleotide sequence of the genome of a genetically distinct strain of maize streak virus from Reunion Island

Summary A complete infectious genome of an isolate of maize streak subgroup 1 geminivirus from Reunion Island (MSV-R) was cloned and sequenced. Using anAgrobacterium tumefaciens Ti plasmid delivery system, the cloned 2.7 kb circular DNA was shown to be infectious in maize. The agroinfected virus could be transmitted byCicadulina mbila, the most common vector species of MSV in Reunion. Analysis of open reading frames (ORFs) revealed seven potential coding regions including the 4 ORFs conserved in all geminiviruses infecting monocotyledonous plants, the 2 on the viral + strand (MP, CP), and the 2 on the complementary – strand (RepA, RepB). The nucleotide sequence of MSV-R was compared to previously determined sequence of three African clones from Nigeria (MSV-N), Kenya (MSV-K), and South Africa (MSV-S). More similarity was found between the African clones (97.0–97.3%) than between these and MSV-R (94.4–95.3%). Nucleotide substitutions were frequent in the large intergenic region, particularly in and around the most likely TATA box for the complementary sense genes, and in the 5 end of ORF V1. The comparison of the predicted peptide sequences of the proteins encoded by ORFs MP, RepA and RepB confirmed the higher similarity between the African clones (97.8–99.3%) than between these and MSV-R (95.1–97.1%). However the amino acid sequences of the protein encoded by ORF CP (capsid protein) were very conserved among all the 4 clones, suggesting a high selection pressure on this ORF.EMBL accession number: X94330     

Analysis of early region 4 of porcine adenovirus type 3

The early region 4 (E4) of porcine adenovirus (PAdV)-3, located at the right-hand end of the genome is transcribed in a leftward direction and has the potential to encode seven (p1-p7) open reading frames (ORFs). To determine the role of each protein in viral replication, we constructed full-length PAdV-3 genomic clones containing deletions of individual E4 ORF or combined deletions of the neighboring ORFs. Transfection of swine testicular (ST) cells with individual E4 mutant plasmid DNAs generated PAdV-3 E4 mutant viruses except with plasmids containing a deletion of ORF p3, ORF p2+ p3 or ORF p3+ p4. Each of the mutants was further analyzed for growth kinetics, and early/late protein synthesis. Mutant viruses carrying deletions in ORF p1, ORF p2 or ORF p4 showed growth characteristics similar to that of wild-type PAdV-3. Early/late protein synthesis was also indistinguishable from that of wild-type PAdV-3. However, mutant viruses carrying deletions in ORF p5, ORF p6 or ORF p7 showed a modest effect in their ability to grow in porcine cells and express early proteins. These results suggest that the E4 ORF p3 (showing low homology with non-essential human adenovirus (HAdV)-9-E4 ORF1 encoded proteins) is essential for the replication of PAdV-3 in vitro. In contrast, the E4 ORF p7 (showing homology to essential HAdV-2 34 kDa protein) is not essential for replication of PAdV-3 in vitro. Moreover, successful deletion of 1.957 kb fragment in E4 region increased the available capacity of replication-competent PAdV-3 (E3 + E4 deleted) to approximately 4.3 kb and that of replication-defective PAdV-3 (E1 + E3 + E4 deleted) to approximately 7 kb. This is extremely useful for the construction of PAdV-3 vectors that express multiple genes and/or regulatory elements for gene therapy and vaccination.     

Characterization of the theta replication plasmid pGR7 from Acetobacter aceti CCM 3610

A cryptic plasmid of Acetobacter aceti CCM 3610, designated pGR7, was sequenced and characterized. It is a 2446-bp circular molecule with a G?+?C content of 30%, which is unusual when compared to the already known plasmids isolated from Acetobacter genera. Sequence analysis of pGR7 revealed three putative open reading frames (ORFs). ORF1 displays low similarity with other Acetobacter plasmid replication proteins. The other two ORFs show similarities only to hypothetical proteins and do not encode any important protein. The replication module comprises a DnaA box-like sequence, indirect repeats, a potential prokaryotic promoter and the rep gene. The rep module organization is similar to that found in other theta-replicating plasmids from acetic acid bacteria that stably maintain in both Acetobacter and Escherichia coli, with two repeated sequences containing modules. Nevertheless, the pGR7 plasmid could replicate and be stably maintained only in Acetobacter strains and not in E. coli, another uncommon feature of this plasmid. The Rep protein was cloned into the pET30a?+?expression vector and purified by high-performance liquid chromatography. The helicase activity was determined and the ability of the protein to bind to the plasmid regulation region was confirmed by an electrophoretic mobility shift assay. The plasmid was stable in the Acetobacter cells after cultivation under nonselective conditions. By real-time polymerase chain reaction, the relative copy number of pGR7 was estimated to be seven copies per host chromosome equivalent.     

F17-like fimbriae from an invasive Escherichia coli strain producing cytotoxic necrotizing factor type 2 toxin.

The F17b fimbriae encoded by the transmissible virulence plasmid Vir, also coding for cytotoxic necrotizing factor type 2, were characterized. A 5.7-kb region of Vir mediates in vitro N-acetylglucosamine-sensitive adhesion to calf intestinal villi. Sequence analysis revealed that this region codes for a structural subunit and an adhesin closely related to the F17-A and F17-G proteins encoded by the F17 fimbrial gene cluster. The F17b-A gene presents an open reading frame of 540 bp encoding a polypeptide of 180 amino acids with a putative signal peptide of 21 residues. The mature protein shows an identity of 74% with the F17-A structural subunit. This 20-kDa protein is recognized by antiserum directed against F17 fimbriae. The F17b-G gene shows an open reading frame of 1,029 bp encoding a polypeptide of 343 amino acids with a putative signal peptide of 22 residues. The F17b-G polypeptide exhibits 95% identity with the F17-G adhesin. The functional homology of the gene products was further confirmed by demonstrating that mutants in the F17-A gene can be complemented by the F17b-A gene and vice versa. These results prove that fimbriae belonging to the F17 family are also found on pathogenic Escherichia coli strains other than enterotoxigenic isolates producing heat-labile or heat-stable enterotoxin.