Plasmids of Chlamydia psittaci: Cloning and comparison of isolates by Southern hybridisation

Abstract A chlamydial plasmid, 6.2 kb in size, was isolated from an avian strain of Chlamydia psittaci and cloned into the Eco RI site of pUC13. A restriction enzyme cleavage map of the resultant clone, pAP¹p, was very similar to the published map of the plasmid cloned from the C. psittaci meningopneumonitis strain Cal-10. Southern hybridisation analyses using pAP¹p as a probe, revealed the presence of plasmids with homologous DNA sequences in avian psittacosis, avian ornithosis, ovine polyarthritis and sporadic bovine encephalomyelitis strains of C. psittaci , as well as the LGV strain of Chlamydia trachomatis . Plasmid was not detected in koala conjunctivitis, ovine abortion or feline conjunctivitis isolates. The plasmid-containing isolates could be grouped according to size (6.2 or 7.2–7.3 kb) and restriction endonuclease pattern. These three plasmid categories correlate with previously reported C. psittaci biotypes, immunotypes and serotypes. The absence of plasmid from three infectious, pathogenic strains of C. psittaci suggests that, in this species at least, plasmid-encoded genes are not essential for survival, infectivity or virulence of the parasite.     

Distribution of plasmid sequences in avian and mammalian strains of Chlamydia psittaci

Plasmid DNA from an avian strain of Chlamydia psittaci was purified and estimated to be 7.9 kb in size using restriction endonuclease analysis. A 5.9 kb fragment of this plasmid was cloned, mapped and used to screen a range of chlamydial strains. Hybridizing DNA was absent from ovine abortion and arthritis isolates and also from the Cal 10 strain but related sequences were detected in C. psittaci strains of feline pneumonitis, guinea-pig inclusion conjunctivitis, ovine conjunctivitis and C. trachomatis serovar L2. The plasmid DNA from the feline strain was shown to have a distinct restriction endonuclease profile. Similar plasmid sequences were detected in all avian isolates tested: thus the clone may have a useful diagnostic role for the detection of the pathogen in its natural host and in zoonotic episodes.     

Evolutionary relationships among members of the genus Chlamydia based on 16S ribosomal DNA analysis.

Nucleotide sequences from strains of the four species currently in the genus Chlamydia, C. pecorum, C. pneumoniae, C. psittaci, and C. trachomatis were investigated. In vitro-amplified RNA genes of the ribosomal small subunit from 30 strains of C. pneumoniae and C. pecorum were subjected to solid-phase DNA sequencing of both strands. The human isolates of C. pneumoniae differed in only one position in the 16S rRNA gene, indicating genetic homogeneity among these strains. Interestingly, horse isolate N16 of C. pneumoniae was found to be closely related to the human isolates of this species, with a 98.9% nucleotide similarity between their 16S rRNA sequences. The type strain and koala isolates of C. pecorum were also found to be very similar to each other, possessing two different 16S rRNA sequences with only one-nucleotide difference. Furthermore, the C. pecorum strains truncated the 16S rRNA molecule by one nucleotide compared to the molecules of the other chlamydial species. This truncation was found to result in loss of a unilaterally bulged nucleotide, an attribute present in all other eubacteria. The phylogenetic structure of the genus Chlamydia was determined by analysis of 16S rRNA sequences. All phylogenetic trees revealed a distinct line of descent of the family Chlamydiaceae built of two main clusters which we denote the C. pneumoniae cluster and the C. psittaci cluster. The clusters were verified by bootstrap analysis of the trees and signature nucleotide analysis. The former cluster contained the human isolates of C. pneumoniae and equine strain N16. The latter cluster consisted of C. psittaci, C. pecorum, and C. trachomatis. The members of the C. pneumoniae cluster showed tight clustering and strain N16 is likely to be a subspecies of C. pneumoniae since these strains also share some antigenic cross-reactivity and clustering of major outer membrane protein gene sequences. C. psittaci and strain N16 branched early out of the respective cluster, and interestingly, their inclusion bodies do not stain with iodine. Furthermore, they also share less reliable features like normal elementary body morphology and plasmid content. Therefore, the branching order presented here is very likely a true reflection of evolution, with strain N16 of the species C. pneumoniae and C. psittaci forming early branches of their respective cluster and with C. trachomatis being the more recently evolved species within the genus Chlamydia.     

Genetic diversity of avian and mammalian Chlamydia psittaci strains and relation to host origin.

Genetic relationships were reported for Chlamydia psittaci derived from psittacine birds, pigeons, turkeys, humans, cats, muskrats, cattle, and sheep and for C. trachomatis, including representative strains of the three biovars, through physical analysis of genomic DNA including DNA fingerprinting with restriction endonuclease SalI, DNA-DNA hybridization in solution with S1 nuclease, and Southern analysis with genomic DNA probes. A total of 26 strains were divided into four groups of C. psittaci and two groups of C. trachomatis, on the basis of DNA fingerprints. The six groups of Chlamydia spp. were related to host origin: two avian groups (Av1 and Av2), one feline and muskrat group (Fe1), one ruminant group (Ru1), one C. trachomatis biovars trachoma and lymphogranuloma group (CtHu), and one C. trachomatis mouse biovar group (CtMo), although an ovine abortion strain belonged to the avian group Av2. DNA-DNA hybridization assay and Southern analysis with genomic DNA probes indicated three DNA homology groups in the genus Chlamydia: an avian-feline group (groups Av1, Av2, and Fe1), a ruminant group (group Ru1), and a C. trachomatis group (groups CtHu and CtMo). Furthermore, the Southern analysis indicated that the homologous sequences (DNA homology of at least 14%) within the avian-feline group were distributed along the whole genome, whereas the homologous sequences (DNA homology of less than 24%) among the three DNA homology groups were localized in distinct regions of the genome DNA. These results suggest that Chlamydia spp. are derived from a common ancestor and have diverged into various groups showing restricted host ranges as a natural characteristic and that the species C. psittaci should be differentiated into groups related to host origin and DNA homology.     

Cloning and antigenic expression of two genes from Chlamydia psittaci avian strain

Abstract: Genes from Chlamydia psittaci P-1041 were cloned into the Bam HI site of pUC19 and were transformed to host Escherichia coli JM109. Two recombinant plasmids that expressed protein antigens of Chlamydia were isolated. The sizes of the DNA fragments were 1350 and 1710 bp, and encoded for polypeptides of M _r 25 and 42 kilodaltons (kDa), respectively. The 25-kDa protein had cross-reactivity with antisera to ten C. psittaci strains and two C. trachomatis strains, whereas the 42-kDa protein reacted only with homologous antiserum to the C. psittaci P-1041 strain. Furthermore, in Southern hybridization analysis these two fragments as probes hybridized with DNA of ten C. psittaci strains and four C. trachomatis strains. These results indicated that the two fragments shared a DNA sequence common to the chlamydial genus.     

Conserved DNA sequences in chlamydial plasmids

Two 7.4-kb plasmids from Chlamydia psittaci have been cloned and characterized. These plasmids are quite distinct from the 6.2-kb C. psittaci and the C. trachomatis plasmids when compared by restriction endonuclease analysis. The plasmids show considerable cross-hybridization, with only a small region highly conserved and identified as a 4 X 22-bp tandemly repeated region. This sequence is identical in the two size categories of C. psittaci plasmids and differs from C. trachomatis plasmids by only 2 bp in the 22-bp motif. AT-rich clusters 5' to the repeat region which are present in C. trachomatis and Escherichia coli plasmids were absent from both classes of C. psittaci plasmids. Extensive regions are less highly conserved but show a sufficient degree of cross-hybridization to suggest that the plasmids are homologous.     

Restriction pattern of the major outer-membrane protein gene provides evidence for a homogeneous invasive group among ruminant isolates of Chlamydia psittaci.

Thirty-six ruminant isolates of Chlamydia psittaci, previously classified as invasive or non-invasive in a mouse model of virulence, were compared by analysing AluI restriction patterns of the major outer-membrane protein (MOMP) gene after DNA amplification by the polymerase chain reaction. The 24 invasive isolates, although from various origins, all belonged to serotype 1 and represented a strictly homogeneous group sharing a specific MOMP-gene restriction pattern that was not observed in the non-invasive strains. On the other hand, the 12 non-invasive strains, although all belonging to serotype 2, constituted a heterogeneous group with eight distinct MOMP-gene restriction patterns. However, all eight patterns shared a 180 bp fragment or the corresponding restricted fragments of 110 and 70 bp. MOMP-gene restriction patterns also clearly distinguished the ruminant strains from an avian C. psittaci isolate, a C. pneumoniae isolate and two C. trachomatis isolates which were studied for comparison. The homogeneous character of the invasive C. psittaci strains argues strongly for their genetic relatedness. Our results illustrate the usefulness of the MOMP-gene restriction mapping in typing chlamydiae.     

Characterization of plasmids from plant pathogenic pseudomonads

Physical characterization of the resident plasmids from Pseudomonas tabaci, P. angulata, and P. coronafaciens strains indicated that they harbored five different plasmid DNA species. Two ATCC strains of P. tabaci contained indistinguishable plasmids that we have named pJP1 and pJP2. An isolate of one of these strains contained a spontaneous variant of pJP1, pJP1¹, which contains an insertion of 3.9 Mdal. This 3.9-Mdal region did not hybridize to pJP1 indicating that the region was foreign DNA and not a duplication of a segment of DNA already present in pJP1. Another P. tabaci strain, PT27881, contained a third plasmid species, pJP27, which had few similarities to pJP1 or pJP2, but was indistinguishable from the plasmids from all three P. angulata strains. pJP27 and pJP1 had a small region, 8.8 Mdal, of sequence homology. The one strain of P. coronafaciens examined contained a plasmid, pJP50, which was different from the P. tabaci plasmids, but had the 8.8-Mdal region and additional regions of sequence homology with pJP1 and pJP27 as well as homology with a portion of the pJP1¹ insertion. A fourth strain of P. tabaci, PTBR-2, a pathogen on beans, contained plasmid pBW, the only plasmid that lacked detectable regions of homology with the other plasmids.     

Plasmid RFLP profiling and DNA homology inThermus isolated from hot springs of different geographical areas

Thirty-four strains belonging to various species of the genus Thermus (T. aquaticus, "T. thermophilus," "T. brockianus," T. scotoductus, and genomic species 2) isolated from hot springs of different geographical areas were examined for plasmid content and restriction fragment length polymorphism (RFLP) of plasmid DNAs. The four strains of the numerical taxonomy cluster E of genomic species 2 did not harbor plasmid DNA. Overall examination of the HindIII-RFLP profiling of plasmid DNA showed considerable variability between and within genomic species, with the exception of presumed clonal isolates. In spite of this heterogeneity, HindIII plasmid digests within a numerical taxonomic cluster gave a subset of restriction fragments of similar or identical length. Strains belonging to genomic species 2 or unclassified isolates from S. Pedro do Sul that harbored plasmid DNA (7 of the 14 strains studied) exhibited strong DNA homology between plasmid regions. No homologous sequences to these plasmid regions were found in chromosomal DNA from strains isolated from S. Pedro do Sul in which no plasmids were detected. The strains belonging to T. scotoductus formed two plasmid DNA homology groups, as estimated by probing with a plasmid fragment that coincided with the two numerical taxonomy clusters proposed previously. Among the other species, homology of plasmid regions was also found between some strains. Strong homology was also found between plasmid regions from some strains of different taxonomic groups, isolated from the same and from different sources, suggesting that these sequences are highly conserved in plasmids present in Thermus. For plasmid-containing strains, results of plasmid RFLP profiling/DNA homology appear promising for the typing of Thermus at the level of biotypes or of individual strains, namely, for monitoring the diversity and frequency of isolates from a particular hot spring. Received: 24 October 1994 / Accepted: 6 March 1995     

Genomic Relatedness of Chlamydia Isolates Determined by Amplified Fragment Length Polymorphism Analysis

The genomic relatedness of 19 Chlamydia pneumoniae isolates (17 from respiratory origin and 2 from atherosclerotic origin), 21 Chlamydia trachomatis isolates (all serovars from the human biovar, an isolate from the mouse biovar, and a porcine isolate), 6 Chlamydia psittaci isolates (5 avian isolates and 1 feline isolate), and 1 Chlamydia pecorum isolate was studied by analyzing genomic amplified fragment length polymorphism (AFLP) fingerprints. The AFLP procedure was adapted from a previously developed method for characterization of clinical C. trachomatis isolates. The fingerprints of all C. pneumoniae isolates were nearly identical, clustering together at a Dice similarity of 92.6% (+/- 1.6% standard deviation). The fingerprints of the C. trachomatis isolates of human, mouse, and swine origin were clearly distinct from each other. The fingerprints of the isolates from the human biovar could be divided into at least 12 different types when the presence or absence of specific bands was taken into account. The C. psittaci fingerprints could be divided into a parakeet, a pigeon, and a feline type. The fingerprint of C. pecorum was clearly distinct from all others. Cluster analysis of selected isolates from all species revealed groups other than those based on sequence data from single genes (in particular, omp1 and rRNA genes) but was in agreement with available DNA-DNA hybridization data. In conclusion, cluster analysis of AFLP fingerprints of representatives of all species provided suggestions for a grouping of chlamydiae based on the analysis of the whole genome. Furthermore, genomic AFLP analysis showed that the genome of C. pneumoniae is highly conserved and that no differences exist between isolates of respiratory and atherosclerotic origins.     

Proposal of Chlamydia pecorum sp. nov. for Chlamydia strains derived from ruminants.

Chlamydia pecorum sp. nov. is proposed as the fourth species of the genus Chlamydia on the basis of the results of a genetic analysis of Chlamydia strains that were isolated from cattle and sheep which had various diseases, including sporadic encephalitis, infectious polyarthritis, pneumonia, and diarrhea. The levels of DNA-DNA homology between C. pecorum and strains of C. psittaci, Chlamydia pneumoniae, and Chlamydia trachomatis were less than 10%. Several DNA probes were used to identify C. pecorum. The C. pecorum strains were distinguished from C. psittaci strains by the results of immunological assays, including an immunofluorescence antibody assay performed with monoclonal antibodies and an immunoblot analysis of the immunological specificity of the major outer membrane protein. Species identification was based on results obtained from DNA analyses and serology. The type strain of C. pecorum is strain ATCC VR628.     

Plasmids in avirulent strains of Agrobacterium.

Twelve strains of Agrobacterium radiobacter isolated from naturally occurring crown galls or soil were found to be avirulent on sunflower, tomato, Kalanchoe, and carrot. Eleven strains contained plasmids of molecular weights 77 X 10(6) to 182 X 10(6) as determined by electron microscopy. One strain contained only a smaller plasmid (50 X 10(6) daltons). Several strains had both large and small (ca. 11 X 10(6) daltons) plasmids; one strain contained two large plasmids (112 X 10(6) and 136 X 10(6) daltons). Hybridization reactions of virulence plasmids from Agrobacterium tumefaciens strains C58 and A6 with plasmids from each of the A. radiobacter strains revealed that some A. radiobacter plasmids had less than 10% homology to either the C58 or A6 plasmids. Plasmids from some strains had approximately 50% homology with the C58 plasmid, but only one A. radiobacter plasmid contained more than 10% homology to the A6 plasmid. The presence of large plasmids in A. radiobacter strains did not correlate with sensitivity to agrocin 84; however, the utilization of the amino acid derivatives octopine and nopaline was generally correlated to partial base sequence homology to the C58 plasmid. We conclude that all large plasmids found in Agrobacterium strains are not virulence associated, although they may share base sequence homology with a virulence-associated plasmid. Further, plasmids from tumorigenic strains may be more closely related by base sequence homology to plasmids from nonpathogenic strains than to plasmids from other pathogenic strains.     

DNA sequencing of a pathogenicity-related plasmid of an avian septicemic Escherichia coli strain

A 43-MDa conjugative plasmid isolated from an avian septicemic Escherichia coli (APEC) strain possessing genes related to the adhesion and invasion capacities of in vitro-cultured cells was sequenced. The results demonstrated that the 43-MDa plasmid harbors bacterial pathogenicity-related sequences which probably allow the wild-type pathogenic strain to adhere to and invade tissues and to cause septicemia in poultry. The existence of homology sequences to sequences belonging to other human pathogenic Enterobacteriaceae like Escherichia coli O157:H7, Shigella and Salmonella was also observed. The presence of these sequences in this plasmid could indicate that there is horizontal genetic transfer between bacterial strains isolated from different host species. In conclusion, the present study suggests that APEC strains harbor high-molecular weight plasmids that present pathogenicity-related sequences and that these are probably responsible for the pathogenicity exhibited by these strains. The presence of human pathogenicity-associated sequences in APEC conjugative plasmids suggests that these strains could represent a zoonotic risk.     

Survey of the plasmid content of group A streptococci

Abstract Examination of 70 M-prototype group A streptococci showed small plasmids (2.0–2.5 MDa) to be present in strains representative of M-types 28, 57, 61, 63, 64 and 69. Identical results were obtained from M _r and restriction endonuclease analyses of a 2.2-MDa plasmid (pDN691) found in the M-type 69 strain and similar plasmids in the M57 prototype and two other independently isolated M-type 57 strains. In all four strains the presence of plasmid correlated with the production of bacteriocin-like inhibitory activity identifiable as P type 614. Similar analysis revealed a possible relationship between a 2.5-MDa plasmid in the prototype M61 and M64 strains and the production of P-type 216 inhibitory activity. A survey of 56 group A streptococci recovered in association with either rheumatic fever or nephritis failed to demonstrate plasmid DNA with the exception of 2.2-MDa plasmids in four nephritis-associated M-type 57 isolates.     

Comparison of Ti plasmids from three different biotypes of Agrobacterium tumefaciens isolated from grapevines.

Twenty-six plasmids from grapevine isolates of Agrobacterium tumefaciens were analyzed by SmaI fingerprinting and by hybridization of nick-translated DNA to DNA of another plasmid. These experiments established that octopine Ti plasmids are not highly conserved, although octopine Ti plasmids from biotype 1 A. tumefaciens strains appeared to be very similar. Octopine Ti plasmids from biotype 3 strains are more variable in terms of host range and SmaI fingerprints, but share extensive DNA homology. Fingerprints of nopaline Ti plasmids from strains of a given biotype resemble each other but not fingerprints of Ti plasmids from strains of the other two biotypes. The wide host range octopine Ti plasmid from the biotype 3 strain Ag86 shares more DNA homology with narrow host range Ti plasmids, nopaline Ti plasmids, and octopine catabolism plasmids than with the wide host range octopine Ti plasmid from biotype 1 strain 20/1. pTiAg86 does share homology with the portion of pTi20/1 integrated and expressed in plant tumor cells. Since all wide host range Ti plasmids studied contain these sequences, we suggest that natural selection for a wide host range resulted in the presence of the common sequences in distantly related plasmids. The lack of homology between this "common DNA" and limited host range Ti plasmids shows that the DNA sequences per se are not required for tumorigenesis.     

Limited-host-range plasmid of Agrobacterium tumefaciens: molecular and genetic analyses of transferred DNA.

A tumor-inducing (Ti) plasmid from a strain of Agrobacterium tumefaciens that induces tumors on only a limited range of plants was characterized and compared with the Ti plasmids from strains that induce tumors on a wide range of plants. Whereas all wide-host-range Ti plasmids characterized to date contain closely linked oncogenic loci within a single transferred DNA (T-DNA) region, homology to these loci is divided into two widely separated T-DNA regions on the limited-host-range plasmid. These two plasmid regions, TA-DNA and TB-DNA, are separated by approximately 25 kilobases of DNA which is not maintained in the tumor. The TA-DNA region resembles a deleted form of the wide-host-range TL-DNA and contains a region homologous to the cytokinin biosynthetic gene. However, a region homologous to the two auxin biosynthetic loci of the wide-host-range plasmid mapped within the TB-DNA region. These latter genes play an important role in tumor formation because mutations in these loci result in a loss of virulence on Nicotiana plants. Furthermore, the TB-DNA region alone conferred tumorigenicity onto strains with an intact set of vir genes. Our results suggest that factors within both the T-DNA and the vir regions contribute to the expression of host range in Agrobacterium species. There was a tremendous variation among plants in susceptibility to tumor formation by various A. tumefaciens strains. This variation occurred not only among different plant species, but also among different varieties of plants within the same genus.     

Physiological studies on the utilization of oleic acid by Saccharomyces cerevisiae in relation to microbody development

Twenty one Chlamydia trachomatis reference strains and 40 clinical isolates belonging to the lymphogranuloma venerum (LGV) and trachoma biovars were genotyped by differential restriction mapping of the major-outer-membrane-protein gene (MOMP) obtained by the polymerase-chain reaction (PCR). AluI digestion of the PCR product distinguishes eight MOMP-genotypes corresponding to 8 serovars. Six additional enzymes (NlaIII, CfoI, EcoRI, HinfI, DdeI and FokI) further permit the discrimination of 10 MOMP-genotypes corresponding to the 10 remaining serovars of the species. AluI alone allows direct typing of 78% of the clinical isolates. AluI digestion patterns of mouse C. trachomatis biovar, a C. pneumoniae and two C. psittaci strains, studied for comparison, were clearly distinguishable from one another and from the C. trachomatis LGV and trachoma strains. These results indicate that MOMP genotyping by PCR is a valuable molecular tool for studying C. trachomatis epidemiology.     

Interspecies structural diversity among chlamydial genes encoding histone H1.

Recently, a eukaryotic histone H1-like protein has been detected in Chlamydia trachomatis serovar L2 [Hackstadt et al., Proc. Natl. Acad. Sci. USA 88 (1991) 3937-3941; Tao et al., J. Bacteriol. 173 (1991) 2818-2822]. We have cloned the corresponding gene from C. trachomatis serovar J and the Chlamydia psittaci strain mn. Sequencing demonstrated absolute gene identity between the two C. trachomatis serovars L2 and J, but divergence in the C. psittaci strain mn. These differences resulted in altered aa residues (in particular no cysteines) and a smaller molecular mass for H1 from C. psittaci strain mn. The amino acid (aa) sequence comparisons with other histone proteins show best alignment to sea urchin H1, notably in the C terminus, for both C. trachomatis and C. psittaci histones. Chlamydial interspecies aa homology, however, is most conserved at the N terminus, suggestive of a bi-functional role for these unique histone proteins.     

Relationship of plasmids responsible for hairy root and crown gall tumorigenicity.

Three strains of Agrobacterium rhizogenes were examined for plasmids. Strains 15834 and A4 contained essentially identical large plasmids, pAr15834c and pArA4c, respectively (approximately 260 x 10(6) daltons). These plasmids can dissociate to two smaller plasmid species. Strain TR105 contained only a single plasmid, which was homologous with the dissociation product of pAr15834c, pAr15834b. Plasmid pAr15834c shared little overall sequence homology with other Ti plasmids. One region of conserved homology between pAr15834c and a region of the octopine type plasmid pTiB6806 which contains oncogenicity functions was detected. Lower levels of homology were detected with sequences which are distributed throughout 65% of pTiB6806. Homology with the so-called common deoxyribonucleic acid in the integrated plasmid deoxyribonucleic acid region was detected only after lowering the stringency of hybridization (Tm, -41 degrees C). Furthermore, the A. rhizogenes plasmid is compatible with other Ti plasmids. Therefore, the results suggest that the virulence plasmids of A. rhizogenes are functionally similar to other Ti plasmids, yet have diverged sufficiently from an ancestral Ti plasmid that they now represent a distinct plasmid type based on homology, compatibility, and virulence.