Insights into the evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis.

Yersinia pestis, the causative agent of plague, is a highly uniform clone that diverged recently from the enteric pathogen Yersinia pseudotuberculosis. Despite their close genetic relationship, they differ radically in their pathogenicity and transmission. Here, we report the complete genomic sequence of Y. pseudotuberculosis IP32953 and its use for detailed genome comparisons with available Y. pestis sequences. Analyses of identified differences across a panel of Yersinia isolates from around the world reveal 32 Y. pestis chromosomal genes that, together with the two Y. pestis-specific plasmids, to our knowledge, represent the only new genetic material in Y. pestis acquired since the the divergence from Y. pseudotuberculosis. In contrast, 149 other pseudogenes (doubling the previous estimate) and 317 genes absent from Y. pestis were detected, indicating that as many as 13% of Y. pseudotuberculosis genes no longer function in Y. pestis. Extensive insertion sequence-mediated genome rearrangements and reductive evolution through massive gene loss, resulting in elimination and modification of preexisting gene expression pathways, appear to be more important than acquisition of genes in the evolution of Y. pestis. These results provide a sobering example of how a highly virulent epidemic clone can suddenly emerge from a less virulent, closely related progenitor.

Murine toxin of Yersinia pestis shows phospholipase D activity but is not required for virulence in mice.

Purified murine toxin (Ymt) of Yersinia pestis is highly toxic for mice and rats but less active in other animals such as guinea pigs, rabbits, dogs and monkeys. This suggested that Ymt contributes to the very low infectious dose of Y. pestis in mice. The gene encoding Ymt (ymt) is localised on the 100-kb plasmid pFra, which is unique for Y. pestis. Sequence analysis revealed that Ymt showed homology to proteins of the phospholipase D (PLD) superfamily of proteins. Y. pestis strains expressing Ymt possessed PLD activity whereas strains carrying deletions in the ymt gene showed no detectable PLD activity. Western blot analysis showed that Ymt was associated with bacteria under normal growth conditions, and immunogold EM revealed that Ymt was mainly localised in the bacterial cytoplasm. Ymt was purified to homogeneity, and the purified toxin showed a dose-dependent PLD activity. Substitution of amino acids in the PLD consensus motif of Ymt essentially abolished the enzymatic activity and these variants of the toxin were no longer toxic to mice. Interestingly, an in-frame deletion mutant of ymt in the Y pestis strain KIM was not significantly attenuated for mouse virulence. Together with the observation that expression of Ymt was higher at room temperature compared to 37 degrees C this prompted us to investigate the role of Ymt in the flea vector. Fleas were infected with isogenic ymt+ or ymt- mutant strains of Y. pestis. Preliminary results suggest that Ymt is important for survival of Y. pestis in the flea and thereby also for the flea-borne route of infection.

The nucleotide sequence of a linear plasmid of Borrelia burgdorferi reveals similarities to those of circular plasmids of other prokaryotes.

A linear plasmid of Borrelia burgdorferi had 16,927 bp, a G+C content of 23.1%, a relative deficiency of CpG dinucleotides, and open reading frames A to O. The OrfC and OrfE proteins were similar to hypothetical proteins encoded by circular plasmids of B. burgdorferi. The OrfM and OrfN proteins were similar to replication proteins of circular plasmids of other bacteria.

Linear plasmids and chromosomes in bacteria.

Linear plasmids and chromosomes were unknown in prokaryotes until recently but have now been found in spirochaetes, Gram-positive bacteria, and Gram-negative bacteria. Two structural types of bacterial linear DNA have been characterized. Linear plasmids of the spirochaete Borrelia have a covalently closed hairpin loop at each end and linear plasmids of the Gram-positive filamentous Streptomyces have a covalently attached protein at each end. Replicons with similar structures are more frequent in eukaryotic cells than in prokaryotes. Linear genomic structures are probably more common in bacteria than previously recognized, however, and some replicons may interconvert between circular and linear isomers. The molecular biology of these widely dispersed elements provides clues to explain the origin of linear DNA in bacteria, including evidence for genetic exchange between prokaryotes and eukaryotes.

New method for plague surveillance using polymerase chain reaction to detect Yersinia pestis in fleas.

Yersinia pestis, the plague bacillus, infects a variety of mammals throughout the world and is transmitted by fleas. We developed a polymerase chain reaction (PCR) test using primers designed from the Y. pestis plasminogen activator gene to directly detect plague-infected fleas. As few as 10 Y. pestis cells were detected, even in the presence of flea tissue, by PCR and then agarose gel electrophoresis and ethidium bromide staining. The feasibility of the assay was demonstrated by using naturally infected Xenopsylla cheopis fleas. The detection of Y. pestis in fleas by PCR provides a rapid and sensitive way to monitor plaque in wild animal populations, allowing public health officials to better assess the potential risk of transmission to humans.

Linear- and circular-plasmid copy numbers in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease agent, and other members of the spirochetal genus Borrelia have double-stranded linear plasmids in addition to supercoiled circular plasmids. The copy number relative to the chromosome was determined for 49- and 16-kb linear plasmids and a 27-kb circular plasmid of the type strain, B31, of B. burgdorferi. All three plasmids were present in low copy number, about one per chromosome equivalent, as determined by relative hybridizations of replicon-specific DNA probes. The low copy number of Borrelia plasmids suggests that initiation of DNA replication and partitioning are carefully controlled during the cell division cycle. The copy numbers of these three plasmids of strain B31 were unchanged after approximately 7,000 generations in continuous in vitro culture. A clone of B. burgdorferi B31 that did not contain the 16-kb linear plasmid was obtained after exposure of a culture to novobiocin, a DNA gyrase inhibitor. The plasmid-cured strain contains only one linear plasmid, the 49-kb plasmid, and thus has the smallest genome reported to date for B. burgdorferi.

Linear plasmids of Borrelia burgdorferi have a telomeric structure and sequence similar to those of a eukaryotic virus.

Spirochetes of the genus Borrelia have double-stranded linear plasmids with covalently closed ends. The physical nature of the terminal connections was determined for the 16-kb linear plasmid of the B31 strain of the Lyme disease agent Borrelia burgdorferi. Native telomeric fragments representing the left and right ends of this plasmid were isolated and subjected to Maxam-Gilbert sequence analysis. At the plasmid ends the two DNA strands formed an uninterrupted, perfectly palindromic, AT-rich sequence. This Borrelia linear plasmid consisted of a continuous polynucleotide chain that is fully base paired except for short single-stranded hairpin loops at each end. The left and right telomeres of the 16-kb plasmid were identical for 16 of the first 19 nucleotide positions and constituted an inverted terminal repeat with respect to each other. The left telomere of the 49-kb plasmid of strain B31 was identical to the corresponding telomere of the 16-kb plasmid. Different-sized plasmids of other strains of B. burgdorferi also contained sequences homologous to the left end of the 16-kb plasmid. When the borrelia telomeres were compared with telomeric sequences of other linear double-stranded DNA replicons, sequence similarities were noted with poxviruses and particularly with the iridovirus agent of African swine fever. The latter virus and a Borrelia sp. share the same tick vector. These findings suggest that the novel linear plasmids of Borrelia originated through a horizontal genetic transfer across kingdoms.

Cloning and sequence analysis of linear plasmid telomeres of the bacterium Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease agent, has double-stranded linear plasmids with covalently closed ends. DNA at the ends, or telomeres, of two linear plasmids of B. burgdorferi strain B31 was examined. Telomeric sequences from both ends of a 16 kb linear plasmid and from one end of a 49 kb linear plasmid were cloned and sequenced. An 18 bp AT-rich inverted repeat was found at each end of the 16 kb linear plasmid. The sequences of the two ends of this plasmid were different beyond these short inverted terminal repeats. The cloned end of the 49 kb linear plasmid had sequence identity with one end of the 16 kb linear plasmid. The end sequence common to both plasmids contained a series of phased, short direct repeats and a 52 bp palindrome adjacent to a highly AT-rich region. These findings indicate that Borrelia linear plasmid telomeres have structural features different from those of other known replicons.