Karin D. E. Everett

Identification of nine species of the Chlamydiaceae using PCR-RFLP.

The family Chlamydiaceae contains two genera and nine species. Rapid and easy identification of these species is essential for taxonomic, epidemiological and clinical determinations. Currently, DNA sequence analysis is the only accepted method that decisively distinguishes all nine species. In this study, a simple and rapid PCR-RFLP procedure was developed by which laboratory-cultured chlamydial specimens could be identified. To accomplish this, conserved oligonucleotide primers and restriction sites were deduced from 16S and 23S rRNA sequence data from > 50 chlamydial strains representing all nine species. DNA from 25 previously characterized chlamydial strains were tested with these primers and restriction enzymes. All nine chlamydial species were reliably distinguished in the tests. The procedure was optimized by adjusting the annealing temperature using both a standard and a heat-activated DNA polymerase to reduce mismatch PCR amplification of mycoplasmas and other bacteria. The result was that a PCR method for species identification of chlamydial isolates and for distinguishing mycoplasmas and chlamydiae was created. This method can be used to rapidly identify known species of the family Chlamydiaceae.

Simkania negevensis strain ZT: growth, antigenic and genome characteristics.

Simkania negevensis is the type species of Simkaniaceae, a recently proposed family in the order Chlamydiales. In the current study, growth, antigenic and genomic characteristics of this intracellular bacterium were investigated and compared to those of members of the family Chlamydiaceae. Growth of the organism, as assessed by infectivity assays, reached a plateau in 2-3 d although by light microscopy the cytopathic effect on the host cells increased for 12 or more days after infection. S. negevensis growth was unaffected by sulfadiazine. Cells infected by S. negevensis strain ZT were not recognized by either of two monoclonal antibodies specific for Chlamydiaceae LPS and several specific Chlamydiaceae ompA primers were unable to PCR amplify a S. negevensis gene. The S. negevensis genome contained one copy of the ribosomal operon. The genome size of S. negevensis strain ZT was determined by PFGE to be 1.7 Mbp, and the G + C content was 42.5 mol%. These data, taken together with other published data, are consistent with the proposal that S. negevensis belongs to a distinct family in the order Chlamydiales.

Characterization of the rnpB gene and RNase P RNA in the order Chlamydiales.

The sequence of the RNase P RNA gene (rnpB) was determined for 60 strains representing all nine species in the family Chlamydiaceae and for the related Chlamydiales species, Parachlamydia acanthamoebae and Simkania negevensis. These sequences were used to infer evolutionary relationships among the Chlamydiaceae. The analysis separated Chlamydophila and Chlamydia into two lineages, with Chlamydophila forming three distinct clusters: the Chlamydophila pneumoniae strains; the Chlamydophila pecorum strains; and a third cluster comprising the species Chlamydophila psittaci, Chlamydophila abortus, Chlamydophila caviae and Chlamydophila felis. The Chlamydia line of descent contained two clusters, with the Chlamydia suis strains distinctly separated from strains of Chlamydia trachomatis and Chlamydia muridarum. This analysis indicated that the rnpB sequence and structure are distinctive markers for species in the Chlamydiaceae. It was also demonstrated that the RNase P RNA derived from Chlamydia trachomatis is able to cleave a tRNA precursor in the absence of protein. These findings are discussed in relation to the structure of Chlamydia RNase P RNA.

Chloroplast genome sequence of the moss Tortula ruralis: gene content, polymorphism, and structural arrangement relative to other green plant chloroplast genomes

BackgroundTortula ruralis, a widely distributed species in the moss family Pottiaceae, is increasingly used as a model organism for the study of desiccation tolerance and mechanisms of cellular repair. In this paper, we present the chloroplast genome sequence of T. ruralis, only the second published chloroplast genome for a moss, and the first for a vegetatively desiccation-tolerant plant.ResultsThe Tortula chloroplast genome is ~123,500 bp, and differs in a number of ways from that of Physcomitrella patens, the first published moss chloroplast genome. For example, Tortula lacks the ~71 kb inversion found in the large single copy region of the Physcomitrella genome and other members of the Funariales. Also, the Tortula chloroplast genome lacks petN, a gene found in all known land plant plastid genomes. In addition, an unusual case of nucleotide polymorphism was discovered.ConclusionsAlthough the chloroplast genome of Tortula ruralis differs from that of the only other sequenced moss, Physcomitrella patens, we have yet to determine the biological significance of the differences. The polymorphisms we have uncovered in the sequencing of the genome offer a rare possibility (for mosses) of the generation of DNA markers for fine-level phylogenetic studies, or to investigate individual variation within populations.

Genotyping of Chlamydophila psittaci by Real-Time PCR and High-Resolution Melt Analysis

ABSTRACT Human infection with Chlamydophila (Chlamydia) psittaci can lead to psittacosis, a disease that occasionally results in severe pneumonia and other medical complications. C. psittaci is currently grouped into seven avian genotypes: A through F and E/B. Serological testing, outer membrane protein A (ompA) gene sequencing, and restriction fragment length polymorphism analysis are currently used for distinguishing these genotypes. Although accurate, these methods are time-consuming and require multiple confirmatory tests. By targeting the ompA gene, a real-time PCR assay has been developed to rapidly detect and genotype C. psittaci by light-upon-extension chemistry and high-resolution melt analysis. Using this assay, we screened 169 animal specimens; 98 were positive for C. psittaci (71.4% genotype A, 3.1% genotype B, 4.1% genotype E, and 21.4% unable to be typed). This test may provide insight into the distribution of each genotype among specific hosts and provide epidemiological and epizootiological data in human and mammalian/avian cases. This diagnostic assay may also have veterinary applications during chlamydial outbreaks, particularly with respect to identifying the sources and tracking the movements of a particular genotype when multiple animal facilities are affected.

The Complete Plastid Genome Sequence of Angiopteris evecta (G. Forst.) Hoffm. (Marattiaceae)

ABSTRACT We have sequenced the complete plastid genome of the fern Angiopteris evecta. This taxon belongs to a major lineage (marattioid ferns) that, in most recent phylogenetic analyses, emerges near the base of the monilophytes. We used fluorescence activated cell sorting (FACS) to isolate organelles, rolling circle amplification (RCA) to amplify the plastid genome, followed by shotgun sequencing to 8X depth coverage, and then we assembled these reads to obtain the plastid genome sequence. The circular genome map has 153,901 bp, containing inverted repeats of 21,053 bp each, a large single-copy region of 89,709 bp, and a small single-copy region of 22,086 bp. Gene order is similar to that of Psilotum. Several unique characters are observed in the Angiopteris plastid genome, such as repeat structure in a pseudogene. We make structural comparisons to Psilotum and Adiantum plastid genomes. However, the overall structural similarity to Psilotum indicates either wholesale conservation of genome organization, or (less likely) repeated convergence to a stable structure. The results are discussed in relation to a growing comparative database of genomic and morphological characters across the green plants.