Molecular genetic characterization of avian Chlamydophila psittaci isolates

Restriction enzyme analysis of the omp1 gene was used to characterize 51 avian Chlamydophila psittaci isolates. The analysis confirmed the predominance of genotype A in parrot C. psittaci isolates and revealed new omp1 genotypes in corvid C. psittaci isolates. The corvid isolates proved to lack an extrachromosomal plasmid. The omp1 and rRNA IGS sequences were determined for the new isolates. Phylogenetic analysis showed that isolate 1V, obtained from a crow, is intermediate in several characters between C. abortus and C. psittaci. The results were compared with data on the phylogenetic relationships of earlier chlamydium isolates.     

Complete genome sequences of 138 mycobacteriophages

Bacteriophages are the most numerous biological entities in the biosphere, and although their genetic diversity is high, it remains ill defined. Mycobacteriophages-the viruses of mycobacterial hosts-provide insights into this diversity as well as tools for manipulating Mycobacterium tuberculosis. We report here the complete genome sequences of 138 new mycobacteriophages, which-together with the 83 mycobacteriophages previously reported-represent the largest collection of phages known to infect a single common host, Mycobacterium smegmatis mc(2) 155.     

Complete genome sequences of three Propionibacterium acnes isolates from the type IA(2) cluster

Propionibacterium acnes is an anaerobic Gram-positive bacterium that has been linked to a wide range of opportunistic human infections and conditions, most notably acne vulgaris (I. Kurokawa et al., Exp. Dermatol. 18:821-832, 2009). We now present the whole-genome sequences of three P. acnes strains from the type IA(2) cluster which were recovered from ophthalmic infections (A. McDowell et al., Microbiology 157:1990-2003, 2011).     

Genome sequence of the zoonotic pathogen Chlamydophila psittaci

We present the first genome sequence of Chlamydophila psittaci, an intracellular pathogen of birds and a human zoonotic pathogen. A comparison with previously sequenced Chlamydophila genomes shows that, as in other chlamydiae, most of the genome diversity is restricted to the plasticity zone. The C. psittaci plasmid was also sequenced.     

Whole genome sequences of four Brucella strains

Brucella melitensis and Brucella suis are intracellular pathogens of livestock and humans. Here we report four genome sequences, those of the virulent strain B. melitensis M28-12 and vaccine strains B. melitensis M5 and M111 and B. suis S2, which show different virulences and pathogenicities, which will help to design a more effective brucellosis vaccine.     

Complete genome sequences of three Pseudomonas aeruginosa isolates with phenotypes of polymyxin B adaptation and inducible resistance

Clinical "superbug" isolates of Pseudomonas aeruginosa were previously observed to be resistant to several antibiotics, including polymyxin B, and/or to have a distinct, reproducible adaptive polymyxin resistance phenotype, identified by observing "skipped" wells (appearance of extra turbid wells) during broth microdilution testing. Here we report the complete assembled draft genome sequences of three such polymyxin resistant P. aeruginosa strains (9BR, 19BR, and 213BR).     

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.     

Complete genome sequences of six strains of the genus methylobacterium

The complete and assembled genome sequences were determined for six strains of the alphaproteobacterial genus Methylobacterium, chosen for their key adaptations to different plant-associated niches and environmental constraints.     

Complete genome sequences of novel canine noroviruses in Hong Kong

We report the complete genome sequences of two novel isolates of norovirus isolated from the fecal swab specimens of dogs in Hong Kong. The complete viral genome is approximately 7.6 kb in length and consists of 3 overlapping open reading frames encoding the ORF1 polyprotein, VP1, and VP2, respectively. Analysis of the VP1 sequence suggested that these noroviruses are divergent from known noroviruses and may represent a novel phylogenetic clade within the genus.     

Monitoring mammalian genome rearrangement with mid-repetitive sequences as probes

By utilization of mid-repetitive sequences, the intracisternal A particle (IAP) gene, as a probe, genome rearrangement involving IAP genes and their neighboring sequences in rodent cells can be monitored. This is based on electrophoretic separation of the twice digested restriction fragments of genomic DNA in a 2-D pattern. The first digestion was done in solution followed by electrophoresis of the restriction fragments in the first dimension. A second restriction enzyme digestion was carried out in situ in the gel followed by electrophoresis in a second dimension perpendicular to the first electrophoresis. After Southern blotting, the DNA on the filter is hybridized with a probe that is a fragment located near the 5' end of the IAP gene, but does not overlap with the 5' long terminal repeat (LTR). The exposed X-ray film revealed about 370 distinct spots in the 2-D maps. In comparing the 2-D maps, genome rearrangement involving IAP was detected.     

Complete sequences of two highly divergent european isolates of TT virus

TT virus is a virus distantly related to the Circoviridae family. We report here the complete genome characterization of two European human isolates (T3PB and TUPB) using a new and simple protocol for sequencing GC-rich genomic regions. Sequence analysis confirmed the existence of two major ORFs, of a CAV-like VP2 motif in ORF2 and of potential stem-loop structures in non-coding regions. Phylogenetic analyses based on complete genomic sequences of human isolates suggested that three different lineages exist at least. The first lineage includes genotypes 1, 2, and 3, and two other lineages include viruses related to the Japanese SANBAN and to the North American TUS01 isolates respectively. Sequence comparison made it possible to assign strain T3PB to genotype 3, and strain TUPB to the TUS01 group. Consequently, this study reports the first full-length sequence of a genotype 3 isolate and demonstrates that viruses belonging to the TUS01 lineage are present in the Old Word.     

三种方法对禽鹦鹉热嗜性衣原体疑似病例病原的流行调查

目的利用免疫荧光抗体法、PCE-ELISA、PCR法分别检测北京市及周边地区禽鹦鹉热嗜性衣原体疑似病例的病原,以了解和评价该病原流行状况。方法收集临床疑似禽衣原体的父母代、商品代家禽、SPF鸡喉头拭子、气囊组织、子宫黏膜,组织标本固定后采用直接免疫荧光染色测定衣原体包含体;喉头拭子、子宫黏膜处理后以PCE-ELISA定量测定衣原体;以衣原体保守性高的序列设计CTU/CTL引物扩增病料组织的omp-1基因片段。结果直接荧光染色法显示家禽衣原体平均阳性率为38.7%,组织检出率依次为子宫黏膜、气囊、喉头拭子;PCE-ELISA检测显示平均阳性率为58.7%,其中SPF鸡阳性率为10.0%,健康肉鸡达到30.0%;PCR法检测家禽衣原体平均阳性率为71.6%,SPF鸡为10.0%。样本的目的基因与标准禽衣原体6BC同源性超过99%。结论种禽场和商品养殖场均发现疑似病例中禽鹦鹉热嗜性衣原体感染情况较为严重。荧光抗体染色、PCE-ELISA可用于临床实践中进行快速、准确检测。     

A monoclonal antibody against a carbohydrate epitope in lipopolysaccharide differentiates Chlamydophila psittaci from Chlamydophila pecorum, Chlamydophila pneumoniae, and Chlamydia trachomatis

Lipopolysaccharide (LPS) of Chlamydophila psittaci but not of Chlamydophila pneumoniae or Chlamydia trachomatis contains a tetrasaccharide of 3-deoxy-alpha-d-manno-oct-2-ulopyranosonic acid (Kdo) of the sequence Kdo(2-->8)[Kdo(2-->4)] Kdo(2-->4)Kdo. After immunization with the synthetic neoglycoconjugate antigen Kdo(2-->8)[Kdo(2-->4)]Kdo(2-->4) Kdo-BSA, we obtained the mouse monoclonal antibody (mAb) S69-4 which was able to differentiate C. psittaci from Chlamydophila pecorum, C. pneumoniae, and C. trachomatis in double labeling experiments of infected cell monolayers and by enzyme-linked immunosorbent assay (ELISA). The epitope specificity of mAb S69-4 was determined by binding and inhibition assays using bacteria, LPS, and natural or synthetic Kdo oligosaccharides as free ligands or conjugated to BSA. The mAb bound preferentially Kdo(2-->8)[Kdo(2-->4)]Kdo(2-->4)Kdo(2-->4) with a K(d) of 10 microM, as determined by surface plasmon resonance (SPR) for the monovalent interaction using mAb or single chain Fv. Cross-reactivity was observed with Kdo(2-->4)Kdo(2-->4) Kdo but not with Kdo(2-->8)Kdo(2-->4)Kdo, Kdo disaccharides in 2-->4- or 2-->8-linkage, or Kdo monosaccharide. MAb S69-4 was able to detect LPS on thin-layer chromatography (TLC) plates in amounts of <10 ng by immunostaining. Due to the high sensitivity achieved in this assay, the antibody also detected in vitro products of cloned Kdo transferases of Chlamydia. The antibody can therefore be used in medical and veterinarian diagnostics, general microbiology, analytical biochemistry, and studies of chlamydial LPS biosynthesis. Further contribution to the general understanding of carbohydrate-binding antibodies was obtained by a comparison of the primary structure of mAb S69-4 to that of mAb S45-18 of which the crystal structure in complex with its ligand has been elucidated recently (Nguyen et al., 2003, Nat. Struct. Biol., 10, 1019-1025).     

Structural analyses of the lipopolysaccharides from Chlamydophila psittaci strain 6BC and Chlamydophila pneumoniae strain Kajaani 6.

Lipopolysaccharides (LPSs) of Chlamydophila psittaci 6BC and Chlamydophila pneumoniae Kajaani 6 contain 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), GlcN, organic bound phosphate, and fatty acids in the molar ratios of approximately 3:2:2.2:4.8 and approximately 2.9:2:2.1:4.9, respectively. The LPSs were immunoreactive with a monoclonal antibody against a family-specific epitope of chlamydial LPS. This finding, together with methylation analyses of both LPSs and MALDI-TOF MS experiments on de-O-, and de-O,N-acylated LPSs, indicate the presence of a Kdo trisaccharide proximal to lipid A having a structure alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo, which appears to be the main component of the core region in the native chlamydial LPSs. In the de-O-acylated LPSs from Chl. psittaci 6BC and Chl. pneumoniae Kajaani 6, two major molecular species are present that differ in distribution of amide-bound hydroxy fatty acids over both GlcN. It appears that either two (R)-3-hydroxy-18-methylicosanoic acids or one (R)-3-hydroxy-18-methylicosanoic acid and one (R)-3-hydroxyicosanoic acid are attached to the GlcN residues. In contrast, the de-O-acylated LPS of Chl. psittaci PK 5082 contains one major molecular species that has two (R)-3-hydroxyicosanoic acid residues attached to two GlcN residues.     

Complete nucleotide sequences and gene organization of mitochondrial genome of Bufo gargarizans

The complete mitochondrial genome of Bufo gargarizans was sequenced using overlapping polymerase chain reaction (PCR) amplicons (GenBank Accession No. DQ275350). The genome is 17,277 base pairs in length, containing 13 protein-coding genes (ATP6, ATP8, COI-III, ND1-6, ND4L, Cyt b), 2 ribosomal RNAs (12S rRNA and 16S rRNA), 22 transfer RNAs and a putative control region. We analyzed the sequence using bioinformatics methods comparing the obtained mtDNA sequence with other toads and frogs. Based on the concatenated nucleotide sequences of protein-coding genes, we constructed a phylogenetic tree with maximum likelihood (ML) and maximum parsimony (MP) methods and discussed the phylogenetic relationships among 11 species of Anura.     

Structural analysis of the lipopolysaccharide from Chlamydophila psittaci strain 6BC.

The lipopolysaccaride of Chlamydophila psittaci 6BC was isolated from tissue culture-grown elementary bodies using a modified phenol/water procedure followed by extraction with phenol/chloroform/light petroleum. Compositional analyses indicated the presence of 3-deoxy-Dmanno-oct-2-ulosonic acid, GlcN, organic bound phosphate and fatty acids in a molar ratio of approximately 3. 3 : 2 : 1.8 : 4.6. Deacylated lipopolysaccharide was obtained after successive microscale treatment with hydrazine and potassium hydroxide, and was then separated by high performance anion-exchange chromatography into two major fractions, the structures of which were determined by 600 MHz NMR spectroscopy as alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcpN -(1 -->6)-alpha-D-GlcpN 1,4'-bisphosphate and alpha-Kdo-(2-->4)-[alpha-Kdo-(2-->8)]-alpha-Kdo-(2-->4)-alpha-Kdo-(2- ->6)-beta-D-GlcpN-(1-->6)-alpha-D-GlcpN 1,4'-bisphosphate. The distribution of fatty acids in lipid A was determined by compositional analyses and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry experiments on lipid A and de-O-acylated lipid A. It was shown that the carbohydrate backbone of lipid A is replaced by a complex mixture of fatty acids, including long-chain and branched (R)-configured 3-hydroxy fatty acids, the latter being exclusively present in an amide linkage.     

Complete sequence of bovine mitochondrial DNA conserved features of the mammalian mitochondrial genome

We present here the complete 16,338 nucleotide DNA sequence of the bovine mitochondrial genome. This sequence is homologous to that of the human mitochondrial genome (Anderson et al., 1981) and the genes are organized in virtually identical fashion. The bovine mitochondrial protein genes are 63 to 79% homologous to their human counterparts, and most of the nucleotide differences occur in the third positions of codons. The minimum rate of base substitution that accounts for the nucleotide differences in the codon third positions is very high: at least 6 × 10^?9 changes per position per year. The bovine and human mitochondrial transfer RNA genes exhibit more interspecies variation than do their cytoplasmic counterparts, with the “TΨC” loop being the most variable part of the molecule. The bovine 12 S and 16 S ribosomal RNA genes, when compared with those from human mitochondrial DNA, show conserved features that are consistent with proposed secondary structure models for the ribosomal RNAs. Unlike the pattern of moderate-to-high homology between the bovine and human mitochondrial DNAs found over most of the genome, the DNA sequence in the bovine D-loop region is only slightly homologous to the corresponding region in the human mitochondrial genome. This region is also quite variable in length, and accounts for the bulk of the size difference between the human and bovine mitochondrial DNAs.     

Methods for identification of epigenetic elements in mammalian long multigenic genome sequences

Epigenetic elements of the genome, i.e. elements that determine stably inherited changes in gene expression without changes in the genomic DNA sequence, are essential tools of genetic regulation in higher eukaryotes. The complete sequencing of the human and other genomes allowed studies to be started on positioning of these elements within long multigenic regions of the genome, which is a prerequisite for a comprehensive functional annotation of genomes. This mini-review considers some recent experimental approaches to the high-throughput identification and mapping of epigenetic elements of mammalian genomes, including the mapping of methylated CpG sites, open and closed chromatin regions, and DNase I hypersensitivity sites.