Direct glutaminyl-tRNA biosynthesis and indirect asparaginyl-tRNA biosynthesis in Pseudomonas aeruginosa PAO1

The genomic sequence of Pseudomonas aeruginosa PAO1 was searched for the presence of open reading frames (ORFs) encoding enzymes potentially involved in the formation of Gln-tRNA and of Asn-tRNA. We found ORFs similar to known glutamyl-tRNA synthetases (GluRS), glutaminyl-tRNA synthetases (GlnRS), aspartyl-tRNA synthetases (AspRS), and trimeric tRNA-dependent amidotransferases (AdT) but none similar to known asparaginyl-tRNA synthetases (AsnRS). The absence of AsnRS was confirmed by biochemical tests with crude and fractionated extracts of P. aeruginosa PAO1, with the homologous tRNA as the substrate. The characterization of GluRS, AspRS, and AdT overproduced from their cloned genes in P. aeruginosa and purified to homogeneity revealed that GluRS is discriminating in the sense that it does not glutamylate tRNA^Gln, that AspRS is nondiscriminating, and that its Asp-tRNA^Asn product is transamidated by AdT. On the other hand, tRNA^Gln is directly glutaminylated by GlnRS. These results show that P. aeruginosa PAO1 is the first organism known to synthesize Asn-tRNA via the indirect pathway and to synthesize Gln-tRNA via the direct pathway. The essential role of AdT in the formation of Asn-tRNA in P. aeruginosa and the absence of a similar activity in the cytoplasm of eukaryotic cells identifies AdT as a potential target for antibiotics to be designed against this human pathogen. Such novel antibiotics could be active against other multidrug-resistant gram-negative pathogens such as Burkholderia and Neisseria as well as all pathogenic gram-positive bacteria.     

Zinc and Chlamydia trachomatis

Zinc was noted to have significant effects upon the infection of McCoy cells by each of two strains of Chlamydia trachomatis. With a high or low Chlamydia inoculant, the number of infected cells increased up to 200% utilizing supplemental zinc (up to 1 X 10(-4) M) in the inoculation media compared with standard Chlamydia cultivation media (8 X 10(-6) M zinc). Ferric chloride and calcium chloride did not effect any such changes. Higher concentrations of zinc, after 2 hr of incubation with Chlamydia, significantly decreased the number of inclusions. This direct effect of zinc on the Chlamydia remained constant after further repassage of the Chlamydia without supplemental zinc, suggesting a lethal effect of the zinc. Supplemental zinc (up to 10(-4)M) may prove to be a useful addition to inoculation media to increase the yield of culturing for Chlamydia trachomatis. Similarly, topical or oral zinc preparations used by people may alter their susceptivity to Chlamydia trachomatis infections.     

Forward Genetic Approaches in Chlamydia trachomatis

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype–phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.     

Developmental-stage-specific plasmid supercoiling in Chlamydia trachomatis

Chlamydia trachomatis elementary body (EB) and reticulate body (RB) developmental stages have polymorphic plasmid DNA. Several plasmid forms separated by gel electrophoresis were identified as topoisomers by treatment with topoisomerase I. Among these topoisomers was one form unique to EBs and one form unique to RBs. The unique EB plasmid topoisomer was characterized as highly supercoiled, on the basis of band migrations by gel electrophoresis and its appearance by electron microscopy. The unusual physical state of this topoisomer was probably mediated, in part, by DNA-specific structural proteins. The unique RB plasmid topoisomer was a supercoiled form of lower superhelical density than the other identified topoisomers. Developmental-stage-specific differences in super-helical density of plasmid DNA suggest cause-and-effect relationships between DNA topology and metabolic activity in RBs and metabolic quiescence in EBs.     

Chlamydia trachomatis infections in infants.

In recent years considerable progress has been made in understanding chlamydial infections. The spectrum of pediatric Chlamydia trachomatis infection includes neonatal inclusion conjunctivitis, infantile pneumonia, occasional respiratory or genital tract infections in older children and sexually transmitted diseases in adolescents. The role of maternal chlamydial infection in prematurity and in perinatal death is currently an area of active study. We outline the current knowledge of the biologic characteristics of C. trachomatis, the epidemiologic features of chlamydial infection, and the clinical aspects, diagnosis and treatment of neonatal chlamydial infections.     

Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39

The genome sequences of Chlamydia trachomatis mouse pneumonitis (MoPn) strain Nigg (1 069 412 nt) and Chlamydia pneumoniae strain AR39 (1 229 853 nt) were determined using a random shotgun strategy. The MoPn genome exhibited a general conservation of gene order and content with the previously sequenced C.trachomatis serovar D. Differences between C.trachomatis strains were focused on an ~50 kb ‘plasticity zone’ near the termination origins. In this region MoPn contained three copies of a novel gene encoding a >3000 amino acid toxin homologous to a predicted toxin from Escherichia coli 0157:H7 but had apparently lost the tryptophan biosyntheis genes found in serovar D in this region. The C.pneumoniae AR39 chromosome was >99.9% identical to the previously sequenced C.pneumoniae CWL029 genome, however, comparative analysis identified an invertible DNA segment upstream of the uridine kinase gene which was in different orientations in the two genomes. AR39 also contained a novel 4524 nt circular single-stranded (ss)DNA bacteriophage, the first time a virus has been reported infecting C.pneumoniae. Although the chlamydial genomes were highly conserved, there were intriguing differences in key nucleotide salvage pathways: C.pneumoniae has a uridine kinase gene for dUTP production, MoPn has a uracil phosphororibosyl transferase, while C.trachomatis serovar D contains neither gene. Chromosomal comparison revealed that there had been multiple large inversion events since the species divergence of C.trachomatis and C.pneumoniae, apparently oriented around the axis of the origin of replication and the termination region. The striking synteny of the Chlamydia genomes and prevalence of tandemly duplicated genes are evidence of minimal chromosome rearrangement and foreign gene uptake, presumably owing to the ecological isolation of the obligate intracellular parasites. In the absence of genetic analysis, comparative genomics will continue to provide insight into the virulence mechanisms of these important human pathogens.     

Reversibility of heat shock in Chlamydia trachomatis

The heat shock effect on chlamydia development was studied. We report here that the reversibility of the heat shock response did not depend on the stage of chlamydial morphogenesis at which transfer to high temperature occurred, and the infectivity of the particles produced was not affected significantly, so long as the heat shock exposure was not prolonged. Exposure to heat shock for more than 9 h resulted in stagnation of the growth cycle, appearance of aberrant reticulate body particles and loss of infectivity. SDS-PAGE analysis of proteins synthesized under prolonged heat shock showed increased relative abundance of heat shock proteins in common with other procaryotic organisms.     

Characterization of trimethoprim- and sulphisoxazole-resistant Chlamydia trachomatis

Trimethoprim and sulphisoxazole were used as selective agents in culture to isolate, by a stepwise procedure, a series of Chlamydia trachomatis L2 populations resistant to the cytotoxic effects of the drugs. Two trimethoprim-resistant populations. L2Tri^R-60 and L2Tri^R-100, and one sulphonamide-resistant population. L2Sulf^R-100, were characterized in more detail. In addition to being resistant to trimethoprim, L2Tri^R-100 was cross-resistant to metho-trexate, sensitive to sulphisoxazole and displayed a ribonucleotide auxotrophy similar to that of its parental wild type, C. trachomatis L2. Surprisingly, L2Tri^R-100 and L2Sulf^R-100 appeared phenotypically identical. Both mutants were highly resistant to trimethoprim, sulphisoxazole, and methotrexate. In contrast to wild-type C. trachomatis L2, these populations were sensitive to 5-fluorouracil L2Tri^R-100 and L2Sulf^R-100 were incapable of taking pyrimidine ribonucleotides from the host cell and no longer synthesized thymidine nucleotides de novo. The pyrimidine requirement of these mutants was met by salvaging host-cell uracil and thymidine, a property which can account for their drug-resistance characteristics. L2Tri^R-100 and L2Sulfⁿ-100 could also salvage adenine and guanine. Using L2Tri^R-100 as a starting stock, a mutant population resistant to the cytotoxic effects of trimethoprim and 5-fluorouracil (L2Tri/5-FU) was selected. L2TFi/5-FU was resistant to 5-fluoro-uracil because it had regained the capacity to take pyrimidine ribonucleotides from the host cell.     

沙眼衣原体质粒蛋白研究进展

沙眼衣原体除含有高度保守的基因组外,也含一个7．5kb的隐蔽性质粒,隐蔽性质粒具有8个开放阅读（ORF1-8）,编码8种质粒蛋白pgpl-8。质粒蛋白在沙眼衣原体致病过程中发挥重要的作用,尤其是新近发现沙眼衣原体的唯一一种分泌到胞浆中的分泌性蛋白pgp3和对毒力相关基因具有转录调节功能的pgp4。对就沙衣原体的质粒蛋白研究现状进行了综述。     

Antibody-neutralizing activity against all urogenital Chlamydia trachomatis serovars in Chlamydia suis-infected pigs

It is known that neutralizing species-specific or serovar-specific antibodies are produced in response to chlamydial infection in humans and in some animal species. In a previous study, a strong in vitro neutralizing activity to Chlamydia suis in 80% of sera from C. suis-infected pigs had been observed. In view of the close relationship between C. suis and Chlamydia trachomatis, in the present study, the neutralizing activity against D-K C. trachomatis and C. suis purified elementary bodies (EBs) in sera collected from C. trachomatis-infected patients and C. suis-infected pigs was evaluated. A neutralizing activity of 50-70% was observed in the human sera against the homologous serovar and one to five heterologous C. trachomatis serovars. These sera were also able to neutralize C. suis EBs. The pig sera showed a strong neutralizing activity (70-100%) against C. suis EBs and all eight urogenital C. trachomatis serovars. These results suggested the presence of common immunogenic antigens in C. trachomatis and C. suis. Immunoblot analysis, performed to elucidate the target of this neutralizing activity, showed a clear reactivity in human and pig sera against two proteins of 150 and 40 kDa MW, when tested either with C. trachomatis or with C. suis EBs.     

Inactivation of Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae by ozone

AIMS: To clarify the inhibitory effects of ozone on Chlamydia trachomatis and C. pneumoniae. METHODS AND RESULTS: Cell culture was performed using HeLa229 cells for C. trachomatis, and Human Line cells for C. pneumoniae. C. trachomatis strain D/UW-3/Cx and C. pneumoniae strain AR-39 were used. Ozone water was generated by an ozone water dispenser and diluted to desired concentration just before each experiment. Preinoculation minimum cidal concentration (MCC) and postinoculation MCC methods were employed. In preinoculation MCC, chlamydial strains were treated with serially diluted ozone water followed by inoculation to cells. In postinoculation method, chlamydial strains were inoculated to cells and incubated for 24 h. Then infected cells were treated with ozone water, followed by additional incubation for 48 h. Complete inactivation was obtained in preinoculation MCC method at 0.5 ppm of ozone water for 30 s, or 4 ppm for 5 s. CONCLUSION: Ozone at a concentration of 4 ppm was enough for immediate inactivation of both C. trachomatis and C. pneumoniae. SIGNIFICANCE AND IMPACT OF THE STUDY: Ozone water at 4 ppm should be applicable for prevention of C. trachomatis urogenital infections.     

Chlamydia trachomatis infection in rural Nova Scotia.

OBJECTIVE: To examine the demographic characteristics of patients who underwent testing for Chlamydia trachomatis and to determine the clinical and behavioural characteristics and the types of treatment for those who had positive test results. DESIGN: Case series. SETTING: Rural county in Nova Scotia. PATIENTS: All residents of the county for whom testing for C. trachomatis was ordered at the regional hospital from Sept. 1, 1990, to Mar. 31, 1991. MAIN OUTCOME MEASURES: Rates of testing and of positive test results by age and sex. Comparison of patient and physician characteristics in relation to testing rates. RESULTS: Of the 1116 patients tested 58 (5.2%) had positive test results. Females accounted for 82.8% of those with positive results whose sex could be determined. Among the females the mean age of those with a positive result was 22.3 years, as compared with 27.5 years for those with a negative result (p < 0.0001). Females 15 to 19 years of age were less likely to have a test performed than women 20 to 29 years and were more likely to have a positive test result than the women in the older groups. Almost 9% of the testing among the females was in those over 39 years of age, although no infection was seen in this age group. The number of tests ordered per general or family practitioner varied from 1 to 154; the physicians'' sex, practice location and length of time in practice did not predict the rates of positive test results. Treatment was most often in keeping with that recommended by national guidelines. Four (8.5%) of the 47 patients with positive results who were interviewed were not aware of their diagnosis, either because they had not returned for follow-up or had not being notified by the physician''s office. CONCLUSIONS: The frequency of testing for C. trachomatis infection may be less than is desirable among young patients, who, if tested, are more likely than older patients to have positive results. More understanding of the diagnostic approach taken by physicians is needed.