Organization of the ribosomal RNA genes in Treponema phagedenis and Treponema pallidum.

The genomic DNA fragment which contains ribosomal RNA (rRNA) genes for Treponema phagedenis was cloned into bacteriophage vector lambda EMBL3. A restriction map of the fragment was constructed and the organization of the rRNA genes was determined. The fragment contained at least one copy of the 16S, 23S and 5S sequences and the genes are arranged in the order 16S-23S-5S. Southern hybridization using radiolabeled rRNA gene probes to genomic DNA from T. phagedenis strain Reiter and T. pallidum strain Nichols showed that these organisms have two radioactive fragments which hybridize to the probes in their genome. These results suggest that both pathogenic and non-pathogenic strains of Treponema may carry at least two sets of rRNA genes on their chromosomes.     

Molecular evolution of the tprC, D, I, K, G, and J genes in the pathogenic genus Treponema

We investigated the evolution of 6 genes from the Treponema pallidum repeat (tpr) gene family, which encode potential virulence factors and are assumed to have evolved through gene duplication and gene conversion events. The 6 loci (tprC, D, G, J, I, and K) were sequenced and analyzed in several members of the genus Treponema, including the 3 subspecies of human T. pallidum (T. pallidum subsp. pallidum, pertenue, and endemicum), Treponema paraluiscuniculi (rabbit syphilis), and the unclassified Fribourg-Blanc (simian) isolate. Phylogenetic methods, recombination analysis, and measures of nucleotide diversity were used to investigate the evolutionary history of the tpr genes. Numerous instances of gene conversion were detected by all 3 methods including both homogenizing gene conversion that involved the entire length of the sequence as well as site-specific conversions that affected smaller regions. We determined the relative age and directionality of the gene conversion events whenever possible. Our data are also relevant to a discussion of the evolution of the treponemes themselves. Higher levels of variation exist between the human subspecies than within them, supporting the classification of the human treponemes into 3 subspecies. In contrast to published theories, the divergence and diversity of T. pallidum subsp. pertenue relative to the other subspecies does not support a much older origin of yaws at the emergence of modern human, nor is the level of divergence seen in T. pallidum subsp. pallidum consistent with a very recent (< 500 years) origin of this subspecies. In general, our results demonstrate that intragenomic recombination has played a significant role in the evolution of the studied tpr genes and emphasize that efforts to infer evolutionary history of the treponemes can be complicated if past recombination events are not recognized.     

Comparative investigation of the genomic regions involved in antigenic variation of the TprK antigen among treponemal species, subspecies, and strains

Although the three Treponema pallidum subspecies (T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, and T. pallidum subsp. endemicum), Treponema paraluiscuniculi, and the unclassified Fribourg-Blanc treponeme cause clinically distinct diseases, these pathogens are genetically and antigenically highly related and are able to cause persistent infection. Recent evidence suggests that the putative surface-exposed variable antigen TprK plays an important role in both treponemal immune evasion and persistence. tprK heterogeneity is generated by nonreciprocal gene conversion between the tprK expression site and donor sites. Although each of the above-mentioned species and subspecies has a functional tprK antigenic variation system, it is still unclear why the level of expression and the rate at which tprK diversifies during infection can differ significantly among isolates. To identify genomic differences that might affect the generation and expression of TprK variants among these pathogens, we performed comparative sequence analysis of the donor sites, as well as the tprK expression sites, among eight T. pallidum subsp. pallidum isolates (Nichols Gen, Nichols Sea, Chicago, Sea81-4, Dal-1, Street14, UW104, and UW126), three T. pallidum subsp. pertenue isolates (Gauthier, CDC2, and Samoa D), one T. pallidum subsp. endemicum isolate (Iraq B), the unclassified Fribourg-Blanc isolate, and the Cuniculi A strain of T. paraluiscuniculi. Synteny and sequence conservation, as well as deletions and insertions, were found in the regions harboring the donor sites. These data suggest that the tprK recombination system is harbored within dynamic genomic regions and that genomic differences might be an important key to explain discrepancies in generation and expression of tprK variants among these Treponema isolates.     

Genetic relationship between Treponema pallidum and Treponema pertenue, two noncultivable human pathogens.

Genetic relationships among two strains of Treponema pallidum (Nichols and KKJ) and a strain of T. pertenue were determined by measuring the degree of deoxyribonucleic acid sequence homology. The results in indicated that these three virulent, noncultivable treponemes were genetically indistinguishable. Like T. pallidum (Nichols), T. pertenue (Gauthier) had no detectable deoxyribonucleic acid sequence homology with T. phagedenis (biotype Reiter), T. refringens (biotype Noguchi), or with salmon sperm.     

Multiple alleles of Treponema pallidum repeat gene D in Treponema pallidum isolates

Two new tprD alleles have been identified in Treponema pallidum: tprD2 is found in 7 of 12 T. pallidum subsp. pallidum isolates and 7 of 8 non-pallidum isolates, and tprD3 is found in one T. pallidum subsp. pertenue isolate. Antibodies against TprD2 are found in persons with syphilis, demonstrating that tprD2 is expressed during infection.     

Cultivation of pathogenic treponema in tissue cultures of SflEp cells

Recently, the successful in vitro cultivation of the Nichols strain of Treponema pallidum was achieved. Afterward, attempts were made to cultivate three other strains of T. pallidum and two strains of T. pertenue. The cultivation of the KKJ, Mexico A, and Bosnia A strains of T. pallidum was somewhat successful; the average increases were 10.8, 9.1, and 7.5-fold, respectively. The range of growth for each of these strains varied dramatically from experiment to experiment. The KKJ strain varied from 14.4 to 8.0-fold; the Mexico A strain from 12.8 to 5.4-fold; and the Bosnia A strain from 11.3 to 3.6-fold. However, the attempts to cultivate the Gauthier and the FB strains of T. pertenue were unsuccessful. The average increases were 1.7 and 1.9-fold, respectively. Although the maximum growth observed was about threefold with either of these strains of T. pertenue, over 50% of the treponemes remained motile for 10 d. These results suggest that although each of these strains of T. pallidum and T. pertenue has been shown to be genetically identical, they are very diverse biologically even among strains of the same species.     

The sequence of the acidic repeat protein (arp) gene differentiates venereal from nonvenereal Treponema pallidum subspecies, and the gene has evolved under strong positive selection in the subspecies that causes syphilis

Despite the completion of the Treponema pallidum genome project, only minor genetic differences have been found between the subspecies that cause venereal syphilis (ssp. pallidum) and the nonvenereal diseases yaws (ssp. pertenue) and bejel (ssp. endemicum). In this paper, we describe sequence variation in the arp gene which allows straightforward differentiation of ssp. pallidum from the nonvenereal subspecies. We also present evidence that this region is subject to positive selection in ssp. pallidum, consistent with pressure from the immune system. Finally, the presence of multiple, but distinct, repeat motifs in both ssp. pallidum and Treponema paraluiscuniculi (the pathogen responsible for rabbit syphilis) suggests that a diverse repertoire of repeat motifs is associated with sexual transmission. This study suggests that variations in the number and sequence of repeat motifs in the arp gene have clinical, epidemiological, and evolutionary significance.     

The endemic treponematoses

Treponemal diseases comprise venereal syphilis (Treponema pallidum subsp. pallidum) and the endemic (non-venereal) treponematoses, i.e. yaws (T. pallidum subsp. pertenue), endemic syphilis (T. pallidum subsp. endemicum) and pinta (T. carateum). Treponemal diseases are distinguished on the basis of epidemiological characteristics and clinical manifestations. They are at present indistinguishable by morphological, immunological or serological methods. Several minor genetic differences have been identified among the subspecies. The endemic treponematoses have not yet been eliminated and are currently thought to affect at least 2.5 million persons. Renewed action towards the elimination of these diseases should be undertaken.     

Complete genome sequence of Treponema paraluiscuniculi, strain Cuniculi A: the loss of infectivity to humans is associated with genome decay

Treponema paraluiscuniculi is the causative agent of rabbit venereal spirochetosis. It is not infectious to humans, although its genome structure is very closely related to other pathogenic Treponema species including Treponema pallidum subspecies pallidum, the etiological agent of syphilis. In this study, the genome sequence of Treponema paraluiscuniculi, strain Cuniculi A, was determined by a combination of several high-throughput sequencing strategies. Whereas the overall size (1,133,390 bp), arrangement, and gene content of the Cuniculi A genome closely resembled those of the T. pallidum genome, the T. paraluiscuniculi genome contained a markedly higher number of pseudogenes and gene fragments (51). In addition to pseudogenes, 33 divergent genes were also found in the T. paraluiscuniculi genome. A set of 32 (out of 84) affected genes encoded proteins of known or predicted function in the Nichols genome. These proteins included virulence factors, gene regulators and components of DNA repair and recombination. The majority (52 or 61.9%) of the Cuniculi A pseudogenes and divergent genes were of unknown function. Our results indicate that T. paraluiscuniculi has evolved from a T. pallidum-like ancestor and adapted to a specialized host-associated niche (rabbits) during loss of infectivity to humans. The genes that are inactivated or altered in T. paraluiscuniculi are candidates for virulence factors important in the infectivity and pathogenesis of T. pallidum subspecies.     

Identification of Escherichia coli through analysis of 16S rRNA and 16S-23S rRNA internal transcribed spacer region sequences

A bacterial strain, designated BzDS03 was isolated from water sample, collected from Dal Lake Srinagar. The strain was characterized by using 16S ribosomal RNA gene and 16S-23S rRNA internal transcribed spacer region sequences. Phylogenetic analysis showed that 16S rRNA sequence of the isolate formed a monophyletic clade with genera Escherichia. The closest phylogenetic relative was Escherichia coli with 99% 16S rRNA gene sequence similarity. The result of Ribosomal database project's classifier tool revealed that the strain BzDS03 belongs to genera Escherichia.16S rRNA sequence of isolate was deposited in GenBank with accession number FJ961336. Further analysis of 16S-23S rRNA sequence of isolate confirms that the identified strain BzDS03 be assigned as the type strain of Escherichia coli with 98% 16S-23S rRNA sequence similarity. The GenBank accession number allotted for 16S-23S rRNA intergenic spacer sequence of isolate is FJ961337.     

Molecular characterization of DNA encoding 16S-23S rRNA intergenic spacer regions and 16S rRNA of pectolytic Erwinia species

Sequences of 16S rDNAs and the intergenic spacer (IGS) regions between the 16S and 23S rDNA of bacterial strains from genus Erwinia were determined. Comparison of 16S rDNA sequences from different species and subspecies clearly revealed intraspecies-subspecies homology and interspecies heterogeneity. Phylogenetic analyses of 16S rDNA sequence data revealed that Erwinia spp. formed a discrete monophyletic clade with moderate to high bootstrap values. PCR amplification of the 16S-23S rDNA regions using primers complementary to the 3' end of 16S and 5' end of 23S rRNA genes generated two DNA fragments. The small 16S-23S rDNA IGS regions of Erwinia spp. examined in this study varied considerably in size and nucleotide sequence. Multiple sequence alignment and phylogenetic analysis of small IGS sequence data showed a consistent relationship among the test strains that was roughly in agreement with the 16S rDNA data that reflected the accepted species and subspecies structure of the taxon. Sequence data derived from the large IGS resolved the strains into coherent groups; however, the sequence information would not allow any phylogenetic conclusion, because it failed to reflect the accepted species structure of the test strains.     

Genome analysis of Treponema pallidum subsp. pallidum and subsp. pertenue strains: most of the genetic differences are localized in six regions

The genomes of eight treponemes including T. p. pallidum strains (Nichols, SS14, DAL-1 and Mexico A), T. p. pertenue strains (Samoa D, CDC-2 and Gauthier), and the Fribourg-Blanc isolate, were amplified in 133 overlapping amplicons, and the restriction patterns of these fragments were compared. The approximate sizes of the genomes investigated based on this whole genome fingerprinting (WGF) analysis ranged from 1139.3-1140.4 kb, with the estimated genome sequence identity of 99.57-99.98% in the homologous genome regions. Restriction target site analysis, detecting the presence of 1773 individual restriction sites found in the reference Nichols genome, revealed a high genome structure similarity of all strains. The unclassified simian Fribourg-Blanc isolate was more closely related to T. p. pertenue than to T. p. pallidum strains. Most of the genetic differences between T. p. pallidum and T. p. pertenue strains were accumulated in six genomic regions. These genome differences likely contribute to the observed differences in pathogenicity between T. p. pallidum and T. p. pertenue strains. These regions of sequence divergence could be used for the molecular detection and discrimination of syphilis and yaws strains.     

Acholeplasma laidlawii has tRNA genes in the 16S-23S spacer of the rRNA operon.

We amplified the 16S-23S rRNA intergenic spacer region of Acholeplasma laidlawii PG8 by polymerase chain reaction (PCR) and obtained two specific PCR products in different sizes. We have sequenced both PCR products and found that one of them has sequence homologous to the spacer tRNA genes in Bacillus subtilis. This is the first evidence of tRNA genes between the 16S-23S rRNA intergenic spacer regions in members of the class Mollicutes.     

16S-23S rDNA intergenic spacer region polymorphism of Lactococcus garvieae,Lactococcus raffinolactis and Lactococcus lactis as revealed by PCR and nucleotide sequence analysis

The intergenic spacer region (ISR) between the 16S and 23S rRNA genes was tested as a tool for differentiating lactococci commonly isolated in a dairy environment. 17 reference strains, representing 11 different species belonging to the genera Lactococcus, Streptococcus, Lactobacillus, Enterococcus and Leuconostoc, and 127 wild streptococcal strains isolated during the whole fermentation process of "Fior di Latte" cheese were analyzed. After 16S-23S rDNA ISR amplification by PCR, species or genus-specific patterns were obtained for most of the reference strains tested. Moreover, results obtained after nucleotide analysis show that the 16S-23S rDNA ISR sequences vary greatly, in size and sequence, among Lactococcus garvieae, Lactococcus raffinolactis, Lactococcus lactis as well as other streptococci from dairy environments. Because of the high degree of inter-specific polymorphism observed, 16S-23S rDNA ISR can be considered a good potential target for selecting species-specific molecular assays, such as PCR primer or probes, for a rapid and extremely reliable differentiation of dairy lactococcal isolates.     

Geobacillus thermodenitrificans subsp. calidus, subsp. nov., a thermophilic and α-glucosidase producing bacterium isolated from Kizilcahamam, Turkey

An α-glucosidase producing, thermophilic, facultatively anaerobic, and endospore-forming, motile, rod-shaped bacterial strain F84b(T) was isolated from a high temperature well-pipeline sediment sample in Kizilcahamam, Turkey. The growth occurred at temperatures, pH and salinities ranging from 45 to 69oC (optimum 60oC), 7.0 to 8.5 (optimum 8.0) and 0 to 5% (w/v) (optimum 3.5%), respectively. Strain F84b(T) was able to grow on a wide range of carbon sources. Starch and tyrosine utilization, amylase, catalase and oxidase activities, nitrate reduction, and gas production from nitrate were all positive. The G+C content of the genomic DNA was 49.6 mol%. The menaquinone content was MK-7. The dominant cellular fatty acids were iso-C17:0, iso-C15:0, and C16:0. In phylogenetic analysis of 16S rRNA gene sequence, strain F84b(T) showed high sequence similarity to Geobacillus thermodenitrificans (99.8%) and to Geobacillus subterraneus (99.3%) with DNA hybridization values of 74.3% and 29.1%, respectively. In addition, the Rep-PCR and the intergenic 16S-23S rRNA gene fingerprinting profiles differentiated strain F84b(T) from the Geobacillus species studied. The results obtained from the physiological and biochemical characters, the menaquinone contents, the borderline DNA-DNA hybridization homology, and the genomic fingerprinting patterns had allowed phenotypic, chemotaxonomic and genotypic differentiation of strain F84b(T) from G. thermodenitrificans. Therefore, strain F84b(T) is assigned to be a new subspecies of G. thermodenitrificans, for which the name Geobacillus thermodenitrificans subsp. calidus, subsp. nov. is proposed (The type strain F84b(T) = DSM 22629(T) = NCIMB 14582(T)).     

Rapid identification,differentiation, and proposed new taxonomic classification of Bifidobacterium lactis

Identification of Bifidobacterium lactis and Bifidobacterium animalis is problematic because of phenotypic and genetic homogeneities and has raised the question of whether they belong to one unique taxon. Analysis of the 16S-23S internally transcribed spacer region of B. lactis DSM10140(T), B. animalis ATCC 25527(T), and six potential B. lactis strains suggested two distinct clusters. Two specific 16S-23S spacer rRNA gene-targeted primers have been developed for specific detection of B. animalis. All of the molecular techniques used (B. lactis or B. animalis PCR primers, enterobacterial repetitive intergenic consensus PCR) demonstrated that B. lactis and B. animalis form two main groups and suggest a revision of the strains assigned to B. animalis. We propose that B. lactis should be separated from B. animalis at the subspecies level.     

2株创伤弧菌的16S-23S rDNA间区的克隆、测序及分析

根据细菌的16SrDNA3’端和23SrDNA5’端的高度保守区设计引物,PCR扩增了2株创伤弧菌（Vibrio vulnificus）的16S-23SrDNA间区（Intergenic spacer,IGS）,克隆到pGEM-T载体上,测序。用BLAST和DNA star软件对16S-23SrDNA间区序列及其内的tRNA基因进行比较分析。结果表明,2株创伤弧菌共测出9条16S-23SrDNA间区序列,其中ZSU006测出5条,间区类型分别为：IGS^GLAV、IGS^GLV、IGS^LA、IGS^A和IGS^G.其中IGS^GLAv最大,包含tRNA^Glu、tRNA^Lys、tRNA^Ala。和tRNA^Val基因;IGS^GLV包含tRNA^Glu、tRNA^Lys。和tRNA^Val基因;IGS^LA,则包含tRNA^Ile和tRNA^Ala基因;IGS^G包含tRNA^Glu基因;而IGS^A仅包含tRNA^Ala基因。菌株CG021测出的16S-23SrDNA IGS序列有4条,除缺少IGS^A外,其余的IGS类型均与ZSU006的相同。与GenBank内的创伤弧菌ATCC27562的IGS序列比较,发现创伤弧菌所有类型的IGS的tRNA基因两端的非编码区具有较高的种内同源性。16S-23SrDNA间区结构的差异为建立一种新的创伤弧菌检测方法奠定了基础。     

Inter- and intraspecies variations of the 16S-23S rDNA intergenic spacer region of various streptococcal species

The 16S-23S rDNA intergenic spacer regions (ISR) of different streptococcal species and subspecies were amplified with primers derived from the highly conserved flanking regions of the 16S rRNA and 23S rRNA genes. The single sized amplicons showed a uniform pattern for S. agalactiae, S. dysgalactiae subsp. dysgalactiae (serogroup C), S. dysgalactiae subsp. equisimilis (serogroup G), S. dysgalactiae subsp. dysgalactiae (serogroup L), S. canis, S. phocae, S. uberis, S. parauberis, S. pyogenes and S. equi subsp. equi, respectively. The amplicons of S. equi subsp. zooepidemicus, S. porcinus and S. suis appeared with 3, 5 and 3 different sizes, respectively. ISR of selected strains of each species or subspecies investigated were sequenced and multiple aligned. This allowed a separation of ISR into regions, with 7 regions for S. agalactiae, S. dysgalactiae subsp. dysgalactiae (serogroup C), S. dysgalactiae subsp. equisimilis (serogroup G), S. dysgalactiae subsp. dysgalactiae (serogroup L), S. canis, S. phocae, S. pyogenes and S. suis, 8 regions for S. uberis and S. parauberis and mostly 9 regions for S. equi subsp. equi, S. equi subsp. zooepidemicus and S. porcinus. Region 4, encoding the transfer RNA for alanine (tRNA(Ala)), was present and identical for all isolates investigated. The size and sequence of ISR appears to be a unique marker for streptococci of various species and subspecies and could be used for bacterial identification. In addition the size and sequence variations of ISR of S. equi subsp. zooepidemicus, S. porcinus and S. suis allows a molecular typing of isolates of these species possibly useful in epidemiological aspects.