groESL Genes Research Articles

Prevalence and molecular characterization of Anaplasmataceae agents in free-ranging Brazilian marsh deer (Blastocerus dichotomus)

Anaplasmataceae organisms comprise a group of obligate intracellular gram-negative, tick-borne bacteria that can infect both animals and humans. In the present work we investigate the presence of Ehrlichia, Anaplasma, and Neorickettsia species in blood samples from Brazilian marsh deer (Blastocerus dichotomus), using both molecular and serologic techniques. Blood was collected from 143 deer captured along floodplains of the Paraná River, near the Porto Primavera hydroelectric power plant. Before and after flooding, marsh deer were captured for a wide range research program under the financial support of São Paulo State Energy Company (CESP), between 1998 and 2001. Samples were divided into four groups according to time and location of capture and named MS01 (n=99), MS02 (n=18) (Mato Grosso do Sul, before and after flooding, respectively), PX (n=9; Peixe River, after flooding), and AGUA (n=17; Aguapeí River, after flooding). The seroprevalences for Ehrlichia chaffeensis and Anaplasma phagocytophilum were 76.76% and 20.2% in MS01, 88.88% and 5.55% in MS02, 88.88% and 22.22% in PX, and 94.12% and 5.88% in AGUA, respectively. Sixty-one animals (42.65% of the total population) were PCR-positive for E. chaffeensis PCR (100.0% identity based on 16S rRNA, dsb, and groESL genes). Seventy deer (48.95% of the total population) were PCR-positive for Anaplasma spp. (99.0% of identity with A. platys, and in the same clade as A. phagocytophilum, A. bovis, and A. platys based on 16S rRNA phylogenetic analysis). Our results demonstrate that Brazilian marsh deer are exposed to E. chaffeensis and Anaplasma spp. and may act as reservoirs for these rickettsial agents, playing a role in disease transmission to humans and other animals.

DNA array with the groESL intergenic sequence to detect Vibrio parahaemolyticus and Vibrio vulnificus

The untranscribed DNA sequences of the intergenic spacer regions (ISRs) in the groESL gene of 23 Vibrio species were determined and compared. ISR sequence length (41–85bp) was variable. Vibrio species could be divided into three groups according to the length and homology of their ISR sequences. DNA array hybridization using ISR-specific probes accurately distinguished Vibrio parahaemolyticus and Vibrio vulnificus from other species.

Isolation of the groESL cluster from Vibrio anguillarum and PCR detection targeting groEL gene

Vibrio anguillarum is a major pathogenic bacterium that causes vibriosis and septicemia in fish and shellfish. In this study, we identified the groESL genes, which encode bacterial chaperonins, from V. anguillarum. The groE gene cluster consisted of a 291-bp groES gene, a 69-bp intergenic spacer region, and a 1,635-bp groEL gene order. Sequence analysis with the groESL gene of Vibrio species exhibited that the groEL gene was more species-specific and suitable than the groES gene for V. anguillarum detection. Owing to the difficulty in distinguishing V. anguillarum from the closely related V. ordalii, we compared the sequences of groEL from V. anguillarum and the groEL homolog hsp60 from V. ordalii, in order to design a primer set based on a region dissimilar between the two. PCR with the groEL primer set produced a clear 195-bp amplicon in six serotypes of V. anguillarum, whereas 23 Vibrio species of 39 samples, including V. ordalii, and 10 species of enteric bacteria gave no bands. PCR using the groEL primers also amplified a unique product from V. anguillarum-infected flounder and oyster tissues. These results demonstrate that the groEL-target PCR assay is a sensitive and species-specific tool for the detection of V. anguillarum infection.

Enterococcus species composition determined by capillary electrophoresis of the groESL gene spacer region DNA

Marine recreational beaches are monitored for fecal contamination by Enterococcus spp. (ENT) counts. Although different ENT species in the environment tend to thrive in and originate from distinct hosts, the current monitoring method does not differentiate among species. Time-consuming isolation-based species identification precludes routine analysis of environmental ENT communities. Therefore, an isolation-independent DNA fingerprinting method was developed to characterize environmental ENT communities using DNA length polymorphism of the spacer region between the groES and groEL genes common to most ENT species. Capillary electrophoresis resulted in distinct peak sizes of PCR products that carried polymorphic groESL spacers (300–335 bp in length) among 8 different ENT species ( Enterococcus avium, Enterococcus gallinarum, Enterococcus casseliflavus, Enterococcus mundtii, Enterococcus hirae, Enterococcus faecium, Enterococcus durans, and Enterococcus faecalis). Distortions in true species ratios observed in electropherograms were caused by PCR biases arising in a mixed ENT community DNA template. E. faecalis was overestimated and E. avium and E. faecium were underestimated compared to the original species ratios in the mixed community. The PCR product bias was constant between species, so good approximation of the species ratio in ENT communities is possible. In environmental samples, a high percentage of E. faecalis (96%) together with high total ENT counts were observed in samples collected from a sewer line and from several sites in a storm drain system where sewage leaks were suspected. In contrast, samples with <400 CFU 100 ml −1 ENT were either dominated by E. mundtii or had 4 or more ENT species. The latter ENT community profiles are considered to be signatures of enterococci rarely associated with animals with low or of non-fecal origin.

Bacteremia caused by non- faecalis and non- faecium enterococcus species at a Medical center in Taiwan, 2000 to 2008

Human infections due to non-faecalis and non-faecium Enterococcus species are emerging but data on the characteristics of these infections are limited. We retrospectively reviewed the computerized database of the bacteriology laboratory at National Taiwan University Hospital from January 2000 through December 2008 to identify patients with non-faecalis and non-faecium enterococcal bacteremia. Enterococcal bacteremia was diagnosed in 1887 patients during the study period and was caused by non-faecalis and non-faecium enterococci in 182 (9.6%) of these patients. The causative organisms included Enterococcus casseliflavus (n = 59, 3.1%), Enterococcus gallinarum (n = 58, 3.0%), Enterococcus avium (n = 45, 2.4%), Enterococcus hirae (n = 9, 0.5%), Enterococcus raffinosus (n = 9, 0.5%), Enterococcus durans (n = 2, 0.1%), Enterococcus cecorum (n = 2, 0.1%), and Enterococcus canintestini (n = 1, 0.5%). A commercially-available phenotypic identification system misidentified six isolates based upon sequence analysis of 16S and groESL genes. Among the 182 patients, 74 (40.7%) had catheter-associated bloodstream infection and 69 (37.9%) presented with biliary tract infection. Healthcare-associated enterococcal bacteremia comprised 99 (54.4%) episodes and a polymicrobial etiology was found in 106 (58.2%) episodes. The clinical manifestations varied between the infecting Enterococcus species. Multivariate logistic regression showed that immunocompromised status is the only risk factor for the all cause mortality. Non-faecalis and non-faecium Enterococcus species can cause protean manifestations which vary with the infecting Enterococcus species. Misidentification of unusual enterococcal species might occur by the commercial identification methods and accurate identification with molecular methods is required.

Diversity of groESL sequences of Anaplasma phagocytophilum among dogs in Slovenia

Anaplasma phagocytophilum, the agent of granulocytic anaplasmosis, has been detected in different hosts (humans, dogs, horses, deer, cattle, sheep, etc.) and ixodid tick vectors [1]. Human anaplasmosis is a mild self-limiting disease [2]. Fatal human cases due to infection with A. phagocytophilum have not been reported in Europe. In contrast, canine granulocytic anaplasmosis may be a more serious disease, taking the form of a chronic infection, and fatal cases have also been described [3]. A vast number of reports describe the genetic diversity of A. phagocytophilum in animals, but only a few refer to domestic dogs. Differences in the nucleotide sequences of the 16S rRNA gene of A. phagocytophilum from dogs in western Washington State were recently described [4]. As the 16S rRNA gene is a conserved gene, the operon groESL might be a better candidate with which to determine the genetic variability. This gene analysis has already been used to delineate genetic variants of A. phagocytophilum among roe and red deer in Slovenia [5]. The focus of the present study is to examine the genetic variability of the groESL gene of A. phagocytophilum in domestic dogs in Slovenia. From 2000 to 2007, 2577 blood samples from dogs with clinical signs of canine anaplasmosis from different locations in Slovenia were tested for the presence of anaplasmal DNA. For the initial screening of samples, amplification of a part of the 16S rRNA of A. phagocytophilum was used [5]. Positive samples were additionally tested with a nested PCR targeting a 1256-bp segment of the groESL operon [5]. To determine the genetic variation of A. phagocytophilum, all amplicons of the groESL operon were further sequenced on both strands and analysed by using TREECON software. A phylogenetic tree was constructed with the neighbour-joining method. Support for the tree nodes was calculated with 1000 bootstrap replicates. Of 2577 samples tested, 103 (4.0%) were positive for the presence of A. phagocytophilum 16S rRNA. The GroESL gene was subsequently amplified from all positive samples. The homology search and the alignment of groESL sequences from 103 dogs revealed two clusters of sequences differing in three nucleotides (99.8% identity). However, translated amino acid sequences were identical, thus indicating silent mutations. One cluster of groESL sequences from dogs (accession no. EU381150) representing 58 of 103 positive dogs (56.3%) showed 100% identity with A. phagocytophilum from a human patient (accession no. AF033101) and from an Ixodes ricinus tick (accession no. EU246961) from Slovenia. Another group of sequences (accession no. EU381151) representing 45 of 103 dogs (43.7%) clustered together with a variant found in German and Slovenian I. ricinus ticks (accession no. AY281847 and EU381152), respectively (Fig. 1). Human and canine anaplasmoses are present in Slovenia. Whereas human cases are rare and present mostly as a mild disease [2], infection with A. phagocytophilum in dogs is a serious disease that is more frequently observed. Interestingly, human A. phagocytophilum shows no genetic variability in groESL sequences, but in this study, two variants of A. phagocytophilum were identified in dogs from Slovenia by PCR and sequence analysis of a part of the groESL operon. A more prevalent variant (accession no. EU381150) demonstrated 100% identity with a variant detected in human cases of anaplasmosis in Slovenia. Given the same genetic variant and different clinical disease in humans and dogs, this could implicate the involvement of specific host factors in granulocytic anaplasmosis. Stuen et al. [6] speculated that certain strains of A. phagocytophilum might induce different severities of the Corresponding author and reprint requests to: T. Avsic Zupanc, Institute of Microbiology and Immunology, Faculty of Medicine, Zaloska 4, SI – 1000 Ljubljana, Slovenia E-mail: tatjana.avsic@mf.uni-lj.si

Detection of Anaplasma phagocytophilum in wild boar in Slovenia

Human granulocytic anaplasmosis (HGA) comprises a group of emerging tick-borne infectious diseases and it is caused by the intracellular bacteria, Anaplasma phagocytophilum. Infections with Anaplasma spp. have been described in humans and animals. Many studies have been performed to elucidate the natural cycle of A. phagocytophilum, but a competent reservoir host has not been clearly established in Europe [1]. Roe and red deer represent a very important host for the adult tick I. ricinus, a vector of A. phagocytophilum, and could therefore also serve as a natural reservoir for A. phagocytophilum in enzootic transmission of this bacterium [1]. Two genetic lineages of groESL operon were described among isolates of A. phagocytophilum from deer in Slovenia. However, both genetic lineages differed from a single variant, found in all Slovenian HGA patients [2]. PCR screening of different wild animals from Austria and the Czech Republic showed that wild boar (Sus scrofa) were infected with A. phagocytophilum [3]. Nucleotide sequences of groESL operon of A. phagocytophilum obtained from these infected animals were identical to the sequences from Slovenian patients [3]. Interestingly, a German horse with equine granulocytic ehrlichiosis was also infected with an identical variant of A. phagocytophilum to that detected in Slovenian patients [4]. In the years 2002 and 2007, 248 wild boars (Sus scrofa) from different locations in Slovenia were shot by professional hunters. Blood and spleen samples collected by hunters were analysed for infection with Anaplasma species in our laboratory. DNA was extracted from 135 spleen (year 2002) and 113 blood (year 2007) samples as described previously [1]. For the initial screening of all samples a segment of the 16S rDNA of A. phagocytophilum was used [1]. All positive samples were additionally amplified with a nested PCR targeting groESL operon of variants of A. phagocytophilum [1]. The gene for 16S rDNA is very conservative, therefore only the amplicons of the groESL operon were further analysed by sequencing. To obtain the genetic variation of A. phagocytophilum, all amplicons of groESL operon were further sequenced on both strands and analysed by using TREECON software. A phylogenetic tree was constructed with the neighbour-joining method. Support for the tree nodes was calculated with 1000 bootstrap replicates. Of 135 spleen samples (2002) and 113 blood (2007) samples, six (4.4%) and four (3.5%) tested positive for the presence of Anaplasmae spp. DNA, respectively. The groESL gene was subsequently amplified from all positive samples. The homology search and the alignment of groESL sequences showed 100% identity with A. phagocytophilum from a human patient (acc. no. AF033101) and from the Ixodes ricinus tick (acc. no. EU246961) from Slovenia (Fig. 1). Genetic sequences from wild boar were also 100% identical among each other. Our study confirms that wild boar is naturally infected with A. phagocytophilum. The Anaplasmae spp. DNA was detected in 10 of 248 (4.0%) samples, collected from different locations in Slovenia in two different years. Anaplasma phagocytophilum was previously detected in wild boar samples. Interestingly, the wild boar were hunted in a different geographical area (Czech Republic) but the results were in accordance with our findings [3]. Scandinavian groESL sequences from dogs and horses differ from Slovenian in six nucleotides, but are identical among each other. These differences might suggest geographical variability of A. phagocytophilum and contribute Corresponding author and reprint requests: Tatjana Avsic Županc, Institute of Microbiology and Immunology, Faculty of Medicine, Zaloska 4, SI–1000 Ljubljana, Slovenia E-mail: tatjana.avsic@mf.uni-lj.si

Reptile Infection with Anaplasma phagocytophilum, the Causative Agent of Granulocytic Anaplasmosis

Granulocytic anaplasmosis (GA) is a potentially fatal tick-borne rickettsial disease that occurs sporadically in the far western United States. We evaluated the prevalence of Anaplasma phagocytophilum in multiple species of lizards and snakes from enzootic sites in northern California, described the infestation prevalence of its tick vector Ixodes pacificus on reptiles, and conducted an experimental challenge of western fence lizards (Sceloporus occidentalis) and Pacific gopher snakes (Pituophis catenifer) with A. phagocytophilum delivered via needle inoculation or tick bite. Both serologically and polymerase-chain reaction (PCR)-positive lizards (seroprevalence = 10.8%, PCR prevalence = 10.2%) and snakes (seroprevalence = 5.8%, PCR prevalence = 11.7%) were detected among wild-caught animals. A DNA sequence of the A. phagocytophilum groESL gene from a PCR-positive snake was 100% homologous to that of the human-derived A. phagocytophilum. Experimental attempts to infect naïve animals were unsuccessful for snakes (n = 2), but 1 of 12 lizards became infected for 1 wk only by tick bite. Xenodiagnostic I. pacificus larvae that fed on a PCR-positive lizard did not acquire or transmit rickettsiae. Our findings suggest that lizards and snakes are exposed to A. phagocytophilum by infected ticks, but that they do not serve as primary reservoir hosts of this rickettsia.

PCR-RFLP assay for species and subspecies differentiation of the Streptococcus bovis group based on groESL sequences

The sequence diversity of groESL genes among Streptococcus bovis group isolates was analysed, including five reference strains and 36 clinical isolates. Phylogenetic analysis of the groES and groEL sequences showed that the isolates that belonged to the same species or subspecies usually clustered together. The intergenic spacer region between groES and groEL was variable in size (67-342 bp) and sequence and appeared to be a unique marker for species or subspecies determination. Sequence similarities of the groESL genes among species and subspecies ranged from 84.2 to 99.0 % in groES, and from 88.0 to 99.0 % in groEL. Based on the sequences determined, a Streptococcus bovis group-specific PCR assay was developed, which may provide an alternative means of distinguishing the bovis group from other viridans streptococci. Restriction digestion of the amplicon with AclI further differentiated the species and subspecies.

Evidence of Anaplasma phagocytophilum and Rickettsia helvetica infection in free-ranging ungulates in central Slovakia

The purpose of this study was to investigate the role of wild animals for Anaplasma phagocytophilum, other ehrlichiae/anaplasmae, Rickettsia helvetica and other rickettsiae and whether different genetic variants of A. phagocytophilum in central Slovakia exist. A total of 109 spleen samples from 49 red deer (Cervus elaphus), 30 roe deer (Capreolus capreolus), 28 wild boar (Sus scrofa) and two mouflon (Ovis musimon) were collected from June 2005 to December 2006. Polymerase chain reaction (PCR) amplification of the16S rRNA gene was used for detection of ehrlichiae/anaplasmae. A nested PCR targeting part (392 bp) of groESL gene was applied for the specific detection of A. phagocytophilum. Fragments of the gltA and ompA genes (381 bp and 632 bp, respectively) were amplified to detect rickettsiae, followed by sequencing. A. phagocytophilum and R. helvetica were detected in wild animals. The prevalence of A. phagocytophilum was 50.0 ± 18.2% in roe deer and 53.1 ± 14.1% in red deer. None of the 28 wild boar was PCR positive for ehrlichiae/anaplasmae. A. phagocytophilum was detected in one mouflon. R. helvetica was found in one roe deer. Our study suggests a role of cervids as a natural reservoir of A. phagocytophilum in Slovakia. However, the role of cervids and wild boars in the circulation of R. helvetica remains unknown. The analysis of sequence variation in the msp4 coding region of A. phagocytophilum showed the presence of different variants previously described in ruminants.

Anaplasma platysin Dogs, Chile

We conducted a 16S rRNA nested PCR for the genus Ehrlichia and Ehrlichia spp. with blood samples from 30 ill dogs in Chile. Phylogenetic analysis was performed by using groESL gene amplification. We identified Anaplasma platys as 1 of the etiologic agents of canine ehrlichiosis.

Molecular Survey ofBabesia microti, EhrlichiaSpecies andCandidatusNeoehrlichia mikurensis in Wild Rodents from Shimane Prefecture, Japan

A significant number of patients are diagnosed with "fevers of unknown origin" (FUO) in Shimane Prefecture in Japan where tick-borne diseases are endemic. We conducted molecular surveys for Babesia microti, Ehrlichia species, and Candidatus Neoehrlichia mikurensis in 62 FUO cases and 62 wild rodents from Shimane Prefecture, Japan. PCR using primers specific for the Babesia 18S small-subunit rRNA (rDNA) gene and Anaplasmataceae groESL amplified products from 45% (28/62) and 25.8% (16/62) of captured mice, respectively. Of the 28 18S rDNA PCR positives, 23 and five samples were positive for Hobetsu- and Kobe-type B. microti, respectively. In contrast, of the 16 groESL PCR positives, eight, one and seven samples were positive for Ehrlichia muris, Ehrlichia sp. HF565 and Candidatus N. mikurensis, respectively. Inoculation of selected blood samples into Golden Syrian hamsters indicated the presence of Hobetsu- and Kobe-type B. microti in four and one sample, respectively. Isolation of the latter strain was considered important as previous studies suggested that the distribution of this type was so far confined to Awaji Island in Hyogo Prefecture, where the first case of transfusion-associated human babesiosis originated. DNA samples from 62 FUO human cases tested negative for B. microti 18S rDNA gene, Anaplasmataceae groESL gene, Rickettsia japonica 17K genus-common antigen gene and Orientia tsutsugamushi 56K antigen gene by PCRs. We also conducted seroepidemiological surveys on 62 human sera collected in Shimane Prefecture from the FUO patients who were suspected of carrying tick-borne diseases. However, indirect immunofluorescent antibody tests using B. microti- and E. muris-infected cells detected IgG against E. muris in only a single positive sample. This study demonstrates the presence of several potentially important tick-borne pathogens in Shimane Prefecture and suggests the need for further study on the causative agents of FUOs.

Cloning and Transcriptional Analysis of groES and groEL in Ethanol-producing Bacterium Zymomonas mobilis TISTR 548

Heat and ethanol had an affect not only on growth and cell viability of an obligatorily fermentative Gram-negative bacterium Zymomonas mobilis, but also on protein synthesis. Analysis by SDS-polyacrylamide gel electrophoresis revealed pronounced increasing of two dominant proteins designated as groES and groEL. Molecular cloning of the gene encoding groES and groEL was performed by PCR technique using specific primers synthesized based on the Z. mobilis groESL gene. Sequencing analysis of 2179 bp led to the detection of two open reading frames encoded for 95 and 549 amino acids, respectively. The deduced amino acid sequence of the Z. mobilis groES and groEL shows a high degree of identity with other. The strongly conserved carboxyl-terminus Gly-Gly-Met motif and two small segments, which appear more conserved between ethanol-producing organisms, were found, suggesting that their may be related to stability of protein under heat or ethanol stress. Induction of groES and groEL occurs in response to heat and ethanol, but not to salt stress.

Neorickettsia helminthoecain Dog, Brazil

<i>Neorickettsia helminthoeca</i>in Dog, Brazil

Species identification of mutans streptococci by groESL gene sequence

The near full-length sequences of the groESL genes were determined and analysed among eight reference strains (serotypes a to h) representing five species of mutans group streptococci. The groES sequences from these reference strains revealed that there are two lengths (285 and 288 bp) in the five species. The intergenic spacer between groES and groEL appears to be a unique marker for species, with a variable size (ranging from 111 to 310 bp) and sequence. Phylogenetic analysis of groES and groEL separated the eight serotypes into two major clusters. Strains of serotypes b, c, e and f were highly related and had groES gene sequences of the same length, 288 bp, while strains of serotypes a, d, g and h were also closely related and their groES gene sequence lengths were 285 bp. The groESL sequences in clinical isolates of three serotypes of S. mutans were analysed for intraspecies polymorphism. The results showed that the groESL sequences could provide information for differentiation among species, but were unable to distinguish serotypes of the same species. Based on the determined sequences, a PCR assay was developed that could differentiate members of the mutans streptococci by amplicon size and provide an alternative way for distinguishing mutans streptococci from other viridans streptococci.

The nitrogen-fixing symbiotic bacteriumMesorhizobium lotihas and expresses the gene encoding pyridoxine 4-oxidase involved in the degradation of vitamin B6

The gene product of mll6785 of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099 was identified as pyridoxine 4-oxidase, the first enzyme in the vitamin B6-degradation pathway. The gene was cloned and ligated into pET-21a(+). Escherichia coli BL21(DE3) was co-transformed with the constructed plasmid plus pKY206 containing groESL genes encoding chaperonins. The overexpressed protein was purified to homogeneity by the ammonium sulfate fractionation and three chromatography steps. The enzymatic properties of the purified protein, such as Km values for pyridoxine (213 ± 19 μM) and oxygen (78 ± 10 μM), were compared to those of pyridoxine 4-oxidase from two bacteria with known vitamin B6-degradation pathway. M. loti grown in a Rhizobium medium showed the enzyme activity. The results suggest that M. loti also contains the degradation pathway of vitamin B6.

The nitrogen-fixing symbiotic bacterium Mesorhizobium loti has and expresses the gene encoding pyridoxine 4-oxidase involved in the degradation of vitamin B 6

The gene product of mll6785 of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099 was identified as pyridoxine 4-oxidase, the first enzyme in the vitamin B 6-degradation pathway. The gene was cloned and ligated into pET-21a(+). Escherichia coli BL21(DE3) was co-transformed with the constructed plasmid plus pKY206 containing groESL genes encoding chaperonins. The overexpressed protein was purified to homogeneity by the ammonium sulfate fractionation and three chromatography steps. The enzymatic properties of the purified protein, such as K m values for pyridoxine (213 ± 19 μM) and oxygen (78 ± 10 μM), were compared to those of pyridoxine 4-oxidase from two bacteria with known vitamin B 6-degradation pathway. M. loti grown in a Rhizobium medium showed the enzyme activity. The results suggest that M. loti also contains the degradation pathway of vitamin B 6.

CIRCE is not involved in heat-dependent transcription of groESL but in stabilization of the mRNA 5'-end in Rhodobacter capsulatus.

The CIRCE element, an inverted DNA repeat, is known to be involved in the temperature-dependent regulation of genes for heat shock proteins in a variety of organisms. The CIRCE element was identified as the target for the HrcA protein, which represses transcription of heat shock genes under normal growth temperature. Our data reveal that the CIRCE element is not involved in the temperature-dependent transcription of the groESL genes in Rhodobacter capsulatus. Apparently, R.capsulatus does not harbour an HrcA protein. The mechanisms of heat shock regulation of the groESL genes in R.capsulatus therefore diverge significantly from the regulatory pathway identified in other organisms. A structural analysis of the CIRCE RNA element revealed a stem of 11 nt pairs and a loop of only 5 nt. This folding differs from a structure with a 9 nt loop suggested previously on the basis of computer analysis. The RNA structure leads to a slight stabilization of the groESL mRNA that is more pronounced at normal growth temperature than under heat shock conditions.

Genetic Characterization of a Chlamydophila pneumoniae isolate from an African Frog and Comparison to Currently Accepted Biovars

The amphibian isolate DE177 identified as Chlamydophila (C.) pneumoniae was sequenced in five genomic regions: 16S ribosomal RNA gene, 16-23S intergenic spacer, ompA, ompB, and groESL genes. Comparison with corresponding sequences of the currently accepted equine, human and koala biovars of C. pneumoniae revealed that koala strains represented the most closely related taxon, although sequence dissimilarities in the ompA (VD4) and ompB gene regions were noted. In this respect, the present isolate is distinct from a previously described frog isolate (Berger et al., 1999) whose sequence analysis yielded identity to the koala biovar. As three of the nucleotide substitutions in ompA (VD4) of DE177 will be translated into two altered amino acids the possible existence of another biovar is discussed.

Cloning, sequencing, and functional expression in Escherichia coli of chaperonin (groESL) genes from Vibrio cholerae.

Using a series of oligonucleotides synthesized on the basis of conserved nucleotide motifs in heat-shock genes, the groESL heat-shock operon from a Vibrio cholerae TSI-4 strain has been cloned and sequenced, revealing that the presence of two open reading frames (ORFs) of 291 nucleotides and 1,632 nucleotides separated by 54 nucleotides. The first ORF encoded a polypeptide of 97 amino acids, GroES homologue, and the second ORF encoded a polypeptide of 544 amino acids, GroEL homologue. A comparison of the deduced amino acid sequences revealed that the primary structures of the V. cholerae GroES and GroEL proteins showed significant homology with those of the GroES and GroEL proteins of other bacteria. Complementation experiments were performed using Escherichia coli groE mutants which have the temperature-sensitive growth phenotype. The results showed that the groES and groEL from V. cholerae were expressed in E. coli, and groE mutants harboring V. cholerae groESL genes regained growth ability at high temperature. The evolutionary analysis indicates a closer relationship between V. cholerae chaperonins and those of the Haemophilus and Yersinia species.