DNA relatedness among strains of Leptospira biflexa

The slot blot method of DNA hybridization was used to study 38 strains of Leptospira biflexa belonging to 38 serovars. Fifteen of these serovars were placed into six groups. The remaining 23 serovars were generally too diverse to show significant DNA relatedness either to these groups or to one another. Serovar thracia was related to Group 5, but it was not included in this group because its percent relatedness was too low. We found that genetically related organisms were antigenically dissimilar. The absence of any significant genetic relationship between Leptonema illini and the Leptospira biflexa serovars tested supports the placement of the former species in a separate genus.     

Occurrence of 3-O-Methylmannose in the Polysaccharide of Leptospira biflexa Urawa

3-O-methylmannose was identified by gas-liquid chromatography-mass spectrometry in the acid hydrolysate of the polysaccharide of Leptospira biflexa Urawa.     

Genome sequence of the saprophyte Leptospira biflexa provides insights into the evolution of Leptospira and the pathogenesis of leptospirosis

Leptospira biflexa is a free-living saprophytic spirochete present in aquatic environments. We determined the genome sequence of L. biflexa, making it the first saprophytic Leptospira to be sequenced. The L. biflexa genome has 3,590 protein-coding genes distributed across three circular replicons: the major 3,604 chromosome, a smaller 278-kb replicon that also carries essential genes, and a third 74-kb replicon. Comparative sequence analysis provides evidence that L. biflexa is an excellent model for the study of Leptospira evolution; we conclude that 2052 genes (61%) represent a progenitor genome that existed before divergence of pathogenic and saprophytic Leptospira species. Comparisons of the L. biflexa genome with two pathogenic Leptospira species reveal several major findings. Nearly one-third of the L. biflexa genes are absent in pathogenic Leptospira. We suggest that once incorporated into the L. biflexa genome, laterally transferred DNA undergoes minimal rearrangement due to physical restrictions imposed by high gene density and limited presence of transposable elements. In contrast, the genomes of pathogenic Leptospira species undergo frequent rearrangements, often involving recombination between insertion sequences. Identification of genes common to the two pathogenic species, L. borgpetersenii and L. interrogans, but absent in L. biflexa, is consistent with a role for these genes in pathogenesis. Differences in environmental sensing capacities of L. biflexa, L. borgpetersenii, and L. interrogans suggest a model which postulates that loss of signal transduction functions in L. borgpetersenii has impaired its survival outside a mammalian host, whereas L. interrogans has retained environmental sensory functions that facilitate disease transmission through water.     

Nucleotide sequence analysis of the Leptospira biflexa serovar patoc rpsL and rpsG genes.

The Leptospira biflexa rpsL and rpsG genes were sequenced. Although similar in many respects, proteins encoded by these L. biflexa genes had several unusual features when compared with homologous proteins of other organisms. Unlike the rpsL genes of other eubacteria, the L. biflexa rpsL gene is adjacent to a rpoC-like gene.     

Identification and nucleotide sequence of the Leptospira biflexa serovar patoc trpE and trpG genes.

Leptospira biflexa is a representative of an evolutionarily distinct group of eubacteria. In order to better understand the genetic organization and gene regulatory mechanisms of this species, we have chosen to study the genes required for tryptophan biosynthesis in this bacterium. The nucleotide sequence of the region of the L. biflexa serovar patoc chromosome encoding the trpE and trpG genes has been determined. Four open reading frames (ORFs) were identified in this region, but only three ORFs were translated into proteins when the cloned genes were introduced into Escherichia coli. Analysis of the predicted amino acid sequences of the proteins encoded by the ORFs allowed us to identify the trpE and trpG genes of L. biflexa. Enzyme assays confirmed the identity of these two ORFs. Anthranilate synthase from L. biflexa was found to be subject to feedback inhibition by tryptophan. Codon usage analysis showed that there was a bias in L. biflexa towards the use of codons rich in A and T, as would be expected from its G + C content of 37%. Comparison of the amino acid sequences of the trpE gene product and the trpG gene product with corresponding gene products from other bacteria showed regions of highly conserved sequence.     

Chemical Studies on the Cell Walls of Leptospira biflexa Strain Urawa and Treponema pallidum Strain Reiter

The preparation and chemical poperties of the cell walls of Leptospira biflexa Urawa and Treponema pallidum Reiter are described. Both cell walls are composed mainly of polysaccharides and peptidoglycans. The data of chemical analysis indicate that the cell wall of L. biflexa Urawa contains rhamnose, arabinose, xylose, mannose, galactose, glucose and unidentified sugars as neutral sugars, and alanine, glutamic acid, α,ε-diaminopimelic acid, glucosamine and muramic acid as major amino acids and amino sugars. As major chemical constituents of the cell wall of T. pallidum Reiter, rhamnose, arabinose, xylose, mannose, galactose, glucose, alanine, glutamic acid, ornithine, glycine, glucosamine and muramic acid have been detected. The chemical properties of protein and polysaccharide fractions prepared from the cells of T. pallidum Reiter were also partially examined.     

Evolution of gp85 gene of subgroup J avian leukosis virus under the selective pressure of antibodies

Subgroup J Avian leucosis virus (ALV-J) strain NX0101 was inoculated into chicken embryo fibroblasts (CEF) monolayers in 6-well plates. The six wells of CEF inoculated with NX0101 were divided into groups A (without anti-ALV-J serum in the medium) and B (with anti-ALV-J serum in the medium), then viruses from each well of both groups were separately passed in CEF every 6 d and formed their independent passage lineages. For each lineage of both groups, gp85 genes of the viruses in the 10th, 20th and 30th passages were amplified, cloned and sequenced. The sequence data indicated that the homologies of gp85 at aa level between the primary virus and the passed viruses of different passages of 3 lineages in group A were 97.7%–99.7%; and the homologies of gp85 between the primary virus and the passed viruses of different passages of 3 lineages in group B were 93.8%–96.1%. Analysis of the ratios of nonsynonium (NS) vs synonium (S) mutations of nucleic acids demonstrated that NS/S in 3 highly variable (hr-) regions at aa#110–120, aa#141–151 and aa#189–194 of gp85 in 3 lineages of group A were 2 (8/4), 1(3/3) and 1.3 (4/3), however, NS/S in the same 3 hr-regions of group B were 4.1 (13/3), 4.7 (14/3) and 3.3 (11/3). This study is the first demonstration of influence of immune selective pressure on evolution of ALV-J gp85 by specific antibodies under the controlled in vitro experiments.     

Evolution of gp85 gene of subgroup J avian leukosis virus under the selective pressure of antibodies

Subgroup J Avian leucosis virus (ALV-J) strain NX0101 was inoculated into chicken embryo fibroblasts (CEF) monolayers in 6-well plates. The six wells of CEF inoculated with NX0101 were divided into groups A (without anti-ALV-J serum in the medium) and B (with anti-ALV-J serum in the medium), then viruses from each well of both groups were separately passed in CEF every 6 d and formed their independent passage lineages. For each lineage of both groups, gp85 genes of the viruses in the 10th, 20th and 30th passages were amplified, cloned and sequenced. The sequence data indicated that the homologies of gp85 at aa level between the primary virus and the passed viruses of different passages of 3 lineages in group A were 97.7%-99.7%; and the homologies of gp85 between the primary virus and the passed viruses of different passages of 3 lineages in group B were 93.8%-96.1%. Analysis of the ratios of nonsynonium (NS) vs synonium (S) mutations of nucleic acids demonstrated that NS/S in 3 highly variable (hr-) regions at aa#110-120, aa#141-151 and aa#189-194 of gp85 in 3 lineages of group A were 2 (8/4), 1(3/3) and 1.3 (4/3), however, NS/S in the same 3 hr-regions of group B were 4.1 (13/3), 4.7 (14/3) and 3.3 (11/3). This study is the first demonstration of influence of immune selective pressure on evolution of ALV-J gp85 by specific antibodies under the controlled in vitro experiments.     

A widely conserved bacterial cytoskeletal component influences unique helical shape and motility of the spirochete Leptospira biflexa

Leptospires and other members of the evolutionarily ancient phylum of Spirochaetes are bacteria often characterized by long, highly motile spiral‐ or wave‐shaped cells. Morphology and motility are critical factors in spirochete physiology, contributing to the ability of these bacteria to successfully colonize diverse environments. However, the mechanisms conferring the helical structure of Leptospira spp. have yet to be fully elucidated. We have identified five Leptospira biflexa bactofilin proteins, a recently characterized protein family with cytoskeletal properties. These five bactofilins are conserved in all species of the Leptospiraceae, indicating that these proteins arose early in the evolution of this family. One member of this protein family, LbbD, confers the optimal pitch distance in the helical structure of L. biflexa. Mutants lacking lbbD display a unique compressed helical morphology, a reduced motility and a decreased ability to tolerate cell wall stressors. The change in the helical spacing, combined with the motility and cell wall integrity defects, showcases the intimate relationship and coevolution between shape and motility in these spirochetes.     

Complete nucleotide sequence of the LE1 prophage from the spirochete Leptospira biflexa and characterization of its replication and partition functions

The first and, to date, only extrachromosomal circular replicon identified in the spirochete Leptospira is the LE1 prophage from Leptospira biflexa. The 74-kb LE1 genome has a GC content of 36%, which is similar to the GC content of Leptospira spp. Most of the 79 predicted open reading frames (ORFs) showed no similarities to known ORFs. However 21 ORFs appeared to be organized in clusters that could code for head and tail structural proteins and immunity repressor proteins. In addition, the pattern of gene expression showed that several LE1 genes are expressed specifically either in LE1 prophage or in L. biflexa late after infection. Since the LE1 prophage replicates autonomously as a circular replicon in L. biflexa, we were able to engineer an L. biflexa-Escherichia coli shuttle vector from a 5.3-kb DNA fragment of LE1 (Saint Girons et al., J. Bacteriol. 182:5700-5705, 2000), opening this genus to genetic manipulation. In this study, base compositional asymmetry confirms the location of the LE1 replication region and suggests that LE1 replicates via a bidirectional Theta-like replication mechanism from this unique origin. By subcloning experiments, the replication region can be narrowed down to a 1-kb region. This minimal replication region consists of a rep encoding a protein of 180 amino acids. Upstream from rep, putative partitioning genes, called parA and parB, were found to be similar to the par loci in Borrelia plasmids. A significant increase of plasmid stability in L. biflexa can be seen only when both parA and parB are present. These results enable the construction of new shuttle vectors for studying the genetics of Leptospira spp. This study will also contribute to a better knowledge of phages unrelated to lambdoid phages.     

Hepatitis A virus mutant spectra under the selective pressure of monoclonal antibodies: codon usage constraints limit capsid variability

Severe structural constraints in the hepatitis A virus (HAV) capsid have been suggested as the reason for the lack of emergence of new serotypes in spite of the occurrence of complex distributions of mutants or quasispecies. Analysis of the HAV mutant spectra under immune pressure by the monoclonal antibodies (MAbs) K34C8 (immunodominant site) and H7C27 (glycophorin binding site) has revealed different evolutionary dynamics. Populations composed of complex ensembles of mutants with very low fitness or single dominant mutants with high fitness permit the acquisition of resistance to each of the MAbs, respectively. Deletion mutants were detected as components of the mutant spectra: up to 61 residues, with an average of 19, and up to 83 residues, with an average of 45, in VP3 and VP1 proteins, respectively. A clear negative selection of those replacements affecting the residues encoded by rare codons of the capsid surface has been detected through the present quasispecies analysis, confirming a certain beneficial role of such clusters. Since these clusters are located near or at the epitope regions, the need to maintain such clusters might prevent the emergence of new serotypes.     

Isolation and characterization of FecA- and FeoB-mediated iron acquisition systems of the spirochete Leptospira biflexa by random insertional mutagenesis

The specific mechanisms by which Leptospira spp. acquire iron from their ecological niches are unknown. A major factor contributing to our ignorance of spirochetal biology is the lack of methods for genetic analysis of these organisms. In this study, we have developed a system for random transposon mutagenesis of Leptospira biflexa using a mariner transposon, Himar1. To demonstrate the validity of Himar1 in vivo transposon mutagenesis in L. biflexa, a screen of mutants for clones impaired in amino acid biosynthesis was first performed, enabling the identification of tryptophan and glutamate auxotrophs. To investigate iron transporters, 2,000 L. biflexa transposon mutants were screened onto media with and without hemin, thus allowing the identification of five hemin-requiring mutants, and the putative genes responsible for this phenotype were identified. Three mutants had distinct insertions in a gene encoding a protein which shares homology with the TonB-dependent receptor FecA, involved in ferric citrate transport. We also identified two mutants with a Himar1 insertion into a feoB-like gene, the product of which is required for ferrous iron uptake in many bacterial organisms. Interestingly, the growth inhibition exhibited by the fecA and feoB mutants was relieved by deferoxamine, suggesting the presence of a ferric hydroxamate transporter. These results confirm the importance of iron for the growth of Leptospira and its ability to use multiple iron sources.     

Fatty acid composition of Spirulina strains grown under various environmental conditions

The fatty acid distribution in 19 strains of Spirulina was studied. All but one contained γ-linolenic acid (GLA). No GLA was found in S. subsalsa, which had a very high content of palmitoleic acid. The fatty acid content of all but one of the tested strains increased with cultivation temperature and the relative amount of polyunsaturated fatty acid decreased. The highest content of GLA was found at 30–35° for most strains. High light intensities at a high temperature (38°), while not affecting the fatty acid composition, had a drastic effect on the fatty acid content, reducing it by as much as 46 %.     

New method for the selective capture of antibodies under physiolgical conditions

Hydrophobic charge induction chromatography is a recently developed method for protein separation based on the use of dual-mode ligands. They are designed in such a way so as to combine a molecular interaction supported by a mild hydrophobic association effect in the absence of salts. When environmental pH is changed, the ligand becomes ionically charged resulting into the desorption of the protein. This method is applied to the separation of antibodies from ascite fluids and culture supernatants from hybridomas cultured in the presence of fetal bovine serum or in protein free environment. Typically adsorption from cell culture supernatants is accomplished without any pH or ionic strength adjustment; the column is then washed with a typical buffer to eliminate protein impurities. Antibodies are then desorbed using acetate buffer, pH 4. Antibody binding capacity is in the range of 30 mg per ml of resin at 10% breakthrough. Antibody purity varies according to the initial feed stock and can reach values higher than 90% in a single pass. One example of antibody purification process involving hydrophobic charge induction chromatography as a capture step followed by a polishing phase with DEAE Ceramic HyperD is described. Longevity and ligand leakage are compatible with large-scale applications.     

Analysis of cloned DNA from Leptospira biflexa serovar patoc which complements a deletion of the Escherichia coli trpE gene.

To analyze the cloned region of the chromosome of the spirochete Leptospira biflexa serovar patoc which complemented a defect in the trpE gene of Escherichia coli, we performed a series of experiments involving subcloning, transposon mutagenesis, and maxicells. By subcloning into pBR322 we were able to isolate the Leptospira genes on a 9.7-kilobase pair plasmid (pYC6). Transposon mutagenesis with Tn5 identified a 2.8-kilobase pair region of this plasmid as being necessary to complement a trpE deletion mutation in E. coli. Transformation of plasmid pYC6 into E. coli cells deleted for trpE and the proximal end of trpD showed that the Leptospira DNA complemented both defects. A maxicell analysis of various transposon-induced mutations of the plasmid revealed that three proteins (53.5, 23.6, and 22 kilodaltons) were encoded by the 2.8-kilobase pair region of the Leptospira genome. Two different promoters controlled the production of these three proteins.     

Antigenic relationship among the eight prototype and new serotype strains of Orientia tsutsugamushi revealed by monoclonal antibodies.

Orientia tsutsugamushi, the etiologic agent of tsutsugamushi disease, exhibits great antigenic variation. Three classical strains (Karp, Gilliam, and Kato) and new antigenic types from Thailand (TA686, TA678, TA716, TA763, and TH1817) have been used as prototype strains of O. tsutsugamushi in many studies. In this study, monoclonal antibodies to the five Thailand strains were produced, and their reactivity against prototype strains and newly identified isolates from Korea and Japan was tested. With a panel of these monoclonal antibodies, we could analyze the antigenic relationship among various strains of O. tsutsugamushi from Thailand, Japan, and Korea. Twelve strains of the O. tsutsugamushi tested showed various reactivities to monoclonal antibodies, and no distinct pattern of reactivity was found according to their location of isolation. Although the Boryong and Kuroki strains were similar in reactivities to most monoclonal antibodies, several monoclonal antibodies could differentiate the two strains. These results indicate that the immunofluorescence antibody test using monoclonal antibodies used in this study is valuable for analyzing the antigenic relationship and classification of O. tsutsugamushi.