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.     

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.     

Identification and analysis of genes present in Leptospira interrogans serovar lai but absent in L. biflexa serovar monvalerio

Genes present in virulent bacterial strains but absent in avirulent close relatives can be of great biologic and clinical interest. This project aimed to identify strain specific DNA sequences of Leptospira interrogens serovar lai, which is absent in the saprophytic L. biflexa serovar monvalerio, via suppression subtractive hybridization with the former as the tester while the latter as the driver. The mixture of PCR amplified DNA fragments from two subtractive hybridization experiments were cloned into pMD 18-T vector and the positive clones were identified by dot blotting against the chromosome DNA of the two strains individually. After DNA sequencing and analysis, the distribution of these genomic fragment sequences in a panel of pathogenic and nonpathogenic leptospires was investigated employing dot blot analysis. Among the 188 positive clones randomly chosen, 24 contained the tester strain specific genomic regions, of which, 5 were non-coding fragments while the others contained 23 distinct protein coding sequences. Besides 9 genes encoding functional proteins, 12 genes encode unknown proteins and the rest two genes encode proteins with recognizable domain structures, one for a putative leucine-rich repeats (LRR) family protein while the other as an outer-membrane protein. Our experiment results indicated that suppression subtractive hybridization is effective for screening specific DNA sequences between two leptospiral strains, and some of these sequences might be responsible for virulence determination. Further analysis of these DNA sequences will provide important information on the pathogenesis of Leptospira.     

Study of a specific polyoside and a group antigen extracted from Leptospira biflexa patoc, patoc I strain]

We extracted from L. biflexa patoc a fraction F, reacting in hemagglutination and ring tests with sera prepared against more than ten different serogroups. This fraction contains mainly a polysaccharide (65 per cent), the role of which was clearly demonstrated in the precipitation reaction with homologous antisera, through periodic oxidation; it also contains lipids (20 per cent) and proteins (10 per cent). We isolated from this fraction F, by Biogel column chromatography, 2 distinct antigens, one, F2, carrying the patoc-type specificity, the other, F 1B, a group specificity shared by many leptospira. These antigens differ not only immunogically, but also in their chemical composition. The type-specific antigen F2 contains mainly a polysaccharide composed of arabinose and glucosamine (possibly an immunodominant sugar). As for the group-specific fraction F 1B, its composition is more complex since lipids and proteins are also found with the polysaccharide. This antigen could therefore be a lipoglycoprotein.     

Cloning and sequence analysis of the rpsL and rpsG genes of Mycobacterium smegmatis and characterization of mutations causing resistance to streptomycin.

The Mycobacterium smegmatis rpsL and rpsG genes, encoding the ribosomal proteins S12 and S7, were cloned, and their DNA sequence was determined. The third nucleotide of the S12 termination codon overlapped the first nucleotide of the S7 translation initiation codon. A collection of 28 spontaneous streptomycin-resistant mutants of M. smegmatis were isolated. All had single-base-pair substitutions in the rpsL gene which were changed to a streptomycin-sensitive phenotype by complementation with a low-copy-number plasmid carrying the wild-type M. smegmatis rpsL gene. A total of eight different mutations were found in two specific regions of the rpsL gene. Fifty-seven percent (16 of 28) altered the Lys codon at position 43. Forty-six percent of the mutations (13 of 28) were due to a transition changing an AAG Lys codon to an AGG Arg codon, with eight changes at codon 43 and five at codon 88.     

Nucleotide sequence of a repetitive element isolated from Leptospira interrogans serovar hardjo type hardjo-bovis.

A repetitive element from the genome of Leptospira interrogans serovar hardjo type hardjo-bovis ('L. hardjo-bovis') was identified, cloned and sequenced. Similar sequences were shown by hybridization to be encoded by a further eight of 32 other leptospiral serovars tested. An undefined number of repetitive elements were located in the L. hardjo-bovis genome; sequence degeneracy of the elements was observed and no significant open reading frames were identified within the AT-rich (60%) 1467 bp repetitive element. The termini encoded a GC-rich 8 bp repeat motif and two variants showed rearrangements centred on these motifs. The nucleotide sequences of the chromosomal regions flanking the repetitive elements were determined but showed no similarities, with one exception which had a GAAC repeat directly adjacent to both termini. Similar hybridization patterns were shown by Southern transfers of L. hardjo-bovis total genomic digests probed with the repetitive element. Oligonucleotide primer pairs designed from sequences internal to the repetitive element and adjacent chromosomal regions were used in polymerase chain reaction experiments. With one primer pair all L. hardjo-bovis isolates, but no other serovar, gave identical amplified products. Evidence that the repetitive element may have derived from an acquired insertion sequence that is now inactive and chromosomally fixed is discussed.     

Identification and Analysis of Genes Present in Leptospira interrogans serovar lai but Absent in L. biflexa serovar monvalerio

Leptospirosis is a globally important zoonotic diseasecaused by the pathogenic species of the spirochete genus,Leptospira including L. interrogans, L. kirschneri, L.noguchii, L. borgpetersenii, L. santarosai, L. weilii andetc. [1]. Pathogenic leptospires …     

The number of large ribosomal RNA genes in Leptospira interrogans and Leptospira biflexa

We determined the number of large ribosomal RNA genes in five strains of Leptospira by hybridization of 15 restriction endonuclease digests of genomic DNA to the [32P]-labeled fragment of 23s rRNA gene. Almost all the restriction gels gave two radioactive bands. The conclusion from these results is that there are at least two rRNA genes in these leptospiral strains. Furthermore, the hybridization patterns of L. icterohaemorrhagiae strains Ictero No. I and RGA are almost identical. The number of rRNA genes and taxonomic relationships of these leptospires were discussed.     

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.     

Phospholipases of Leptospira. I. Presence of phospholipase A1 and lysophospholipase in Leptospira biflexa

The hydrolysis of phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and trioleoylglycerol by Leptospira biflexa strain Urawa was studied in vitro. Phospholipase A₁ was identified by the formation of ³²P- and ¹⁴C-labeled lyso-derivatives from ³²P-phosphatidylcholine, ³²P-phosphatidylethanolamine, or 1-acyl-2-[1-¹⁴C]oleoyl-sn-glycero-3-phosphorylcholine. Phospholipase A₁ activity was independent of lipase in the microorganism since ¹⁴C-labeled trioleoylglycerol was scarcely attacked under the same conditions in which the phospholipids were hydrolyzed. Lysophospholipase activity was also demonstrated using ³²P- and non-labeled lysophosphatidylcholine. The activity of phospholipase A₁ was found in a broad range of pH but no optimal pH was determined. The pH optimum of lysophospholipase was 8.0. Both enzymes were labile to heat. Phospholipase C activity, however, could not be detected because no radioactive di- and monoacylglycerol was found in the experiment with 1-acyl-2-[1-¹⁴C]-oleoyl-sn-glycero-3-phosphorylcholine as the substrate. It was inferred that phosphatidylethanolamine, which was the major component of phospholipids in leptospirae, was hydrolyzed serially by phospholipase A (A₁ and/or A₂?) and lysophospholipase to glycerophosphorylethanolamine via 2-acyl-type-lyso-derivative as one metabolic pathway of the substrate.     

Contamination of Bacteriological Media by Leptospira biflexa

Contamination of media with a strain of Leptospira biflexa was traced to the deionized water supply. The leptospiral contaminant appeared in media sterilized by filtration through 0.45- and 0.22-mum pore size membrane filters.     

Occurrence of [--> 3)-beta-D-Manp-(1 --> 4)-beta-D-Manp-(1 -->]n units in the antigenic polysaccharides from Leptospira biflexa serovar patoc strain Patoc I

In this study, we isolated three kinds of antigenic polysaccharide components (tentatively designed as AP-1-3) from cells of Leptospira biflexa serovar patoc strain Patoc I (L. biflexa patoc Patoc I) by the hot phenol-water procedure, followed by treatment with mild acid and column chromatography. Two of them (AP-1 and AP-2) were recovered from the phenol-soluble fraction whereas another (AP-3) was recovered from the aqueous fraction. All of them reacted toward an anti-L. biflexa serum and also cross-reacted in similar extents toward most of the other leptospiral antisera tested. Such immunoreactions were specifically inhibited by a beta-(1 --> 4)-linked mannobiose, but were not by any mono- and oligosaccharide tested. From their structural analyses including 1H and 13C NMR spectrometry, Smith degradation and methylation analysis, it was revealed that all of these antigenic polysaccharides had the same disaccharide unit --> 3)-beta-D-Manp-(1 --> 4)-beta-D-Manp-(1 --> in their major polysaccharide parts, but they differed in the acyl substituents. Therefore it is most likely that such mannobiose unit is a candidate for the antigenic epitopes of L. biflexa polysaccharides.     

Nucleotide sequence of Klebsiella pneumoniae lac genes.

The nucleotide sequences of the Klebsiella pneumoniae lacI and lacZ genes and part of the lacY gene were determined, and these genes were located and oriented relative to one another. The K. pneumoniae lac operon is divergent in that the lacI and lacZ genes are oriented head to head, and complementary strands are transcribed. Besides base substitutions, the lacZ genes of K. pneumoniae and Escherichia coli have suffered short distance shifts of reading frame caused by additions or deletions or both during evolutionary divergence from a common ancestral gene. Relative to corresponding E. coli sequences, the nucleotide sequences of the lacZ and lacY genes are 61 and 67% conserved, and the lacI genes are 49% conserved. A comparison of both nucleotide and amino acid sequences revealed that the K. pneumoniae and E. coli lacI genes and lac repressor proteins each are related to the galR gene and gal repressor of E. coli to about the same extent. In terms of evolutionary relationships, the divergence of the forerunner of the galR gene from an ancestral lac repressor gene preceded separation and differentiation of the K. pneumoniae and E. coli lac repressor genes.     

Nucleotide sequence analysis of purH and purD genes from Salmonella typhimurium

The purH and purD genes coding for the 5'-phosphoribosyl 5-amino-imidazole-4-carboxamide (AICAR) transformylase and 5'-phosphoribosyl-glycinamide (GAR) synthetase, respectively, were identified on a 4.8 kb Eco RI fragment of chromosomal DNA from Salmonella typhimurium. Nucleotide sequence analysis of the cloned fragment revealed the presence of two large open reading frames (O.R.F.), which were separated by 11 base pairs (bp). Substantial DNA and amino acid sequence homology was noted between the purH and purD genes of S. typhimurium and Escherichia coli. Expression of the Salmonella purD gene in a T7 polymerase/promoter system revealed the presence of a 49 kDa protein band by SDS-PAGE and subsequent autoradiography. The purH gene of Salmonella was not expressed since the 5' end of this gene was not cloned.     

Nucleotide sequence of the two rat cellular rasH genes.

We present the nucleotide sequence of the coding region of the rat c-rasH-1 gene and a partial sequence analysis of the rat c-rasH-2 gene. By comparing these sequences with the Harvey murine sarcoma virus ras gene, we predict that the p21 protein encoded by the Harvey virus differs from the cellular c-rasH-1-encoded p21 at only two amino acids; those at positions 12 and 59. Alterations at each of these positions may play a role in activating the viral p21 protein. The c-rasH-2 gene is likely to be a nonfunctional pseudogene because it lacks introns, cannot be activated to transform NIH 3T3 cells, and differs in sequence from both c-rasH-1 and v-rasH at several base pair positions.     

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.