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3-O-methylmannose was identified by gas-liquid chromatography-mass spectrometry in the acid hydrolysate of the polysaccharide of Leptospira biflexa Urawa.
Representative serovars and strains of the seven genetic groups of Leptospira interrogans, and two previously studied serovars, were all found to form exclusively right-handed helices as determined by scanning electron microscopy. No change in handedness occurred in cells grown in a minimal medium (Tween-80 albumin) compared to cells grown in a rich medium (rabbit serum). The right-handedness of the organisms was related to the evolution, cell wall structure, and the mechanism of motility of L. interrogans.
We cloned Sau3AI fragments containing the rRNA genes for Leptospira interrogans serovar canicola strain Moulton in the BamHI site of lambda EMBL3 bacteriophage DNA. Physical maps of the fragments were constructed, and the locations of the rRNA genes were determined by Southern blot hybridization and S1 protection. Each fragment of the 23S or the 16S rRNA gene contained at least one copy of the 23S or the 16S sequence. Genomic hybridization showed that there were two genes for the 23S rRNA and the 16S rRNA but only one gene for the 5S rRNA on the chromosome of L. interrogans. The results revealed the important fact that each rRNA gene is located far from the other rRNA genes. Our findings, accordingly, also suggest that these rRNA genes are expressed independently in this organism.
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.
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.
The gene encoding the 5S rRNA for Leptospira interrogans serovar canicola strain Moulton was isolated and sequenced. The 5S rRNA gene occurs as a single copy within the genome and encodes a 117-nucleotide-long RNA molecule. The 5S rRNA gene is flanked at both the 5' and 3' ends by regions of A + T-rich sequences, and the 5'-flanking region contains a promoter sequence. L. interrogans has a unique and remarkable organization of the 5S rRNA gene. The 5S rRNA molecule exhibits a strong similarity to typical eubacterial 5S rRNA in terms of overall secondary structure, while the primary sequence is conserved to a lesser degree. Restriction analysis of the 5S rRNA gene indicated that the DNA sequence including the 5S rRNA gene is highly conserved in the genomes of parasitic leptospires.
All known ribosomes of procaryotic organisms are made up of three rRNA components that are 23, 16, and 5S in size. We now report that in some Leptospira interrogans strains, the classical 23S rRNA is further processed to generate 14 and 17S rRNAs. This processing step was previously known to occur only in some eucaryotes and in a small group of procaryotes. The implications of this finding are discussed.
Reference strains for each of the 23 serogroups of Leptospira interrogans yielded different pulsed-field gel electrophoresis patterns of NotI digestion products. This was also the case for the 14 serovars belonging to serogroup Icterohaemorrhagiae (with one exception). The NotI restriction patterns of 45 clinical leptospiral isolates belonging to serovar icterohaemorrhagiae were analyzed and compared with those of type strains. No differences were observed between isolates from countries of different continents, namely, France, French Guiana, New Caledonia, and Tahiti. The pattern was indistinguishable from that of the reference strain of serovar icterohaemorrhagiae.
The structure and composition of periplasmic flagella (PF) from Leptospira interrogans serovar pomona type kennewicki were characterized. Electron microscopic observations showed that leptospiral PF were complex structures composed of an 11.3-nm-diameter core surrounded by two sheath layers with 21.5- and 42-nm diameters. Two-dimensional gel electrophoresis of isolated PF showed the presence of seven different proteins ranging in mass from 31.5 to 36 kDa. Rabbit polyclonal and mouse monoclonal antibodies against PF proteins were prepared and were used to localize specific proteins to portions of the PF structure by immunoelectron microscopy. A 34-kDa protein was associated with the 11.3-nm-diameter core filament, while a 36-kDa protein was associated with a PF sheath (21.5-nm-diameter filament). The amino termini of the 34- and 35.5-kDa proteins were homologous to PF core proteins of other spirochetes. The experimental data suggested that L. interrogans PF contains 2 proteins (34 and 35.5 kDa) in the PF core.
Leptospira interrogans is a pathogenic bacterium with a low G+C content (34 to 39%). The restriction enzymes NotI, AscI, and SrfI cut the chromosome of L. interrogans serovar icterohaemorrhagiae into 13, 3, and 5 fragments separable by one- and two-dimensional pulsed-field gel electrophoresis (PFGE). The genome is composed of a circular 4.6-Mbp chromosome and a 0.35-Mbp extrachromosomal element. A physical map of the chromosome was constructed for NotI, AscI, and SrfI by using single and double digests, or partial NotI digests obtained at random or by cross-protection of NotI sites by FnuDII methylase, and linking clones. rRNA genes were found to be widely scattered on the chromosome.
Genetic maps were constructed for Leptospira interrogans serovars icterohaemorrhagiae and pomona. Previously we independently constructed physical maps of the genomes for these two serovars. The genomes of both serovars consist of a large replicon (4.4 to 4.6 Mb) and a small replicon (350 kb). Genes were localized on the physical maps by using Southern blot analysis with specific probes. Among the probes used were genes encoding a variety of essential enzymes and genes usually found near bacterial chromosomal replication origins. Most of the essential genes are on the larger replicon of each serovar. However, the smaller replicons of both serovars contain the asd gene. The asd gene encodes aspartate beta-semialdehyde dehydrogenase, an enzyme essential in amino acid and cell wall biosyntheses. The finding that both L. interrogans replicons contain essential genes suggests that both replicons are chromosomes. Comparison of the genetic maps of the larger replicons of the two serovars showed evidence of large rearrangements. These data show that there is considerable intraspecies heterogeneity in L. interrogans.
The Leptospira meyeri serovar semaranga metX gene was identified by complementation of an Escherichia coli metA mutant, i.e., devoid of homoserine O-succinyltransferase. However, the MetX protein exhibited a homoserine O-acetyltransferase activity in agreement with its similarity to homoserine O-acetyltransferases. Reverse transcription-PCR analysis demonstrated that metX is the second gene of an operon.
Immunity to leptospirosis is principally humorally mediated and involves opsonization of leptospires for phagocytosis by macrophages and neutrophils. The only protective antigen identified to date is the leptospiral lipopolysaccharide (LPS), which biochemically resembles typical gram-negative LPS but has greatly reduced endotoxic activity. Little is known about the structure of leptospiral LPS. A 2.1-kb EcoRI fragment from the chromosome of serovar Copenhageni was cloned in pUC18 in Escherichia coli, after which flanking regions were cloned from a genomic library constructed in bacteriophage lambda GEM12. Sequence analysis identified four open reading frames which showed similarity to the rfbC, rfbD, rfbB, and rfbA genes, transcribed in that order, which encode the four enzymes involved in the biosynthesis of dTDP-rhamnose for the assembly of LPS in Salmonella enterica, E. coli, and Shigella flexneri. An additional open reading frame downstream of the rfbCDBA locus showed similarity with the rhamnosyltransferase genes of Shigella and Yersinia enterocolitica but not Salmonella. Comparison of deduced amino acid sequences showed up to 85% similarity of the leptospiral proteins with those of other gram-negative bacteria. Polyacrylamide gel electrophoresis of recombinant clones identified the putative RfbCDBA proteins, while reverse transcriptase-mediated PCR analysis indicated that the rfbCDBA gene cluster was expressed in Leptospira. Moreover, it could restore normal LPS phenotype to a defined rfbB::Tn5 mutant of S. flexneri which was deficient in all four genes, thereby confirming the functional identification of a part of the leptospiral rfb locus.
We report the first evidence of a chromosome-encoded toxin-antitoxin locus in spirochetes. This locus has been found in the pathogenic spirochete Leptospira interrogans and exhibits homologies with the pem/chp loci. The L. interrogans chp locus consists of two genes: chpK (for "killer protein") and its upstream partner chpI (for "inhibitory protein"). Expression of ChpK in Escherichia coli results in the inhibition of bacterial growth. The coexpression of ChpI neutralizes ChpK toxicity. By Southern blot analysis, chp homologs were found in all representative pathogenic strains of L. interrogans.
Three leuA-like protein-coding sequences were identified in Leptospira interrogans. One of these, the cimA gene, was shown to encode citramalate synthase (EC 4.1.3.-). The other two encoded alpha-isopropylmalate synthase (EC 4.1.3.12). Expressed in Escherichia coli, the citramalate synthase was purified and characterized. Although its activity was relatively low, it was strictly specific for pyruvate as the keto acid substrate. Unlike the citramalate synthase of the thermophile Methanococcus jannaschii, the L. interrogans enzyme is temperature sensitive but exhibits a much lower K(m) (0.04 mM) for pyruvate. The reaction product was characterized as (R)-citramalate, and the proposed beta-methyl-d-malate pathway was further confirmed by demonstrating that citraconate was the substrate for the following reaction. This alternative pathway for isoleucine biosynthesis from pyruvate was analyzed both in vitro by assays of leptospiral isopropylmalate isomerase (EC 4.2.1.33) and beta-isopropylmalate dehydrogenase (EC 1.1.1.85) in E. coli extracts bearing the corresponding clones and in vivo by complementation of E. coli ilvA, leuC/D, and leuB mutants. Thus, the existence of a leucine-like pathway for isoleucine biosynthesis in L. interrogans under physiological conditions was unequivocally proven. Significant variations in either the enzymatic activities or mRNA levels of the cimA and leuA genes were detected in L. interrogans grown on minimal medium supplemented with different levels of the corresponding amino acids or in cells grown on serum-containing rich medium. The similarity of this metabolic pathway in leptospires and archaea is consistent with the evolutionarily primitive status of the eubacterial spirochetes.
Footprinting and mutagenesis experiments demonstrated that Leptospira interrogans LexA binds the palindrome TTTGN(5)CAAA found in the recA promoter but not in the lexA promoter. In silico analysis revealed that none of the other canonical SOS genes is under direct control of LexA, making the leptospiral lexA gene the first described which is not autoregulated.
The cytochrome content of three leptospiral strains grown in several media was investigated after it was shown that respiratory inhibitors suppressed oxygen consumption of a water isolate, B(16), and that two pathogenic serotypes, pomona and schueffneri, were active catalase producers, whereas B(16) lacked catalase activity. Reduced minus oxidized difference spectra disclosed cytochromes of the a, c, and c(1) types in all strains. Although no spectral evidence suggested the existence of cytochrome b components, they could have been masked by cytochrome c, and their presence cannot be ruled out. Carbon monoxide difference spectra revealed peaks indicative of a cytochrome oxidase of the o type in all strains. Carbon monoxide spectra further suggested that a cytochrome a oxidase, possibly a(1) or a(3), and a pigment with absorption spectra different from those of previously characterized cytochromes existed in the two pathogens and not in the water isolate. Physiological reduction of the cytochromes by various metabolic substrates implied that the cytochrome system in Leptospira was functional. No effect of the various growth media on the cytochrome patterns of the three strains was observed, indicating that all three strains were capable of synthesis of cytochrome components and did not require heme prosthetic groups present in serum.
Metabolic studies were performed on three representative serotypes of Leptospira: a water isolate designated B(16) and two pathogenic serotypes, pomona and schueffneri. Examination of whole cells of B(16) for their ability to oxidize various substrates revealed that oleate significantly stimulated oxygen uptake. The respiratory quotient of 0.7 implied that oleate was degraded to carbon dioxide and water. Other substrates, such as carbohydrates, alcohols, intermediates of the citric acid cycle, and short-chain acids, including selected amino acids, did not stimulate endogenous respiration of whole cells. No oxygen uptake could be measured when cell-free extracts were tested with the substrates used with whole cells. Enzymatic analyses of cell-free extracts of the three strains demonstrated enzymes of the citric acid cycle, enzymes of the glycolytic and pentose pathways, and the general acyl coenzyme A dehydrogenase required for beta-oxidation of fatty acids. Strain B(16) and the two pathogenic serotypes appeared to possess similar metabolic capabilities. Enzymatic data might also explain the apparent inability of B(16) to oxidize other substrates; kinases necessary for activation of common nonphosphorylated compounds were not detected in leptospiral extracts. These findings emphasized the dependence of leptospiral growth upon long-chain fatty acids.