Homologues of helicase genes priA and ruvAB of Borrelia burgdorferi, the Lyme borreliosis agent.

A DNA library of strain HB19 from Borrelia burgdorferi sensu stricto, an agent of Lyme borreliosis, was constructed in the cosmid pLA2917. Genes involved in initiation of DNA replication and resolution of recombination intermediates (Holliday junctions) were found on a 23-kbp region up to 0.7 kbp of the "left" extremity of the linear chromosome in representative species of B. burgdorferi sensu lato. The potential ruvB gene, located at 22 kbp from the left telomere, was identified by the similarity of its deduced amino acid sequence to RuvB (helicases) of other bacteria. B. burgdorferi ruvB is part of an operon which comprises the homologues of ruvA, queA and pfbB. Expression of the B. burgdorferi ruvB and ruvA genes renders a wild-type Escherichia coli sensitive to UV light and mitomycin, indicative of negative complementation. priA, which encodes the potential recognition factor for the primosome assembly site, was found at 15 kbp from the left telomere. RuvB and PriA sequences have motifs characteristic of helicases: a DExH box and an ATP binding site.

Molecular characterization of a large Borrelia burgdorferi motility operon which is initiated by a consensus sigma70 promoter.

A large motility operon, referred to as the flgB operon, was identified, characterized, and mapped at 310 to 320 kb on the linear chromosome of the spirochete Borrelia burgdorferi. This is the first report that a sigma70-like promoter rather than a sigma28-like promoter is involved in the transcription of a major motility operon in bacteria. From these results in conjunction with results from a previous study (Y. Ge and N. W. Charon, Gene, in press), we have identified 26 genes in this operon that are relevant to motility and flagellar synthesis. With few exceptions, the gene order and deduced gene products were most similar to those of other spirochetes and Bacillus subtilis. Primer extension analysis indicated that transcription initiated from a conserved sigma70-like promoter immediately upstream of flgB; this promoter mapped within the heat-shock-induced protease gene hslU. Reverse transcriptase PCR analysis indicated that a single transcript of 21 kb initiated at this promoter and extended through flgE and (with our previous results) onto the putative motility gene flbE. The flgB promoter element had strong activity in both Escherichia coli and Salmonella typhimurium. As expected, a mutant of S. typhimurium with an inactivated flagellum-specific sigma28 factor did not affect the function of this promoter. Western blot analysis indicated that B. burgdorferi recombinant FliG and FliI were antigenically similar to those of E. coli and other spirochetes. Although complementation of E. coli or S. typhimurium fliG or fliI mutants with the B. burgdorferi genes was unsuccessful, B. burgdorferi recombinant FliI completely inhibited flagellar synthesis and motility of wild-type E. coli and S. typhimurium. These results show that spirochete motility genes can influence flagellar synthesis in other species of bacteria. Finally, Western blot analysis with sera from infected humans and animals indicated a weak or nondetectable response to recombinant FliG and FliI. These results indicate that these antigens are not favorable candidate reagents to be used in the diagnosis of Lyme disease.

A cheA cheW operon in Borrelia burgdorferi, the agent of Lyme disease.

Borrelia burgdorferi sensu stricto homologues of cheA and cheW were cloned and characterized. A combination of strategies such as polymerase chain reaction (PCR) using degenerate primers, random-primed gene walking PCR and construction of a lamda library were used to identify the putative cheA gene. Sequence analysis of the DNA fragments obtained from the CT strain identified a 2,592-bp open reading frame (ORF) encoding an 864-amino-acid protein with significant similarity (53-64.6% identical residues) to the CheA of several genera of eubacteria. In particular, hallmarks of a histidine kinase family were found such as the location of the histidine autophosphorylation domain very close to the NH2 terminus and the nucleotide-binding site. A second ORF located immediately downstream from the putative borrelial cheA gene encoded a 195-amino-acid protein which displayed a high level of similarity to bacterial CheW. Using reverse transcription PCR, we demonstrated that cheA and cheW form an operon with an upstream, unidentified ORF. The cheA and cheW homologues were located at 722-737 kbp, 738-768 kbp and 743-824 kbp on the linear chromosomes of B. burgdorferi sensu stricto, B. garinii and B. afzelii, respectively. Identification of cheA and cheW is the first step toward elucidation of a possible role of chemotaxis in virulence of the Lyme disease borreliae.

FliH and fliI of Borrelia burgdorferi are similar to flagellar and virulence factor export proteins of other bacteria.

Two motility genes (fliH and fliI) of the Lyme disease spirochete Borrelia burgdorferi were cloned, physically mapped and sequenced, FliH and FliI showed extensive homology to the proteins involved in the export of flagellar components and to virulence factors found in both animal and plant bacterial pathogens. The results suggest that the flagellar apparatus and associated protein export pathway are well conserved in evolution.

Microbial genes homologous to the peptidyl-tRNA hydrolase-encoding gene of Escherichia coli.

We have cloned and determined the nucleotide (nt) sequences of the genes encoding peptidyl-tRNA hydrolase (Pth) homologues of Salmonella typhi (St) and the Lyme disease spirochaete, Borrelia burgdorferi (Bb). We also completed the nt sequence of a pth homologous gene contained in a Chlamydia trachomatis (Ct) clone identified in the databanks. The open reading frames (ORFs) of the Pth homologues encode putative polypeptides of 194 (St), 188 (Bb) and 194 (Ct) amino acids exhibiting significant identity with Escherichia coli (Ec) Pth. Together with the products of two previously unidentified ORFs from Bacillus subtilis and Saccharomyces cerevisiae, and the recently recognized Haemophilus influenzae and Mycoplasma genitalium pth genes, these seven putative polypeptides and the Ec Pth form a group of homologous basic proteins spanning eubacteria and eukaryota which can be defined by at least three conserved regions. Previously known Ec pth mutations were located in highly conserved residues.

Conservation of gene arrangement and an unusual organization of rRNA genes in the linear chromosomes of the Lyme disease spirochaetes Borrelia burgdorferi, B. garinii and B. afzelii.

Physical maps of the chromosomes of the Lyme disease spirochaetes Borrelia garinii and Borrelia afzelii have been elucidated for the enzymes CspI, SgrAI, I-CeuI, SmaI, EagI, BssHII, MluI and ApaI by two-dimensional pulsed-field gel electrophoresis techniques. The maps contain 42 sites for B. garinii and 32 for B. afzelii. The mapping studies showed that the two chromosomes are linear DNA molecules of 953 and 948 kbp, respectively. A comparison of the physical maps of B. garinii and B. afzelii and the published map of the other Lyme disease spirochaete, Borrelia burgdorferi [Davidson, B.E., MacDougall, J. & Saint Girons, I. (1992) J Bacteriol 174, 3766-3774] revealed that the three chromosomes have few endonuclease sites in common, apart from a cluster in rrl (encoding 23S rRNA) and rrs (encoding 16S rRNA). Cloned borrelial genes were used as specific hybridization probes to construct genetic maps, using the physical maps as a basis. The resulting maps contain 41 genetic loci for B. burgdorferi, 39 for B. garinii, and 33 for B. afzelii. In contrast to the physical maps, the three genetic maps are closely related, with no detectable differences in gene order along the entire length of the chromosome. It is concluded that the chromosomes of these three borrelial species have undergone no major rearrangements, deletions or insertions during their evolution from a common ancestor. Detailed mapping of the region of the B. garinii and B. afzelii chromosomes that encodes rRNA revealed that each chromosome contains one copy of rrs separated by 5 kbp from two copies each of rrl and rrf (encoding 5S rRNA). (ABSTRACT TRUNCATED AT 250 WORDS)

The Met repressor-operator complex: DNA recognition by beta-strands.

The crystal structure of the E. coli met repressor in complex with a synthetic 19-base pair oligonucleotide reveals two dimeric repressor molecules bound to adjacent sites on the DNA. The oligonucleotide contains two adjacent repeats of an 8-mer known as a met-box, which represents the consensus of the met operator sites. Each met repressor dimer is centered on a met box and interacts with the adjacent dimer through antiparallel alpha-helices, which explained the observed cooperative nature of the binding. DNA binding takes place through the insertion of a beta-ribbon into the major groove of B-form DNA, representing a novel DNA binding motif. Sequence specificity arises from direct interactions between side chains of the beta-strands and the edges of the bases in the major groove. The local DNA conformation confers additional specificity through interactions between protein and the phosphate backbone. The repressor is activated through binding of S-adenosyl methionine (SAM), the corepressor, to the face opposite to that used for DNA binding. The lack of significant conformational change upon SAM binding, together with electrostatic calculations, suggests that DNA binding enhancement occurs through long-range electrostatic interactions.

Unique genetic arrangement in the dnaA region of the Borrelia burgdorferi linear chromosome: nucleotide sequence of the dnaA gene.

The complete nucleotide sequence of the Borrelia burgdorferi dnaA gene (encoding the initiator protein of chromosome replication) and its flanking regions was determined. The putative DnaA polypeptide exhibited 29-42% identity with those of other eubacteria. The gene order in the dnaA region at the centre of the B. burgdorferi linear chromosome is rnpA-rpmH-dnaN-dnaA-gyrB-gyrA in contrast to the consensus eubacterial order of rnpA-rpmH-dnaA-dnaN-recF-gyrB, suggesting a rearrangement during the evolution of the Borrelia chromosome. We did not detect the multiple 9-nucleotide repeats known as DnaA boxes, which characterise origin of replications, in the dnaA-gyrB and dnaA-dnaN intergenic regions. In addition B. burgdorferi DnaA protein differs considerably from those of other eubacteria in a normally highly conserved region at the C-terminus of the polypeptide which may be involved in DNA binding.

Physical mapping of the scattered methionine genes on the Escherichia coli chromosome.

Methionine is an important amino acid which acts not only as a substrate for protein elongation but also as the initiator of protein synthesis. The genes of the met regulon, which consists of 10 biosynthetic genes (metA, metB, metC, metE, metF, metH, metK, metL, metQ, and metX), two regulatory genes (metJ and metR), and the methionyl tRNA synthetase gene (metG), are scattered throughout the chromosome. The only linked genes are metK and metX at 63.6 min, metE and metR at 86.3 min, and the metJBLF gene cluster at 89 min. metBL form the only met operon.

Mapping of genes on the linear chromosome of the bacterium Borrelia burgdorferi: possible locations for its origin of replication.

Molecular clones of Borrelia burgdorferi, aetiologic agent of Lyme borreliosis, were isolated and analysed by DNA sequence determination. This procedure yielded B. burgdorferi homologues of gidA, gyrB, gyrA, ftsA and ftsZ. The genes were located on the physical map of the B. burgdorferi linear chromosome. Also mapped were the genes fla and p60 while dnaA was mapped using a heterologous probe. gyrA and gyrB were found to be in tandem and were mapped, along with dnaA at the centre of the chromosome. gidA was located close to the left hand extremity of the chromosome. Because gyrB, dnaA and gidA are normally located within 50 kb of the origin of replication (oriC), we propose two possible sites for oriC in the B. burgdorferi linear chromosome.

Probing met repressor-operator recognition in solution.

The three-dimensional crystal structure of the Escherichia coli methionine repressor, MetJ, complexed with a DNA operator fragment is described in an accompanying article. The complex exhibits several novel features of DNA-protein interaction. DNA sequence recognition is achieved largely by hydrogen-bond contacts between the bases and amino-acid side chains located on a beta-ribbon, a mode of recognition previously hypothesized on the basis of modelling of idealized beta-strands and DNA, and mutagenesis of the Salmonella phage P22 repressors Arc and Mnt. The complex comprises a pair of MetJ repressor dimers which bind to adjacent met-box sites on the DNA, and contact each other by means of a pair of antiparallel alpha-helices. Here we assess the importance of these contacts, and also of contacts that would be made between the C-helices of the protein and DNA in a previous model of the complex, by studying mutations aimed at disrupting them. The role of the carboxy-terminal helix face in operator binding was unclear, but we demonstrate that recognition of operator sequences occurs through side chains in the beta-strand motif and that dimer-dimer interactions are required for effective repression.

Overproduction and characterization of the iclR gene product of Escherichia coli K-12 and comparison with that of Salmonella typhimurium LT2.

The iclR gene of Escherichia coli K-12, which encodes a regulatory protein (repressor) for the aceBAK operon, is located between that operon and metH in the 91-min region of the chromosome. The iclR gene was cloned and expressed in a coupled T7 RNA polymerase/promoter system and the gene product was identified by specific binding to a fragment containing the aceBAK operator region. The iclR gene product is a polypeptide of 274 amino acids (aa) with a calculated Mr of 29,741. Comparison of the deduced IclR aa sequence to that of Salmonella typhimurium revealed that the two IclR repressors exhibit 89% identity. A possible helix-turn-helix motif characteristic of DNA-binding proteins was found within the IclR sequence. A search in protein data banks revealed that IclR has a score of similarity of 43.7% with GylR, a transcriptional regulator of the glycerol operon of Streptomyces coelicolor.

Nucleotide sequence of the metH gene of Escherichia coli K-12 and comparison with that of Salmonella typhimurium LT2.

The Escherichia coli K-12 metH gene, encoding the vitamin B12-dependent homocysteine transmethylase, is located between iclR and lysC in the 91-min region of the chromosome. The metH gene has been sequenced and reveals an open reading frame of 3600 bp encoding a polypeptide of 1200 amino acids (aa) with a calculated Mr of 132 628. The first 414 aa of the deduced polypeptide sequence are 92% identical to the 414 aa deduced from the partially sequenced Salmonella typhimurium LT2 metH gene. In-frame fusions of metH to lacZ were used to confirm the reading frame of the metH gene and to study its regulation. metH was repressed tenfold, presumably indirectly, by L-methionine and the metJ gene product, while vitamin B12 did not induce de novo synthesis of MetH.

Cloning and characterization of the genes for the two homocysteine transmethylases of Escherichia coli.

We have cloned the genes for the two homocysteine transmethylases of Escherichia coli K12. The vitamin B12-independent enzyme is encoded by the metE gene while the metH gene codes for the vitamin B12-requiring enzyme. Overexpression of the gene products and Tn1000 mutagenesis have enabled the metE and metH gene products to be identified as 99 kDa and 130 kDa polypeptides, respectively. The truncated polypeptides generated by Tn1000 insertion were used to determine the direction of transcription of the metE and metH genes. Negative complementation suggests that the MetH enzyme exists as an oligomer. Investigation of the expression of the chromosomal- and plasmid-encoded gene products confirms that metE is subject to negative control by vitamin B12 and methionine, and that metH is under positive control by the cofactor and negative control by methionine. For vitamin B12 and methionine to act as regulatory effectors in metE control, functional metH and metJ genes are required, respectively. The use of stable Tn1000-generated fragments of the metE product as electrophoretic markers for the plasmid-encoded metE gene product demonstrated that the two regulatory proteins involved in negative control of metE are present in excess. Under conditions whereby both forms of negative metE control are non-functional, the metE gene product represented about 90% of the total protein, and cell growth was severely impaired.