Sequence diversity of a gene encoding a putative primary sigma factor among Borrelia burgdorferi sensu lato strains

Twenty-six strains of Borrelia burgdorferi sensu lato were subjected to polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis for assessing the sequence divergence of rpoD gene encoding the primary sigma factor. Four and five RFLP patterns were observed from two fragments of rpoD gene. Sequence analysis of a subgenic fragment covering region 1 through 4 from 13 strains of Borrelia burgdorferi s. l. revealed that 21 of 450 deduced amino acid residues were diverged. These results indicate that the sequence heterogeneity of rpoD is present in different strains of Borrelia burgdorferi s. l., and agreed well with the current classification of genospecies.     

Analysis and expression of the Borrelia burgdorferi P/Gau fla gene: Identification of heterogeneity with the B31 strain

The flagellin gene from the P/Gau strain of Borrelia burgdorferi was cloned and sequenced. The translated P/Gau flagellin protein differed from the flagellin of the B31 strain at 13 of 336 amino acids. This includes seven differences between amino acids 190-234, an immunodominant and specific region for B. burgdorferi. The entire flagellin molecule, as well as peptides of the internal portion of the protein which is more specific for B. burgdorferi, has been expressed in Escherichia coli using a pET7HIS.2 expression system. These peptides may be of great value for the development of sensitive and specific recombinant-based serological assays.     

Sequence of the complete osp operon encoding two major outer membrane proteins of a European Borrelia burgdorferi isolate (B29)

The nucleotide sequence of the operon encoding the major outer surface proteins, OspA and OspB, of a European isolate of Borrelia burgdorferi (strain B29) was determined and compared to the osp operon of the American strain, B31. An amino acid (aa) identity of 80.7% was found when comparing the OspA of B29 with that of B31, whereas the aa sequence of the OspB of B29 reveals only 61.3% identity with the OspB of B31. Thus, strains B31 and B29 can be regarded as representatives of different B. burgdorferi groups.     

Sequence and analysis of the rpoN sigma factor gene of rhizobium sp. strain NGR234, a primary coregulator of symbiosis.

We report the nucleotide sequence of the rpoN gene from broad-host-range Rhizobium sp. strain NGR234 and analyze the encoded RPON protein, a sigma factor. Comparative analysis of the deduced amino acid sequence of RPON from NGR234 with sequences from other gram-negative bacteria identified a perfectly conserved RPON box unique to RPON sigma factors. Symbiotic regulatory phenotypes were defined for a site-directed internal deletion within the coding sequence of the rpoN gene of Rhizobium strain NGR234: they included quantitative nodulation kinetics on Vigna unguiculata and microscopic analysis of the Fix- determinate nodules of V. unguiculata and Macroptilium atropurpureum. RPON was a primary coregulator of nodulation and was implicated in establishment or maintenance of the plant-synthesized peribacteroid membrane. Phenotypes of rpoN in Rhizobium strain NGR234 could be grouped as symbiosis related, rather than simply pleiotropically physiological as in free-living bacteria such as Klebsiella pneumoniae and Pseudomonas putida.     

Identification of a 47 kDa fibronectin-binding protein expressed by Borrelia burgdorferi isolate B31

The attachment of pathogenic microorganisms to host cells and tissues is often mediated through the expression of surface receptors recognizing components of the extracellular matrix (ECM). Here, we investigate the ability of Borrelia spirochaetes to bind the ECM constituent, fibronectin. Borrelia lysates were separated by SDS–PAGE, transferred to nitrocellulose and probed with alkaline phosphatase-labelled fibronectin (fibronectin-AP). Five of six Borrelia species and four of eight B. burgdorferi sensu lato isolates expressed one or more fibronectin-binding proteins. Borrelia burgdorferi isolate B31 expressed a 47 kDa (P47) fibronectin-binding protein that was localized to the outer envelope based on susceptibility to proteinase K. The interaction of P47 with fibronectin was specific, and the region of fibronectin bound by P47 mapped to the gelatin/collagen binding domain. P47 was purified by affinity chromatography, digested with endoproteinase Lys-C, and the peptide fragments analysed by liquid chromatography/tandem mass spectroscopy. A search of protein databases disclosed that the P47 peptide mass profile matched that predicted for the bbk32 gene product of B. burgdorferi isolate B31. The bbk32 gene was cloned into Escherichia coli , and the ability of recombinant BBK32 to bind fibronectin and inhibit the attachment of B. burgdorferi was demonstrated. The identification of BBK32 as a receptor for fibronectin binding may enhance our understanding of the pathogenesis and chronic nature of Lyme disease.     

A new gene, sigG, encoding a putative alternative sigma factor of Streptomyces coelicolor A3(2)

An oligonucleotide probe encoding a peptide motif conserved in all sigma factors was used to isolate a new gene, sigG, from a Streptomyces coelicolor A3(2) genomic library. The deduced protein of 263 amino acids with an M(r) of 29,422 showed the greatest similarity to the previously identified sporulation sigma factor (sigma F) of Streptomyces coelicolor, and general stress response sigma factor (sigma B) of Bacillus subtilis, mostly in domains suggested to be involved in recognition of -10 and -35 promoter regions. Southern-blot hybridization with DNA from several Streptomyces spp. revealed the presence of a similar gene in all strains tested. Disruption of the S. coelicolor sigG gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin.     

Recombination between genes encoding major outer surface proteins A and B of Borrelia burgdorferi

Borrelia burgdorferi causes Lyme disease, a multisystem illness that can persist in humans for many years. We describe recombination between homologous genes encoding the major outer surface proteins (Osps) A and B of B. burgdorferi which both deletes osp gene sequences and creates chimaeric gene fusions. Recombinant osp genes occur in multiple strains and encode unique proteins that lack some characteristic Osp epitopes. Antigenic variation in Osp through recombination may be relevant to the persistence of B. burgdorferi in an infected host, and has important implications for the utility of OspA and OspB as diagnostic or vaccine candidates for Lyme disease. We also describe Osp variation arising from nonsense mutations and sequence divergence, which may also represent significant sources of Osp polymorphism.     

High-throughput plasmid content analysis of Borrelia burgdorferi B31 by using Luminex multiplex technology

Borrelia burgdorferi, the causative agent of Lyme disease in North America, is an invasive pathogen that causes persistent multiorgan manifestations in humans and other mammals. Genetic studies of this bacterium are complicated by the presence of multiple plasmid replicons, many of which are readily lost during in vitro culture. The analysis of B. burgdorferi plasmid content by plasmid-specific PCR and agarose gel electrophoresis or other existing techniques is informative, but these techniques are cumbersome and challenging to perform in a high-throughput manner. In this study, a PCR-based Luminex assay was developed for determination of the plasmid content of the strain B. burgdorferi B31. This multiplex, high-throughput method allows simultaneous detection of the plasmid contents of many B. burgdorferi strains in a 96-well format. The procedure was used to evaluate the occurrence of plasmid loss in 44 low-passage B. burgdorferi B31 clones and in a library of over 4,000 signature-tagged mutagenesis (STM) transposon mutant clones. This analysis indicated that only 40% of the clones contained all plasmids, with (in order of decreasing frequency) lp5, lp56, lp28-1, lp25, cp9, lp28-4, lp28-2, and lp21 being the most commonly missing plasmids. These results further emphasize the need for careful plasmid analysis in Lyme disease Borrelia studies. Adaptations of this approach may also be useful in the evaluation of plasmid content and chromosomal gene variations in additional Lyme disease Borrelia strains and other organisms with variable genomes and in the correlation of these genetic differences with pathogenesis and other biological properties.     

Cloning and expression of a gene from Streptomyces scabies encoding a putative pathogenicity factor.

We cloned a 9.4-kb DNA fragment from Streptomyces scabies ATCC 41973 that allows the nonpathogen Streptomyces lividans 66 TK24 to necrotize and colonize potato tuber slices and produce scab-like symptoms on potato minitubers. Deletion analysis demonstrated that activity was conferred by a 1.6-kb DNA region. Sequence analysis of a 2.4-kb DNA fragment spanning the DNA region necessary for activity revealed three open reading frames (ORFs). The deduced amino acid sequence of ORF1, designated ORFtnp, showed high levels of identity with the first 233 amino acids of the putative transposases of the IS1164 elements from Rhodococcus rhodochrous (71%) and Mycobacterium bovis (68%), members of the Staphylococcus aureus IS256 family of transposases. No significant homologies to ORF2 and ORF3 were found in the nucleic acid and protein databases. ORFtnp is located 5' of ORF3. ORF2 is incomplete and is located 3' of ORF3. Subcloning of the individual ORFs demonstrated that ORF3, designated nec1, is sufficient for necrotizing activity in S. lividans 66 TK24. S. lividans 66 TK24 expressing nec1 does not produce thaxtomin A but produces an unidentified extracellular water-soluble compound that causes necrosis on potato tuber discs. The G+C content of nec1 suggests that it has moved horizontally from another genus. Southern analysis of ORFtnp and nec1 demonstrate that these genes are physically linked in Streptomyces strains, including S. scabies and Streptomyces acidiscabies strains, that are pathogenic on potato and that produce the phytotoxin thaxtomin A. These data suggest that nec1 may have been mobilized into S. scabies through a transposition event mediated by ORFtnp.     

Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi

Borrelia burgdorferi is a spirochete pathogen transmitted among warm- blooded hosts by ixodid ticks. Frequency-dependent selection for variant outer-surface proteins might be expected to arise in this species, since rare variants are more likely to avoid immune surveillance in previously infected hosts. We sequenced the OspA and OspB genes of nine North American strains and compared them with nine strains previously described. For each gene, the mean number of synonymous substitutions per synonymous site and the mean number of nonsynonymous substitutions per nonsynonymous site show only a twofold excess of silent mutations. Synonymous rates vary widely along the OspB protein. Some regions show a significant excess of silent substitutions, while divergence in other regions is constrained by biased base composition or selection. The presence, in antigenically important regions of the protein, of significant variation among strains, as well as evidence for recombination among strains, should be considered in attempts to develop vaccines against this disease.      

Disruption of the Borrelia burgdorferi gac gene, encoding the naturally synthesized GyrA C-terminal domain

The C-terminal domain of the A subunit of DNA gyrase, which we term Gac, is naturally synthesized in Borrelia burgdorferi as an abundant DNA-binding protein. Full-length GyrA, which includes the C-terminal domain, is also synthesized by the spirochete and functions as a subunit of DNA gyrase. We have disrupted synthesis of Gac as an independent protein and demonstrated that it is not essential for growth in a coumarin-resistant background. We detected no alterations in DNA maintenance, condensation, or topology in B. burgdorferi lacking this small DNA-binding protein.     

A second allele of eppA in Borrelia burgdorferi strain B31 is located on the previously undetected circular plasmid cp9-2

Although sequence analysis of Borrelia burgdorferi isolate B31 was recently declared "complete," we found that cultures of this strain can contain a novel 9-kb circular plasmid, cp9-2. The newly described plasmid contains both sequence similarities with and differences from the previously identified B31 plasmid cp9-1 (formerly cp9). cp9-1 and cp9-2 each encode a unique allele of EppA, a putative membrane protein synthesized by B. burgdorferi during mammalian infection.