Infectious cycle analysis of a Borrelia burgdorferi mutant defective in transport of chitobiose, a tick cuticle component.

Chitobiose is the dimer subunit of chitin, a component of tick cuticle and peritrophic matrix, which is not found in mammals. The Borrelia burgdorferi chbC gene is required for the use of chitobiose as a source of the essential nutrient N-acetyl glucosamine during growth in vitro. In order to investigate the role of chitobiose transport in the infectious cycle, we constructed isogenic chbC mutant and wild-type strains in an infectious B. burgdorferi background and confirmed that the mutants were defective in chitobiose utilization. The defect in the mutants was shown to be in chitobiose transport, consistent with the predicted function of the ChbC protein as the membrane component of a phosphotransferase transporter for chitobiose. We then tested whether this locus is also required for any stage of the experimental mouse-tick infectious cycle. We found that both wild-type and mutant bacteria successfully infect both mice and ticks and are transmitted between the two hosts. These results demonstrate that B. burgdorferi growth in vivo is independent of chitobiose transport, even in an environmental niche in which the sugar is likely to be present.

Borrelia burgdorferi OspC protein required exclusively in a crucial early stage of mammalian infection.

This study demonstrates a strict temporal requirement for a virulence determinant of the Lyme disease spirochete Borrelia burgdorferi during a unique point in its natural infection cycle, which alternates between ticks and small mammals. OspC is a major surface protein produced by B. burgdorferi when infected ticks feed but whose synthesis decreases after transmission to a mammalian host. We have previously shown that spirochetes lacking OspC are competent to replicate in and migrate to the salivary glands of the tick vector but do not infect mice. Here we assessed the timing of the requirement for OspC by using an ospC mutant complemented with an unstable copy of the ospC gene and show that B. burgdorferi's requirement for OspC is specific to the mammal and limited to a critical early stage of mammalian infection. By using this unique system, we found that most bacterial reisolates from mice persistently infected with the initially complemented ospC mutant strain no longer carried the wild-type copy of ospC. Such spirochetes were acquired by feeding ticks and migrated to the tick salivary glands during subsequent feeding. Despite normal behavior in ticks, these ospC mutant spirochetes did not infect naive mice. ospC mutant spirochetes from persistently infected mice also failed to infect naive mice by tissue transplantation. We conclude that OspC is indispensable for establishing infection by B. burgdorferi in mammals but is not required at any other point of the mouse-tick infection cycle.

Relapsing fever spirochaetes produce a serine protease that provides resistance to oxidative stress and killing by neutrophils.

The spirochaetes that cause tick-borne relapsing fever and Lyme disease are closely related human pathogens, yet they differ significantly in their ecology and pathogenicity. Genome sequencing of two species of relapsing fever spirochaetes, Borrelia hermsii and Borrelia turicatae, identified a chromosomal open reading frame, designated bhpA, not present in the Lyme disease spirochaete Borrelia burgdorferi. The predicted amino acid sequence of bhpA was homologous with the HtrA serine proteases, which are involved with stress responses and virulence in other bacteria. B. hermsii produced an active serine protease that was recognized by BhpA antibodies and the recombinant BhpA protein-degraded beta-casein. bhpA was transcribed in vitro at all growth temperatures and transcription levels were slightly elevated at higher temperatures. These results correlated with the synthesis of BhpA during B. hermsii infection in mice. With the exception of Borrelia recurrentis, the bhpA gene, protein and enzymatic activity were found in all relapsing fever spirochaetes, but not in Lyme disease or related spirochaetes. Heterologous expression of bhpA in B. burgdorferi increased the spirochaete's resistance to both oxidative stress and killing by human neutrophils. Therefore, we propose that bhpA encodes a unique and functional serine protease in relapsing fever spirochaetes. This periplasmic enzyme may prevent the accumulation of proteins damaged by the innate immune response and contribute to the ability of the relapsing fever spirochaetes to achieve high cell densities in blood.

Genome-wide transposon mutagenesis of Borrelia burgdorferi for identification of phenotypic mutants.

The spirochete Borrelia burgdorferi is the causative agent of Lyme disease, the leading vector-borne illness in the United States. Many of the genetic factors affecting spirochete morphology and physiology are unknown due to the limited genetic tools available and the large number of open reading frames with unknown functions. By adapting a mariner transposon to function in B. burgdorferi, we have developed a random mutagenesis system that tags the mutated locus for rapid identification. Transposition occurs at saturating levels in B. burgdorferi and appears to be random, targeting both linear and circular replicons. By combining the transposon system with a screen for factors affecting growth rate, mutations were readily identified in genes putatively involved in cell division and chemotaxis and a hypothetical open reading frame involved in outer membrane integrity. The successful adaptation of a mariner transposon to function in B. burgdorferi should aid in identifying virulence factors and novel gene products related to spirochete physiology.

Kinetic and microcalorimetric analysis of substrate and cofactor interactions in epoxyalkane:CoM transferase, a zinc-dependent epoxidase.

Epoxyalkane:CoM transferase (EaCoMT) is a key enzyme of bacterial propylene metabolism, catalyzing the nucleophilic attack of coenzyme M (CoM, 2-mercaptoethanesulfonic acid) on epoxypropane to form the thioether conjugate 2-hydroxypropyl-CoM. The biochemical and molecular properties of EaCoMT suggest that the enzyme belongs to the family of alkyltransferase enzymes for which Zn plays a key role in activating an organic thiol substrate for nucleophilic attack on an alkyl-donating substrate. In the present work, the role of Zn in the EaCoMT-catalyzed reactions is established by removing Zn from EaCoMT, resulting in loss of catalytic activity that was restored upon addition of Zn back to the enzyme, and by expressing an inactive and Zn-deficient form of the enzyme that was activated by addition of ZnCl(2) or CoCl(2). Site-directed mutagenesis of one of the predicted Zn ligands (C220A) resulted in the formation of a largely catalytically inactive protein (0.06% of wild-type activity) that, when purified, contained a substoichiometric complement of Zn. EaCoMT was kinetically characterized and found to follow a random sequential mechanism with kinetic parameters K(m,epoxypropane) = 1.8 microM, K(m,CoM) = 34 microM, and k(cat) = 6.5 s(-1). The CoM analogues 2-mercaptopropionate, 2-mercaptoethanol, and cysteine substituted poorly for CoM as the thiol substrate, with specific rates of epoxyalkane conjugation that were at best 0.6% of the CoM-dependent rate, while ethanethiol, propanethiol, glutathione, homocysteine, and lipoic acid provided no activity. 2-Mercaptoethanol was a weak competitive inhibitor vs CoM with a K(I) of 192 mM. Isothermal titration calorimetry was used to investigate the thermodynamic binding determinants for the interaction of CoM and analogues with holo, Zn-deficient, and C220A EaCoMT variants. The stoichiometry of CoM binding correlated directly with the Zn content rather than monomer content of protein samples, reinforcing the importance of Zn in CoM binding. The binding of CoM to EaCoMT occurred with DeltaG = -7.5 kcal/mol (K(d) = 3.8 microM) and was driven by a large release of enthalpy. The thermodynamic contributors (K(a), DeltaG, DeltaH, DeltaS) to the individual binding of CoM, ethanesulfonate, and ethanethiol were determined and used to assess the contributions of the thiol, alkyl, and sulfonate moieties to total binding energy in the E x CoM binary complex.

Pleiotropic effects of inactivating a carboxyl-terminal protease, CtpA, in Borrelia burgdorferi.

A gene encoding a putative carboxyl-terminal protease (CtpA), an unusual type of protease, is present in the Borrelia burgdorferi B31 genome. The B. burgdorferi CtpA amino acid sequence exhibits similarities to the sequences of the CtpA enzymes of the cyanobacterium Synechocystis sp. strain PCC 6803 and higher plants and also exhibits similarities to the sequences of putative CtpA proteins in other bacterial species. Here, we studied the effect of ctpA gene inactivation on the B. burgdorferi protein expression profile. Total B. burgdorferi proteins were separated by two-dimensional gel electrophoresis, and the results revealed that six proteins of the wild type were not detected in the ctpA mutant and that nine proteins observed in the ctpA mutant were undetectable in the wild type. Immunoblot analysis showed that the integral outer membrane protein P13 was larger and had a more acidic pI in the ctpA mutant, which is consistent with the theoretical change in pI for P13 not processed at the carboxyl terminus. Matrix-assisted laser desorption ionization-time of flight data indicated that in addition to P13, the BB0323 protein may serve as a substrate for carboxyl-terminal processing by CtpA. Complementation analysis of the ctpA mutant provided strong evidence that the observed effect on proteins depended on inactivation of the ctpA gene alone. We show that CtpA in B. burgdorferi is involved in the processing of proteins such as P13 and BB0323 and that inactivation of ctpA has a pleiotropic effect on borrelial protein synthesis. To our knowledge, this is the first analysis of both a CtpA protease and different substrate proteins in a pathogenic bacterium.

Biochemical, molecular, and genetic analyses of the acetone carboxylases from Xanthobacter autotrophicus strain Py2 and Rhodobacter capsulatus strain B10.

Acetone carboxylase is the key enzyme of bacterial acetone metabolism, catalyzing the condensation of acetone and CO(2) to form acetoacetate. In this study, the acetone carboxylase of the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus was purified to homogeneity and compared to that of Xanthobacter autotrophicus strain Py2, the only other organism from which an acetone carboxylase has been purified. The biochemical properties of the enzymes were virtually indistinguishable, with identical subunit compositions (alpha(2)beta(2)gamma(2) multimers of 85-, 78-, and 20-kDa subunits), reaction stoichiometries (CH(3)COCH(3) + CO(2) + ATP-->CH(3)COCH(2)COO(-) + H(+) + AMP + 2P(i)), and kinetic properties (K(m) for acetone, 8 microM; k(cat) = 45 min(-1)). Both enzymes were expressed to high levels (17 to 25% of soluble protein) in cells grown with acetone as the carbon source but were not present at detectable levels in cells grown with other carbon sources. The genes encoding the acetone carboxylase subunits were identified by transposon mutagenesis of X. autotrophicus and sequence analysis of the R. capsulatus genome and were found to be clustered in similar operons consisting of the genes acxA (beta subunit), acxB (alpha subunit), and acxC (gamma subunit). Transposon mutagenesis of X. autotrophicus revealed a requirement of sigma(54) and a sigma(54)-dependent transcriptional activator (AcxR) for acetone-dependent growth and acetone carboxylase gene expression. A potential sigma(54)-dependent promoter 122 bp upstream of X. autotrophicus acxABC was identified. An AcxR gene homolog was identified 127 bp upstream of acxA in R. capsulatus, but this activator lacked key features of sigma(54)-dependent activators, and the associated acxABC lacked an apparent sigma(54)-dependent promoter, suggesting that sigma(54) is not required for expression of acxABC in R. capsulatus. These studies reveal a conserved strategy of ATP-dependent acetone carboxylation and the involvement of transcriptional enhancers in acetone carboxylase gene expression in gram-negative acetone-utilizing bacteria.

New antibiotic resistance cassettes suitable for genetic studies in Borrelia burgdorferi.

In this report we describe two distinct approaches to develop new antibiotic resistance cassettes that allow for efficient selection of Borrelia burgdorferi transformants. The first approach utilizes fusions of borrelial flagellar promoters to antibiotic resistance markers from other bacteria. The AACC1 gene, which encodes a gentamicin acetyltransferase, conferred a high level of gentamicin resistance in B. Burfdorferi when expressed from these promoters. No cross-resistance occurred between this cassette and the kanamycin resistance cassette, which was previously developed in an analogous fashion. A second and different approach was taken to develop an efficient selectable marker that confers resistance to the antibiotic coumermycin A1. A synthetic gene was designed from the GYRB301 allele of the coumermycin-resistant B. Burgdorferi strain B31-NGR by altering the coding sequence at the wobble position. The resulting gene, GYRB(SYN), encodes a protein identical to the product of GYRB301, but the genes share only 66% nucleotide identity. The nucleotide sequence of GYRB(SYN)is sufficiently divergent from the endogenous B. Burgdorferi GYRB gene to prevent recombination between them. The cassettes described in this paper improve our repertoire of genetic tools in B. Burgdorferi. These studies also provide insight into parameters governing recombination and gene expression in B. Burgdorferi.

Outer-surface protein C of the Lyme disease spirochete: a protein induced in ticks for infection of mammals.

Environmentally responsive synthesis of surface proteins represents a hallmark of the infectious cycle of the Lyme disease agent, Borrelia burgdorferi. Here we created and analyzed a B. burgdorferi mutant lacking outer-surface protein C (OspC), an abundant Osp that spirochetes normally synthesize in the tick vector during the blood meal and down-regulate after transmission to the mammal. We demonstrate that B. burgdorferi strictly requires OspC to infect mice but not to localize or migrate appropriately in the tick. The induction of a spirochetal virulence factor preceding the time and host in which it is required demonstrates a developmental sequence for transmission of this arthropod-borne pathogen.