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.

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.

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. burgdorferi 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_synis sufficiently divergent from the endogenous B. burgdorferigyrB 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.

The bacterial metabolism of short-chain aliphatic alkenes occurs via oxidation to epoxyalkanes followed by carboxylation to ␤-ketoacids. Epoxyalkane carboxylation requires four enzymes (components I-IV), NADPH, NAD ؉ , and a previously unidentified nucleophilic thiol. In the present work, coenzyme M (2-mercaptoethanesulfonic acid), a compound previously found only in the methanogenic Archaea where it serves as a methyl group carrier and activator, has been identified as the thiol and central cofactor of aliphatic epoxide carboxylation in the Gram-negative bacterium Xanthobacter strain Py2. Component I catalyzed the addition of coenzyme M to epoxypropane to form a ␤-hydroxythioether, 2-(2-hydroxypropylthio)ethanesulfonate. Components III and IV catalyzed the NAD ؉ -dependent stereoselective dehydrogenation of R-and S-enantiomers of 2-(2-hydroxypropylthio) ethanesulfonate to form 2-(2-ketopropylthio)ethanesulfonate. Component II catalyzed the NADPH-dependent cleavage and carboxylation of the ␤-ketothioether to form acetoacetate and coenzyme M. These findings evince a newfound versatility for coenzyme M as a carrier and activator of alkyl groups longer in chain-length than methane, a function for coenzyme M in a catabolic pathway of hydrocarbon oxidation, and the presence of coenzyme M in the bacterial domain of the phylogenetic tree. These results serve to unify bacterial and Archaeal metabolism further and showcase diverse biological functions for an elegantly simple organic molecule.

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 (␣ 2 ␤ 2 ␥ 2 multimers of 85-, 78-, and 20-kDa subunits), reaction stoichiometries (CH 3 COCH 3 ؉ CO 2 ؉ ATP3CH 3 COCH 2 COO ؊ ؉ H ؉ ؉ AMP ؉ 2P i ), and kinetic properties (K m for acetone, 8 M; 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 (␤ subunit), acxB (␣ subunit), and acxC (␥ subunit). Transposon mutagenesis of X. autotrophicus revealed a requirement of 54 and a 54 -dependent transcriptional activator (AcxR) for acetonedependent growth and acetone carboxylase gene expression. A potential 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 54 -dependent activators, and the associated acxABC lacked an apparent 54 -dependent promoter, suggesting that 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 acetoneutilizing bacteria.In addition to its importance as an industrial solvent, acetone is a major fermentation product of certain anaerobic bacteria (19,51), an intermediate in the microbial metabolism of propane and isopropanol (7,32,49), and one of the ketone bodies produced under ketogenic conditions (i.e., fasting or diabetes) in mammals. Acetone is known to undergo metabolic transformations in mammals, where the physiological importance is not fully understood (4, 25), and in diverse microbes which are capable of growth using acetone as the primary source of carbon and energy (20). The mammalian metabolism of acetone is believed to be mediated largely by cytochrome P450 isozyme 2E1, sequentially producing acetol and methylglyoxal as gluconeogenic intermediates (8,11,28). The carbon atoms originating from acetone are incorporated into glucose in starved mice, suggesting that acetone may be an intermediate in the only mammalian pathway allowing net synthesis of glucose from fatty acids (4,25,26,30).Two distinct transformation...

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.Lyme disease is the leading vector-borne illness in the United States and is caused by the spirochete Borrelia burgdorferi. The class Spirochaetes contains many significant pathogens of humans and other animals, including Treponema pallidum (agent of syphilis), Treponema denticola (associated with periodontal disease), Brachyspira hyodysenteriae (associated with swine dysentery), Brachyspira pilosicoli (associated with porcine intestinal spirochetosis), Leptospira interrogans (associated with leptospirosis), and Borrelia spp. (associated with relapsing fever) (27). However, many of the genetic tools available for members of the Enterobacteriaceae have not yet been developed for the spirochetes, limiting the experimental identification of virulence factors and genes specific to spirochete physiology. Identifying new virulence factors and functions of hypothetical open reading frames (ORFs) in B. burgdorferi has been further hindered by the low efficiency of targeted allelic exchange (for a review, see reference 25).An efficient and random mutagenesis system would facilitate functional identification of the large number of unknown ORFs identified in the genome sequence of B. burgdorferi (8,12). Some transposon systems are capable of achieving saturation mutagenesis and are nearly random in their insertion sites, and the mutated locus is marked by the transposon insertion and therefore easily identified. Specifically, transposons of the mariner family have been used successfully for mutagenesis of a diverse range of organisms, including eukaryotes, archaea, and both gram-positive and gram-negative bacteria (1,14,16,21,29). The mariner elements do not require host cofactors for transposition, likely contributing to their wide host range (15). Further, mariner transposition is virtually random, requiring only a TA dinucleotide for target specificity. In addition, Lampe and colleagues derived hyperactive transposase mutants of Himar1 (a member of the mariner family), increasing...

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.

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.