BmpA Is a Surface-Exposed Outer-Membrane Protein of Borrelia Burgdorferi

BmpA is an immunodominant protein of Borrelia burgdorferi as well as an arthritogenic factor. Rabbit antirecombinant BmpA (rBmpA) antibodies were raised, characterized by assaying their cross reactivity with rBmpB, rBmpC and rBmpD, and then rendered monospecific by absorption with rBmpB. This monospecific reagent reacted only with rBmpA in dot immunobinding and detected a single 39 kDa, pI 5.0, spot on two-dimensional immunoblots. It was used to assess the BmpA cellular location. BmpA was present in both detergent-soluble and -insoluble fractions of Triton X-114 phase-partitioned borrelial cells, suggesting that it was a membrane lipoprotein. Immunoblots of proteinase K-treated intact and Triton X-100 permeabilized cells showed digestion of BmpA in intact cells, consistent with surface exposure. This exposure was confirmed by dual-label immunofluorescence microscopy of intact and permeabilized borrelial cells. Conservation and surface localization of BmpA in all B. burgdorferi sensu lato genospecies could point to its playing a key role in this organism\u27s biology and pathobiology

Functional Analysis of Borrelia Burgdorferi uvrA in DNA Damage Protection

Bacterial pathogens face constant challenges from DNA-damaging agents generated by host phagocytes. Although Borrelia burgdorferi appears to have much fewer DNA repair enzymes than pathogens with larger genomes, it does contain homologues of uvrA and uvrB (subunits A and B of excinuclease ABC). As a first step to exploring the physiologic function of uvrA(Bbu) and its possible role in survival in the host in the face of DNA-damaging agents, a partially deleted uvrA mutant was isolated by targeted inactivation. While growth of this mutant was markedly inhibited by UV irradiation, mitomycin C (MMC) and hydrogen peroxide at doses that lacked effect on wild-type B. burgdorferi, its response to pH 6.0-6.8 and reactive nitrogen intermediates was similar to that of the wild-type parental strain. The sensitivity of the inactivation mutant to UV irradiation, MMC and peroxide was complemented by an extrachromosomal copy of uvrA(Bbu). We conclude that uvrA(Bbu) is functional in B. burgdorferi

Rational Design of a Plasmid Origin That Replicates Efficiently in Both Gram-Positive and Gram-Negative Bacteria

Background: Most plasmids replicate only within a particular genus or family

Oligonucleotides and PCR primers used in the study.

<p>Oligonucleotides and PCR primers used in the study.</p

Heterologous expression of <i>lux</i> operon from pBAV1K-T5-<i>luxABCDE</i>.

<p>Five bacterial species (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013244#pone-0013244-g002" target="_blank">Fig. 2</a>) were transformed with pBAV1K-T5-<i>lux</i>. The transformants were propagated in liquid culture (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013244#pone-0013244-t003" target="_blank">Table 3</a>). The luminescence from equal volumes of culture (100 microliters) was measured in a luminometer. The values (plotted on a log scale) represent the averages of three independent experiments.</p

Plasmid stability assays.

<p>(A) Plasmids pBAV1K-T5-<i>gfp</i>, pGK12, pLZ12-T5-<i>gfp</i>, pQBAV3Cm-T5-<i>gfp</i> and pIMBB-T5-<i>gfp</i> were separately propagated in <i>E. coli</i> for 80 generations without antibiotic selection. Plasmid stability was determined by replica plating onto selective media and presented as a percentage of cells that retain antibiotic resistance. (B) Plasmids pBAV1K-T5-<i>gfp</i>, pGK12, pLZ12-T5-<i>gfp</i>, were propagated in <i>B. subtilis</i> for 80 generations without antibiotic selection. Plasmid stability was determined by the plasmid content comparison in the total DNA pools between different generations. Error bars represent standard error.</p

Bacterial strains and transformation procedures used in the study.

<p>Bacterial strains and transformation procedures used in the study.</p

Plasmid pBAV1K-T5-<i>gfp</i> replicates to high copy number in <i>Escherichia coli</i>.

<p>(A) <i>E. coli</i> was transformed with plasmids pBAV1K-T5-<i>gfp</i>, pLZ12-T5-<i>gfp</i>, pGK12 (two other RCR plasmids), pQBAV3Cm-T5-<i>gfp</i> or pIMBB-T5-<i>gfp</i> (two ColE1 derived plasmids). The transformants were propagated in liquid LB cultures supplemented with the appropriate antibiotics. The plasmids were purified, and 2 microliters of each were analyzed on a 0.8% agarose gel. The higher yield and faster mobility of the pBAV1k relative to the larger pWV01 derivatives indicates supercoiling. (B) Five different species of bacteria (namely <i>Agrobacterium tumefaciens</i>, <i>Streptococcus pneumoniae</i>, <i>Bacillus subtilis</i>, <i>Acinetobacter baylyi</i> ADP1 and <i>E. coli</i>) were transformed with pBAV1K-T5-<i>luxABCDE</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013244#pone-0013244-t003" target="_blank">Table 3</a>). The APH(3′)-IIIa gene present on the plasmid was used as a target to estimate the copy number in reference to the chromosomal <i>relA</i>/<i>spoT</i> gene (or its homolog) by quantitative real-time PCR. Each bar represents the average of three replicates. Error bars represent standard error.</p

Probes and Primers for Real Time PCR used in the study.

<p>Probes and Primers for Real Time PCR used in the study.</p

Construction of pBAV1K-T5-<i>lux</i>, a very broad host range expression vector.

<p>The cryptic plasmid, pWV01, exhibits broad host range but is unstable in many species. The ORF D, and inverted repeats IV, V and VI were deleted from its plasmid origin; terminators t0 and T1 were inserted on opposite ends of the shortened origin (upper right). The selectable marker, the <i>Enterococcus</i> 3′,5″-aminoglycoside phosphotransferase type III, and a T5 promoter within a BioBrick multiple cloning site were cloned into the plasmid (top circle). The lux genes of <i>Photorhabdus luminescens</i> were individually PCR amplified, cloned, assembled with ribosome binding sites (middle) and cloned into the plasmid to create pBAV1k-T5-<i>luxABCDE</i> (bottom circle).</p