Changes in Bacterial Growth Rate Govern Expression of the \u3cem\u3eBorrelia burgdorferi\u3c/em\u3e OspC and Erp Infection-Associated Surface Proteins

The Lyme disease spirochete controls production of its OspC and Erp outer surface proteins, repressing protein synthesis during colonization of vector ticks but increasing expression when those ticks feed on vertebrate hosts. Early studies found that the synthesis of OspC and Erps can be stimulated in culture by shifting the temperature from 23°C to 34°C, leading to a hypothesis that Borrelia burgdorferi senses environmental temperature to determine its location in the tick-mammal infectious cycle. However, borreliae cultured at 34°C divide several times faster than do those cultured at 23°C. We developed methods that disassociate bacterial growth rate and temperature, allowing a separate evaluation of each factor\u27s impacts on B. burgdorferi gene and protein expression. Altogether, the data support a new paradigm that B. burgdorferi actually responds to changes in its own replication rate, not temperature per se, as the impetus to increase the expression of the OspC and Erp infection-associated proteins

Eubacterial SpoVG homologs constitute a new family of site-specific DNA-binding proteins

A site-specific DNA-binding protein was purified from Borrelia burgdorferi cytoplasmic extracts, and determined to be a member of the highly conserved SpoVG family. This is the first time a function has been attributed to any of these ubiquitous bacterial proteins. Further investigations into SpoVG orthologues indicated that the Staphylococcus aureus protein also binds DNA, but interacts preferentially with a distinct nucleic acid sequence. Site-directed mutagenesis and domain swapping between the S. aureus and B. burgdorferi proteins identified that a 6-residue stretch of the SpoVG α-helix contributes to DNA sequence specificity. Two additional, highly conserved amino acid residues on an adjacent β-sheet are essential for DNA-binding, apparently by contacts with the DNA phosphate backbone. Results of these studies thus identified a novel family of bacterial DNA-binding proteins, developed a model of SpoVG-DNA interactions, and provide direction for future functional studies on these wide-spread proteins

\u3cem\u3eBorrelia burgdorferi\u3c/em\u3e cp32 BpaB Modulates Expression of the Prophage NucP Nuclease and SsbP Single-Stranded DNA-Binding Protein

The Borrelia burgdorferi BpaB proteins of the spirochete\u27s ubiquitous cp32 prophages are DNA-binding proteins, required both for maintenance of the bacteriophage episomes and for transcriptional regulation of the cp32 erp operons. Through use of DNase I footprinting, we demonstrate that BpaB binds the erp operator initially at the sequence 5′-TTATA-3′. Electrophoretic mobility shift assays indicated that BpaB also binds with high affinity to sites located in the 5′ noncoding regions of two additional cp32 genes. Characterization of the proteins encoded by those genes indicated that they are a single-stranded DNA-binding protein and a nuclease, which we named SsbP and NucP, respectively. Chromatin immunoprecipitation indicated that BpaB binds erp, ssbP, and nucP in live B. burgdorferi. A mutant bacterium that overexpressed BpaB produced significantly higher levels of ssbP and nucP transcript than did the wild-type parent

\u3cem\u3eBorrelia burgdorferi\u3c/em\u3e SpoVG DNA- and RNA-Binding Protein Modulates the Physiology of the Lyme Disease Spirochete

The SpoVG protein of Borrelia burgdorferi, the Lyme disease spirochete, binds to specific sites of DNA and RNA. The bacterium regulates transcription of spoVG during the natural tick-mammal infectious cycle and in response to some changes in culture conditions. Bacterial levels of spoVG mRNA and SpoVG protein did not necessarily correlate, suggesting that posttranscriptional mechanisms also control protein levels. Consistent with this, SpoVG binds to its own mRNA, adjacent to the ribosome-binding site. SpoVG also binds to two DNA sites in the glpFKD operon and to two RNA sites in glpFKD mRNA; that operon encodes genes necessary for glycerol catabolism and is important for colonization in ticks. In addition, spirochetes engineered to dysregulate spoVG exhibited physiological alterations

BpaB, a Novel Protein Encoded by the Lyme Disease Spirochete\u27s Cp32 Prophages, Binds to Erp Operator 2 DNA

Borrelia burgdorferi produces Erp outer surface proteins throughout mammalian infection, but represses their synthesis during colonization of vector ticks. A DNA region 5′ of the start of erp transcription, Operator 2, was previously shown to be essential for regulation of expression. We now report identification and characterization of a novel erp Operator 2-binding protein, which we named BpaB. erp operons are located on episomal cp32 prophages, and a single bacterium may contain as many as 10 different cp32s. Each cp32 family member encodes a unique BpaB protein, yet the three tested cp32-encoded BpaB alleles all bound to the same DNA sequence. A 20-bp region of erp Operator 2 was determined to be essential for BpaB binding, and initial protein binding to that site was required for binding of additional BpaB molecules. A 36-residue region near the BpaB carboxy terminus was found to be essential for high-affinity DNA-binding. BpaB competed for binding to erp Operator 2 with a second B. burgdorferi DNA-binding protein, EbfC. Thus, cellular levels of free BpaB and EbfC could potentially control erp transcription levels

BpaB, a novel protein encoded by the Lyme disease spirochete’s cp32 prophages, binds to erp Operator 2 DNA

Borrelia burgdorferi produces Erp outer surface proteins throughout mammalian infection, but represses their synthesis during colonization of vector ticks. A DNA region 5′ of the start of erp transcription, Operator 2, was previously shown to be essential for regulation of expression. We now report identification and characterization of a novel erp Operator 2-binding protein, which we named BpaB. erp operons are located on episomal cp32 prophages, and a single bacterium may contain as many as 10 different cp32s. Each cp32 family member encodes a unique BpaB protein, yet the three tested cp32-encoded BpaB alleles all bound to the same DNA sequence. A 20-bp region of erp Operator 2 was determined to be essential for BpaB binding, and initial protein binding to that site was required for binding of additional BpaB molecules. A 36-residue region near the BpaB carboxy terminus was found to be essential for high-affinity DNA-binding. BpaB competed for binding to erp Operator 2 with a second B. burgdorferi DNA-binding protein, EbfC. Thus, cellular levels of free BpaB and EbfC could potentially control erp transcription levels

Alanine mutagenesis determined residues required for binding DNA.

<p>Lanes 1, 2, 5 and 6 contain 2 nM labeled <i>vlsE</i> probe. Lanes 3,4, and 7–12 contain 2 nM labeled <i>cap41</i> probe. Additional ingredients of each EMSA are: Lane 2, 1.5 µM mutant SpoVG<i>_Bb</i> R53A-R54A; Lane 4, 1.5 µM mutant SpoVG<i>_Sa</i> K50A-R51A; Lane 6, 500 nM wild-type SpoVG<i>_Bb</i>; Lane 8, 1.5 µM mutant SpoVG<i>_Sa</i> K50A: Lane 10, 1.5 µM mutant SpoVG<i>_Sa</i> R51A; Lane 12, 500 µM wild-type SpoVG<i>_Sa</i>.</p

Site directed mutagenesis did not influence SpoVG oligomerization.

<p>Results of panels A through D illustrate HPLC sizing column chromatography of wild type and mutant SpoVG<i>_Sa</i> proteins. For some preparations, proteins eluted with a retention time of approximately 7 minutes, which corresponds with a molecular mass >440 kDa and are composed of protein aggregates. (<b>A</b>) Wild-type SpoVG<i>_Sa</i>; (<b>B</b>) SpoVG<i>_Sa</i> K50A-R51A; (<b>C</b>) SpoVG<i>_Sa</i> K50A; (<b>D</b>) SpoVG<i>_Sa</i> R51A. Peaks marked with red asterisks indicate retention volumes corresponding with approximately 55–60 kDa. Panels E and F illustrates proteins separated following native of denaturing PAGE, respectively. M, Molecular mass standards; Lane 1, 1.5 µM mutant SpoVG<i>_Sa</i> K50A-R51A; Lane 2, 1 µM mutant SpoVG<i>_Sa</i> K50A; Lane 3, 1 µM mutant SpoVG<i>_Sa</i> R51A; Lane 4, 1 µM wild-type SpoVG<i>_Sa</i>.</p

DNA-affinity chromatographically purified SpoVG<i>_Bb</i>. <i>vlsE</i> probe affixed to magnetic beads was incubated with cell-free extracts and bound protein were eluted by titration with increasing concentrations of NaCl.

<p>Eluates were separated by SDS PAGE, stained with Sypro Ruby. M. Molecular mass standards. Lanes 1–3. Proteins eluted with 500, 750, and 1000 mM NaCl elution, respectively.</p