Borrelia burgdorferi rel is responsible for generation of guanosine-3'-diphosphate-5'-triphosphate and growth control

Abstract

The global transcriptional regulator (p)ppGpp (guanosine-3'-diphosphate-5'-triphosphate and guanosine-3',5'-bisphosphate, collectively) produced by the relA and spoT genes in Escherichia coli allows bacteria to adapt to different environmental stresses. The genome of Borrelia burgdorferi encodes a single chromosomal rel gene (BB0198) (B. burgdorferi rel [rel(Bbu)]) homologous to relA and spoT of E. coli. Its role in (p)ppGpp synthesis, bacterial growth, and modulation of gene expression has not been studied in detail. We constructed a rel(Bbu) deletion mutant in an infectious B. burgdorferi 297 strain and isolated an extrachromosomally complemented derivative of this mutant. The mutant did not synthesize rel(Bbu) mRNA, Rel(Bbu) protein, or (p)ppGpp. This synthesis was restored in the complemented derivative, confirming that rel(Bbu) is necessary and sufficient for (p)ppGpp synthesis and degradation in B. burgdorferi. The rel(Bbu) mutant grew well during log phase in complete BSK-H but reached lower cell concentrations in the stationary phase than the wild-type parent, suggesting that (p)ppGpp may be an important factor in the ability of B. burgdorferi to adapt to stationary phase. Deletion of rel(Bbu) did not eliminate the temperature-elicited OspC shift, nor did it alter bmp gene expression or B. burgdorferi antibiotic susceptibility. Although deletion of rel(Bbu) eliminated B. burgdorferi virulence for mice, which was not restored by complementation, we suggest that rel(Bbu)-dependent accumulation of (p)ppGpp may be important for in vivo survival of this pathogen.

FIG. 1.

Production of the B. burgdorferi 297 rel_Bbu deletion mutation by replacement of central region of rel_Bbu with the kanamycin resistance gene aph(3′)-IIIa (Km) from Enterococcus faecalis (43). Numbers showing the positions of nucleotides on B. burgdorferi chromosome indicate the start and the end of rel_Bbu gene and positions of flanking rel_Bbu fragments used in inactivation construction. Primers used for analysis of rel_Bbu deletion are shown by arrows. wt, wild type.

FIG. 2.

Confirmation of rel_Bbu deletion and complementation in B. burgdorferi. (A) PCR analysis of the B. burgdorferi wild type (wt), rel_Bbu mutant (Δrel_Bbu), vector-transformed rel_Bbu mutant (Δrel_Bbu/pKFSS1), and complemented mutant (Δrel_Bbu/pKFSS1-rel_Bbu) with primers R/SpPROT1S and R/SpPROT1EndA (Fig. 1; Table 1). −, reaction with DNA omitted. (B) RT-PCR analysis of mRNA expression for BB0199 gene in the B. burgdorferi wild-type (wt) and rel_Bbu mutant (Δrel_Bbu). −, reaction with mRNA omitted; +RT, reaction with reverse transcriptase; −RT, reaction with reverse transcriptase omitted. (C) BamHI-PstI-digested plasmid DNA isolated from E. coli transformed with DNA from vector-transformed (Δrel_Bbu/pKFSS1) or complemented (Δrel_Bbu/pKFSS1-rel_Bbu) rel_Bbu mutant and BamHI-PstI-digested plasmids used for original transformation of B. burgdorferi (pKFSS1, pKFSS1-rel_Bbu). (D) Competitive PCR analysis of rel_Bbu gene copy number between B. burgdorferi wild-type (wt) and complemented rel_Bbu mutant (Δrel_Bbu/pKFSS1-rel_Bbu). Reaction mixtures contained the same amount of total B. burgdorferi DNA and six serial twofold dilutions of competitor (lanes 1 to 6). Lane 4 shows equal signal intensities from target DNA (upper band) and competitor (lower band) for the wild type and the complemented rel_Bbu mutant, indicating that they are present in equal concentrations in this reaction mixture. −, negative control.

FIG. 3.

rel_Bbu mRNA synthesis in the B. burgdorferi wild type (wt), rel_Bbu mutant (Δrel_Bbu), vector-transformed rel_Bbu mutant (Δrel_Bbu/pKFSS1), and complemented mutant (Δrel_Bbu/pKFSS1-rel_Bbu). RT-PCR for constitutively expressed flaB gene was used as a control to show the presence of RNA in a sample. −, RT-PCR in the absence of mRNA; +RT, reaction containing reverse transcriptase; −RT, reaction in the absence of reverse transcriptase. Arrows indicate PCR fragments corresponding to rel_Bbu or flaB mRNA.

FIG. 4.

Rel_Bbu protein synthesis in the B. burgdorferi wild type (wt), rel_Bbu mutant (Δrel_Bbu), vector-transformed rel_Bbu mutant (Δrel_Bbu/pKFSS1), and complemented mutant (Δrel_Bbu/pKFSS1-rel_Bbu). (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver stain) of total B. burgdorferi lysates shows that the load of total protein is similar for each lane. (B) Western blot analysis with anti-Rel_Bbu antibodies. (C) Relative Rel_Bbu band intensities estimated from six independent Western blots and presented as means ± standard deviations.

FIG. 5.

(p)ppGpp synthesis in the B. burgdorferi wild type (wt), rel_Bbu mutant (Δrel_Bbu), vector-transformed rel_Bbu mutant (Δrel_Bbu/pKFSS1), and complemented mutant (Δrel_Bbu/pKFSS1-rel_Bbu). (A) One-dimensional TLC; (B) two-dimensional TLC. The identities of B. burgdorferi pppGpp and ppGpp were determined by comigration on chromatograms with (p)ppGpp of E. coli (3). Pi, inorganic phosphate.

FIG. 6.

Growth of the B. burgdorferi wild type (wt), rel_Bbu mutant (Δrel_Bbu), vector-transformed rel_Bbu mutant (Δrel_Bbu/pKFSS1), and complemented mutant (Δrel_Bbu/pKFSS1-rel_Bbu) in BSK-H at 34°C. (A) Growth curves of the B. burgdorferi wild type (closed circles), rel_Bbu mutant (open squares), vector-transformed rel_Bbu mutant (open diamonds), and complemented mutant (closed squares). The numbers of cells/ml of culture were calculated from the results from two independent experiments in duplicate and presented as means ± standard deviations for the corresponding day of incubation. (B) Survival index (as a percentage) = viable cells/ml_{Day 30} (by limiting dilution)/total cells/ml_{day 14} (by microscopy) × 100. The survival index is presented as the mean ± standard deviation calculated from the results from two independent experiments in duplicate.

FIG. 7.

Temperature shift in the B. burgdorferi wild type (wt), rel_Bbu mutant (Δrel_Bbu), vector-transformed rel_Bbu mutant (Δrel_Bbu/pKFSS1), and complemented mutant (Δrel_Bbu/pKFSS1-rel_Bbu). (A) Western blots for FlaB, OspA, and OspC in B. burgdorferi wild-type and rel_Bbu mutant mid-log-phase cultures in BSK-H at 23°C, 34°C, and 37°C. OspC levels in the B. burgdorferi wild type and rel_Bbu mutant in mid-log-phase cultures in BSK-H at 23°C, 34°C, and 37°C were estimated from the results from four independent Western blots and presented as means ± standard deviations. (B) Western blots for FlaB and OspC in wild-type, rel_Bbu mutant, and complemented B. burgdorferi from early-stationary-phase cultures grown in BSK-H at 37°C. OspC levels in wild-type, rel_Bbu mutant, and complemented B. burgdorferi from early-stationary-phase cultures grown in BSK-H at 37°C were compared from four different Western blots and presented as means ± standard deviations. Similar intensities of FlaB bands in panels A and B show similar protein loads for each lane.