Characterization of the stringent response and rel(Bbu) expression in Borrelia burgdorferi

Abstract

The stringent response is a global bacterial response to nutritional stress mediated by (p)ppGpp. We previously found that both noninfectious Borrelia burgdorferi strain B31 and infectious B. burgdorferi strain N40 produced large amounts of (p)ppGpp during growth in BSK-H medium and suggested that the stringent response was triggered in B. burgdorferi under these conditions. Here we report that (p)ppGpp levels in B. burgdorferi growing in BSK-II or BSK-H medium are not further increased by nutrient limitation or by serine hydroxamate-induced inhibition of protein synthesis and that the presence of (p)ppGpp during growth of N40 in BSK-H medium is not associated with decreased 16S rRNA synthesis. Decreased 16S rRNA synthesis was associated with the decreased growth rate of N40 seen during coculture with tick cells, which are growth conditions that were previously shown to decrease (p)ppGpp levels. One-half as much of the mRNA of the gene encoding the Rel protein of B. burgdorferi (rel(Bbu)) was produced by B31 as by N40 during in vitro growth (2 +/- 0.5 and 4 +/- 0.8 fg of rel(Bbu) mRNA/ng of total Borrelia RNA, respectively). Although the amounts of N40 rel(Bbu) mRNA were identical during growth in vitro and in rat peritoneal chambers, they were markedly decreased during growth in nymphal ticks. In contrast to the lack of change in rel(Bbu) mRNA levels, larger amounts of a 78-kDa protein that was cross-reactive with antibodies to Bacillus subtilis Rel(Bsu) were detected in immunoblots of N40 lysates after growth in rat peritoneal chambers than after growth in vitro. Differences in the level of production of (p)ppGpp between B31 and N40 could not be explained by differences in rel(Bbu) promoters since identical transcriptional start sites 309 nucleotides upstream from the B31 and N40 rel(Bbu) ATG start codon and identical sigma(70)-like promoters were identified by primer extension and sequencing analysis. rel(Bbu) complemented an Escherichia coli CF1693 relA spoT double mutant for growth on M9 minimal medium, and the transformed cells produced rel(Bbu) mRNA. These results indicate that rel(Bbu) is functional and that its transcription and translation and production of (p)ppGpp are affected by environmental conditions in strains N40 and B31. They also suggest that in B. burgdorferi, an organism with few rRNA operons that grows slowly, the role of (p)ppGpp may differ from the classic role played by this molecule in E. coli and that (p)ppGpp may not be responsible for growth rate control.

FIG. 1.

Inhibition of growth of B. burgdorferi B31 in BSK-II medium (•) by 0.1 mg of serine hydroxamate per ml (□) and by 1 mg of serine hydroxamate per ml (○). See Materials and Methods for details.

FIG. 2.

(p)ppGpp accumulation in B. burgdorferi under various culture conditions. (A) B. burgdorferi B31 was grown in BSK-II medium containing [³²P]orthophosphate for 36 h and then centrifuged and resuspended in the same amount of BSK-II medium (lanes 1 and 6), in BSK-II medium containing 10-fold-lower levels of rabbit serum (final concentration, 0.6%) (lanes 2 and 7), yeastolate (lanes 3 and 8), or neopeptone (lanes 4 and 9), or in BSK-II medium containing 1 mg of dl-serine hydroxamate per ml (lanes 5 and 10) (all resuspension media contained [³²P]orthophosphate) for 10 min or 1 day. (B) B. burgdorferi N40 was grown in BSK-H medium containing [³²P]orthophosphate for 2 days, centrifuged, and resuspended in the same amount of BSK-H medium (lane 11), in BSK-H medium containing 10-fold-lower levels of rabbit serum (lane 12), or in BSK-H medium containing 1 mg of dl-serine hydroxamate per ml (lane 13) (all resuspension media contained [³²P]orthophosphate) for 1 day. Cells were collected by centrifugation after growth for 10 min or 1 day, and (p)ppGpp was extracted as described in Materials and Methods.

FIG. 3.

RT-PCR analysis of B. burgdorferi rel_Bbu mRNA. (A) Relative concentrations of rel_Bbu mRNA in B. burgdorferi N40 grown in BSK-H medium or in BSK-H medium in rat peritoneal chambers or in B. burgdorferi B31 grown in BSK-H medium. rel_Bbu mRNA was detected by competitive RT-PCR. All reaction mixtures contained 1 ng of B. burgdorferi total RNA. The amounts of competitor were 16, 8, 4, 2, and 1 fg in lanes 1 to 5, respectively. Lane 3 for N40 and lane 4 for B31 showed equal signal intensities for rel_Bbu mRNA (upper band) and the competitor (lower band), indicating that in these reaction mixtures they were present at equal concentrations. In lane C RNA was omitted. (B) rel_Bbu mRNA in B. burgdorferi N40 in BSK-H medium and in ticks. −, reverse transcriptase omitted; +, complete reaction mixture for specific RNA detection.

FIG. 4.

Rel_Bbu protein in B. burgdorferi N40 grown in BSK-H medium at 37°C (lanes 1) or at 23°C (lanes 2) or in rat peritoneal chambers containing BSK-H medium (lanes 3). (A) Silver staining of B. burgdorferi proteins. Downmodulation of OspA and OspB and upregulation of OspC (lane 3) indicated that there was host adaptation of B. burgdorferi in rat peritoneal chambers. (B) Immunoblot analysis of B. burgdorferi proteins with anti-B. burgdorferi FlaB monoclonal antibodies (loading control), anti-B. burgdorferi OspA monoclonal antibodies (host adaptation control), and anti-B. subtilis Rel_Bsu polyclonal antibodies. The positions of protein molecular mass standards are indicated on the right. In the FlaB panel the similar intensities of FlaB in lanes 1, 2, and 3 indicate that the amounts of total B. burgdorferi protein loaded in lanes 1, 2, and 3 were the same. In the OspA panel the absence of OspA in lane 3 is characteristic of host-adapted B. burgdorferi growing in rat peritoneal chambers (; Caimano et al., unpublished).

FIG. 5.

Primer extension analysis of B. burgdorferi B31 (lane B31) and N40 (lane N40) mRNAs and sequencing ladder of the homologous region of the B31 rel_Bbu gene (lanes T, G, C, and A). Products having similar sizes were generated with AMV reverse transcriptase from B31 and N40. The arrow indicates the position of the primer extension products in relation to the sequencing ladder. +1, rel_Bbu transcription site. The putative −10 promoter sequence and the putative −35 promoter sequence are underlined in the nucleotide sequence of the region upstream of rel_Bbu. Homology of the deduced rel_Bbu promoter with the E. coli σ⁷⁰ promoter (21) is indicated by asterisks.

FIG. 6.

Complementation of E. coli CF1693 (relA⁻ spoT⁻) with B. burgdorferi rel_Bbu homologue. (A) E. coli CF1693 was transformed with pBluescript IISK+ (left side) or pBlue-ST plasmid DNA (right side) and grown on a 1.5% agar-M9 medium plate. (B) B. burgdorferi rel_Bbu mRNA synthesis by CF1693 containing pBlue-ST following growth in MOPS starvation medium or LB medium. −, reverse transcriptase omitted; +, complete reaction mixture for specific RNA detection. In lane C RNA was omitted. (C) (p)ppGpp accumulation in E. coli, as determined with wild-type strain CF1648 and the CF1693 relA⁻ spoT⁻ double mutant containing pBlue-ST in MOPS starvation medium or LB medium.