Figure 1.
Construction and characterization of the hk2 mutant.
(A) Strategy for insertional inactivation of hk2. wt, genomic structure of hk2 and the surrounding region in wild-type B. burgdorferi. pHX-hk2-Kan, the suicide vector used for generating the hk2 mutant. Only the relevant portion of the plasmid is shown. hk2, the diagram showing the genomic structure of the hk2 mutant. Small labeled arrows denote positions of oligonucleotide primers used for PCR analyses. (B) Confirmation of the hk2 mutant by PCR analyses. Letter combinations denote primer pairs used for PCR. kb: kilobase DNA ladder, wt, wild type strain; hk2, hk2 mutant. (C) Immunoblot analyses of the hk2 mutant. Cultures were grown at 35°C to late logarithmic phase (5×107 spirochetes/ml) and subjected to immunoblotting with monoclonal antibodies against Hk2, Rrp2, RpoS, OspC or FlaB (loading control).
Table 1.
Strain description.
Figure 2.
hk2 mutant spirochetes cultivated in DMCs are capable of activating the Rrp2-RpoN-RpoS pathway.
Wild-type (wt), hk2 mutant (hk2), or rpoS mutant (rpoS) spirochetes were cultivated in DMCs, after which whole cell lysates were separated by SDS-PAGE and visualized with silver stain. The bands corresponding to OspA and OspC are indicated by arrowheads on the right. Molecular mass markers in kilodaltons are shown on the left.
Table 2.
Mouse infectivity of the hk2 mutant.
Figure 3.
The hk1 mutant remains capable of activating the Rrp2-RpoN-RpoS pathway.
(A) Strategy for insertional inactivation of hk1. wt, genomic structure of hk1 in wild-type B. burgdorferi. pXY245, the suicide vector used for generating the hk1 mutant. Only the relevant portion of the plasmid is shown. (B) Confirmation of the hk1 mutant by RT-PCR analyses. RT-PCR was performed using primers specific for ospA, hk1, or rrp1 (labeled on the top). kb: the kilobase DNA ladder. RT indicates the absence (-) or presence (+) of reverse transcriptase in the reaction. (C) Production of OspC by the hk1 mutant. Various strains of spirochetes (labeled on the top) were grown at 35°C and harvested in the late logarithmic phase (5×107 spirochetes/ml) and subjected to immunoblot analysis using a mixture of monoclonal antibodies specific for OspC and FlaB, respectively. A strain harboring a G239C point mutation within Rrp2 [18], serves as a negative control for OspC expression. The bands corresponding to OspC or FlaB are indicated by the arrowhead on the right.
Figure 4.
The hk1 hk2 double mutant and the cheA1 or cheA2 mutant have normal level of Rrp2 activation.
Various strains of spirochetes (labeled on the top) were grown at 35°C, harvested in the late logarithmic phase (5×107 spirochetes/ml), and subjected to immunoblot analysis using monoclonal antibodies specific against Hk2, OspC or FlaB as indicated.
Figure 5.
Influence of overexpression of wild-type or mutated version of the Rrp2 N-terminal receiver domain on Rrp2 activation.
(A) Schematic diagram of predicted Rrp2 domain structure and various versions of overexpressed N-terminal receiver domains. D52 is the putative phosphorylation site. (B) Immunoblot of wild-type strain (lane 1), the strain carrying the shuttle vector only (lane 2), the strain with overexpression of Rrp2-N (lane 3), the strain with overexpression of Rrp2-N(D52A) (lane 4), and the strain with overexpression of Rrp2-N(D52E) (lane 5). Cultures were grown to late logarithmic phase at 35°C. Pooled antibodies/antisera against Rrp2, FlaB, and OspC were used. Bands corresponding to each protein were labeled on the right. (C) qRT-PCR analysis of ospC expression in various strains shown in (B). Levels of ospC transcript were normalized per 1000 copies of flaB in each sample.
Table 3.
Mouse infectivity of Borrelia burgdorferi with overproduction of Rrp2-N or Pta.
Figure 6.
Inactivation of the carbamoyl-P biosynthesis pathway does not affect Rrp2 activation.
(A) Diagram of the arginine fermentation pathway in B. burgdorferi. The arcA (bb0841) and arcB (bb0842) genes are predicted to encode arginine deaminase and ornithine carbamoyltransferase, respectively. (B) Immunoblot analysis of whole cell lysates of wild-type (wt), the rrp2 mutant [rrp2(G239C)], and the arcA mutant (arcA) with a mixture of antibodies against OspC and FlaB. Spirochetes were cultured at 35°C and harvested at late logarithmic growth. The bands corresponding to FlaB and OspC are indicated on the right.
Figure 7.
Acetyl∼P plays an important role in Rrp2 activation under in vitro cultivation conditions.
(A) Diagram of the ACK-PTA pathway in B. burgdorferi. ack (bb0622) encodes acetate kinase (Ack), which converts acetate to the intermediate acetyl∼P, while pta (bb0589) encodes phosphate acetyltransferase (Pta), which synthesizes acetyl-CoA from acetyl∼P and CoASH [17]. In B. burgdorferi, the Ack-Pta pathway appears to be the sole pathway for biosynthesis of acetyl-CoA, a molecule required for cell membrane biosynthesis (see Results and Discussion for details). (B) Acetate induces activation of the Rrp2-RpoN-RpoS pathway. Wild-type B. burgdorferi strain B31-A3 was cultivated in the BSK-H medium supplemented with 0–90 mM NaOAc with a final media pH value of 7.0. Cells were harvested at the early-logarithmic phase (5×106 spirochetes/ml). Cell lysates were subjected to SDS-PAGE (top panel) or immunoblot (bottom panels) analysis. The bands corresponding to OspC, RpoS and FlaB were labeled on the right. (C) Overexpression of Pta reduces acetate-induced Rrp2 activation. Wild-type B. burgdorferi strain B31 13A (-) or the strain carrying flaBp-pta (+) were cultivated in the BSK-H medium supplemented with 15 mM NaOAc at pH 7. Cells were harvested at the cell density of 5×106 and then subjected to immunoblot analyses with a mixture of antibodies against RpoS, OspC, or FlaB (internal control). The bands corresponding to each protein are indicated on the right. (D) Overexpression of Pta reduces temperature and cell density-induced activation of the Rrp2-RpoN-RpoS pathway. Wild-type B. burgdorferi strain B31 13A (-) or the strain carrying flaBp-pta (+) were cultivated either at 23 or 35°C in the standard BSK-H medium. Cells were harvested at the late-logarithmic growth phase (5×107 spirochetes/ml) and then subjected to immunoblot analyses. (E) In vitro phosphorylation of recombinant Rrp2 by acetyl∼P. Different quantities of purified recombinant Rrp2 or various versions of Rrp2-N were incubated with [32P]acetyl phosphate and the reactions were terminated at 15 or 30 min. The reaction mixtures were separated by SDS-PAGE followed by exposure on Kodak X-ray film.