Expression of Borrelia burgdorferi OspC and DbpA is controlled by a RpoN-RpoS regulatory pathway - PubMed (original) (raw)

Expression of Borrelia burgdorferi OspC and DbpA is controlled by a RpoN-RpoS regulatory pathway

A Hübner et al. Proc Natl Acad Sci U S A. 2001.

Abstract

RpoS and RpoN are two alternative sigma factors typically associated with general stress responses in bacteria. To date, there has been no experimental evidence that RpoS and RpoN can directly control the expression of one another. Herein, using a combined strategy of gene disruption and genetic complementation targeting rpoN and rpoS in Borrelia burgdorferi strain 297, we describe a regulatory network for B. burgdorferi. In this network, RpoN controls the expression of RpoS, which, in turn, governs the expression of two important membrane lipoproteins, outer surface protein C and decorin-binding protein A, and likely other proteins of B. burgdorferi. Our findings provide a foundation for elucidating further key regulatory networks that potentially impact many aspects of B. burgdorferi's parasitic strategy, host range, and virulence expression.

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Figures

Figure 1

Strategy for gene inactivation and complementation of rpoN (a) and rpoS (b) in 297. rpoN and rpoS (black solid boxes) were first cloned in pGEM-T (pALH364 and pALH362, respectively). Only the relevant portions of the plasmids are shown (labeled at the left). rpoN and rpoS were insertionally disrupted with ermC (diagonal stripes) (pALH394 and pALH386, respectively). For complementation of rpoN, suicide plasmid pALH400 was constructed as described in Materials and Methods. Relevant restriction sites are shown, and relevant promoters are indicated by light gray boxes. Short arrows denote primers used for PCR (Table 2).

Figure 2

Construction of pALH251 for complementation of inactivated rpoN with constitutively expressed rpoS. (a) A 965-bp fragment was excised from pJRS525 and a 1,158-bp fragment containing a P_flaB_ -kan promoter fusion was inserted into the _Alw_NI and Fsp_I sites, yielding pALH227. (b) pALH251 was obtained on ligating a P_flgB -rpoS fusion construct (1,224 bp) into the _Bam_HI and _Nco_I sites of the multiple cloning site of pALH227.

Figure 3

PCR analysis of rpoN and rpoS mutants. (a) Schematic of PCR primer pairs (short arrows). (b and c) Agarose gel patterns of amplicons for wild type (lanes 2–4), (b) a rpoN mutant, or (c) a rpoS mutant (lanes 5–7). Lanes 1 contain DNA markers of ΦX174/_Hae_III. Gene disruption by ermC results in an increased size of the amplicons (compare lanes 2 and 5). A combination of _ermC_-specific and flanking primers yielded only products for the mutants (lanes 6 and 7), but not for 297 (lanes 3 and 4). Lanes 3 and 6: priAH102 (arrow 102) in combination with (b) priAH59 (arrow 59) or (c) priAH27 (arrow 27). Lanes 4 and 7: priAH104 (arrow 104) in combination with (b) priAH60 (arrow 60) or (c) priAH131 (arrow 131).

Figure 4

RT-PCR analysis of a rpoN mutant. Borreliae were cultivated in BSK-H at pH 6.8. (a and c) Comparative RT-PCR; 10-fold dilutions of RNA from the wild type (strain 297) or the mutant were tested for rpoN (a) and rpoS (c) transcripts. Buf, buffer only; −RT, lacking RT. (b and d) Quantitative competitive RT-PCR; competitor RNA (cRNA) was used at exponential dilution, as indicated above the panels. The expression of constitutive flaB was unaffected by the rpoN mutation (b), whereas the expression of ospC was abolished (d).

Figure 5

SDS/PAGE (Coomassie blue stain) (a) and immunoblots (b_–_g) of whole cell lysates (39) from wild-type, mutant, and complemented strains of 297. Total protein from about 2 × 107 spirochetes (late-logarithmic phase of growth) was loaded per gel lane, except when probing for RpoS (10-fold more material). Monoclonal antibodies against OspA and FlaB and polyclonal antisera against all other proteins have been described (23, 51). Cultivation pH and temperature are indicated above each lane. Lanes 1 and 2, wild-type 297. Lane 3, rpoN mutant. Lanes 4–6, rpoN mutant complemented with wild-type rpoN. Lane 7, rpoN mutant complemented with pALH251 (rpoS). Lane 8, rpoS mutant.

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Expression of Borrelia burgdorferi OspC and DbpA is controlled by a RpoN-RpoS regulatory pathway - PubMed (original) (raw)