REFERENTES TEÓRICOS

Strains

WT Wild-type B. bronchiseptica strain RB50; Smr ₍₁₂₎

ΔplrS RB50 containing an in-frame deletion in plrS removing the codons for

amino acids 5 to 198; Smr (18)

RB515 RB50ΔcyaA (51)

bvgSC _{RB50 containing a R570H point mutation in bvgS; Sm}r ₍₁₂₎

ΔplrS-bvgSC RB50 containing a R570H point mutation in bvgS and a deletion in plrS

removing the codons for amino acids 5 to 198; Smr This Study

ΔbscN RB50::pSJ23; RB50 containing a plasmid insertion mutation in bscN

(BB1628); Gmr This Study

B. pertussis

Strains

BP536 Wild-type B. pertussis strain; Smr ₍₅₈₎

BP536ΔplrS B. pertussis strain containing an in-frame deletion in plrS from residues 9

thru 768; Smr This Study

Plasmids

pMD11 pSS4245-based allelic exchange vector containing a 0.8-kb DNA fragment

deleting the codons for amino acids 5 to 198 of plrS This study

pGFLIP-PflaA pUC18-based vector with PS12-gfp and nptII flanked by FRT sequences

and flp recombinase driven by the RB50 flaA promoter; Apr _Kmr* (15)

pGFLIP-PptxA pUC18-based vector with PS12-gfp and nptII flanked by FRT sequences

and flp recombinase driven by the BPSM ptxA promoter; Apr_{, Km}r* (15)

pTNS3 Tn7 transposase expression vector containing tnsABCD; Apr ₍₅₇₎

pBam Plasmid that integrates into the Bordetella chromosome at the bvgAS locus

and allows for generation of LCPs; Apr_{, Gm}r (29)

pEG7 Suicide plasmid for B. bronchiseptica; Gmr ₍₉₎

pSJ23 pEG7-based suicide plasmid containing a B. bronchiseptica DNA fragment

from bscN (BB1628); Gmr This Study

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CHAPTER 3. THE BVGS PAS DOMAIN: AN INDEPENDENT SENSORY PERCEPTION MODULE IN THE BORDETELLA BRONCHISEPTICA BVGAS

PHOSPHORELAY2 Introduction

Pertussis, also known as whooping cough, is a severe, highly contagious, reemerging respiratory disease (1). It is caused by the bacterium Bordetella pertussis, which infects only humans and appears to be capable of surviving only transiently outside the human respiratory tract (2). By contrast, the closely related species Bordetella bronchiseptica infects nearly all mammals and can survive long periods of time in ex vivo environments, including those with scarce nutrients such as filtered pond water and buffered saline (3, 4). Despite these differences,

B. pertussis and B. bronchiseptica produce a nearly identical set of virulence factors that are regulated at the level of transcription by functionally interchangeable BvgAS Two-Component Regulatory Systems (TCS) (5). By activating and repressing at least four classes of genes, BvgAS controls transition of the bacteria between three different phenotypic phases: the Bvg+ phase, which is necessary for virulence in mammals (4), the Bvg– phase, which, in B.

bronchiseptica, is required for survival under nutrient-limiting conditions (4) and for interactions with non-mammalian hosts (6), and the Bvgi phase, which has been hypothesized to be important for bacterial transmission (7).

2 _{M. Ashley Sobran and Peggy A. Cotter. “The BvgS PAS domain: an independent sensory perception module in the}

Bordetella bronchiseptica BvgAS phosphorelay”. Accepted at Journal of Bacteriology. MAS contributed to this manuscript by assisting in the design, performance, and analysis of all presented research. MAS compiled and formatted all figures and tables, wrote all sections of the manuscript with the assistance of PAC, and addressed all

The BvgAS system is composed of an unorthodox sensor kinase, BvgS, and a response regulator, BvgA (8). BvgS contains tandem, N-terminal, periplasmic Venus Flytrap (VFT) domains, followed by a transmembrane domain, a cytoplasmic Per/Arnt/Sim (PAS) domain, a histidine kinase (HK) domain, a receiver (REC) domain, and a C-terminal, histidine

phosphotransfer (Hpt) domain (Fig. 3.1A). BvgA contains an N-terminal REC domain and a C- terminal helix-turn-helix (HTH) DNA binding domain. When Bordetella are grown on Bordet- Gengou (BG) blood agar or in Stainer-Scholte (SS) broth at 37C, BvgS autophosphorylates, and, via a His-Asp-His-Asp phosphorelay, trans-phosphorylates BvgA. BvgA~P forms

homodimers capable of binding DNA and activating or repressing gene transcription (9). Under these conditions, BvgA~P levels are high and the bacteria express the Bvg+ phase, which is characterized by transcription of all known vir-activated genes (vags), including those encoding critical virulence factors (10). When Bordetella are grown at 25°C or at 37°C in the presence of millimolar concentrations of nicotinic acid (NA) or magnesium sulfate (MgSO4), BvgA is unphosphorylated (11), presumably due to the reversal of the BvgAS phosphorelay, resulting in the Bvg– phase. The Bvg– phase is characterized by the expression of genes that are repressed by BvgA~P (termed vir-repressed genes (vrgs)) and lack of expression of vags (10). In B.

bronchiseptica, flagellar regulon genes, including the flagellin-encoding gene flaA, are vrgs (12). When Bordetella transition from the Bvg– phase to the Bvg+ phase, or when the bacteria are cultured in the presence of low concentrations of NA or MgSO4, BvgA~P levels in the cell are low, resulting in the Bvgi phase, which is characterized by repression of the vrgs, maximal expression of the intermediate phase gene bipA, and expression of vags with promoters containing high affinity BvgA~P binding sites (such as bvgAS itself and fhaB, encoding the adhesin filamentous hemagglutinin (FHA)) but not vags with promoters containing low affinity

BvgA~P binding sites (such as the ptx operon, which encodes pertussis toxin and its export system) (9, 13, 14). The transition from Bvg+ phase to Bvg– phase is termed phenotypic modulation.

Although the BvgAS phosphorelay and transcriptional control of the BvgAS regulon are well characterized, the natural signals that impact BvgS activity and the mechanism by which these signals are perceived and integrated by this system are unknown. Of BvgS’s three putative sensory perception domains, the periplasmic VFTs have been studied most. Structure-function analyses support a model in which VFT2 (the domain closest to the membrane) is in a closed conformation in the absence of modulating signals, and that this conformation renders BvgS active (15, 16). Hence the ‘default’ state for BvgS is one in which it is an active kinase. Binding of NA to VFT2 alters its conformation and this change is apparently transduced across the membrane, which is proposed to cause reversal of the phosphorelay, resulting in the dephosphorylation of BvgA and a shift to the Bvg– phase (17).

The role of the PAS domain in regulating BvgS activity is less understood. PAS domains are abundant in prokaryotic signal transduction proteins and have a conserved structure, which includes a core binding pocket composed of a central, five-stranded antiparallel beta sheet and flanking alpha helices (18). Despite their conserved structure, PAS domains can perceive a multitude of stimuli, either by directly binding a signal molecule or by sensing with the

assistance of a co-factor, in addition to promoting protein-protein interactions that mediate signal transduction (19–22). The PAS domain of BvgS contains two perception motifs that are critical for PAS domain function in other regulatory proteins; a quinone binding motif [aliphatic-(X)3-H- (X)2,3-(L/T/S)] and a highly conserved cysteine residue (C607) (23, 24). In vitro studies with the cytoplasmic portion of BvgS from B. pertussis showed that kinase activity was decreased in the

presence of an oxidized ubiquinone analog and that the histidine in the quinone binding motif (H643) contributed to this effect (23). Jacob-Dubuisson and colleagues hypothesized that H643 may be involved in binding heme, but were unable to detect heme, or other cofactors, bound to the BvgS PAS domain (25). Their characterization of an H643A mutant of B. pertussis showed no defect in activation of the ptx promoter under aerobic conditions, but, instead, a defect in responding to NA and MgSO4. This group also investigated the importance of the conserved cysteine (C607) and found that a C607A mutant was similarly defective only in its ability to modulate in response to NA (25). Approximately one-third of BvgS homologs contain coiled- coil domains instead of PAS domains. Jacob-Dubuisson and colleagues showed that a B.

pertussis mutant in which the BvgS PAS domain was replaced with the coiled-coil domain from the BvgS homolog of Enterobacter cloacae was similar to wild-type bacteria in its ability to activate ptx expression and to respond to NA (26). Taking these and other observations together, this group concluded that the role of the BvgS PAS domain is primarily to transduce signals from the VFTs to the kinase domain and that it may not function as a sensory perception domain to