bioRxiv preprint doi: https://doi.org/10.1101/614891; this version posted April 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 The BvgS PAS domain: an independent sensory perception module in the Bordetella BvgAS 2 phosphorelay 3 4 M. Ashley Sobran1 and Peggy A. Cotter1,* 5 6 1 Department of Microbiology and Immunology, University of North Carolina Chapel Hill, Chapel 7 Hill, NC 27599 8 9 *Corresponding author: [email protected] 10 11 Keywords: Bordetella, two-component system, PAS domain, BvgAS, PlrSR, respiratory infection 12 Running Title: The BvgS PAS domain is an independent signal perception domain 13 14 15 16 17 18 19 20 21 22 bioRxiv preprint doi: https://doi.org/10.1101/614891; this version posted April 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 23 24 Abstract 25 26 To detect and respond to the diverse environments they encounter, bacteria often use two- 27 component regulatory systems (TCSs) to coordinate essential cellular processes required for 28 survival. In pathogenic Bordetella species, the BvgAS TCS regulates expression of hundreds of 29 genes, including those encoding all known protein virulence factors, and its kinase activity is 30 essential for respiratory infection. Maintenance of BvgS kinase activity in the lower respiratory 31 tract (LRT) depends on the function of another TCS, PlrSR. While the periplasmic venus fly-trap 32 domains of BvgS have been implicated in responding to so-called modulating signals in vitro 33 (nicotinic acid and MgSO4), a role for the cytoplasmic Per-Arnt-Sim (PAS) domain in signal 34 perception has not previously been demonstrated. By comparing B. bronchiseptica strains with 35 mutations in the PAS domain-encoding region of bvgS with wild-type bacteria in vitro and in 36 vivo, we found that although the PAS domain is not required to sense modulating signals in 37 vitro, it is required for the inactivation of BvgS that occurs in the absence of PlrS in the LRT of 38 mice, suggesting that the BvgS PAS domain functions as an independent signal perception 39 domain. Our data also indicate that the BvgS PAS domain is important for controlling absolute 40 levels of BvgS kinase activity and the efficiency of the response to modulating signals in vitro. 41 Our results indicate that BvgS is capable of integrating sensory inputs from both the periplasm 42 and the cytoplasm to control precise gene expression patterns in diverse environmental 43 conditions. 44 bioRxiv preprint doi: https://doi.org/10.1101/614891; this version posted April 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 45 Importance 46 47 Despite high rates of vaccination, Pertussis, a severe, highly contagious respiratory disease, 48 caused by the bacterium Bordetella pertussis, has reemerged as a significant health threat. In 49 Bordetella pertussis and the closely related species, Bordetella bronchiseptica, activity of the 50 BvgAS two-component regulatory system is critical for colonization of the human respiratory 51 tract and other mammalian hosts, respectively. Here we show that the cytoplasmic PAS domain 52 of BvgS can function as an independent signal perception domain that is capable of integrating 53 environmental signals that influence overall BvgS activity. Our work is significant as it reveals a 54 critical, yet previously unrecognized role, for the PAS domain in the BvgAS phosphorelay and 55 provides a greater understanding of virulence regulation in Bordetella. 56 57 Introduction 58 59 Pertussis, also known as whooping cough, is a severe, highly contagious, reemerging respiratory 60 disease (1). It is caused by the bacterium Bordetella pertussis, which infects only humans and 61 appears to be capable of surviving only transiently outside the human respiratory tract (2). By 62 contrast, the closely related species Bordetella bronchiseptica infects nearly all mammals and 63 can survive long periods of time in ex vivo environments, including those with scarce nutrients 64 such as filtered pond water and buffered saline (3, 4). Despite these differences, B. pertussis 65 and B. bronchiseptica produce a nearly identical set of virulence factors that are regulated at 66 the level of transcription by functionally interchangeable BvgAS Two-Component Regulatory bioRxiv preprint doi: https://doi.org/10.1101/614891; this version posted April 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 67 Systems (TCS) (5). By activating and repressing at least four classes of genes, BvgAS controls 68 transition of the bacteria between three different phenotypic phases: the Bvg+ phase, which is 69 necessary for virulence in mammals (6), the Bvg– phase, which, in B. bronchiseptica, is required 70 for survival under nutrient-limiting conditions (4) and for interactions with non-mammalian 71 hosts (7), and the Bvgi phase, which has been hypothesized to be important for bacterial 72 transmission (8). 73 74 The BvgAS system is composed of a hybrid sensor kinase, BvgS, and a response regulator, BvgA 75 (9). BvgS contains tandem, N-terminal, periplasmic Venus Flytrap (VFT) domains, followed by a 76 transmembrane domain, a cytoplasmic Per/Arnt/Sim (PAS) domain, a histidine kinase (HK) 77 domain, a receiver (REC) domain, and a C-terminal, histidine phosphotransfer (Hpt) domain 78 (Fig. 1A). BvgA contains an N-terminal REC domain and a C-terminal helix-turn-helix (HTH) DNA 79 binding domain. When Bordetella are grown on Bordet-Gengou (BG) blood agar or in Stainer- 80 Scholte (SS) broth at 37C, BvgS autophosphorylates, and, via a His-Asp-His-Asp phosphorelay, 81 trans-phosphorylates BvgA. BvgA~P forms homodimers capable of binding DNA and activating 82 or repressing gene transcription (10). Under these conditions, BvgA~P levels are high and the 83 bacteria express the Bvg+ phase, which is characterized by transcription of all known virulence- 84 activated genes (vags), including those encoding critical virulence factors (11). When Bordetella 85 are grown at 25°C or at 37°C in the presence of millimolar concentrations of nicotinic acid (NA) 86 or magnesium sulfate (MgSO4), the BvgS phosphorelay is reversed and BvgS dephosphorylates 87 BvgA, resulting in the Bvg– phase. The Bvg– phase is characterized by the expression of genes 88 that are repressed by BvgA~P (termed virulence-repressed genes (vrgs)) and lack of expression bioRxiv preprint doi: https://doi.org/10.1101/614891; this version posted April 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 89 of vags (11). In B. bronchiseptica, flagellar regulon genes, including the flagellin-encoding gene 90 flaA, are vrgs (12). When Bordetella transition between the Bvg– phase to the Bvg+ phase or 91 when the bacteria are cultured in the presence of low concentrations of NA or MgSO4, BvgA~P 92 levels in the cell are low, resulting in the Bvgi phase, which is characterized by repression of the 93 vrgs, maximal expression of the intermediate phase gene bipA, and expression of vags with 94 promoters containing high affinity BvgA~P binding sites (such as bvgAS itself and fhaB, encoding 95 the adhesin filamentous hemagglutinin (FHA)) but not vags with promoters containing low 96 affinity BvgA~P binding sites (such as the ptx operon, which encodes pertussis toxin and its 97 export system) (10, 13, 14). The transition from Bvg+ phase to Bvg– phase is termed phenotypic 98 modulation. 99 100 Although the BvgAS phosphorelay and transcriptional control of the BvgAS regulon are well 101 characterized, the natural signals that impact BvgS activity and the mechanism by which these 102 signals are perceived and integrated by this system are unknown. Of BvgS’s three putative 103 sensory perception domains, the periplasmic VFTs have been studied most. Structure-function 104 analyses support a model in which VFT2 (the domain closest to the membrane) is in a closed 105 conformation in the absence of modulating signals, and that this conformation renders BvgS 106 active (15, 16). Hence the ‘default’ state for BvgS is one in which it is an active kinase. Binding of 107 NA to VFT2 alters its conformation and this change is apparently transduced across the 108 membrane, causing BvgS to reverse the phosphorelay and dephosphorylate BvgA, resulting in a 109 shift to the Bvg– phase (17). 110 bioRxiv preprint doi: https://doi.org/10.1101/614891; this version posted April 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 111 The role of the PAS domain in regulating BvgS activity is less understood. PAS domains are 112 abundant in prokaryotic signal transduction proteins and have a conserved structure, which 113 includes a core binding pocket composed of a central, five-stranded antiparallel beta sheet and 114 flanking alpha helices (18). Despite their conserved structure, PAS domains can perceive a 115 multitude of stimuli, either by directly binding a signal molecule or by sensing with the 116 assistance of a co-factor, in addition to promoting protein-protein interactions that mediate 117 signal transduction (19–22). The PAS domain of BvgS contains two perception motifs that are 118 critical for PAS domain function in other regulatory proteins; a quinone binding motif [aliphatic- 119 (X)3-H-(X)2,3-(L/T/S)] and a highly conserved cysteine residue (C607) (23, 24).