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. 2018 Oct 22;14(10):e1007714.
doi: 10.1371/journal.pgen.1007714. eCollection 2018 Oct.

Suppressor mutations reveal an NtrC-like response regulator, NmpR, for modulation of Type-IV Pili-dependent motility in Myxococcus xanthus

Daniel J Bretl  1 , Kayla M Ladd  2 , Samantha N Atkinson  1   3 , Susanne Müller  1 , John R Kirby  1
Affiliations

Suppressor mutations reveal an NtrC-like response regulator, NmpR, for modulation of Type-IV Pili-dependent motility in Myxococcus xanthus

Daniel J Bretl et al. PLoS Genet. .

Abstract

Two-component signaling systems (TCS) regulate bacterial responses to environmental signals through the process of protein phosphorylation. Specifically, sensor histidine kinases (SK) recognize signals and propagate the response via phosphorylation of a cognate response regulator (RR) that functions to initiate transcription of specific genes. Signaling within a single TCS is remarkably specific and cross-talk between TCS is limited. However, regulation of the flow of information through complex signaling networks that include closely related TCS remains largely unknown. Additionally, many bacteria utilize multi-component signaling networks which provide additional genetic and biochemical interactions that must be regulated for signaling fidelity, input and output specificity, and phosphorylation kinetics. Here we describe the characterization of an NtrC-like RR that participates in regulation of Type-IV pilus-dependent motility of Myxococcus xanthus and is thus named NmpR, NtrC Modulator of Pili Regulator. A complex multi-component signaling system including NmpR was revealed by suppressor mutations that restored motility to cells lacking PilR, an evolutionarily conserved RR required for expression of pilA encoding the major Type-IV pilus monomer found in many bacterial species. The system contains at least four signaling proteins: a SK with a protoglobin sensor domain (NmpU), a hybrid SK (NmpS), a phospho-sink protein (NmpT), and an NtrC-like RR (NmpR). We demonstrate that ΔpilR bypass suppressor mutations affect regulation of the NmpRSTU multi-component system, such that NmpR activation is capable of restoring expression of pilA in the absence of PilR. Our findings indicate that pilus gene expression in M. xanthus is regulated by an extended network of TCS which interact to refine control of pilus function.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Characterization of M. xanthus ΔpilR suppressor mutants.
(A) Representative suppressor mutations were observed after prolong incubation (7–14 days) on 0.5% agar. (B) Nine suppressor mutants were characterized for T4P-dependent motility related-phenotypes including motility rate (mm/day), sedimentation in static cultures reported as the percentage of lost absorbance at OD600 after 2 hours, and extracellular polysaccharide (EPS) production measured by the binding of Trypan blue to whole cells in solution and normalized to wild-type DZ2 which was set at 100%. All data represent the averages of at least 3 biological replicates. All suppressor mutant (C1-C9) measurements were significantly different (t-test, p<0.05) from wild-type DZ2 and ΔpilR with the exception of the EPS production of C6, C8, and C9 which were not different from that of the wild-type DZ2. Western blot analysis of total PilA from whole cell lysates was normalized to total protein determined by Bradford assay. All lysates were on the same blot, developed together, and can be directly compared to each other.
Fig 2
Fig 2. Suppressor mutations identified in uncharacterized TCS signaling genes.
Suppressor strains were sequenced with the Illumina MiSeq platform and compared to the annotated M. xanthus DK1622 genome [27]. (A) Genomic organization of the locus containing the RR gene mxan_4240 (nmpR) (B) and SK gene mxan_4246 (nmpU) (C). This locus also contains a gene encoding a protein with two RR receiver domains (mxan_4245, nmpT, RR-RR) and an atypical hybrid response regulator/sensor kinase (mxan_4244, nmpS, RR-SK). The results of the mutations are depicted (B and C): a missense mutation in nmpR (V87E) and premature stop codons (stop signs) or frameshifts (Δ-1) in nmpU. The location of the mutations within each protein is to scale. The positions of each domain of these proteins is also indicated by amino acid number (RR = response regulator receiver domain, Sigma54 activation = central ATPase that interacts with σ54, HTH_8 = DNA-binding helix-turn-helix, Protoglobin = putative heme binding sensor domain, HisKA = domain of histidine phosphorylation and SK dimerization, HK CA:9 = ATPase. All domain nomenclatures are based on the Mist 2.2 database [87] and Pfam (http://pfam.xfam.org, [93]). The strain designation (C1-C9) is the same as in Table 1 and Fig 1.
Fig 3
Fig 3. The NmpR V87E suppressor strain has a gain-of-function mutation.
(A) In-frame nmpR deletions were constructed in the indicated strains and assayed for motility on 0.5% agar. All images depicted are after two days of motility. (B) Single copy complementation vectors were integrated at the Mx9 phage attB site and resulting strains assayed for motility. The pNat = 585 bp upstream of mxan_4236 (See Fig 2A) and pHigh = 623 bp upstream of mxan_4894, groES [43, 44] (See S1 Fig). D54A is an unphosphorylatable form of NmpR and D54E is a phosphomimetic.
Fig 4
Fig 4. Additional gain-of-function suppressor mutations identified in nmpR.
The M. xanthus ΔpilR strain was transformed with pHigh-nmpR, and suppressor mutants with restored motility were isolated and subjected to targeted sequencing of the over-expression construct. The identified mutations are indicated in a linear depiction of the NmpR domains (A), in the primary amino acid sequence of NmpR (B and C), or unbiasedly modeled on the ribbon structure of NtrX of Brucella abortus [51] with α-helices H1-H5 indicated (D and E). In panels B and C, the E. coli NtrC amino acid sequence is included for comparison and regions of interest in the protein sequence are indicated in red text and/or underlined including: the α-helix 4 of the RR receiver domain, the nearly 100% conserved RR amino acid pair of KP at 104/105, the variable Q-linker, the sensor II motif necessary for ATPase activity, and the low-homology linker between the sigma54 activation domain and helix-turn-helix. Mutations indicated with a * were identified in suppressor mutants resulting from the NmpU Q283Stop ΔnmpR strain (See text and Fig 5C). Finally, in panels D and E the same suppressor mutations of NmpR are indicated in red, the conserved aspartic acid that becomes phosphorylated in green, and the specificity residues [11] of NmpR in blue.
Fig 5
Fig 5. SK NmpU, RR-SK NmpS, and RR NmpR are in a single signaling pathway.
Epistasis analysis demonstrates these signaling proteins are in a shared pathway. Mutations in various strains were constructed as depicted and motility assayed as before. Note especially that deletion of nmpR or nmpS is epistatic to a deleted or non-functional nmpU (B and D); that suppressor mutations in nmpR arose in the NmpUQ283Stop ΔnmpS strain (B and Fig 4); and that deletion of nmpU is sufficient to restore motility in the ΔpilR parental strain (D). As before, over-expression of a phosphomimetic (D54E) NmpR is necessary to restore motility of a ΔnmpR strain (C). The motility image of the parental NmpRV87E strain (A) is a representative image reproduced from Fig 3A.
Fig 6
Fig 6. In vitro phosphotransfer in the NmpRSTU multi-component pathway.
(A) The kinases NmpS and NmpU autophosphorylate in the presence of ATP-γP32 as seen by the accumulation of the radioactive phosphoryl group. NmpU specifically phosphorylates the first receiver domain of NmpT (RR1) and the receiver domain of NmpS (B and C). In contrast, NmpS only phosphorylates NmpR (D and E). Note the rapid kinetics of the phosphorylation of NmpR by NmpS (E). Also, the V87E amino acid change of NmpR does not affect the specificity of the phosphotransfer (C and E). The specificity residues [10, 11, 96] of the SK domains of NmpS and NmpU and the corresponding specificity residues of the receiver domains of NmpR, NmpS, and NmpT further supports the observed phosphotransfer patterns (F).
Fig 7
Fig 7. The conserved aspartic acid of the response regulator domain of NmpS is not necessary for kinase activity.
(A) Full-length wild-type NmpS and a construct in which the conserved aspartic acid was substituted with an alanine (D59A) were used in an autokinase assay as in Fig 6. The aspartic acid residue was not necessary for activity, supporting the conclusion that the unphosphorylated form of NmpS is the active state. A Coomasie stained gel is included demonstrating the same amount of kinase was in each reaction. (B and C) M. xanthus wild-type or the ΔpilR strain were transformed with NmpS or NmpS D59A over-expression constructs and only the NmpS D59A construct was able to rescue motility of ΔpilR. This again is consistent with the in vitro kinase activity (A) that supports the conclusion that the unphosphorylated form of NmpS is active.
Fig 8
Fig 8. NmpR binds specific promoters upstream of its own putative operon and upstream of pilR.
(A) NmpR binds to the promoter regions of mxan_4236 (referred to elsewhere as pNat—585 bp upstream of mxan_4236) (See also Fig 2A), but not to that of the pilA promoter (217 bp upstream of pilA) or pgroES (referred to elsewhere as pHigh—623 bp upstream of mxan_4894, groES). (B) NmpR binds specifically to a probe encompassing 800 bp upstream of pilR that includes a putative σ54 in the pilS open reading frame. The σ54 in the ppilA known to be utilized by PilR in a wild-type genomic context is also indicated. (C) Finally, NmpR binds only to a region within pilS upstream of the putative σ54 binding site. The sequence containing this NmpR-dependent promoter is necessary for the restored motility in the suppressor strains because deletion of pilS returned these strains to a non-motile phenotype (D and E). All shift assays depicted contained 200 fmol of DNA probe and increasing quantities of NmpR (0, 2, 8, 16, and 32 pmol, left to right). The exception is the pgroES shift that includes the same quantities of NmpR but with a broader range (0, 2, 4, 8, 12, 16, 20, 24, 32 pmol, left to right). All gene lengths and locations, as well as probe length and location are to scale.
Fig 9
Fig 9. Model of the NmpRSTU multi-component signaling pathway.
The presence of oxygen is sensed by the protoglobin domain of the SK NmpU. During high oxygen conditions, NmpU is “on”, autophosphorylating and subsequently phosphorylating the receiver domain of the hybrid RR-SK NmpS. We propose this would maintain NmpS in an “off” state. Additionally, the first receiver domain of NmpT serves as a phospho-sink for NmpU. When oxygen is limiting, perhaps in certain soil types/depths or during multi-cellular development, NmpU switches to an “off” state, leading to the loss of phosphorylation of NmpS and turning “on” the second branch of the pathway. NmpS phosphorylation of the RR NmpR under these conditions activates NmpR leading to modulation of the expression of pilR, which in turn regulates pilA expression influencing T4P-dependent motility.
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