Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: The extended CTT of WalK is unique in S. mutans and required for its interaction with WalR. (A) Phylogenetic analysis of S. mutans HKs. Evolutionary relationship of all 14 HKs is shown in a circular tree, which are grouped into two based on six key residues following their catalytic histidine with conservation for each group below. HKs with a conserved HisKA motif are colored in green. HKs with HisKA_3 motif are grouped in orange. HKs are named with four digits in their protein ID: NP_72****.1. (B) Alignment of streptococcus WalK C-terminal sequences. Completely conserved residues are shown in white with a red background and boxed in blue. Highly conserved residues are in red with a white background and boxed in blue. Marked on top are protein secondary structures and residue numbers in S. mutans . (C) Mutations in the CTT disrupt the WalRK interaction. A GST fusion protein with full-length S. mutans WalR was used to pull-down S. mutans WalK (196–450) WT and mutant proteins (top gel). As a negative control, GST alone was used to pull down WalK WT and mutant proteins (middle gel). Shown in the bottom gel are 10% of the WalK protein levels used above. CK shows GST-WalR or GST used in the pull-down. (D–F) Quantification of the WalRK interaction by ITC experiments. WalK (196–450) WT and mutant proteins were titrated against the full-length WalR, resulting in raw titration curves at the top and their global fittings at the bottom. The derived thermodynamic parameters are shown within.

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Residue, Mutagenesis, Negative Control, Titration, Derivative Assay

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: The CTT of S. mutans WalK is indispensable for its enzymatic activities. (A) Autokinase activity of WalK (31–450) and its tail mutants. The WalK loadings were shown in the lower gel stained by CBB, while the phosphorylation of WalK was detected by ATPγS and anti-thiophosphate antibodies shown in the upper gel. H217A is an autokinase inactive mutant. (B) Phosphotransferase of WalK. The phosphotransferase activity was examined by the reduced phosphorylation of WalK incubated with WalR and detected using ATPγS and anti-thiophosphate antibody over time. Quantitative analysis of phosphotransferase activity normalized to 0 min is shown below the gel. (C) Phosphatase of WalK. Phosphorylated WalR was incubated with WalK and its derivatives at 1:5 (WalK:WalR), separated from the dephosphorylated WalR in a Phos-tag gel and stained with CBB. Quantitative analysis of phosphatase activity is below the gel. The phosphorylated/dephosphorylated WalR was estimated, normalized to its initial amount at 0 s of the gel. Data presented are means ± standard deviations (error bars) for three independent experiments. Student’s t -tests were used to compare mutants to WT at each time point (*** p < 0.001 and **** p < 0.0001).

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Activity Assay, Staining, Phospho-proteomics, Mutagenesis, Incubation

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: The CTT of S. mutans WalK contributes to the interaction with the WalR DBD. (A) The domain architectures of WalK and WalR. (B) WalK (196–450) interacts with WalR in GST pull-down experiments. The 10% loading controls for WalK, GST, GST-WalR full-length (FL), GST-RD, and GST-DBD are shown in lanes 1, 2, 4, 6, and 8, respectively. (C) Mutations in the WalK CTT disrupt the interaction with the WalR DBD. As a negative control, GST alone was used to pull down WalK (middle gel). Shown in the bottom gel are 10% of the WalK protein used above. CK shows GST-DBD or GST used in the pull-down. (D) Phosphotransferase activity of WalK is diminished toward the RD of WalR alone. Phosphorylated WalK detected by anti-thiophosphate antibody was incubated with WalR full-length, RD, DBD, and D52A, shown from top to bottom, respectively.

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Negative Control, Activity Assay, Incubation

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: The CTT of S. mutans WalK is important for WalK in competition with promoter DNA. (A,B) WalK (196–450) competes off fluorescein labeled 25-mer promoter DNA from binding to WalR or DBD in a dose-dependent manner. (C) Comparison of the relative ability of CTT mutants (W443A, Δtail) of WalK to compete with DBD in promoter binding. All samples were mixed and incubated for 15 min at RT before loading onto gels. Final concentrations of proteins and DNA used in the reactions were marked above the panels. All EMSA gels were imaged under UV to show DNA in the upper panel and stained with CBB to visualize protein loading in the lower panel.

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Labeling, Binding Assay, Comparison, Incubation, Staining

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: Effect of WalK mutations on biofilm development of S. mutans . (A–C) SEM analyses of mature biofilms grown for 72 h. (D–F) Quantification of biofilms by fluorescent staining. All biofilms were quantified for their thickness and horizontal growth shown below each 3D image. (G) Phosphorylation state of WalR in vivo . Phosphorylated WalR was separated from its non-phosphorylated state in a Phos-tag gel and detected using an anti-WalR antibody. The non-phosphorylated (CK) and phosphorylated WalR (His-tagged, treated with AcP) were loaded in the first two lanes. The cytoplasmic phosphorylation ratio of WalR shown in the bar chart was determined by band intensities and averaged from three independent experiments with error bars showing standard deviation.

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Staining, Phospho-proteomics, In Vivo, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: Protein profiling in S. mutans biofilms. (A) Protein profiling of S mutans WT and Δtail strains from a quantitative mass spectroscopy experiment. The x -axis indicates the fold change of LFQ in the Δtail strain. The y -axis of log ( P ) indicates a significance level of the t- test. The black curves separate those proteins at a level of false discovery rate (FDR) = 0.01 and minimal fold changes (S0) = 0.1. Below the curve in gray are those unchanged proteins. Those proteins that were upregulated are colored in red, and those that were downregulated are colored in blue. Total and altered proteins are listed in MS Dataset. (B) Proteins that were most altered in the Δtail strain. Proteins were selected at a difference cutoff of > 1.5 except GbpA and GbpB. Five short, functionally unknown, peptides were excluded. The functional annotations of the proteins are shown in . (C,D) qRT-PCR analyses of key genes known to be regulated by WalRK. Transcriptional profiles of the genes gtfBCD and gbpABC were normalized to 16S RNA. Data presented are means ± standard deviations (error bars) for three independent experiments. Student’s t -tests were used to compare Δ tail strain to WT strain (** p < 0.005 and *** p < 0.001).

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Mass Spectrometry, Functional Assay, Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: The W-Acidic Motif of Histidine Kinase WalK Is Required for Signaling and Transcriptional Regulation in Streptococcus mutans

doi: 10.3389/fmicb.2022.820089

Figure Lengend Snippet: The CTT of WalK is required for S. aureus WalRK interaction and enzymatic activity. (A) Conservation of the WalK CTT across Gram-positive bacteria. Representative sequences of WalK C-termini were aligned and boxed. The residues are numbered according to S. mutans WalK. Completely conserved residues are colored in white in a red background. The less conserved residues are highlighted in yellow. (B) Alignment of staphylococcus WalK tail sequences. Completely conserved residues are shown in white in a red background and boxed in blue. Highly conserved residues are in red in a white background and boxed in blue. Marked on top are protein secondary structures and residue numbers in S. aureus . (C) Mutations in the CTT disrupt WalRK interaction. A GST fusion protein with full-length S. aureus WalR was used to pull-down S. aureus WalK (364–608) WT and mutant derivatives shown in the top panel. As a negative control, GST alone was used to pull down WalK WT and its mutants shown on the center panel. Shown in the bottom panel are 10% levels of WalK proteins used in each lane. CK shows GST-WalR or GST used in the pull-down. (D) Phosphotransferase of WalK (364–608) was disrupted by mutations in the WalK CTT. The phosphotransferase activity was represented by the reduction in WalK phosphorylation relative to T0, the initial phos-WalK. After the addition of WalR, the mixtures were incubated for the indicated time and stopped by the addition of SDS-loading buffer. Loading controls are shown in the bottom gel. The enzymatic activity was quantified shown below. (E) Phosphatase of WalK (364–608) was disrupted by mutations in the WalK CTT. Phos-tag gel is shown in the top, and a regular SDS-PAGE below shows the total protein used. The phosphatase activity was quantified by the reduction in the phos-WalR relative to the amount of T0, the initial phos-WalR. The reactions were incubated for the indicated time and stopped by the addition of SDS-loading buffer. Data presented are means ± standard deviations (error bars) for three independent experiments. Student’s t -tests were used to compare mutants to WT at each time point (** p < 0.005 and * p < 0.05).

Article Snippet: The acquired wiff files were searched with Maxquant V1.5 against S. mutans serotype C (strain UA159, ATCC 700610) in UniProt.

Techniques: Activity Assay, Bacteria, Residue, Mutagenesis, Negative Control, Phospho-proteomics, Incubation, SDS Page