Fig 1.
S. mutans encodes at least five sets of autoregulatory LRS.
A) Schematic representation of the putative LRS encoded by S. mutans. Open reading frames are drawn to scale and color-coded as follows: LytTR Family regulator (brown), LRS membrane protein (blue), ABC transporter (yellow), other ORFs (grey). ORFs are numbered according to the terminal portions of their respective NCBI Gene Locus Tags (SMU_xxxx). B) Luciferase ORFs were inserted immediately downstream of each putative LRS to create transcription fusion reporters. Luciferase activity was normalized using optical density (OD600) and then compared between the wild-type (WT) and LRS membrane protein mutant reporter strains (ΔM). Data for each of the LRS reporter strains are color-coded as follows: SMU_294/295 (purple), SMU_433/434 (blue), SMU_1070c/1069c (green), HdrRM (orange), and BrsRM (red). C) Each of the putative LRS was tested pairwise for potential cross-regulation of other LRS operons. Luciferase activity of the mutant reporter strains was normalized to optical density (OD600) and then expressed relative to the parental reporter strain values, which were arbitrarily assigned values of 1. LRS reporter strains are color-coded as follows: SMU_294/295 (purple), SMU_433/434 (blue), SMU_1070c/1069c (green), HdrRM (orange), and BrsRM (red). Genes mutated in each of these reporter backgrounds are listed beneath each corresponding column. Unnamed genes are listed by the terminal portions of their respective NCBI Gene Locus Tags (SMU_xxxx). Statistical significance was assessed for each of the reporter strains exhibiting ≥2-fold difference in reporter activity relative to its parental reporter. D) Summary of cross-regulatory interactions between all five LRS. All luciferase data are expressed as means ± s.d. (indicated by error bars) derived from three or four biological replicates. ***P<0.001, **P<0.01, *P<0.05 Unpaired two-tailed Student’s t-test with Welch’s correction.
Table 1.
S. mutans LRS operons are part of the core genome.
Fig 2.
HdrRM operon regulatory elements mediate an autoregulatory positive feedback loop.
A) Partial sequence of the intergenic region upstream of the hdrRM ORFs. The operon promoter is shown in blue font, the direct repeats are shown in red font, and the hdrR operon transcription start site (+1) is shown in green font. ORFs are shaded in solid colors, whereas intergenic regions are striped. B) An hdrRM luciferase reporter was created by replacing the hdrRM ORFs with that of luciferase. The luciferase activity of this parent reporter strain (RM-) was then compared after ectopic hdrR overexpression (ROE), mutation of the operon promoter -10 site (-10), and after combining hdrR ectopic overexpression with a mutant operon promoter -10 site (ROE/-10). Data are presented relative to the parent reporter strain, which was arbitrarily assigned a value of 1. C) Electrophoretic mobility shift assays (EMSAs) were performed with recombinant HdrR and 1 ng of a labeled DNA probe (hdrRMP) encompassing the direct repeat region upstream of the hdrRM operon promoter. To confirm the specificity of HdrR binding to the direct repeats, an unlabeled wild-type DNA probe (hdrRMP) and an unlabeled direct repeat mutant DNA probe (Mutant) were added to the EMSA reactions as competitors. The sequences of both competitor probes are presented under the EMSA image with the direct repeats shown in red. HdrR abundance per reaction: Lane 1 (0 μg), Lane 2 (10 μg), Lane 3 (20 μg), and Lanes 4–8 (30 μg). Wild-type competitor DNA (hdrRMP) abundance per reaction: Lane 5 (50 ng) and Lane 6 (200 ng). Mutant competitor DNA (Mutant) abundance per reaction: Lane 7 (50 ng) and Lane 8 (200 ng). D) An hdrRM luciferase reporter was created by placing a luciferase ORF immediately downstream of the hdrRM ORFs. The luciferase activity of this parent reporter strain (WT) was then compared after mutating hdrM (M-) and after doubly mutating hdrM and the operon direct repeats (M-/DR-). Data are presented relative to the parent reporter strain, which was arbitrarily assigned a value of 1. E) An hdrRM luciferase reporter was created by replacing the hdrRM ORFs with that of luciferase and then ectopically overexpressing hdrR in a single copy on the chromosome, while hdrM was ectopically expressed from a multicopy plasmid (i.e. uncoupled hdrRM expression). The luciferase activity of this reporter strain (RMOE) was then compared to an hdrR ectopic overexpression reporter strain (ROE) and an hdrR ectopic overexpression reporter strain with mutated operon direct repeats (ROE/DR-). Data are presented relative to the reporter strain RMOE, which was arbitrarily assigned a value of 1. All luciferase data are expressed as means ± s.d. (indicated by error bars) derived from four biological replicates. ***P<0.001, **P<0.01, and *P<0.05, Unpaired two-tailed Student’s t-test with Welch’s correction, significance compared to RM- (B), WT (D), and RMOE (E).
Table 2.
S. mutans LRS all encode direct repeat-mediated autoregulation.
Fig 3.
LRS are distinct from TCSTS and ECF systems.
A) Comparison of the domain architectures of TCSTS response regulators, ECF σ factors, and LRS regulators. The illustrated proteins are from S. mutans with the exception of σW (B. subtilis) and σE (E. coli) and are all drawn to scale. Individual protein domains are labeled accordingly. B) Comparison of the domain architectures of TCSTS sensor kinases, ECF anti-σ factors, and LRS membrane proteins. The illustrated proteins are from S. mutans with the exception of RsiW (B. subtilis) and RseA (E. coli) and are all drawn to scale. Individual protein domains are labeled accordingly. Blue rectangles indicate transmembrane segments that are not located within identified conserved protein domains. All proteins were illustrated and annotated using the SMART webserver (http://smart.embl-heidelberg.de) [63].
Fig 4.
Global distribution of putative LRS among prokaryotes. The membrane proteins from the five S. mutans LRS were used as queries to identify potential LRS within the genome data of the NCBI non-redundant nucleotide collection (nr/nt) and whole genome shotgun (wgs) databases. The sizes of the filled circles are proportional to the number of identified genera encoding putative LRS matching to each of the corresponding S. mutans-type LRS.
Fig 5.
Comparison of LRS operons among diverse prokaryotes.
A set of three representative operons matching to each of the five S. mutans LRS was randomly selected from the master table of putative LRS (S3 Table) and illustrated for comparison. In each set of three operons, their descending order in the figure is indicative of their relative homologies to the corresponding S. mutans LRS (Top = high homology, middle = medium homology, and bottom = low homology). Open reading frames are drawn to scale and color-coded as follows: LytTR Family regulator (brown), LRS membrane protein (blue), ABC transporter (yellow), and other ORFs (grey). ORFs are numbered according to the terminal portions of their respective NCBI Gene Locus Tags. Species and Gene Locus Tags are listed from top to bottom as: St (Streptococcus troglodytae; SRT_xxxxx), Ra (Rothia aeria; RA11412_0xxx), Pr (Pseudobutyrivibrio ruminis; CSX00_RSxxxxx), Sa (Streptococcus anginosus; SAIN_RS0xxxx), Lp (Lactobacillus plantarum; LPST_RS0xxxx), Oo (Oenococcus oeni; OEOE_0xxx), Sp (Streptococcus pantholopis; A0O21_RS00xxx), Bf (Butyrivibrio fibrisolvens; G624_RS01100xx), Am (Anaerosporobacter mobilis; BUB90_RSxxxxx), Sc (Streptococcus caviae; BMI76_0xxxx), Cs (Clostridium sciendens; CLOSCI_RS0xxxx), Ct (Chlamydia trachomatis; ERS095036_xxxxx), Sr (Streptococcus ratti; SRA_0xxxx), Sc (Staphylococcus carnosus; VV61_0xxxx), and Ta (Thermoplasmatales archaeon; TALC_RS0xxxx).
Table 3.
Autoregulatory LRS are encoded by diverse bacteria and archaea.
Fig 6.
Purines mediate activation of the BrsRM LRS.
A) The brsRM-gusA reporter was spotted onto chemically defined medium agar plates ± adenine (A) or guanine (G) and grown for four days. The concentrations of the purines supplemented in the agar plates are listed above or below the respective images. In addition, each of the brsRM-inducing transposon insertion mutations was introduced into the brsRM-gusA reporter strain and spotted onto chemically defined medium agar plates containing either B) no purines (–AG), C) 0.132 mM guanine (+G), D) 0.15 mM adenine (+A), or E) 0.132 mM guanine + 0.15 mM adenine (+AG). Strains are listed from left to right as: WT (parent reporter strain), 1 (tilS mutant), 2 (rpoB mutant), 3 (SMU_2060/2061 IGR insertion), 4 (rgpD mutant), 5 (ssuE mutant), 6 (SMU_1406c mutant), 7 (prfC mutant), 8 (SMU_1193 mutant), 9 (mnmE mutant), 10 (SMU_1297 mutant), and 11 (comE mutant). The strains were incubated for two days before imaging.
Table 4.
brsRM-inducing transposon mutations.