3'-Phosphoadenosine-5'-phosphate phosphatase activity is required for superoxide stress tolerance in Streptococcus mutans

Abstract

Aerobic microorganisms have evolved different strategies to withstand environmental oxidative stresses generated by various reactive oxygen species (ROS). For the facultative anaerobic human oral pathogen Streptococcus mutans, the mechanisms used to protect against ROS are not fully understood, since it does not possess catalase, an enzyme that degrades hydrogen peroxide. In order to elucidate the genes that are essential for superoxide stress response, methyl viologen (MV)-sensitive mutants of S. mutans were generated via ISS1 mutagenesis. Screening of approximately 2,500 mutants revealed six MV-sensitive mutants, each containing an insertion in one of five genes, including a highly conserved hypothetical gene, SMU.1297. Sequence analysis suggests that SMU.1297 encodes a hypothetical protein with a high degree of homology to the Bacillus subtilis YtqI protein, which possesses an oligoribonuclease activity that cleaves nano-RNAs and a phosphatase activity that degrades 3'-phosphoadenosine-5'-phosphate (pAp) and 3'-phosphoadenosine-5'-phosphosulfate (pApS) to produce AMP; the latter activity is similar to the activity of the Escherichia coli CysQ protein, which is required for sulfur assimilation. SMU.1297 was deleted using a markerless Cre-loxP-based strategy; the SMU.1297 deletion mutant was just as sensitive to MV as the ISS1 insertion mutant. Complementation of the deletion mutant with wild-type SMU.1297, in trans, restored the parental phenotype. Biochemical analyses with purified SMU.1297 protein demonstrated that it has pAp phosphatase activity similar to that of YtqI but apparently lacks an oligoribonuclease activity. The ability of SMU.1297 to dephosphorylate pApS in vivo was confirmed by complementation of an E. coli cysQ mutant with SMU.1297 in trans. Thus, our results suggest that SMU.1297 is involved in superoxide stress tolerance in S. mutans. Furthermore, the distribution of homologs of SMU.1297 in streptococci indicates that this protein is essential for superoxide stress tolerance in these organisms.

FIG. 1.

Verification of superoxide-sensitive phenotype. ISS1 transposon mutants that displayed an initial MV-sensitive phenotype were further verified by spotting of 7.5 μl from a 10-fold dilution series, with a starting optical density (A₆₀₀) of 2.0 made in 0.85% NaCl, onto THY agar plates containing 5 mM MV (THY+MV). As a control, cultures were also spotted on plain THY agar plates with no additions (THY). Experiments were repeated at least three times, and the relevant areas of the representative plates are shown. UA159 is the wild-type strain, while IBS921, IBS922, and IBS923 are independent MV-sensitive mutants.

FIG. 2.

(A) Construction of a SMU.1297 deletion mutant. The direction of transcription of SMU.1297 and adjacent genes in the chromosome of S. mutans is indicated by block arrows. The SMU.1297 deletion strain, IBS934, was constructed using a Cre-loxP-based markerless gene deletion system (see Materials and Methods) that removed the central part of the SMU.1297 gene (hatched box), and inserted a 34-bp lox72 sequence to disrupt the gene (vertical white line). The bar below the chromosome indicates the region of SMU.1297 that was cloned into the complementing plasmid pIB1297. The site of the ISS1 insertion is shown by an inverted triangle. The bent arrow indicates a putative promoter region upstream of SMU.1297 gene. The diagram is not drawn to scale. (B) Transcriptional linkage analysis of the SMU.1297 locus. RNA, isolated from S. mutans UA159, was used as a template to produce cDNA, on which PCR was performed (lanes 2 and 5). RNA (lanes 3 and 6), and chromosomal DNA (lanes 1 and 4) were included as controls. Primers 1 and 2 (5′-GCCGGATCGACTGGACTGAAAGAATCGCTCAG-3′ and 5′-GCCCTCGAGCTGACTGCCTAATGCGTCAGGG-3′, respectively), shown by arrowheads in panel A, were used for the linkage analysis between SMU.1296 and SMU.1297, whereas primers 3 and 4 (5′-GGCCATCCATTAGCAAGTGGTG-3′ and 5′-TTGGTAACCCGTTGAAGTATCC-3′, respectively) were used for the linkage analysis between SMU.1297 and rpmE.

FIG. 3.

Phenotypic characterization of the SMU.1297 deletion mutants. Overnight cultures of the wild type (UA159), the ISS1 insertion mutant (IBS921), the clean Cre-loxP deletion mutant (IBS934), and the complemented strain (IBS934/pIB1297) were grown in THY medium with appropriate antibiotics. Different dilutions of the overnight culture, with a starting optical density (A₆₀₀) of 5.0 made in 0.85% NaCl, were spotted on THY agar (THY) or THY agar supplemented with 5 mM MV (THY+MV) or 25 μg/ml menadione (THY+MND) and incubated overnight under microaerophilic conditions at 37°C. Experiments were repeated at least three times, and relevant areas of the representative plates are shown.

FIG. 4.

SMU.1297 is not required for general stress tolerance in S. mutans. (A) Acid stress. Dilutions of fresh overnight cultures were spotted on THY agar plates in the absence (THY) or presence of sodium citrate buffer to generate a pH of 5.5 to mimic acid stress; the pH 7.0 culture served as a control. (B) Oxidative and thermal stress. Cultures were prepared and spotted as described above, on THY plates containing 1 mM hydrogen peroxide (H₂O₂), for oxidative stress, or on plates containing 0.75 μg/ml puromycin (PU), for thermal stress. Plates were incubated overnight under microaerophilic conditions at 37°C. Experiments were repeated at least three times, and the relevant areas of the representative plates are shown.

FIG. 5.

Genomic context and sequence alignment of SMU.1297. (A) Organization of open reading frames in the SMU.1297 region in various members of the Firmicutes. The SMU.1297 locus (black arrow) appears to be variable in different Firmicutes. However, the gene immediately downstream of SMU.1297, rpmE, appears to be conserved in streptococci. A putative GST gene (dark gray arrows) is located immediately upstream of SMU.1297 in S. mutans and S. thermophilus only. Smu. S. mutans; Sag, Streptococcus agalactiae; Spr, Streptococcus pneumoniae; Spy, S. pyogenes; Bsu, B. subtilis; Str, S. thermophilus; Sgo, Streptococcus gordonii; Lac, Lactococcus lactis; Efa, Enterococcus faecalis; and Sau, S. aureus. (B) Comparison of the amino acid sequence of SMU.1297 with homologous proteins identified by BLASTP search. Clustal-W was used to align the sequences from different Firmicutes.

FIG. 6.

SMU.1297 is required for growth under cysteine starvation conditions. (A) Complementation of an E. coli cysQ mutant by the expression of SMU.1297. E. coli UM285, a cysQ-deficient strain, was transformed with vector plasmid pOri23 or pOri23 carrying the full-length wild-type SMU.1297 gene (pIB1297). As a positive control, UM285 containing pUM412, which contains the B. subtilis ytqI gene, was also included. Cultures were prepared as described in Materials and Methods, 10-fold serially diluted, and spotted on a semisynthetic minimum agar medium (CDM) with or without 0.2% cysteine. Plates were incubated overnight at 37°C under aerobic conditions. Experiments were repeated three times, and relevant areas of the representative plates are shown. (B) Growth of S. mutans under cysteine-depleted conditions. Fresh overnight cultures were washed with 0.85% NaCl, 10-fold serially diluted, and spotted on CDM with or without 0.2% cysteine. Plates were incubated 48 h at 37°C under microaerophilic conditions. Experiments were repeated four times, and relevant areas of the representative plates are shown. (C) Growth curve of S. mutans strains in CDM broth containing 0.075% cysteine.

FIG. 7.

pAp phosphatase activity of S. mutans SMU.1297 protein. (A) pAp phosphatase activity as a function of time. Reaction mixtures containing 5 μg of protein and 40 μM pAp were incubated for various lengths of time at room temperature. The reaction buffer contained either MnCl₂ or MgCl₂, as described in the text. Reactions were stopped by the addition of an equal volume of 100 mM EDTA, the liberated phosphates were quantified using a phosphate colorimetric assay kit (malachite green method) as described in Materials and Methods, and the absorbance was measured using a BioTek Synergy HT reader, at 600 nm. (B) pAp phosphatase activity as a function of protein concentration. The assay for phosphatase activity was carried out for 20 min, at room temperature, in the presence of increasing amounts of His-SMU.1297 protein and 40 μM pAp. The liberated phosphate was quantified as described for panel A. For both the graphs, the y axis shows percent conversion of pAp to AMP and inorganic phosphate.