3′-Phosphoadenosine-5′-Phosphate Phosphatase Activity Is Required for Superoxide Stress Tolerance inStreptococcus 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.

Characterization of NrnA homologs from Mycobacterium tuberculosis and Mycoplasma pneumoniae

“…This protein was previously identified in a screen for proteins that confer resistance to superoxide stress in this organism. SMU.1297 was shown to have pAp-phosphatase activity in vitro and to rescue an E. coli cysQ mutant when expressed from a plasmid (Zhang and Biswas 2009). However, nanoRNase activity could not be detected in vitro in that study.…”

“…In addition, we demonstrate that SMU.1297 from Streptococcus mutans, which was previously shown to dephosphorylate pAp in vitro and to complement an E. coli cysQ mutant (Zhang and Biswas 2009), can also complement a conditional E. coli orn mutant. Our results suggest that the ability to hydrolyze both pAp and nanoRNA is a common feature of NrnA homologs.…”

“…We also show proof for bifunctionality of SMU.1297, the NrnA homolog from S. mutans, by showing the ability of this protein to complement a conditional mutant of E. coli orn. This protein was previously shown to have pAp-phosphatase activity, but activity on nanoRNA could not be detected in vitro (Zhang and Biswas 2009).…”

Role of RecJ-like Protein with 5′-3′ Exonuclease Activity in Oligo(deoxy)nucleotide Degradation

“…SMU.1297 (family N_142143, Fig. 1A) from Streptococcus mutans, a facultative anaerobe, hydrolyzed pAp, but not short ssRNA, and complemented an E. coli cysQ mutant (55). A smu.1297 mutant was sensitive to superoxide stress, suggesting that SMU.1297 was involved in superoxide stress tolerance.…”

Role for cytoplasmic nucleotide hydrolysis in hepatic function and protein synthesis

“…In addition, the identification of a phenotypically related human skeletal disease resulting from mutations in gPAPP confirms that Golgi-resident 3′-nucleotidase activity is essential for sulfation-dependent processes such as cartilage formation and long bone growth (14). Conversely, functional studies in bacteria and yeast have implicated the more ancient cytosolic 3′-nucleotidase Bpnt1 in numerous sulfation-independent processes, including salt tolerance and methionine biosynthesis (15)(16)(17)(18)(19)(20)(21)(22)(23). Thus, although it is clear that gPAPP is essential for proper Golgimediated sulfation, the role of Bpnt1 in mammals and its potential connection to cytosolic sulfation remains unclear.…”