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. 2012 Apr;194(8):1968-78.
doi: 10.1128/JB.00037-12. Epub 2012 Feb 17.

Transcriptional organization and physiological contributions of the relQ operon of Streptococcus mutans

Jeong Nam Kim  1 , Sang-Joon Ahn, Kinda Seaton, Steven Garrett, Robert A Burne
Affiliations

Transcriptional organization and physiological contributions of the relQ operon of Streptococcus mutans

Jeong Nam Kim et al. J Bacteriol. 2012 Apr.

Abstract

The molecular alarmone (p)ppGpp functions as a global regulator of gene expression in bacteria. In Streptococcus mutans, (p)ppGpp synthesis is catalyzed by three gene products: RelA, RelP, and RelQ. RelA is responsible for (p)ppGpp production during a stringent response, and RelP is the primary source of (p)ppGpp during exponential growth, but the role of RelQ has not been thoroughly investigated. In this study, we analyzed the four-gene relQ operon to establish how these gene products may affect homeostasis and stress tolerance in the dental caries pathogen S. mutans. Northern blotting and reverse transcriptase PCR demonstrated that relQ is cotranscribed with the downstream genes ppnK (NAD kinase), rluE (pseudouridine synthase), and pta (phosphotransacetylase). In addition, a promoter located within the rluE gene was shown to drive transcription of pta. Inactivation of relQ, ppnK, or rluE did not significantly affect growth of or stress tolerance by S. mutans, whereas strains lacking pta were more sensitive to acid and oxidative stresses. Interestingly, introduction of an rluE deletion into the pta mutant reversed the deleterious effects of the pta mutation on growth and stress tolerance. Accumulation of (p)ppGpp was also decreased in a pta mutant strain, whereas inactivation of relQ caused enhanced (p)ppGpp synthesis in exponential-phase cells. The results reveal an important role for the relQ operon in the expression of traits that are essential for persistence and pathogenesis by S. mutans and provide evidence for a molecular connection of acetate and (p)ppGpp metabolism with tolerance of acid and oxidative stresses.

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Figures

Fig 1
Fig 1
Schematic representation of the relQ operon. RelQ has been demonstrated to be a small (p)ppGpp synthetase. The ppnK is predicted to encode an NAD+ kinase, rluE codes for a pseudouridine synthase, and pta codes for a phosphotransacetylase. SMU.1047c and SMU.1048 (GenBank designations) encode a hypothetical small protein and a protein with similarity to adenylate cyclases, respectively. The prs gene encodes a predicted phosphoribosyl pyrophosphate synthetase. Data were retrieved from the Oral Pathogen Sequence Database (http://www.oralgen.lanl.gov).
Fig 2
Fig 2
(A) Promoters predicted by BPROM (see text) to drive transcription of the relQ operon (top two lines) and pta (bottom three lines). Underlined letters indicate −35 and −10 regions for the putative promoters. Bold letters indicate the predicted start codons (ATG) of relQ and pta. (B) Northern blot analysis with a biotin-labeled probe specific for the pta transcript. (C) Real-time PCR results measuring the copy numbers of pta transcripts in the wild type and relQ operon mutations. (D) Promoter-cat fusion activity from the pta promoter located within the rluE gene. Cells were grown to mid-exponential phase in BHI broth at 37°C in a 5% CO2 atmosphere, and CAT activity was measured as described in Materials and Methods. *, data differ from the wild-type genetic background data at P < 0.001; **, data differ from the wild-type genetic background data at P < 0.01 (Student's t test). WT, wild type.
Fig 3
Fig 3
Growth curves of wild-type and mutant strains obtained during growth in BHI medium. The wild-type (◆), ΔrelQ (■), Δpta (▲), ΔrluEΩ (×), and ΔrluE ΔptaE (●) strains were grown at 37°C with a mineral oil overlay (A) or without a mineral oil overlay (B). Optical density at 600 nm was monitored every 30 min at 37°C using the Bioscreen C Lab system. The results represent the mean values of triplicate growth curves for three independent isolates.
Fig 4
Fig 4
Biofilm formation. S. mutans strains were grown to an OD600 of 0.5 in BHI broth, diluted 1:50 in BM semidefined medium supplemented with 10 mM sucrose (A) or with 20 mM glucose (B) in a 96-well microtiter plate, and incubated at 37°C with 5% CO2 for 48 h. To quantify biofilm formation, the plates were washed twice, stained with 0.1% crystal violet, and resuspended with an ethanol-acetone (8:2 [vol/vol]) mixture. The optical density of the stained biofilm was measured at OD575. Data represent the means and standard deviations (error bars) of the results obtained with three separate isolates assayed in triplicate. *, data differ from the wild-type genetic background data at P ≤ 0.001 (Student's t test); **, data differ from the Δpta strain data at P < 0.005. WT, wild type.
Fig 5
Fig 5
Complementation analysis of the S. mutans Δpta mutant. (A) The copy numbers of the pta transcripts were determined using qRT-PCRs. 16S rRNA was used as a reference transcript for normalization of the data. Values shown represent the means ± standard deviations for RNA data from three separate cultures. *, data differ from the wild-type genetic background data at P < 0.001 (Student's t test). (B) The wild-type strain (◆), Δpta strain (▲), pta strain harboring pDL278 (■), and complemented Δpta strain harboring pBK101 (●) were grown to mid-exponential phase in BHI medium and diluted 1:100 into fresh BHI medium with a mineral oil overlay. Optical density at 600 nm was monitored every 30 min at 37°C using the Bioscreen C Lab system. The results are expressed as means of the results determined with triplicate experiments performed with three independent isolates.
Fig 6
Fig 6
Growth under various stress conditions. The wild-type (◆), ΔrelQ (■), Δpta (▲), ΔrluEΩ (×), and ΔrluE ΔptaE (●) strains were grown in triplicate to mid-exponential phase in BHI medium and diluted to 1:100 into fresh BHI medium supplemented with HCl to lower the pH 5.5 under anaerobic conditions (A), with 25 mM paraquat under anaerobic conditions (B), with 0.003% hydrogen peroxide (H2O2) under anaerobic conditions (C), or with 50 mM sodium acetate under aerobic conditions (D). Optical density at 600 nm was monitored every 30 min at 37°C using the Bioscreen C Lab system. Data are expressed as means of the results determined with triplicate wells for three independent isolates.
Fig 7
Fig 7
Accumulation of (p)ppGpp in S. mutans strains in the presence or absence of hydrogen peroxide. (p)ppGpp accumulation of the wild-type strain compared (A) with that of the ΔrelP, ΔrelQ, and ΔrelPQ mutants and (B) with that of the Δpta, ΔrluEΩ, and ΔrluE ΔptaE mutants. Cells growing exponentially in FMC medium were labeled with [32P]orthophosphate and subjected to the stress conditions for 1 h. Extracts derived from treatment of the cells with 13 M formic acid were normalized to counts per minute (CPM) and spotted onto PEI-cellulose plates for TLC using 1.5 M KH2PO4 buffer.
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