Genetics and Physiology of Acetate Metabolism by the Pta-Ack Pathway of Streptococcus mutans

Abstract

In the dental caries pathogen Streptococcus mutans, phosphotransacetylase (Pta) catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate (AcP), which can be converted to acetate by acetate kinase (Ack), with the concomitant generation of ATP. A ΔackA mutant displayed enhanced accumulation of AcP under aerobic conditions, whereas little or no AcP was observed in the Δpta or Δpta ΔackA mutant. The Δpta and Δpta ΔackA mutants also had diminished ATP pools compared to the size of the ATP pool for the parental or ΔackA strain. Surprisingly, when exposed to oxidative stress, the Δpta ΔackA strain appeared to regain the capacity to produce AcP, with a concurrent increase in the size of the ATP pool compared to that for the parental strain. The ΔackA and Δpta ΔackA mutants exhibited enhanced (p)ppGpp accumulation, whereas the strain lacking Pta produced less (p)ppGpp than the wild-type strain. The ΔackA and Δpta ΔackA mutants displayed global changes in gene expression, as assessed by microarrays. All strains lacking Pta, which had defects in AcP production under aerobic conditions, were impaired in their abilities to form biofilms when glucose was the growth carbohydrate. Collectively, these data demonstrate the complex regulation of the Pta-Ack pathway and critical roles for these enzymes in processes that appear to be essential for the persistence and pathogenesis of S. mutans.

FIG 1

Schematic of the Pta-Ack pathway that produces an AcP from acetyl-CoA and then acetate and ATP. BCAA, branched-chain amino acids; PDH, pyruvate dehydrogenase; PFL, pyruvate formate lyase; P_i, inorganic phosphate; CoASH, coenzyme A.

FIG 2

(A) 2D-TLC. AcP levels in acid extracts of cells that were labeled with [³²P]orthophosphate were monitored. Arrows, the position to which AcP migrates. Strains include the wild-type (WT) and ΔackA, Δpta, and Δpta ΔackA double mutant strains, as indicated by the labels. (B) Complementation of ackA and pta mutations. Accumulation of AcP in the wild-type and ΔackA mutant strains compared with that in the Δpta and ΔackA mutants in which the defective gene was complemented with a wild-type copy of pta (Δpta + com) or ackA (ΔackA + com). (C) Measurement of ATP. The wild-type, ΔackA, Δpta, and Δpta ΔackA strains were grown aerobically to early exponential phase in FMC medium, harvested, and resuspended in 250 μl cold buffer A (see details in Materials and Methods). Cells were lysed, and 50 μl of the lysates was reacted with an equal volume of the luminescent reagent. Luminescence intensity was measured using a Synergy 2 multimode microplate reader. The values shown are the means ± standard deviations for cell lysates from three separate cultures. *, the result differs significantly (P < 0.05, Student's t test) from that obtained in the wild-type genetic background. The results are expressed as means from triplicate assays for three independent isolates.

FIG 3

Growth and accumulation of AcP under stress conditions. (A) Growth of the wild-type, ΔackA, Δpta, and Δpta ΔackA strains was monitored in medium supplemented with 0.003% (vol/vol) H₂O₂ under relatively anaerobic conditions (see Materials and Methods for details) at 37°C using a Bioscreen C lab system. Data are expressed as the means of the results for three independent isolates grown in triplicate wells. (B) AcP accumulation of the wild-type, ΔackA, Δpta, and Δpta ΔackA strains was detected when cells were grown in the presence of 0.003% hydrogen peroxide. Cells were grown to the early exponential phase (OD₆₀₀ = 0.4) in FMC medium, and cells were concurrently labeled with [³²P]orthophosphate and subjected to the stress conditions for 1 h. AcP and P_i derived from S. mutans extracts by formic acid were spotted onto PEI-cellulose plates for 2D-TLC using first-dimension buffer (0.52 M LiCl and 1% [vol/vol] glacial acetic acid) and second-dimension buffer (1 M ammonium acetate and 0.35 M ammonium chloride). Arrows, the spot corresponding to AcP.

FIG 4

Accumulation of (p)ppGpp and cellular ATP generation under stress conditions. (A) The (p)ppGpp accumulation of the wild-type strain was compared with that of the ΔackA, Δpta, and Δpta ΔackA strains grown in FMC medium supplemented with (+) and without (−) 0.003% hydrogen peroxide. Strains were labeled with [³²P]orthophosphate in FMC medium. ³²P-labeled nucleotides were extracted by adding an equal volume of 13 M formic acid, followed by three freeze-thaw cycles in a dry ice-ethanol bath. Acid extracts were spotted onto PEI-cellulose plates for TLC in 1.5 M KH₂PO₄ buffer. (B) The amount of ATP in the wild-type strain and the ΔackA, Δpta, and Δpta ΔackA mutants was measured after they were grown to early exponential phase in FMC medium supplemented with and without 0.003% hydrogen peroxide (see details in Materials and Methods). Fifty microliters of cell lysate was reacted with an equal volume of the luminescent reagent. Luminescence intensity was measured using a Synergy 2 multimode microplate reader. Values shown are the means ± standard deviations for cell lysates from three separate cultures. *, the result differs significantly (P < 0.05, Student's t test) from that for the wild-type genetic background. The results are expressed as means from triplicate assays for three independent isolates.

FIG 5

Confocal microscopic images of biofilms. Biofilms of S. mutans UA159 and mutant strains were grown in BM supplemented with 20 mM glucose for 24 h (A) or 48 h (B) at 37°C in a 5% CO₂ atmosphere. The data presented are representative of those from three independent experiments. The distance between two asterisks is 5 μm per side. Small images at the bottom present biofilm thickness. See the Materials and Methods section for details on labeling and image analysis.

FIG 6

Numbers of genes grouped by functional categories that were differently expressed between wild-type and ΔackA strains (A), between wild-type and Δpta ΔackA strains (B), and between ΔackA and Δpta ΔackA strains (C). Gene annotations are based on information provided by the Los Alamos National Laboratory (http://www.oralgen.org). aa, amino acid.

FIG 7

Current model for the role of the Pta-Ack pathway in S. mutans. The activities of products encoded by the ackA or pta genes are primarily responsible for the generation of AcP, the levels of which strongly influence physiology and gene expression in S. mutans. In particular, the role of the Pta-Ack pathway in modulating the expression of genes involved in carbohydrate flux at the pyruvate node, as well as those contributing to biofilm formation and stress tolerance, is significant. Moreover, a metabolic linkage between acetate metabolism and (p)ppGpp production in response to oxidative stress is established and is probably mediated by the small (p)ppGpp synthase RelP and the two-component signal transduction system RelRS. Red arrow, high level of accumulation of AcP; dashed arrows, the observations and models.