Analysis of an Agmatine Deiminase Gene Cluster in Streptococcus mutans UA159

An operon encoding enzymes of the agmatine deiminase system(AgDS) has been identified in the cariogenic bacterium Streptococcusmutans UA159. The AgDS is regulated by agmatine induction andcarbohydrate catabolite repression. Ammonia is produced fromagmatine at low pH, suggesting that the AgDS could augment acidtolerance.

INTRODUCTION

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Introduction

Streptococcus mutans is the etiological agent of dental caries(12). A key virulence attribute of this organism is acid tolerance,since catabolism of dietary carbohydrates lowers the pH of dentalplaque to values below 4.0 (5). A primary determinant of acidtolerance is the membrane-bound F₁F₀-ATPase, although reductionin the proton permeability of the cell membrane and inductionof DNA repair pathways and stress proteins also contribute tothe acid tolerance of S. mutans (17). Another widely used mechanismfor acid resistance in less acid-tolerant oral streptococciis the generation of ammonia by the arginine deiminase system(ADS) (6).

S. mutans metabolizes a wider variety of carbohydrates thanmany gram-positive bacteria, but the ADS is not present in thisorganism (1, 15). Consequently, when a cluster of genes withsimilarity to ADS genes (1) was identified in the S. mutansUA159 genome, it was proposed that the genes encoded enzymesof the agmatine deiminase system (AgDS). The AgDS is analogousto the ADS but is involved in catabolism of agmatine (Fig. 1)(1), a decarboxylated derivative of arginine (19). In lacticacid bacteria and Enterococcus faecalis, the AgDS resemblesthe ADS. However, in Pseudomonas aeruginosa and other organisms,agmatine is produced from arginine via an intracellular argininedecarboxylase, and putrescine is converted to spermidine orbroken down to succinate. The identification of an ADS-likegene cluster in S. mutans is significant because agmatine catabolismmay contribute to acid tolerance. Production of alkali fromagmatine may have critical implications for the pathogenicityof S. mutans and may profoundly affect the ecology of oral biofilms.Thus, we have initiated an analysis of the AgDS gene cluster.

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FIG. 1. Comparison of the ADS and AgDS pathways. Agmatine can be produced from arginine by arginine decarboxylase. AD, arginine deiminase; CK, carbamate kinase.

Analysis of the sequence of the AgD gene cluster.

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Analysis of the sequence...

The first gene in the operon, otcA, encodes a putative putrescinecarbamoyltransferase (PTC) and has been redesignated aguB. AguBis 80% identical to ArgF-2, annotated as an ornithine carbamoyltransferase(OTC) in E. faecalis V583 (Fig. 2) (14). ArgF-2 is one of twoputative OTCases encoded by the E. faecalis genome (14), althoughargF-2 is located in an operon encoding an apparent AgD, carbamatekinase, and antiporter. The other OTC gene is in the ADS operon(4), so ArgF-2 may be a PTC (18). The PTC amino acid sequenceis not available, but the enzyme is well characterized and distinctfrom OTC (21).

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FIG. 2. Putative AgDS gene cluster in S. mutans UA159 and percent identity to similarly organized clusters in other bacteria, based on NCBI Blast using sequences obtained from GenBank (National Center for Biotechnology Information). ${alpha}$ Port, agmatine:putrescine antiporter; CK, carbamate kinase; N/A, xasA sequence is not available.

According to Prosite (http://www.expasy.org/prosite), the carbamoyltransferaseconsensus is F-X-(E/K)-X-S-(G/T)-R-T, with the third residuedifferentiating aspartate (E) and ornithine (K) carbamoyltransferases.This consensus is present in the amino acid sequence of theS. mutans aguB product, as well as E. faecalis ArgF-2, Lactobacilluslactis subsp. lactis OtcA, Listeria monocytogenes LMO0036, andLactobacillus sakei ArgF, with a highly conserved Q in the thirdposition, perhaps reflecting a preference for putrescine. TheHPTQ residues at positions 143 to 146, which are involved incarbamoylphosphate binding, are present in AguB, as are theHCLP residues at positions 281 to 284, which facilitate ornithinebinding (11). Since the structures of ornithine and putrescineare identical at the position of cleavage, this conservationis not surprising. The second gene in the operon, SMU.263, encodesa putative amino acid antiporter and has been redesignated aguD.Similar to the case of other amino acid antiporters, 11 transmembranehelices were predicted for the amino acid sequence of AguD byusing the "DAS" - Transmembrane Prediction server at http://www.sbc.su.se/ $~$ miklos/DAS(9). The third gene, SMU.264, encodes AgD and has been redesignatedaguA. The GGGNIHCITQQ sequence (13) was identified at the Cterminus of AguA. The final gene, arcC, encodes a carbamatekinase and has been redesignated aguC to reflect its associationwith the AgDS.

In addition to aguBDAC, a transcriptional regulator of the LuxRfamily is located 239 bp upstream of aguB and transcribed inthe opposite direction. LuxR-type proteins belong to the FixJ-NarLsuperfamily, mainly composed of two-component response regulators(10). Homologs of this regulatory protein were identified 399and 234 bp upstream of the AgDS gene clusters in E. faecalisand L. lactis subsp. lactis, respectively. Involvement of thisprotein in AgDS regulation is under investigation.

Cotranscription of aguBDAC.

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Cotranscription of agubdac.

No terminator-like sequences were identified in the agu intergenicregions. To demonstrate cotranscription of aguBDAC, a polarmutation in aguB was constructed by insertion of an ${Omega}$ kan cassetteharboring strong transcription/translation termination signals(16). Northern blotting was performed (3) with 10 µg oftotal RNA extracted (8) from wild-type and aguB mutant cellsgrown in low-carbohydrate tryptone-vitamin-based (TV) broth(7) supplemented with 0.5% galactose and 10 mM agmatine. TheRNA was hybridized to an aguB probe and labeled by using theBright Star labeling kit (Ambion Inc., Austin, Tex.). The probehybridized to a 4.6-kb wild-type mRNA, consistent with the sizeof the operon (Fig. 3). No transcript was detected in the aguBmutant strain.

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FIG. 3. Northern blot analysis of total RNA isolated from wild-type and aguB mutant S. mutans strains grown in TV broth containing 0.5% galactose and 10 mM agmatine. RNA (10 µg) was hybridized to an aguB DNA probe.

AgDS expression in S. mutans.

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Agds expression in s....

To quantify mRNA under different growth conditions, slot blotanalysis was used. Total RNA extracted from cells grown in TVbroth plus 0.5% glucose or 0.5% galactose, with or without 10mM agmatine, was transferred to a 0.45-µm nitrocellulosemembrane. The RNA was hybridized to an aguB probe. AgDS-specificmRNAs were detected under all conditions, although expressionwas severalfold higher when cells were grown in medium containingagmatine (Fig. 4).

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FIG. 4. Slot blot analysis of mRNA isolated from wild-type S. mutans grown in TV broth containing 0.5% glucose (Glu), 0.5% glucose and 10 mM agmatine (GluAg), 0.5% galactose (Gal), or 0.5% galactose and 10 mM agmatine (GalAg). The mRNA was transferred to a nitrocellulose membrane and hybridized to an aguB DNA probe. "RNased" indicates a control in which 2 µg of RNA was treated with RNase prior to application to the membrane.

AgD activity in permeabilized cells grown under different growthconditions was measured by colorimetric determination of N-carbamoylputrescineproduction from agmatine (Fig. 5A) (2). In wild-type S. mutans,peak AgD activity was observed in cells grown in TV containingthe non-catabolite-repressing sugar galactose and agmatine,whereas activity was 65% lower in cells grown in the repressingsugar glucose and agmatine. Thus, the operon is inducible byagmatine and under the control of carbohydrate catabolite repression.No AgD activity was measurable in the strain carrying the polarinsertion in aguB, confirming that the operon encodes the AgDenzyme and transcription of the genes occurs from a promoter5' to aguB.

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FIG. 5. AgD activity (A) and production of ammonia from agmatine (B) in wild-type (WT) S. mutans grown in TV containing 0.5% glucose (Glu), 0.5% glucose and 10 mM agmatine (GluAg), 0.5% galactose (Gal), or 0.5% galactose and 10 mM agmatine (GalAg). Activity of a polar mutant (AguB^-) of S. mutans grown in TV containing 0.5% galactose and 10 mM agmatine was measured.

Ammonia production was measured after cells were incubated withbuffer and 10 mM agmatine, using an ammonia detection kit (DiagnosticChemicals Limited, Charlottetown, Canada). Consistent with AgDactivity, ammonia production was observed only in cells grownwith agmatine (Fig. 5B). Ammonia production increased two-thirdsin cells grown in galactose and agmatine, compared to glucoseand agmatine. The optimum pH for ammonia production from agmatinewas 4 (Fig. 6).

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FIG. 6. Ammonia production from agmatine at various pHs by wild-type S. mutans grown in TV broth containing 0.5% galactose and 10 mM agmatine.

Summary.

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Summary.

AgDS of S. mutans is functional and under tight genetic control.The system is expressed at a low level relative to urease andarginine deiminase of other oral streptococci, and it is unlikelythat agmatine catabolism would cause significant environmentalalkalinization. However, ammonia production by the AgDS at lowpH would increase ${Delta}$ pH and provide ATP, which could be used forgrowth or to extrude protons, and the system is capable of functioningat pH values that are considered extreme in dental plaque (20).Therefore, the AgDS may represent a significant contributorto acid tolerance and thus to the virulence of S. mutans invivo.

Nucleotide sequence accession number.

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Nucleotide sequence accession...

The sequences of these genes have been deposited with GenBankand bear accession number BK004003.

ACKNOWLEDGMENTS

This research was supported by Public Health Service grant DE10362from the National Institute of Dental and Craniofacial Research.

FOOTNOTES

* Corresponding author. Mailing address: Department of Oral Biology, University of Florida, P.O. Box 100424, 1600 SW Archer Rd., Gainesville, FL 32610-0424. Phone: (352) 392-4370. Fax: (352) 392-7357. E-mail: rburne{at}dental.ufl.edu.

REFERENCES

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