Cloning and nucleotide sequence analysis of the Streptococcus mutans membrane-bound, proton-translocating ATPase operon

GENE AN INTERNATIONAL

JOURNAL

ON

GENES AND OENOME5

ELSEVIER

Gene183(1996) 87-96

Cloning and nucleotide sequence analysis of the Streptococcus mutans membrane-bound, proton-translocating ATPase operon Alan J. Smith a, Robert G. Quivey, Jr. a'b'*, Roberta C. Faustoferri

a

a Department of Dental Research, School of Medicine and Dentistry, University of Rochester, Box 611, 601 Elmwood Ave., Rochester, N Y 14642, USA b Department of Microbiology and Immunology, School of Medicine and Dentistry, University of Rochester, Rochester, N Y 14642, USA

Received 17 January 1996; revised 6 May 1996; accepted 10 May 1996

Abstract

The function of the membrane-bound ATPase in S. mutans is to regulate cytoplasmic pH values for the purpose of maintaining ApH. Previous studies have shown that as part of its acid-adaptive ability, S. mutans is able to increase H+-ATPase levels in response to acidification. As part of the study of ATPase regulation in S. mutans, we have cloned the ATPase operon and determined its genetic organization. The structural genes from S. mutans were found to be in the order: c, a, b, delta, alpha, gamma, beta, and epsilon; where c and a were reversed from the more typical bacterial organization. The operon contained no I gene homologue but was preceded by a 239-bp intergenic space. Deduced aa sequences from open reading frames indicated that genes encoding homologues of glycogen phosphorylase and nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase flank the H ÷-ATPase operon, 5' and 3' respectively. Sequence analysis indicated the presence of three invertedrepeat nt sequences in the glgP-uncE intergenic space. Primer extension analysis of mRNAs prepared from batch-grown or steadystate cultures demonstrated that the transcriptional start site did not change as a function of culture pH value. The data suggest that potential stem-and-loop structures in the promoter region of the operon do not function to alter the starting position of ATPase-specific mRNA transcription. Keywords: Streptococci; ATPase; Adaptation; Acidurance; Caries

I. Introduction S. mutans is an etiologic agent of dental caries which adheres to tooth surfaces and participates in the development of the microbiological niche referred to as dental plaque (Loesche, 1986). The metabolism of dietary sugars by S. mutans results in the production and secretion of lactic acid, which in turn reduces the p H value of the surrounding milieu. As plaque p H values * Corresponding author at address a. Tel. + 1 716 2750382; Fax + 1 716 4732679; e-mail: [email protected] Abbreviations: aa, amino acid(s); Ap, ampicillin; ATPase, membranebound, proton-translocating ATPase; B., Bacillus; BHI, Brain heart infusion (medium); Cm, chloramphenicol; E., Escherichia; En., Enterococcus; gapN, nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene; glgP, glycogen phosphorylase; kb, kilobase(s) or 1000bp; LB, Luria-Bertani (medium); nt, nucleotide(s); oligo, oligodeoxyribonucleotide; PCR, polymerase chain reaction; R, resistance/resistant; S., Streptococcus; V., Vibrio. 0378-1119/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0378-1119(96)00502-1

are lowered, tooth enamel begins to demineralize and the caries process is initiated (Nikiforuk, 1985). S. mutans is able to survive acidification of its environment due to adaptive mechanisms that appear to include the membrane-bound, proton-translocating ATPase (Belli and Marquis, 1991; Hamilton and Buckley, 1991) and a D N A repair system (Quivey et al., 1995). The ATPase functions to regulate the internal p H (pHi) of oral streptococci (and En. hirae), such that a ApH is maintained between the extracellular environment and the interior of the cell, with the interior being more alkaline (Kobayashi, 1985; Bender et al., 1986; Kobayashi et al., 1986; Dashper and Reynolds, 1992). When cultures of S. mutans become acidified, the organism increases levels of ATPase activity in order to both remove protons from the cytoplasm and to protect relatively acid-sensitive glycolytic enzymes (Belli and Marquis, 1991; Hamilton and Buckley, 1991). Acidurance of S. mutans depends, therefore, mainly on its ability to regulate ATPase levels.

88

A.J. Smith et al./Gene 183 (1996) 87-96

Previous studies have shown that the adaptation can occur in the time-frame of one generation (Belli and Marquis, 1991). Thus, it appears that specific mechanisms may act to influence ATPase levels in S. mutans. As part of the ongoing study to understand the mechanisms of acid adaptation in S. mutans, and the means by which this organism increases its levels of ATPase activity, we report here the cloning of the genes encoding the ATPase operon from S. mutans. The operon contained eight structural genes in the order of c, a, b, delta, alpha, gamma, beta, and epsilon. An intergenic space, containing three pairs of inverted-repeats, was found between the first structural gene and the preceding open-reading frame. Thus, the organization of the structural genes is similar to reported partial operon sequences from other streptococcal species but dissimilar to that from En. hirae (formerly S. faecalis) (Shibata et al., 1992). The promoter from the S. mutans operon was most homologous to that reported for En. hirae, which is also able to regulate its ATPase levels (Kobayashi, 1985), thereby suggesting a conserved mechanism of ATPase regulation.

2. Results and discussion 2.1. Cloning o f the S. mutans ATPase operon

PCR amplification of S. mutans GS-5 chromosomal DNA, using degenerate primers from conserved [3-subunit aa sequences, generated an approximately 750-bp DNA fragment. The nt sequence of the fragment was found to be highly homologous, at the DNA level, to the fragment which we had previously identified from S. sobrinus (Quivey et al., 1991). Using the S. mutans GS-5 13-subunit fragment as a probe, an approximately 8.5-kb EcoRI fragment was identified in digests of genomic DNA. The larger fragment was subsequently cloned into an intermediate copy-number vector, pSU20, to form the plasmid pSMA40. Nucleotide sequence of the 5' end of the cloned EcoRI fragment revealed significant homology to the partial c subunit coding information previously reported for S. oralis (Fenoll et al., 1994). On the basis of that homology, the deduced aa sequence from the 5'-terminus of the insert in pSMA40 was aligned with other species, such that we had tentatively identified the coding sequence for the c subunit homologue from S. mutans (Fig. 1). Subclones of pSMA40, and nested deletions thereof, were used to obtain the complete nt sequence of the operon (see Table 1 for subclones; Fig. 2 for nt sequence). The results of the sequence determination showed that pSMA40 contained complete ATPase structural genes in the order c, a, b, delta, alpha, gamma, beta, and epsilon (Fig. 2). The gene order was similar to that reported for En. hirae (formerly S. faecalis)

(Shibata et al., 1992), S. oralis, and S. pneumoniae (Fenoll et al., 1994). Interestingly, the c and a subunits were reversed from what has been typically found in most bacterial species. The calculated molecular weights for each of the subunits are shown with the estimated isoelectric points for each of the subunits (Fig. 3). The calculated weights were similar to previous estimates, arrived at following SDS-polyacrylamide gel electrophoresis of the F1 subunits (Sutton and Marquis, 1987). 2.2. ATPase flanking regions identified as glgP and gapN

Since the insert in pSMA40 contained only nine bases upstream of the c subunit initiation ATG codon, it was necessary to identify restriction fragments of genomic DNA which might contain potential regulatory sequences for the operon. PCR was used to amplify a 145-bp fragment from the c subunit coding region, which was then used to probe restriction digests of S. mutans DNA. Southern blot analysis identified an approximately 1.6-kb PstI fragment of genomic DNA which hybridized to the c subunit probe. DNA fragments of the approximate size range were gel fractionated, purified, cloned, and screened by colony hybridization using the 145-bp c subunit fragment as the probe. Nucleotide data of one resulting clone, designated pGP78, were analyzed for possible sequence homologies using a BLAST search of the GenBank databases (Altschul et al., 1990). The search results suggested that the 5'-terminus of the insert contained the coding information for the carboxy-terminus of the S. mutans homologue of glgP, based on a high degree of homology of the deduced aa sequence to that from B. subtilis (Kiel et al., 1994). Nucleotide data from the 3'-terminus of the insert showed sequences which originated in the c subunit and extended upstream from the ATPase structural genes into the glgP locus. The results suggested that we had obtained the region of S. rnutans GS-5 chromosome most likely to contain the ATPase promoter region. Sequences downstream from epsilon, the last structural gene of the operon, were also used to BLAST search the GenBank database (Altschul et al., 1990). The results of that search revealed a high level of homology to the recently reported sequence for gapN from S. mutans strain NG5 (Boyd et al., 1995). The genetic map resulting from nt determinations of the S. mutans ATPase operon, with flanking regions, is shown (Fig. 3). 2.3. Relationship o f S. mutans ATPase to other bacterial species

Comparisons between homologies of the S. mutans ATPase structural genes to established sequences and

89

A.J. Smith et al./Gene 183 (1996) 87 96

S. E. B. B. E.

1 mutans ......... ML hirae ......... MNY megaterium ...... MGL subtilis ....... M N L coli MENLNMDLLY

S. E. B. B. E.

mutans hirae megaterium subtilis coli

NLKILALGIA IAAAIAIMGA IASAIAIGLA IAAAIAIGLG MAAAVMMGLA

51 TLMIMGVAFI TTMFIGVALV SMMFVGVALV TLMFMGIALV TQFFIVMGLV

VLGVSLGEGI AIGAGYGNGQ ALGAGIGNGL ALGAGIGNGL AIGAAIGIGI

EGTFFVLLAS EAVPILGVVI EALPIIAVVI EALPIIAVVI DAIPMIAVGL

LVANIAKSAA VISKTIESMA IVSKTIEGTA IVSRTVEGIA LGGKFLEGAA

50 RQPEMYGKLQ RQPEMSGQLR RQPEARGTLT RQPEAGKELR RQPDLIPLLR

80 TFFVG* .... ALILVFAV*. AFMVQGK*.. AFLAFFG*.. GLYVMFAVA*

Fig. 1. Deduced aa sequence alignment of the S. mutans ATPase c subunit with those from other bacterial species. Sources of nt sequence were as follows: En. hirae (Shibata et al., 1992); B. megaterium (Brusilow et al., 1989); B. subtilis (Shibata et al., 1992); E. coli (Walker et al., 1984). Completely conserved residues are shown in boldface type.

other representative bacteria are shown (Table 2). As expected, the areas of greatest homology were generally found in the subunits encoding the cytoplasmic domain, F 1, where ATP catalysis occurs. Significantly lower homologies were seen for the subunits of the membranebound domain (Fo). The exception to this observation was the delta subunit, part of the F1 domain, which exhibited the lowest subunit homology to those included in the comparison. Previous work had indicated that differences in ATPase-specific activity from different oral organisms were reduced when the catalytic domain was separated from the membrane-bound domain (Sturr and Marquis, 1992). The data suggested that differences in the aciduric

qualities of the ATPases may lie primarily in the speciesspecific alterations of the membrane-bound subunits and in their interactions with membrane components. Since the order of c and a were reversed in the S. mutans operon, with respect to the more commonly found order, it was of interest to examine the similarities of those genes to other well-characterized membrane-bound subunits. The estimated hydrophilicities of five bacterial c and a subunits were compared to see whether the c and a subunits of S. mutans retained the character seen in enzymes from other sources. Examination of the c subunit sequences revealed that the S. mutans c subunit is more similar, in the first 30 residues, to the subunits from Bacillus than those from either En. hirae or E. coli

Table 1 Plasmids used or constructed during this study Plasmid

Phenotype

Source

pSU20

Cmr; intermediate copy-number plasmid derived from pACYC184, contains multiple cloning site in lacZ for X-Gal screening of insertions Ap r Cmr; An ~8.5 kbp EcoRI fragment of S. mutans GS-5 chromosomal DNA cloned into the EcoRI site of pSU20. The insert contains the ATPase structural genes and the amino terminal portion of gapN. Cmr; pSMA40 subclone in pSU20 containing a 2.9 kbp HindIII fragment bearing the c, a, b, delta subunits and the first 906 bases of alpha. Cmr; pSMA40 subclone in pSU20 containing a 3.2 kbp HindIII fragment (bp 3997 to 7172) bearing the last 281 bases of alpha, gamma, beta and epsilon Apt; subclone of pSM40 similar to pHSU259 used to create nested deletions for sequencing. A 2.9 DraI-HindIII fragment (positions 767-3679 bp, respectively) cloned into pGEM7Zf(+) digested with SmaI and HindIII. The insert contains c, a, b, delta and the first 906 bases of alpha. Apt; subclone of pHSD34 used to create nested deletions for sequencing. A 1.3 kbp HindIII-NsiI fragment (positions 3997-5290, respectively) cloned into pGEMTZf(+) digested with HindIII and NsiI. The insert contains 281 bases of alpha, gamma and the first 90 bases of beta. Apt; A subclone of pHSD34 used to create nested deletions for sequencing. A 1.8 kbp NsiI-HindIII fragment (positions 5290-7170) cloned into pGEM7Zf(+) digested with HindIII and NsiI. The insert contains 1310 bases of beta and epsilon. Apt; A 1.6 kbp PstI fragment of S. mutans GS-5 chromosomal DNA cloned into the NsiI site of pGEMTZf(+). The insert contains promoter sequences for the ATPase operon, the first 91 bases of the c subunit, and the carboxy-terminus of glgP

Bartolom6 et al. (1991)

pGEM pSMA40

pHSU259 pHSD34 pGDH2

pGHN5

pGHN4

pGP78

Promega this study

this study this study this study

this study

this study

this study

90 CA

A.J. Smith et al./Gene 183 (1996) 87-96 GCC A

AAT N

ATT I

TCT GAA S E

CAG Q

ATC I

TCT S

CTA L

GCT A

TCC S

AAG K

GAA E

GCC A

TCC S

GGA G

ACG T

TCT S

AAC N

ATG M

AAA K

TTC F

ATG M

ATG M

ACG T

GGT G

GCT A

GTT V

ACT T

TTA L

GCA A

ACG T>

98

CTT L

GAC D

GGT G

GCT A

AAT N

ATT I

GAG E

ATC I

AAA K

GAT D

GAA E

GTT V

GGT G

GAT D

GAG E

AAT N

ATT 1

GTC V

ATC I

TTT F

GGT G

ATG M

ACC T

AAG K

GAT D

GAT D

GTC V

TAC Y

CGT R

CAT H

TAT Y

GAA E

AAT N>

197

CAT H

GAT D

TAT Y

TAC Y

TCA S

CGC R

GGT G

GTC V

TAT Y

GAA E

TCT S

AAT N

CCT P

GTT V

ATC I

AAA K

CGT R

GTT V

GTT V

GAT D

ACC T

TTT F

ATC I

AAT N

GGA G

ACG T

ATT I

CCT P

AAT N

AGT S

CAA Q

AGT S

GAA E>

296

GGG G

ACT T

GAA E

ATT I

TAT Y

GAA E

GCC A

CTG L

ATT I

ACC T

CAT H

AAT N

GAT D

GAA E

TAT Y

TTC F

TTA L

CTT L

GAA E

GAT D

TTC F

ATA I

GCT A

TAT Y

GTG V

CAG Q

GCT A

CAA Q

GAA E

AAG K

ATT I

GAT D

GCT A>

395

CTT L

TAT Y

CGT R

GAT D

AAA K

GAA E

ACT T

TGG W

TCG S

CGT R

ATG M

AGT S

TTG L

TGT C

AAT N

ATT I

GCT A

AAC N

TCT S

GAT D

AAA K

TTT F

ACT T

TCA S

GAT D

GAT D

ACG T

ATT I

ACA T

CAA Q

TAT Y

GCT A

AAA K>

494

GAA E

ATT I

TGG W

CAT H

TTA L

GAA E

ATT I

TAA GC CTTCCCAATA *> ~ g l g P

AGCGTCTTCT

ATAATGTTGA

GAAGTGCTAT

CTAAACAAGT

A ~ T T T

TGAAGGAGAA

TGATAATTTA

AGAGACTTAG

AAAGAATAGG

ACAAAAGCGT

TTAGGAAAAG

CTAAGTCTCT

TCTAAGAAAA

TTTTTGACCT

CGCAACCAAA

AATATTTATA

TTTCTTGTTT

AGATAAGTAA

TCATTGACAA

TCGAATTAAA

ACCTGCAAAA

600

TAGGACTAAA

720

TTCATT uUm~l~

ATG M

TTG L

AAT N

TTA L

AAG K

ATT I

TTA L

GCA A

CTT L

GGG G

ATT I

GCT A

GTT V

TTA L

GGC G

GTT V

AGC S

CTT L

GGT G

GAA E

GGA G

ATT I

TTA L>

825

GAA E

ATG M

TAT Y

GGT G

A~ K

TTA L

CAA Q

ACG T

CTC L

ATG M

ATT I

ATG M

GGT G

GTT V

GCC A

TTT F

ATT I

GAA E

GGT G

ACC T

TTT F>

924

TTG L

GAA E

AAA K

ACA T

ATA I

AAT N

CCA P

ACG T

GTT V

AAA K

TTC F

TTA L

GGT G>

1028

GTT V

GCT A

AAT N

ATT I

GCA A

AAA K

TCT S

GCA A

GCT A

CGT R

CAG Q

CCT P

TTC F

GTG V

CTT L

CTT L

GCT A

TCA S

ACA T

TTC F

TTT F

GTT V

GGC G

TGA TTTCATAATA *>

ATT I

GAG E

TTT F

GAC D

TTA L

ACC T

ATC I

TTG L

ATG M

ATG M

TCT S

CTC L

TTA L

GTT V

GTT V

CTT L

ATT I

GCA A

TTC F

TTA L

TTT F

GTC V

TTT F

TGG W

ACA T

AGC S

CGC R

CAT H

CTG L

AAA K

ATA I

AAG K

CCT P>

1127

ACG T

GGC G

AGA R

CAA Q

AAT N

GTT V

TTA L

GAA E

TGG W

ATC I

TAT Y

GAT D

TTT F

GTC v

CTT L

GGA G

ATT I

ATC I

AAG K

CCT AAT P N

TTA L

GGT G

TCT S

TAT Y

ACT T

AAA K

AAT N

TAC Y

AGC S

CTA L

TTT F

GCT A>

1226

TTT F

TGT C

CTC L

TTC F

CTT L

TTT F

GTT v

TTT F

GTT V

GCT A

AAC N

AAT N

ATT I

GGT G

TTA L

TTA L

ACA T

AAG K

ATT I

CAA Q

GTT V

AAA K

GAT D

TAT Y

AAT N

TTA 5

TGG W

ACT T

TCC S

CCA P

ACA T

GCA A

AAT N>

1325

TTT F

GCA A

GTT V

GAT D

TTT F

GGT G

CTT L

TCT S

TTA L

ATG M

GTG V

GCG A

GTA V

ATC I

TGT C

CAC H

TTT F

GAA E

GGT G

ATT I

CGT R

AAG K

CAT H

GGC G

TTG L

AAA K

ACA T

TAC Y

TTA L

AAG K

GAT D

TAT Y

TTA L>

1424

GAA E

CCG P

ACA T

GCA A

GCT A

ATG M

TTG L

CCT P

ATG M

AAT N

CTC L

TTA L

GAA E

GAA E

CTA L

ACG T

AAT N

ATT I

ATT I

TCA S

CTG L

TCT S

CTT L

CGT R

TTA L

TAT Y

GGT G

AAT N

ATT I

TAT Y

GCT A

GGT G

GAA E>

1523

GTT V

GTT V

ATG M

GCG A

CTT L

TTG L

GTA V

CAG Q

TTT F

GCT A

GAT D

TTT F

AGC S

CCA P

TAT Y

GCG A

ACA T

CCA P

ATA I

GCC A

TTT F

CTG L

CTT L

AAC N

ATG M

GCT A

TGG W

ATT I

GGA G

TTT F

TCT S

ATT I

TTC F>

1622

ATC I

TCA S

GGA G

ATA I

CAA Q

GCC A

TAT Y

GTC V

TTT F

GTT V

CTT L

TTA L

ACG T

ACG T

ACT T

TAT Y

ATT I

GGT G

AAA K

AAG K

GTC V

AAT N

ATT I

GAT D

ACT T

AAA K

GGC G

AAT N

TAA G AAAGGAGCAG *>

TGATTT ATG ul~cF ~ M

TCA S

ACA T

CTT L

ATT I

AAT N

GGA G

ACA T

AGT S

CTA L

GGC G

AAT N

TTG L

AGA~GGGG

CTT L

ATC I

GAATTAGAG Lzmc~ *

GTG V

ACA T

GGA G

TCT S

TTT F

ATC I

CTT L

TTA L

TTA L

CTT L

CTG L

GTT V

AAG K

~ K

TTC F

1720

GCT A>

1819

TGG W

TCT S

CAG Q

CTG GCA L A

GCT A

ATT I

TTC F

AAA K

ACA T

CGA R

GAA E

GAA E

AAA K

ATT I

GCA A

AAG K

GAT D

ATT I

GAT D

GAT D

GCT A

GAA E

AAT N

TCA S

CGT R

CAA Q

AAT N

GCT A

CAG Q

GTT v

TTA L

GAG E>

1918

AAT N

AAA K

CGT R

CAA Q

GAG E

CTT L

AAC N

CAA Q

GCT A

AAG K

GAT D

GAA E

GCT A

GCC A

CAA Q

ATT I

ATT I

GAT D

AAC N

GCT A

AAG K

GAA E

ACT T

GGT G

A~ K

GCT A

CAA Q

GAG E

TCT S

AAG K

ATT i

ATA I>

2017

GTT V

Fig. 2. Nucleotide sequence of the S. mutans ATPase operon and flanking regions with deduced aa sequences in single letter format. Invertedrepeat sequences are shown as arrows above the codons. The transcriptional start site for the ATPase operon is shown as an enlarged G at 729 bp. The --10 region is shown in single-underline beginning at 717 bp. The - 3 5 region is shown in double-underline beginning at 694 bp. Methods: Laboratory stock strains of S. mutans GS-5 were maintained on brain heart infusion medium (Difco Laboratories, Detroit, MI) containing 1.5% (w/v) agar at 37°C, in an atmosphere of 5% (v/v) CO2 and stored at - 8 0 ° C as frozen stocks. E. coli strain DH10B (BRL/Life Technologies, Bethesda, MD; Grant et al., 1990) was maintained on LB medium (Sambrook et al., 1989). Transformants harboring ATPase-containing plasmids were maintained on LB agar plates containing an appropriate antibiotic (100 p.g/ml Ap or 20 ~tg/ml Cm) at 37°C. The principal plasmids used or constructed in this study are shown in Table 1. Previously, we had amplified a 600-bp portion of the S. sobrinus proton-translocating ATPase 13-subunit by PCR (Quivey et al., 1991). Using similar methodology, we designed degenerate primers based on En. hirae (Shibata et al., 1992) and B. megaterium (Hawthorne and Brusilow, 1988) ATPase [3-subunit sequences to amplify a slightly larger fragment from S. mutans GS-5 chromosomal DNA, prepared as previously described (Quivey et al., 1991). Primer sequences were as follows: 5'-ATTACC/GCAAATTGTTGCIGG-3' and 5'-ACCTGTCAGGGCAACCCGCAT-3', for the upstream and downstream primers, respectively. The PCR product, of 748 bp, was isolated and cloned into the vector pCRII (Invitrogen, San Diego, CA). Sequence analysis confirmed the cloned DNA was homologous to the ATPase [3-subunit sequences of other bacteria (data not shown). The 13-subunit fragment was then used to probe a Southern blot of S. mutans GS-5 genomic DNA digested to completion with various restriction endonucleases. An approximately 7 10-kbp EcoRI fragment was identified and cloned into the intermediate copy-number plasmid, pSU20 (Bartolom6 et al., 1991 ). Positive transformants of E. coli were screened by colony hybridization using the 13-subunit fragment as a probe. Probe labeling, colony hybridization, and autoradiography were as reported previously (Quivey et al., 1991). One hybridizing colony, harboring a plasmid designated pSMA40, contained an approximately 8.5-kbp insert. Initial sequence analysis of this clone showed strong homology with the S. oralis ATPase c subunit (Fenoll et al., 1994). To facilitate sequencing of the entire insert, several subclones of pSMA40 were constructed (Table 1). A 2912-bp DraI-HindIII fragment of pSMA40 was cloned into SmaI and HindIII-digested pGEM7Z-f(+) (Promega, Madison, WI) to obtain pGDH2. A 3175-bp HindIII fragment of pSMA40 was cloned into the HindIII site of pSU20 to obtain pHSD34. Additional subclones were constructed by NsiI-HindIII digestion of pHSD34, resulting in the release of two fragments, 1293 bp

91

A.J. Smith et al./Gene 183 (1996) 87-96 unc glgP

"I

> B

E

F

I I

H

H

I

1

A

2

'

3

'

a

b

7059 m,w.

~

a

17950 27069

gapN :>

It

6

'

7 kbp

'

'

~ 7

54351

20317

C

I

S

~

c

D

11

I4

'

I~ subunlt

G

II

X

H 13

E

£

51767 32349

15586

8.88 pl

8.59

4.83

5.03

4.66

5.44

5. I0

6.08

Fig. 3. Genetic organization of the S. mutans ATPase operon and flanking regions. The map shows the position and relative size of the ATPase subunits in the open boxes. The unc designation is shown above the boxes in italicized letters. The common designation of the subunits is shown below the scale. Restriction enzyme sites are shown on the kbp scale and the abbreviations were as follows: D, DraI; E, EcoRI; P, PstI; H, HindllI; N, NsiI. The estimated molecular weights from deduced aa sequences are shown below the scale as are the estimated isoelectric points for each of the subunit proteins. Arrows indicate the direction of transcription for the ATPase operon, glgP and gapN. Table 2 Percent homologies" of the deduced S. mutans ATPase-subunit amino acid sequences compared to those from En. hirae, B. megaterium, B. subtilis and E. coli b Subunit

En. hirae

a c b

47.8 32.3 42.8 28.4 81.9 65.4 76.3 58.9

7 e

(69) c (62) (63) (53) (90) (76) (87) (78)

B. megaterium

B. subtil~

38.0 33.8 34.3 23.8 75.5 53.5 67.9 38.3

39.4 34.3 38.6 19.3 74.7 49.5 67.4 39.9

(63) (62) (60) (47) (86) (70) (80) (68)

E coli

(65) (65) (63) (46) (85) (69) (80) (68)

32.0 26.5 27.4 23.3 51.9 33.7 59.2 27.0

(61) (48) (48) (48) (70) (57) (77) (53)

" % homologies were calculated as described by Needleman and Wunsch (1970). b Sources used as the basis for the deduced amino acid sequences were: En. hirae (Shibata et al., 1992); B. megaterium (Brusilow et al., 1989); B. subtilis (Santana et al., 1994); E. coli (Walker et al., 1984). c Numbers in parentheses represent the percentage, to the nearest integer, of similar amino acids.

(Fig. 4). An identical evaluation of the deduced amino acid sequences from uncBgenes indicated that the S. mutans a subunit was more similar to the En. hirae form (Fig. 5). In particular, a pronounced difference between the E. coli subunit (a Gram-negative representative) and the Gram-positive forms was found to be centered around amino acid residue 140 (Fig. 5). The functional implications of the gene reversal in the streptococci are not clear at this time. It had been earlier postulated that the gene order of a, then c, seen in Gram-negative and other Gram-positive species, was a function of the assembly of the enzyme. However, considerable data now exist to show that the assembly of a functional m e m b r a n e domain is dependent on the presence of the delta subunit (Brusilow, 1993), such that the order c, then a, as it is seen in S. mutans, m a y or may not have significance. Hydrophilicity comparisons suggested that the S. mutans c subunit was more similar to the Bacillus c subunits; whereas, the a subunit

and 1882 bp. The NsiI-HindlII fragments were subcloned into pGEM7Z-f(+) to obtain pGHN5 and pGNH4, respectively. A series of nested deletions varying in size from 150 to 200 bp was created by exonuclease III digestion of pGDH2, pGHN5 and pGNH4 (Erase-a-base, Promega, Madison, WI). Sequences upstream of the ATPase operon structural genes were identified in genomic DNA using an approach similar to that by which the structural genes were obtained. S. mutans GS-5 genomic DNA was digested with various restriction enzymes and subjected to Southern blot analysis using a PCR-amplified c subunit fragment (776-921 bp, Fig. 1), under labeling and hybridization conditions as previously reported (Quivey et al., 1991). An approximately 1.6-kbp PstI fragment was identified, isolated and cloned into pGEM7Z-f(+) cleaved with NsiI. The resulting clone, pGP78, was then analyzed for its nt sequence. The nt sequence of cloned S. mutans GS-5 DNA fragments was determined by dideoxynucleotide sequencing, modified for use with Bca DNA polymerase (Umeori et al., 1992) (PanVera Corp., Madison, WI) and [aSS]dATP (NEN, Boston, MA) according to the manufacturer's recommendations. For each sequencing reaction, plasmid DNA was isolated from 10 ml cultures of E. coli DH10B using QIAprep-spin columns (Qiagen, Chatsworth, CA). Analysis and alignment of nt sequence was performed using MacVector and Assemblylign software (Kodak, New Haven, CT) and the Wisconsin Genetics Computer Group program for the VAX (Devereux et al., 1984). Sequences not obtained from the nested deletions were determined using primers homologous to sequences flanking the region in question. Universal and custom oligonucleotide primers were either synthesized on an Applied Biosystems Model 391A synthesizer or purchased commercially (BRL/Life Technologies, Gaithersburg, MD). All PCR experiments were carried out in a Hybaid Omnigene Thermal Cycler (National Labnet Co., Woodbridge, NJ). PCR products were amplified from approximately 50 ng template DNA in a reaction containing lx PCR buffer (Quivey et al., 1991), 20 ~tM dNTPs, 1 ~tM each appropriate primer, and 1 Unit Taq polymerase (BRL/Life Technologies, Gaithersburg, MD). The reaction mixtures were heated for 1 min prior to the addition of enzyme. Amplification was allowed to proceed for 35 cycles consisting of: 94°C, 1 min; 55°C, 2 min and 72°C, 2 min. The amplified products were agarose gel purified prior to use. The nt sequence reported here for the ATPase operon and flanking regions is available as accession No. U31170 from GenBank.

92

A.J. Smith et al./Gene 183 (1996) 87 96

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Fig. 4. Kyte-Doolittle hydrophilicity comparisons of the S. mutans uncE (c subunit) deduced aa sequences with those from En. hirae (Shibata et al., 1992); B. megaterium (Brusilow et al., 1989); B. subtilis (Shibata et al., 1992); E. coli (Walker et al., 1984). The x-axis represents aa residues measured from the N-termini. The y-axis was the arbitrary scale of hydropathy previously described (Kyte and Doolittle, 1982), as modified to represent hydrophilicity (Devereux et al., 1984).

appeared to be more similar to the En. h i r a e form. It could be speculated that the unusual relationship of c, and a, m a y extend to the delta subunit, which was found to be the least homologous subunit to other species.

Since delta is known to participate in the interaction of the relatively highly conserved membrane-extrinsic domain (F1) and the much less conserved membraneintrinsic domain (F0), the lack of conservation in delta

93

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m a y be due, in part, to the species-specific nature o f the c and a subunits (Brusilow, 1993). 2.4. Transcriptional start site o f A T P a s e is not affected by culture p H value

In addition to the c o m p a r i s o n s o f the first two subunits o f the Fo domain, we also began a study on the potential function o f the intergenic space between glgP and the c subunit coding region. Nucleotide sequence analysis indicated several regions o f D N A in which

inverted-repeat sequences occurred (Fig. 2). The first inverted-repeat was seen at 12-43 bp following the term i n a t i o n c o d o n o f glgP and m a y be involved in the termination o f glgP transcription. A subsequent pair o f inverted-repeats occurred at 7 7 - 1 1 6 bp and 148-174 bp d o w n s t r e a m o f glgP. The last inverted-repeat to be considered here was f o u n d at position 7042-7078, immediately d o w n s t r e a m o f the epsilon subunit o f the ATPase, and was t h o u g h t to be involved in the termination o f ATPase o p e r o n transcription (Fig. 2). The presence o f the pair o f inverted-repeats preceding the structural

94

A.J. Smith et al./Gene 183 (1996) 87 96

the - 3 5 region b e g a n two bases from the p r o x i m a l inverted-repeat u p s t r e a m o f the coding region. These data strongly suggest that we have identified the prom o t e r region of the S. m u t a n s GS-5 ATPase operon. The S. m u t a n s o p e r o n did n o t c o n t a i n an u n c I gene, a n element seen in the o r g a n i z a t i o n of the operons from E. coli a n d the B a c i l l u s species. The intergenic region between the first s u b u n i t o f S. m u t a n s , c, a n d from En. hirae, a, are similarly m u c h larger t h a n those seen in o r g a n i s m s where U n c I is present. T h o u g h the f u n c t i o n of I is n o t yet clear, the hypothesis that the intergenic region o f E n . hirae a n d S. r n u t a n s c o n t a i n s the regulatory region for b o t h o p e r o n s is attractive. Inverted-repeat D N A sequences, such as those f o u n d in this study for the S. m u t a n s ATPase, have been shown to be involved in the regulation of genes in G r a m - p o s i t i v e organisms (Stewart, 1993). Recent data o n the r e g u l a t i o n of the fructosyltransferase gene ( f t f ) indicate the clear involvem e n t of inverted-repeats in sucrose-mediated regulation of f t f t r a n s c r i p t i o n a l activity ( K i s k a a n d M a c r i n a , 1994). C a t a b o l i t e repression appears to have little to do

genes suggested the possibility that they were involved in the r e g u l a t i o n o f the S. m u t a n s ATPase. The initial a p p r o a c h to the p r o b l e m was to ask whether the m R N A e n c o d i n g the A T P a s e s u b u n i t s was affected by changes in the p H value of the cultures. P r i m e r extension experim e n t s were c o n d u c t e d on total R N A samples extracted f r o m b a t c h g r o w n cultures where the p H value of the culture was n o t fixed a n d from cultures g r o w n at steadystate with p H values of 5.2 a n d 7.0. The results indicated that the t r a n s c r i p t i o n a l start site of the ATPase o p e r o n was at a g u a n i n e residue at - 2 8 b p of the p r e s u m e d t r a n s l a t i o n start site. F u r t h e r , n o differences were observed between the start site o n m R N A isolated from b a t c h g r o w n cultures or those at a p H value of 5.2 or 7 (Fig. 6). A t - 1 2 b p from the t r a n s c r i p t i o n a l start, a putative P r i b n o w box was identified by the sequence T A A A C T , representing a 66% identity as c o m p a r e d to the consensus T A T A A T - 1 0 sequence from E. coli. A t - 3 5 b p from the start site, S. m u t a n s GS-5 c o n t a i n e d the sequence T T G A C A which is identical to the c a n o n i cal sequence for - 3 5 regions f r o m E. coli. Interestingly,

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A Fig. 6. Primer extension analysis of ATPase-specificmRNA extracted from batch-grown and steady-state cultures of S. mutans GS-5. Nucleotide sequence ladder is shown vertically, with the lanes marked for base content. Lanes containing cDNA resulting from the primer extensions are as follows: B, cDNA resulting from the extension of mRNA extracted from batch-grown cultures; 5, cDNA resulting from the extension of mRNA extracted from steady-state-grown cultures maintained at a pH value of 5.2; and 7, cDNA resulting from the extension of mRNA extracted from steady-state-grown cultures maintained at a pH value of 7.0. The nt sequence shown was obtained with the primer used for the primer extension experiments and is complementary to the coding strand sequence. Methods: Batch cultures of S. mutans were grown overnight in BHI medium at 37°C. Steady-state cultures, at fixed pH values, were grown as previously reported (Quivey et al., 1995). Dilution rates for the continuous culture of S. mutans were D = 0.09 h 1 (equivalent to an approximately 8 h gen. time). Total cellular RNA was extracted from S. mutans GS-5 essentially as described previously (McDonald et al., 1984; Hollingshead et al., 1987)• RNA samples were prepared from 100 ml overnight cultures of batch grown cells or 100 ml of chemostat-grown cultures, maintained at a pH value of 5.2 or 7.0. RNAs were sedimented through 5.7 M CsC1 (Glisin et al., 1974) and pellets were resuspended in sterile water. Primer extension analysis of the RNA was performed using a kit (Promega, AMV Reverse Transcriptase Primer Extension System) as recommended by the manufacturer with approximately 50 gg total RNA and 100 fmol labeled primer. The primer was composed of the following sequence, 5'-ACGAGCTGCAGATTTTGC-3', located at bp position 855-838 in the c subunit gene (see Fig. 1). 10 pmol primer was end-labeled with [7-a2P]ATP(3000 Ci/mmol) (NEN, Boston, MA) and T4 polynucleotide kinase, according to the manufacturer's instructions (BRL/Life Technologies, Gaithersburg, MD).

A.J. Smith et al./Gene 183 (1996) 87-96

with ATPase regulation in E. coil, but it is involved with repression of ATPase in V. parahaemolyticus (SakaiTomita et al., 1992). Therefore, the role of catabolite repression, mediated through inverted-repeat D N A structures, is still an open question with regard to the S. mutans ATPase operon. The results of our primer extension experiments indicated that the transcriptional start site does not change with alterations in culture p H values. These data suggest that m R N A encoding the ATPase m a y not be altered as a function of changes in the cell's environment. However, additional experiments to completely evaluate the stability of the S. mutans ATPase m R N A are warranted in light of the data from E. coli showing a possible role for selective degradation of ATPase-specific message in regulation of subunit stoichiometry (Dallman and Dunn, 1994; Patel and Dunn, 1995). Experiments using reporter-gene fusions to the inverted-repeat structures, such as those described for the f t f gene ( K i s k a and Macrina, 1994), will likely provide information on the transcriptional activity of the S. mutans ATPase under varying growth conditions. The structural genes for the S. mutans operon were isolated on a single, continuous fragment. In contrast, the operon from En. hirae proved to be unstable in E. coli such that deleted regions had to be amplified by P C R for identification and sequencing (Shibata et al., 1992). In previous attempts to clone the S. mutans ATPase genes, we experienced similar difficulty in isolating a stable clone of the intact operon using high copynumber vectors. However, using the lower copy-number vector, pSU20, we were able to stably maintain the entire sequence. Previous studies have shown that high levels of membrane subunits from ATPase can be lethal to cells (von Meyenburg et al., 1985). These reported observations, and our own attempts to clone the ATPase in high copy-number, argue that the genes were expressed in E. coli. Furthermore, based on the strong similarities of the - 10 and - 3 5 regions of the S. mutans ATPase, it seems likely that the operon can be readily expressed in E. coli. At presently, studies with E. eoli and S. mutans promoter regions fused to the structural genes are being conducted to determine the ability of the S. mutans ATPase gene products to function productively in E. coli.

3. Conclusions (1) We have cloned and determined the nt sequence for the ATPase operon and flanking regions of S. mutans. The D N A sequence contains the complete coding information for the eight structural genes of the operon, the 3'-terminus of glgP, and the 5'-terminus of gapN. The operon did not contain an uncI gene homologue.

95

(2) The gene order for the ATPase operon was c, a, b, delta, alpha, gamma, beta, and epsilon. The first two genes, c and a, were reversed with respect to the typical genetic organization found in other species. Hydrophilicity comparisons indicated that the S. mutans c and a subunits were more similar to those from Bacillus species than those from En. hirae. The Bacillus and En. hirae species both feature the a, then c, organization. However, the Bacillus species contain an uncI gene; whereas En. hirae does not. These data indicate that uncI is not related to the correct functioning of the a and c subunits and that the intergenic space likely evolved independently of the uncI gene and the order of the first two subunit genes. (3) The intergenic space between the S. mutans c subunit gene and glgP contained three inverted-repeats of D N A sequence. Primer extension analysis showed that the pair of inverted-repeats, close to the structural genes of the operon, did not affect the start site for transcription of the operon as a function of culture p H value. (4) The cloning and isolation of an intact ATPase operon from S. mutans will facilitate studies into the role of the enzyme in acid-adaptation by this oral pathogen.

Acknowledgement We thank members of the R.A. Burne laboratory for their assistance with the R N A extraction procedures. This work was supported in part by PHS P01-DE-11549 and the Rochester Cariology Training Program (T32-DE-07165) (A.J.S.).

References Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215,403-410. Bartolom6, B., Jubete, Y., Martinez, E. and de la Cruz, F. (1991) Construction and properties of a family of pACYC 184-derivedcloning vectors compatible with pBR322 and its derivatives. Gene 102, 75-78. Belli, W.A. and Marquis, R.E. (1991) Adaptation of Streptococcus mutans and Enterococcus hirae to acid stress in continuous culture. Appl. Environ. Microbiol. 57, 1134-1138. Bender, G.R., Sutton, S.V.W. and Marquis, R.E. (1986) Acid tolerance, proton permeabilities, and membrane ATPases of oral streptococci. Infect. Immun. 53, 331-338. Boyd, D.A., Cvitkovitch, D.G. and Hamilton, I.R. (1995) Sequence, expression, and function of the gene for the nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase of Streptococcus mutans. J. Bacteriol. 177, 2622-2627. Brusilow, W.S.A. (1993) Assembly of the Escherichia coli F1FOATPase, a large multimeric membrane-bound enzyme. Mol. Microbiol. 9, 419-424. Brusilow, W.S.A., Scarpetta, M.A., Hawthorne, C.A. and Clark, W.P.

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A.J. Smith et al./Gene 183 (1996) 87-96

(1989) Organization and sequence of the genes coding for the proton-translocating ATPase of Bacillus megaterium. J. Biol. Chem. 264, 1528-1533. Dallman, H.G. and Dunn, S.D. (1994) Translation through an uncDC mRNA secondary structure governs the level of uncC expression in Escherichia coli. J. Bacteriol. 176, 1242-1250. Dashper, S.G. and Reynolds, E.C. (1992) pH Regulation by Streptococcus mutans. J. Dent. Res. 71, 1159-1165. Devereux, J., Haberli, P. and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387-395. Eenoll, A., Munoz, R., Garcia, E. and de la Campa, A.G. (1994) Molecular basis of the optochin-sensitive phenotype of pneumococcus: characterization of the genes encoding the F0 complex of the Streptococcus pneumoniae and Streptococcus oralis H(+)-ATPases. Mol. Microbiol. 12, 587-598. Glisin, V., Crkvenjakov, R. and Byus, C. (1974) Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13, 2633 2637. Grant, S.G., Jessee, J., Bloom, F.R. and Hanahan, D. (1990) Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc. Natl. Acad. Sci. USA 87, 4645 4649. Hamilton, I.R. and Buckley, N.D. ( 1991 ) Adaptation by Streptococcus mutans to acid tolerance. Oral Microbiol. Immun. 6, 65-71. Hawthorne, C.A. and Brusilow, W.S.A. (1988) Sequence of the genes for the b and e subunits of the ATP synthase of Bacillus megaterium QM B1551. Biochem. Biophys. Res. Commun. 151,926 931. Hollingshead, S.K., Fischetti, V.A. and Scott, J.R. (1987) A highly conserved region present in transcripts encoding heterologous M proteins of group A streptococci. Infect. Immun. 55, 3237-3239. Kiel, J.A., Boels, J.M., Beldman, G. and Venema, G. (1994) Glycogen in Bacillus subtilis: molecular characterization of an operon encoding enzymes involved in glycogen biosynthesis and degradation. Mol. Microbiol. 11,203 218. Kiska, D.L. and Macrina, F.L. (1994) Genetic regulation of fructosyltransferase in Streptococcus mutans. Infect. lmmun. 62, 1241-1251. Kobayashi, H. (1985) A proton-translocating ATPase regulates pH of the bacterial cytoplasm. J. Biol. Chem. 260, 72-76. Kobayashi, H., Suzuki, T. and Unemoto, T. (1986) Streptococcal cytoplasmic pH is regulated by changes in amount and activity of a proton-translocating ATPase. J. Biol. Chem. 261,627 630. Kyte, J. and Doolittle, R.F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105 132. Loesche, W.J. (1986) Role of Streptococcus mutans in human dental decay. Microbiol. Rev. 50, 353-380. McDonald, P.M., Kutter, E. and Mosig, G. (1984) Regulation of a bacteriophage T4 late gene, SOC, which maps in an early region. Genetics 106, 17-27.

Needleman, S.B. and Wunsch, C.D. (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol. 48, 443-453. Nikiforuk, G. (1985) Understanding Dental Caries, Vol. 1: Etiology and Mechanisms. S. Karger, Basel, Switzerland, 303 pp. Patel, A. and Dunn, S.D. (1995) Degradation of Escherichia coli uneB mRNA by multiple endonucleolytic cleavages. J. Bacteriol. 177, 3917 3922. Quivey, R.G., Faustoferri, R.C., Belli, W.A. and Flores, J.S. (1991) Polymerase chain reaction amplification, cloning, sequence determination and homologies of streptococcal ATPase-encoding DNAs. Gene 97, 63 68. Quivey, R.G., Faustoferri, R.C., Clancy, A.K. and Marquis, R.E. (1995) Acid adaptation in Streptococcus mutans alleviates sensitization to environmental stress due to RecA deficiency. FEMS Microbiol. Lett. 126, 257-262. Sakai-Tomita, Y., Moritani, C., Kanazawa, H., Tsuda, M. and Tsuchiya, T. (1992) Catabolite repression of the H(+)-translocating ATPase in Vibrio parahaemolyticus. J. Bacteriol. 174, 6743 6751. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY. Santana, M., lonescu, M., Vertes, A., Longin, R., Kunst, F., Danchin, A. and Glaser, P. (1994) Bacillus subtilis FOF1 ATPase: DNA sequence of the atp operon and characterization of atp mutants. J. Bacteriol. 176, 6802 6811. Shibata, C., Ehara, T., Tomura, K., Igarashi, K. and Kobayashi, H. (1992) Gene structure of Enterococcus hirae (Streptococcus faecalis) FIF0-ATPase, which functions as a regulator of cytoplasmic pH. J. Bacteriol. 174, 6117 6124. Stewart, G.C. (1993) Catabolite repression in the Gram-positive bacteria: Generation of negative regulators of transcription. J. Cell. Biochem. 51, 25 28. Sturr, M.G. and Marquis, R.E. (1992) Comparative acid tolerances and inhibitor sensitivities of isolated F-ATPases of oral lactic acid bacteria. Appl. Environ. Microbiol. 58, 2287-2291. Sutton, S.V. and Marquis, R.E. (1987) Membrane-associated and solubilized ATPases of Streptococcus mutans and Streptococcus sanguis. J. Dent. Res. 66, 1095-1098. Umeori, T., Ishino, Y., Fujita, K., Asada, K. and Kato, I. (1992) Cloning of the DNA polymerase gene product of Bacillus caldotenax and characterization of the gene product. J. Biochem. 113,401 410. von Meyenburg, K., Jorgensen, B.B., Michelsen, O., Sorensen, L. and McCarthy, J.E. (1985) Proton conduction by subunit a of the membrane-bound ATP synthase of Escherichia coli revealed after induced overproduction. Embo J. 4, 2357 2363. Walker, J.E., Saraste, M. and Gay, N.J. (1984) The Unc operon. Nucleotide sequence, regulation and structure of ATP-synthase. Biochim. Biophys. Acta Bioenerg. 768, 164-200.