An OmpA Family Protein, a Target of the GinI/GinR …jb.asm.org/content/190/14/5009.full.pdf · a...

JOURNAL OF BACTERIOLOGY, July 2008, p. 5009–5019 Vol. 190, No. 140021-9193/08/$08.00�0 doi:10.1128/JB.00378-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

An OmpA Family Protein, a Target of the GinI/GinR Quorum-SensingSystem in Gluconacetobacter intermedius, Controls

Acetic Acid Fermentation�†Aya Iida, Yasuo Ohnishi, and Sueharu Horinouchi*

Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo,Bunkyo-ku, Tokyo 113-8657, Japan

Received 15 March 2008/Accepted 2 May 2008

Via N-acylhomoserine lactones, the GinI/GinR quorum-sensing system in Gluconacetobacter intermediusNCI1051, a gram-negative acetic acid bacterium, represses acetic acid and gluconic acid fermentation. Two-dimensional polyacrylamide gel electrophoretic analysis of protein profiles of strain NCI1051 and ginI and ginRmutants identified a protein that was produced in response to the GinI/GinR regulatory system. Cloning andnucleotide sequencing of the gene encoding this protein revealed that it encoded an OmpA family protein,named GmpA. gmpA was a member of the gene cluster containing three adjacent homologous genes, gmpA togmpC, the organization of which appeared to be unique to vinegar producers, including “Gluconacetobacterpolyoxogenes.” In addition, GmpA was unique among the OmpA family proteins in that its N-terminal mem-brane domain forming eight antiparallel transmembrane �-strands contained an extra sequence in one of thesurface-exposed loops. Transcriptional analysis showed that only gmpA of the three adjacent gmp genes wasactivated by the GinI/GinR quorum-sensing system. However, gmpA was not controlled directly by GinR butwas controlled by an 89-amino-acid protein, GinA, a target of this quorum-sensing system. A gmpA mutantgrew more rapidly in the presence of 2% (vol/vol) ethanol and accumulated acetic acid and gluconic acid ingreater final yields than strain NCI1051. Thus, GmpA plays a role in repressing oxidative fermentation,including acetic acid fermentation, which is unique to acetic acid bacteria and allows ATP synthesis via ethanoloxidation. Consistent with the involvement of gmpA in oxidative fermentation, its transcription was alsoenhanced by ethanol and acetic acid.

Many gram-negative bacteria use N-acylhomoserine lactone(AHL)-dependent quorum-sensing systems to regulate geneexpression in concert with cell density (6, 7). Quorum sensingis used to regulate diverse physiological functions and charac-teristics, including secondary metabolite production, swimmingand swarming motility, conjugal plasmid transfer, biofilm for-mation, and virulence (25, 26). Two important proteins areinvolved in AHL-dependent quorum sensing: a LuxR-type pro-tein, which is an AHL-dependent transcriptional regulator,and a LuxI-type protein, which directs the synthesis of AHLsfrom S-adenosylmethionine and diverse �-ketoacyl-acyl carrierprotein intermediates in fatty acid biosynthesis. In general,LuxR-type proteins bind their cognate AHLs once the concen-tration of the AHL reaches a critical level, and the resultingcomplex activates the transcription of specific target genes bybinding the promoter recognition sequences, termed lux boxes.

Acetic acid bacteria are gram-negative, obligatory aerobicbacteria with the ability to oxidize ethanol and sugars into thecorresponding organic acids. Acetobacter and Gluconaceto-bacter species are used commercially to produce vinegar be-cause of their high-level abilities to oxidize ethanol into aceticacid and their strong resistance to acetic acid and ethanol. Two

membrane-bound enzymes, alcohol dehydrogenase and alde-hyde dehydrogenase, catalyze the oxidation of ethanol. Wepreviously found that several acetic acid bacteria, includingGluconacetobacter intermedius NCI1051, contain a pair of luxIand luxR homologues (10). The LuxI and LuxR homologues,named GinI and GinR, comprise a typical quorum-sensingsystem, in which GinI directs the synthesis of three AHLs withdifferent acyl chains and GinR serves as a transcriptional reg-ulator using the AHLs as its ligands to control the lux box-containing promoter of ginI. Downstream of ginI, a small pro-tein consisting of 89 amino acids, named GinA, is encoded, andginA and ginI are cotranscribed. GinA, showing no homologyto any known proteins, represses (i) oxidative fermentation,including acetic acid fermentation and gluconic acid fermen-tation; (ii) growth in the presence of ethanol; and (iii) theantifoam activity of cells. These findings clearly show that theginI/ginR quorum-sensing system in G. intermedius controlsthe phenotypes that are characteristic of acetic acid bacteria.Oxidative fermentation and growth in the presence of ethanolare essential for acetic acid bacteria to generate ATP via eth-anol oxidation by means of acetic acid fermentation.

In order to reveal how GinA affects the phenotypes charac-teristic of acetic acid bacteria and what target genes other thanginA are under the control of the ginI/ginR regulatory system,we performed two-dimensional polyacrylamide gel electro-phoresis (2D-PAGE) analysis for the comparison of proteinprofiles of the wild-type strain and mutants deficient in theginI/ginR regulatory system. As a result, we found an OmpAfamily protein that was induced in response to the GinI/GinR

* Corresponding author. Mailing address: Department of Biotech-nology, Graduate School of Agriculture and Life Sciences, The Uni-versity of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan. Phone: 81 3 58415123. Fax: 81 3 5841 8021. E-mail: [email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

� Published ahead of print on 16 May 2008.

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quorum-sensing system. This OmpA family protein was foundto repress oxidative fermentation, including acetic acid andgluconic acid fermentation. The genome sequences of severalacetic acid bacteria have revealed the presence of multiplecopies of ompA that appeared during evolution through geneduplication in this specific group of bacteria. In fact, in addi-tion to two ompA genes, perhaps playing a structural role in theintegrity of the surfaces of gram-negative bacteria by providinga physical linkage between the outer membrane and the un-derlying peptidoglycan layer, vinegar producers, such as G.intermedius and “Gluconacetobacter polyoxogenes,” additionallycontain triplicate ompA-like genes in a locus, one of whichcorresponded to the ompA-like gene that was regulated by theGinI/GinR system. In this report, we describe the identificationof the ompA-like gene, named gmpA (Gluconacetobacter outermembrane protein A), as a target of the GinI/GinR quorum-sensing system via GinA and its negative effects on oxidativefermentation, including acetic acid and gluconic acid fermen-tation. GmpA, an outer membrane protein possibly specific toacetic acid bacteria and members of related genera, appears toplay an important role in oxidative fermentation by this uniquegroup of bacteria.

MATERIALS AND METHODS

Bacterial strains and plasmids. G. intermedius NCI1051 and G. polyoxogenesNCI1028 were obtained from stock cultures at the Central Research Institute,Mizkan Group Co., Ltd. An Acetobacter/Gluconacetobacter-Escherichia coli shut-tle vector, pMV24, was described previously (5). A ginI-disrupted mutant(ginI::Km), a ginR-disrupted mutant (ginR::Km), and a ginA-disrupted mutant(ginA::Km) were described previously (10). pGinR, pGinI, pGinIA, and pGinA,all of which were pMV24-derived plasmids, contained the intact ginR, ginI, ginIand ginA, and ginA sequences, as described previously (10). pMGinA containeda ginA sequence with a frameshift mutation (10). E. coli JM109 and plasmidpUC19, used for DNA manipulation, were purchased from Takara Bio. Agrobac-terium tumefaciens NTL4(pZLR4), obtained from S. K. Farrand, was used toassay AHL production.

Media and culture conditions. YPG medium (pH 6.5) consisted of 5 g of yeastextract (Wako Pure Chemicals), 3 g of polypeptone (Wako Pure Chemicals), and30 g of glucose in 1 liter of water. The Gluconacetobacter strains were grown at30°C in YPG medium with 1% (vol/vol) cellulase (Celluclast 1.5L; Novozymes)and with or without 2% (vol/vol) ethanol.

For acetic acid fermentation tests, the Gluconacetobacter strains were firstcultured at 30°C for 24 h in 5 ml of YPG medium with 1% (vol/vol) cellulase ina test tube with shaking. A portion (5 ml) of this culture was inoculated into 1.5liters of YPG medium supplemented with 3% (vol/vol) ethanol, 1% (vol/vol)cellulase, and 0.001% (vol/vol) silicone (KM72; Shin-Etsu Chemical Co., Ltd.) ina 3-liter mini-jar fermentor (Bioneer 300 3L; B. E. Marubishi Co., Ltd.) andcultured at 30°C with agitation at 500 rpm and aeration at a rate of 1.0 liter/min.The ethanol concentration was automatically maintained at 2% (vol/vol) by theaddition of ethanol during cultivation. The acetic acid and gluconic acid concen-trations in the culture broths were determined by high-performance liquid chro-matography with a Shodex RSpak KC-811 column and a Shimadzu CDD-10Aconductivity detector. Bacterial growth was monitored by measuring the turbid-ities of the cultures at 660 nm by a photometer.

E. coli was cultured at 37°C in Luria-Bertani medium. A. tumefaciensNTL4(pZLR4) was cultured at 30°C in AB medium containing 0.2% (wt/vol)glucose, 0.1% (wt/vol) yeast extract, and 5 �g of gentamicin/ml (15). Ampicillinand kanamycin were used at a final concentration of 100 �g/ml, when necessaryto maintain the plasmids.

DNA manipulation. Restriction enzymes, T4 DNA ligase, and other DNA-modifying enzymes were purchased from Takara Bio. All DNA manipulations inE. coli were performed as described previously (1, 16). The Gluconacetobacterstrains were transformed by electroporation (27). Chromosomal DNA fromGluconacetobacter strains was isolated with the genomicPrep cell and tissueDNA isolation kit (GE Healthcare). Chromosomal DNA from G. intermediusNCI1051 and G. polyoxogenes was used as the templates for PCR. All nucleotide

sequences were determined with a CEQ dye terminator cycle sequencer using aquick-start kit (Beckman Coulter).

2D-PAGE. Gluconacetobacter strains were grown at 30°C for 12 h in YPGmedium containing 1% (vol/vol) cellulase and 2% (vol/vol) ethanol in a shakingflask. Cells were washed with 10 mM Tris buffer (pH 7.5), resuspended in thesame buffer, and then disrupted twice using a French pressure cell at 20,000lb/in2. The cell lysates were centrifuged at 8,000 � g and 4°C for 10 min, and thesupernatants were further ultracentrifuged at 370,000 � g for 1 h. The superna-tants were used as soluble fractions. The pellets were resuspended in ReadyPrepReagent 3 (Bio-Rad Laboratories), and the suspensions were used as membranefractions. 2D-PAGE was carried out using the PROTEAN isoelectric focusingcell (Bio-Rad Laboratories) with immobilized pH gradients (precast IPGReadyStrip gel, pH 3 to 10; 11 cm) in the first dimension and sodium dodecylsulfate–12.5% polyacrylamide gel in the second dimension, according to theinstructions of the manufacturer. The protein concentrations were determinedwith the DC protein assay kit (Bio-Rad Laboratories) using bovine serum albu-min as a standard.

N-terminal amino acid sequencing. After 2D-PAGE, the proteins were blottedonto a polyvinylidene difluoride membrane (Millipore) with a semidry blottingsystem (HorizBlot; ATTO Corporation) and analyzed by Edman degradation onan Applied Biosystems model 492cLC protein sequencer.

Cloning of gmpA, gmpB, and gmpC. A 0.4-kb fragment containing a portion ofthe GP1736 gene sequence from G. polyoxogenes NCI1028 was amplified by PCRwith primers AF and AR (Table 1). The amplified fragment was used for the32P-labeled probe for Southern hybridization against the restriction enzyme-digested chromosomal DNA from G. intermedius NCI1051. Standard DNA ma-nipulation, including colony hybridization using this 0.4-kb fragment as theprobe, gave three DNA fragments, a 3.4-kb SmaI fragment, a 4.0-kb SphI frag-ment, and a 4.2-kb EcoRV fragment, which revealed the presence of threeompA-like genes, gmpA to gmpC.

Disruption of gmpA. The 3.4-kb SmaI fragment cloned in pUC19 was digestedby NruI. A SmaI fragment carrying the neomycin-kanamycin resistance genefrom Tn5 (2) was then inserted into the NruI site within the gmpA codingsequence. This plasmid was introduced into G. intermedius NCI1051 by electro-poration, and kanamycin-resistant colonies were selected as candidates forgmpA-disrupted mutants. Correct gmpA-disrupted mutants were selected bySouthern hybridization with a 0.4-kb fragment that had been amplified by PCRwith primers AF and AR (Table 1) as the 32P-labeled probe.

Plasmid construction. For the construction of a plasmid for the expression ofgmpA alone, a 1.8-kb fragment containing gmpA was amplified by PCR withprimers P1F, containing an EcoRI site, and P1R, containing a SmaI site (Table1), and placed between the EcoRI and SmaI sites of pMV24, generating plasmidpGmpA. Nucleotide sequencing confirmed that no errors occurred during PCR.

For the construction of a mutagenic plasmid, a 0.5-kb fragment containing an

TABLE 1. Primers used in this study

Primer Sequence (5� to 3�)a

AF .............................GCCTGCAGATGACCACCGAATACAR.............................GCCGTCACGGATCAGTTCAGP1F ............................CCGGAATTCGACCCCAACACGTCCGTGTGP1R............................GCGCCCGGGCCGCATGCCCATGAGGTGTGMR ............................GCCATGCTGCATGCGTTCGATCTTCCCGCTCTCGCMF.............................GCGAGAGCGGGAAGATCGAACGCATGCAGCATGGCS1-IF..........................CCGGAATTCGGATATGTCGCTCCCCATTCS1-IR.........................GGGGACTTCCCACTGCTCATTAGS1-AF........................GCGGAATTCGACCCCAACACGTCCGTGTGS1-AR .......................CAGATGGGGGCATGTTCCACS1-BF ........................GCGGAATTCCGAATCGTTTTGGGACGCGTCAGS1-BR........................CTGTTGTTGCAGGGCAAAGCCS1-CF ........................GCGGAATTCTCCCGACCAACCGGTCTCAGS1-CR........................CTTCCGTATCGGCGAAGTGACF0...............................GCGGAATTCGACCCCAACACGTCCGTGTGF1...............................CAGCATGGCGATGGCTGGACF2...............................CGAGCATAGTGCTGACATCAAGF3...............................GTCACTTCGCCGATACGGAAGR1..............................GCCACCGGGAACGACGGTGCR2..............................CGACATGCCATCCATGGTGCR3..............................CGACCGGGGGAGTTGCCCACR0..............................CGTTCAGGCTGGAAGGGAAC

a An EcoRI site in P1F and a SmaI site in P1R are indicated by underlining.

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upstream region of gmpA was amplified by PCR using primers P1F and MR(Table 1), and a 1.2-kb fragment containing a downstream region of gmpA wasamplified by PCR using primers MF and P1R (Table 1). A 1.7-kb fragment wasamplified by splice overlap extension-PCR (9) with primers P1F and P1R byusing the 0.5-kb fragment and the 1.2-kb fragment as the templates, and theproduct was placed between the EcoRI and SmaI sites of pMV24, generatingplasmid pGmpA�. Nucleotide sequencing confirmed that no errors occurredduring PCR.

S1 nuclease mapping. Total RNA was isolated by the hot-phenol method (20)from cells grown at 30°C for 8 h in YPG medium containing 2% (vol/vol) ethanoland 1% (vol/vol) cellulase in a shaking flask. S1 nuclease mapping was conductedas described previously (12). The hybridization probes were prepared by PCRusing pairs of 32P-labeled and nonlabeled primers. The primer pairs were asfollows: S1-AF and S1-AR for gmpA, S1-BF and S1-BR for gmpB, S1-CF andS1-CR for gmpC, and S1-IF and S1-IR for ginI (Table 1). Primers S1-AR, S1-BR,S1-CR, and S1-IR were labeled with 32P at their 5� ends by using T4 polynucle-otide kinase before PCR. All sequencing reactions were performed using aBcaBEST dideoxy sequencing kit (Takara Bio).

Northern blot analysis. Total RNA was isolated by the hot-phenol method(20) from cells grown at 30°C for 6, 8, 10, 12, 16, and 24 h in YPG mediumcontaining 2% (vol/vol) ethanol and 1% (vol/vol) cellulase in a shaking flask. A0.4-kb fragment containing part of gmpA was amplified by PCR with primers AFand AR (Table 1) using the 3.4-kb SmaI fragment as the template. The PCRproduct was verified by nucleotide sequencing. The amplified fragment was usedfor the 32P-labeled probe for Northern hybridization against the total RNA. ThegmpA probe specifically bound the gmpA coding sequence, as determined bySouthern hybridization (data not shown).

Reverse transcription (RT)-PCR. Total RNA was isolated by the hot-phenolmethod (20) from cells grown at 30°C for 8 h in YPG medium containing 2%(vol/vol) ethanol and 1% (vol/vol) cellulase in a shaking flask. RT was performedusing the total RNA with SuperScript III reverse transcriptase (Invitrogen). ThecDNA was then amplified by PCR using the RT reaction mixture as the templateand specific primers (Table 1). The RNA samples were tested with and withoutreverse transcriptase to verify the complete removal of genomic DNA. PCRusing the genomic DNA from G. intermedius NCI1051 as the template wasperformed to ensure the fidelity for each primer pair.

AHL assay. The Gluconacetobacter strains were grown at 30°C for 24 h in YPGmedium containing 1% (vol/vol) cellulase and 2% (vol/vol) ethanol. Cells wereremoved by centrifugation, and AHLs were extracted from culture supernatantswith acidified ethyl acetate, as described previously (23). The AHL productionwas evaluated by measuring the �-galactosidase activity of an AHL indicatorstrain, A. tumefaciens NTL4(pZLR4), as described by Luo et al. (15).

Nucleotide sequence accession number. The nucleotide sequence of gmpA togmpC has been deposited in the DDBJ, EMBL, and GenBank databases underaccession number AB426240.

RESULTS

Comparison of protein profiles of the wild-type strainNCI1051, mutant ginI::Km, and mutant ginR::Km. As the firststep to identify targets of the GinI/GinR quorum-sensing sys-tem in G. intermedius NCI1051, we employed 2D-PAGE toanalyze soluble and membrane proteins isolated from wild-type strain NCI1051, the ginI::Km mutant, and the ginR::Kmmutant, all of which harbored pMV24 and were grown at 30°Cfor 12 h in YPG medium containing 2% (vol/vol) ethanol and1% (vol/vol) cellulase. The comparison of the patterns of mem-brane protein spots on the 2D gels revealed that the levels ofa protein with an observed molecular mass of 35 kDa, namedM1, in the ginI::Km and ginR::Km mutants were apparentlydecreased (Fig. 1B and D) compared to that in the wild-typestrain NCI1051 (Fig. 1A). The introduction of the intact ginIAgene on pGinIA into the ginI::Km mutant restored the expres-sion of protein M1 to the wild-type level (Fig. 1C). Similarly,the introduction of ginR on pGinR into the ginR::Km mutantalso restored the wild-type expression of protein M1 (Fig. 1E).The N-terminal amino acid sequence of protein M1 was de-termined by Edman degradation to be TTITGPYVGIGGG.

BLAST analysis of the genome sequence of G. polyoxogenesNCI1028, which was determined by a research group at MizkanGroup Co., Ltd. (K. Kondo, personal communication), predictedthat one or more of the three products (the GP1736, GP1737, andGP1740 proteins) encoded by three adjacent homologous genesthat are clustered at a locus was protein M1. All three proteins,belonging to the OmpA family, contain N-terminal amino acidsequences almost the same as the established sequence of proteinM1. Although two additional genes encoding OmpA family pro-teins exist far from the nested ompA-like gene locus on the chro-mosome of G. polyoxoxgenes NCI1028, the sequences of the twoproteins show no significant similarity to the N-terminal aminoacid sequence of M1. As described below, protein M1 was foundto be GmpA, because mutant gmpA::Km lacked protein M1 andthe introduction of pGmpA into this mutant restored the produc-tion of M1.

Cloning of gmpA to gmpC. We prepared 32P-labeled probeon the basis of the nucleotide sequence of the GP1736 gene inG. polyoxogenes NCI1028 (Kondo, personal communication).The probe consisted of 0.4 kb corresponding to Leu216 throughGly362 of the GP1736 protein. Southern hybridization with thisprobe against SmaI-, SphI-, or EcoRV-digested chromosomalDNA from G. intermedius NCI1051 revealed the presence ofnucleotide sequences homologous to the probe in strainNCI1051 (data not shown). We used the same probe to clonea 3.4-kb SmaI fragment, a 4.0-kb SphI fragment, and a 4.2-kbEcoRV fragment from G. intermedius NCI1051, all giving apositive signal, by standard DNA manipulation methods in-cluding colony hybridization. All three fragments were clonedin pUC19.

The nucleotide sequences of the cloned fragments and theassembly of the fragments indicated the presence of threeadjacent homologous open reading frames (ORFs) oriented inthe same direction (Fig. 2A), as was found for G. polyoxogenes.We named these three ORFs gmpA, gmpB, and gmpC, becausethe deduced amino acid sequences of the C-terminal regions ofGmpA, GmpB, and GmpC encoded by these ORFs showedsimilarity to the so-called OmpA motif of E. coli OmpA, themajor outer membrane protein (Fig. 2B). PSORTb analysis(http://www.psort.org/psortb/) of GmpA, GmpB, and GmpCpredicted that they were outer membrane proteins with a pu-tative 22-amino-acid signal peptide (the most likely cleavagesite being between amino acids Ala22 and Thr23) (Fig. 2B), aspredicted by SignalP (http://www.cbs.dtu.dk/services/SignalP/).The amino acid sequence from Thr24 to Gly35 (Fig. 2B) justafter this cleavage site in GmpA and GmpB completelymatched the amino acid sequence of protein M1 determinedby N-terminal amino acid sequencing, but that of GmpC didnot match. As described below, protein M1 corresponded toGmpA, as determined by its response to the GinI/GinR regu-latory system.

GmpA (405 amino acids), GmpB (373 amino acids), andGmpC (364 amino acids) are highly homologous to one an-other, especially in their C termini containing the OmpA motif.The OmpA motif is believed to interact specifically with thepeptidoglycan layer (14). The N-terminal regions of GmpA,GmpB, and GmpC all contain eight �-stranded transmem-brane sequences, �1 to �8 (Fig. 2B), as predicted by Jpred(http://www.compbio.dundee.ac.uk/�www-jpred/). The predic-tion of the structures of these proteins by FUGUE (http:

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//tardis.nibio.go.jp/fugue/prfsearch.html) gave almost the samesecondary structures as the prediction by Jpred. Referring tothe crystal structure of E. coli OmpA (14, 21), we assume thatthe N-terminal domains serve as a membrane anchorconsisting of eight antiparallel �-barrels, with the N-terminaland C-terminal ends in the periplasm. The spacing patterns ofthe eight �-strands of E. coli OmpA and the Gmp proteins arealmost identical, except that part of the �4 region of GmpAwas predicted to contain both a �-strand and an �-helix (Fig.2B). GmpA contains an extra hydrophilic sequence in loop 1 inthe N terminus compared to GmpB and GmpC (Fig. 2B).Loop 1 is exposed to the surface. As described below, this extraamino acid sequence was found only in vinegar producers andwas essential for the function of GmpA.

It should be noted that the primary amino acid sequences ofGmpA to GmpC show only 32% identity to that of OmpAfrom E. coli (corresponding to DNA database accession no.NP_415477) and have no significant similarity to OmpA intheir N termini, compared to the level of similarity of their Ctermini containing the OmpA motif. However, GmpA toGmpC are homologous to the OmpA family proteins found inacetic acid bacteria. GmpA to GmpC show 60 to 64% identityin amino acid sequence to the OmpA family GOX1787 protein(corresponding to GenBank accession no. YP_192182) fromGluconobacter oxydans 621H (22) and about 45 to 48% identity

to the OmpA family proteins encoded by Gb0359 (GenBankaccession no. YP_744180) from Granulibacter bethesdensisCGDNIH1 (8), GOX1260 (GenBank accession no. YP_191677) from Gluconobacter oxydans 621H (22), and Acry_0174 (GenBank accession no. YP_001233321) and Acry_0173(GenBank accession no. YP_001233320) from Acidiphiliumcryptum JF-5. The OmpA family proteins are grouped intothree subfamilies (see Fig. S1A in the supplemental material):(i) so-called OmpA-like proteins containing �-barrels as anouter membrane anchor in their N termini; (ii) rather smallproteins, with fewer than 250 amino acid residues, containingno transmembrane domains; and (iii) functionally unknownproteins containing �-helices instead of �-barrels as a mem-brane anchor. Figure S1B in the supplemental material is aphylogenetic tree, constructed by a neighbor-joining method,for the entire sequences of the OmpA family proteins anddelineates the three classes. Figure S1C in the supplementalmaterial is a phylogenetic tree for the OmpA-like proteinscontaining �-barrels as a membrane anchor. The evolutionaryanalysis of the OmpA family proteins gave us no hints topredict or speculate on the function of GmpA, although aGmpA-containing group of proteins distant from the E. coliOmpA (see Fig. S1C in the supplemental material) may haveevolved to exert some specific functions in a particular speciesof bacteria to inhabit a given niche.

FIG. 1. 2D-gel images of the membrane fractions prepared from the wild-type (w.t.) strain NCI1051 harboring pMV24 and the ginR::Kmmutant strain harboring pMV24 (upper panels) and parts of the 2D gels including GmpA (A to E) and corresponding to the gmpA mutant strains(F to H). The membrane fractions from G. intermedius strains, which were grown at 30°C for 12 h in YPG medium containing ethanol, wereprepared as described in Materials and Methods. These proteins were analyzed by 2D-PAGE and stained with Coomassie brilliant blue. Thetriangles indicate proteins M1 (black), M2 (white), and M3 (gray).


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In addition to the rather high level of homology in aminoacid sequence among the OmpA family proteins, the numbersand organization of the ompA family genes are unique to aceticacid bacteria, especially to those that produce acetic acid athigh yields. Among the bacteria, including acetic acid bacteria,whose genome sequences are available, only G. intermediusNCI1051 and G. polyoxogenes NCI1028 (Kondo, personal com-munication), both of which were isolated from vinegar, contain

three adjacent omp genes, like gmpA to gmpC, as a gene clus-ter. Other Acetobacteraceae, such as Gluconobacter oxydans621H, Granulibacter bethesdensis CGDNIH1, and Acidiphiliumcryptum JF-5 (Fig. 2B), contain one or two ompA-like genesthat encode GmpB- and GmpC-type proteins without the N-terminal extra sequence in GmpA. The genes located up-stream and downstream of gmpA to gmpC in G. intermediusNCI1051, as well as in G. polyoxogenes NCI1028, and the

FIG. 2. Gene organization of the gmpA-to-gmpC locus in G. intermedius, G. polyoxogenes, and Gluconobacter oxydans (A) and amino acidalignment of the OmpA family proteins (B). (A) The organization of gmpA to gmpC and their neighbors in G. intermedius NCI1051 and of thecorresponding regions in G. polyoxogenes NCI1028 and Gluconobacter oxydans 621H is illustrated. gmpA to gmpC are located between orf1(encoding a putative bifunctional shikimate kinase) and orf2 (encoding a putative alanyl-tRNA synthetase) in G. intermedius. The GP1736, GP1737,and GP1740 genes, encoding OmpA family proteins in G. polyoxogenes, are located between the GP1735 gene, encoding a bifunctional shikimatekinase, and the GP1743 gene, encoding an alanyl-tRNA synthetase. The GOX1787 gene (GenBank gene identification no. 3250051), encoding anOmpA family protein in Gluconobacter oxydans, is located between the GOX1788 gene (identification no. 3250052), encoding a bifunctionalshikimate kinase, and alaS (identification no. 3250050), encoding an alanyl-tRNA synthetase. (B) Alignment of the primary sequences of OmpAfamily proteins. These include GmpA, GmpB, and GmpC from G. intermedius NCI1051; the products of GOX1787 (GenBank accession no.YP_192182) and GOX1260 (GenBank accession no. YP_191677) from Gluconobacter oxydans 621H; the product of Gb0359 (GenBank accessionno. YP_744180) from Granulibacter bethesdensis CGDNIH1; the products of Acry_0174 (GenBank accession no. YP_001233321) and Acry_0173(GenBank accession no. YP_001233320) from Acidiphilium cryptum JF-5; and OmpA (corresponding to GenBank accession no. NP_415477) fromE. coli K-12. These amino acid sequences were aligned with ClustalW (http://www.ebi.ac.uk/Tools/clustalw/) and presented by BOXSHADE 3.21(http://www.ch.embnet.org/software/BOX_form.html). The putative signal peptide comprising Met1 to Ala22 is indicated by a line. The amino acidsequence of GmpA, which matched the N-terminal sequence of protein M1, is indicated by a dashed line. The extra region of GmpA, which isabsent from GmpB and GmpC, is indicated by double lines. The OmpA motif is indicated by an arrow. The �-strands (�1 to �8, shown in red)and loops (L1 to L4) of the transmembrane domain of E. coli OmpA, as determined by X-ray crystallography (21), are indicated. The �-strandsin GmpA to GmpC, as predicted by Jpred (http://www.compbio.dundee.ac.uk/�www-jpred/), are also shown in red. Part of the fourth �-strandregion of GmpA, corresponding to �4 in E. coli OmpA, was predicted to contain an �-helix (shown in green). Identical amino acids are shadedin black, and similar amino acids are shaded in gray.


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GOX1787 gene, encoding an OmpA family protein in Glu-conobacter oxydans 621H, are conserved (Fig. 2A), which sug-gests that the duplication of an ancestral omp gene, GOX1787,has occurred twice in strains NCI1051 and NCI1028. Becausethe GOX1787 protein contains no extra N-terminal sequencein loop 1, gmpA probably acquired the extra sequence duringthe duplication.

Dependence of gmpA transcription on ginI and ginR. Theabove-described 2D-PAGE analysis suggested that eithergmpA or gmpB or both were under the control of the GinI/GinR quorum-sensing system. We first determined the tran-scriptional organization of the gmp genes. RNA was isolated bythe hot-phenol method from cells grown at 30°C for 8 h inYPG medium containing 1% (vol/vol) cellulase and 2% (vol/vol) ethanol. High-resolution S1 nuclease mapping identified asingle transcriptional start point for each of the gmpA, gmpB,and gmpC genes (Fig. 3A), indicating that each gene was tran-scribed from its own promoter (Fig. 3B). The transcriptionalstart point of gmpA was, for example, 174 bp upstream of thefirst letter of the translational start codon. In front of theirtranscriptional start points, neither probable promoter ele-ments (5�-TATAAT-3� for 10 and 5�-TTGACA-3� for 35)nor a lux box, a 20-bp inverted repeat at approximate position45 with respect to the transcriptional start point (7, 10), wasfound.

RT-PCR analysis to detect read-through transcription usingcDNA synthesized by RT from the RNA prepared from thewild-type strain gave a distinct amplified fragment for eachgene: fragment F1R1, with 309 bp, synthesized with primers F1and R1 for gmpA; fragment F2R2, with 349 bp, for gmpB; andfragment F3R3, with 328 bp, for gmpC (Fig. 3C). A gmpA-gmpB-spanning region was also amplified by RT-PCR usingprimers F1 and R2, yielding fragment F1R2, with 1,866 bp,which indicated the occurrence of read-through transcriptionfrom the gmpA promoter into gmpB. In contrast, a gmpB-gmpC-spanning region was not amplified by RT-PCR usingprimers F2 and R3. The RT-PCR analysis was also performedwith the RNA prepared from mutant ginI::Km to show thedependence of the gmpA promoter on the GinI/GinR regula-tory system (see below). These high-resolution S1 mapping andRT-PCR analyses showed that each of the gmpA, gmpB, andgmpC genes had its own single promoter, although there wastranscriptional read-through from gmpA to gmpB but no read-through from gmpB to gmpC. Despite the finding that gmpAtranscription is under the control of the GinI/GinR regulatorysystem, no lux box-like sequence was present in the promoterregions of gmpA to gmpC. The absence of a lux box is consis-tent with the fact that gmpA is not directly controlled by GinRbut is controlled via GinA (see below).

We next assessed the transcription of gmpA to gmpC in thewild-type strain NCI1051, the ginI::Km mutant, and theginR::Km mutant, all of which harbored the vector plasmidpMV24, by low-resolution S1 nuclease mapping (Fig. 3D).RNA was isolated by the hot-phenol method from cells grownat 30°C for 8 h in YPG medium containing 1% (vol/vol) cel-lulase and 2% (vol/vol) ethanol. rRNA was used to check theamount of RNA used. gmpA in strain NCI1051 was activelytranscribed, but the levels of transcription in the ginI::Km andginR::Km mutants were markedly decreased (Fig. 3D). Theintroduction of pGinIA and pGinR into the ginI::Km and

ginR::Km mutants, respectively, restored gmpA transcription tothe wild-type level (Fig. 3D), showing that GinI and GinR wereessential for gmpA transcription. On the other hand, gmpB andgmpC in the ginI::Km and ginR::Km mutants were transcribedat the same levels as those in the parental strain. Northern blotanalysis of gmpA by using RNA isolated from cells grown for 6to 24 h confirmed the results of the S1 mapping; the intactginIA and ginR genes on the vector pMV24 complemented theginI and ginR mutations and restored the wild-type level ofgmpA transcription in the mutants (Fig. 3E). The major signalof 1.5 kb represented the gmpA transcript starting from thegmpA promoter, and a minor signal of 3.5 kb represented theread-through transcript from the gmpA promoter into gmpB.The RT-PCR analysis with the RNA from mutant ginI::Km(Fig. 3C) supported these results; only the gmpA promoterdepended on the GinI/GinR regulatory system, and there wasread-through from the gmpA promoter to gmpB. From thesedata, we concluded that each of the genes gmpA, gmpB, andgmpC had its own transcription start site and that only thegmpA promoter was positively regulated by the GinI/GinRquorum-sensing system.

These transcriptional analyses suggested that GmpA corre-sponded to protein M1, which was produced in response to theGinI/GinR quorum-sensing system, as observed by 2D-PAGE(Fig. 1). An additional spot, M2, which was about 5 kDasmaller than M1, was observed at almost the same levels on the2D gels for all strains (Fig. 1). The N-terminal amino acidsequence of protein M2 also matched the sequences of GmpAand GmpB. Because the calculated molecular mass of GmpBwas about 5 kDa smaller than that of GmpA, protein M2presumably corresponds to GmpB.

Dependence of gmpA transcription on ginA. The absence ofany lux box-like sequence in the gmpA promoter excluded thepossibility that GinR directly controlled gmpA transcription.Our previous study showed that GinA as a target of the GinI/GinR regulatory system plays an important role in the controlof oxidative fermentation, growth in medium containing etha-nol, and antifoam activity (10). To determine whether ginA wasinvolved in the control of gmpA transcription, we introducedginA on pGinA into the ginI::Km mutant and examined gmpAtranscription in this mutant by S1 nuclease mapping. ginA onpGinA was transcribed from a heterologous promoter, the E.coli lac promoter on pMV24. The decreased gmpA transcrip-tion in the ginI::Km mutant was restored to the wild-type levelby the introduction of ginA on pGinA (Fig. 4). The ginI mu-tation caused no effects on the transcription of gmpB or gmpC,as described above. We next introduced a frameshift mutationinto the ginA gene on pGinA, generating pMGinA as describedpreviously (10). The introduction of pMGinA into the ginI::Kmmutant gave no restoration of the phenotype of the mutant(Fig. 4), indicating that the GinA protein function was essen-tial for restoring the gmpA transcription in the ginI::Km mu-tant.

To clarify the involvement of ginA in gmpA transcription, weexamined gmpA transcription in the ginA::Km mutant. ThegmpA transcription in this mutant was greatly reduced (Fig. 4).No such decrease was observed for gmpB or gmpC. The intro-duction of pGinA into the ginA::Km mutant restored the wild-type level of gmpA transcription (Fig. 4). These data furtherconfirmed that GinA was essential for gmpA transcription. We


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checked that the introduction of ginA on pGinA into theginI::Km mutant or the loss of ginA had no significant effects onAHL production, as determined by using the A. tumefaciensreporter system (data not shown). We thus concluded thatgmpA transcription was positively regulated by GinA, but in astill unknown manner.

Growth repression by gmpA in the presence of ethanol. Todetermine the involvement of gmpA in the repression of oxi-dative fermentation, including acetic acid and gluconic acidfermentation, growth in the presence of ethanol, and the an-tifoam activity of cells, we disrupted the chromosomal gmpAgene in G. intermedius NCI1051, generating mutant gmpA::

FIG. 3. Transcriptional analysis of gmpA, gmpB, and gmpC. (A) Determination of the transcriptional start points of gmpA, gmpB, and gmpCby high-resolution S1 nuclease mapping. The resultant DNA fragments in the S1 mapping (left) were analyzed by PAGE with the sequencingladders (A, C, G, and T) of the probes. The transcriptional start points are indicated by arrows. (B) Nucleotide sequences upstream of thetranscriptional start points (�1; corresponding letters are in bold) of gmpA to gmpC. No promoter elements similar to the 35 (5�-TTGACA-3�)and 10 (5�-TATAAT-3�) elements are present. M, start codon. (C) Agarose gel electrophoresis analysis of RT-PCR products. RT-PCR wasperformed using total RNA from the wild-type strain NCI1051 (w.t.) and the ginI::Km mutant. The RNA samples were analyzed with (�RT) andwithout (RT) reverse transcriptase to verify the absence of genomic DNA. Lane P was a control lane in which the genomic DNA of G. intermediusNCI1051 was amplified with the respective pair of primers. The combinations of the primers, F0 and R1, for example, are given in the form F0R1.The positions of gmpA to gmpC and PCR products are also shown schematically. (D) Transcription of gmpA, gmpB, and gmpC in wild-type strainNCI1051 harboring pMV24, the ginI::Km mutant harboring pMV24, the ginI::Km mutant harboring pGinIA, the ginR::Km mutant harboringpMV24, and the ginR::Km mutant harboring pGinR, as determined by low-resolution S1 mapping. (E) Northern blot analysis of gmpA in wild-typestrain NCI1051 harboring pMV24 (lane 1), the ginI::Km mutant harboring pMV24 (lane 2), the ginI::Km mutant harboring pGinIA (lane 3), theginR::Km mutant harboring pMV24 (lane 4), and the ginR::Km mutant harboring pGinR (lane 5).


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Km. The gmpA::Km mutant grew in medium containing noethanol with the same time course and cell mass as the parentalstrain (Fig. 5A). In YPG medium containing 2% (vol/vol)ethanol, however, the growth rate and cell mass of mutantgmpA::Km during the exponential growth phase were higherthan those of the parental strain (Fig. 5B). This growth profileof mutant gmpA::Km was the same as those of mutantsginI::Km, ginR::Km, and ginA::Km (10). The higher growthrate of the mutant was due to the mutation in gmpA, becausethe introduction of pGmpA into the mutant decreased thegrowth rate to the same level as that of the parental strain(Fig. 5B).

The importance of the extra region in the N-terminal por-tion of GmpA was confirmed by constructing plasmidpGmpA�, in which the sequence encoding the extra region wasdeleted, and introducing this plasmid into mutant gmpA::Km.The production and localization of the N-terminal mutantGmpA protein GmpA�, in which the extra region was deleted,were evaluated by 2D-PAGE analysis of the membrane frac-tion of mutant gmpA::Km harboring pGmpA� after this mu-tant was grown at 30°C for 12 h in YPG medium containing 2%(vol/vol) ethanol and 1% (vol/vol) cellulase (Fig. 1F to H). Thecomparison of the 2D-PAGE patterns of the membrane pro-tein spots revealed that protein M1 (GmpA) disappeared inthe gmpA::Km mutant harboring pMV24 (Fig. 1F) but that theintroduction of the intact gmpA gene on pGmpA into thegmpA::Km mutant restored the production of GmpA (Fig.1G). A spot, M3, which was about 2 kDa smaller than GmpA(calculated molecular mass, 41.4 kDa; pI 6.05) and presumablyrepresented the GmpA� protein (39.3 kDa; pI 5.99), was ob-served in the membrane fraction of the gmpA::Km mutantharboring pGmpA� (Fig. 1H). These data showed that theGmpA� protein was produced and localized in the membrane.The growth rate of mutant gmpA::Km harboring pGmpA� inmedium containing 2% (vol/vol) ethanol was the same as thatof the gmpA::Km mutant harboring the vector pMV24 (Fig.5B). Thus, the extra region in the N terminus of GmpA wasimportant for the protein to exert its function of repressing thegrowth of strain NCI1051 in the presence of ethanol.

Our previous study showed that GinI/GinR quorum sensingrepresses antifoam activity; mutants ginI::Km, ginR::Km, andginA::Km form foams of smaller volumes on the culture broththan those formed by the wild-type strain (10). However, thegmpA::Km mutant formed foams of almost the same volume asthose formed by the wild-type strain NCI1051 (data not shown).Therefore, gmpA had no effects on the antifoam activity.

Repression of oxidative fermentation by gmpA. We nextmeasured acetic acid and gluconic acid production by mutantgmpA::Km. The strain was cultured at 30°C in YPG mediumcontaining 3% (vol/vol) ethanol in a 3-liter mini-jar fermentor.Figure 6A and B show the acetic acid and gluconic acid fer-mentation profiles, respectively, of the wild-type strainNCI1051, the ginI::Km mutant, and the gmpA::Km mutant, allof which harbored the vector pMV24. Like mutant ginI::Km,the gmpA::Km mutant accumulated acetic acid and gluconicacid at higher yields than the parental strain NCI1051. Thefinal yields of acetic acid (mean standard deviation, 4.17% 0.07% [wt/vol]) and gluconic acid (2.26% 0.03%) producedby mutant gmpA::Km were also higher than those (acetic acid,2.78% 0.11%; gluconic acid, 1.89% 0.08%) produced bythe parental strain NCI1051. The increased production ratesand final yields of acetic acid and gluconic acid in thegmpA::Km mutant were restored to almost the wild-type levelsby introducing gmpA on pGmpA into this mutant (data notshown).

Consistent with the observation that the gmpA mutation ledto the production of acetic acid and gluconic acid at highyields, the introduction of gmpA under the control of both itsown promoter and the E. coli lac promoter on the pMV24-derived plasmid pGmpA into the wild-type strain NCI1051

FIG. 5. Growth of G. intermedius strains in YPG medium (A) andYPG medium containing 2% (vol/vol) ethanol (B). Wild-type strainNCI1051 harboring pMV24 (F), the gmpA::Km mutant harboringpMV24 (�), the gmpA::Km mutant harboring pGmpA (‚), and thegmpA::Km mutant harboring pGmpA� (}) were cultured as describedin Materials and Methods. The inset shows an enlarged profile ofgrowth from 6 to 14 h. OD660, optical density at 660 nm.

FIG. 4. Dependence of gmpA transcription on ginA. S1 nucleasemapping of gmpA to gmpC in wild-type (w.t.) strain NCI1051 harbor-ing pMV24, the ginI::Km mutant harboring pMV24, the ginI::Km mu-tant harboring pGinA, the ginI::Km mutant harboring pMGinA, theginA::Km mutant harboring pMV24, and the ginA::Km mutant harbor-ing pGinA is shown.


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reduced the final yields of acetic acid (2.52% 0.03% [wt/vol])and gluconic acid (1.70% 0.02%) below those of the paren-tal strain (Fig. 6A and B). The copy number of pMV24 is about10 (10). These findings suggested that GmpA, as one of thetargets of the GinI/GinR quorum-sensing system in G. inter-medius NCI1051, repressed oxidative fermentation, includingacetic acid and gluconic acid fermentation, and played an im-portant role in the regulation of oxidative fermentation.

Mutant gmpA::Km continued to produce acetic acid in thestationary phase, although the production of acetic acid by theginI::Km and ginA::Km mutants ceased in the stationary phase(Fig. 6A) (10). Therefore, the phenotypes of the gmpA::Kmmutant were not completely the same as those of the ginI::Kmand ginA::Km mutants. This finding raised a possibility thatsome other target gene(s) of GinA was present, affecting thesephenotypes in some unknown way.

Dependence of gmpA transcription on ethanol and aceticacid. Because gmpA affected acetic acid fermentation, we ex-pected that ethanol and acetic acid would affect gmpA tran-scription. We examined gmpA transcription in the wild-typestrain NCI1051 harboring pMV24 by low-resolution S1 nucle-ase mapping (Fig. 7A). RNA was isolated by the hot-phenolmethod from cells grown to early exponential, late exponential,

and stationary phases at 30°C in YPG medium, YPG mediumcontaining 2% (vol/vol) ethanol, or YPG medium containing1% (vol/vol) acetic acid (Fig. 7B). rRNA was used to check theamount of RNA used. gmpA transcription was greatly en-hanced by the addition of ethanol and acetic acid. On the otherhand, the addition of ethanol or acetic acid had no significanteffects on the transcription of gmpB, gmpC, or ginI. These datasuggested that gmpA transcription was positively regulated notonly by the GinI/GinR quorum-sensing system via GinA butalso by ethanol and acetic acid in the medium.

Lack of detectable changes in vesicle formation by or anti-biotic susceptibility of the gmpA mutant. Pgm6 and Pgm7, bothof which show a high level of similarity to E. coli OmpA in theC-terminal region and form eight-stranded �-barrels in the N-terminal region, are major outer membrane proteins in Porphy-romonas gingivalis, one of the most important bacteria in theprogression of chronic periodontal disease. Because the heterolo-gously trimeric complex Pgm6-Pgm7 functions as a stabilizer ofthe cell wall, Pgm6-Pgm7 mutants have a wavy, uneven, discon-tinuous outer membrane and release more membrane vesiclesfrom cells than wild-type strains (11). These mutants also showincreased permeability for solutes compared to wild-type strains

FIG. 6. Courses of acetic acid (A) and gluconic acid (B) fermentationby G. intermedius strains, together with their growth (C). The growth ofthe strains was measured by monitoring the optical densities at 660 nm(OD660) of the culture broths. Wild-type strain NCI1051 harboringpMV24 (F), the ginI::Km mutant harboring pMV24 (Œ), the gmpA::Kmmutant harboring pMV24 (�), and wild-type strain NCI1051 harboringpGmpA (�) were cultured as described in Materials and Methods.

FIG. 7. Dependence of gmpA transcription on ethanol and aceticacid. (A) S1 nuclease mapping of ginI and gmpA to gmpC in wild-typestrain NCI1051 harboring pMV24 cultured in YPG medium (YPG),YPG medium containing and 2% (vol/vol) ethanol (� ethanol), andYPG medium containing 1% (vol/vol) acetic acid (� acetic acid) forthe indicated hours. All media contained 1% (vol/vol) cellulase.(B) Growth of wild-type strain NCI1051 harboring pMV24 in YPGmedium (F), YPG medium containing 2% (vol/vol) ethanol (Œ), andYPG medium containing 1% (vol/vol) acetic acid (f). All media con-tained 1% (vol/vol) cellulase. OD660, optical density at 660 nm.


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(18). These phenotypes of Pgm6-Pgm7 mutants, together with thefact that outer membrane vesicles are found in various gram-negative bacteria, including acetic acid bacteria (3), prompted usto examine the structure of the outer membrane by electronmicroscopy and the antibiotic susceptibilities of mutants with dis-ruptions in the GinI/GinR regulatory system. We examined mu-tant ginI::Km, instead of mutant gmpA::Km, because almost noGmpA was produced in mutant ginI::Km (Fig. 1B). However,scanning and transmission electron microscopy showed no appar-ent difference in membrane structure or in the formation andmorphology of membrane vesicles (see Fig. S2 in the supplemen-tal material).

Some OmpA family proteins, located in the outer mem-brane, form nonspecific diffusion channels that allow the pen-etration of various solutes, although the rate of solute diffusionthrough OmpA in E. coli is about 2 orders of magnitude lowerthan that through the OmpF or OmpC porin (24). To assessthe function of GmpA as a diffusion pump, we measured theantibiotic susceptibility of the gmpA mutant and determinedMICs of apramycin, erythromycin, gentamicin, novobiocin,polymyxin B, rifampin, and streptomycin by the standard dilu-tion method with a 96-well microtiter plate. Serial dilutions ofthe corresponding antibiotics were added to YPG mediumcontaining 1% (vol/vol) cellulase with or without 2% (vol/vol)ethanol. After 24 h of incubation with shaking at 30°C, theturbidities of the cultures at 660 nm were measured by aphotometer. There was no significant difference in MICs forthe wild-type strain NCI1051 and the gmpA::Km mutant (seeTable S1 in the supplemental material). Because strong diffu-sion channels, OmpF and OmpC, are still active in mutantgmpA::Km and because perhaps 99% of the penetration ofsolutes across the outer membrane would occur through thetrimeric OmpF and OmpC porins (19), the difference in sus-ceptibility to antibiotics would be observed only when GmpAserves as a specific, strong diffusion channel. The lack of dif-ference in antibiotic susceptibility between the wild-type strainand mutant gmpA::Km suggested that GmpA did not serve asa special, efficient diffusion channel.

DISCUSSION

We have demonstrated that the GinI/GinR quorum-sensingsystem in G. intermedius controls the expression of gmpA viaGinA, which in turn represses oxidative fermentation, includingacetic acid and gluconic acid fermentation, and growth in mediumcontaining ethanol. The OmpA family proteins play a structuralrole in the maintenance of gram-negative bacterial membraneintegrity by embedding their N-terminal transmembrane �-strands into the outer membrane and providing their C-terminaldomains in the periplasmic space as a physical linkage betweenthe outer membrane and the peptidoglycan layer (4, 13, 14). Thepresence of an extra sequence in one of the surface-exposed loopsin the N-terminal transmembrane domain, which turned out to beessential for the repression of oxidative fermentation, appears tobe specific to so-called vinegar producers that accumulate aceticacid as a waste product in large amounts, as a result of ATPgeneration by means of ethanol oxidation. In addition, the genecluster containing three ompA family genes is also specific to G.intermedius and G. polyoxogenes, both of which accumulate aceticacid at high yields. These findings suggest that GmpA plays a

specific and important role in acetic acid fermentation. Consistentwith this idea, gmpA transcription was regulated not only by theGinI/GinR quorum-sensing system via GinA but also by ethanolas the substrate of the oxidative fermentation and acetic acid asthe final product. Although the function of the 89-amino-acidprotein GinA, with no known motifs, is unclear at present, GinAmay control gmpA transcription by protein-protein interactionwith an unknown activator.

Various functions controlled by a quorum-sensing system re-flect the needs of a particular species of bacteria to inhabit a givenniche. The gene cluster containing three adjacent omp genes, oneof which encodes a GmpA-type protein containing an extra re-gion in one of the loops in the N-terminal membrane domain, hasbeen found so far only in G. intermedius NCI1051 and G. poly-oxogenes NCI1028, high-yield vinegar producers. Because thesubstrate, ethanol, and the product, acetic acid, of acetic acidfermentation are both toxic to the cell, acetic acid bacteria syn-thesize ATP by oxidative fermentation under extremely unfavor-able conditions. It is conceivable that acetic acid bacteria havedeveloped the GinI/GinR system to control the GmpA expressionvia GinA, which in turn controls the rates of growth and aceticacid production in the presence of ethanol, where the bacteria ofthis group generate ATP by ethanol oxidation via a specific elec-tron transport system. The vinegar producer strains NCI1051 andNCI1028 have probably duplicated the GOX1787 gene twice toadapt to acetic acid fermentation during evolution, which is ap-parent from the gene organization around the ancestral gmpAgene, GOX1787, in Gluconobacter oxydans 621H and the twovinegar producers (Fig. 2A). Concerning the function of GmpAas a stabilizer of the cell wall, we speculate that GmpA functionsas a permeability barrier for environmental factors, such as anorganic solvent, ethanol, that is toxic to the cell, although nodifferences in ethanol resistance between the wild-type strain andmutant gmpA::Km were detectable by a routine test in which bothstrains were challenged in medium containing various amounts ofethanol (data not shown). The wild-type strain could reduce ace-tic acid production from ethanol, compared to that in thegmpA::Km mutant, because the outer membrane containingGmpA prevents the ethanol from being imported into theperiplasm, where alcohol dehydrogenase and aldehyde dehydro-genase, both of which are anchored to the inner membrane (17)and are required for ethanol oxidation into acetic acid, arepresent.

The disruption of gmpA resulted in an increase in both therates of acetic acid and gluconic acid production and the finalyields of acetic acid and gluconic acid (Fig. 6A and B), asobserved for the ginI::Km, ginR::Km, and ginA::Km mutants(10). However, the gmpA::Km mutant continued to produceacetic acid in the stationary phase (Fig. 6A), unlike theginI::Km and ginA::Km mutants (10). Thus, the phenotypes ofthe gmpA::Km mutant were not completely the same as thoseof the ginI::Km and ginA::Km mutants. Some other targetgene(s) of GinA is perhaps present, affecting these phenotypesin some unknown way.

Our previous study showed that GinA represses an antifoamactivity, which suggested that ginA has some effects on cellsurface hydrophobicity. Because GmpA is an outer membraneprotein, we expected that GmpA might be involved in theantifoam activity of cell. However, the disruption of gmpA hadno effects on antifoam activity. These findings show that


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GmpA is not involved in the antifoam activity and that GinAhas another unknown target related to the antifoam activity.

In conclusion, we have revealed that GinA, a target of theGinI/GinR quorum-sensing system in the acetic acid bacteriumG. intermedius and perhaps G. polyoxogenes, too, repressesacetic acid fermentation via an outer membrane protein,GmpA (Fig. 8). In other words, the GinI/GinR quorum-sens-ing system controls the most important and specific function ofacetic acid bacteria, i.e., ATP generation by means of aceticacid fermentation in the presence of ethanol. However, thereare several black boxes to be elucidated in the hierarchicalregulatory scheme presented in Fig. 8. First, how the 89-amino-acid protein GinA, with no known motifs, activates gmpA tran-scription should be clarified. Second, how GmpA, with an extrahydrophilic sequence in the surface-exposed loop, repressesoxidative fermentation is also unknown. Third, how GinA re-presses antifoam activity is still unclear. Notwithstanding theunknown steps in the regulatory hierarchy in Fig. 8, our find-ings hitherto would be useful in strain improvement for indus-trial vinegar fermentation by acetic acid bacteria. In fact, forexample, we successfully increased the final yield of acetic acidby disrupting gmpA in G. intermedius.

ACKNOWLEDGMENTS

We thank Stephen K. Farrand (University of Illinois) for providingthe AHL reporter strain. We also thank Shigeru Nakano (MizkanGroup Co., Ltd.) for helpful advice on 2D-PAGE.

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ginR ginI ginA

R I

A

AHL

P

Oxidative fermentation

- acetic acid fermentation - gluconic acid fermentationGrowth in ethanol-containing medium

Antifoam activity

P

R

R

PgmpA

GmpA

EthanolAcetic acid

FIG. 8. Possible model for the quorum-sensing system in G. inter-medius NCI1051. The GinI/GinR quorum-sensing system activates thetranscription of ginA, encoding an 89-amino-acid protein, which in turnactivates the transcription of gmpA in a still unknown manner. Thetranscription of gmpA, encoding an OmpA outer membrane familyprotein, is also activated by ethanol and acetic acid. Thus induced,GmpA represses oxidative fermentation, including acetic acid andgluconic acid fermentation, and growth in the presence of ethanol inan unknown manner. GinA also represses the antifoam activity of cells(10). R, GinR; I, GinI; P, promoter; A, GinA.


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