The �-Glucoside (bgl) Operon of Escherichia coli Is Involved in theRegulation of oppA, Encoding an Oligopeptide Transporter

Dharmesh Harwani,* Parisa Zangoui, and S. Mahadevan

Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India

We report that the bgl operon of Escherichia coli, encoding the functions necessary for the uptake and metabolism of aryl-�-glucosides, is involved in the regulation of oligopeptide transport during stationary phase. Global analysis of intracellular pro-teins from Bgl-positive (Bgl�) and Bgl-negative (Bgl�) strains revealed that the operon exerts regulation on at least 12 down-stream target genes. Of these, oppA, which encodes an oligopeptide transporter, was confirmed to be upregulated in the Bgl�

strain. Loss of oppA function results in a partial loss of the growth advantage in stationary-phase (GASP) phenotype of Bgl�

cells. The regulatory effect of the bgl operon on oppA expression is indirect and is mediated via gcvA, the activator of the glycinecleavage system, and gcvB, which regulates oppA at the posttranscriptional level. We show that BglG destabilizes the gcvA mRNAin vivo, leading to reduced expression of gcvA in the stationary phase. Deletion of gcvA results in the downregulation of gcvB andupregulation of oppA and can partially rescue the loss of the GASP phenotype seen in �bglG strains. A possible mechanism bywhich oppA confers a competitive advantage to Bgl� cells relative to Bgl� cells is discussed.

In natural environments, bacteria live in close associations, mostof the time under nutrient scarcity. This in turn leads to compe-

tition within populations for the limited resources that are avail-able. Bacteria have evolved distinct mechanisms to extract utiliz-able substrates from available resources and consequently acquirea fitness advantage over competitors. One of the strategies is theexploitation of cryptic cellular functions encoded by genetic sys-tems that are silent under laboratory conditions, such as the bgl(�-glucoside) operon of Escherichia coli, involved in the uptakeand metabolism of the plant-derived aromatic �-glucosides sali-cin and arbutin (20).

The three genes bglG, bglF, and bglB of the bgl operon are es-sential for the transport and hydrolysis of �-glucosides (15, 27).The regulatory sequences involved in transcription initiation arelocated within the region bglR upstream of the structural genes.The product of bglG, the first gene of the operon, functions as anantiterminator at two rho-independent terminators flanking it(14, 25). BglG is a sequence-specific RNA binding protein thatinteracts with a 37-nucleotide target sequence overlapping the ter-minator (10) known as the ribonucleic antiterminator (RAT) (3).The product of the second gene, BglF, is the bgl-specific compo-nent of the phosphotransferase system (PTS), which is also a neg-ative regulator of the operon. BglF phosphorylates BglG in theabsence of �-glucosides, leading to its inactivation (1, 26). Thus,the BglG-BglF combination that mediates induction of the bgloperon in response to the presence of �-glucosides resembles two-component signaling systems prevalent in bacteria. The thirdgene, bglB, encodes a phospho-�-glucosidase B that can hydrolyzethe phosphorylated forms of salicin and arbutin.

In spite of encoding a functional permease and a phospho-�-glucosidase, wild-type E. coli strains are unable to grow on salicinand arbutin because the bgl operon is transcriptionally silent as aresult of the presence of negative elements within bglR (11, 17, 24,31). Mutations that disrupt the negative elements activate theoperon, a major class of which consists of transposable elementssuch as IS1 and IS5 (21). The operon is also activated by mutationswithin the hns locus, encoding the histone-like nucleoid structur-ing protein H-NS (8).

Maintenance of the silent bgl operon over evolutionary time,without the accumulation of inactivating mutations within thestructural genes, has prompted the speculation that, apart fromfacilitating the utilization of �-glucosides, it is also involved inregulating an additional function(s) in the cell (18). This is con-sistent with the observations that Bgl-positive (Bgl�) strains inwhich the operon has been transcriptionally activated show agrowth advantage in stationary phase (GASP) phenotype over theparent Bgl-negative (Bgl�) strain (12) and the operon is expressedat elevated levels in the stationary phase (13). The precise mecha-nism by which the activated bgl operon confers a growth advan-tage remains unknown.

In an attempt to identify putative downstream target genesregulated by the bgl operon, proteomes of Bgl� and Bgl� culturesgrown for 24 h were compared by 2-dimensional (2D) polyacryl-amide gel electrophoresis (PAGE). A minimum of 12 genes werefound to be differentially regulated between Bgl� and Bgl� strains.In the present communication, we report the detailed analysis ofthe bgl-mediated regulation of oppA, which encodes a subunit ofan oligonucleotide-peptide transporter. Our studies indicate thatthe effect of the bgl operon on oppA is exerted via its posttranscrip-tional regulator, gcvB, which in turn is positively regulated bygcvA. We show that BglG decreases the half-life of the gcvA mRNAin the stationary phase, leading to elevated levels of oppA due tothe downregulation of gcvB. Possible fitness advantages gained asa result of oppA overexpression are discussed.

Received 20 July 2011 Accepted 7 October 2011

Published ahead of print 21 October 2011

Address correspondence to S. Mahadevan, mahi@mrdg.iisc.ernet.in.

* Present address: Department of Microbiology, Maharaja Ganga Singh University,Bikaner, India.

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

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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MATERIALS AND METHODSBacterial strains and plasmids. The E. coli strains and plasmids used inthis study are listed in Table 1.

Media and growth conditions. Strains were grown in Luria-Bertani(LB) liquid medium or LB agar (1.5%) (16) at 37°C. Salicin utilization wastested by growth on MacConkey indicator medium supplemented with1% salicin. Antibiotics were added at the following final concentrations:ampicillin, 100 �g ml�1; chloramphenicol, 15 �g ml�1; kanamycin, 20 �gml�1; rifampin, 200 �g ml�1; and tetracycline, 15 �g ml�1.

DNA manipulations. All standard DNA manipulations were car-ried out as described previously (22). Sequencing reactions were car-ried out at Macrogen, South Korea, using specific primers. Transduc-tions using P1 phage were performed as described previously (16).DNA and protein sequences were analyzed using the E. coli databaseECDC (www.uni-giessen.de/�gx1052/ECDC/ecdc.htm).

Preparation of protein extracts and 2D PAGE. One-day-old Luriabroth-grown cultures of strains ZK819-97T (Bgl�) and ZK819-Tn10(Bgl�) were resuspended in 200 ml lysis solution (7 M urea, 2 Mthiourea, 0.5% [wt/vol] 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate [CHAPS], 1% [vol/vol] Triton X-100) containing aprotease inhibitor cocktail (Sigma). The suspension was chilled on ice for30 min and subsequently subjected to three freeze-thaw cycles, followedby sonication on ice 5 times for 10 s each time at low output. Samples werecentrifuged for 30 min at 12,000 rpm at 4°C to collect the supernatant(protein extract). For isoelectric focusing (IEF), 100 �g of the proteinextract was added to the rehydration buffer (7 M urea, 2 M thiourea, 0.5%[wt/vol] CHAPS, 10 mM dithiothreitol [DTT], 0.5% [vol/vol] Pharma-lyte carrier ampholytes, 0.02% [wt/vol] bromophenol blue), and the mix-ture was incubated at room temperature for 60 min. Immobilized pH gradi-ent (IPG; GE Healthcare) strips (13 cm, pH 3 to 11) were rehydrated at 20°Cfor 16 h. Isoelectric focusing was performed at 20°C on EttanIPGphorII iso-electric focusing system (GE Healthcare Bio-Sciences). Before separation onthe second dimension, the gel strips were equilibrated for 15 min in SDSequilibration buffer (6 M urea, 50 mM Tris [pH 8.8], 30% [vol/vol] glyc-erol, 2% [wt/vol] SDS, 2% [wt/vol] DTT). Strips were then reequilibratedin SDS equilibration buffer (6 M urea, 50 mM Tris [pH 8.8], 30% [vol/vol]glycerol, 2% [wt/vol] SDS, 2.5% [wt/vol] iodoacetamide, 0.02% [wt/vol]bromophenol blue). Electrophoresis was performed using a precastpolyacrylamide gel (12%). Gels were stained with Coomassie blueR-250, and protein spots were compared visually. Protein spots thatdisplayed reproducible patterns in three replicates were selected forfurther identification.

Tryptic in-gel digestion and MALDI-TOF MS analysis. The proteinspots of interest were excised from the 2D gels, minced into �1-mm3

pieces, washed three times with 500 �l of a solution containing 50% (vol/vol) acetonitrile and 50 mM ammonium bicarbonate for 15 min withgentle agitation, and dehydrated in 100% acetonitrile for 5 min. Trypsinstock was prepared in 50 mM ammonium bicarbonate to a final concen-tration of 20 �g/100 �l. The gel pieces were rehydrated with 2 �l of thetrypsin solution and were incubated overnight at 37°C. The supernatantscontaining the extracted peptides were concentrated by centrifugal evap-oration to near dryness. The dried peptides were resuspended in 5 �l ofthe resuspension solution (50% [vol/vol] acetonitrile, 0.1% [vol/vol] tri-fluoroacetic acid) and analyzed by matrix-assisted laser desorption ion-ization–time of flight (MALDI-TOF) tandem mass spectrometry (MS/MS) (Ultraflex, BrukerDaltonics). The MS spectra were identified usingthe Mascot database (Matrix Science, United Kingdom), and hits withscores greater than 60 were considered significant.

Quantitative real-time reverse transcription-PCR (qRT-PCR). TotalRNA was isolated by following the acid phenol method as described pre-viously (31), qualitatively analyzed on a 1% (wt/vol) morpholinepropane-sulfonic acid-HCHO agarose gel, and quantified using a Bio-Rad spectro-photometer. Purified RNA (2 �g) was treated with DNase I (MBIFermentas) to remove genomic DNA and reverse transcribed using ran-dom hexamer primer and murine leukemia virus (MMLV) reverse trans-criptase (MBI Fermentas) as per the manufacturer’s protocol. The PCRproducts were analyzed on a 0.8% agarose gel. cDNA equivalent to 10 ngof total RNA was used for all the real-time PCRs. The following primerssequences were used to analyze various transcript levels by real-time-PCR:forward primer for oppA, 5=-CGA GCT CGG GAC CCA GCC TGG TAATAT C-3=; reverse primer for oppA, 5=-CTT AA C AAG GTC TTC GACTTC CTT AAG G-3=; forward primer for bglG, 5=-CCC ATA TGA TTAATT TCC GAA CCT GGA T-3=; reverse primer for bglG, 5=-CGG GATCCC CAG TAT TCT CTG GTT ATG T-3=; forward primer for gcvA,5=-CCC ATA TGG GAA AAA CTG TAC GCC GAA T-3=; reverse primerfor gcvA, 5=-CGG GAT CCC GTA TGT TTA GCC AGA TCT T-3=; for-ward primer for gcvB, 5=-CGA GCT CGA CTT CCT GAG CCG GAA CGAA-3=; reverse primer for gcvB, 5=-AAA AAG GTA GCT TTG CTA CCATGG TCT GA-3=. Each 20-�l reaction mixture contained Dynamo SYBRgreen mix (Finnzymes, Finland), carboxy-X-rhodamine reference, cDNAtemplate, and forward and reverse primers. Reactions were carried outusing an ABI Prism 7900HT sequence detection system (Applied Biosys-tems). Reaction tubes were incubated for 2 min at 50°C and then 10 minat 95°C, followed by 40 cycles of 15 s at 95°C, 30 s at 60°C, and 30 s at 72°C.

TABLE 1 Bacterial strains and plasmids used in the study

Strain or plasmid GenotypeReferenceor source

StrainsZK819 ZK126 (W3110�lacU169) rpoS819 Smr (Bgl�) 35ZK819-97T ZK819 tna::Tn10 bglR (Bgl�) (Tetr) 12ZK819-Tn5 ZK819 bglR0 (Bgl�) (Kanr) 12ZK819-Tn10 ZK819 bglR0 (Bgl�) (Tetr) 12ZK819-97T�oppA ZK819 tna::Tn10 bglR �oppA (Bgl�) This studyZK819-97T�bglG ZK819 tna::Tn10 bglR �bglG (Bgl�) This studyZK819-97T�gcvA ZK819 tna::Tn10 bglR �gcvA (Bgl�) This studyZK81997T�bglG�gcvA ZK819 tna::Tn10 bglR �bglG �gcvA (Bgl�) This studyZK819R�bglF ZK819 bglR::IS1 �bglF [BglG(Con) Kanr] This studyDH5� F= endA1 hsdR17 (rK

� mK�) supE44 thi-1 recA1 gyrA Nalr relA1 �(lacZYA argF)U169 deoF �80dlac �(lacZ)M15 33

MA200 F� �lacX174 thi bglR1(bglR::IS1) srl::Tn10 recA56 �dbglR7 bglG= lacZ lacY �(Bgl� lac) (Bgl�) 15MA200-1 MA200 bglF201 [BglG(Con)] 15

PlasmidspKD46 Ampr beta exo gam (lambda red) ori(Ts) 5pKD3 pANTS� derivative, Camr 5pCP20 flp bla cat rep101(Ts) Ampr Camr 4

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All reactions were performed in triplicate for two biological replicates. ART-minus control was used to test for the presence of residual DNA for allRNA samples analyzed. Thresholds were set manually in the middle of thelinear phase of the amplification curves. The fold change for the geneunder observation, relative to the calibrator, was determined by the2���CT (where CT is threshold cycle) method as described earlier (23)using rrnC (16S rRNA) for normalization. Analysis of data was carried outusing SDS (version 2.1) software (Applied Biosystems).

Construction of �oppA, �bglG, �gcvA, �bglG �gcvA, and �bglFmutants. Gene knockouts were carried out using the bacteriophage � redgam recombination method (5, 34). A linear cam cassette was prepared byPCR using hybrid primers for oppA (region spanning from positions�186 to �1732), bglG (region spanning from positions �25 to �1074),and gcvA (region spanning from positions �72 to �1019) and the tem-plate plasmid pKD3 (forward primer for oppA, 5=-CCG CAG GCG TCACAC TGG CGG AAA AAC AAA CAC TGG GTG TAG GCT GGA GCTGCT TCG-3=; reverse primer for oppA, 5=-CCA TTA GTG CTT CAC AAT

GTA CAT ATT CCG GGT ATA CAT ATG AAT ATC CTC CTT A-3=;forward primer for bglG, 5=-CCA TTA ATA AAT GAC TGG ATT GTTACT GCA TTC GCA GTG TAG GCT GGA GCT GCT TCG-3=; reverseprimer for bglG, 5=-CTT GCC CTC TAC CGC TTT GCG GCA AAA CTCCAA AAA CAT ATG AAT ATC CTC CTT A-3=; forward primer for gcvA,5=-ATG TCT AAA CGA TTA CCA CCG CTA AAT GCC TTA CGA GTTTTT GAT GTG TAG GCT GGA GCT GCT TCG-3=, reverse primer forgcvA, 5=-TTA TTG TTC ATA ACG AAA GCG GAA TTT TTC TTG TTCAGC AGC GGC CAT ATG AAT ATC CTC CTT A-3=). Hybrid primersfor bglG gene deletion were designed in such a way that the terminatorstructures flanking bglG (t1 and t2) were deleted. The �bglG �gcvA mu-tant was constructed by transducing the gcvA::kan allele from ZK819-97TgcvA::kan into ZK819-97T�bglG. The antibiotic resistance gene waseliminated by using the FLP plasmid pCP20 expressing the Flp recombi-nase. The bglF deletion was generated using the forward hybrid primer5=-GGG CGC AGA TAA CAT TGT GAG TCT GAT GCA TTG CGC GTGTAG GCT GGA GCT GCT TCG-3= and the reverse primer 5=-CGC TAT

FIG 1 Proteome analysis of whole-cell lysates of ZK819-Tn5 (Bgl� left) and ZK19-97T (Bgl� right) strains by 2D PAGE. Proteins were visualized by Coomassieblue R-250 stain, and differences between the Bgl� and Bgl� gels were visually analyzed. Black circles (circles 1 and 2), proteins significantly overexpressed in theBgl� proteome; gray circles (circles 3 to 12), proteins significantly overexpressed in the Bgl� proteome (n � 3).

TABLE 2 Proteins identified in Bgl� and Bgl� proteome

Proteinspot

Proteinidentified Function of identified protein

1a FkpA Heat shock peptidyl-prolyl isomerase (PPIase) having chaperone function2a SecD Membrane component of Sec protein secretion complex3 OppA Periplasmic component of oligopeptide transporter4 SdhA Succinate dehydrogenase5 ManX Mannose-specific (EIIAB) PTS permease involved in translocating exogenous hexoses including N-acetylglucosamine6 TrpS Tryptophanyl-tRNA synthetase7 SucD Succinyl coenzyme A synthetase, � subunit8 AdhE Alcohol dehydrogenase9 FabI Enoyl-acyl carrier protein reductase10 NusG Required for rho-dependent transcription termination as well as for antitermination during lambda phage transcription11 BetI Repressor of glycine betaine synthesis from choline12 YjgF Hypothetical proteina Protein overexpression picked up in Bgl� proteome.

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TAC TGA TTA ATA CCG GCG TCG TCA GAT CAA CAT ATG AATATC CTC CTT A-3= in the ZK819 parent carrying a bgl operon activatedby an IS1 insertion in bglR.

Competition assays. Cultures were grown in Luria-Bertani broth (HiMedia) with aeration in a New Brunswick shaker at 37°C. Mixed cultureswere started after individual cultures were grown for 24 h, and appropri-ate volumes of the two stationary-phase cultures were mixed in a freshtube with a total final volume of 3 ml without addition of fresh medium.A reciprocal cell ratio of 1:1,000 was used between the competing cultures.Long-term cultures were replenished with sterile distilled water when nec-essary to compensate for evaporation. The titers of the cultures at differentpoints of time were determined by counting the numbers of CFU ml�1 onLB agar plates containing appropriate antibiotics.

Measurement of mRNA half-life. To determine the half-life of thegcvA transcript, levels of mRNA remaining were determined at differenttime points after transcription arrest by qRT-PCR. Strains MA200 andMA200-1 were grown to stationary phase (24 h), and transcription initi-ation was arrested using rifampin at a final concentration of 200 �g ml�1.Total RNA was isolated from shaking cultures at intervals of 1.5 min afterthe addition of rifampin and treated with DNase I to remove genomicDNA. Two micrograms of RNA was reverse transcribed using randomhexamer primer and MMLV reverse transcriptase. cDNA equivalent to 10ng of total RNA was used for all the real-time PCRs. Spectrophotometricmeasurements (Bio-Rad) at 260 nm were used to assess the concentrationof cDNA. A standard curve was prepared by plotting known concentra-tions of gcvA and rrnC cDNAs (input amount, reverse transcribed from

FIG 2 Steady-state expression of oppA mRNA in Bgl� (ZK819-Tn5) and Bgl� (ZK19-97T) strains under exponential and stationary phase. The y axis representsthe fold change of oppA gene expression relative to the Bgl� (ZK819-Tn5) calibrator measured by qRT-PCR (n � 2). The rrnC (16S rRNA) gene was used as acontrol for normalization. See Materials and Methods for details.

FIG 3 Competition between stationary-phase-grown (24 h in LB medium) cultures of ZK819-Tn5 (Bgl�) (□) versus ZK819-97T (Bgl�) (�) strains in a 1,000:1ratio (A) and a 1:1,000 ratio (B). ZK819-Tn5 (Bgl�) (�) versus ZK819-97T�oppA (Bgl�) (�) strains in a 1,000:1 ratio (C) and 1:1,000 ratio (D). Viable counts(numbers of CFU ml�1) were monitored for the days indicated on the x axis (n � 2). The mixed cultures were maintained in the original LB medium withoutaddition of fresh medium.

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the RNA isolated from MA200 and MA200-1 strains) as the x values andCT as the y values (see Fig. S3 in the supplemental material). RNA con-centrations of the experimental samples were extrapolated from the stan-dard curves. The percentages of mRNA remaining at each time intervalafter rifampin treatment were calculated relative to the signal obtained at0 min and were plotted on the y axis versus time on the x axis. The timepoint at which 50% of the gcvA mRNA has been degraded was extrapo-lated from the decay plot to determine the half-life.

RESULTSIdentification of downstream target genes regulated by bgloperon. Total proteins from strains ZK819-97T (Bgl�) andZK819-Tn5 (Bgl�) grown to stationary phase (24 h) were ana-lyzed by 2D PAGE to identify putative candidates whose expres-sion is regulated by the bgl operon. Comparison of the proteomeprofiles indicated the presence of a minimum of 10 proteins inZK819-97T (Bgl�) and 2 proteins in ZK819-Tn5 (Bgl�) that wereoverexpressed (Fig. 1). The overexpressed protein spots wereidentified by MALDI-TOF MS. The peptide mass fingerprintsmatched those of 12 known proteins (Table 2). Among these,OppA, an oligonucleotide-peptide transporter that showed con-sistent overexpression in the Bgl� strain, was selected for furtherinvestigations. The overexpression at the protein level was vali-dated by examining the transcript levels of oppA.

Steady-state level of oppA mRNA is higher in Bgl� cells. Thesteady-state levels of oppA mRNA from Bgl� and Bgl� strains atexponential phase (4 h) and stationary phase (24 h) were com-pared by qRT-PCR. These studies showed that there is a marginaldecrease of oppA gene expression in the Bgl� strain grown for 4 hcompared to its wild-type Bgl� parent. However, expression of the

oppA gene was about 25-fold higher in the Bgl� cells grown for 24h (Fig. 2). Therefore, the enhanced level of OppA seen in theproteome of the Bgl� strains is also reflected in the steady-statetranscript level.

FIG 4 Steady-state expression of gcvB (A) and gcvA (B) transcripts in Bgl� (ZK819-Tn5) and Bgl� (ZK19-97T) strains under exponential and stationary phase.The y axis represents the fold change of gcvA/gcvB gene expression relative to the Bgl� (ZK819-Tn5) calibrator measured by qRT-PCR (n � 2). The rrnC (16SrRNA) gene was used as a control for normalization. See Materials and Methods for details.

FIG 5 qRT-PCR analysis of the half-life of gcvA mRNA in MA200 (BglG�)and MA200-1 [BglG(Con)] strains after inhibiting transcription initiationwith rifampin (200 �g ml�1). The percentage of bgl mRNA remaining at eachtime interval was calculated from the standard curve prepared for knowncDNA concentrations, as described in Materials and Methods. The signal ob-tained at 0 min was considered 100%, and the percentage of mRNA remainingat each time point was plotted on the y axis versus time on the x axis. The timepoint at which 50% of the gcvA mRNA has been degraded was calculated fromthe decay plot to determine the half-life (t1/2).

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Loss of oppA function leads to partial loss of GASP pheno-type of Bgl� cells. Bgl� cells exhibit a fitness advantage in thestationary phase when competed against the Bgl� parent in mixedculture experiments (12) (Fig. 3). Since oppA has been shown to beupregulated in the Bgl� strain, it is conceivable that the functionsencoded by oppA contribute to the GASP phenotype exhibited byBgl� strains. To test this possibility, the oppA locus was deleted inBgl� strain ZK819-97T and the �oppA mutant was competedagainst its wild-type parent, ZK819-Tn5 (Bgl�), in a cocultureexperiment. Data from competition assays indicated that ZK819-97T�oppA cells have lost the strong fitness advantage shown byparent strain ZK819-97T (Fig. 3). The decrease in the GASP phe-

notype in the �oppA strain suggests that a part of the growthadvantage of the Bgl� strain is contributed by OppA.

RAT-like motif is present within the protein-coding se-quence of gcvA. One possible way by which the bgl operon couldexert regulation on a downstream target gene is via its regulators,BglG and BglF. BglG binds to the RAT element located at positions�40 to �77 within the 5= leader region of the bgl mRNA, where itacts as an antiterminator of transcription, enabling the expressionof the bgl structural genes (10). Examination of the oppA DNAsequence did not reveal the presence of a RAT-like motif withinthe gene, suggesting that the regulation of oppA by the bgl operonis likely to be indirect.

FIG 6 Competition between stationary-phase-grown ZK819-97 T (Bgl�) (�) and ZK819R�bglF [BglG(Con) Bgl�] (□). Culture conditions were similar tothose described in the legend to Fig. 3 (n � 3).

FIG 7 Competition between stationary-phase-grown ZK819-Tn5 (Bgl�) (□) and ZK819-97T�bglG (Bgl�) (�) strains in a 1,000:1 ratio (A) and a 1:1,000 ratio(B). ZK819-Tn5 (Bgl�) (�) versus ZK819-97T�bglG�gcvA (Bgl�) (�) strains in a 1,000:1 ratio (C) and a 1:1,000 ratio (D). Viable counts (numbers of CFUml�1) were monitored for the days indicated on the x axis (n � 2).

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Expression of oppA has been shown to be negatively regulatedat the posttranscriptional level by the small RNA (sRNA) gcvB(28), which in turn is transcriptionally activated by the product ofthe gcvA gene, involved in the regulation of the glycine cleavagesystem (32). Measurements of the steady-state transcript levels ofgcvB and its positive regulator, gcvA, by qRT-PCR showed amarked reduction in the stationary phase in the Bgl� strain (Fig.4A and B). The pronounced reduction in the levels of gcvB andgcvA transcripts in the presence of higher levels of bglG in the Bgl�

strain (see Fig. S1 in the supplemental material) suggests that BglGis involved in their regulation. This is consistent with the observa-tion that gcvA carries a RAT-like sequence within the protein-coding region (480 bp downstream of position �1) that has 46%sequence identity to the bgl-specific RAT sequence (see Fig. S2 inthe supplemental material).

Stability of gcvA mRNA is reduced in presence of functionalBglG. Apart from functioning as an antiterminator, BglG has beenshown to stabilize the bgl leader transcript upstream of the termi-nator (6). To examine whether BglG has any effect on the stabilityof the gcvA transcript, the levels of gcvA mRNA remaining at dif-ferent time points after blocking transcription initiation in two

isogenic strains that express different levels of bglG were deter-mined by qRT-PCR (Fig. 5). The half-life of the gcvA transcriptwas 9.7 min in Bgl� strain MA200. In contrast, the half-life of gcvAmRNA was 4.3 min in strain MA200-1, in which the steady-statebglG transcript levels are substantially higher as a result of a mu-tation in the negative regulator bglF (see Fig. S3 in the supplemen-tal material). Enhanced levels of bglG therefore lead to a significantreduction in the half-life of the gcvA transcript.

The comparison of the stability of the gcvA mRNA was madeusing the two strains MA200 (Bgl�) and MA200-1 (bglF) essen-tially to amplify the effect of bglG. To determine if the elevatedlevels of bglG in the bglF strain confer a growth advantage in sta-tionary phase, strain ZK819R�bglF, carrying a deletion of bglF,was competed against Bgl� strain ZK819-97T. These studiesshowed that the �bglF (Bgl�) strain had a distinct growth advan-tage over the Bgl� strain (Fig. 6), indicating that the GASP phe-notype is correlated with the higher levels of bglG.

BglG is necessary for the GASP phenotype of Bgl� strains.The results described above show a direct correlation betweenhigher levels of bglG and the downregulation of gcvA. If BglG isupstream of OppA in conferring the GASP phenotype to Bgl� cells

FIG 8 Steady-state expression of gcvA (A), gcvB (B), and oppA (C) transcripts in Bgl� (ZK819-Tn5) and Bgl� (ZK19-97T�bglG) strains under exponential- andstationary-phase growth conditions. The y axis represents the fold change of gcvA/gcvB/oppA gene expression relative to the Bgl� (ZK819-Tn5) calibratormeasured by qRT-PCR (n � 2). The rrnC (16S rRNA) gene was used as a control for normalization. See Materials and Methods for details.

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via regulating the expression of gcvA, loss of BglG should result inthe loss of the GASP phenotype. Deletion of bglG in strain ZK819-97T resulted in the complete loss of the GASP phenotype of thestrain when competed against the wild-type parent, ZK819-Tn5(Fig. 7A and B).

Loss of BglG function results in upregulation of gcvA. If BglGdestabilizes the gcvA transcript, gcvA levels are expected to in-crease in a bglG mutant. The loss of BglG function resulted in theupregulation of gcvA and gcvB expression and a concomitantdownregulation of the steady-state levels of oppA, as detected byqRT-PCR (Fig. 8). The upregulation of gcvA in the �bglG strain isconsistent with the proposed role of BglG as a negative regulator ofgcvA. The loss of the GASP phenotype in the �bglG strain may bepartly correlated to the decrease in the expression of OppA, due toelevated levels of its negative regulators, gcvA and gcvB.

Deletion of gcvA partly rescues loss of the GASP phenotypein �bglG strains. As gcvA is known to mediate negative regula-tion of oppA via gcvB, loss of gcvA function is expected to resultin an increase in oppA gene expression. Real-time PCR mea-surements of mRNA levels in a strain carrying a deletion ofgcvA showed reduced levels of gcvB and elevated levels of oppAcompared to the wild-type strain (Fig. 9). Interestingly, dele-tion of gcvA could also rescue the loss of oppA expression seenin a �bglG background. Measurements of oppA mRNA in a�gcvA �bglG double mutant showed elevated levels even in theabsence of BglG (Fig. 10). More importantly, the loss of theGASP phenotype seen in the �bglG strain could be partly res-cued when gcvA is deleted (Fig. 7C and D).

DISCUSSION

Retention of silent genetic systems such as the bgl operon of E. coli,without the loss of function of the structural genes due to theaccumulation of inactivating mutations, is an enigma. In this con-text, the following two observations are significant. First, strainsthat carry an activated bgl operon can outcompete the isogenicwild-type strain in competition experiments (12) (Fig. 3), evenwhen �-glucosides are not supplemented in the medium. Second,transcription from the wild-type bgl promoter is enhanced in thestationary phase even in the absence of activating mutations (13),though this increase is insufficient to allow growth on�-glucosides. These observations suggest the possibility that thebgl operon exerts a regulatory effect on downstream target genesother than those implicated in �-glucoside catabolism, expressionof which provides a fitness advantage in the stationary phase. Themajority of upregulated proteins identified in the Bgl� proteomeeither are known to participate in transport functions or are en-zymes involved in cellular metabolism. As a result, Bgl� cells arelikely to be better equipped than Bgl� cells to scavenge availablenutrient substrates by activating additional metabolic functions.

Among the 10 candidate genes overexpressed in the Bgl� pro-teome, oppA was selected to study the role of the bgl operon indownstream regulation. OppA (60.9 kDa) is an oligonucleotide-peptide transporter subunit encoded by the oppABCDF operonand is a member of the ATP-binding cassette (ABC) superfamilyof transporters (19). The system has been shown to be involved infunctions related to oligonucleotide-peptide uptake and the recy-

FIG 9 Steady-state expression of gcvB (A) and oppA (B) transcripts in Bgl� (ZK819-Tn5) and Bgl� (ZK19-97T�gcvA) strains under exponential- and stationary-phase growth conditions. The y axis represents the fold change of gcvB/oppA gene expression relative to the Bgl� (ZK819-Tn5) calibrator measured by qRT-PCR(n � 2). The rrnC (16S rRNA) gene was used as a control for normalization. See Materials and Methods for details.

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cling of cell wall peptides (9). The upregulation of OppA in theBgl� proteome was further validated by qRT-PCR, which showedsignificantly high steady-state transcript levels in the stationaryphase in Bgl� cells compared to its Bgl� parent.

OppA, which is known to assist in the transport of small pep-tides up to 5 amino acids in length (7), could be one of the factorsthat contribute toward the GASP phenotype of Bgl� strains byenabling the uptake of potential nutrients from the environment.The partial loss of the GASP phenotype observed in the competi-tion assay between Bgl� (�oppA) and Bgl� strains is consistentwith this possibility. The GASP phenotype of Bgl� strains is de-pendent on the presence of the rpoS819 allele, which results in theattenuated expression of RpoS (12). The presence of the rpoS819allele has also been shown to enable faster growth in the presenceof certain amino acids (36). Expression from the oppA promoter isupregulated in the absence of RpoS (29). Therefore, the GASPphenotype conferred by oppA is likely to be the combined effect ofincreased transcription from the oppA promoter due to the pres-ence of the rpoS819 allele and the involvement of the activated bgloperon in its expression.

The involvement of the bgl operon in the regulation of oppAexpression could be direct or indirect. OppA is regulated nega-tively by a small regulatory RNA (sRNA), gcvB (2), which has beenshown to inhibit translation initiation by binding to the oppAmRNA (28). In turn, the transcription of gcvB is positively regu-lated by the GcvA protein, the major transcription factor of theglycine cleavage system (32). Expression of gcvB is high duringearly log phase, but its level decreases during cell growth (2). Thisreduction in gcvB expression was much more pronounced in Bgl�

cells. Similarly, a significant decrease in gcvA transcription in Bgl�

cells was also registered in the stationary phase. These observa-tions suggest that the regulation of oppA by the bgl operon is via itsregulators, gcvA and gcvB.

The search for a possible mechanism by which the bgl operonexerts a regulatory effect on oppA led to the identification of aRAT-like motif present within the protein-coding region of thegcvA gene. Since the gcvARAT motif present within the gcvA openreading frame is not associated with a transcription terminatorstructure, BglG is unlikely to function as an antiterminator oftranscription in this case. BglG could alter the translation of gcvAby acting as a roadblock for the movement of ribosomes. Alterna-tively, BglG could destabilize the gcvA transcript, which could neg-atively impact GcvA expression. This is supported by the observa-tion that the half-life of the gcvA transcript is reduced in a strain inwhich bglG expression is high. Enhanced expression of oppA inBgl� cells is lost when bglG is deleted, indicating a positive role forBglG in its expression. The requirement for bglG for oppA expres-sion can be overcome by deletion of gcvA, consistent with theassumption that gcvA is the target of bglG-mediated regulation ofoppA.

Our earlier work had shown that bglB, which encodes the hy-drolytic enzyme phospho-�-glucosidase B, is involved in theGASP phenotype, as a deletion of bglB led to the loss of the GASPphenotype (12). The results presented in this communication in-dicate that bglG is epistatic over bglB, as a deletion of bglG retain-ing bglF and bglB function results in the loss of the GASP pheno-type. These studies also show that the relative levels of bglG areimportant, as a �bglF (Bgl�) strain expressing bglG constitutivelycan outcompete a Bgl� strain, just as a Bgl� strain can outcompetea Bgl� strain.

Based on the observations described above, we propose thatthe ability to transport oligonucleotide-peptides mediated by theoverexpression of oppA is partly responsible for the GASP pheno-type of Bgl� strains, consistent with the observation that the�oppA strain shows a partial loss of the GASP phenotype. Down-regulation of oppA in a strain carrying a deletion of bglG may be

FIG 10 Steady-state expression of gcvB (A) and oppA (B) transcripts in Bgl� (ZK819-Tn5) and Bgl� (ZK19-97T�bglG�gcvA) strains under exponential- andstationary-phase growth conditions. The y axis represents the fold change of gcvB/oppA gene expression relative to the Bgl� (ZK819-Tn5) calibrator measuredby qRT-PCR (n � 2). The rrnC (16S rRNA) gene was used as a control for normalization. See Materials and Methods for details.

Harwani et al.

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one of the reasons for the loss of the GASP phenotype of the �bglGstrain. The complete loss of the GASP phenotype in the �bglGmutant and its partial rescue in the �bglG �gcvA double mutantsuggest that BglG is a master regulator involved in modulating theexpression of downstream genes important in stationary-phasesurvival, and oppA is one such locus. The validation of the addi-tional loci detected in the Bgl� proteome is expected to provide amore complete picture of the involvement of the bgl operon instationary-phase survival.

The involvement of the bgl operon in functions unrelated to thecatabolism of �-glucosides suggests that selection for elevated expres-sion of the operon can occur even in the absence of �-glucosides. Thiscould be achieved either by mutations or by overriding its negativeregulation under specific growth conditions such as stationary phase.Our earlier work has shown enhancement of bgl expression in sta-tionary phase (13). Though such elevated expression may not be suf-ficient to allow utilization of �-glucosides, it may be sufficient for theregulation of the downstream target genes. If this requirement is for aprolonged period of time, there is likely to be selection for mutationalactivation of the operon under these conditions. Involvement of thebgl operon in the regulation of additional functions provides a selec-tive force for the maintenance of the bgl genes over evolutionary time.

ACKNOWLEDGMENTS

We are thankful to D. Chatterji and Manish Kumar for their help withtwo-dimensional electrophoresis and Imran Khan for help with real-timePCR experiments.

The proteomic analysis was carried out at the institutional facilitysupported by the Department of Biotechnology (DBT), Government ofIndia. D.H. is thankful to DBT for a postdoctoral research fellowship. Thiswork was supported by a grant to S.M. from the Department of Scienceand Technology (DST), Government of India.

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