1

The β-glucoside (bgl) Operon of Escherichia coli is Involved in the

Regulation of oppA Encoding an Oligo-peptide Transporter

Dharmesh Harwani#, Parisa Zangoui, and S. Mahadevan*

Department of Molecular Reproduction, Development and Genetics,

Indian Institute of Science, Bangalore 560 012, INDIA

*Corresponding author Address for correspondence: Department of Molecular Reproduction, Development, and Genetics, Indian Institute of Science,

Bangalore 560 012, INDIA

#Present address: Department of Microbiology, Maharaja Ganga Singh University, Bikaner 334 001, INDIA

E-mail: mahi@mrdg.iisc.ernet.in Tel: +91 80 2293 2607 Fax: +91 80 2360 0999 Keywords: β-glucosides, post-transcriptional regulation, mRNA stability, BglG, oppA, gcvA

Running title: The bgl operon of E. coli is involved in the regulation of oppA Abbreviations: GASP (Growth Advantage in Stationary Phase), RAT (Ribonucleic

Antiterminator Sequence), 2DE (Two Dimensional Gel Electrophoresis), MALDI-TOF-MS

(Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry)

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.05837-11 JB Accepts, published online ahead of print on 21 October 2011

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We report that the bgl operon of E. coli, encoding the functions necessary for the uptake and 1

metabolism of aryl-β-glucosides, is involved in the regulation of oligo-peptide transport 2

during stationary phase. Global analysis of intracellular proteins from Bgl+ and Bgl- strains 3

revealed that the operon exerts regulation on at least twelve downstream target genes. Of 4

these, oppA, which encodes an oligo-peptide transporter, was confirmed to be up-regulated 5

in the Bgl+ strain. Loss of oppA function results in a partial loss of the Growth Advantage in 6

Stationary Phase (GASP) phenotype of Bgl+ cells. The regulatory effect of the bgl operon on 7

oppA expression is indirect and is mediated via gcvA, the activator of the glycine cleavage 8

system, and gcvB that regulates oppA at the translational level. We show that BglG 9

destabilizes the gcvA mRNA in vivo leading to reduced expression of gcvA in the stationary 10

phase. Deletion of gcvA results in the down-regulation of gcvB and up-regulation of oppA 11

and can partially rescue the loss of the GASP phenotype seen in ΔbglG strains. A possible 12

mechanism by which oppA confers a competitive advantage to Bgl+ cells relative to Bgl- cells 13

is discussed. 14

In natural environments bacteria live in close associations, most of the times under nutrient 15

scarcity. This in turn leads to competition within populations for the limited resources that are 16

available. Bacteria have evolved distinct mechanisms to extract utilizable substrates from 17

available resources and consequently acquire fitness advantage over competitors. One of the 18

strategies is the exploitation of cryptic cellular functions encoded by genetic systems that are silent 19

under laboratory conditions, such as the bgl (β-glucoside) operon of E. coli involved in the uptake 20

and metabolism of the plant derived aromatic β-glucosides salicin and arbutin (21). 21

The three genes bglG, bglF, and bglB of the bgl operon are essential for the transport and 22

hydrolysis of β-glucosides (16, 27). The regulatory sequences involved in transcription initiation 23

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are located within the region bglR upstream of the structural genes. The product of bglG, the first 24

gene of the operon, functions as an anti-terminator at two rho-independent terminators flanking it 25

(15, 25). BglG is a sequence-specific RNA binding protein that interacts with a 37-nucleotide 26

target sequence overlapping the terminator (10) known as the ribonucleic antiterminator (RAT) 27

(3). The product of the second gene, BglF, is the bgl-specific component of the phospho-28

transferase system (PTS), which is also a negative regulator of the operon. BglF phosphorylates 29

BglG in the absence of β-glucosides, leading to its inactivation (1, 26). Thus the BglG-BglF 30

combine that mediates induction of the bgl opern in response to the presence of β-glucosides, 31

resembles two-component signalling systems prevalent in bacteria. The third gene bglB encodes a 32

phospho-β-glucosidase B that can hydrolyze the phosphorylated forms of salicin and arbutin. 33

In spite of encoding a functional permease and a phospho-β-glucosidase, wild-type E. coli 34

strains are unable to grow on salicin and arbutin because the bgl operon is transcriptionally silent 35

as a result of the presence of negative elements within bglR (12, 24, 31, 18). Mutations that disrupt 36

the negative elements activate the operon, a major class of which consists of transposable elements 37

such as IS1 and IS5 (22). The operon is also activated by mutations within the hns locus encoding 38

the histone-like nucleoid structuring protein H-NS (8). 39

Maintenance of the silent bgl operon over evolutionary time, without the accumulation of 40

inactivating mutations within the structural genes, has prompted the speculation that, apart from 41

facilitating the utilization of β-glucosides, it is also involved in regulating additional function/s in 42

the cell (19). This is consistent with the observations that Bgl+ strains in which the operon has 43

been transcriptionally activated show a Growth Advantage in Stationary Phase (GASP) phenotype 44

over the parent Bgl- strain (13) and the operon is expressed at elevated levels in the stationary 45

phase (14). The precise mechanism by which the activated bgl operon confers a growth advantage 46

remains unknown. 47

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In an attempt to identify putative downstream target genes regulated by the bgl operon, 48

proteomes of Bgl+ and Bgl- cultures grown for 24hours were compared by 2-Dimensional 49

Polyacrylamide Gel Electrophoresis (2D-PAGE). A minimum of twelve genes were found to be 50

differentially regulated between Bgl+ and Bgl- strains. In the present communication we report the 51

detailed analysis of the bgl-mediated regulation of oppA that encodes a subunit of an oligo-peptide 52

transporter. Our studies indicate that the effect of the bgl operon on oppA is exerted via its 53

translational regulator gcvB, which in turn is positively regulated by gcvA. We show that BglG 54

decreases the half life of the gcvA mRNA in the stationary phase, leading to elevated levels of 55

oppA due to the down-regulation of gcvB. Possible fitness advantages gained as a result of oppA 56

over-expression are discussed. 57

Materials and Methods 58

Bacterial strains and plasmids 59

The E. coli strains and plasmids used in this study are listed in Table 1 and 2. 60

Media and growth conditions 61

Strains were grown in Luria-Bertani (LB) liquid medium or LB agar (1.5%) at 37°C. Salicin 62

utilization was tested by growth on MacConkey indicator medium supplemented with 1% salicin. 63

Antibiotics were added at the following final concentrations: Ampicillin 100μg ml-1, 64

Chloramphenicol 15μg ml-1, Kanamycin 20μg ml-1, Rifampicin 200μg ml-1, and Tetracycline 15μg 65

ml-1. 66

DNA manipulations 67

All standard DNA manipulations were carried out as described previously (28). Sequencing 68

reactions were carried out at Macrogen, South Korea, using specific primers. Transductions using 69

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P1 phage were performed as described previously (17). DNA and protein sequences were 70

analyzed using the E. coli database ECDC (www.uni-giessen.de/~gx1052/ECDC/ecdc.htm). 71

Preparation of protein extracts, 1DE and 2D-PAGE 72

One day old Luria Broth grown cultures of ZK819-97T (Bgl+) and ZK819-Tn10 (Bgl-) strains 73

were re-suspended in 200ml lysis solution (7M urea, 2M thiourea, 0.5% w/v CHAPS, 1% v/v 74

triton X) containing a protease inhibitor cocktail (Sigma). The suspension was chilled on ice for 75

30 min and subsequently subjected to three freeze-thaw cycles followed by sonication on ice for 76

10s x 5 at low output. Samples were centrifuged for 30minutes at 12,000rpm at 4oC to collect the 77

supernatant (protein extract). For isoelectric focusing (IEF), 100µg of the protein extract was 78

added to the rehydration buffer (7M urea, 2M thiourea, 0.5% w/v CHAPS, 10mM DTT, 0.5% v/v 79

pharmalytes, 0.02% w/v bromophenol blue) and incubated at room temperature for 60minutes. 80

Immobilized pH gradient (IPG, GE Healthcare) strips (13 cm, pH 3-11) were rehydrated at 20oC 81

for 16hours. Isoelectric focusing was performed at 20oC on EttanIPGphorII (GE Healthcare Bio-82

Sciences). Before separation on the second dimension, the gel strips were equilibrated for 15 83

minutes in SDS equilibration buffer (6M urea, 50mM Tris (pH 8.8), 30% v/v glycerol, 2% w/v 84

SDS, 2% w/v DTT). Strips were then re-equilibrated in SDS equilibration buffer (6M urea, 50mM 85

Tris (pH 8.8), 30% v/v glycerol, 2% w/v SDS, 2.5% w/v iodoacetamide, 0.02% w/v bromophenol 86

blue). Electrophoresis was performed using pre-cast polyacrylamide gel (12%). Gels were 87

stained with Coomassie Blue R-250 and protein spots were compared visually. Protein spots that 88

displayed reproducible patterns in three replicates were selected for further identification. 89

Tryptic in-gel digestion and MALDI-TOF-MS analysis 90

The protein spots of interest were excised from the 2D gels, minced into ~1 mm3 pieces, washed 91

three times with 500µl of a solution containing 50% v/v acetonitrile and 50mM amonium 92

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bicarbonate for 15 minutes with gentle agitation, and dehydrated in 100% acetonitrile for 93

5minutes. Trypsin stock was prepared in 50mM ammonium bicarbonate to a final concentration 94

of 20µg/100µl. The gel pieces were rehydrated with 2µl of the trypsin solution and were incubated 95

overnight at 37oC. The supernatants containing the extracted peptides were concentrated by 96

centrifugal evaporation to near dryness. The dried peptides were resuspended in 5μl of the re-97

suspension solution (50% v/v acetonitrile, 0.1% v/v TFA) and analyzed by MALDI-MS-MS 98

(Ultraflex, BrukerDaltonics). The MS spectra were identified using the Mascot database (Matrix 99

Science, UK) and hits with scores greater than 60 were considered significant. 100

Quantitative Real-Time Reverse Transcription-PCR (qRT-PCR) 101

Total RNA was isolated by following the acid phenol method as described previously (31), 102

qualitatively analyzed on 1% w/v MOPS-HCHO agarose gel, and quantified using a BIORAD 103

spectrophotometer. Purified RNA (2µg) was treated with DNaseI (MBI Fermentas) to remove 104

genomic DNA, reverse transcribed using random hexamer primer and MMuLv reverse 105

transcriptase (MBI Fermentas) as per the manufacturer’s protocol. The PCR products were 106

analyzed on 0.8% agarose gel. Complementary DNA (cDNA) equivalent to 10ng of total RNA 107

was used for all the real-time polymerase chain reactions (RT-PCR). The following primers 108

sequences were used to analyze various transcript levels by RT-PCR (forward primer for oppA: 109

5’- cga gct cgg gac cca gcc tgg taa tat c -3’; reverse primer for oppA: 5’- ctt aa c aag gtc ttc 110

gac ttc ctt aag g -3’;forward primer for bglG: 5’- ccc ata tga tta att tcc gaa cct gga t -3’; reverse 111

primer for bglG: 5’- cgg gat ccc cag tat tct ctg gtt atg t -3’; forward primer for gcvA: 5’- ccc ata 112

tgg gaa aaa ctg tac gcc gaa t -3’; reverse primer for gcvA: 5’- cgg gat ccc gta tgt tta gcc aga tct t-113

3’; forward primer for gcvB: 5’- cga gct cga ctt cct gag ccg gaa cga a-3’; reverse primer for gcvB: 114

5’-aaa aag gta gct ttg cta cca tgg tct ga-3’). Each 20μl reaction contained Dynamo SYBR Green 115

mix (Finnzymes, Finland), ROX reference, cDNA template, forward and reverse primers. 116

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Reactions were carried out using an ABI Prism 7900HT sequence detection system (Applied 117

Biosystems, USA). Reaction tubes were incubated for 2 minutes at 50°C and then 10 minutes at 118

95°C followed by 40 cycles of 15s at 95°C, 30s at 60°C and 30s at 72°C. All reactions were 119

performed in triplicates for two biological replicates. An RT-minus control was used to test for the 120

presence of residual DNA for all RNA samples analyzed. Thresholds were set manually in the 121

middle of the linear phase of the amplification curves. The fold change for the gene under 122

observation, relative to the calibrator, was determined by the 2-∆∆Ct method as described earlier 123

(16) using rrnC (16S-RNA) for normalization. Analysis of data was carried out using SDS 2.1 124

software (Applied Biosystems, USA). 125

Construction of the ∆oppA, ∆bglG, ∆gcvA, ∆bglG∆gcvA and ΔbglF mutants 126

Gene knock outs were carried out using the λ red gam recombination method (8, 40). Linear cam 127

cassette was prepared by PCR using hybrid primers for oppA (region spanning from +186 to 128

+1732), bglG (region spanning from +25 to +1074), gcvA (region spanning from +72 to +1019) 129

and the template plasmid pKD3 (forward primer for oppA: 5’- ccg cag gcg tca cac tgg cgg aaa aac 130

aaa cac tgg gtg tag gct gga gct gct tcg - 3’; reverse primer for oppA: 5’- cca tta gtg ctt cac aat gta 131

cat att ccg ggt ata cat atg aat atc ctc ctt a - 3’; forward primer for bglG: 5’- cca tta ata aat gac tgg 132

att gtt act gca ttc gca gtg tag gct gga gct gct tcg - 3’; reverse primer for bglG: 5’- ctt gcc ctc tac 133

cgc ttt gcg gca aaa ctc caa aaa cat atg aat atc ctc ctt a - 3’; forward primer for gcvA: 5’- atg tct aaa 134

cga tta cca ccg cta aat gcc tta cga gtt ttt gat gtg tag gct gga gct gct tcg -3’, reverse primer for gcvA: 135

5’-tta ttg ttc ata acg aaa gcg gaa ttt ttc ttg ttc agc agc ggc cat atg aat atc ctc ctt a - 3’). Hybrid 136

primers for bglG gene deletion were designed in such a way that the terminator structures flanking 137

bglG (t1 and t2) were deleted. The ∆bglG∆gcvA mutant was constructed by transducing the 138

gcvA::kan allele from ZK819-97TgcvA::kan into ZK819-97T∆bglG. The antibiotic resistance 139

gene was eliminated by using the FLP plasmid pCP20 expressing the Flp recombinase. The bglF 140

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deletion was generated using the forward hybrid primer 5’ ggg cgc aga taa cat tgt gag tct gat gca 141

ttg cgc gtg tag gct gga gct gct tcg 3’ and the reverse primer 5’ cgc tat tac tga tta ata ccg gcg tcg tca 142

gat caa cat atg aat atc ctc ctt a 3’ in the ZK 819 parent carrying a bgl operon activated by an IS1 143

insertion in bglR. 144

Competition Assays 145

Cultures were grown in Luria-Bertani broth (Hi Media) with aeration in a New Brunswick shaker 146

at 37°C. Mixed cultures were started after growing individual cultures for 24 hours and mixing 147

appropriate volumes of the two stationary phase cultures in a fresh tube with a total final volume 148

of 3 ml without addition of fresh medium. A reciprocal cell ratio of 1:1000 was used between the 149

competing cultures. Long-term cultures were replenished with sterile distilled water when 150

necessary to compensate for evaporation. The titres of the cultures at different points of time were 151

determined by counting colony forming units (CFU) ml-1 on LB agar plates containing appropriate 152

antibiotics. 153

Measurement of mRNA half life 154

To determine the half-life of the gcvA transcript, levels of mRNA remaining were determined at 155

different time points after transcription arrest by quantitative RT-PCR. The strains MA200 and 156

MA200-1 were grown to stationary phase (24hours) and transcription initiation was arrested using 157

rifampicin at a final concentration of 200µg ml-1. Total RNA was isolated from shaking cultures at 158

intervals of 1.5 minutes after the addition of rifampicin and treated with DNaseI to remove 159

genomic DNA. 2μg of RNA was reverse transcribed using random hexamer primer and MMuLv 160

reverse transcriptase. Complementary DNA (cDNA) equivalent to 10ng of total RNA was used for 161

all the real-time PCR (RT-PCR) reactions. Spectrophotometric measurements (BIORAD) at 162

260nm were used to assess the concentration of cDNA. A standard curve was prepared by plotting 163

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known concentrations of gcvA and rrnC cDNAs (input amount, reverse transcribed from the RNA 164

isolated from MA200 and MA200-1 strains) as the X values and Ct as the Y values (Fig. S3). 165

RNA concentrations of the experimental samples were extrapolated from the standard curves. The 166

percentages of mRNA remaining at each time interval after rifampicin treatment were calculated 167

relative to the signal obtained at 0 minute and were plotted on the Y axis versus time on the X 168

axis. The time point at which 50% of the gcvA mRNA has been degraded was extrapolated from 169

the decay plot to determine the t1/2. 170

RESULTS 171

Identification of downstream target genes regulated by the bgl operon 172

Total proteins from the strains ZK819-97T (Bgl+) and ZK819-Tn5 (Bgl-), grown to stationary 173

phase (24hours), were analyzed by 2D-PAGE to identify putative candidates whose expression is 174

regulated by the bgl operon. Comparison of the proteome profiles indicated the presence of a 175

minimum of ten proteins in ZK819-97T (Bgl+) and two proteins in ZK819-Tn5 (Bgl-) that were 176

over-expressed (Fig. 1). The over-expressed protein spots were identified by Matrix Assisted 177

Laser Desorption Ionization Time of Flight (MALDI-TOF). The peptide mass fingerprints 178

matched that of twelve known proteins (Table 2). Amongst these, OppA, an oligo-peptide 179

transporter that showed consistent over-expression in the Bgl+ strain, was selected for further 180

investigations. The over-expression at the protein level was validated by examining the transcript 181

levels of oppA. 182

Steady-state level of oppA mRNA is higher in Bgl+ cell 183

The steady-state levels of oppA mRNA from Bgl– and Bgl+ strains were compared at exponential 184

phase (4hours) and stationary phase (24hours) by Real-Time Polymerase Chain Reaction (qRT-185

PCR). These studies showed that, there is a marginal decrease of oppA gene expression in the Bgl+ 186

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strain grown for 4hours compared to its wild type Bgl- parent. However, expression of the oppA 187

gene was about twenty five fold higher in the Bgl+ cells grown for 24hours (Fig. 2). Therefore, the 188

enhanced level of OppA seen in the proteome of the Bgl+ strains is also reflected in the steady-189

state transcript level. 190

Loss of oppA function leads to partial loss of the GASP phenotype of Bgl+ cells 191

Bgl+ cells exhibit a fitness advantage in the stationary phase when competed against the Bgl- 192

parent in mixed culture experiments (13, Fig. 3). Since oppA has been shown to be up-regulated in 193

the Bgl+ strain, it is conceivable that the functions encoded by oppA contribute to the GASP 194

phenotype exhibited by Bgl+ strains. To test this possibility, the oppA locus was deleted in the 195

Bgl+ strain ZK819-97T and the ΔoppA mutant was competed against its wild type parent ZK819-196

Tn5 (Bgl-) in a co-culture experiment. Data from competition assays indicated that ZK819-197

97T∆oppA cells have lost the strong fitness advantage shown by the parent strain ZK819-97T 198

(Fig. 3). The decrease in the GASP phenotype in the ∆oppA strain suggests that a part of the 199

growth advantage of the Bgl+ strain is contributed by OppA. 200

RAT-like motif is present within the protein-coding sequence of gcvA 201

One possible way by which the bgl operon could exert regulation on a downstream target gene is 202

via its regulators BglG and BglF. BglG binds to the RAT element located at position +40 to +77 203

within the 5’ leader region of the bgl mRNA where it acts as an anti-terminator of transcription, 204

enabling the expression of the bgl structural genes (10). Examination of the oppA DNA sequence 205

did not reveal the presence of a RAT-like motif within the gene, suggesting that the regulation of 206

oppA by the bgl operon is likely to be indirect. 207

Expression of oppA has been shown to be negatively regulated at the translational level by the 208

small RNA (sRNA) gcvB (28) which in turn is transcriptionally activated by the product of the 209

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gcvA gene involved in the regulation of the glycine cleavage system (32). Measurements of the 210

steady-state transcript levels of gcvB and its positive regulator gcvA by qRT-PCR showed a 211

marked reduction in the stationary phase in the Bgl+ strain (Fig. 4A and 4B). The pronounced 212

reduction in the levels of gcvB and gcvA transcripts in the presence of higher levels of bglG in the 213

Bgl+ strain (Fig. S1) suggests that BglG is involved in their regulation. This is consistent with the 214

observation that gcvA carries a RAT-like sequence within the protein-coding region (480bp 215

downstream to +1) that has 46% sequence identity to the bgl-specific RAT sequence (Fig. S2). 216

Stability of the gcvA mRNA is reduced in the presence of functional BglG 217

Apart from functioning as an antiterminator, BglG has been shown to stabilize the bgl leader 218

transcript upstream of the terminator (6). To examine whether BglG has any effect on the stability 219

of the gcvA transcript, the levels of gcvA mRNA remaining at different time points after blocking 220

transcription initiation were determined by qRT-PCR in two isogenic strains that express different 221

levels of bglG (Fig. 5). The half-life of the gcvA transcript was 9.7 minutes in the Bgl+ strain 222

MA200. In contrast, the half-life of the gcvA mRNA was 4.3 minutes in the strain MA200-1 in 223

which the steady-state bglG transcript levels are substantially higher as a result of a mutation in 224

the negative regulator bglF (Fig. S3). Enhanced levels of bglG therefore lead to a significant 225

reduction in the half-life of the gcvA transcript. 226

The comparison of the stability of the gcvA mRNA was made using the two strains MA200 (Bgl+) 227

and MA200-1 (bglF) essentially to amplify the effect of bglG. To determine if the elevated levels 228

of bglG in the bglF strain confer a growth advantage in stationary phase, the strain ZK819RΔbglF 229

carrying a deletion of bglF was competed against the Bgl+ strain ZK819-97T. These studies 230

showed that the ΔbglF (Bgl-) strain had a distinct growth advantage over the Bgl+ strain (Fig. 6), 231

indicating that the GASP phenotype is correlated with the higher levels of bglG. 232

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BglG is necessary for the GASP phenotype of Bgl+ Strains 233

The results described above show a direct correlation between higher levels of bglG and the down-234

regulation of gcvA. If BglG is upstream of OppA in conferring the GASP phenotype to Bgl+ cells 235

via regulating the expression of gcvA, loss of BglG should result in the loss of the GASP 236

phenotype. Deletion of bglG in the strain ZK819-97T resulted in the complete loss of the GASP 237

phenotype of the strain when competed against the wild-type parent ZK819-Tn5 (Fig. 7A and 7B). 238

Loss of BglG function results in the up-regulation of gcvA 239

If BglG destabilizes the gcvA transcript, gcvA levels are expected to increase in a bglG mutant. 240

The loss of BglG function resulted in the up-regulation of gcvA and gcvB expression and a 241

concomitant down-regulation of the steady state levels of oppA, as detected by qRT-PCR (Fig. 8). 242

The up-regulation of gcvA in the ∆bglG strain is consistent with the proposed role of BglG as a 243

negative regulator of gcvA. The loss of the GASP phenotype in the ∆bglG strain may be 244

correlated partly to the decrease in the expression of OppA, due to elevated levels of its negative 245

regulators gcvA and gcvB. 246

Deletion of gcvA partly rescues the loss of the GASP phenotype in ∆bglG strains 247

As gcvA is known to mediate negative regulation of oppA via gcvB, loss of gcvA function is 248

expected to result in an increase in oppA gene expression. Real Time-PCR measurements of 249

mRNA levels in a strain carrying a deletion of gcvA showed reduced levels of gcvB and elevated 250

levels of oppA compared to the wild type strain (Fig. 9). Interestingly, deletion of gcvA could also 251

rescue the loss of oppA expression seen in a ∆bglG background. Measurements of oppA mRNA 252

in a ∆gcvA ∆bglG double mutant showed elevated levels even in the absence of BglG (Fig. 10). 253

More importantly, the loss of the GASP phenotype seen in the ∆bglG strain could be partly 254

rescued when gcvA is deleted (Fig. 7C and 7D). 255

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DISCUSSION 256

Retention of silent genetic systems such as the bgl operon of E. coli, without the loss of function 257

of the structural genes due to the accumulation of inactivating mutations, is an enigma. In this 258

context, the following two observations are significant. First, strains that carry an activated bgl 259

operon can out-compete the isogenic wild-type strain in competition experiments (13, Fig. 3) even 260

when β-glucosides are not supplemented in the medium. Second, transcription from the wild-type 261

bgl promoter is enhanced in the stationary phase even in the absence of activating mutations (14), 262

though this increase is insufficient to allow growth on β-glucosides. These observations suggest 263

the possibility that the bgl operon exerts a regulatory effect on downstream target genes other than 264

those implicated in β-glucoside catabolism, expression of which provides a fitness advantage in 265

the stationary phase. The majority of up-regulated proteins identified in the Bgl+ proteome are 266

known to participate either in transport functions or are enzymes involved in cellular metabolism. 267

As a result, Bgl+ cells are likely to be better equipped to scavenge available nutrient substrates by 268

activating additional metabolic functions, compared to Bgl- cells. 269

Among the ten candidate genes over-expressed in the Bgl+ proteome, oppA was selected to 270

study the role of the bgl operon in downstream regulation. OppA (60.9 kDa) is an oligo-peptide 271

transporter subunit encoded by the oppABCDF operon and is a member of the ATP-Binding 272

Cassette (ABC) super family of transporters (20). The system has been shown to be involved in 273

functions related to oligo-peptide uptake and the recycling of cell wall peptides (9). The up-274

regulation of OppA in the Bgl+ proteome was further validated by qRT-PCR that showed 275

significantly high steady-state transcript levels in the stationary phase in Bgl+ cells compared to its 276

Bgl- parent. 277

OppA, which is known to assist in the transport of small peptides up to five amino acids in 278

length (7) could be one of the factors that contribute towards the GASP phenotype of Bgl+ strains 279

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by enabling the uptake of potential nutrients from the environment. The partial loss of the GASP 280

phenotype observed in the competition assay between Bgl+ (∆oppA) and Bgl- strains is consistent 281

with this possibility. The GASP phenotype of Bgl+ strains is dependent on the presence of the 282

rpoS819 allele that results in the attenuated expression of RpoS (13). Presence of the rpoS819 283

allele has also been shown to enable faster growth in the presence of certain amino acids (36). 284

Expression from the oppA promoter is up-regulated in the absence of RpoS (29). Therefore, the 285

GASP phenotype conferred by oppA is likely to be the combined effect of increased transcription 286

from the oppA promoter due to the presence of the rpoS819 allele and the involvement of the 287

activated bgl operon in its expression. 288

The involvement of the bgl operon in the regulation of oppA expression could be direct or 289

indirect. OppA is regulated negatively by a small regulatory RNA (sRNA) gcvB (2) which has 290

been shown to inhibit translation initiation by binding to the oppA mRNA (28). In turn, the 291

transcription of gcvB is positively regulated by the GcvA protein, the major transcription factor of 292

the glycine cleavage system (32). Expression of gcvB is high during early log phase, but its level 293

decreases during cell growth (2). This reduction in gcvB expression was much more pronounced 294

in Bgl+ cells. Similarly, a significant decrease in gcvA transcription in Bgl+ cells was also 295

registered in the stationary phase. These observations suggest that the regulation of oppA by the 296

bgl operon is via its regulators gcvA and gcvB. 297

The search for a possible mechanism by which the bgl operon exerts a regulatory effect on 298

oppA led to the identification of a RAT-like motif present within the protein-coding region of 299

gcvA gene. Since the gcvARAT motif present within the gcvA ORF is not associated with a 300

transcription terminator structure, BglG is unlikely to function as an anti-terminator of 301

transcription in this case. BglG could alter the translation of gcvA by acting as a road-block for the 302

movement of ribosomes. Alternatively BglG could destabilize the gcvA transcript, which could 303

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negatively impact GcvA expression. This is supported by the observation that the half-life of 304

gcvA transcript is reduced in a strain in which bglG expression is high. Enhanced expression of 305

oppA in Bgl+ cells is lost when bglG is deleted, indicating a positive role for BglG in its 306

expression. The requirement for bglG for oppA expression can be overcome by deletion of gcvA, 307

consistent with the assumption that gcvA is the target of bglG-mediated regulation of oppA. 308

Our earlier work had shown that bglB that encodes the hydrolytic enzyme phosho-β-309

glucosidase B is involved in the GASP phenotype as a deletion of bglB led to the loss of the 310

GASP phenotype (13). The results presented in this communication indicate that bglG is epistatic 311

over bglB as a deletion of bglG retaining bglF and bglB function results in the loss of the GASP 312

phenotype. These studies also show that the relative levels of bglG are important as a ΔbglF (Bgl-) 313

strain expressing bglG constitutively can out-compete a Bgl+ strain just as a Bgl+ strain can out-314

compete a Bgl- strain. 315

Based on the observations described above, we propose that the ability to transport oligo-316

peptides mediated by the over expression of oppA is partly responsible for the GASP phenotype of 317

Bgl+ strains, consistent with the observation that the ΔoppA strain shows a partial loss of the 318

GASP phenotype. Down-regulation of oppA in a strain carrying a deletion of bglG may be one of 319

the reasons for the loss of the GASP phenotype of the ΔbglG strain. The complete loss of the 320

GASP phenotype in the ΔbglG mutant and its partial rescue in the ΔbglGΔgcvA double mutant 321

suggest that BglG is a master regulator involved in modulating the expression of downstream 322

genes important in stationary phase survival and oppA is one such locus. The validation of the 323

additional loci detected in the Bgl+ proteome is expected to provide a more complete picture of the 324

involvement of the bgl operon in stationary phase survival. 325

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The involvement of the bgl operon in functions unrelated to the catabolism of β-glucosides 326

suggests that selection for elevated expression of the operon can occur even in the absence of β-327

glucosides. This could be achieved by either by mutations or by overriding its negative regulation 328

under specific growth conditions such as stationary phase. Our earlier work has shown 329

enhancement of bgl expression in stationary phse (14). Though such elevated expression may not 330

be sufficient to allow utilisation of β-glucosides, it may be sufficient for the regulation of the 331

downstream target genes. If this requirement is for a prolonged period of time, there is likely to be 332

selection for mutational activation of the opern under these conditions. Involvement of the bgl 333

operon in the regulation of additional functions provides a selective force for the maintenance of 334

the bgl genes over evolutionary time. 335

ACKNOWLEDGEMENTS 336

The authors are thankful to D. Chatterji, and Manish Kumar for their help with two dimensional 337

electrophoresis, and Imran Khan for help with RT-PCR experiments. The proteomic analysis was 338

carried out at the institutional facility supported by the Department of Biotechnology (DBT), 339

Govt. of India. DH is thankful to DBT for a postdoctoral research fellowship. This work was 340

supported by a grant to SM from the Department of Science and Technology (DST), Govt. of 341

India. 342

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440

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TABLE 1 441

Bacterial strains and plasmids used in the study 442

Strains Genotype Source

ZK819

ZK126 (W3110ΔlacU169) rpoS819 SmR (Bgl-)

41

ZK819-97T ZK819 tna::Tn10 bglR (Bgl+) (TetR) 18

ZK819-Tn5 ZK819 bglR0 (Bgl-) (KanR) 18

ZK819-Tn10 ZK819 bglR0 (Bgl-) (TetR) 18

ZK819-97T∆oppA ZK819 tna::Tn10 bglR ∆oppA (Bgl+) This Study

ZK819-97T∆bglG ZK819 tna::Tn10 bglR ∆bglG (Bgl+) This Study

ZK819-97T∆gcvA ZK819 tna::Tn10 bglR ∆gcvA (Bgl+) This Study

ZK81997T∆bglG∆gcvA ZK819RΔbglF

ZK819 tna::Tn10 bglR ∆bglG ∆gcvA (Bgl+)

ZK819 bglR::IS1 ΔbglF (BglGC, KanR)

This Study

This study

DH5α F’endA1 hsdR17 (rk-mk+) supE44 thi-1 recA1 gyrA (NalR) relA1 ∆(lacZYA argF)U169 deoF (Φ80dlac Δ(lacZ) M15)

39

MA200

F– ΔlacX174 thi bglR1(bglR::IS1) srl::Tn10 recA56 λdbglR7 bglG’lacZ lacY Φ(bgl-lac) (Bgl+)

21

MA200-1

MA200, bglF201 (BglGc)

21

Plasmids Genotype Source

pKD46 AmpR, beta,exo,gam (lambda red),orits 8

pKD3 pANTSγ derivative, CamR 8

pCP20 flp, bla, cat, rep101ts AmpR CamR 4

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TABLE 2 443

The summary of proteins identified in Bgl+ and Bgl- proteome 444

Protein spots

Proteins identified

Function of the identified proteins

1a FkpA Heat shock peptidyl-prolyl isomerase (PPIase) having chaperone function

2a SecD A membrane component of Sec protein secretion complex

3 OppA Periplasmic component of the oligo-peptide transporter

4 SdhA Succinate dehydrogenase

5 ManX

Mannose-specific (EIIAB) PTS permease involved in translocating exogenous hexoses including N-acetylglucosamine

6 TrpS Tryptophanyl-tRNA synthetase

7 SucD Succinyl-CoA synthetase, α subunit

8 AdhE Alcohol dehydrogenase

9 FabI Enoyl-acyl carrier protein reductase

10 NusG Required for Rho-dependent transcription termination as well as for antitermination during lambda phage transcription

11 BetI

Repressor of glycine betaine synthesis from choline

12 YjgF Hypothetical protein

aProtein over-expression picked up in Bgl- proteome 445

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FIGURES AND LEGENDS 446

FIG. 1 447

FIG. 1 Proteome analysis of whole cell lysates of ZK819-Tn5 (Bgl- left) and ZK19-97T (Bgl+ 448

right) strains by 2D-PAGE. Proteins were visualized by Coomassie Blue R-250 stain, and 449

differences between the Bgl- and Bgl+ gels were visually analyzed. Black circles (1, 2) represent 450

proteins significantly over-expressed in the Bgl- proteome and grey circles (3-12) represent 451

proteins significantly over-expressed in the Bgl+ proteome (n=3). 452

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FIG. 2 453

FIG.2. Steady-state expression of oppA mRNA in Bgl- (ZK819-Tn5) and Bgl+ (ZK19-97T) strains 454

under exponential and stationary phase. The Y axis represents fold change of oppA gene 455

expression relative to the Bgl- (ZK819-Tn5) calibrator as measured by qRT-PCR (n=2). The rrnC 456

(16S-RNA) gene was used as a control for normalization. See Materials and Methods for details. 457

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FIG. 3A FIG. 3B 458

FIG. 3C FIG. 3D 459

FIG. 3 Competition between stationary phase grown (24hours in LB medium) cultures of ZK819-460

Tn5 (Bgl-) ( ) vs. ZK819-97T (Bgl+) (■) strains in 1000:1 ratio (A) 1:1000 ratio (B). ZK819-Tn5 461

(Bgl-) (◊) vs. ZK819-97T∆oppA (Bgl+) (♦) strains in 1000: 1 ratio (C) and 1:1000 ratio (D). 462

Viable counts (CFU ml-1) were monitored for the days indicated on the X axis (n=2). The mixed 463

cultures were maintained in the original LB medium without addition of fresh medium. 464

0

2

4

6

8

10

0 2 4 6 8 10

Log

cfu

/ml

Days

0

2

4

6

8

10

0 2 4 6 8 10

Log

cfu

/ml

Days

0

2

4

6

8

10

0 2 4 6 8 10

Log

cfu

/ml

Days

0

2

4

6

8

10

0 2 4 6 8 10

Log

cfu

/ml

Days

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FIG. 4A 465

FIG. 4B 466

FIG. 4 Steady-state expression of gcvB (A) and gcvA (B) transcripts in Bgl- (ZK819-Tn5) and 467

Bgl+ (ZK19-97T) strains under exponential and stationary phase. The Y axis represents fold 468

change of gcvA/gcvB gene expression relative to the Bgl- (ZK819-Tn5) calibrator as measured by 469

qRT-PCR (n=2). The rrnC (16S-RNA) gene was used as control for normalization. See Materials 470

and Methods for details. 471

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FIG. 5 472

1

10

100MA200 t1/2 =9.7 (min)

MA200-1 t1/2 =4.3 (min)

2 4 6 8 10 12 14

50

Time (min)

gcvA

mR

NA

Rem

ain

ing

(%

)

FIG. 5 Real-time PCR (qRT-PCR) analysis of the half-life of gcvA mRNA in MA200 (BglG+) 473

and MA200-1 (BglGc) strains after inhibiting transcription initiation with rifampicin (200µg ml-1). 474

The percentage bgl mRNA remaining at each time interval was calculated from the standard curve 475

prepared for known cDNA concentrations as described in Materials and Methods. The signal 476

obtained at 0 minute was considered as 100% and the percentage mRNA remaining at each time 477

point was plotted on the Y axis versus time on the X axis. The time point at which 50% of the 478

gcvA mRNA has been degraded was calculated from the decay plot to determine the half-life. 479

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Fig. 6

Fig. 6 Competition between stationary phase grown ZK819-97 T (Bgl+) (■) and ZK819R∆bglF 480

(BglGC Bgl-) ( ). Culture conditions were similar to those described in Fig. 3. (n=3). 481

0

2

4

6

8

10

0 1 2 3 4 5 6

LogC

FU/m

l

Number of days

0

2

4

6

8

10

0 1 2 3 4 5 6Lo

gCFU

/ml

Number of days

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FIG. 7A FIG. 7B 482

FIG. 7C FIG. 7D 483

FIG. 7 Competition between stationary phase-grown ZK819-Tn5 (Bgl-) ( ) and ZK819-484

97T∆bglG (Bgl+) (■) strains in 1000:1 ratio (A) and 1: 1000 ratio (B). ZK819-Tn5 (Bgl-) (◊) vs. 485

ZK819-97T∆bglG∆gcvA (Bgl+) (♦) strains in 1000: 1 ratio (C) and 1:1000 ratio (D). Viable CFU 486

ml-1 counts were monitored for the days indicated on the X axis (n=2). 487

0

2

4

6

8

10

0 2 4 6 8 10

Log

cfu

/ml

Days

0

2

4

6

8

10

0 2 4 6 8 10L

og c

fu/m

l

Days

0

2

4

6

8

10

0 2 4 6 8

Log

cfu

/ml

Days

0

2

4

6

8

10

0 2 4 6 8

Log

Cfu

/ml

Days

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FIG. 8A 488

FIG. 8B 489

FIG. 8C 490

FIG. 8 Steady-state expression of gcvA (A), gcvB (B) and oppA (C) transcripts in Bgl- (ZK819-491

Tn5) and Bgl+ (ZK19-97T∆bglG) strains under exponential and stationary phase growth 492

conditions. The Y axis represents fold change of gcvA/gcvB/oppA gene expression relative to the 493

Bgl- (ZK819-Tn5) calibrator as measured by qRT-PCR (n=2). The rrnC (16S-RNA) was used as 494

a control for normalization. See Materials and Methods for details. 495

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FIG. 9A 496

FIG. 9B 497

FIG. 9 Steady-state expression of gcvB (A) and oppA (B) transcripts in Bgl- (ZK819-Tn5) and 498

Bgl+ (ZK19-97T∆gcvA) strains under exponential and stationary phase growth conditions. The Y 499

axis represents fold change of gcvB/oppA gene expression relative to the Bgl- (ZK819-Tn5) 500

calibrator as measured by qRT-PCR (n=2). The rrnC (16S-RNA) was used as a control for 501

normalization. See Materials and Methods for details. 502

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FIG. 10A 503

FIG. 10B 504

FIG. 10 Steady-state expression of gcvB (A) and oppA (B) transcripts in Bgl- (ZK819-Tn5) and 505

Bgl+ (ZK19-97T∆bglG∆gcvA) strains under exponential and stationary phase growth conditions. 506

The Y axis represents fold change of gcvB/oppA gene expression relative to the Bgl- (ZK819-Tn5) 507

calibrator as measured by qRT-PCR (n=2). The rrnC (16S-RNA) was used as a control for 508

normalization. See Materials and Methods for details. 509

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