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Journal of Bacteriology, January 2006, p. 587-598, Vol. 188, No. 2
0021-9193/06/$08.00+0     doi:10.1128/JB.188.2.587-598.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

YdgG (TqsA) Controls Biofilm Formation in Escherichia coli K-12 through Autoinducer 2 Transport

Moshe Herzberg,1 Ian K. Kaye,1 Wolfgang Peti,2 and Thomas K. Wood1*

Departments of Chemical Engineering and Molecular & Cell Biology, University of Connecticut, 191 Auditorium Road, Storrs, Connecticut 06269-3222,1 Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, 70 Ship Street GE3, Providence, Rhode Island 029122

Received 1 September 2005/ Accepted 27 October 2005


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ABSTRACT
 
YdgG is an uncharacterized protein that is induced in Escherichia coli biofilms. Here it is shown that deletion of ydgG decreased extracellular and increased intracellular concentrations of autoinducer 2 (AI-2); hence, YdgG enhances transport of AI-2. Consistent with this hypothesis, deletion of ydgG resulted in a 7,000-fold increase in biofilm thickness and 574-fold increase in biomass in flow cells. Also consistent with the hypothesis, deletion of ydgG increased cell motility by increasing transcription of flagellar genes (genes induced by AI-2). By expressing ydgG in trans, the wild-type phenotypes for extracellular AI-2 activity, motility, and biofilm formation were restored. YdgG is also predicted to be a membrane-spanning protein that is conserved in many bacteria, and it influences resistance to several antimicrobials, including crystal violet and streptomycin (this phenotype could also be complemented). Deletion of ydgG also caused 31% of the bacterial chromosome to be differentially expressed in biofilms, as expected, since AI-2 controls hundreds of genes. YdgG was found to negatively modulate expression of flagellum- and motility-related genes, as well as other known products essential for biofilm formation, including operons for type 1 fimbriae, autotransporter protein Ag43, curli production, colanic acid production, and production of polysaccharide adhesin. Eighty genes not previously related to biofilm formation were also identified, including those that encode transport proteins (yihN and yihP), polysialic acid production (gutM and gutQ), CP4-57 prophage functions (yfjR and alpA), methionine biosynthesis (metR), biotin and thiamine biosynthesis (bioF and thiDFH), anaerobic metabolism (focB, hyfACDR, ttdA, and fumB), and proteins with unknown function (ybfG, yceO, yjhQ, and yjbE); 10 of these genes were verified through mutation to decrease biofilm formation by 40% or more (yfjR, bioF, yccW, yjbE, yceO, ttdA, fumB, yjiP, gutQ, and yihR). Hence, it appears YdgG controls the transport of the quorum-sensing signal AI-2, and so we suggest the gene name tqsA.


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INTRODUCTION
 
Escherichia coli biofilms have hundreds of genes differentially expressed (5, 48, 56); hence, it is important to understand how the biofilm lifestyle is regulated so that bacteria may be controlled (4). In the absence of conjugation plasmids (47), it appears type I fimbriae are required for cells to attach and that motility is essential for biofilm formation, probably for initial interaction (overcoming repulsive forces) and swimming along the surface (43). Following attachment, extracellular polymers and adhesive factors are produced, including curli and the outer membrane protein Ag43 (flu) (11, 44). The microcolonies mature into complex architecture by synthesis of an extracellular polysaccharide matrix such as colanic acid in E. coli biofilms (12).

Cell signaling (quorum sensing) also is involved in biofilm formation as it controls the production and secretion of exopolysaccharides for Vibrio cholerae biofilms (21). Vibrio harveyi uses three quorum-sensing signals, including N-(3-hydroxybutanoyl)homoserine lactone, furanosyl borate diester (autoinducer 2 [AI-2]), and a signal, synthesized by CqsA, whose structure is unknown (23). N-(3-Oxododecanoyl)-L-homoserine lactone controls biofilm formation in Pseudomonas aeruginosa (14). A homoserine lactone signal was also found to control cell aggregation and biofilm formation of Serratia liquefaciens (32). AI-2 was found to regulate mixed-species biofilm formation by Streptococcus gordonii and Porphyromonas gingivalis (39) and also to increase the amount of microcolonies in early stages of biofilm formation by Klebsiella pneumoniae and to increase the total mature biofilm formed by 30 to 50% (2). We have found that the quorum-sensing disrupter (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone of the alga Delisea pulchra inhibits E. coli biofilms by repressing the same suite of genes that are induced by AI-2 (49), which argues that AI-2 should directly stimulate E. coli biofilm formation. This has recently been shown by using enzymatically synthesized AI-2, which increases biofilm formation 30-fold in 96-well biofilm assays and in flow cells (19).

AI-2 induces transcription of the lsrACDBFGE operon of Salmonella enterica serovar Typhimurium, in which the first four genes encode an ATP-binding cassette-type import protein with high homology to the ribose transporter (62, 63). AI-2 is phosphorylated inside the cell by LsrK, and phosphorylated AI-2 induces inactivation of the lsr operon repressor LsrR (62, 69). Addition of sugars that are part of the phosphotransferase system increases AI-2 extracellular activity to its maximum level in the mid- to late-exponential phase (60) through a decrease in the cyclic AMP (cAMP) concentration (65). Once glucose is depleted, cAMP concentrations increase, the lsr operon is activated by cAMP, and AI-2 is internalized by cells (60). However, the means by which AI-2 is exported has not been identified, assuming that secretion of hydrophilic AI-2 requires active transport.

There are three studies describing E. coli global gene expression in biofilms (5, 48, 56). Beloin et al. (5) found biofilm cells have genes induced for stress response, cell biogenesis and transport, and energy and carbohydrate metabolism and that genes were repressed involving amino acid, carbohydrate, and inorganic ion transport; in all, 2% of all genes were differentially expressed more than twofold. Schembri et al. (56) found that 5% or 14% of the genes were differentially expressed compared to exponential- or stationary-phase suspended cultures, respectively. By comparing cells from the same reactor, we found that genes for stress response, type 1 fimbriae, and genes with unknown functions, including ydgG (b1601, 344 amino acids [aa]), are induced in biofilms after 7 h (48). The ydgG mutant was chosen for further study since this mutation increased biofilm formation upon addition of glucose to the medium.

To determine more rigorously the role of YdgG in biofilm formation, DNA microarrays were used here to study gene expression in a biofilm for a biofilm up mutant versus that of the isogenic wild-type strain. Previously, Zhu and Mekalanos (72) used microarrays to study genome expression in V. cholerae for a biofilm up mutant and found induced expression of Vibrio polysaccharide synthesis operons. We assumed that the differential gene expression of a poor biofilm-forming strain compared to its mutant that forms robust biofilms (e.g., E. coli ydgG) in a mature biofilm will show a wider view of the gene expression pattern in biofilm cells than an experiment that compares mature biofilm cells versus planktonic cells (33). Since many of the differentially expressed genes identified in the biofilm of the ydgG mutant are controlled by AI-2, we investigated the possibility that YdgG transports AI-2.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, growth media, and growth rate measurements. The strains and plasmids used in this study are listed in Table 1. For E. coli K-12 BW25113 {Delta}ydgG, the insertion of the kanamycin resistance gene and the complete ydgG deletion were corroborated with five sets of PCRs (three primers homologous to sequences internal to the kanamycin gene and two primers homologous to a sequence upstream and downstream of ydgG) (1, 13). For plasmid selection, 100 µg/ml of ampicillin, 50 µg/ml of kanamycin, 30 µg/ml of chloramphenicol, or 300 µg/ml of erythromycin was added. Luria Bertani (LB) medium (55) was used for preculturing the E. coli strains. LM agar plates (18) were used for enumerating V. harveyi BB170, which was cultured in AB medium (3). For the 96-well biofilm assays, LB medium, LB medium supplemented with 0.2% glucose (LB glu), and M9 medium supplemented with 0.4% glucose and 0.4% Casamino Acids (M9C glu) (53) were used. M9C glu was also used for the E. coli flow cell experiment. The specific growth rates of wild-type and {Delta}ydgG strains were determined by measuring turbidity at 600 nm (optical density at 600 nm [OD600]) and calculated by using the linear portion of the natural logarithm of OD600 versus time (OD600 from 0.05 to 0.5) in LB, LB glu, and M9C glu.


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  		TABLE 1. E. coli strains and plasmids used in this study

Ninety-six-well biofilm assay. The 96-well biofilm assay was conducted as described previously (52). Briefly, two independent overnight E. coli cultures were diluted to an OD600 of 0.05 and grown in M9C glu, LB, or LB glu and the biofilms were stained with 0.1% crystal violet (Fisher, Hanover Park, IL) for 20 min to quantify the total biofilm mass (for both the biofilms at the bottom and those at the air-liquid interface). Each datum point was averaged from two independent experiments, each with four replicate wells.

Flow cell biofilm experiments and image analysis. M9C glu supplemented with 300 µg/ml erythromycin to maintain constitutive green fluorescent protein plasmid pCM18 (22) was used to form biofilms at 37°C in a continuous-flow cell as described previously (19). The biofilm was visualized at 24 h with a TCS SP2 scanning confocal laser microscope (Leica Microsystems, Heidelberg, Germany) with a 40x N PLAN L dry objective with a correction collar and a numerical aperture of 0.55. Color confocal flow cell images were analyzed with COMSTAT image-processing software (24) as described previously (52). At 24 h, nine different positions were chosen for microscope analysis and 25 images were processed for each position; in total, 225 images were analyzed. The values reported are means of data from the different positions at the same time point, and standard deviations were calculated based on these mean values for each position. Simulated three-dimensional images were obtained with IMARIS (BITplane, Zurich, Switzerland). Twenty-five pictures were processed for each three-dimensional image.

Motility assay. The motility assay was adapted from Sperandio et al. (59); LB overnight cultures were used to assay motility in plates containing 1% (wt/vol) tryptone, 0.25% (wt/vol) NaCl, and 0.3% (wt/vol) agar (where indicated, motility plates were supplemented with glucose or chloramphenicol). Motility halos were measured at 16 h, and five to eight plates were used to compare motility between the strains. For the motility complementation experiment, motility ratios between the ydgG mutant and the wild-type strain are used in order to eliminate the small density differences between the motility plates. On each plate, both the wild type and the ydgG mutant were inoculated.

Promoter transcriptional assays. E. coli cultures with plasmids containing the flhD::lacZ, fliA::lacZ, fliC::lacZ, motA::lacZ, and qseB::lacZ fusions (Table 1) were cultured overnight in LB medium supplemented with ampicillin (100 µg/ml) and then diluted 1:100 in LB and LB glu (both supplemented with 100 µg/ml ampicillin) to create exponentially growing cells that were harvested at an OD600 of 1. ß-Galactosidase activity was assayed as described previously (68). All activities were calculated based on a protein concentration of 0.24 mg protein/ml/OD600 unit. Each experiment was performed twice with two different cultures for each strain in LB and LB glu.

Complementation of motility, biofilm, drug resistance, and extracellular AI-2 phenotypes. The ydgG deletion was complemented in trans with pCA24N ydgG+ (1, 13), which has isopropyl-ß-D-thiogalactopyranoside (IPTG; Sigma, St. Louis)-inducible expression of ydgG+. Control of ydgG+ transcription was tight due to the presence of lacIq. The same plasmid without the ydgG+ insertion, pCA24N, was used as a negative control. Overnight cultures with IPTG (0, 0.5, 0.75, 1.0, and 1.5 mM) were used for the 96-well biofilm assay, motility assay, drug resistance assay, and extracellular AI-2 activity assay. Chloramphenicol (30 µg/ml) was added to the growth media and motility agar, except for the 96-well assay, where 50 µg/ml chloramphenicol was used in the biofilm assay. For complementing the extracellular AI-2 activity, cells were harvested at an OD600 of 0.6, washed twice (10 min at 8,820 x g) to remove chloramphenicol (inhibits the growth of the Vibrio reporter), and resuspended in the same volume of 20 ml LB glu. Then cells were grown to an OD600 of 0.9 for AI-2 production in the absence of chloramphenicol.

AI-2 assays. To determine extracellular AI-2 concentrations, overnight E. coli cultures were diluted 1:100 and grown to exponential phase (OD600 of 0.6) in LB and LB glu. Filter-sterilized supernatants were prepared and assayed as described previously with the reporter V. harveyi BB170 (51). To determine the intracellular AI-2 concentration, overnight cultures of E. coli containing lsrACDBFG::lacZ (Table 1) were diluted to create both exponentially growing and stationary-phase cells and so were harvested at OD600 values of 0.6 and 6, respectively, and then ß-galactosidase activity was assayed as described previously (68). Taga et al. (62) showed that as AI-2 is internalized, higher ß-galactosidase activity is measured from the lsr::lacZ fusion.

Drug transport measurements. Analysis of the effect of the ydgG mutation on the MICs of various antibiotics was conducted as described previously (35). Briefly, strains were grown overnight in LB glu and for the ydgG mutant, the medium was supplemented with 50 µg/ml kanamycin. The overnight culture was diluted 1:100 in LB glu, grown to an OD600 of 0.4, and cooled to 4°C. The cells were grown again, after dilution of the cultures (OD600 of 0.4) to a cell density of 5 x 104 cells/ml in LB glu with 50- to 500-fold increasing concentrations of drugs (streptomycin sulfate and spectinomycin at 1 to 50 µg/ml, crystal violet at 10 to 500 µg/ml, chloramphenicol at 0.1 to 10 µg/ml, amoxicillin at 0.1 to 10 µg/ml, 2,6-dichloroquinone-4-chloroimide at 5 to 500 µg/ml, penicillin G at 1 to 500 µg/ml, erythromycin at 5 to 500 µg/ml, sodium dodecyl sulfate at 10 to 1,000 µg/ml, ampicillin at 0.1 to 10 µg/ml, and tetracycline at 0.1 to 10 µg/ml). The OD600 was measured after 18 h of incubation at 250 rpm at 37°C.

Biofilm total RNA isolation for DNA microarrays. Overnight cultures were diluted 100-fold into 1-liter shake flasks containing 250 ml of LB glu and 10 g of glass wool. The cells were shaken at 250 rpm at 37°C for 24 h (final OD600 of 5 for the suspended cells) to form a biofilm on the glass wool, and RNA was isolated from the biofilm as described previously (48).

DNA microarrays. The E. coli GeneChip antisense genome array (P/N 900381; Affymetrix) was used to study the differential gene expression profile of the ydgG mutant compared to that of the isogenic wild type in a mature biofilm as described in the Gene Expression Technical Manual and as previously published (19). The data were inspected for quality and analyzed as described in Data Analysis Fundamentals, which includes using premixed polyadenylated transcripts of the B. subtilis genes (lys, phe, thr, and dap) at different concentrations. Also, as expected, there was insignificant ydgG mRNA signal in the biofilm of the ydgG mutant (60-fold lower than the mean signal and 13-fold lower than the signal accepted from the ydgG open reading frame [ORF] in the wild-type strain), and the genes which are known to be completely deleted from E. coli K-12 BW25113 (e.g., araA and rhaA) showed insignificant mRNA levels. To ensure the reliability of the induced-repressed gene list, genes were identified as differentially expressed if the P value was less than 0.05 and if the expression ratio was greater than 2 since the standard deviation for the expression ratio for all the genes was 2.2 (GEO accession number GSE3514). The gene functions were obtained from the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/); the Institute for Genomic Research, University of California at San Diego; and the UNAM database (http://biocyc.org/ECOLI/).


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RESULTS
 
YdgG represses biofilm formation without affecting growth. Almost no change in the growth rate was observed when ydgG was deleted from E. coli K-12; the growth rates in LB medium were 1.49 ± 0.05 versus 1.67 ± 0.05/h, in LB glu they were 1.73 ± 0.0 versus 1.80 ± 0.02/h, and in M9C glu they were 1.01 ± 0.01 versus 1.15 ± 0.03/h for the wild-type strain and the ydgG mutant, respectively. However, this deletion increased biofilm formation in 96-well plates when glucose was added to the medium (3.8-fold ± 0.2-fold for LB glu and 1.6-fold ± 0.1-fold for M9C glu) and decreased biofilm formation for LB medium (0.5-fold ± 0.1-fold).

The increase in biofilm formation in M9C glu measured by the crystal violet assay was corroborated in the more rigorous continuous-flow system, where the ydgG deletion dramatically increased biofilm formation (Fig. 1). The wild-type biofilm architecture in the flow cell consists of only a few individual cells on less than 1% of the observable surface, while the ydgG mutant formed a flat biofilm covering at least 30% of the viewable area and was composed of large, irregularly shaped, smooth masses containing random protrusions (Fig. 1). These changes in biofilm architecture were quantified with the COMSTAT computer program for quantifying biofilm structures (24); compared to the wild type, after 24 h, the biomass increased 574-fold, the substratum coverage increased 10-fold, the mean thickness was increased 7,000-fold, and the roughness coefficient was decreased 12-fold. Although different biofilm systems were used to measure the effect of YdgG on biofilm formation (one involves static conditions, and the other involves flow), an enhancement of biofilm formation with the ydgG mutant was obtained consistently and the difference in biofilm formation between the two systems may be attributed to the differences in the biofilm assays.



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  		FIG. 1. Effect of ydgG deletion (A) on biofilm formation in a flow chamber with M9C glu versus wild-type E. coli (B). Biomass was measured after 24 h, and images were analyzed with IMARIS. Scale bars, 10 µm.

To confirm that the increase in biofilm formation is due to the deletion of ydgG, the mutant strain was complemented in trans and biofilm formation was measured in a 96-well biofilm assay with M9C glu and LB glu. As expected, increasing ydgG expression decreased the biofilm formation of the ydgG mutant in both M9C glu and LB glu to that of the wild-type strain. In M9C glu, at IPTG concentrations of 0.5, 0.75, 1.0, and 1.5 mM, the relative values of biofilm formation by the ydgG mutant expressing YdgG in trans were 1.9 ± 0.3, 1.8 ± 0.3, 1.1 ± 0.2, and 1.1 ± 0.2, respectively. In LB glu, at the same IPTG concentrations, the relative values of biofilm formation by the ydgG mutant expressing YdgG in trans were 1.8 ± 0.2, 1.6 ± 0.2, 1.4 ± 0.2, and 0.9 ± 0.2, respectively. Hence, YdgG represses biofilm formation.

YdgG represses motility. Compared to the wild-type strain, deletion of ydgG resulted in a sixfold increase in swimming motility; the motility diameter for the wild-type strain was 0.3 ± 0.2 cm versus 2.0 ± 0.5 cm for the ydgG mutant (there was no effect of glucose). Similar to complementing biofilm formation, as ydgG+ was induced in trans, the motility of E. coli {Delta}ydgG/pCA24N ydgG+ became equal to that of the wild-type strain containing plasmid pCA24N, indicating the motility phenotype could be complemented. At IPTG concentrations of 0.1, 0.25, 0.5, 0.75, and 1.0 mM, the relative values for the motility of the ydgG mutant expressing YdgG in trans were 3.8 ± 0.2, 3.4 ± 0.1, 1.8 ± 0.1, 1.6 ± 0.0, and 1.0 ± 0.1, respectively. Hence, YdgG represses motility.

To determine the cause of this increase in motility seen upon deletion of ydgG, transcription of the quorum-sensing response regulator of flagellum genes (qseB), flagellar synthesis genes (flhD, fliA, and fliC), and the motility gene (motA) was investigated. The ydgG mutation increased the expression of these flagellum and motility genes 15- to 120-fold in both LB and LB glu (Fig. 2).



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  		FIG. 2. Effect of deletion of ydgG on the transcription of flhD, fliA, fliC, motA, and qseB in LB (A) and LB glu (B) for cells in suspension. The experiment was done in duplicate, and 1 standard deviation is shown. ONPG, o-nitrophenyl-ß-D-galactopyranoside.

YdgG increases extracellular AI-2 activity. Significantly higher extracellular AI-2 activity was measured for the wild-type strain compared to the ydgG mutant in both LB (13-fold ± 9-fold) and LB glu (4-fold ± 2-fold) under exponential growth conditions (relative light units measured per cell of V. harveyi BB170 were (3.4 ± 0.6) x 10–5 and (8.0 ± 0.1) x 10–5 for the wild-type strain, respectively). Hence, YdgG appears to have a role in either enhancing AI-2 export from the cell or inhibition of AI-2 uptake. As expected (60), extracellular AI-2 activity was higher for both the wild type and the ydgG mutant upon the addition of glucose to LB medium (2-fold ± 0.5-fold and 7-fold ± 5-fold, respectively).

Similar to complementing biofilm formation and motility, by expressing ydgG+ in trans, we were able to increase the extracellular AI-2 activity of the E. coli ydgG mutant to that of the wild-type strain (containing control plasmid pCA24N) by expressing ydgG+ in trans in LB glu with pCA24N ydgG+. At IPTG concentrations of 0.5, 0.75, 1.0, and 1.5 mM, the relative extracellular AI-2 concentrations of the ydgG mutant expressing YdgG in trans were 0.3 ± 0.3, 0.5 ± 0.4, 0.8 ± 0.2, and 1.0 ± 0.3, respectively.

YdgG reduces intracellular AI-2 activity. Significantly higher intracellular AI-2 activity was measured for the ydgG mutant compared to the wild-type strain in LB glu (16-fold ± 5-fold and 10-fold ± 5-fold under exponential-growth and stationary-phase conditions, respectively) (Fig. 3), as determined by comparing transcription of the lsrACDBFG promoter region. This operon is induced by internal phosphorylated AI-2 produced by the cytoplasmic kinase LsrK and intracellular AI-2 (62). Hence, once again, YdgG appears to either enhance AI-2 export from the cell or inhibit AI-2 uptake. As expected, addition of glucose to the wild-type strain repressed lsr transcription via catabolic repression by 4.0-fold ± 0.3-fold and 4.0-fold ± 0.7-fold during the exponential and stationary phases, respectively (65). Since transport of AI-2 is altered in the ydgG mutant, addition of glucose had no effect on the ydgG mutant during stationary phase (Fig. 3); some increase (2.7-fold ± 0.3-fold) in the intracellular AI-2 concentration was observed upon glucose addition during exponential growth of the ydgG mutant (Fig. 3). Future experiments with a luxS ydgG double mutant should be performed to investigate whether the ydgG mutation affects lsr expression.



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  		FIG. 3. Effect of deletion of ydgG on transcription of the lsrACDBFG operon with exponentially growing and stationary-phase cells in suspension in LB and LB glu. The experiment was done in duplicate, and 1 standard deviation is shown. ONPG, o-nitrophenyl-ß-D-galactopyranoside.

YdgG increases drug susceptibility. If YdgG is a transporter, one might expect it to affect drug resistance (27); hence, we tested the impact of the ydgG deletion with 11 different antimicrobials. Higher resistance of the ydgG mutant was observed with the following drugs compared to the wild-type strain: crystal violet (MICs of 100 and 250 µg/ml, respectively), spectinomycin (MICs of 20 and 50 µg/ml, respectively), streptomycin sulfate (MICs of 5 and 10 µg/ml, respectively), 2,6-dichloroquinone-4-chloroimide (MICs of 50 and 100 µg/ml, respectively), chloramphenicol (MICs of 5 and 10 µg/ml, respectively), and amoxicillin (MICs of 5 and 10 µg/ml, respectively). No change in resistance to the other drugs used was observed. Also, when expressing ydgG in trans in the ydgG mutant with pCA24N ydgG+, the phenotype could be complemented in that a higher susceptibility to crystal violet was observed in the mutant as the IPTG concentration was increased; BW25113 {Delta}ydgG/pCA24N ydgG+ grew to an OD600 of 0.9 after 18 h in LB glu supplemented with 30 µg/ml chloramphenicol, but addition of 0.5 mM IPTG caused no growth (no growth was observed in the controls BW25113/pCA24N and BW25113/pCA24N ydgG+).

Prediction of YdgG structure and function. Multiple secondary-structure prediction programs, such as PSIPRED (38), JPRED (10), and PHD (54), were used to identify the secondary structure of YdgG as almost exclusively all {alpha}-helical. Additionally, a ProDom (7) and BLAST (37) search indicated that YdgG most likely functions as a transporter and is predicted to be a membrane protein. Therefore, the membrane protein topology prediction programs MEMSTAT-2 (25, 26), TMHMM (31, 57), and PHDhtm (54) were used to predict seven to eight transmembrane helices, and the topology of the {alpha}-helices is shown in Fig. 4. The results from these programs correlated very well, and all predict (with 50 to 75% reliability) that the first loop is out of the membrane and the N and the C termini are cytosolic. Between residues 83 and 145 and residues 164 and 201, large loops are predicted, which could potentially play a role in regulating the transport of YdgG.



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  		FIG. 4. Schematic of YdgG combining data predicted by different bioinformatic programs.

Differential gene expression in biofilms due to deletion of ydgG. To explore further the mechanism by which YdgG represses biofilms, differential gene expression in the biofilms (rather than in suspension) as a result of deletion of ydgG was investigated; it was found that 1,330 (31%) of the E. coli genes were differentially induced and 327 (8%) were differentially repressed when a twofold cutoff was used (292 genes were induced and 99 genes were differentially repressed when a threefold cutoff was used). The most significantly induced and repressed genes (more than fourfold) are summarized in Tables 2 and 3.


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  		TABLE 2. Genes induced more than fourfold in LB glu medium biofilms upon deleting ydgG


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  		TABLE 3. Genes repressed more than fourfold in LB glu medium biofilms upon deleting ydgG

Deletion of ydgG increased AI-2 intracellular activity during both the exponential and stationary phases in shake flasks (Fig. 3); hence, many genes that are induced by AI-2 synthesis via active LuxS (49, 58) were induced in the ydgG mutant. For example, 87% of the genes induced by AI-2 (49) were also induced here in the biofilm culture of the ydgG mutant more than 1.5-fold, and 41% of AI-2-repressed genes (49) were repressed here more than 1.5-fold in the biofilm of the ydgG mutant. Also, 94% of the flagellum and motility genes reported to be induced by AI-2 by Sperandio et al. (58) were induced in the biofilm of the ydgG mutant (1.5-fold cutoff), and 70% of the differentially expressed genes in the study of Sperandio et al., which are involved in growth and cell division, showed the same expression (induced and repressed more than 1.5-fold) in the biofilm of the ydgG mutant.

The most significantly induced genes in the biofilm due to deletion of ydgG are those related to the cell envelope, transport, polysialic acid production, phage functions, methionine biosynthesis, biotin and thiamine biosynthesis, anaerobic metabolism, and unknown function (Table 2). The most significant cell envelope transport genes induced were acrEF (24- and 5.7-fold). AcrEF is a multidrug efflux system in E. coli which secretes indole, the product of tryptophan degradation (27). Indole was found to act as an extracellular signal in E. coli and to regulate biofilm formation (16) and amino acid degradation (64). An unknown stationary-phase signal has been found previously that is related to indole (50, 64). This stationary-phase signal was found to repress AI-2 extracellular activity and to induce genes related to biofilm formation such as fliK, tnaA, and ybaJ (50).

Genes involved in biofilm formation (43) were found to be induced due to ydgG deletion. For example, the four operons for flagellar synthesis (8) were induced in the ydgG mutant biofilm. The class 1 master flagellar regulator flhC was induced 2.1-fold, which led to overexpression of flhDC-induced class 2 flagellum genes, including the flgABCDEFGHIJ operon, where the most significant increase in expression was observed in flgI (induced 6.5-fold). Other class 2 flagellum operons were also induced in the ydgG mutant, including flhA and flhB (both induced twofold). The fliEFGHIJK operon was also induced 2.5-fold, and fliLMNOPQR was induced 2.1- to 3.2-fold. The sigma factor FliA, {sigma}28, was induced 2.1-fold (activates class 3 flagellum operons [8]), and the force generator gene motA and its motility and chemotaxis operon (motAB and cheAW) were induced around twofold. The chemotactic response gene cheZ was also induced 2.5-fold. fliD (encoding the filament cap FliD) was induced 2.5-fold. fliS and fliT encoding FliD chaperones were both induced 2.1-fold. Other class 3 flagellum genes were also induced; flgN, encoding the hook-filament chaperone proteins FlgK and FlgL, was induced 2.3-fold. The lower relative induction of the flagellum genes in the biofilms of the ydgG mutant (determined via microarrays) compared to the induction of flagellum genes observed in the exponential-phase suspension cultures of the ydgG mutant (Fig. 2) may be attributed to the two different modes of bacterial growth.

Adhesion determinants induced in the ydgG biofilm include the type 1 fimbria operon, other putative type 1 fimbria operons, and Ag43. Type 1 fimbriae are an important determinant of E. coli adhesion to both biotic and abiotic surfaces (43) during the first step of cell attachment to a surface. In the ydgG biofilm, fimB and fimE were induced 2.6- and 2-fold, respectively. Another eight putative type 1 fimbria-like predictive operons (http://biocyc.org/ECOLI/) were induced, with at least two genes from each operon induced more than twofold. The most highly induced was the sfm gene cluster, where sfmD, encoding a putative outer membrane protein, was induced fourfold. fimZ, an activator of FimA (located in the sfm gene cluster, which is involved in type 1 fimbria gene expression) (71), was also induced fourfold. The surface adhesin autotransporter protein Ag43 (encoded by flu) was induced 2.5-fold and is also known to promote biofilm formation (29). The two curli production operons in charge of producing the major (CsgA) and minor (CsgB) subunits of these fibronectin tentacle filaments were induced; csgBAC was induced 2.1- to 2.5-fold, and csgDEFG was induced 2- to 4-fold. Curli fibers are required both for adhesion and biofilm maturation (44). Also, the pgaABCD operon was induced 2- to 2.8-fold; this operon produces the ß-1,6-N-acetyl-D-glucosamine polysaccharide adhesin, which affects biofilm formation (66).

The colanic acid operon (wza wzb wzc wcaABCDEFG), involved in polysaccharide production, was induced (two- to sevenfold) when the highest expression ratio was observed in wcaC, a putative glycosyltransferase. Colanic acid is important for biofilm three-dimensional structure rather than the initial attachment of cells to a surface (44).

Other induced genes in the ydgG biofilm include those which encode uncharacterized proteins, including yihR (11-fold, putative aldose-1-epimerase), yihN (12-fold, putative proton-driven sugar phosphate uptake protein), yihP (11-fold, putative transport protein for galactosides-pentoses-hexuronides), and yfjR (16-fold, putative protein related to phage).

Biofilm formation after deletion of the 15 most significantly induced genes. To investigate the genes which may affect biofilm formation, 15 isogenic E. coli K-12 strains with mutations in the highly induced genes were assayed for biofilm formation after 24 h of growth in the 96-well biofilm assay. As expected, 14 of the 15 had reduced biofilm formation and 10 out of the 15 mutations resulted in 40% or more biofilm removal (Fig. 5). Deletion of the gene that encodes YfjR, a putative transcriptional repressor probably related to phage, showed the largest decrease in biofilm formation (65%). Other genes which affect biofilm formation were a biotin synthesis-related gene (bioF), a gene that encodes a putative methyltransferase (yccW), genes that encode putative regulatory proteins with an unknown function (yjbE and yceO), genes related to anaerobic metabolism (ttdA and fumB), a gene that encodes an uncharacterized protein (yjiP, probably transposon related), a gene that encodes a putative aldose-1-epimerase (yihR), and a polysialic acid production-related gene (gutQ). The acrE deletion did not seem to affect biofilm formation after 24 h of growth, which is probably due to AcrAB complementing AcrEF (30). Therefore, 10 genes (yfjR, bioF, yccW, yjbE, yceO, ttdA, fumB, yjiP, gutQ, and yihR) that were not previously linked with biofilms were shown here to be involved in biofilm formation, including 1 that suggests a role for polysialic acid (via gutQ) in E. coli biofilms.



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  		FIG. 5. Effects of selected deletions on E. coli biofilm formation in 96-well plates after 24 h in LB glu. The experiment was done in duplicate, and 1 standard deviation is shown.


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DISCUSSION
 
In this study, the following lines of evidence show that YdgG is a putative transport protein that either enhances AI-2 secretion or inhibits AI-2 uptake in E. coli. (i) Since external AI-2 addition increases biofilm formation (19), if YdgG promotes AI-2 export or inhibits AI-2 uptake, it is expected that deletion of ydgG should increase biofilms since more AI-2 is internalized, and this was indeed the case (570-fold, Fig. 1). (ii) Deletion of ydgG led to both a decrease in external AI-2 (13-fold) and an increase (10-fold) in internal AI-2 (Fig. 3); both results indicate that YdgG influences the export of AI-2 or inhibits AI-2 uptake. A higher intracellular AI-2 concentration may lead to more AI-2 species being phosphorylated by LsrK, leading to lsr operon induction. (iii) The gene expression pattern in the biofilm observed here upon deletion of ydgG resembles the gene expression pattern observed for restoring luxS (net effect of AI-2 addition to suspended cultures) (49, 58); hence, YdgG influences a large number of genes, which is expected for the transporter of AI-2. (iv) The quorum-sensing regulator qseB is clearly observed to be induced upon deletion of ydgG under exponential growth conditions (Fig. 2), and qseB has been shown in our lab to be induced by enzymatically synthesized AI-2 (19); hence, if YdgG is inactive, either less AI-2 leaves the cell or more AI-2 is internalized, and one would expect AI-2-controlled genes like qseB to be up-regulated. (v) AI-2 controls motility and chemotaxis genes (49, 58); hence, if YdgG influences AI-2 secretion, upon deletion of ydgG, larger intracellular AI-2 concentrations should lead to higher motility, and sixfold more motility was found with the ydgG mutant here. (vi) The predicted secondary protein structure shows that YdgG is most likely to be a transport membrane protein. (vii) YdgG is highly conserved in other bacteria (72 genera have homologs), and 9 of the 11 genera that have the most-closely related YdgG homologs have LuxS homologs (e.g., Shigella, Salmonella, Yersinia, Shewanella, Pasteurella, Haemophilus, Mannheimia, Actinobacillus, and Chromohalobacter). (viii) Corroborating the prediction of YdgG as a transport protein, an increase in drug resistance was observed upon deletion of ydgG. (ix) The proximity of ydgG to both ydgEF, which encodes a multidrug resistance efflux protein (this locus is adjacent to ydgG) (40), and ydgI, which encodes an AcrD protein similar to an arginine-ornithine transporter in Pseudomonas aeruginosa (6) (four genes downstream from ydgG), supports the notion that YdgG is involved in transport. These results suggest that YdgG controls the efflux of AI-2 and fit well with the suggestion that AcrAB of E. coli effluxes quorum signals (45) and with the evidence that the MexAB-OprM multidrug efflux system of P. aeruginosa effluxes the quorum signal N-(3-oxododecanoyl)-L-homoserine lactone (17, 42).

The increase in biofilm formation by the ydgG mutant only when glucose was added to the medium may be attributed to greater differences in intracellular AI-2 production between the ydgG mutant and the wild-type strain (Fig. 3) than when glucose was absent. This result agrees with the increase in AI-2 synthesis upon addition of glucose to the growth medium due to catabolic repression by decreasing the levels of cAMP-cAMP receptor protein (65). Previous reports (60, 65) have shown that adding glucose increases extracellular AI-2 concentrations in mid- to late-exponential phase. Further study of differential gene expression of ydgG mutant biofilm cells in the absence of glucose may reveal the reason for the increase in biofilm formation only when glucose was added to the medium.

The greater biofilm formation of the ydgG mutant may also be due to induction of the indole secretion system acrEF, since similar genes have been related to biofilms previously; for example, Maira-Litrán et al. (36) showed that constitutive expression of acrAB protected E. coli biofilms from ciprofloxacin at 4 µg/liter. Additionally, it has been shown that genes related to the cell envelope and secretion are induced in biofilm cells compared to suspended cultures (5). The induction of acrEF, stationary-phase indole efflux-related genes, upon ydgG deletion and alteration of AI-2 secretion, fits well with our hypothesis that different signals control gene expression at different growth stages in E. coli: AI-2 for exponential-phase growth and an indole-related molecule for stationary-phase growth (50). Indeed, it appears that intracellular indole represses biofilms.

It should be pointed out that ydgG was found to be induced in E. coli JM109 biofilms containing the F' conjugative plasmid (48) whereas the strains used in this study are F–. Also, it has been reported that AI-2 is not necessary for biofilm formation in strains carrying conjugative plasmids (47). However, in this study, since AI-2 is necessary for biofilm formation (19), deletion of ydgG is shown to alter AI-2 transport and consequently to increase biofilm formation.

Gene expression in the biofilm of the ydgG mutant is consistent with the effect of adding AI-2 on the global gene expression in E. coli for suspension cells during exponential growth (49, 58) in that both conditions induce flagellum, motility, and chemotaxis genes. In addition, AI-2-repressible genes involved in growth and cell division (58) were also repressed in the ydgG mutant biofilms; for example, ribosomal genes such as rplX, rplN, rplP, rplS, rplR, rpsK, and rpsT. Also, growth-related genes such as ftsA, ftsQ, fmt (methionyl-tRNA formyltransferase), and miaA [tRNA delta(2)-isopentenylpyrophosphate] were repressed in both studies.

Typical related regulatory genes that were differentially expressed in the biofilm of the ydgG mutant are rpoS (repressed 2.8-fold, encodes the sigma factor {sigma}38) and barA (induced 3.24-fold, encodes a sensor kinase) (4). The RpoS regulon controls gene expression required for survival during starvation periods, transition between the exponential and stationary phases, heat shock, and cold shock (34); more than 100 genes are positively regulated by RpoS (41). We found 75% of the genes induced by RpoS in a stationary-phase suspended culture were repressed in the ydgG biofilm by more than 1.5-fold, and 72% of the genes repressed by RpoS were induced more than 1.5-fold in the ydgG biofilm.

In addition to the known biofilm-related genes induced in this study and the good agreement of the regulatory circuits involved in their expression, some uncharacterized genes related to biofilm formation in E. coli were identified in biofilms of the ydgG mutant. One highly induced operon in the ydgG mutant biofilm is the gut operon, which is related to the production of polysialic acid. gutM and gutQ were induced roughly 10-fold in the biofilm upon deletion of ydgG. gutM encodes the glucitol operon activator (70), and GutQ is similar to KpsF in E. coli K1, which plays a role in assembling the polysialic acid capsule virulence factor (9). Greiner et al. (20) found that 5-N-glycolylneuraminic acid (Neu5Ac), one of the building blocks of polysialic acid, is a constituent of Haemophilus influenzae biofilms. Also, Swords et al. (61) showed that sialylation promotes biofilm formation by H. influenzae. Further study may reveal that polysialic acid as an important component in E. coli and other species biofilms.

Genes related to anaerobic respiration were also highly induced upon deletion of ydgG and include those that are formate transport related (focB and hyfACDR). The significantly higher biomass in the ydgG mutant biofilm is likely to cause anaerobic conditions. Other genes related to anaerobic metabolism that are induced include pflD, encoding pyruvate formate-lyase (13.9-fold); ttdA, encoding L-tartrate dehydratase (13.9-fold); and fumB encoding fumarase B (9.2-fold). TtdA and FumB are enzymes for converting tartrate to oxaloacetate and malate to fumarate, respectively. There are three fumarase isozymes in E. coli, fumarases A, B, and C (products of fumA, fumB, and fumC, respectively). The cell adapts to changing environmental oxygen conditions by utilizing the different isozymes; hence, in the ydgG mutant biofilm, more biofilm may cause anaerobic conditions and fumB is relatively activated. TtdA is a stereospecific enzyme that is known to be induced during anaerobic growth with glycerol (46).

Among the induced genes (Table 2), more than 20 regulatory proteins were identified. The most induced was yjiP (103 aa, 20-fold). alpA was induced 6.5-fold and encodes a transcriptional activator related to phage functions (28). Differential gene expression of phage-related genes in biofilms has been reported for Xylella fastidiosa by de Souza et al. (15) on the basis of DNA microarrays. Also, Webb et al. (67) related phage functions and biofilms of P. aeruginosa. Further study needs to be done to establish the relationship of phage-related functions and biofilm formation in E. coli.

Also, 80% of the genes differentially expressed in a continuous reactor after 6 days of operation at a growth rate of 0.03/h (48) were also differentially expressed in the 24-h biofilm upon deletion of ydgG. This indicates the ydgG mutation causes the genes that are required for steady-state biofilm formation to be expressed, which results in greater biofilm formation.

We found 31% of the E. coli genome was differentially induced more than twofold in the ydgG mutant biofilm compared to the wild-type biofilm culture, and 8% of the genes were differentially repressed more than twofold. YdgG was found to repress cell surface determinants (genes related to flagellum, type 1 fimbria, Ag43, curli, and polysaccharide production), and it appears it controls these genes through AI-2 transport. Additionally, 10 genes previously not linked to biofilms were found here to influence biofilm formation in E. coli, and polysialic acid appears to have a role in the biofilm matrix. The results presented in this paper show that YdgG either enhances secretion of AI-2 or inhibits AI-2 uptake and that altering AI-2 intracellular concentrations affects global gene expression in the biofilms. Hence, we propose a new name for this locus: transport of quorum-sensing signal or tqsA. These results corroborate previous results that AI-2 is directly involved in biofilm formation (19) and imply that AI-2 must be actively transported from cells for cell signaling.


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ACKNOWLEDGMENTS
 
We thank A. Heydorn of the Technical University of Denmark for kindly providing COMSTAT; S. Molin of the Technical University of Denmark for sending plasmid pCM18; Hirotada Mori of the Nara Institute of Science in Japan for sending the E. coli K-12 BW25113 mutants; James Kaper of the University of Maryland for sending plasmids pVS159, pVS176, pVS175, pVS182, and pVS183; and William E. Bentley of the University of Maryland for sending plasmid pLW11.


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FOOTNOTES
 
* Corresponding author. Mailing address: Departments of Chemical Engineering and Biology, Texas A & M University, 220 Jack E. Brown Building, College Station, TX 77843-3122. Phone: (979) 862-1588. Fax: (979) 845-6884. E-mail: Thomas.Wood{at}chemail.tamu.edu. Back


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Journal of Bacteriology, January 2006, p. 587-598, Vol. 188, No. 2
0021-9193/06/$08.00+0     doi:10.1128/JB.188.2.587-598.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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