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Gene expression in Escherichia coli biofilms

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Gene expression in Escherichia coli biofilms

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     Appl Microbiol Biotechnol (2004) 64: 515?C524 DOI

    10.1007/s00253-003-1517-y

     APPLIED GENE TICS AN D MO LECULA R BIO TECH NOLOGY

     D. Ren . L. A. Bedzyk . S. M. Thomas . R. W. Ye . T. K. Wood

     Gene expression in Escherichia coli biofilms

     Received: 28 July 2003 / Revised: 31 October 2003 / Accepted: 21 November 2003 / Published online: 16 January 2004 # Springer-Verlag 2004

     Abstract DNA microarrays were used to study the gene expression profile of Escherichia coli JM109 and K12 biofilms. Both glass wool in shake flasks and mild steel 1010 plates in continuous reactors were used to create the biofilms. For the biofilms grown on glass wool, 22 genes were induced significantly (p?Ü0.05) compared to suspension cells, including several genes for the stress response (hslS, hslT, hha, and soxS), type I fimbriae (fimG), metabolism (metK), and 11 genes of unknown function (ybaJ, ychM, yefM, ygfA, b1060, b1112, b2377, b3022, b1373, b1601, and b0836). The DNA microarray results were corroborated with RNA dot blotting. For the biofilm grown on mild steel plates, the DNA microarray data showed that, at a specific growth rate of 0.05/h, the mature biofilm after 5 days in the continuous reactors did not exhibit differential gene expression compared to suspension cells although genes were induced at 0.03/h. The present study suggests that biofilm gene expression is strongly associated with environmental conditions and that stress genes are involved in E. coli JM109 biofilm formation.

     Introduction

     Bacterial biofilms?ªsessile microbial communities formed on solid surfaces?ªare ubiquitous in natural environments as well as those related to medicine and engineering (Elvers and Lappin-Scott 2000). Due to their high resistance to antibiotics (Nickel et al. 1985), biofilms cause serious problems to human health such as lung infections, dental disease, and urinary tract infections (Potera 1999). It is estimated that biofilms are involved in 65% of human bacterial infections (Potera 1999). Biofilms are also problematic in industry since they can stimulate microbial-induced corrosion on the surfaces of pipes, reduce the efficiency of heat exchangers, and cause food spoilage (Elvers and Lappin-Scott 2000). Biofilm formation is a dynamic process including attachment of the cells to the surface, increase in cell population, and maturation of the biofilm (Elvers and Lappin-Scott 2000; Kuchma and O??Toole 2000). The fully developed biofilm has a three-dimensional structure made up of a polysaccharide matrix that

    contains water channels for the transfer of nutrients and for the removal of wastes (Elvers and Lappin-Scott 2000). Biofilms have attracted extensive research; however, the understanding of biofilm formation at the genetic level lags behind what is known of their physical properties (Kolter and Losick 1998). Recently, random insertion mutagenesis and screening has been used successfully to show that motility and type I fimbriae are important for Escherichia coli early biofilm formation (Pratt and Kolter 1998), and that flagellar and twitching motility are necessary for Pseudomonas aeruginosa early biofilm formation (O??Toole and Kolter 1998). It has also been suggested that the sporulation gene spo0A is important for biofilm formation of Bacillus subtilis (Hamon and Lazazzera 2001) and that the quorum-sensing system (bacterial gene expression controlled by sensing their population; Bassler 1999) luxI/luxR is important for biofilm formation with P. aeruginosa (Davies et al. 1998); however, this is controversial as several reports dispute the importance of quorum sensing in biofilm

     D. Ren . T. K. Wood (*) Departments of Chemical Engineering and Molecular and Cell Biology, University of Connecticut, 191 Auditorium Road, Storrs, CT 06269?C3222, USA e-mail: twood@engr.uconn.edu Tel.: +1-860-486-2483 Fax: +1-860-486-2959 L. A. Bedzyk . S. M. Thomas . R. W. Ye Experimental Station E328/B33, DuPont Central Research and Development, Wilmington, DE 19880, USA Present address: D. Ren Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA

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     formation of Gram-negative strains (Sauer and Camper 2001; Heydorn et al. 2002; Purevdorj et al. 2002). Compared to the traditional methods of studying individual genes, proteomics provides a global study of gene expression and has been used successfully to study biofilm formation of Bacillus cereus (Oosthuizen et al. 2001, 2002; Steyn et al. 2001). By using 2-D electrophoresis, it was found that 15 proteins were uniquely expressed in 2-h B. cereus biofilms and 7 proteins in 18h biofilms (Oosthuizen et al. 2002). Moreover, due to the dynamic character of biofilms, the green fluorescent protein system has been used to study gene expression in vivo and in three dimensions (Kievit et al. 2001; Heydorn et al. 2002). With this approach, it was found that the P. aeruginosa quorum sensing genes lasI and rhlI were most expressed at the attachment surface of a liquid-solid interface biofilm, and the expression of lasI decreased with time while rhlI was more consistent during biofilm development (Kievit et al. 2001). DNA microarrays provide a means to monitor the global gene expression profile in response to different stimuli (Whiteley et al. 2001). They have been widely used to study microbial physiology, including response

    to heat shock and other stresses (Wilson et al. 1999; Helmann et al. 2001; Zheng et al. 2001), quorum sensing (DeLisa et al. 2001; Sperandio et al. 2001), anaerobic metabolism (Ye et al. 2000), sporulation (Fawcett et al. 2000), and biofilm formation (Whiteley et al. 2001; Schembri et al. 2003; Stanley et al. 2003). DNA microarrays have advantages for understanding biofilm formation because this mode of growth requires a significant change in gene expression for cell attachment and structure development (Kuchma and O??Toole 2000). Recently, Schembri et al. (2003) studied the early stages of E. coli MG1655 biofilm formation (on glass slides in flow chambers) with DNA microarrays, and reported that the biofilm cells have 5.4% or 13.5% of genes differentially expressed compared to exponentially growing suspension cells or stationary suspension cells, respectively. While DNA microarrays are a promising approach for studying biofilms, there are some challenges, including the short half-life of E. coli mRNA (3?C5 min; Lodish et al. 1999); hence, sampling and cell lysis should be rapid so that transcription can be stopped before significant mRNA degradation occurs. This is very important for biofilm experiments because harvesting biofilm cells takes longer than harvesting regular suspension cells. In the present study, transcription was terminated within seconds to 1 min. Also, a rigorous criterion of 23S rRNA/16S rRNA >2 was used to check RNA integrity as prescribed by the Qiagen RNeasy Mini Kit. Since biofilm formation is a dynamic procedure and is sensitive to many environmental factors, growth conditions were optimized to ensure adequate yields of total RNA from the biofilm and suspension cells in each individual reactor. Hence, gene expression in the biofilm was not compared to separately grown suspension cells and no combined parallel samples were analyzed as in a previous study (Schembri et al. 2003).

     Compared to the genes involved in cell attachment and biofilm development, little is known about the genes involved in maintaining biofilms. However, this information is important since biofilm infections are strongly associated with the antibiotic resistance of mature biofilms (Nickel et al. 1985; Potera 1999). In the present study, both 7-h batch and 5-day continuous E. coli biofilms were studied for differential gene expression, and it is one of the first reports biofilm formation studied with DNA microarrays. Of the 22 genes found as important biofilm genes of E. coli JM109, 17 were identified for the first time.

     Materials and methods

     Bacterial strains and growth medium E. coli JM109 (recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi ??(lac-proAB) F?ä[traD36 proAB+ lacIq lacZ??M15]) (Yanisch-Perron et al. 1985) and E. coli K12 (F? ?Ë? ilvG rfb50 rph1) (ATCC 25404) were used to grow biofilms on glass wool and

    metal surfaces, respectively. LB medium (Sambrook et al. 1989) containing 10 g/l tryptone, 5 g/l yeast extract, and 10 g/l NaCl was used to grow the strains and develop the biofilms.

     E. coli JM109 biofilm formation on glass wool in batch reactors E. coli JM109 was grown in LB medium overnight, and 1 ml overnight culture was inoculated into a 250 ml shake flask containing 100 ml fresh LB and 0.5 g untreated glass wool (Corning Glass Works, Corning, N.Y.). The cells were incubated with shaking (250 rpm in a C25 incubator shaker; New Brunswick Scientific, Edison, N.J.) at 37?ãC to form the biofilm on the glass wool. Seven hours after inoculation, the glass wool was taken from the culture and quickly and gently washed two times in 100 ml 0?ãC 0.85% NaCl buffer (within 30 s). Then the biofilm cells were removed from the glass wool by sonication in 200 ml 0?ãC 0.85% NaCl buffer for 2 min. The buffer containing biofilm cells was centrifuged (10,000 g in a J2-HS centrifuge; Beckman, Palo Alto, Calif.) for 3 min at ?2?ãC to precipitate the cells. The biofilm cells were then resuspended in 6 ml 0?ãC 0.85% NaCl buffer, transferred to mini bead beater tubes, and centrifuged (10,000 g) for 15 s at room temperature; the cell pellets were flash-frozen in a dry ice-ethanol bath. While sonicating the biofilm cells, suspension cells with an optical density at 600 nm (OD) of 2 were harvested by centrifugation (10,000 g) at room temperature for 15 s in mini bead beater tubes. The cell pellets were then flash-frozen in a dry ice-ethanol bath and kept at ?80?ãC until RNA isolation.

     E. coli K12 biofilm formation on metal in continuous reactors Biofilms were developed on mild steel 1010 plates in continuous reactors that contained 150 ml LB medium and in which the temperature was controlled at 34?ãC. Each autoclavable reactor consists of a 5.5 cm conical glass cell, a mild steel 1010 plate at the bottom, and a Teflon top (?rnek et al. 2002). Air was filtered and supplied to the reactors at 200 ml/min. The reactors were inoculated with a 1:150 dilution of an overnight culture of E. coli K12. Continuous nutrient addition commenced 1 day after inoculation at 8 ml/h LB medium [dilution rate (?Ì)=0.05 h?1]. Biofilm and suspension cells were sampled 5 days after inoculation. After opening the reactor quickly, the metal plate was washed quickly and gently in 0?ãC 0.85% NaCl buffer to remove the contaminating suspension cells and sonicated (FS3 sonicator; Fisher, Hanover Park, Ill.) for 2 min in 0?ãC 0.85% NaCl buffer to remove the biofilm

     517 cells from the metal surface. The buffer containing biofilm cells was centrifuged for 3 min at ?2?ãC (10,000 g in a Beckman J2-HS centrifuge), and the precipitated biofilm cells were resuspended in 6 ml 0?ãC 0.85% NaCl buffer, transferred to cold mini bead beater tubes (Biospec, Bartlesville, Okla.), and centrifuged (10,000 g in a Hermle

    microcentrifuge; Labnet, Woodbridge, N.J.) for 15 s at room temperature. The cell pellets were frozen immediately by putting the tubes in a dry ice-ethanol bath. Cell samples were kept at ?80?ãC until RNA isolation. During the sonication of the metal plates, suspension cells were taken by pipetting into mini bead beater tubes. Cells were centrifuged (10,000 g in a Hermle microcentrifuge) for 15 s at room temperature. The cell pellets were then frozen in a dry ice-ethanol bath and kept at ?80?ãC until RNA isolation. Hybridization and washing The suspension and the biofilm cDNA samples (6 ?Ìg of each) were each labeled with both Cy3 and Cy5 dyes to remove artifacts related to different labeling efficiencies; hence, each experiment needed two slides. The Cy3-labeled suspension sample and Cy5-labeled biofilm sample were hybridized on the first slide. Similarly, the Cy5-labeled suspension sample and Cy3-labeled biofilm sample were hybridized on the second slide. Since each gene has two spots on a slide, the two hybridizations generated eight data points for each gene (four points for the suspension sample and four points for the biofilm sample). The DNA microarrays were incubated in prehybridization solution [3.5?Á SSC (Invitrogen), 0.1% SDS (Invitrogen), 0.1% bovine serum albumin (Invitrogen)] at 45?ãC for 20 min. The arrays were rinsed with double-distilled water (ddH2O) and spun dry by centrifugation. Labeled cDNA (6 ?Ìg) was concentrated to 10 ?Ìl total volume and mixed with 10 ?Ìl 4?Á cDNA hybridization solution (Full Moon Biosystems) and 20 ?Ìl formamide (EM Science, Gibbstown, N.J.). The hybridization mix was heated to 95?ãC for 2 min and added to the DNA microarrays; each array was covered with a coverslip (Corning) and incubated overnight at 37?ãC for hybridization. When the hybridization was finished, the coverslips were removed in 1?Á SSC, 0.1% SDS at room temperature, and the arrays were washed once for 5 min in 1?Á SSC, 0.1% SDS at 40?ãC, twice for 10 min in 0.1?Á SSC, 0.1% SDS at 40?ãC, and twice for 1 min in 0.1?Á SSC at 40?ãC. The arrays were quickly rinsed by dipping in ddH2O at room temperature and then spun dry by centrifugation.

     Total RNA isolation To lyse the cells, 1.0 ml RLT buffer (Qiagen, Valencia, Calif.) and 0.2 ml 0.1 mm zirconia/silica beads (Biospec) were added to the frozen bead beater tubes containing the cell pellets. The tubes were closed tightly and beat for 30 s at the maximum speed in a mini bead beater (Cat. No. 3110BX, Biospec). Total RNA was isolated following the protocol of the RNeasy Mini Kit (Qiagen), including an on-column DNase digestion with RNase-free DNase (Qiagen). An OD reading at 260 nm was used to quantify the RNA yield. OD260/OD280 and 23S/16S rRNA were measured to check the purity and integrity of RNA (RNeasy Mini handbook, Qiagen).

     DNA microarrays E. coli DNA microarrays were prepared as described previously (Wei et al. 2001). Each gene probe was synthesized by

    polymerase chain reaction (PCR) as the full open reading frame, 200?C2,000 nt. The double-strand PCR products were denatured in 50% dimethyl sulfoxide and spotted onto aminosilane slides (Full Moon Biosystems, Sunnyvale, Calif.). Total RNA isolated from the experimental samples was converted by reverse transcription to complimentary DNA (cDNA), which was hybridized to the denatured DNA probes on the microarray slides to quantify the expression level of each gene. It has been shown that each array can detect 4,228 of the 4,290 E. coli ORFs (Wei et al. 2001). Each gene has two spots per slide.

     Image and data analysis The hybridized slides were scanned with a Generation III Array Scanner (Molecular Dynamics, Sunnyvale, Calif.); 570 nm and 670 nm were used to quantify the probes labeled with Cy3 and Cy5, respectively. The signal was quantified with Array Vision 4.0 or 6.0 software (Imaging Research, Ontario, Canada). Genes were identified as differentially expressed in the biofilm if the expression ratio was greater than 2.5 and the P-value (t-test) was less than 0.05. P-Values were calculated on log-transformed, normalized intensities. Including the P-value criterion ensures the reliability of the induced/repressed gene list. Normalization was relative to the median total fluorescent intensity per slide per channel. The gene functions were obtained from the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/).

     Synthesis of Cy3- or Cy5-labeled cDNA To convert the total RNA into labeled cDNA, reverse transcription was performed in 1.5 ml microcentrifuge tubes (Fisher) to which 6 ?Ìg total RNA and 6 ?Ìg random hexamer primers (Invitrogen, Carlsbad, Calif.) were added; the volume was adjusted to 24 ?Ìl with RNase-free water (Invitrogen). The mixture was incubated for 10 min at 70?ãC followed by 10 min at room temperature for annealing, then the reaction components were added, consisting of 8 ?Ìl 5?Á SuperScript II reaction buffer (Invitrogen), 4 ?Ìl 0.1 M dithiothreitol (DTT) (Invitrogen), 1 ?Ìl deoxynucleoside triphosphates (dNTPs) mix (2 mM each of dATP, dGTP, dTTP and 1 mM dCTP), 1 ?Ìl 0.5 mM Cy3- or Cy5-labeled dCTP (Amersham Biosciences, Piscataway, N.J.), and 2 ?Ìl SuperScript II reverse transcriptase (10 U/?Ìl, Invitrogen). cDNA synthesis was conducted at 42?ãC for 2 h and stopped by heating at 94?ãC for 5 min. After cDNA synthesis, the RNA template was removed with 2 ?Ìl 2.5 M NaOH. The pH was neutralized with 10 ?Ìl 2 M HEPES buffer, and the cDNA was purified with a Qiaquick PCR Mini Kit (Qiagen). The efficiency of labeling was checked via absorbance at 260 nm for the cDNA concentration, 550 nm for Cy3 incorporation, and 650 nm for Cy5 incorporation. RNA dot blotting Digoxigenin (DIG)-labeled DNA probes of six genes, b3022, hha, soxS, yhaJ, hslT, and b0753, were synthesized using the PCR DIG Probe Synthesis Kit (Roche Applied Science, Mannheim, Germany). PCR was performed in 30 cycles at 95?ãC for 30 sec,

    60?ãC for 30 sec, and 72?ãC for 40 sec. The final extension was at 72?ãC for 7 min. The probes have lengths of between 172 bp and 400 bp (see Table 3 for specific primers). Total RNA (1.25, 2.5, or 5 ?Ìg) from independent cell cultures (different experiments than those used for the DNA microarrays but identical culture conditions) was blotted on positively-charged nylon membranes (Boehringer Ingelheim, Ridgefield, Conn.) using a Bio-Dot Microfiltration Apparatus (BioRad, Richmond, Calif.). Total RNA was fixed by baking for 2 h at 80?ãC. DNA probes (about 400 ng, a serial dilution of RNA samples was tested to ensure excess of the DNA probes) were denatured in boiling water for 5 min before hybridizing to RNA. Hybridization (50?ãC, 16 h) and washing were conducted by following the protocol for DIG labeling and detection (Roche Applied Science). To detect the signal, disodium

    3-(4-methoxyspiro {1,2-dioxetane-3,2-(5-chloro)tricycle [3.3.1.1,7] decan}-4-yl) phenyl phosphate (Roche Applied Science) was used as a substrate to give chemiluminescence, and the light was recorded by Biomax X-ray film (Kodak, Rochester, N.Y.).

     518 Microscopy observation of E. coli JM109 biofilm on glass wool The biofilm was developed as for the microarray experiments. The glass wool containing the biofilm was gently washed two times in 0.85% NaCl buffer and stained with 0.1% crystal violet for 20 min, and the extra dye was washed off with 0.85% NaCl buffer (twice). The cells on the surface of glass wool were imaged using optical microscopy at 400?Á total magnification (Zeiss Axioskop, Zeiss, Oberkochen, Germany).

     Confocal laser scanning microscopy Metal plates with attached E. coli K12 biofilms were immersed in 0.85% NaCl buffer to remove the bulk supernatant cells. The Live/ Dead Baclight bacteria viability assay kit (L-7007; Molecular Probes, Eugene, Ore.) was used to stain the biofilm at a concentration of 1.125 ?Ìl/ml (for both kit components A and B). The staining process was performed in the dark at room temperature for 30 min. The stained plates were protected with a cover glass and observed with confocal microscopy (MRC 600, Bio-Rad, Hercules, Calif.), which was conducted using the dual channel mode (K1/K2 filter block combination); the sample was excited at 488 nm and biofilm thickness was measured by focusing through the biofilm from the solid-liquid interface to the metal surface and averaging 5?C 6 positions. The images were processed with National Institutes of Health Image 1.6 and Adobe Photoshop 5.5 (Adobe, San Jose, Calif.). Z-section images (single image of the sectioning plane parallel to the metal surface) and vertical section images (a collection of images orthogonal to the metal surface at one position) were obtained.

     Fig. 1 Escherichia coli JM109 biofilm formed on glass wool in shake flasks 7 h after inoculation. Total magnification 400?Á. Bar 10 ?Ìm

     Table 1 Genes induced in the Escherichia coli JM109 biofilm grown

    on glass wool. Each sample set contained one biofilm RNA sample and one suspension RNA sample from the same independent reactor. Genes consistently induced in both data sets are in bold Gene Known hslS soxS hha glnA rhoL hslT fimG rho tus metK b-number genes b3686 b4062 b0460 b3870 b3782 b3687 b4319 b3783 b1610 b2942 Expression ratio (set 1) Expression ratio (set 2) Description

     48 35 20 12 5 4 3 3 3 2 2 37 21 17 8 5 5 4 3 3 2 2

     16 63 30 1.5 6 4 1.4 5 4 3 4 35 4 7 8 6 8 5 1.3 3 3 3

     trpE b1264 Unknown genes ybaJ b0461 b2377 b2377 b1112 b1112 b3022 b3022 b1601 ygfA b1060 b1373 b0836 ychM yefM b1601 b2912 b1060 b1373 b0836 b1206 b2017

     Heat shock protein Regulation of superoxide response regulon, global regulator Haemolysin expression modulating protein, regulator Glutamine synthetase Leader, RNA synthesis, modification, DNA transcription Heat shock protein Fimbrial morphology Transcription termination factor Rho, polarity suppressor Factor, DNA-replication, repair, restriction/modification Methionine adenosyltransferase 1 (adomet synthetase), methyl and propylamine donor, corepressor of met genes Anthranilate synthase component I Unknown Unknown Unknown, possible stress response (Zheng et al. 2001) Unknown (protein 36% identical to Sinorhizobium melilotiLrp, leucine-responsive regulatory protein, for LPS synthesis) Unknown Putative ligase Unknown Unknown Putative receptor Unknown Unknown

     519

     Results

     E. coli JM109 biofilm formation on glass wool in batch reactors E. coli JM109 was tested and found to yield good biofilms when grown on glass wool for 7 h (about 40 ?Ìg total RNA was obtained from biofilm cells in each culture). Observation with a microscope at 400?Á indicated the cells attached well to the glass wool and cell clusters were clearly seen (Fig. 1). This condition was therefore used for further study. Two independent sets of samples (each set had a suspension RNA sample and a biofilm RNA sample harvested from the same individual flask) were harvested and analyzed with DNA microarrays. In the first set, 13 genes (7 with unknown functions) were induced in the biofilm (expressed more than 2.5-fold over suspension cells, P-value <0.05), while 252 genes were repressed more than 2.5-fold. Hence, there are more repressed genes than induced genes in the biofilm. Similarly, in the second set, 19 genes (9 with unknown functions) were induced in the biofilm, while 718 genes were repressed more than 2.5-fold. These two sets of samples agreed well except that the second set has more repressed genes. Consistently, 9 genes (5 with unknown functions) were induced and 201 genes were repressed in both sets of samples. Hence, 69% of the induced genes and 79% of the repressed genes from the first set were identified

    in the second set. Interestingly, although there were only 9 induced genes shared by these

     two sets of samples, the other 4 induced genes from the first set sample and 9 of the other 10 induced genes (except for insB_6) from the second set were also upregulated in the other sample set (either the levels of induction were lower than the 2.5 threshold or the P-value was higher than 0.05). Hence, we purpose that all of these 22 consistently up-regulated genes are candidates for biofilm-formation genes and they are shown in Table 1. Table 2 lists the 20 consistently most-repressed genes. Induction of stress genes in the glass wool biofilm The microarray results from the glass wool biofilms identified several induced genes related to the stress response (hslS, hslT, hha, and soxS), type I fimbriae (fimG), metabolism (metK), and some genes with unknown functions (ybaJ, ychM, yefM, ygfA, b1060, b1112, b2377, b3022, b1373, b1601, and b0836) (Table 1). hslT (alternate name ibpA) and hslS (alternate name ibpB) encode small proteins for response to heat shock and superoxide stresses (Kitagawa et al. 2000), and soxS encodes the regulator SoxS in response to superoxide (Zheng et al. 2001; Mich??n et al. 2002). E. coli cells overexpressing hslT and hslS are more resistant to different stresses including heat, ethanol, and superoxide (Kitagawa et al. 2000). Three genes found induced in the glass wool biofilm in the present study, b1112, soxS, and hslS, were also found induced in E. coli in response to hydrogen peroxide (Zheng et al. 2001). Hence, upregulation of hslT,

     Table 2 Genes repressed in the E. coli JM109 biofilm grown on glass wool (the 20 consistently most-repressed genes). Each sample set contained one biofilm RNA sample and one suspension RNA sample from the same independent reactor Gene b-number Expression ratio (set 1) Expression ratio (set 2)

    Description ?9 ?8 ?7 ?7 ?7 ?7 ?6 ?14 ?9 ?8 ?7 ?7 ?7 ?7 ?7 ?7 ?6 ?6

     ?6 ?6 ?7 ?7 ?6 ?5 ?5 ?5 ?5 ?3 ?4 ?6 ?6 ?3 ?4 ?6 ?8 ?4 ?5 ?5 ?8 ?6

     Known genes gabD b2661 lacZ b0344 ygaF b2660 artI b0863 artP b0864 hyaA b0972 fruK b2168 Unknown genes b0725 b0725 yhfG b3362 b0753 b0753 b1444 b1444 yhiD b3508 b1836 b1836 b0334 b0334 yceK b1050 ybgF b0742 yhiM b3491 b1747 b1747 b1824 b1824 b0333 b0333

     Succinate-semialdehyde dehydrogenase, NADP-dependent activity Enzyme, degradation of small molecules: carbon compounds Unknown Transport, transport of small molecules: amino acids, amines Transport, transport of small molecules: amino acids, amines Enzyme, energy metabolism, carbon: aerobic respiration Enzyme, energy metabolism, carbon: glycolysis Unknown Unknown Putative regulator, not classified Putative aldehyde dehydrogenase Putative transport ATPase Unknown Unknown Unknown Unknown Unknown Unknown Unknown Putative enzyme, not classified

     520 Table 3 Confirmation of gene expression in the E. coli JM109 biofilm grown on glass wool with RNA dot blotting. Each sample set contained one biofilm RNA sample and one suspension RNA sample from the same independent reactor Gene Primers used for probe synthesis Expression ratio (DNA microarray) Set 1 b3022 hha soxS ybaJ hslT b0753 5?ä-ATGGAAAAACGCACACCACATACAC-3?ä 5?ä-AAGCCTGGGTCTGTAAACATCCTGC-3?ä 5?ä-GTCCGAAAAACCTTTAACGAAAACC-3?ä 5?ä-TTTATTCATGGTCAATTCGGCGAGG-3?ä 5?ä-TTCAAAGTGGTACTTGCAACGAATG-3?ä 5?ä-TAATCGCTGGGAGTGCGATCAAACT-3?ä 5?ä-ATGGATGAATACTCACCCAAAAGAC-3?ä 5?ä-TCCATTTCTGAAGATCCTGCATATT-3?ä 5?ä-CGATTTATCCCCACTGATGCGTCAA-3?ä 5?ä-ACGTTCGCTGATAGCGATACGCTGC-3?ä 5?ä-ACTGGCCACATTATTTCTGACTGCC-3?ä 5?ä-TTACTGCGTGGTACCGTCGGTTTTG-3?ä +8 +20 +35 +37 +4 ?8 Set 2 +8 +30 +63 +35 +4 ?6 +5 +10 +20 +10 +10 ?5 Expression ratio (RNA dot blotting)

     hslS, soxS, and b1112 here suggests the E. coli JM109 biofilm on glass wool needs expression of these stress genes. Also induced in the biofilm are the regulator hha and the downstream gene ybaJ with unknown function. The hha gene plays a role in a temperature and

    osmolaritydependent regulation of expression of E. coli virulence factors including hemolysin and Vir antigen (Mourino et al. 1996). Induction of genes with unknown functions in the glass wool biofilm Of the 22 candidate biofilm genes (Table 1), 11 have unknown functions. One of these, b3022, was induced in both sets of E. coli JM109 glass wool biofilm samples compared to suspension samples. Also, it was expressed in all three sets of the E. coli K12 biofilm samples grown in continuous reactors (Table 4, discussed later). A BLAST search (NCBI database, http://www.ncbi.nlm.nih.gov/) indicates it encodes a protein that has homology to proteins from other strains such as a hypothetical protein from Yersinia pestis (67% identity) and a hypothetical protein from Ralstonia eutropha (60% identity). Interestingly, it has 36% identity to Lrp of Sinorhizobium meliloti, which is a

    leucine-responsive regulatory protein used for lipopolysaccharide (LPS) synthesis (Lagares et al. 2001). Further study with knockout mutation of this gene may generate information for understanding E. coli biofilms. Repression of genes in the glass wool biofilm Previous reports showed E. coli CsrA is a repressor of biofilm formation and expression of csrA is decreased during biofilm formation (Jackson et al. 2002). In agreement with this, csrA was found repressed in our glass wool biofilm cells compared to suspension cells (?1.6?Á in sample set 1 and ?2.9?Á in sample set 2). Table 2

     shows the 20 consistent, most-repressed genes including those with functions for metabolism, transport, and some with unknown functions. Validation of the DNA microarray results with RNA dot blotting To corroborate the gene expression results of the glass wool biofilm, total RNA was isolated from independent reactor samples, prepared as for the

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