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< or =0.05) 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.

The quorum sensing disrupter (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) of the alga Delisea pulchra was previously found by us (Environ Microbiol 3:731-736, 2001) to inhibit quorum sensing in Escherichia coli via autoinducer-2 (AI-2, produced by LuxS). In this study, DNA microarrays were used to study the genetic basis of this natural furanone inhibition of AI-2 signaling (significant values with p < 0.05 are reported). Using DNA microarrays, the AI-2 mutant Escherichia coli DH5alpha was compared with the AI-2 wild-type strain, E. coli K12, to determine how AI-2 influenced gene expression. Escherichia coli K12 was also grown with 0 and 60 microg/mL furanone to study the inhibition of quorum sensing gene expression. It was found that 166 genes were differentially expressed by AI-2 (67 were induced and 99 were repressed) and 90 genes were differentially expressed by furanone (34 were induced and 56 were repressed). Importantly, 79% (44 out of 56) of the genes repressed by furanone were induced by AI-2, which indicated that furanone inhibited AI-2 signaling and influenced the same suite of genes as a regulon. Most of these genes have functions of chemotaxis, motility, and flagellar synthesis. Interestingly, the aerotaxis genes aer and tsr were discovered to be induced by AI-2 and repressed by furanone. Representative microarray results were confirmed by RNA dot blotting. Furthermore, the E. coli air-liquid interface biofilm formation was repressed by furanone, supporting the results that taxis and flagellar genes were repressed by furanone. The autoinducer bioassay indicated that 100 microg/mL furanone decreased the extracellular concentration of AI-2 2-fold, yet luxS and pfs transcription were not significantly altered. Hence, furanone appeared to alter AI-2 signaling post-transcriptionally.

No abstract

Quorum sensing via autoinducer-2 (AI-2) has been identified in different strains, including those from Escherichia, Vibrio, Streptococcus, and Bacillus species, and previous studies have suggested the existence of additional quorum-sensing signals working in the stationary phase of Escherichia coli cultures. To investigate the presence and global effect of these possible quorum-sensing signals other than AI-2, DNA microarrays were used to study the effect of stationary-phase signals on the gene expression of early exponential-phase cells of the AI-2-deficient strain E. coli DH5␣. For statistically significant differential gene expression (P < 0.05), 14 genes were induced by supernatants from a stationary culture and 6 genes were repressed, suggesting the involvement of indole (induction of tnaA and tnaL) and phosphate (repression of phoA, phoB, and phoU). To study the stability of the signals, the stationary-phase supernatant was autoclaved and was used to study its effect on E. coli gene expression. Three genes were induced by autoclaved stationary-phase supernatant, and 34 genes were repressed. In total, three genes (ompC, ptsA, and btuB) were induced and five genes (nupC, phoB, phoU, argT, and ompF) were repressed by both fresh and autoclaved stationary-phase supernatants. Furthermore, supernatant from E. coli DH5␣ stationary culture was found to repress E. coli K-12 AI-2 concentrations by 4.8-fold ؎ 0.4-fold, suggesting that an additional quorum-sensing system in E. coli exists and that gene expression is controlled as a network with different signals working at different growth stages.Quorum sensing, the regulation of gene expression by producing and responding to secreted autoinducers (AIs) whose concentrations reflect the population density (2), commonly exists in bacteria. Gram-negative bacteria use acylated homoserine lactones as AIs, and gram-positive bacteria use oligopeptides (2, 16). When the cell density is high, the binding of AIs to cell receptors regulates gene expression for a variety of phenotypes, such as production of virulence factors (4), protein production (7), siderophore synthesis (29), bioluminescence (5), biofilm formation (6), and plasmid conjugation (15). Generally, each bacterial species uses its own signal; however, a common AI-2 signal has been discovered for interspecies communication (30,31,37). Escherichia coli does not produce acylated homoserine lactone, but it possesses the AI-2 quorum-sensing system (30). Recent studies have found that E. coli O157:H7 uses AI-2 to control the expression of virulence factors, type III secretion, chemotaxis, flagellar synthesis, and motility (24-26) and that E. coli K-12 uses AI-2 to control chemotaxis, motility, and flagellar synthesis (D. Ren, A. Bedzyk, S. M. Thomas, R. W. Ye, and T. K. Wood, submitted for publication). In addition, E. coli RP37 uses AI-2 to control cell aggregation (17). In Salmonella enterica serovar Typhimurium, AI-2 concentrations are maximal in mid-exponential-phase growth, and it is degraded in the stationary phase ...

Biological oxidation of cyclic ketones normally results in formation of the corresponding dicarboxylic acids, which are further metabolized in the cell. Rhodococcus ruber strain SC1 was isolated from an industrial wastewater bioreactor that was able to utilize cyclododecanone as the sole carbon source. A reverse genetic approach was used to isolate a 10-kb gene cluster containing all genes required for oxidative conversion of cyclododecanone to 1,12-dodecanedioic acid (DDDA). The genes required for cyclododecanone oxidation were only marginally similar to the analogous genes for cyclohexanone oxidation. The biochemical function of the enzymes encoded on the 10-kb gene cluster, the flavin monooxygenase, the lactone hydrolase, the alcohol dehydrogenase, and the aldehyde dehydrogenase, was determined in Escherichia coli based on the ability to convert cyclododecanone. Recombinant E. coli strains grown in the presence of cyclododecanone accumulated lauryl lactone, 12-hydroxylauric acid, and/or DDDA depending on the genes cloned. The cyclododecanone monooxygenase is a type 1 Baeyer-Villiger flavin monooxygenase (FAD as cofactor) and exhibited substrate specificity towards long-chain cyclic ketones (C 11 to C 15 ), which is different from the specificity of cyclohexanone monooxygenase favoring short-chain cyclic compounds (C 5 to C 7 ).

Biological oxidation of cyclic alcohols normally results in formation of the corresponding dicarboxylic acids, which are further metabolized and enter the central carbon metabolism in the cell. We isolated an Acinetobacter sp. from an industrial wastewater bioreactor that utilized cyclohexanol as a sole carbon source. A cosmid library was constructed from Acinetobacter sp. strain SE19, and oxidation of cyclohexanol to adipic acid was demonstrated in recombinant Escherichia coli carrying a SE19 DNA segment. A region that was essential for cyclohexanol oxidation was localized to a 14-kb fragment on the cosmid DNA. Several putative open reading frames (ORFs) that were expected to encode enzymes catalyzing the conversion of cyclohexanol to adipic acid were identified. Whereas one ORF showed high homology to cyclohexanone monooxygenase from Acinetobacter sp. strain NCIB 9871, most of the ORFs showed only moderate homology to proteins in GenBank. In order to assign functions of the various ORFs, in vitro transposon mutagenesis was performed using the cosmid DNA as a target. A set of transposon mutants with a single insertion in each of the ORFs was screened for cyclohexanol oxidation in E. coli. Several of the transposon mutants accumulated a variety of cyclohexanol oxidation intermediates. The in vitro transposon mutagenesis technique was shown to be a powerful tool for rapidly assigning gene functions to all ORFs in the pathway.

The technique of mRNA differential display was used to identify simultaneously two metabolic genes involved in the degradation of cyclohexanone in a new halotolerant Brevibacterium environmental isolate. In a strategy based only on the knowledge that cyclohexanone oxidation was inducible in this strain, the mRNA population of cells exposed to cyclohexanone was compared to that of control cells using reverse transcription-PCR reactions primed with a collection of 81 arbitrary oligonucleotides. Three DNA fragments encoding segments of flavin monooxygenases were isolated with this technique, leading to the identification of the genes of two distinct cyclohexanone monooxygenases, the enzymes responsible for the oxidation of cyclohexanone. Each monooxygenase was expressed in Escherichia coli and characterized. This work validates the application of mRNA differential display for the discovery of new microbial metabolic genes.It is now widely accepted that the diversity of microorganisms extends far beyond the few thousand species in culture collections (15). This diversity of microbes and their metabolism constitutes a vast source of enzymes and genes for biotechnology applications. The identification of useful metabolic genes has traditionally proceeded either through a direct genetic approach or by the reverse genetics approach, starting with the purification of the enzyme of interest followed by identification of its gene through the use of antibodies or amino acid sequence information obtained from the pure protein.Although both strategies are routinely used, they are often limited by technical problems. The direct genetic approach can be used only for organisms that have a developed genetic system or whose genes can be expressed in heterologous hosts. The reverse genetics approach requires purification of the protein of interest, which often takes a long time, and the successful amplification of a DNA probe from degenerate primers, a technique that sometimes fails. Recently mRNA techniques have made it possible to access regulated genes directly without the purification of their gene products and in the absence of a genetic system. These approaches are based on comparison of the mRNA population between two cultures or tissues and identification of the subset of genes whose mRNA is more abundant under conditions of induction. These techniques rely on the hybridization of labeled mRNAs onto arrays of DNA on membranes (4) or DNA microarrays (9), large-scale sample sequencing of expressed sequence tag libraries (28), or the sampling of mRNA by the production of randomly amplified DNA fragments by reverse transcription (RT) followed by PCR (RT-PCR) (19,20,35). Because it can easily be done by individual scientists at low cost, the latter approach has been used extensively since it was first described.Two variations of this RT-PCR method have been published. The first, called differential display (DD) (19,20), begins with the synthesis of cDNAs by RT of mRNA using a poly(dT) primer that hybridizes to the poly(A) tail o...

mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. It is now well recognized that the diversity of microbial species and their metabolic capabilities constitute a tremendous source of biocatalysts (6,10,39). Only a small fraction of microorganisms in most environments can be readily isolated (1, 58); therefore, gene discovery techniques which overcome the need for strain isolation provide access to the diversity of microbial chemistry. Direct cloning approaches can be very successful (21,27,28,48), but they require a genetic selection or an easy screen as well as the efficient expression of the cloned DNA in an appropriate host (15). Other approaches, based on PCR amplification from environmental DNA, target only highly conserved gene families (50). While these techniques are powerful, they often are not applicable. Differential display (DD) is an alternate technique that can be used for the discovery of bacterial genes, requiring neither a genetic selection or screen nor the presence of highly conserved genes. This technique of DD involves the reproducible amplification of DNA fragments from the mRNA population at arbitrary sites by reverse transcription (RT) followed by PCR (RT-PCR) (36,37,57). DD is used to compare the mRNA pools from cells grown under different physiological conditions. Genes expressed at the same level in all cultures will be amplified equally from all cultures, while genes expressed only under a specific condition will give rise to RT-PCR bands only under that condition. DD is a gene discovery technique that can be applied to identify differentially expressed genes. It does not rely on prior knowledge of the genes targeted or on a genomic sequence but only on the fact that the activity that these genes encode is inducible.DD has been applied extensively to eukaryotic systems and takes advantage of the poly(A) tails of eukaryotic mRNA by using poly(dT) primers to synthesize cDNAs by RT (36,37,57). This approach of DD cannot be applied to prokaryotes, which lack stable poly(A) tails. A second variation of DD uses arbitrary oligonucleotide primers to initiate RT of the message at random sites (57) and thus can be applied to archaeal and bacterial s...