Flow cytometric analysis of microorganisms.

The application of flow cytometry to microorganisms is as old as the technique itself, but it has historically been underexploited for microbial applications. This is now being reversed and microbiologists are ideally placed to benefit from recent technological advances. While earlier papers demonstrated the use of flow cytometry for studies of viability and taxonomy, recent developments in bioinformatics and reporter gene technologies are leading to novel applications in microbiology. Variants of green fluorescent protein have been used for the study of conditional microbial gene regulation in medically important host-pathogen interactions and fluorescence-activated cell sorting is being applied to the isolation of novel mutants in directed evolution studies. This paper reviews the reasons for the delay in the application of flow cytometry to microbial problems, the range of applications, and their limitations and considers the progress made in developing new strategies for use in microbiological investigations.

Rapid analysis of high-dimensional bioprocesses using multivariate spectroscopies and advanced chemometrics.

There are an increasing number of instrumental methods for obtaining data from biochemical processes, many of which now provide information on many (indeed many hundreds) of variables simultaneously. The wealth of data that these methods provide, however, is useless without the means to extract the required information. As instruments advance, and the quantity of data produced increases, the fields of bioinformatics and chemometrics have consequently grown greatly in importance. The chemometric methods nowadays available are both powerful and dangerous, and there are many issues to be considered when using statistical analyses on data for which there are numerous measurements (which often exceed the number of samples). It is not difficult to carry out statistical analysis on multivariate data in such a way that the results appear much more impressive than they really are. The authors present some of the methods that we have developed and exploited in Aberystwyth for gathering highly multivariate data from bioprocesses, and some techniques of sound multivariate statistical analyses (and of related methods based on neural and evolutionary computing) which can ensure that the results will stand up to the most rigorous scrutiny.

Systematic functional analysis of the yeast genome.

The genome sequence of the yeast Saccharomyces cerevisiae has provided the first complete inventory of the working parts of a eukaryotic cell. The challenge is now to discover what each of the gene products does and how they interact in a living yeast cell. Systematic and comprehensive approaches to the elucidation of yeast gene function are discussed and the prospects for the functional genomics of eukaryotic organisms evaluated.

Chitinolytic activity in Chromobacterium violaceum: substrate analysis and regulation by quorum sensing.

Quorum sensing control mediated by N-acyl homoserine lactone (AHL) signaling molecules has been established as a key feature of the regulation of exoenzyme production in many gram-negative bacteria. In Chromobacterium violaceum ATCC 31532 a number of phenotypic characteristics, including production of the purple pigment violacein, hydrogen cyanide, antibiotics, and exoproteases are known to be regulated by the endogenous AHL N-hexanoyl-L-homoserine lactone (HHL). In this study we show that C. violaceum produces a set of chitinolytic enzymes whose production is regulated by HHL. The chitinolytic activity was induced in strains grown in the presence of chitin as the sole carbon source and quantitated in the secreted proteins by using p-nitrophenol analogs of disaccharide, trisaccharide, and tetrasaccharide oligomers of N-acetylglucosamine. By using 4-methylumbelliferyl analogs of the same oligomers of N-acetylglucosamine as substrates for proteins separated and renatured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, at least six enzymes were detected: a chitobiase with high specificity to a dimeric substrate of 87 kDa, two N-acetylglucosaminidases with apparent molecular masses of 162 and 133 kDa, two endochitinases of 108 and 67 kDa, and a chitobiosidase of 56 kDa. In addition, two unidentified bands of >205 kDa were found where a tetrameric chitin derivative was used as a substrate. A pleiotropic mini-Tn5 mutant of C. violaceum (CV026) that is defective in HHL production and other quorum-sensing-regulated factors was also found to be completely deficient in chitinolytic activity. Growth of this mutant on minimal medium with chitin supplemented with culture supernatant from the C. violaceum wild-type strain or 10 microM synthetic HHL restored chitinase production to the level shown by the parental strain. These results constitute the most complete evidence so far for regulation of chitinolytic activity by AHL signaling in a gram-negative bacterium.

Construction and analysis of luxCDABE-based plasmid sensors for investigating N-acyl homoserine lactone-mediated quorum sensing.

Plasmid reporter vectors have been constructed which respond to activation of LuxR and its homologues LasR and RhlR (VsmR) by N-acyl homoserine lactones (AHLs). The expression of luxCDABE from transcriptional fusions to PluxI, PlasI and PrhlI respectively, occurs in the presence of activating AHLs. A profile of structure/activity relationships is seen where the natural ligand is most potent. The characterisation of individual LuxR homologue/AHL combinations allows a comprehensive evaluation of quorum sensing signals from a test organism.

Engineering the luxCDABE genes from Photorhabdus luminescens to provide a bioluminescent reporter for constitutive and promoter probe plasmids and mini-Tn5 constructs.

The luxCDABE operon of Photorhabdus luminescens has been cloned and engineered as an easily mobilisable cassette flanked by sites for commonly used restriction enzymes. Constitutive and promoter probe plasmids utilising the P. luminescens luxCDABE have been constructed using a number of compatible replicons and antibiotic markers. Complementary to these plasmids, a range of promoterless and constitutive luxCDABE mini-Tn5 derivatives has been constructed. The potential of coupling mini-Tn5 luxCDABE promoter probe transposons with automated luminometry and photometry to screen for mutants that exhibit growth phase variation in gene expression is demonstrated.

Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules.

Spent culture supernatants from both Aeromonas hydrophila and Aeromonas salmonicida activate a range of biosensors responsive to N-acylhomoserine lactones (AHLs). The genes for a quorum sensing signal generator and a response regulator were cloned from each Aeromonas species and termed ahyRI and asaRI, respectively. Protein sequence homology analysis places the gene products within the growing family of LuxRI homologs. ahyR and asaR are transcribed divergently from ahyI and asaI, respectively, and in both Aeromonas species, the genes downstream have been identified by DNA sequence and PCR analysis. Downstream of both ahyI and asaI is a gene with close homology to iciA, an inhibitor of chromosome replication in Escherichia coli, a finding which implies that in Aeromonas, cell division may be linked to quorum sensing. The major signal molecule synthesized via both AhyI and AsaI was purified from spent culture supernatants and identified as N-(butanoyl)-L-homoserine lactone (BHL) by thin-layer chromatography, high-pressure liquid chromatography analysis, and mass spectrometry. In addition, a second, minor AHL, N-hexanoyl-L-homoserine lactone, was identified. Transcriptional reporter studies with ahyI::luxCDABE fusions indicate that AhyR and BHL are both required for ahyI transcription. For A. salmonicida, although the addition of exogenous BHL gives only a small stimulation of the production of serine protease with comparison to the control culture, the incorporation of a longer-chain AHL, N-(3-oxodecanoyl)-L-homoserine lactone, reduced the final level (by approximately 50%) and delayed the appearance (from an A650 of 0.9 in the control to an A650 of 1.2 in the test) of protease in the culture supernatant. These data add A. hydrophila and A. salmonicida to the growing family of gram-negative bacteria now known to control gene expression through quorum sensing.

The construction and application of a lux-based nitrate biosensor.

A plasmid-borne transcriptional fusion between the Escherichia coli nitrate reductase (narG) promoter and the Photorhabdus luminescens lux operon provides E. coli with a highly bioluminescent phenotype in the presence of nitrate. This E. coli biosensor can detect nitrate to a level of 5 x 10(-5) mol l-1 (0.3 ppm), levels relevant to those levels encountered in brewing water. Since induction of the narG promoter requires NarL, the plasmid-based sensor can also be used to interrogate enteric bacteria for the presence of functional homologues of this E. coli regulatory protein. Obesumbacterium proteus, an important bacterial brewery contaminant, failed to provide nitrate-dependent bioluminescence demonstrating divergence in this organism from E. coli in the mechanism of nitrate reductase regulation.

Involvement of N-acyl-L-hormoserine lactone autoinducers in controlling the multicellular behaviour of Serratia liquefaciens.

Several bacterial species possess the ability to differentiate into highly motile swarmer cells capable of rapid surface colonization. In Serratia liquefaciens, we demonstrate that initiation of swarmer-cell differentiation involves diffusible signal molecules that are released into the growth medium. Using high-performance liquid chromatography (HPLC), high resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, we identified N-butanoyl-L-homoserine lactone (BHL) and N-hex anoyl-L-homoserine lactone (HHL) in cell-free Serratia culture supernatants. BHL and HHL are present in a ratio of approximately 10:1 and their structures were unequivocally confirmed by chemical synthesis. The swrl (swarmer initiation) gene, the predicted translation product of which exhibits substantial homology to the LuxI family of putative N-acyl homoserine lactone (AHL) synthases is responsible for directing synthesis of both BHL and HHL. In an swrl mutant, swarming motility is abolished but can be restored by the addition of an exogenous AHL. These results add swarming motility to the rapidly expanding list of phenotypes known to be controlled through quorum sensing.

Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces a spectrum of exoproducts many of which have been implicated in the pathogenesis of human infection. Expression of some of these factors requires cell-cell communication involving the interaction of a small diffusible molecule, an "autoinducer," with a positive transcriptional activator. In P. aeruginosa PAO1, LasI directs the synthesis of the autoinducer N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), which activates the positive transcriptional activator, LasR. Recently, we have discovered a second signaling molecule-based modulon in PAO1, termed vsm, which contains the genes vsmR and vsmI. Using HPLC, mass spectrometry, and NMR spectroscopy we now establish that in Escherichia coli, VsmI directs the synthesis of N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone (HHL). These compounds are present in the spent culture supernatants of P. aeruginosa in a molar ratio of approximately 15:1 and their structures were unequivocally confirmed by chemical synthesis. Addition of either BHL or HHL to PAN067, a pleiotropic P. aeruginosa mutant unable to synthesize either of these autoinducers, restored elastase, chitinase, and cyanide production. In E. coli carrying a vsmR/vsmI'::lux transcriptional fusion, BHL and HHL activated VsmR to a similar extent. Analogues of these N-acyl-L-homoserine lactones in which the N-acyl side chain has been extended and/or oxidized at the C-3 position exhibit substantially lower activity (e.g., OdDHL) or no activity (e.g., dDHL) in this lux reporter assay. These data indicate that multiple families of quorum sensing modulons interactively regulate gene expression in P. aeruginosa.

Multiple homologues of LuxR and LuxI control expression of virulence determinants and secondary metabolites through quorum sensing in Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa PAO1, expression of elastase is dependent upon an interaction between the positive transcriptional activator LasR and the auto-inducer molecule N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), the synthesis of which is directed by LasI. Previously we have shown that in PAN067, an elastase-negative mutant of PAO1, elastase production can be restored to some extent by addition of exogenous N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). Here we report that PAN067 is also defective in the production of alkaline protease, haemolysin, cyanide, pyocyanin and autoinducer(s). As neither addition of exogenous OdDHL nor introduction of lasR restored PAN067 to the parental phenotype, we sought to complement PAN067 with PAO1 DNA. From a cosmid library, a 2 kb DNA fragment was identified which re-established production of autoinducer(s) and exoproducts in PAN067. From the nucleotide sequence of this fragment, two genes termed rhIR and rhII were identified. RhII is responsible for autoinducer synthesis and shares 31% homology with LasI; RhIR has been previously identified in P. aeruginosa strain DSM2659 as a regulator of rhamnolipid biosynthesis and shares 28% identity with LasR. These data provide clear evidence that multiple families of quorum-sensing modulons interactively regulate gene expression in P. aeruginosa.

Characterisation of the yenI/yenR locus from Yersinia enterocolitica mediating the synthesis of two N-acylhomoserine lactone signal molecules.

Yersinia enterocolitica produces compounds capable of transcriptionally activating the Photobacterium fischeri bioluminescence (lux) operon. Using high-performance liquid chromatography, high resolution tandem mass spectrometry in conjunction with chemical synthesis, two signal molecules were identified and shown to be N-hexanoyl-L-homoserine lactone (HHL) and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). A gene (yenI) was isolated from Y. enterocolitica and demonstrated to direct the synthesis of both HHL and OHHL. DNA sequence analysis revealed an open reading frame (ORF) of 642 bp encoding a protein (YenI) of 24.6 kDa with approximately 20% identity to the LuxI family of proteins. Northern blot analysis of yenI expression indicated yenI is transcribed as a single gene and 5' transcript mapping of yenI identified a transcriptional start site 89 bp upstream of the ORF. DNA sequence analysis of the region downstream of yenI located a second ORF, termed yenR, with significant homology to the LuxR family of transcriptional activators. An insertion mutation of yenI abolishes HHL and OHHL production, indicating its central role in N-acylhomoserine lactone synthesis in Y. enterocolitica. Transcriptional analysis using a chromosomal yenI::luxAB fusion has demonstrated that yenI is not subject to autoinduction but is expressed constitutively. Whilst production of the Yop proteins in the wild type and in yenI mutants is indistinguishable, two-dimensional SDS-PAGE analysis of total cell proteins indicated that a number of proteins lack the yenI mutant.

Gram-negative bacterial communication by N-acyl homoserine lactones: a universal language?

The ability of bacterial cells to use small signalling molecules to monitor population growth may help them to react rapidly to environmental change. Regulatory systems made up of a small sensor molecule, an N-acyl homoserine lactone and a protein effector have been identified recently in a wide range of Gram-negative bacteria. These mediate signal cascades that amplify and coordinate the induction of single or multiple regulons.

A novel strategy for the isolation of luxI homologues: evidence for the widespread distribution of a LuxR:LuxI superfamily in enteric bacteria.

The pheromone N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) regulates expression of bioluminescence in the marine bacterium Vibrio fischeri, the production of carbapenem antibiotic in Erwinia carotovora and exoenzymes in both E. carotovora and Pseudomonas aeruginosa. A characteristic feature of this regulatory mechanism in V. fischeri is that it is cell density-dependent, reflecting the need to accumulate sufficient pheromone to trigger the induction of gene expression. Using a lux plasmid-based bioluminescent sensor for OHHL, pheromone production by E. carotovora, Enterobacter agglomerans, Hafnia alvei, Rahnella aquatilis and Serratia marcescens has been demonstrated and shown also to be cell density-dependent. Production of OHHL implies the presence in these bacteria of a gene equivalent to luxI. Chromosomal banks from all five enteric bacteria have yielded clones capable of eliciting OHHL production when expressed in Escherichia coli. The luxI homologue from both E. carotovora (carI) and E. agglomerans (eagI) were characterized at the DNA sequence level and the deduced protein sequences have only 25% identity with the V. fischeri LuxI. Despite this, carI, eagI and luxI are shown to be biologically equivalent. An insertion mutant of eagI demonstrates that this gene is essential for OHHL production in E. agglomerans.

A comparison of the multiple alleles of xylS carried by TOL plasmids pWW53 and pDK1 and its implications for their evolutionary relationship.

Both of the independently isolated TOL plasmids pWW53 and pDK1 contain multiple regions homologous to the xylS regulatory gene of the archetypal TOL plasmid pWW0. The three homologues on pWW53 vary in the extent of their homology to xylSpWW0, xylS1pWW53 is 99% identical to xylSpWW0 and is located relative to the single copy of xylRpWW53 in exactly the same way as xylS and xylR on pWW0. The DNA sequence of xylS3pWW53 is 87% identical to the xylSpWW0 sequence within the coding region but the non-coding DNA upstream is not homologous. There is a frame-shift change at the end of the coding region which causes the C terminus of XylS3pWW53 to be extended by an additional 10 amino acids relative to XylSpWW0. xylS2pWW53 is anomalous and appears to encode a truncated pseudogene lacking the first 525 bases found in the other xylS genes. Evidence is presented to show that both xylS1pWW53 and xylS3pWW53 act as regulators of meta pathway operons. Plasmid pDK1 carries two homologues of xylS. xylS1pDK1 is functional and is a hybrid gene: its 5' end and the upstream sequences are highly homologous to both xylS1pWW53 and xylSpWW0, whereas its 3' end is identical to xylS3pWW53. The sequence of xylS2pDK1 is identical to that of the anomalous truncated xylS2pWW53. Comparison of the organization and the restriction maps of the xyl catabolic operons on pDK1 and pWW53, together with the nucleotide sequences presented here, indicates that the catabolic DNA on pDK1 has derived from a replicon on which the xyl genes are organized similarly to pWW53 and that a genetic rearrangement has taken place involving a reciprocal recombination internal to two of its xylS homologues.

Small molecule-mediated density-dependent control of gene expression in prokaryotes: bioluminescence and the biosynthesis of carbapenem antibiotics.

Sophisticated signal transduction systems enable prokaryotes to sense their growth environment and mount an appropriate adaptive response. Signal transduction and gene regulation through the phosphorylation of two regulatory components is now recognised as one of the major global regulatory networks in bacteria. However, not all types of sensor-regulator circuits relay information via phosphoryl transfer. The Vibrio fischeri LuxR protein which has previously been characterised as a member of the response-regulator superfamily responds to a small diffusible signal molecule N-(3-oxohexanoyl)homoserine lactone (HSL). Biosynthesis of HSL in V. fischeri is dependent on the expression of the luxI gene. Until recently, the role of HSL as an 'autoinducer' was thought to be restricted to V. fischeri and a few related marine bacteria in which it controls the onset of bioluminescence. However, we have discovered that a diverse group of terrestrial bacteria: (1) produce HSL; (2) possess genes analogous to luxI; and (3) exhibit cell density-dependent induction of bioluminesence when transformed with a recombinant plasmid carrying V. fischeri lux genes but lacking luxI. In one of these, Erwinia carotovora, HSL is shown to mediate the cell density-dependent biosynthesis of a carbapenem antibiotic.