Generalized schemes for high-throughput manipulation of the Desulfovibrio vulgaris genome.

The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high-throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA "parts" to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications, including gene replacement and the creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.

Genome-wide transposon mutagenesis identifies a role for host neuroendocrine stress hormones in regulating the expression of virulence genes in Salmonella.

Bacterial sensing of environmental signals plays a key role in regulating virulence and mediating bacterium-host interactions. The sensing of the neuroendocrine stress hormones epinephrine (adrenaline) and norepinephrine (noradrenaline) plays an important role in modulating bacterial virulence. We used MudJ transposon mutagenesis to globally screen for genes regulated by neuroendocrine stress hormones in Salmonella enterica serovar Typhimurium. We identified eight hormone-regulated genes, including yhaK, iroC, nrdF, accC, yedP, STM3081, and the virulence-related genes virK and mig14. The mammalian alpha-adrenergic receptor antagonist phentolamine reversed the hormone-mediated effects on yhaK, virK, and mig14 but did not affect the other genes. The beta-adrenergic receptor antagonist propranolol had no activity in these assays. The virK and mig14 genes are involved in antimicrobial peptide resistance, and phenotypic screens revealed that exposure to neuroendocrine hormones increased the sensitivity of S. Typhimurium to the antimicrobial peptide LL-37. A virK mutant and a virK mig14 double mutant also displayed increased sensitivity to LL-37. In contrast to enterohemorrhagic Escherichia coli (EHEC), we have found no role for the two-component systems QseBC and QseEF in the adrenergic regulation of any of the identified genes. Furthermore, hormone-regulated gene expression could not be blocked by the QseC inhibitor LED209, suggesting that sensing of hormones is mediated through alternative signaling pathways in S. Typhimurium. This study has identified a role for host-derived neuroendocrine stress hormones in downregulating S. Typhimurium virulence gene expression to the benefit of the host, thus providing further insights into the field of host-pathogen communication.

Global analysis of heat shock response in Desulfovibrio vulgaris Hildenborough.

Desulfovibrio vulgaris Hildenborough belongs to a class of sulfate-reducing bacteria (SRB) and is found ubiquitously in nature. Given the importance of SRB-mediated reduction for bioremediation of metal ion contaminants, ongoing research on D. vulgaris has been in the direction of elucidating regulatory mechanisms for this organism under a variety of stress conditions. This work presents a global view of this organism's response to elevated growth temperature using whole-cell transcriptomics and proteomics tools. Transcriptional response (1.7-fold change or greater; Z >/= 1.5) ranged from 1,135 genes at 15 min to 1,463 genes at 120 min for a temperature up-shift of 13 degrees C from a growth temperature of 37 degrees C for this organism and suggested both direct and indirect modes of heat sensing. Clusters of orthologous group categories that were significantly affected included posttranslational modifications; protein turnover and chaperones (up-regulated); energy production and conversion (down-regulated), nucleotide transport, metabolism (down-regulated), and translation; ribosomal structure; and biogenesis (down-regulated). Analysis of the genome sequence revealed the presence of features of both negative and positive regulation which included the CIRCE element and promoter sequences corresponding to the alternate sigma factors sigma(32) and sigma(54). While mechanisms of heat shock control for some genes appeared to coincide with those established for Escherichia coli and Bacillus subtilis, the presence of unique control schemes for several other genes was also evident. Analysis of protein expression levels using differential in-gel electrophoresis suggested good agreement with transcriptional profiles of several heat shock proteins, including DnaK (DVU0811), HtpG (DVU2643), HtrA (DVU1468), and AhpC (DVU2247). The proteomics study also suggested the possibility of posttranslational modifications in the chaperones DnaK, AhpC, GroES (DVU1977), and GroEL (DVU1976) and also several periplasmic ABC transporters.

Alleviation of insulitis and moderation of diabetes in NOD mice following treatment with a synthetic Pseudomonas aeruginosa signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone.

Quorum sensing signal molecules (QSSMs) from the bacterium Pseudomonas aeruginosa control bacterial population density and the expression of virulence determinants. Coincidentally, and possibly to allow this pathogen to gain a foothold in the human body, certain signal molecules also downregulate immunological responses in an apparently T-helper 1-selective manner, which would suggest their application as therapeutics to some autoimmune diseases. In the present paper, experiments are described that indicate that one particular signal molecule, a synthetic N-(3-oxododecanoyl)-L-homoserine lactone, can be used to alleviate insulitis and diabetes in non-obese diabetic (NOD) mice, suggesting that bacterial signal molecules may represent a novel source of immune modulatory compounds for the treatment of type 1 diabetes, which afflicts more than 2 million individuals in Europe and North America.

raiIR genes are part of a quorum-sensing network controlled by cinI and cinR in Rhizobium leguminosarum.

Analysis of N-acyl-L-homoserine lactones (AHLs) produced by Rhizobium leguminosarum bv. viciae indicated that there may be a network of quorum-sensing regulatory systems producing multiple AHLs in this species. Using a strain lacking a symbiosis plasmid, which carries some of the quorum-sensing genes, we isolated mutations in two genes (raiI and raiR) that are required for production of AHLs. The raiIR genes are located adjacent to dad genes (involved in D-alanine catabolism) on a large indigenous plasmid. RaiR is predicted to be a typical LuxR-type quorum-sensing regulator and is required for raiI expression. The raiR gene was expressed at a low level, possibly from a constitutive promoter, and its expression was increased under the influence of the upstream raiI promoter. Using gene fusions and analysis of AHLs produced, we showed that expression of raiI is strongly reduced in strains carrying mutations in cinI or cinR, genes which determine a higher-level quorum-sensing system that is required for normal expression of raiIR. The product of CinI, N-(3-hydroxy-7-cis tetradecenoyl) homoserine lactone, can induce raiR-dependent raiI expression, although higher levels of expression are induced by other AHLs. Expression of raiI in a strain of Agrobacterium that makes no AHLs resulted in the identification of N-(3-hydroxyoctanoyl)-L-homoserine lactone (3OH,C(8)-HSL) as the major product of RaiI, although other AHLs that comigrate with N-hexanoyl-, N-heptanoyl-, and N-octanoyl-homoserine lactones were also made at low levels. The raiI gene was strongly induced by 3OH,C(8)-HSL (the product of RaiI) but could also be induced by other AHLs, suggesting that the raiI promoter can be activated by other quorum-sensing systems within a network of regulation which also involves AHLs determined by genes on the symbiotic plasmid. Thus, the raiIR and cinIR genes are part of a complex regulatory network that influences AHL biosynthesis in R. leguminosarum.

Galactomannanases Man2 and Man5 from Thermotoga species: growth physiology on galactomannans, gene sequence analysis, and biochemical properties of recombinant enzymes.

The enzymatic hydrolysis of mannan-based hemicelluloses is technologically important for applications ranging from pulp and paper processing to food processing to gas and oil well stimulation. In many cases, thermostability and activity at elevated temperatures can be advantageous. To this end, the genes encoding beta-mannosidase (man2) and beta-mannanase (man5) from the hyperthermophilic bacteria Thermotoga neapolitana 5068 and Thermotoga maritima were isolated, cloned, and expressed in Escherichia coli. The amino acid sequences for the mannosidases from these organisms were 77% identical and corresponded to proteins with an M(r) of approximately 92 kDa. The translated nucleotide sequences for the beta-mannanase genes (man5) encoded polypeptides with an M(r) of 76 kDa that exhibited 84% amino acid sequence identity. The recombinant versions of Man2 and Man5 had similar respective biochemical and biophysical properties, which were also comparable to those determined for the native versions of these enzymes in T. neapolitana. The optimal temperature and pH for the recombinant Man2 and Man5 from both organisms were approximately 90 degrees C and 7.0, respectively. The presence of Man2 and Man5 in these two Thermotoga species indicates that galactomannan is a potential growth substrate. This was supported by the fact that beta-mannanase and beta-mannosidase activities were significantly stimulated when T. neapolitana was grown on guar or carob galactomannan. Maximum cell densities increased by at least tenfold when either guar or carob galactomannan was added to the growth medium. For T. neapolitana grown on guar at 83 degrees C, Man5 was secreted into the culture media, whereas Man2 was intracellular. These localizations were consistent with the presence and lack of signal peptides for Man5 and Man2, respectively. The identification of the galactomannan-degrading enzymes in these Thermotoga species adds to the list of biotechnologically important hemicellulases produced by members of this hyperthermophilic genera.

Haemodynamic effects of the bacterial quorum sensing signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone, in conscious, normal and endotoxaemic rats.

N-acylhomoserine lactones (AHLs) are small, diffusible signalling molecules, employed by Gram-negative bacteria to coordinate gene expression with cell population density. Recent in vitro findings indicate that AHLs may function as virulence determinants per se, through modification of cytokine production by eukaryotic cells, and by stimulating the relaxation of blood vessels. In the present study, we assessed the influence of AHLs on cardiovascular function in conscious rats, and draw attention to the ability of the N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL), a signal molecule produced by P. aeruginosa, to cause marked bradycardia. This bradycardic effect was blocked by atropine and atenolol, and did not occur in vitro. Furthermore, modification of the acyl side chain length resulted in the loss of activity, whereas removal of the homoserine lactone ring, did not. The bradycardic effect of 3-oxo-C12-HSL was also observed in endotoxaemic animals, albeit attenuated. In normal rats, 3-oxo-C12-HSL caused initial mesenteric and hindquarters vasoconstriction, but only slight, and delayed signs of vasodilatation in the renal and mesenteric vascular beds. Furthermore, administration of 3-oxo-C12-HSL (pre-treatment or 2 h post-treatment) together with LPS, did not modify the established regional haemodynamic effects of the LPS, 6 h after the onset of its infusion. Our observations do not provide any clear evidence for an ability of 3-oxo-C12-HSL to modify the haemodynamic responses to LPS infusion. However, they are not inconsistent with the hypothesis that some of the cardiovascular sequelae of bacterial infection may be modulated by an influence of bacterial quorum sensing signalling molecules on the host.

The Pseudomonas aeruginosa quorum-sensing signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone, inhibits porcine arterial smooth muscle contraction.

The Pseudomonas aeruginosa quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been shown to suppress cytokine production in macrophages. We have examined the effect of OdDHL and related compounds on constrictor tone of porcine blood vessels. OdDHL (1-30 microM) caused a concentration-dependent inhibition of U46619-induced contractions of the coronary artery through a largely endothelium-independent mechanism, but was markedly less effective in the pulmonary artery. Quantitively similar effects to those produced by OdDHL were observed with N-(3-oxododecanoyl)-L-homocysteine thiolactone, a thiolactone derivative, while N-3-oxododecanamide, a lactone-free acyl analogue, possessed 1/3rd the potency as a vasorelaxant. Neither N-butanoyl-L-homoserine lactone nor L-homoserine lactone (up to 30 microM) were active. Our findings indicate that OdDHL inhibits vasoconstrictor tone of both pulmonary and coronary blood vessels from the pig. The vasorelaxant action of OdDHL appears to be primarily determined by the N-acyl chain length, with a minor contribution by the homoserine lactone moiety.

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.

In vitro biosynthesis of the Pseudomonas aeruginosa quorum-sensing signal molecule N-butanoyl-L-homoserine lactone.

In Pseudomonas aeruginosa, synthesis of the quorum-sensing signal molecules N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone (HHL) requires the Luxl homologue Rhll(Vsml). By using thin-layer chromatography in conjunction with high-performance liquid chromatography (HPLC) and mass spectrometry, we show that purified Rhll can catalyse the biosynthesis of BHL and HHL using either S-adenosylmethionine (SAM) or homoserine lactone (HSL) but not homoserine as the source of the homoserine lactone moiety. As we were unable to detect homoserine lactone in cytoplasmic extracts of Escherichia coli, we conclude that SAM is the natural substrate for Rhll-directed N-acylhomoserine lactone (AHL) biosynthesis. The N-acyl chain of BHL and HHL can be supplied by the appropriately charged coenzyme A derivative (either n-butanoyl-CoA or n-hexanoyl-CoA). The specificity of Rhll for charged CoA derivatives is demonstrated as Rhll was unable to generate AHLs detectable in our bioassays from acetyl-CoA, malonyl-CoA, n-octanoyl-CoA, n-decanoyl-CoA, DL-beta-hydroxybutanoyl-CoA or crotonoyl-CoA. Rhll was also unable to use N-acetyl-S-3-oxobutanoylcysteamine, a chemical mimic for 3-oxobutanoyl-CoA. Furthermore, the Rhll-catalysed synthesis of BHL and HHL was most efficiently driven when NADPH was included in the reaction mixture.

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.

Cell density-regulated recovery of starved biofilm populations of ammonia-oxidizing bacteria.

The speed of recovery of cell suspensions and biofilm populations of the ammonia oxidizer Nitrosomonas europaea, following starvation was determined. Stationary-phase cells, washed and resuspended in ammoniumfree inorganic medium, were starved for periods of up to 42 days, after which the medium was supplemented with ammonium and subsequent growth was monitored by measuring nitrite concentration changes. Cultures exhibited a lag phase prior to exponential nitrite production, which increased from 8.72 h (no starvation) to 153 h after starvation for 42 days. Biofilm populations of N. europaea colonizing sand or soil particles in continuous-flow, fixed column reactors were starved by continuous supply of ammonium-free medium. Following resupply of ammonium, starved biofilms exhibited no lag phase prior to nitrite production, even after starvation for 43.2 days, although there was evidence of cell loss during starvation. Biofilm formation will therefore provide a significant ecological advantage for ammonia oxidizers in natural environments in which the substrate supply is intermittent. Cell density-dependent phenomena in a number of gram-negative bacteria are mediated by N-acyl homoserine lactones (AHL), including N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). Addition of both ammonium and OHHL to cell suspensions starved for 28 days decreased the lag phase in a concentration-dependent manner from 53.4 h to a minimum of 10.8 h. AHL production by N. europaea was detected by using a luxR-luxAB AHL reporter system. The results suggest that rapid recovery of high-density biofilm populations may be due to production and accumulation of OHHL to levels not possible in relatively low-density cell suspensions.

Quorum sensing in Vibrio anguillarum: characterization of the vanI/vanR locus and identification of the autoinducer N-(3-oxodecanoyl)-L-homoserine lactone.

Certain gram-negative pathogens are known to control virulence gene expression through cell-cell communication via small diffusible signal molecules termed autoinducers. This intercellular signal transduction mechanism termed quorum sensing depends on the interaction of an N-acylhomoserine lactone (AHL) auto-inducer molecule with a receptor protein belonging to the LuxR family of positive transcriptional activators. Vibrio anguillarum is a gram-negative pathogen capable of causing a terminal hemorrhagic septicemia known as vibriosis in fish such as rainbow trout. In this study, we sought to determine whether V. anguillarum employs AHLs to regulate virulence gene expression. Spent V. anguillarum culture supernatants stimulated bioluminescence in a recombinant lux-based Escherichia coli AHL biosensor strain, whereas they both stimulated and inhibited AHL-mediated violacein pigment production in Chromobacterium violaceum. This finding suggested that V. anguillarum may produce multiple AHL signal molecules. Using high-performance liquid chromatography and high-resolution tandem mass spectrometry, we identified the major V. anguillarum AHL as N-(3-oxodecanoyl)-L-homoserine lactone (ODHL), a structure which was unequivocally confirmed by chemical synthesis. The gene (vanI) responsible for ODHL synthesis was cloned and sequenced and shown to belong to the LuxI family of putative AHL synthases. Further sequencing downstream of vanI revealed a second gene (vanR) related to the LuxR family of transcriptional activators. Although deletion of vanI abolished ODHL synthesis, no reduction of either metalloprotease production or virulence in a fish infection model was observed. However, the vanI mutant remained capable of weakly activating both bioluminescence and violacein in the E. coli and C. violaceum biosensors, respectively, indicating the existence of additional layers of AHL-mediated regulatory complexity.

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.

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.

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.

The lux autoinducer regulates the production of exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa.

Erwinia carotovora and Pseudomonas aeruginosa secrete exoenzymes that contribute to the pathogenesis of plant and mammalian infections respectively. E.carotovora mutants defective in synthesis of the pectinase, cellulase and protease exoenzymes were isolated and classified into two groups. Group 2 mutants were found to be defective in the production of a small freely diffusible molecule, N-3-(oxohexanoyl)-L-homoserine, lactone (HSL), and were avirulent. Addition of exogenous HSL to these group 2 mutants restores synthesis of the exoenzymes and virulence in planta. Of the exoenzymes of P.aeruginosa the metalloprotease, elastase, is an established virulence determinant. Mutants of P.aeruginosa that are defective in elastase production have been isolated and were again found to fall into two groups. Analogous to the group 2 mutants of E.carotovora, group 2 mutants of P. aeruginosa are defective in the synthesis of HSL and exogenous HSL restores elastase production. HSL has now been linked to the control of bioluminescence in Vibrio fischeri, carbapenem antibiotic production of E.carotovora and the above exoenzyme virulence determinants. This information significantly enhances our understanding of the extent and nature of pheromone mediated gene expression control in prokaryotes.

Synthesis of 2'-deoxyuridine and 5-fluoro-2'-deoxyuridine derivatives and evaluation in antibody targeting studies.

Derivatives of 2'-deoxyuridine and of the anticancer agent 5-fluoro-2'-deoxyuridine (FdUR) were linked indirectly via a human serum albumin carrier (HSA) to the murine antiosteosarcoma monoclonal antibody 791T/36. Starting from the 2'-deoxyuridines 1a and 1b, the new nucleosides containing 5'-succinamic acid 7 and 5'-maleamic acid 8 spacers were synthesized from the key intermediate 5'-aminonucleoside 4, and the ribofuronamidobenzoic acid 13 from ribofuranuronic acid 10. These nucleosides were linked via their spacer functionality to HSA. High molar substitution ratios (MSR: moles of drug/mole of HSA) of 25-40 for these derivative-HSA conjugates were achieved. All derivatives were less cytotoxic than the parent drug against both antigen positive osteogenic sarcoma 791T and antigen negative bladder carcinoma T24 cell lines; no IC50 was achieved with any derivative against 791T cells. The fluorodeoxyuridine-HSA conjugates were then further linked via a stable thioether bond to the mouse monoclonal antibody 791T/36. The optimum fluorinated 5'-succinamic acid immunoconjugate exhibited an IC50 of 1 microM against 791T and T24 cells, slightly better than that of fluorodeoxyuridine. The unconjugated derivative 7 was much less cytotoxic than immunoconjugate, with an IC50 of 62 microM on T24 cells, and failed to reach 50% inhibition of 791T cell growth at 290 microM concentration. Derivative 7-HSA conjugate was 10-fold less cytotoxic than the immunoconjugate against both cell lines. Immunoconjugates synthesized with the other 5-fluoro derivatives were unable to effect 50% inhibition of growth of cell lines. Nonfluorinated derivatives and their HSA conjugates and immunoconjugates exhibited no cytotoxicity.