A novel and sensitive test for rapid determination of water toxicity.

The performance of a novel, rapid, and sensitive test for detecting chemical toxicants in water is described in this article. The bioassay utilizes a highly sensitive variant of the luminescent bacterium Photobacterium leiognathi that allows the detection in water at levels below milligrams per liter of diverse groups of toxicants, including heavy metals, pesticides, PCBs, polycyclic aromatic hydrocarbons, and fuel traces. For most toxic agents reported in this study, the new assay was markedly more sensitive than the Microtox(trade mark) Vibrio fischeri assay according to the bacterial bioluminescence toxicity data reported in the literature. Additional features of the new bioassay include the ability to discriminate between cationic heavy metals and organic toxicants and the option of being run at ambient temperatures (18 degrees C-27 degrees C), thereby enabling on-site testing with low-cost luminometers. In addition, the stability of the freeze-dried bacterial reagent preparation at ambient temperatures precludes the need for refrigeration or freezing during shipment, which contributes to further reducing overall operational costs.

Highly sensitive and rapid detection of antibody catalysis by luminescent bacteria.

A highly sensitive, inexpensive, and facile bioluminescent assay for the detection of catalytic antibodies has been developed. This assay may be used for the early detection of antibody catalysis. The efficiency of this technique was exemplified by the use of the luminescent bacterium VhM42 for monitoring an antibody-catalyzed retroaldol fragmentation reaction with aldolase antibodies 38C2 and 24H6.

In vivo and in vitro function of GroEL mutants with impaired allosteric properties.

Escherichia coli cells that produce only plasmid-encoded wild-type or mutant GroEL were generated by bacteriophage P1 transduction. Effects of mutations that affect the allosteric properties of GroEL were characterized in vivo. Cells containing only GroEL(R197A), which has reduced intra-ring positive cooperativity and inter-ring negative cooperativity in ATP binding, grow poorly upon a temperature shift from 25 to 42 degrees C. This strain supports the growth of phages T4 and T5 but not phage lambda and produces light at 28 degrees C when transformed with a second plasmid containing the lux operon. In contrast, cells containing only GroEL(R13G, A126V) which lacks negative cooperativity between rings but has intact intra-ring positive cooperativity grow normally and support phage growth but do not produce light at 28 degrees C. In vitro refolding of luciferase in the presence of this mutant is found to be less efficient compared with wild-type GroEL or other mutants tested. Our results show that allostery in GroEL is important in vivo in a manner that depends on the physiological conditions and is protein substrate specific.

Identification and quantification of toxic chemicals by use of Escherichia coli carrying lux genes fused to stress promoters.

The luxCDABE bioluminescence genes of the Vibrio fischeri lux system have been used as a reporter system for different stress and regulatory promoters of Escherichia coli. Selected E. coli strains carrying lux genes fused to different promoters were exposed to various toxic chemicals, and the recorded luminescence was used for the characterization of the biologic signature of each compound. Analysis of these data with the aid of a proper algorithm allowed quantitative and qualitative assessment of toxic chemicals. Of the 25 tested chemicals, 23 were identified by this novel strategy in a 3-h procedure. This system can also be adapted for the identification of simple mixtures of toxic agents when the biologic signatures of the individual compounds are known. This biologic recognition strategy also provides a tool for evaluating the degree of similarity between the modes of action of different toxic agents.

H-NS controls the transcription of three promoters of Vibrio fischeri lux cloned in Escherichia coli.

We have recently proposed that the expression of V. fischeri right lux operon is controlled by two promoters; the first one located upstream of the luxl gene, while the second one seems to be located upstream of the luxC gene. The transcription from both promoters is negatively controlled by H-NS protein. Escherichia coli MC4100 rpoS hns mutant that carried the V. fischeri lux system with a deletion in either the luxl or luxR gene showed a constitutive mode and more than 10,000-fold higher luminescence than the control cells. The present study shows that neither luxR nor luxl are required for the transcription of the luxCDABE genes in an H-NS deficient strain of E. coli. The MC4100 rpoS hns mutant harbouring the luxCDABE-carrying plasmid showed constitutive mode and 70,000-fold higher luminescence than the wild-type cells. The question whether both the left and the right operons of V. fischeri lux system are controlled by H-NS was addressed with the aid of plasmids harbouring the lacZ gene fused with luxR or luxl. In MC4100 hns rpoS background, luxR and luxl genes were very early and actively transcribed, as judged by the strong beta-galactosidase activity that was developed at early stage of growth. The beta-galactosidase activity in the wild-type cells was 20-40 times lower and occurred mainly during the second half of the growth cycle. It thus appears that H-NS inhibits the transcription of three promoters of the lux system of V. fischeri; the left operon that codes for LuxR protein and two promoters located upstream and downstream to luxl gene.

LuxR controls the expression of Vibrio fischeri luxCDABE clone in Escherichia coli in the absence of luxI gene.

We have recently suggested that the expression of V. fischeri right lux operon is initiated from two sites, the first located upstream of the luxI gene, while the second seems to be located upstream of the luxC gene. The transcription from both sites is negatively controlled by H-NS protein. E. coli MC4100 rpoS hns mutant harbouring the V. fischeri luxCDABE genes showed constitutive mode and 70,000-fold higher luminescence than the wild-type cells. The present study shows that the expression of luxCDABE genes in E. coli MC4100 wild-type cells is also controlled by LuxR protein in the absence of the autoinducer. The co-presence of a ptac-controlled luxR gene in a trans position to a plasmid carrying the luxCDABE genes resulted in 100,000 times higher luminescence. In the absence of the autoinducer, the presence of the luxR gene under its own regulated control resulted in about 100-200-fold increase of luminescence from the luxC upstream site. Taken together, it seems that the LuxR protein initiates the formation of the V. fischeri lux system cloned in E. coli from two sites located upstream and downstream of the luxI gene. Only the activation of the first site requires the presence of the autoinducer, whereas the second site is fully activated by LuxR protein in the absence of the autoinducer.

H-NS protein represses transcription of the lux systems of Vibrio fischeri and other luminous bacteria cloned into Escherichia coli.

High expression in Escherichia coli of the lux system cistron of a luminous bacteria under its own control has been accomplished only for the Vibrio fischeri lux system at high cell density. Mutation of the hns gene in E. coli has resulted in strong expression of the V. fischeri lux system at low cell density even in an rpoS-deleted strain of E. coli that emits very low levels of luminescence. The E. coli double mutant, MC4110 hns::kan rpoS::tet carrying the lux system of V. fischeri, developed high luminescence from the very early stages of cellular growth, regardless of the presence of deletion mutations in the luxI or luxR genes. Moreover, autoinducer synthesis was restored in the double mutant with the luxR-deleted system. plac-controlled V. fischeri luxCDABE genes missing luxI and luxR were dim in E. coli rpoS mutant cells, but had wild-type levels of light in the hns-deleted strain [MC4110 hns rpoS], showing that expression was independent of lux regulators in the absence of H-NS. DNA gyrase inhibitors and DNA intercalating agents also brought about the restoration of luminescence in the rpoS-deficient strain. High expression of the lux systems of Vibrio harveyi, Photobacterium leiognathi, and Xenorhabdus luminescens in E. coli MC4110 hns rpoS cells compared with that in wild-type or rpoS mutants was also accomplished. Taken together, these data suggest that the H-NS protein inhibits transcription in E. coli of the lux systems of all or most luminous bacteria at the luxC gene as well as in the luxRI region of the V. fischeri lux operon. These DNA regions are highly enriched with homopolymeric stretches of poly d(A) and poly d(T) characterizing curved DNA, a preferable site of H-NS binding. The significance of the new findings in understanding the regulatory control of the bacterial lux system is discussed.

Bacterial toxicity of cyclodextrins: luminuous Escherichia coli as a model.

The effect of various concentrations of natural and chemically modified cyclodextrins on the luminescence of an Escherichia coli suspension was investigated. All cyclodextrins were found to reduce, albeit to a varying degree, the luminescence level of the bacterial cells, thus suggesting a direct interaction between the cyclodextrins and cells. The inhibitory concentrations IC20 and IC50 of the various cyclodextrins were determined and taken to represent their toxicity effects upon the bacterial cells. Among the natural cyclodextrins, gamma- and alpha-CD interfered minimally with the bacterial luminescence and consequently were essentially non-toxic. The following descending order of toxicity was observed: beta-CD >> alpha-CD > gamma-CD. Among the chemically modified cyclodextrins, Dimeb was clearly toxic while Trimeb and the hydroxylated derivatives (hydroxypropyl-alpha-CD, HPACD; -beta-CD, HPBCD; -gamma-CD, HPGCD) were essentially non-toxic. The following descending order of toxicity was observed: Dimeb >> HPBCD > Trimeb > HPACD > HPGCD.

GroESL proteins facilitate binding of externally added inducer by LuxR protein-containing E. coli cells.

htpR- (rpoH, sigma 32 minus) strain of E. coli harbouring the whole lux system of Vibrio fischeri is very dim. We have recently shown that GroESL proteins fully recover the expression of the lux system in this strain. This work has been undertaken to study our assumption that the GroESL proteins stabilize the LuxR protein, thus enhancing the formation of LuxR-Inducer complex. E. coli htpR- cells harbouring the luxR gene were unable to bind extracellularly added inducer, while late logarithmically growing htpR+ strain bound small quantities of the inducer. Reduction in the nutrient content of the growth medium resulted in a large increase in the capability of these cells to bind the inducer. htpR+ or htpR- E. coli strains harbouring both the luxR and the groESL genes bound large quantities of the inducer. The molecular and ecological significance of these results is discussed.

A stable Escherichia coli-Mycobacterium smegmatis plasmid shuttle vector containing the mycobacteriophage D29 origin.

A plasmid shuttle vector for Escherichia coli and mycobacteria was constructed from an E. coli plasmid containing the ColE1 origin, a 2.6-kb PstI fragment from bacteriophage D29 that grows in numerous mycobacterial species, and the kanamycin resistance gene either of Tn903 or of Tn5. The resultant plasmid is 7.63 kb and can be introduced via transformation into Mycobacterium smegmatis with high efficiency. In M. smegmatis the plasmid is stable and apparently present in multiple copies. Bioluminescence (luxA and luxB of Vibrio harveyi and fischeri) has been expressed in M. smegmatis from the aminoglycoside transferase promoter of Tn5. The D29 fragment should carry an origin of replication and some associated genes that act on it since various mutations destroy the ability of this fragment to replicate in M. smegmatis. The fragment was localized on the D29 genome map.

Formation of the LuxR protein in the Vibrio fischeri lux system is controlled by HtpR through the GroESL proteins.

The transcription of the luminescence (lux) system of Vibrio fischeri is regulated by the LuxR protein and an autoinducer. We previously showed that apart from these regulatory elements, the transcription of the lux system is negatively controlled by the LexA protein and positively controlled by the HtpR protein (sigma 32). This study was conducted in order to elucidate the mode of action of the HtpR protein. Using luxR-lacZ fused genes, we showed that the HtpR protein is essential for the maximum expression of beta-galactosidase activity in Escherichia coli lac mutant cells. Using this construct, we also demonstrated that luxR is preferentially expressed toward the end of the logarithmic phase of growth. Starvation and addition of ethanol significantly advanced the appearance of beta-galactosidase activity in htpR+ cells. The luminescence system of E. coli htpR+ cells harboring the pChv1 plasmid with a deletion in the luxI gene is induced in the presence of low and constant concentrations (150 pg/ml) of the inducer only at a late stage of the logarithmic phase of growth. When the cellular LuxR content is reduced, following 23 generations of exponential growth in Luria broth, a mid-log-phase culture does not respond to the inducer (150 pg/ml). On the basis of the above observations we suggest that the HtpR protein controls the formation of V. fischeri LuxR protein. Preliminary findings indicate that the HtpR protein acts through the chaperonins GroESL. E. coli htpR/pChv1 cells retained their full level of in vivo and in vitro luciferase activities in the presence of multiple copies of groESL genes. The possibility that GroESL proteins stabilize the native form of LuxR protein is discussed.

Citrate synthase from Mycobacterium smegmatis. Cloning, sequence determination and expression in Escherichia coli.

A Mycobacterium smegmatis PstI library was constructed by cloning these fragments downstream from the lac promoter of the expression vector pHG171. Three identically sized clones were isolated by complementation of an Escherichia coli strain (chi 2338) deficient in citrate synthase. One insert (pBL265) was used in hybridization experiments with DNA from E. coli and M. smegmatis and it was demonstrated that the clones were indeed from M. smegmatis. The transcription of the M. smegmatis citrate synthase gene in E. coli relied upon the lac promoter. In translation experiments performed in vitro pBL265 gave rise to a novel protein of about 42 kDa. This band was not seen in 'opposite-orientation' subclones. Various subclones in which the 5'-end was shortened nevertheless complement E. coli chi 2338 and produce the 42 kDa protein. This demonstrates that the M. smegmatis citrate synthase gene uses its own ribosome-binding site in E. coli. The relevant 1.8 kb of the 2.8 kb insert was sequenced. A consensus E. coli ribosome-binding site was found centred precisely 10 bp upstream of the methionine codon. Other interesting features revealed by the sequence are discussed. Citrate synthase activity was assayed in vitro and the mycobacterial enzyme was found to be similar to those of the Gram-positive bacteria.

Mass transfer studies using cloned-luminous strain of Xanthomonas campestris.

Measurements of mass transfer in a highly viscous pseudoplastic broth, which is typical to Xanthomonas campestris fermentations, are difficult to obtain by conventional methods and little data is available. A novel research method that uses bioluminescence for mass transfer studies has been developed. A plasmid carrying the luminescence operon of marine luminous bacteria is introduced into an industrial bacteria, X. campestris. Besides producing the polysaccharide xanthangum, the bioluminescent X. campestris emits measurable light. Monitoring the luminescence is a simple, noncontaminating nondestructive and very sensitive indicator of the metabolic activity of the culture during fermentation. Energy drain due to bioluminescence is very low; growth rate and polysaccharide production rate are close to those of the wild-type strain.Oxygen and substrate mass transfer are determined by inducing step or periodic fluctuations in their concentration and measuring the resultant luminescence response. Oxygen mass transfer coefficients show linear dependence on Reynolds number and an exponential dependence on the average shear rate. Viscosity effect is small at high viscosities but increases rapidly below 10 Pa-s. The influence of oxygen uptake rate is studied.Mass transfer of the limiting component (ammonium ions) is analyzed under pulsating feed conditions. The luminescence declines, following a feed pulse, due to energy investment in active transport of ammonium ions through the cell membrane, it regenerates then to its baseline. The relation between mass transfer and luminescence fluctuation is elucidated.

The regulatory control of the bacterial luminescence system--a new view.

We have recently shown that the transcription of the PR lux operon for Vibrio fischeri luminescence is positively controlled by the htpR (sigma 32) protein. It was suggested that the LexA protein might negatively control the lux genes. This paper extends these findings. It was found that Escherichia coli cells that contain the entire lux operon (pChv1) in RecA or LexA mutants which are unable to remove the LexA protein are considerably dimmer than the wild-type strain. Mutants that do not make LexA or form a weakly bound LexA are very bright. The role of sigma 32 protein was studied on luxR-luxI genes that are fused to beta-galactosidase. The addition of V. fischeri inducer brings about the formation of beta-galactosidase activity in htpR+ but not in htpR- strains of E. coli/pMJ3. Similar to the effect of starvation on the induction of luminescence in marine bacteria and in E. coli/pChv1 cells, beta-galactosidase activity in such constructs is preferentially induced by low nutrient concentrations. A new model for the regulatory control of the V. fischeri luminescence system is discussed.

Determination of oil oxidation by an aldehyde-requiring mutant of luminous bacteria.

A simple, rapid bioluminescence test (BT) for the determination of lipid oxidation is described. The test utilizes an aldehyde-requiring dark mutant of Vibrio harveyi (M42) that emits light in the presence of long chain (C8-C16) aliphatic aldehydes. The procedure consists of treating the oil or fat with CO2+ ion in ethanolic medium at alkaline pH. This treatment facilitates the decomposition of the hydroperoxides into long-chain aldehydes, part of which is used by the bacteria to produce light. The test was evaluated with corn, soybean and safflower oils, and shows excellent correlation with the commonly used peroxide value assay.

Detection of genotoxicity of metallic compounds by the bacterial bioluminescence test.

Twenty metallic compounds were assayed for their genotoxic mutagenic activity by the bioluminescence test restoration of the luminescence of dark mutant of the luminous bacterium Photobacterium fischeri). The activity of the metals was tested in a liquid medium as well as on a solid medium. K2Cr2O7, MnCl2, BeCl2, KH2AsO4, ZnCl2 and Na2WO4 showed strong activity in liquid medium while AgNO3, Cd(OOCCH3)2, CoCl2, CuCl2, HgCl2, Na2SeO3 and Pb(NO3)2 were more active in the solid medium test. BaCl2, Na2MoO4, NaAsO2, NiSO4, Na2SeO4, RbCl, and SnCl2 were not active in the bioluminescence test. The correlation between the genotoxic activity of the tested metallic compounds in the bioluminescence test and other bacterial tests for genotoxic agents as well as the correlation between these results and the carcinogenicity of these compounds is discussed.

The transcription of bacterial luminescence is regulated by sigma 32.

Luminescence in the marine bacterium, Vibrio fischeri, is regulated by a small molecule, the autoinducer. The transcription of the V. fischeri lux genes also requires a regulatory protein, (luxR), cAMP and CRP. We show that, apart from these components, the transcription of the PR lux operon is also controlled by the activity of sigma 32 (htpR protein). In luminescent Escherichia coli (E. coli/pChv1), as well as in different marine luminous bacteria and their naturally occurring dark (K) variants, the luminescence system can be induced by starvation under microaerophilic conditions. Heat shock also induces luminescence in htpR+ but not in htpR- strains of E. coli/pChv1. An htpR- mutant of E. coli containing pChv1 is very dim and its luminescence is not induced by starvation or heat shock. The addition of a plasmid bearing the gene for htpR+ into such cells restores their response to starvation and heat shock. Cells of wild type E. coli/pChv1 that have been starved or heat shocked respond to lower concentrations of V. fischeri inducer than untreated cells. These cultures also produce more extracellular inducer than untreated cells. Starvation, heat shock and the presence of sigma 32 do not induce luminescence in luxl deleted E. coli/pChv1 cells. SOS-inducing agents advance the onset of luminescence in both htpR+ and htpR- strains but not in luxl deleted E. coli/pChvi cells. DNA sequencing of the luxR-luxl region reveals the presence of a promoter region of the kind typical for sigma 32 at the beginning of the luxl gene. In addition we find a LexA protein-DNA binding site in the non-consensus sequence for the -35 region of the PR operon. It is proposed that the regulatory protein-inducer complex displaces the LexA protein and allows the transcription of the right operon. SOS-inducing agents result in proteolysis of LexA protein and advance the onset of luminescence. sigma 32 enhances the transcription from the PR operon and thus initiates a positive control circuit. It seems that sigma 32 is the major controlling element in determining the onset of luminescence both in vivo and in vitro.

Use of bacterial luciferase to establish a promoter probe vehicle capable of nondestructive real-time analysis of gene expression in Bacillus spp.

We report the construction and use of a new promoter probe vehicle capable of allowing extremely sensitive measurements of transcriptional activity promoted from random, chromosomal DNA fragment inserts. Coupled with the advantage of sensitivity, the detection system is noninvasive, nondestructive, and provides real-time reportage of expression potential. These latter aspects make it an especially valuable system for a continuing analysis of the complex transcriptional regulation patterns now recognized as a dominant control feature during the differentiation and morphogenesis characteristic of the sporulation cycle in Bacillus species. In this respect we describe the isolation of DNA fragments from B. megaterium and B. subtilis capable of initiating transcription in both the respective parent organisms and, in certain instances, also in Escherichia coli. Detailed luminescence studies showed that several promoter regions which are entirely or substantially developmentally controlled were isolated.