Conditions that influence bacterial luminescence in the presence of blood serum

Conditions that influence the luminescence of natural and recombinant luminescent bacteria in the presence of blood serum were studied. In general, blood serum quenched the luminescence of the marine Photobacterium phosphoreum and the recombinant Escherichia coli strains harboring the luminescent system genes of Photobacterium leiognathi, but enhanced the luminescence of the soil bacterium Photorhabdus luminescens Zm1 and the recombinant E. coli strain harboring the lux operon of P. luminescens Zm1. The quenching effect of blood serum increased with its concentration and the time and temperature of incubation. The components of blood serum that determine the degree and specificity of its action on bacterial luminescence were identified.     

Cloning and insertion mutagenesis of DNA fragment coding for the luminescent system of Photobacterium leiognathi

Fragments of DNA, obtained from the luminescent bacterium Photobacterium leiognathi and inserted into the plasmid pBR322, were found to code for the luminescence expressed in E. coli cells. The genetic functions necessary for light production in E. coli are localized on a DNA fragment of about 7 kbp. The insertion mutagenesis was used to define the luminescence functions encoded by the hybrid plasmid.     

The lux genes of the luminous bacterial symbiont, Photobacterium leiognathi, of the ponyfish. Nucleotide sequence, difference in gene organization, and high expression in mutant Escherichia coli

The lux genes required for light expression in the luminescent bacterium Photobacterium leiognathi (ATCC 25521) have been cloned and expressed in Escherichia coli and their organization and nucleotide sequence determined. Transformation of a recombinant 9.5-kbp chromosomal DNA fragment of P. leiognathi into an E. coli mutant (43R) gave luminescent colonies that were as bright as those of the parental strain. Moreover, expression of the lux genes in the mutant E. coli was strong enough so that not only were high levels of luciferase detected in crude extracts, but the fatty-acid reductase activity responsible for synthesis of the aldehyde substrate for the luminescent reaction could readily be measured. Determination of the 7.3-kbp nucleotide sequence of P. leiognathi DNA, including the genes for luciferase (luxAB) and fatty-acid reductase (luxCDE) as well as a new lux gene (luxG) found recently in luminescent Vibrio species, showed that the order of the lux genes was luxCDABEG. Moreover, luxF, a gene homologous to luxB and located between luxB and luxE in Photobacterium but not Vibrio strains, was absent. In spite of this different lux gene organization, an intergenic stem-loop structure between luxB and luxE was discovered to be highly conserved in other Photobacterium species after luxF.     

Isolation of the lux genes from Photobacterium leiognathi and expression in Escherichia coli

Genes necessary for luminescence (lux genes) in the marine bacterium Photobacterium leiognathi, strain PL721, were isolated and expressed in Escherichia coli. A 15-kb fragment obtained from a partial digestion of PL721 DNA with HindIII was cloned into the plasmid pACYC184, resulting in the hybrid plasmid pSD721. When pSD721 was transformed into E. coli ED8654, the resulting transformants were luminous with no additions to the cells, indicating that it contained the structural genes coding for the alpha and beta subunits of luciferase (luxA and luxB), and for components involved in aldehyde biosynthesis. Hybridization analysis with luxA and luxB 32P probes confirmed the location of these two genes on the 15-kb insert. When pSD721 was transformed into four different strains of E. coli, luminescence expression varied widely in amount and in pattern. In some strains, luminescence developed like an autoinducible system, and at maximum induction was very bright, even with no addition of aldehyde, while in others, luminescence was 100-fold less, and no induction was seen. In no case was luminescence affected by shifts in temperature, osmolarity, or iron concentration. These results indicate that, while the complete lux regulon is apparently contained on the 15-kb cloned fragment, the regulation of the lux regulon in pSD721 is subject to host controls by E. coli, controls which vary widely among different E. coli strains.     

Expression of Photobacterium leiognathi bioluminescence system genes in Escherichia coli

Expression of Photobacterium leiognathi bioluminescence genes under the control of lac, tac, tet promoters in Escherichia coli cells has been studied. The position of the genes for aliphatic aldehyde biosynthesis and for the synthesis of luciferase subunits was identified. The plasmid pBRPL1 has been constructed containing the system of bioluminescence genes devoid of promoter following the polylinker DNA fragment. The plasmid can be used for selection of promoter containing DNA sequences as well as for studying the promoters regulation in process of Escherichia coli cells growth.     

Mathematical modeling of population dynamics of unstable plasmid-containing bacteria during continuous cultivation in a chemostat

A structural approach to studying the regularities of the population dynamics of unstable recombinant bacterial strains in a chemostat was elaborated. The approach is based on the mathematical modeling of cell distribution in a population with different numbers of plasmid copies. The effect of decreased selective preference of plasmidless variants of the recombinant strain in the chemostat, which is related to a decrease in the number of plasmid copies in cells upon long-term incubation was analyzed. It is shown that the time of half-elimination of plasmids from the bacterial population in the steady state in the chemostat T1/2 does not depend on the maximum number of plasmid copies in cells N but is determined only by the mean time of generation g and the probability of the loss of one plasmid copy tau. The dependence of the preference of bacterial plasmidless variants on the efficiency of expression of genes cloned into plasmids in chemostat was analyzed using the recombinant strain E. coli Z905, whose plasmids pPHL-7 contain cloned genes for the luminescence system of marine luminescing bacteria Photobacterium leiognathi.     

Quantitative criteria for the estimation of the effectiveness of bioluminescence expression in natural and transgenic luminescent bacteria

Computation coefficients for estimating the effectiveness of bioluminescence expression in natural luminescent bacteria P. leiognathi 54 and transgenic strain E. coli Z905/pPHL7 bearing lux-operon in multicopy plasmid are suggested, and their use on the molecular, cell, and population levels was considered. It was shown that, on the population level, all transgenic variants got the better of natural variants of P. leiognathi 54 irrespective of the type of lux-operon regulation. On the cell level, in the bright and dim variants of the transgenic strain, the effectiveness of bioluminescence expression increases by several orders. On the level of one lux-operon, the effectiveness of expression of the bright variant of transgenic strain is substantially higher than in the natural bright variant; in dim variants, the efficiency values are similar, and the effectiveness of bioluminescence expression in the dark variant of E. coli Z905-2 /pPHL7 is by two orders lower than that in the dark variant of P. leiognathi 54.     

Effect of salts on luminescence of natural and recombinant luminescent bacterial biosensors

Effect of cations K+, Na+, Mg2+, and Ca2+ and anions SO4(2-), HCO3(-), and CO3(2-) on the luminescence intensity of the marine luminescent bacterium Photobacterium phorphoreum (Microbiosensor B-17 677f) and the recombinant strain Escherichia coli with cloned lux operon of P. leiognathi (Ekolyum-9). It is found that small concentrations of chlorides and sulfates of the cations studied had a concentration-dependent stimulatory effect on bacterial bioluminescence; as the concentration of agents increased, activation was succeeded by quenching. The strength of the inhibitory effect, which is characterized by EC50, decreased in the series Ca2+ > Na+ > Mg2+ > K+. Carbonates and hydrocarbonates had a pronounced inhibitory effect on the bioluminescence intensity, determined by an increase in pH. We showed that some types of highly mineralized water with a high hydrocarbonate content have a marked inhibitory effect on the luminescence intensity of microbial luminescent biosensors, mimicking the effect of chemical pollutants.     

Biotests for mineral waters with natural and recombinant luminescent microorganisms

We have developed methods of biotesting mineral waters involving use of natural or recombinant luminescent strains with elimination of the effect of salt concentration and pH. To overcome the adverse effect of high salt concentrations, disguising the action of chemical pollutants, a special method of mineral water sample preparation is proposed. In this method, the marine luminescent bacterium Photobacterium phosphoreum (Microbiosensor B17 677f) is used as a test object. Samples to be analyzed are supplemented with NaCl depending on their natural salt concentration to adjust it to 3 g/l. Another approach, more universal and efficient, involves pH adjustment in the samples to 7.5. This value is suitable for application of both Microbiosensor B17 677f and the recombinant Escherichia coli strain harboring the cloned lux operon of P. leiognathi (Ecolum 9). It has been shown that this treatment, retaining the natural luminescence level of the bacterial biosensors, allows bioluminescent detection of exogenous pollutants added to the samples, including benzene and Cr(VI).     

Immobilization of Escherichia coli expressing the lux genes of Xenorhabdus luminescens.

The luxCDABE operon of Xenorhabdus luminescens was cloned into pUC18 to make pLITE27. Expression of the lux genes from the lac promoter resulted in strong constitutive light emission by Escherichia coli DH5 carrying the recombinant lux plasmid, pLITE27. When strain DH5(pLITE27) was immobilized with sodium alginate-CaCl2, the embedded cells retained their luminescence up to 2 weeks under appropriate storage conditions.     

Heterogeneity of the populations of marine luminescent bacteria Photobacterium leiognathi under different conditions of cultivation

Manifestation of pleiotropic effects in the isogenic variants of luminescent bacteria Photobacterium leiognathi 54 was investigated. The decrease or increase of the expression level of bioluminescence was caused by changes in lux operon regulation. The dynamics of the bioluminescence of dark and dim variants did not differ from the dynamics of the initial luminescent variant, but dependence of the level of luminescence intensity on the exogenous autoinductor of the lux operon was revealed. The investigated variants of P. leiognathi 54 inherited fairly stable morphological characteristics, colony architectonics, level of luminescence, and activity of some enzymes; variants with reduced bioluminescence formed colonies of the S type. Stable bright variants with S- and R-type colonies appeared both in the initial strain population and in the dark variant population, but with smaller frequency. Populations of the bright variant with R-type colonies were most heterogeneous; this can be determined by the lack of glucose repression of the bioluminescence in contrast to other investigated variants of P. leiognathi.     

Cloning of pectate-lyase genes of Erwinia chrysanthemi in Escherichia coli cells

Erwinia chrysanthemi DNA fragment digested by restriction endonuclease EcoRI and carrying the gene EC16 determining the synthesis of pectatelyase with Rf 0.20 and mol. mass 40kD has been cloned in plasmid pUC 9 plasmid in Escherichia coli HB101 cells. Three genes for pectatelyases of Erwinia chrysanthemi ENA49 have been cloned in vector phage lambda 47.1 in Escherichia coli cells. Two genes determining the synthesis of pectatelyases with Rf 0.06 and 0.19 and mol. masses 40 kD and 39 kD have been cloned as a part of an 7 kb Eco RI-fragment, that suggested their close location on the chromosome of Erwinia chrysanthemi ENA49. All of the cloned pectatelyase genes are expressed constitutively with pectatelyases accumulating in periplasm and being unable to secret into the cultural medium.     

Localization of a genetic region involved in McrB restriction by Escherichia coli K-12.

A 5,500-base-pair BglII-EcoRI fragment proximal to the hsd genes of Escherichia coli K-12 has been cloned in the plasmid vector pUC9. The resultant hybrid plasmid was shown to complement the mcrB mutation of E. coli K802. The presence of the hybrid plasmid in strain K802 caused an 18.3-fold drop in transformation efficiency with AluI-methylated pACYC184 relative to unmethylated pACYC184. These results indicate that the cloned DNA is involved in the McrB system restriction of 5-methylcytosine DNA.     

Cloning and expression of the Photobacterium phosphoreum luminescence system demonstrates a unique lux gene organization

The organization of the lux structural genes (A-E) in Photobacterium phosphoreum has been determined and a new gene designated as luxF discovered. The P. phosphoreum luminescence system was cloned into Escherichia coli using a pBR322 vector and identified by cross-hybridization with Vibrio fischeri lux DNA. The lux genes were located by specific expression of P. phosphoreum DNA fragments in the T7-phage polymerase/promoter system in E. coli and identification of the labeled polypeptide products. The luxA and luxB gene products (luciferase subunits) were shown to catalyze light emission in the presence of FMNH2, O2, and aldehyde. The luxC, luxD, and luxE gene products (fatty acid reductase subunits) responsible for aldehyde biosynthesis could be specifically acylated with 3H-labeled fatty acids. The order of the lux genes in P. phosphoreum was found to be luxCDABFE with luxF coding for a new polypeptide of 26 kDa. The presence of a new gene in the P. phosphoreum luminescence system between luxB and luxE as compared to the organization of the lux structural gene in V. fischeri and Vibrio harveyi (luxCDABE) demonstrates that the luminescent systems in the marine bacteria have significantly diverged. The discovery of the luxF gene provides the basis for elucidating the role of its gene product in the expression of luminescence in different marine bacteria.     

Comparative biochemistry of the cell envelopes of Photobacterium leiognathi and Escherichia coli

Photobacterium leiognathi closely resembles Escherichia coli with respect to cell lysis by lysozyme, and the fractionation of outer and cytoplasmic membranes. The two organisms differ in their phospholipid contents and, more significantly, in outer membrane protein compositions.     

Coexistence of transgenic Escherichia coli strains and natural microorganisms in experimental aquatic microcosms

In experimental aquatic microcosms (AMCs), the population of the Escherichia coli strain Z905 harboring the recombinant plasmid pPHL7 (AprLux+) was found to gradually accumulate AMC-adapted cells, which retained the plasmid but differed from the original cells in some biochemical and physiological characteristics. Both the original and the AMC-adapted E. coli cells could coexist with the native AMC microflora for a year or longer. When introduced into AMCs together with native pseudomonads, the AMC-adapted E. coli Z905-33 (pPHL7) cells were more competitive than nonadapted cells.     

Energetic aspects of malate and lactate fermentation by Acetobacterium malicum

The complete nucleotide sequence of two genes from Clostridium thermosulfurogenes EM1 homologous to E. coli genes encoding transport proteins was determined by the dideoxy procedure. The genes were cloned from plasmid pCT4, which contains the alpha-amylase gene from C. thermosulfurogenes EM1 as a 2.9-kbp XbaI fragment, inserted into the XbaI site of pUC18, to yield plasmid pCT401. The proteins encoded by the two identified complete ORFs are very hydrophobic and thus are probably integral membrane proteins. They show over 50% similarity to the maltose transport proteins MalF and MalG and to the glycerol-3-phosphate uptake proteins UgpA and UgpE of Escherichia coli. Since these genes are located immediately upstream of the alpha-amylase gene (amyA) of C. thermosulfurogenes EM1, the encoded proteins might be involved in transport of starch degradation products. The genes were tentatively designated amyC and amyD.