Expression of bioluminescence by Escherichia coli containing recombinant Vibrio harveyi DNA.

When isogenic strains of Escherichia coli, RR1 (rec+) and HB101 (recA), were transformed with mapped recombinant plasmids known to contain Vibrio harveyi luciferase genes and large regions of DNA flanking on both sides, a small percentage (0.005%) of the colonies expressed high levels of luminescence (up to 10(12) quanta s-1 ml-1) in the absence of added aldehyde. The altered ability to express light was found to be due to a mutation in the host and not to an alteration in the recombinant DNA. When these bright colonies were cured of plasmid, they could be retransformed with cloned V. harveyi gene fragments in cis and in trans to yield luminescent colonies at 100% frequency. The maximum length of V. harveyi DNA required to produce light-emitting E. coli was shorter (6.3 kilobase pairs) than that required for expression of the V. fischeri system in E. coli. Cell extracts from bright clones contained wild-type levels of activity for the heteropolymeric (alpha beta) luciferase; fatty acid labeling revealed the presence of the three acylated polypeptides of the fatty acid reductase system which is involved in aldehyde biosynthesis for the luminescence reaction. The increased light emission in the mutant bacteria appeared to arise in part from production of higher levels of polycistronic mRNAs coding for luciferase.     

Active site hydrophobicity is critical to the bioluminescence activity of Vibrio harveyi luciferase

Vibrio harveyi luciferase is an alphabeta heterodimer containing a single active site, proposed earlier to be at a cleft in the alpha subunit. In this work, six conserved phenylalanine residues at this proposed active site were subjected to site-directed mutations to investigate their possible functional roles and to delineate the makeup of luciferase active site. After initial screening of Phe --> Ala mutants, alphaF46, alphaF49, alphaF114, and alphaF117 were chosen for additional mutations to Asp, Ser, and Tyr. Comparisons of the general kinetic properties of wild-type and mutated luciferases indicated that the hydrophobic nature of alphaF46, alphaF49, alphaF114, and alphaF117 was important to luciferase V(max) and V(max)/K(m), which were reduced by 3-5 orders of magnitude for the Phe --> Asp mutants. Both alphaF46 and alphaF117 also appeared to be involved in the binding of reduced flavin substrate. Additional studies on the stability and yield of the 4a-hydroperoxyflavin intermediate II and measurements of decanal substrate oxidation by alphaF46D, alphaF49D, alphaF114D, and alphaF117D revealed that their marked reductions in the overall quantum yield (phi( degrees )) were a consequence of diminished yields of luciferase intermediates and, with the exception of alphaF114D, emission quantum yield of the excited emitter due to the replacement of the hydrophobic Phe by the anionic Asp. The locations of these four critical Phe residues in relation to other essential and/or hydrophobic residues are depicted in a refined map of the active site. Functional implications of these residues are discussed.     

Mobilization of cloned luciferase genes into Vibrio harveyi luminescence mutants

A recombinant plasmid which carried a 5 kb fragment of Vibrio harveyi DNA containing the luxA and luxB genes was mobilized from Escherichia coli into luminescence-deficient mutants of V. harveyi. The cloned genes complemented a temperature sensitive luciferase mutation, but failed to complement lesions in two different aldehyde deficient mutants. Expression of the cloned genes was not subject to autoinduction in either E. coli or in V. harveyi.     

Cloning and expression in Escherichia coli of a recA-like gene from Vibrio cholerae

Summary A library containing more than 80% of the Vibrio cholerae genome was constructed by cloning BamH1 restriction fragments into pBR322. Using interspecific complementation of an Escherichia coli recA mutant with plasmids containing the gene bank of V. cholerae, a recA-like gene was identified. The recombinant plasmid, designated as pDP145, contained a 1.45 kb segment of V. cholerae DNA which codes for a protein of molecular weight 39,000. The product of this gene confers methyl methane sulphonate resistance on the E. coli recA mutant, suppresses its ultraviolet (UV) light sensitive phenotype and has proteolytic activity on the phage repressor. Induction of a 39,000 dalton protein in UV-irradiated V. cholerae cells was demonstrated.     

Heterogeneity in quorum sensing-regulated bioluminescence of Vibrio harveyi

Quorum sensing (QS) refers to the ability of bacterial populations to read out the local environment for cell density and to collectively activate gene expression. Vibrio harveyi , one of the best characterized model organisms in QS, was used to address the question how single cells behave within a QS-activated community in a homogeneous environment. Analysis of the QS-regulated bioluminescence of a wild type strain revealed that even at high cell densities only 69% of the cells of the population produced bioluminescence, 25% remained dark and 6% were dead. Moreover, light intensities greatly varied from cell to cell at high population density. Addition of autoinducer to a bright liquid culture of V. harveyi increased the percentage of luminescent cells up to 98%, suggesting that V. harveyi produces and/or keeps the autoinducers at non-saturating concentrations. In contrast, all living cells of a constitutive QS-active mutant (Δ luxO ) produced light. We also found that QS affects biofilm formation in V. harveyi . Our data provide first evidence that a heterogeneous population produces more biofilm than a homogeneous one. It is suggested that even a QS-committed population of V. harveyi takes advantage of heterogeneity, which extends the current view of QS-regulated uniformity.     

Crystals of luciferase from Vibrio harveyi. A preliminary characterization

Bacterial luciferase from Vibrio harveyi, the 77,000-dalton light-emitting enzyme of the marine bacterium, has been crystallized into a two million cubic Angstrom cell with P212121 symmetry. The cell constants are a = 59.6 +/- 0.4 A, b = 112 +/- 0.7 A, and c = 302 +/- 2 A. The reflections corresponding to the 302-A cell edge can be resolved by suitable collimation of the incident beam, without resorting to focusing mirrors. The crystals diffract to better than 3-A resolution and are large enough (0.7 mm) for data collection. The crystallization conditions are presented and general crystallization characteristics are discussed.     

Cloning and expression of the beta-galactosidase genes from Lactobacillus reuteri in Escherichia coli

Heterodimeric beta-galactosidase of Lactobacillus reuteri L103 is encoded by two overlapping genes, lacL and lacM. The lacL (1887bp) and lacM (960bp) genes encode polypeptides with calculated molecular masses of 73,620 and 35,682Da, respectively. The deduced amino acid sequences of lacL and lacM show significant identity with the sequences of beta-galactosidases from other lactobacilli and Escherichia coli. The coding regions of the lacLM genes were cloned and successfully overexpressed in E. coli using an expression system based on the T7 RNA polymerase promoter. Expression of lacL alone and coexpression of lacL and lacM as well as activity staining of both native and recombinant beta-galactosidases suggested a translational coupling between lacL and lacM, indicating that the formation of a functional beta-galactosidase requires both genes. Recombinant beta-galactosidase was purified to apparent homogeneity, characterized and compared with the native beta-galactosidase from L. reuteri L103.     

Cloning and expression of tryptophan genes from Brevibacterium lactofermentum in Escherichia coli

A gene bank from the amino acid producer Brevibacterium lactofermentum has been prepared in Escherichia coli using pBR322 as vector. Four clones containing genetic information needed to complement mutations in A,B,C and D genes from E. coli have been isolated. The cloned fragments range between 4.3 kb (pULT61) and 7.9 kb (pULT62). All the four clones contain genetic information that complements trpB gene from E. coli. The cloned trpB gene is very stable and is maintained extrachromosomally in E. coli. It is expressed very efficiently showing high levels of tryptophan synthetase activity.     

Cloning and expression in Escherichia coli of dextranase genes from Bacteroides thetaiotaomicron

A genomic bank was constructed in Escherichia coli HB101, consisting of DNA fragments from Bacteroides thetaiotaomicron strain 489 inserted within the vector pBR322. By screening on complex medium containing blue dextran, 10 stable dextranase-positive (Dex+) clones were isolated. Seven groups of Dex+ inserts were identified on the basis of their restriction maps and hybridization responses. Dextanase activity of the recombinant clones was weak, and was revealed on the selection medium after 15 days. Subcloning of a Sau3AI partially digested 3.2-kb insert in the expression vector pDR720 greatly enhanced dextranse activity on blue dextran plates in one clone, but the delay remained unaltered. This suggested that the enzyme was released by cell lysis. Expression of this 0.7-kb subcloned insert was dependent on the promoter region of tryptophan operon carried by pDR720.     

Cloning and expression of pca genes from Pseudomonas putida in Escherichia coli

Beta-Ketoadipate elicits expression of five structural pca genes encoding enzymes that catalyse consecutive reactions in the utilization of protocatechuate by Pseudomonas putida. Three derivatives of P. putida PRS2000 were obtained, each carrying a single copy of Tn5 DNA inserted into a separate region of the genome and preventing expression of different sets of pca genes. Selection of Tn5 in or near the pca genes in these derivatives was used to clone four structural pca genes and to enable their expression as inserts in pUC19 carried in Escherichia coli. Three of the genes were clustered as components of an apparent operon in the order pcaBDC. This observation indicates that rearrangement of the closely linked genes accompanied divergence of their evolutionary homologues, which are known to appear in the order pcaDBC in the Acinetobacter calcoaceticus pcaEFDBCA gene cluster. Additional evidence for genetic reorganization during evolutionary divergence emerged from the demonstration that the P. putida pcaE gene lies more than 15 kilobase pairs (kbp) away from the pcaBDC operon. An additional P. putida gene, pcaR, was shown to be required for expression of the pca structural genes in response to beta-ketoadipate. The regulatory pcaR gene is located about 15 kbp upstream from the pcaBDC operon.     

In vivo translational inaccuracy in Escherichia coli: missense reporting using extremely low activity mutants of Vibrio harveyi luciferase

A convenient, sensitive assay for measurement of in vivo missense translational errors is reported that uses luciferase activity generated by mistranslation of a gene encoding an inactive mutant alpha chain of the Vibrio harveyi enzyme. Mutations were introduced at alpha45 His, a position known to be highly intolerant of amino acids other than histidine. To normalize for any variations in expression level, the concentration of wild-type luciferase alphabeta dimer was determined by a novel assay using co-refolding of active/wild-type beta enzyme subunits with inactive alpha subunits in lysate with an excess of exogenously added active alpha subunits. Four His alpha45 missense mutants of luciferase encoded by leucine codons (CUC, CUU, CUG, and UUG) had histidine misincorporation rates of 2.0 x 10(-6), 1.3 x 10(-6), 9.0 x 10(-8), and 1.5 x 10(-8) respectively, a variation of over 133-fold among synonymous codons. Any substantial contribution of mutation was ruled out by a Luria-Delbrück fluctuation test. The two leucine codons with the highest rates, CUU and CUC, have a single central-mismatch to the histidyl-tRNAQUG anticodon. Aminoglycoside antibiotics known to enhance mistranslation increased the error rate of the CUC codon more than those of the CUU and CUG codons, consistent with the hypothesis that CUC codon mistranslation arises primarily from miscoding events such as the selection of noncognate histidyl-tRNAQUG at the central position of the codon.     

Survival of Escherichia coli and Vibrio harveyi in Dead Sea water

Abstract When suspensions of Escherichia coli or the marine luminescent bacterium Vibrio harveyi were mixed with Dead Sea water, the number of viable bacteria decreased by 90% in a time varying from less than 1 h to several hours, depending on the bacterial strain tested. Survival was better at low temperatures, and diluting the Dead Sea water permitted prolonged survival of both coliform bacteria and V. harveyi . The death rate of E. coli in Dead Sea water was comparable to that in water from the Great Salt Lake (Utah). The high concentrations of calcium and magnesium in Dead Sea water, rather than the high total salinity, was identified as the main factor responsible for the rapid die-off. Exposure to direct solar irradiation significantly increased the die-off rate of E. coli in Dead Sea water.
Large numbers of coliform bacteria were recovered from the lake at distances of at least 20 m from a sewage discharge site on the western shore of the Dead Sea.     

Cloning and expression of the Vibrio cholerae neuraminidase gene nanH in Escherichia coli

A cosmid gene bank of Vibrio cholerae 395, classical Ogawa, was screened in Escherichia coli HB101 for expression of the vibrio neuraminidase (NANase) gene nanH (N-acylneuraminate glycohydrolase). Positive clones were identified by their ability to cleave the fluorogenic NANase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Seven NANase-positive clones were detected after screening 683 cosmid isolates with a rapid, qualitative plate assay method. The nanH gene was subcloned from one of the cosmids and was located within a 4.8-kilobase-pair BglII restriction endonuclease fragment. Evidence that nanH was the NANase structural gene was obtained by transposon mutagenesis and by purification and comparison of the cloned gene product with the secreted NANase purified from the parent V. cholerae strain. The sequence of the first 20 amino-terminal amino acids of the secreted NANase purified from V. cholerae was determined by automated Edman degradation and matched perfectly with the amino acid sequence predicted from nucleotide sequencing of nanH. The sequence data also revealed the existence of a potential signal peptide that was apparently processed from NANase in both V. cholerae and E. coli. In contrast to V. cholerae, E. coli nanH+ clones did not secrete NANase into the growth medium, retaining most of the enzyme in the periplasmic compartment. Kinetic studies in V. cholerae showed that nanH expression and NANase secretion were temporally correlated as cells in batch culture entered late-exponential-phase growth. Similar kinetics were observed in at least one of the E. coli nanH+ clones, suggesting that nanH expression in E. coli might be controlled by some of the same signals as in the parent V. cholerae strain.     

Cloning, sequencing, and functional expression in Escherichia coli of chaperonin (groESL) genes from Vibrio cholerae.

Using a series of oligonucleotides synthesized on the basis of conserved nucleotide motifs in heat-shock genes, the groESL heat-shock operon from a Vibrio cholerae TSI-4 strain has been cloned and sequenced, revealing that the presence of two open reading frames (ORFs) of 291 nucleotides and 1,632 nucleotides separated by 54 nucleotides. The first ORF encoded a polypeptide of 97 amino acids, GroES homologue, and the second ORF encoded a polypeptide of 544 amino acids, GroEL homologue. A comparison of the deduced amino acid sequences revealed that the primary structures of the V. cholerae GroES and GroEL proteins showed significant homology with those of the GroES and GroEL proteins of other bacteria. Complementation experiments were performed using Escherichia coli groE mutants which have the temperature-sensitive growth phenotype. The results showed that the groES and groEL from V. cholerae were expressed in E. coli, and groE mutants harboring V. cholerae groESL genes regained growth ability at high temperature. The evolutionary analysis indicates a closer relationship between V. cholerae chaperonins and those of the Haemophilus and Yersinia species.