Hyperactivity and interactions of a chimeric myristoryl-ACP thioesterase from the lux system of luminescent bacteria

A chimeric myristoyl-ACP thioesterase with much higher catalytic efficiency than the parental enzymes has been generated by ligating the N-terminal half of the lux-specific thioesterase (LuxD) from Photobacterium phosphoreum with the C-terminal half of LuxD from Vibrio harveyi. The LuxD chimera had the same rate-limiting step and specificity, but cleaved esters and thioesters over eight times faster than the native enzymes. LuxD, along with acyl-protein synthetase (LuxE) and reductase (LuxC), comprise a multienzyme complex channeling activated fatty acids into the aldehyde substrate for the bacterial bioluminescence reaction. As P. phosphoreum LuxD and LuxE modulate each of their respective activities, the effects of mixing V. harveyi and the chimeric LuxD with P. phosphoreum LuxE were investigated. The chimeric LuxD stimulated acylation of LuxE to the same extent as V. harveyi LuxD, but to a lower level than that caused by P. phosphoreum LuxD. Conversely, P. phosphoreum LuxE stimulated the thioesterase activity of V. harveyi LuxD by 30% and the chimeric LuxD by 20% while the activity of P. phosphoreum LuxD was increased by over 140%. These results show that the stimulatory effects are unrelated to the level of thioesterase activity and indicate that the carboxyl terminal region of LuxD interacts with LuxE and causes a conformational change.     

Ultrastructural organization of marine luminescent bacteria and their mutants

The aim of this work was to study the submicroscopic organization of luminescent bacteria belonging to the genera Photobacterium and Lucibacterium as well as that of their "dark" mutants incapable of luminescence. The ultrastructural organization of all studied bacteria is typical of gram-negative species. The luminescent bacteria are characterized by the presence, in their cytoplasm, of osmophilic formations 22--110 nm in size. The cells of "dark" mutants accumulate volutin and contain complex membrane systems which are related to decelerated growth of the cultures.     

Genome organization of repetitive elements in the rodent,Peromyscus leucopus

To document the frequency and distribution of repetitive elements in Peromyscus leucopus, the white-footed mouse, a cosmid genomic library was examined. Two thousand thirteen randomly chosen recombinants, with an average insert size of 35 kb and representing 2.35% of the haploid genome of P. leucopus, were screened with probes representing microsatellites, tandem repeats, and transposable elements. Of the four dinucleotides, (GT)_n was present in 87% of the clones, (CT)_n was present in 59% of the clones, and (AT)_n and (GC)_n each was represented in our sample by a single clone (0.05%). (TCC)_n was present in 8% of the clones. Of the tandem repeats, the 28S ribosomal probe and the (TTAGGG)_n telomere probe were not represented in the library, whereas a heterochromatic fragment was present in 9% of the clones. A transposable element, mys, was estimated to occur in 4700 copies, whereas a long interspersed element (LINE) was estimated to occur in about 41,000 copies per haploid genome. LINE and mys occurred together in the same clones more frequently than expected on the basis of chance. Hybridizing the library to genomic DNA from P. leucopus, Reithrodontomys fulvescens, Mus musculus, and human produced general agreement between phylogenetic relatedness and intensity of hybridization. However, dinucleotide repeats appeared to account for a disproportionately high number of positive clones in the more distantly related taxa.     

Sequence organization of repetitive elements in the flanking regions of the chloroplast ribosomal unit of Chlamydomonas reinhardii.

The flanking regions and the end of the chloroplast ribosomal unit of Chlamydomonas reinhardii have been sequenced. The upstream region of the ribosomal unit contains three open reading frames coding for 111, 117 and 124 amino acids, respectively. The latter polypeptide is partially related to the ribosomal protein L16 of E. coli. Two of the open reading frames overlap each other and are oriented in opposite direction. The region between these open reading frames and the 5' end of the 16S rRNA gene contains numerous short direct and inverted repeats which can be folded into large stem-loop structures. Sequence elements that resemble prokaryotic promoters are found in the same region. Several of the repeated elements are distributed throughout the non-coding regions of the chloroplast inverted repeat. Sequence comparison between the 5S rRNA and its gene does not reveal any significant sequence heterogeneity between the chloroplast 5S rRNA genes.     

Structural organization of interspersed repetitive elements present in the DNA of Mus musculus

Two-dimensional displays of the restriction fragments from the DNA of Mus musculus revealed a complex species-specific pattern produced from nonsatellite repetitive sequences. The patterns have been used as a guide in the direct purification of a group of broadly interspersed repeated DNA sequences (characterized by a 1350-bp Eco-Bam fragment) that have been studied by molecular cloning, restriction mapping and genomic Southern blotting. These studies show that the cloned representatives originate from an abundant group of sequences that share homology with about 2% of the mouse genome. The sequences do not appear to share homology with mouse-interspersed-family-1 (MIF-1) nor with the major AT-rich satellite sequences of mouse. They appear to be part of a group of larger repetitive elements that is both broadly interspersed and heavily methylated in normal mouse tissue.     

Comparison of the spectral emission of lux recombinant and bioluminescent marine bacteria

The purpose of the present paper was to study the influence of bacteria harbouring the luciferase‐encoding Vibrio harveyi luxAB genes upon the spectral emission during growth in batch‐culture conditions. In vivo bioluminescence spectra were compared from several bioluminescent strains, either naturally luminescent (Vibrio fischeri and Vibrio harveyi) or in recombinant strains (two Gram‐negative Escherichia coli::luxAB strains and a Gram‐positive Bacillus subtilis::luxAB strain). Spectral emission was recorded from 400 nm to 750 nm using a highly sensitive spectrometer initially devoted to Raman scattering. Two peaks were clearly identified, one at 491–500 nm (± 5 nm) and a second peak at 585–595 (± 5 nm) with the Raman CCD. The former peak was the only one detected with traditional spectrometers with a photomultiplier detector commonly used for spectral emission measurement, due to their lack of sensitivity and low resolution in the 550–650 nm window. When spectra were compared between all the studied bacteria, no difference was observed between natural or recombinant cells, between Gram‐positive and Gram‐negative strains, and growth conditions and growth medium were not found to modify the spectrum of light emission. Copyright © 2003 John Wiley & Sons, Ltd.     

"Quorum sensing" regulation of lux gene expression and the structure of lux operon in marine bacteria Alivibrio logei

A group of luminescent strains of marine bacteria Alivibrio logei has been isolated (basins of the Okhotsk, White and Bering Seas). Strains A. logei were shown to be psycrophiic bacteria with an optimal growth temperature of approximately 15 degrees C. Biolumiscent characteristics of strains were studied, and the expression of lux genes was shown to be regulated by the "quorum sensing" system. The A. logei lux operon was cloned in Escherichia coli cells and the structure of this operon and its nucleotide sequence were determined. The structure of A. logei lux operon differs markedly from that in the closely related species of luminescent marine bacteria A. fischeri. In the structure of the A. logei lux operon, the the luxI gene is absent in front of luxC, and a fragment containing luxR2-luxI genes is located immediately after luxG gene. Luminescent psycrophiic marine bacteria of A. logei are assumed to be widely distributed in cold waters of northern seas.     

Database of natural luminescent bacteria

The database of luminescent bacteria stored in the IBSO collection is one of the metasections of BIOLUMBASE. A logical schema of the metasection “Natural luminescent organisms”, classification of entities, and methods of attribute presentation have been developed. The database of luminescent bacteria maintained in the IBSO collection is being widened by findings of the collection staff as well as by information from scientific literature. The expectant contents of the database will be useful for resolving various problems of microbial ecology and biotechnology which deal with luminescent bacteria, luminescent system derived from them, and lux-genes cloned to other organisms. A potential user would be able not only to access cataloged data on strains but also to get information on properties, functions, use, and bibliography and to perform an attribute-match search of a strain.     

The classification of luminescent bacteria

Summary The luminescent bacteria are logically placed in two genera. The common coccoid and frequently non-motile species placed byBeije-rinck first in his genusPhotobacterium, 1889 under the namePhotobacterium phosphorescens syn.Bacterium phosphorescens Fischer, should be recognized as the type species ofPhotobacterium. Other characters indicate that this genus should be placed in the FamilyPseudomonadaceae Winslowet al.. and should include other straight, rod-shaped, luminescent, polar flagellate bacteria that ferment glucose without, however, necessarily producing gas (H₂ and CO₂) as does the type species. The species that have the form of vibrios should be accepted as members of the genusVibrio as suggested by several previous investigators. They have characters much like those ofVibrio comma, the type species of the genusVibrio.     

Near on-line detection of enteric bacteria using lux recombinant bacteriophage

The potential for a revolution in microbial testing can be perceived with the near on-line detection of indicator microorganisms. By definition, these are microorganisms present in significant numbers within a food which, while not pathogenic, can be related through increasing count to the increased probability of pathogen contamination. We have used recombinant lux+ bacteriophage to detect enteric indicator bacteria without recovery or enrichment in 50 min, provided that they are present at levels greater than 10(4) g-1 or cm-2. After a 4-h enrichment, samples having enteric counts of 10 g-1 or cm-2 can be distinguished from background.     

Recombinant luminescent bacteria for measuring bioavailable arsenite and antimonite.

Luminescent bacterial strains for the measurement of bioavailable arsenite and antimony were constructed. The expression of firefly luciferase was controlled by the regulatory unit of the ars operon of Staphylococcus aureus plasmid pI258 in recombinant plasmid pTOO21, with S. aureus RN4220, Bacillus subtilis BR151, and Escherichia coli MC1061 as host strains. Strain RN4220(pTOO21) was found to be the most sensitive for metal detection responding to arsenite, antimonite, and cadmium, the lowest detectable concentrations being 100, 33, and 330 nM, respectively. Strains BR151(pTOO21) and MC1061(pTOO21) responded to arsenite, arsenate, antimonite, and cadmium, the lowest detectable concentrations being 3.3 and 330 microM and 330 and 330 nM with BR151(pTOO21), respectively, and 3.3, 33, 3.3, and 33 microM with MC1061(pTOO21), respectively. In the absence of the mentioned ions, the expression of luciferase was repressed and only a small amount of background light was emitted. Other ions did not notably interfere with the measurement in any of the strains tested. Freeze-drying of the cells did not decrease the sensitivity of the detection of arsenite; however, the induction coefficients were somewhat lower.     

Expansion of repetitive DNA into cytogenetically visible elements.

Two cases of amplified repetitive elements accidentally identified in cancer samples are reported. In both cases, repeated DNA that is normally not visible by traditional chromosome banding had increased in amount to become cytogenetically visible. In one case, an addition to the short arm of chromosome 1 was originally diagnosed. However, upon molecular analysis the diagnosis could be corrected to an amplification of the D1Z2 repeat. In the second case, a strongly DAPI-positive band was visible at the top of the short arm of chromosome 22, and the original diagnosis was add(22). Staining for telomeric repeats revealed their presence inside the DAPI-positive element, thus confirming that the element in question was truly added to the end of the chromosome. Curiously, no telomeric repeats could be detected distal to the DAPI-positive element. The identity of the DAPI-positive element could not be established, as it was not stained by any of the specific probes applied, nor in a scanning hybridization with labeled Cot-1 DNA. It thus seems to represent an expansion from some lowly repetitive AT-rich DNA translocated to the tip of chromosome 22.     

Analysis of repetitive sequence elements containing tRNA-like sequences.

Several repetitive sequence elements from diverse species share extensive sequence homology with tRNA molecules. Analysis of the tRNA-like sequences within these elements suggest that they have originated from authentic tRNA sequences. Elements containing tRNA-like sequences can be divided into three distinct groups whose members share extensive sequence homology, have similar sequence organization and have unique species distribution. We suggest that these three groups represent independent examples of retroposon families that have originated from tRNAs.     

Novel families of interspersed repetitive elements from the human genome.

Six novel families of interspersed repetitive elements have been detected in the available human DNA sequences using computer-assisted analyses. The estimated total number of elements in the reported six families is over 17,000. Sequences representative for each family range from approximately 150 to 650 base pairs (bp) in length and are predominantly (A + T)-rich. Sequences from four families contain stretches of patchy complementarity up to 45 bp long. Member of one of the families is likely be directly involved in a multigene deletion on chromosome 14. Two of the six sequence families are homologous to 'low reiteration frequency sequences' from monkey cells, detected first in defective variants of simian virus 40. Like Alu and L1 families, the newly discovered families are probably composed of pseudogenes derived from functional genes.     

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.