Reporter genes lucFF, luxCDABE, gfp, and dsred have different characteristics in whole-cell bacterial sensors

The selection of a genetic reporter can be difficult because of the wide range of genes available. In order to reduce the selection, we compared the performance of different reporter genes: firefly luciferase (Photinus pyralis lucFF), bacterial luciferase operon (Photorhabdus luminescens luxCDABE), green fluorescent protein (Aequorea victoria gfp), and red fluorescent protein (Discosoma sp. dsred) in whole-cell bacterial sensors. Escherichia coli sensor bacteria were engineered to contain a reporter plasmid that carries the reporter gene under the control of mercury- (mer from Tn21) or arsenite- (ars from R773) responsive regulatory units. Characteristics of the strains were studied by using different arsenite or mercury concentrations and incubation times. The lowest detectable concentration of analytes and the fastest responses were achieved with lucFF or luxCDABE as reporter genes. The fluorescent proteins, GFP and DsRed, gave responses at higher analyte concentrations and after significantly longer incubation times. The results indicate that luciferases are better reporters in whole-cell sensor bacteria.     

Efficient Insertion Mutagenesis Strategy for Bacterial Genomes Involving Electroporation of In Vitro-Assembled DNA Transposition Complexes of Bacteriophage Mu

An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction was developed. Incubation of MuA transposase protein with artificial mini-Mu transposon DNA in the absence of divalent cations in vitro resulted in stable but inactive Mu DNA transposition complexes, or transpososomes. Following delivery into bacterial cells by electroporation, the complexes were activated for DNA transposition chemistry after encountering divalent metal ions within the cells. Mini-Mu transposons were integrated into bacterial chromosomes with efficiencies ranging from 10⁴ to 10⁶ CFU/μg of input transposon DNA in the four species tested, i.e., Escherichia coli, Salmonella enterica serovar Typhimurium, Erwinia carotovora, and Yersinia enterocolitica. Efficiency of integration was influenced mostly by the competence status of a given strain or batch of bacteria. An accurate 5-bp target site duplication flanking the transposon, a hallmark of Mu transposition, was generated upon mini-Mu integration into the genome, indicating that a genuine DNA transposition reaction was reproduced within the cells of the bacteria studied. This insertion mutagenesis strategy for microbial genomes may be applicable to a variety of organisms provided that a means to introduce DNA into their cells is available.     

Cyclic digene system as a control element of a bacterial biosensor

The Escherichia coli JC158(pCIA12/pGFK5) strain carrying a cyclic digene system with a negative feedback on the pCIA12 plasmid reacting to the DNA damage by changing the synthesis level of reporter genes—GFP and β-galactosidase—was tested. The acquired phenotype was inherited by the next generations after the removal of the genotoxic action when the concentration of the DNA-damaging compounds was above the threshold level. A potential has been shown for the application of bacterial biosensors to monitor the presence of genotoxicants in the environment and to test the consequences of short-term exposures to toxic compounds.     

Efficient insertion mutagenesis strategy for bacterial genomes involving electroporation of in vitro-assembled DNA transposition complexes of bacteriophage mu

An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction was developed. Incubation of MuA transposase protein with artificial mini-Mu transposon DNA in the absence of divalent cations in vitro resulted in stable but inactive Mu DNA transposition complexes, or transpososomes. Following delivery into bacterial cells by electroporation, the complexes were activated for DNA transposition chemistry after encountering divalent metal ions within the cells. Mini-Mu transposons were integrated into bacterial chromosomes with efficiencies ranging from 10(4) to 10(6) CFU/microg of input transposon DNA in the four species tested, i.e., Escherichia coli, Salmonella enterica serovar Typhimurium, Erwinia carotovora, and Yersinia enterocolitica. Efficiency of integration was influenced mostly by the competence status of a given strain or batch of bacteria. An accurate 5-bp target site duplication flanking the transposon, a hallmark of Mu transposition, was generated upon mini-Mu integration into the genome, indicating that a genuine DNA transposition reaction was reproduced within the cells of the bacteria studied. This insertion mutagenesis strategy for microbial genomes may be applicable to a variety of organisms provided that a means to introduce DNA into their cells is available.     

GENETIC STRUCTURE AND DIVERSITY WITHIN LOCAL POPULATIONS OF BACILLUS MYCOIDES

Sixty strains of Bacillus mycoides were isolated from each of two sites and characterized by their responses to standard metabolic tests used in bacterial taxonomy, by multilocus enzyme electrophoresis (MLEE), and by restriction-fragment-length polymorphism (RFLP) analysis of Southern blots probed with both a conserved DNA fragment derived from a Salmonella typhimurium ribosomal cistron and with two cosmid probes derived from B. mycoides ATCC strain 6463. Both MLEE and RFLP analyses indicated that the collection contained two genetically distinct sets of strains (I and II); one of these sets was further differentiated genetically by the same analyses (IIA and IIB). Standard taxonomic analysis did not distinguish these sets of strains; biochemical test profiles were similar for all isolates. The genetic distance between groups I and II is as great as that observed for recognized species of bacteria. It is proposed that these groups are sibling species having a common evolutionary descent and that their metabolic phenotype has been conserved, whereas their DNA and protein sequences have diverged. No strong evidence of geographic differentiation between strains from the two sites appeared in either genetic or phenetic characters.     

Cyclic digene system as a control element of a bacterial biosensor

The Escherichia coil JC1 58(pCIA12/pG FK5) strain carrying a cyclic digene system with a negative feedback on the pCIA12 plasmid reacting to the DNA damage by changing the synthesis level of reporter genes-GFP and beta-galactosidase-was tested. The acquired phenotype was inherited by the next generations after the removal of the genotoxic action when the concentration of the DNA-damaging compounds was above the threshold level. A potential has been shown for the application of bacterial biosensors to monitor the presence of genotoxicants in the environment and to test the consequences of short-term exposures to toxic compounds.     

Detection and characterization of quorum sensing signal molecules in Acinetobacter strains

Quorum sensing is a widespread regulatory mechanism among Gram-negative bacteria. In this study, Acinetobacter strains were assayed for the presence of quorum sensing signal molecules capable of activating N-acylhomoserine lactone biosensors. By using an Agrobacterium tumefaciens reporter strain it was shown that all the cultures produced two to four detectable signal molecules with different chromatographic patterns. In A. calcoaceticus BD413 supernatants four compounds were detected in a time-dependent manner, and maximal activity was reached at stationary phase. The number of signal molecules was dependent on medium composition; typically, cultures in minimal medium displayed one or two more signals, as compared to complex medium. None of the Acinetobacter supematants showed autoinduction activity with an Chromobacterium violaceum reporter strain, neither in direct or competition assays.     

A Vibrio splendidus strain is associated with summer mortality of juvenile oysters Crassostrea gigas in the Bay of Morlaix (North Brittany, France).

Juvenile oysters Crassostrea gigas cultured in the Bay of Morlaix (France) have suffered unexplained summer mortalities for over a decade. In the present study, we tested the hypothesis that a bacterial pathogen could be responsible for this phenomenon. A first attempt failed to isolate a bacterial pathogen from moribund or weak oysters. Only non-pathogenic, probably opportunistic, bacteria were isolated. As an alternative approach, we focused on oysters presenting reduced stress-response capacities (determined by circulating noradrenaline measurements), a characteristic of juvenile oysters entering an early phase of the disease. Cultures of bacterial isolates on TCBS plates revealed that a Vibrio strain was present in diseased oysters and scarce or absent in healthy oysters. Experimental infections indicated that this Vibrio can cause mortalities of juvenile oysters when injected at concentrations ranging from 10(4) to 10(8) CFU oyster(-1). Similarly to the summer mortality disease, the Vibrio isolate caused higher mortalities at higher temperatures; apparently, it could not be transmitted horizontally, it did not affect adult oysters and it induced stress-response dysfunctions in juvenile oysters. Phenotypic and genotypic characterizations identified the pathogen as Vibrio splendidus. Taken together, the present results satisfy Koch's postulate and suggest that this bacterial strain is probably responsible for the juvenile oyster summer mortalities in the Bay of Morlaix.     

Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast

Luciferase reporter genes have been successfully used in a variety of organisms to examine gene expression in living cells, but are yet to be successfully developed for use in chloroplast. Green fluorescent protein (gfp) has been used as a reporter of chloroplast gene expression, but because of high auto-fluorescence, very high levels of GFP accumulation are required for visualization in vivo. We have developed a luciferase reporter for chloroplast by synthesizing the two-subunit bacterial luciferase (lux)AB, as a single fusion protein in Chlamydomonas reinhardtii chloroplast codon bias. We expressed a chloroplast luciferase gene, luxCt, in C. reinhardtii chloroplasts under the control of the ATPase alpha subunit (atpA) or psbA promoter and 5' untranslated regions (UTRs) and the rubisco large subunit (rbcL) 3' UTR. We show that luxCt is a sensitive reporter of chloroplast gene expression, and that luciferase activity can be measured in vivo using a charge coupled device (CCD) camera or in vitro using a luminometer. We further demonstrate that luxCt protein accumulation, as measured by Western blot analysis, is proportional to luminescence, as determined both in vivo and in vitro, and that luxCt is capable of reporting changes in chloroplast gene expression during a dark to light shift. These data demonstrate the utility of the luxCt gene as a versatile and sensitive reporter of chloroplast gene expression in living cells.     

Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome.

An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.     

Autoinducer-2 detection among commensal oral streptococci is dependent on pH and boric acid

Autoinducer-2, considered a universal signaling molecule, is produced by many species of bacteria; including oral strains. Structurally, autoinducer-2 can exist bound to boron (borated autoinducer-2). Functionally, autoinducer-2 has been linked to important bacterial processes such as virulence and biofilm formation. In order to test production of autoinducer-2 by a given bacterial strain, a bioassay using marine bioluminescent bacteria Vibrio harveyi as a reporter for autoinducer-2 has been designed. We hypothesize that pH adjustment and addition of boron are required for optimal bioluminescence and accurate autoinducer-2 detection. Using this reporter strain we tested autoinducer-2 activity from two oral commensal species, Streptococcus gordonii DL1 and Streptococcus oralis 34. Spent broth was collected and adjusted to pH 7.5 and supplemented with boric acid prior to measuring autoinducer- 2 activity. Results show that low pH inhibits bioluminescence of the reporter strain, but pH 7.5 allows for bioluminescence induction and proper readings of autoinducer-2 activity. Addition of boric acid also has a positive effect on bioluminescence allowing for a more sensitive detection of autoinducer-2 activity. Our data suggests that although autoinducer-2 is present in spent broth, low pH and/or low levels of boric acid become an obstacle for proper autoinducer-2 detection. For proper autoinducer-2 detection, we propose a protocol using this bioassay to include pH adjustment and boric acid addition to spent broth. Studies on autoinducer-2 activity in several bacteria species represent an important area of study as this universal signaling molecule is involved in critical bacterial phenotypes such as virulence and biofilm formation.     

Characterization of the Streptomyces violaceoruber SANK95570 plasmids pSV1 and pSV2

The gene coding for recA in the oral pathogen Actinobacillus actinomycetemcomitans SUNY 465 was cloned and sequenced. The DNA sequence coded for a 352-amino acid protein that was homologous to RecA of a variety of bacterial species. A derivative of a non-replicating mobilizable plasmid was constructed for directed mutagenesis in A. actinomycetemcomitans. A recA-deficient strain of A. actinomycetemcomitans was developed by homologous recombination of an internal recA fragment contained on the mobilizable suicide vector. The recA mutant strain was more sensitive to UV radiation and showed a reduced recombinatorial proficiency than the isogenic parent strain. These data suggest that recA of A. actinomycetemcomitans SUNY 465 is involved in the repair of DNA damage caused by UV irradiation and homologous recombination as determined for other bacteria.     

Bacterial strain typing in the genomic era

Bacterial strain typing, or identifying bacteria at the strain level, is particularly important for diagnosis, treatment, and epidemiological surveillance of bacterial infections. This is especially the case for bacteria exhibiting high levels of antibiotic resistance or virulence, and those involved in nosocomial or pandemic infections. Strain typing also has applications in studying bacterial population dynamics. Over the last two decades, molecular methods have progressively replaced phenotypic assays to type bacterial strains. In this article, we review the current bacterial genotyping methods and classify them into three main categories: (1) DNA banding pattern-based methods, which classify bacteria according to the size of fragments generated by amplification and/or enzymatic digestion of genomic DNA, (2) DNA sequencing-based methods, which study the polymorphism of DNA sequences, and (3) DNA hybridization-based methods using nucleotidic probes. We described and compared the applications of genotyping methods to the study of bacterial strain diversity. We also discussed the selection of appropriate genotyping methods and the challenges of bacterial strain typing, described the current trends of genotyping methods, and investigated the progresses allowed by the availability of genomic sequences.     

Vectors to express foreign genes and techniques to monitor gene expression in Pseudomonads

Improved tools for Pseudomonas research include small, broad-host-range vectors that allow regulated expression from the lac operon and T7 promoters whose biology is well understood and adaptable to many bacteria. To facilitate studies on gene regulation, tracking and monitoring of bacteria in diverse environments, and the construction of biosensors, various reporter genes with versatile assay formats have been developed that can be delivered on plasmid, transposon and integration-proficient vectors.     

SPEX, a System for the Expression of Recombinant Proteins from Gram-Positive Bacterial Vectors

Using a conserved pathway for surface protein extrusion, a system has been developed for the expression and secretion of proteins from gram-positive bacteria. As proof-of-concept, theStreptococcus gordoniiChallis strain has been engineered to express a series of recombinant proteins fused to the conserved region of the M6 protein ofStreptococcus pyogenes.In the prototype surface protein expression system, the recombinant M6 protein is anchored to the surface ofS. gordoniicells expressing it. In order to overexpress the protein and easily purify it away from the bacteria, the protein was modified to enable it to be secreted into the medium. To accomplish this, a stop codon was introduced into the gene just prior to the anchor region using site-directed mutagenesis. Using enzyme-linked immunosorbent assays, it was possible to quantitate the amount of protein expressed using this system. With little or no optimization, 3 mg of protein per liter of culture was expressed and secreted into the medium of a bacterial culture grown to an OD₆₀₀equal to 1.0. This system should be broadly applicable for the expression and secretion of a variety of proteins (antigens, hormones, and enzymes) directly into the medium.