Occurrence and Expression of Luminescence in Vibrio cholerae

Several species of the genus Vibrio, including Vibrio cholerae, are bioluminescent or contain bioluminescent strains. Previous studies have reported that only 10% of V. cholerae strains are luminescent. Analysis of 224 isolates of non-O1/non-O139 V. cholerae collected from Chesapeake Bay, MD, revealed that 52% (116/224) were luminescent when an improved assay method was employed and 58% (130/224) of isolates harbored the luxA gene. In contrast, 334 non-O1/non-O139 V. cholerae strains isolated from two rural provinces in Bangladesh yielded only 21 (6.3%) luminescent and 35 (10.5%) luxA⁺ isolates. An additional 270 clinical and environmental isolates of V. cholerae serogroups O1 and O139 were tested, and none were luminescent or harbored luxA. These results indicate that bioluminescence may be a trait specific for non-O1/non-O139 V. cholerae strains that frequently occur in certain environments. Luminescence expression patterns of V. cholerae were also investigated, and isolates could be grouped based on expression level. Several strains with defective expression of the lux operon, including natural K variants, were identified.     

Relationship of the luminous bacterial symbiont of the Caribbean flashlight fish,Kryptophanaron alfredi (family Anomalopidae) to other luminous bacteria based on bacterial luciferase (luxA) genes

Flashlight fishes (family Anomalopidae) have light organs that contain luminous bacterial symbionts. Although the symbionts have not yet been successfully cultured, the luciferase genes have been cloned directly from the light organ of the Caribbean species, Kryptophanaron alfredi. The goal of this project was to evaluate the relationship of the symbiont to free-living luminous bacteria by comparison of genes coding for bacterial luciferase (lux genes). Hybridization of a luxAB probe from the Kryptophanaron alfredi symbiont to DNAs from 9 strains (8 species) of luminous bacteria showed that none of the strains tested had lux genes highly similar to the symbiont. The most similar were a group consisting of Vibrio harveyi, Vibrio splendidus and Vibrio orientalis. The nucleotide sequence of the luciferase subunit gene luxA of the Kryptophanaron alfredi symbiont was determined in order to do a more detailed comparison with published luxA sequences from Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi. The hybridization results, sequence comparisons and the mol% G+C of the Kryptophanaron alfredi symbiont luxA gene suggest that the symbiont may be considered as a new species of luminous Vibrio related to Vibrio harveyi.The nucleotide sequence reported in this article has been deposited in Genbank under accession number M36597     

Stimulation of DNA repair as an evolutionary drive for bacterial luminescence

It was demonstrated recently that luminescence of a free‐living marine bacterium, Vibrio harveyi, stimulates DNA repair, most probably by activation of the photoreactivation process. Here, we ask whether the stimulation of DNA repair could be an evolutionary drive that ensured maintenance and development of early bacterial luminescent systems. To test this hypothesis, we cultivated V. harveyi lux⁺ bacteria and luxA mutants in mixed cultures. Initial cultures were mixed to obtain a culture consisting of roughly 50% lux⁺ cells and 50% luxA mutants. Then bacteria were cultivated for several days and ratio of luminescent to dark bacteria was measured. Under these conditions, luxA mutants became highly predominant within a few days of cultivation. This indicates that, without a selective pressure, the luminescence is a disadvantage for bacteria, perhaps due to consumption of significant portion of cell energy. However, when the same experiments were repeated but cultures were irradiated with low UV doses, luminescent bacteria started to predominate shortly after the irradiation. Therefore, we conclude that stimulation of photoreactivation may be an evolutionary drive for bacterial bioluminescence. Copyright © 2003 John Wiley & Sons, Ltd.     

Codon optimization of bacterial luciferase (lux) for expression in mammalian cells

Expression of the bacterial luciferase (lux) system in mammalian cells would culminate in a new generation of bioreporters for in vivo monitoring and diagnostics technology. Past efforts to express bacterial luciferase in mammalian cells have resulted in only modest gains due in part to low overall expression of the bacterial genes. To optimize expression, we have designed and synthesized codon-optimized versions of the luxA and luxB genes from Photorhabdus luminsecens. To evaluate these genes in vivo, stable HEK293 cell lines were created harboring wild type luxA and luxB (WTA/WTB), codon-optimized luxA and wild type luxB (COA/WTB), and codon-optimized versions of both luxA and luxB genes (COA/COB). Although mRNA levels within these clones remained approximately equal, LuxA protein levels increased significantly after codon optimization. On average, bioluminescence levels were increased by more than six-fold [5×10⁵ vs 2.9×10⁶ relative light units (RLU)/mg total protein] with the codon-optimized luxA and wild type luxB. Bioluminescence was further enhanced upon expression of both optimized genes (2.7×10⁷ RLU/mg total protein). These results show promise toward the potential development of an autonomous light generating lux reporter system in mammalian cells     

“Quorum sensing” regulation and the structure of <Emphasis Type="Italic">lux</Emphasis> the operon in marine bacteria <Emphasis Type="Italic">Aliivibrio logei</Emphasis>

A group of luminescent strains of marine bacteria Aliivibrio logei has been isolated (basins of the Okhotsk, White and Bering Seas). Strains A. logei were shown to be psycrophilic bacteria with an optimal growth temperature of approximately 15°C. Bioluminescent 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 luxI gene is absent in front of luxC, and a fragment containing luxR2-luxI genes is located immediately after luxG gene. Luminescent psycrophilic marine bacteria of A. logei are assumed to be widely distributed in cold waters of northern seas.     

Tn5/7-lux: a versatile tool for the identification and capture of promoters in Gram-negative bacteria

Background

The combination of imaging technologies and luciferase-based bioluminescent bacterial reporter strains provide a sensitive and simple non-invasive detection method (photonic bioimaging) for the study of diverse biological processes, as well as efficacy of therapeutic interventions, in live animal models of disease. The engineering of bioluminescent bacteria required for photonic bioimaging is frequently hampered by lack of promoters suitable for strong, yet stable luciferase gene expression.

Results

We devised a novel method for identification of constitutive native promoters in Gram-negative bacteria. The method is based on a Tn5/7 transposon that exploits the unique features of Tn5 (random transposition) and Tn7 (site-specific transposition). The transposons are designed such that Tn5 transposition will allow insertion of a promoter-less bacterial luxCDABE operon downstream of a bacterial gene promoter. Cloning of DNA fragments from luminescent isolates results in a plasmid that replicates in pir⁺ hosts. Sequencing of the lux-chromosomal DNA junctions on the plasmid reveals transposon insertion sites within genes or operons. The plasmid is also a mini-Tn7-lux delivery vector that can be used to introduce the promoter-lux operon fusion into other derivatives of the bacterium of interest in an isogenic fashion. Alternatively, promoter-containing sequences can be PCR-amplified from plasmid or chromosomal DNA and cloned into a series of accompanying mini-Tn7-lux vectors. The mini-Tn5/7-lux and mini-Tn7-lux vectors are equipped with diverse selection markers and thus applicable in numerous Gram-negative bacteria. Various mini-Tn5/7-lux vectors were successfully tested for transposition and promoter identification by imaging in Acinetobacter baumannii, Escherichia coli, and Burkholderia pseudomallei. Strong promoters were captured for lux expression in E. coli and A. baumannii. Some mini-Tn7-lux vectors are also equipped with attB sites for swapping of the lux operon with other reporter genes using Gateway technology.

Conclusions

Although mini-Tn5-lux and mini-Tn7-lux elements have previously been developed and used for bacterial promoter identification and chromosomal insertion of promoter-lux gene fusions, respectively, the newly developed mini-Tn5/7-lux and accompanying accessory plasmids streamline and accelerate the promoter discovery and bioluminescent strain engineering processes. Availability of vectors with diverse selection markers greatly extend the host-range of promoter probe and lux gene fusion vectors.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0354-3) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Aliivibrio logei</Emphasis> KCh1 (Kamchatka isolate): Biochemical and bioluminescence characteristics and cloning of the <Emphasis Type="Italic">lux</Emphasis> operon

A new strain of luminescent marine bacteria of the genus Aliivibrio (formerly Vibrio) was isolated from the intestines of a goby Cottida sp. (Sea of Okhotsk basin; the strain was marked as KCh1). The basic conditions of growth on laboratory media were determined for the strain. Strain KCh1 was shown to be a psychrophilic bacterium with an optimal growth temperature of approximately 15°C. The nucleotide sequence of the 16S rRNA gene was determined and shown to be almost identical to the 16S rRNA gene sequence of Aliivibrio logei and A. salmonicida but considerably different from that of A. fischeri. The biochemical characteristics (nitrate reduction, lysine decarboxylation, and D-galactose fermentation) of strain KCh1 matched those of A. logei and A. fischeri strains. An antibioticogram for strain KCh1 was determined. The lux operon of the strain was cloned in Escherichia coli cells and partially sequenced, the results show a high homology with the lux operon of A. salmonicida.     

Vibrios dominate as culturable nitrogen-fixing bacteria of the Brazilian coral Mussismilia hispida

Taxonomic characterization was performed on the putative N₂-fixing microbiota associated with the coral species Mussismilia hispida, and with its sympatric species Palythoa caribaeorum, P. variabilis, and Zoanthus solanderi, off the coast of São Sebastião (São Paulo State, Brazil). The 95 isolates belonged to the Gammaproteobacteria according to the 16S rDNA gene sequences. In order to identify the isolates unambiguously, pyrH gene sequencing was carried out. The majority of the isolates (n=76) fell within the Vibrio core group, with the highest gene sequence similarity being towards Vibrio harveyi and Vibrio alginolyticus. Nineteen representative isolates belonging to V. harveyi (n=7), V. alginolyticus (n=8), V. campbellii (n=3), and V. parahaemolyticus (n=1) were capable of growing six successive times in nitrogen-free medium and some of them showed strong nitrogenase activity by means of the acetylene reduction assay (ARA). It was concluded that nitrogen fixation is a common phenotypic trait among Vibrio species of the core group. The fact that different Vibrio species can fix N₂ might explain why they are so abundant in the mucus of different coral species.     

Identification of Vibrio splendidus as a Member of the Planktonic Luminous Bacteria from the Persian Gulf and Kuwait Region with luxA Probes

Hybridization probes specific for the luxA genes of four groups of luminous bacteria were used to screen luminous isolates obtained from the Persian Gulf, near Al Khiran, Kuwait Nine of these isolates were identified as Vibrio harveyi, a commonly encountered planktonic isolate, while three others showed no hybridization to any of the four probes (V. harveyi, Vibrio fischeri, Photobacterium phosphoreum, or Photobacterium leiognathi) under high-stringency conditions. Polymerase chain reaction amplification was used to prepare a luxA probe against one of these isolates, K-1, and this probe was screened under high-stringency conditions against a collection of DNAs from luminous bacteria; it was found to hybridize specifically to the DNA of the species Vibrio splendidus. A probe prepared against the type strain of V. splendidus (ATCC 33369) was tested against the collection of luminous bacterial DNA preparations and against the Kuwait isolates and was found to hybridize only against the type strain and the three unidentified Kuwait isolates. Extensive taxonomic analysis by standard methods confirmed the identification of the 13 isolates.     

Association of a luminous Vibrio sp., taxonomically related to Vibrio harveyi, with Clytia linearis (Thornely, 1900) (Hydrozoa, Cnidaria)

A previously unknown association between a luminous Vibrio sp., taxonomically related to the species Vibrio harveyi and a common member of the shallow/mid water communities of the Mediterranean Sea, the hydrozoan Clytia linearis is described. All the specimens of C. linearis observed under blue light excitation showed both a natural luminescence appearing as a series of fine dots due to clytin, and a clear fluorescence on the external side of the perisarc around the colonies due to the presence of luminous bacteria. Luminous bacteria were isolated from the surface of C. linearis, their phenotypic characterization as isolates was performed by several morphological, biochemical, and cultural tests, completed with 16S rDNA sequence analysis. All the isolates were referred to a Vibrio sp. taxonomically related to V. harveyi. The association of the V. harveyi-related species with C. linearis, as already suggested for another hydroid, Aglaophenia octodonta, could be explained with the activity of these bacteria of feeding on the chitinous structures present in these hydroids. Moreover, the adhesion of the luminous bacterium (here referred to as Vibrio sp. CL1) on C. linearis may contribute to the survival of this Vibrio species in the marine environment providing a suitable growth habitat.     

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

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

Regulatory mutants of the Vibrio harveyi lux system

Regulatory mutants of the luminescent bacterium, Vibrio harveyi, have been isolated whose light emission can be stimulated by extracts of the growth media. Chloroform extracts of conditioned media in which V. harveyi has been grown can increase light emission in one of the dark mutants, D34, over 10³-fold. An increase in the level of the mRNA and the enzymes associated with the lux system can also be demonstrated. Analysis of the expression of the lux system in Escherichia coli transformed with DNA from the D34 regulatory mutant demonstrates that the mutation resides outside the luciferase structural genes. The results suggest that the decrease in light emission in the regulatory mutants may be due to a mutation in synthesis of an autoinducer analogous to that found for the Vibrio fischeri lux system.     

Detection of the Light Organ Symbiont, Vibrio fischeri, in Hawaiian Seawater by Using lux Gene Probes

Symbiotic bacteria that inhabit the light-emitting organ of the Hawaiian squid Euprymna scolopes are distinctive from typical Vibrio fischeri organisms in that they are not visibly luminous when grown in laboratory culture. Therefore, the abundance of these bacteria in seawater samples cannot be estimated simply by identifying them among luminous colonies that arise on nutrient agar plates. Instead, we have used luxR and polymerase chain reaction generated luxA gene probes to identify both luminous and non-visibly luminous V. fischeri colonies by DNA-DNA hybridization. The probes were specific, hybridizing at least 50 to 100 times more strongly to immobilized DNAs from V. fischeri strains than to those of pure cultures of other related species. Thus, even non-visibly luminous V. fischeri colonies could be identified among colonies obtained from natural seawater samples by their probe-positive reaction. Bacteria in seawater samples, obtained either within or distant from squid habitats, were collected on membrane filters and incubated until colonies appeared. The filters were then observed for visibly luminous V. fischeri colonies and hybridized with the lux gene probes to determine the number of total V. fischeri colonies (both luminous and non-visibly luminous). We detected no significant differences in the abundance of luminous V. fischeri CFU in any of the water samples observed (≤1 to 3 CFU/100 ml). However, probe-positive colonies of V. fischeri (up to 900 CFU/100 ml) were found only in seawater collected from within the natural habitats of the squids. A number of criteria were used to confirm that these probe-positive strains were indistinguishable from symbiotic V. fischeri. Therefore, the luxA and luxR gene probes were species specific and gave a reliable estimate of the number of culturable V. fischeri colonies in natural water samples.     

Predatory Bacteria as Natural Modulators of Vibrio parahaemolyticus and Vibrio vulnificus in Seawater and Oysters

This study shows that naturally occurring Vibrio predatory bacteria (VPB) exert a major role in controlling pathogenic vibrios in seawater and shellfish. The growth and persistence of Vibrio parahaemolyticus and Vibrio vulnificus were assessed in natural seawater and in the Eastern oyster, Crassostrea virginica. The pathogens examined were V. vulnificus strain VV1003, V. parahaemolyticus O1:KUT (KUT stands for K untypeable), and V. parahaemolyticus O3:K6 and corresponding O3:K6 mutants deficient in the toxRS virulence regulatory gene or the rpoS alternative stress response sigma factor gene. Vibrios were selected for streptomycin resistance, which facilitated their enumeration. In natural seawater, oysters bioconcentrated each Vibrio strain for 24 h at 22°C; however, counts rapidly declined to near negligible levels by 72 h. In natural seawater with or without oysters, vibrios decreased more than 3 log units to near negligible levels within 72 h. Neither toxRS nor rpoS had a significant effect on Vibrio levels. In autoclaved seawater, V. parahaemolyticus O3:K6 counts increased 1,000-fold over 72 h. Failure of the vibrios to persist in natural seawater and oysters led to screening of the water samples for VPB on lawns of V. parahaemolyticus O3:K6 host cells. Many VPB, including Bdellovibrio and like organisms (BALOs; Bdellovibrio bacteriovorus and Bacteriovorax stolpii) and Micavibrio aeruginosavorus-like predators, were detected by plaque assay and electron microscopic analysis of plaque-purified isolates from Atlantic, Gulf Coast, and Hawaiian seawater. When V. parahaemolyticus O3:K6 was added to natural seawater containing trace amounts of VPB, Vibrio counts diminished 3 log units to nondetectable levels, while VPB increased 3 log units within 48 h. We propose a new paradigm that VPB are important modulators of pathogenic vibrios in seawater and oysters.     

Inactivation of Bacteria in Seawater by Low-Amperage Electric Current

Seawater used in mariculture has been suspected of being a potential source of infection. In this study, the lethal effects of low-amperage electric treatment on microorganisms were examined in natural seawater and in seawater inoculated with Vibrio parahaemolyticus. In both cases, bacteria including V. parahaemolyticus in seawater were completely eliminated in 100 ms by a 0.5-A, 12-V direct current. Electron microscopic investigation of the electrically treated bacteria revealed substantial structural damage at the cellular level. In conclusion, our results indicate that low-amperage electric treatment is effective for rapid inactivation of microorganisms in seawater.     

Quorum Sensing in Burkholderia cepacia: Identification of the LuxRI Homologs CepRI

Burkholderia cepacia has emerged as an important pathogen in patients with cystic fibrosis. Many gram-negative pathogens regulate the production of extracellular virulence factors by a cell density-dependent mechanism termed quorum sensing, which involves production of diffusible N-acylated homoserine lactone signal molecules, called autoinducers. Transposon insertion mutants of B. cepacia K56-2 which hyperproduced siderophores on chrome azurol S agar were identified. One mutant, K56-R2, contained an insertion in a luxR homolog that was designated cepR. The flanking DNA region was used to clone the wild-type copy of cepR. Sequence analysis revealed the presence of cepI, a luxI homolog, located 727 bp upstream and divergently transcribed from cepR. A lux box-like sequence was identified upstream of cepI. CepR was 36% identical to Pseudomonas aeruginosa RhlR and 67% identical to SolR of Ralstonia solanacearum. CepI was 38% identical to RhlI and 64% identical to SolI. K56-R2 demonstrated a 67% increase in the production of the siderophore ornibactin, was protease negative on dialyzed brain heart infusion milk agar, and produced 45% less lipase activity in comparison to the parental strain. Complementation of a cepR mutation restored parental levels of ornibactin and protease but not lipase. An N-acylhomoserine lactone was purified from culture fluids and identified as N-octanoylhomoserine lactone. K56-I2, a cepI mutant, was created and shown not to produce N-octanoylhomoserine lactone. K56-I2 hyperproduced ornibactin and did not produce protease. These data suggest both a positive and negative role for cepIR in the regulation of extracellular virulence factor production by B. cepacia.     

Contribution of Many Charged Residues at the Stator-Rotor Interface of the Na+-Driven Flagellar Motor to Torque Generation in Vibrio alginolyticus

In torque generation by the bacterial flagellar motor, it has been suggested that electrostatic interactions between charged residues of MotA and FliG at the rotor-stator interface are important. However, the actual role(s) of those charged residues has not yet been clarified. In this study, we systematically made mutants of Vibrio alginolyticus whose charged residues of PomA (MotA homologue) and FliG were replaced by uncharged or charge-reversed residues and characterized the motilities of those mutants. We found that the members of a group of charged residues, 7 in PomA and 6 in FliG, collectively participate in torque generation of the Na⁺-driven flagellar motor in Vibrio. An additional specific interaction between PomA-E97 and FliG-K284 is critical for proper performance of the Vibrio motor. Our results also reveal that more charged residues are involved in the PomA-FliG interactions in the Vibrio Na⁺-driven motor than in the MotA-FliG interactions in the H⁺-driven one. This suggests that a larger number of conserved charged residues at the PomA-FliG interface contributes to the robustness of the Vibrio motor against mutations. The interaction surfaces of the stator and rotor of the Na⁺-driven motor seem to be more complex than those previously proposed in the H⁺-driven motor.     

Transcriptomic profiling of the oyster pathogen Vibrio splendidus opens a window on the evolutionary dynamics of the small RNA repertoire in the Vibrio genus

Work in recent years has led to the recognition of the importance of small regulatory RNAs (sRNAs) in bacterial regulation networks. New high-throughput sequencing technologies are paving the way to the exploration of an expanding sRNA world in nonmodel bacteria. In the Vibrio genus, compared to the enterobacteriaceae, still a limited number of sRNAs have been characterized, mostly in Vibrio cholerae, where they have been shown to be important for virulence, as well as in Vibrio harveyi. In addition, genome-wide approaches in V. cholerae have led to the discovery of hundreds of potential new sRNAs. Vibrio splendidus is an oyster pathogen that has been recently associated with massive mortality episodes in the French oyster growing industry. Here, we report the first RNA-seq study in a Vibrio outside of the V. cholerae species. We have uncovered hundreds of candidate regulatory RNAs, be it cis-regulatory elements, antisense RNAs, and trans-encoded sRNAs. Conservation studies showed the majority of them to be specific to V. splendidus. However, several novel sRNAs, previously unidentified, are also present in V. cholerae. Finally, we identified 28 trans sRNAs that are conserved in all the Vibrio genus species for which a complete genome sequence is available, possibly forming a Vibrio “sRNA core.”