Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy

Detecting the folding/unfolding pathways of biological macromolecules is one of the urgent problems of molecular biophysics. The unfolding of bacterial luciferase from Vibrio harveyi is well-studied, unlike that of Photobacterium leiognathi, despite the fact that both of them are actively used as a reporter system. The aim of this study was to compare the conformational transitions of these luciferases from two different protein subfamilies during equilibrium unfolding with urea. Intrinsic steady-state and time-resolved fluorescence spectra and circular dichroism spectra were used to determine the stages of the protein unfolding. Molecular dynamics methods were applied to find the differences in the surroundings of tryptophans in both luciferases. We found that the unfolding pathway is the same for the studied luciferases. However, the results obtained indicate more stable tertiary and secondary structures of P. leiognathi luciferase as compared to enzyme from V. harveyi during the last stage of denaturation, including the unfolding of individual subunits. The distinctions in fluorescence of the two proteins are associated with differences in the structure of the C-terminal domain of α-subunits, which causes different quenching of tryptophan emissions. The time-resolved fluorescence technique proved to be a more effective method for studying protein unfolding than steady-state methods.

Generation of Fluorescent Bacteria with the Genes Coding for Lumazine Protein and Riboflavin Biosynthesis

Lumazine protein is a member of the riboflavin synthase superfamily and the intense fluorescence is caused by non-covalently bound to 6,7-dimethyl 8-ribityllumazine. The pRFN4 plasmid, which contains the riboflavin synthesis genes from Bacillus subtilis, was originally designed for overproduction of the fluorescent ligand of 6,7-dimethyl 8-ribityllumazine. To provide the basis for a biosensor based on the lux gene from bioluminescent bacteria of Photobacterium leiognathi, the gene coding for N-terminal domain half of the lumazine protein extending to amino acid 112 (N-LumP) and the gene for whole lumazine protein (W-LumP) from P. leiognathi were introduced by polymerase chain reaction (PCR) and ligated into pRFN4 vector, to construct the recombinant plasmids of N-lumP-pRFN4 and W-lumP-pRFN4 as well as their modified plasmids by insertion of the lux promoter. The expression of the genes in the recombinant plasmids was checked in various Escherichia coli strains, and the fluorescence intensity in Escherichia coli 43R can even be observed in a single cell. These results concerning the co-expression of the genes coding for lumazine protein and for riboflavin synthesis raise the possibility to generate fluorescent bacteria which can be used in the field of bio-imaging.

An Intact Cell Bioluminescence-Based Assay for the Simple and Rapid Diagnosis of Urinary Tract Infection

Urinary tract infection (UTI) is one of the most common infections, accounting for a substantial portion of outpatient hospital and clinic visits. Standard diagnosis of UTI by culture and sensitivity can take at least 48 h, and improper diagnosis can lead to an increase in antibiotic resistance following therapy. To address these shortcomings, rapid bioluminescence assays were developed and evaluated for the detection of UTI using intact, viable cells of Photobacterium mandapamensis USTCMS 1132 or previously lyophilized cells of Photobacterium leiognathi ATCC 33981™. Two platform technologies—tube bioluminescence extinction technology urine (TuBETUr) and cellphone-based UTI bioluminescence extinction technology (CUBET)—were developed and standardized using artificial urine to detect four commonly isolated UTI pathogens—namely, Escherichia coli, Proteus mirabilis, Staphylococcus aureus, and Candida albicans. Besides detection, these assays could also provide information regarding pathogen concentration/level, helping guide treatment decisions. These technologies were able to detect microbes associated with UTI at less than 105 CFU/mL, which is usually the lower cut-off limit for a positive UTI diagnosis. Among the 29 positive UTI samples yielding 105–106 CFU/mL pathogen concentrations, a total of 29 urine specimens were correctly detected by TuBETUr as UTI-positive based on an 1119 s detection window. Similarly, the rapid CUBET method was able to discriminate UTIs from normal samples with high confidence (p ≤ 0.0001), using single-pot conditions and cell phone-based monitoring. These technologies could potentially address the need for point-of-care UTI detection while reducing the possibility of antibiotic resistance associated with misdiagnosed cases of urinary tract infections, especially in low-resource environments.

Cloning and expression of the flavin reductase LuxG from Photobacterium leiognathi YL and its improvement for NADH detection

LuxG from P. leiognathi YL was successfully purified and characterized. Based on this, a coupled pure enzyme bioluminescent system was established and used for NADH detection, which showed higher sensitivity than existing bioluminescent systems.

Complete Genome Sequence of Photobacterium leiognathi Strain JS01

ABSTRACT Photobacterium leiognathi is a bioluminescent symbiont of fish of the Leiognathidae family. Here, we present the full-genome sequence of P. leiognathi strain JS01, a strain isolated from a nonluminescent Loligo sp. squid of Singaporean origin. No finished genome sequence of this species is currently publicly available.