luxA Gene From Enhygromyxa salina Encodes a Functional Homodimeric Luciferase.

Several clades of luminescent bacteria are known currently. They all contain similar lux operons, which include the genes luxA and luxB encoding a heterodimeric luciferase. The aldehyde oxygenation reaction is presumed to be catalyzed primarily by the subunit LuxA, whereas LuxB is required for efficiency and stability of the complex. Recently, genomic analysis identified a subset of bacterial species with rearranged lux operons lacking luxB. Here, we show that the product of the luxA gene from the reduced luxACDE operon of Enhygromyxa salina is luminescent upon addition of aldehydes both in vivo in Escherichia coli and in vitro. Overall, EsLuxA is much less bright compared with luciferases from Aliivibrio fischeri (AfLuxAB) and Photorhabdus luminescens (PlLuxAB), and most active with medium-chain C4-C9 aldehydes. Crystal structure of EsLuxA determined at the resolution of 2.71 Å reveals a (ß/α)8 TIM-barrel fold, characteristic for other bacterial luciferases, and the protein preferentially forms a dimer in solution. The mobile loop residues 264-293, which form a ß-hairpin or a coil in Vibrio harveyi LuxA, form α-helices in EsLuxA. Phylogenetic analysis shows EsLuxA and related proteins may be bacterial protoluciferases that arose prior to duplication of the luxA gene and its speciation to luxA and luxB in the previously described luminescent bacteria. Our work paves the way for the development of new bacterial luciferases that have an advantage of being encoded by a single gene.

ClpL Chaperone as a Possible Component of the Disaggregase Activity of Limosilactobacillus fermentum U-21.

The L. fermentum U-21 strain, known for secreting chaperones into the extracellular milieu, emerges as a promising candidate for the development of novel therapeutics termed disaggregases for Parkinson's disease. Our study focuses on characterizing the secreted protein encoded by the C0965_000195 locus in the genome of this strain. Through sequence analysis and structural predictions, the protein encoded by C0965_000195 is identified as ClpL, homologs of which are known for their chaperone functions. The chaperone activity of ClpL from L. fermentum U-21 is investigated in vivo by assessing the refolding of luciferases with varying thermostabilities from Aliivibrio fischeri and Photorhabdus luminescens within Escherichia coli cells. The results indicate that the clpL gene from L. fermentum U-21 can compensate for the absence of the clpB gene, enhancing the refolding capacity of thermodenatured proteins in clpB-deficient cells. In vitro experiments demonstrate that both spent culture medium containing proteins secreted by L. fermentum U-21 cells, including ClpL, and purified heterologically expressed ClpL partially prevent the thermodenaturation of luciferases. The findings suggest that the ClpL protein from L. fermentum U-21, exhibiting disaggregase properties against aggregating proteins, may represent a key component contributing to the pharmabiotic attributes of this strain.

LitR directly upregulates autoinducer synthesis and luminescence in Aliivibrio logei.

LitR is a master-regulator of transcription in the ainS/R and luxS/PQ quorum sensing (QS) systems of bacteria from Vibrio and Aliivibrio genera. Here, we for the first time directly investigated the influence of LitR on gene expression in the luxI/R QS system of psychrophilic bacteria Aliivibrio logei. Investigated promoters were fused with Photorhabdus luminescens luxCDABE reporter genes cassette in a heterological system of Escherichia coli cells, litR A. logei was introduced into the cells under control of P lac promoter. LitR has been shown to upregulate genes of autoinducer synthase (luxI), luciferase and reductase (luxCDABE), and this effect doesn't depend on presence of luxR gene. To a much lesser degree, LitR induces luxR1, but not the luxR2 - the main luxI/R regulator. Enhanced litR expression leads to an increase in a LuxI-autoinducer synthesis and a subsequent LuxR-mediated activation of the luxI/R QS system. Effect of LitR on luxI transcription depends on lux-box sequence in luxI promoter even in absence of luxR (lux-box is binding site of LuxR). The last finding indicates a direct interaction of LitR with the promoter in the lux-box region. Investigation of the effect of LitR A. logei on luxI/R QS systems of mesophilic Aliivibrio fischeri and psychrophilic Aliivibrio salmonicida showed direct luxR-independent upregulation of luxI and luxCDABE genes. To a lesser degree, it induces luxR A. fischeri and luxR1 A. salmonicida. Therefore, we assume that the main role of LitR in cross-interaction of these three QS systems is stimulating the expression of luxI.

Influence of the luxR Regulatory Gene Dosage and Expression Level on the Sensitivity of the Whole-Cell Biosensor to Acyl-Homoserine Lactone.

Aliivibrio fischeri LuxR and Aliivibrio logei LuxR1 and LuxR2 regulatory proteins are quorum sensing transcriptional (QS) activators, inducing promoters of luxICDABEG genes in the presence of an autoinducer (3-oxo-hexanoyl-l-homoserine lactone). In the Aliivibrio cells, luxR genes are regulated by HNS, CRP, LitR, etc. Here we investigated the role of the luxR expression level in LuxI/R QS system functionality and improved the whole-cell biosensor for autoinducer detection. Escherichia coli-based bacterial lux-biosensors were used, in which Photorhabdus luminescensluxCDABE genes were controlled by LuxR-dependent promoters and luxR, luxR1, or luxR2 regulatory genes. We varied either the dosage of the regulatory gene in the cells using additional plasmids, or the level of the regulatory gene expression using the lactose operon promoter. It was shown that an increase in expression level, as well as dosage of the regulatory gene in biosensor cells, leads to an increase in sensitivity (the threshold concentration of AI is reduced by one order of magnitude) and to a two to threefold reduction in response time. The best parameters were obtained for a biosensor with an increased dosage of luxRA. fischeri (sensitivity to 3-oxo-hexanoyl-l-homoserine lactone reached 30-100 pM).