Rozprawy doktorskie na temat „Vibrio. luxR”

Quorum sensing is a mechanism used by many proteobacteria to regulate expression of target genes in a population-dependent manner. The quorum sensing system of Vibrio fischeri activates the luminescence (lux) operon when the autoinducer signaling molecule (N-3-oxohexanoyl homoserine lactone) is recognized and bound by the activator protein LuxR. LuxR subsequently binds to the lux box centered at à 42.5 bp upstream of the transcription initiation site and activates transcription from the lux operon promoter, resulting in the emission of light at high cell densities. LuxR consists of 250 amino acids arranged into an N-terminal (regulatory) domain and a C-terminal (activation) domain, and is thought to function as an ambidextrous activator capable of making multiple contacts with the alpha and sigma subunits of RNA polymerase (RNAP). Published work describing the results of alanine scanning mutagenesis performed on the C-terminal domain of LuxR (residues 190-250) has identified residues (K198, W201 and I206) that appear to play a role in positive control of transcription initiation. Additional mutagenesis of residues 180-189 has been undertaken via a three-primer or four-primer PCR-based method in this study. Variants of LuxR were screened for their ability to activate luciferase production and to repress transcription from an artificial promoter, and production of full-length LuxR was measured, in an attempt to identify additional positive control variants. No additional positive control variants were found in this study. Work has also been undertaken to identify intergenic suppressors between positive control variants of LuxR and the RNAP alpha subunit, RpoA. Starting with a recombinant Escherichia coli strain encoding the lux operon and LuxR variant I206E, a random chemical mutagenesis was performed on a vector encoding RpoA. Following transformation of the mutated plasmids encoding RpoA, high throughput luminescence assays were used to identify isolates with phenotypes brighter than the control. Isolation of an intergenic suppressor will confirm the existence of protein-protein interactions between LuxR and RpoA within the transcription initiation complex. The ability of other LuxR family members to establish productive protein-protein interactions with RNAP necessary for transcription initiation was also examined. LuxR homologues EsaR of Pantoea stewarti ssp. stewartii, a repressor of known function, and ExpR of Erwinia carotovora subsp. carotovora were also analyzed for their ability to activate the lux operon, as well as to repress transcription from an artificial promoter containing the lux box.
Master of Science

Quorum sensing is a bacterial signaling system that controls gene expression in a population density-dependent manner. In Gram-negative proteobacteria, the cell density control of luminescence was first observed in the symbiotic marine bacterium Vibrio fischeri and this system is one of the best studied quorum sensing systems. Two-dimensional sodium dodecyl sulfate-polyacrylamide (2D-SDS) gel electrophoresis analysis previously identified several non-Lux proteins in V. fischeri MJ-100 whose expression was dependent on LuxR and 3-oxo-hexanoyl-L-homoserine lactone (3-oxo-C6-HSL). A lacZ reporter was used to show that the promoters for qsrP, acfA, and ribB were directly activated via LuxR-3-oxo-C6-HSL in recombinant Escherichia coli. The sites of transcription initiation were established via primer extension analysis. Based on the position of the lux box-binding site near position â 40, all three promoters appear to have a class II-type promoter structure. Real-time reverse transcription-PCR was used to study the temporal expression of qsrP, acfA, and ribB during the exponential and stationary phases of growth, and electrophoretic mobility shift assays were used to compare the binding affinities of LuxR to the promoters under investigation. In order to fully characterize the LuxR regulon in V. fischeri ES114, microarray analysis was performed in the Greenberg lab (University of Washington) and 18 LuxR-3-oxo-C6-HSL regulated promoters were found including 2 genes (qsrP and acfA) identified previously in MJ-100 in addition to the well-studied lux operon. In collaboration with them, full-length purified LuxR protein was used to show direct interaction between the LuxR protein and 7 genes/operons newly identified out of 13 genes/operons examined. The binding affinity between LuxR proteins and those genes was also measured. Based on the sequence of the lux boxes of the known genes regulated by LuxR and LitR, a position specific weight matrix (PSWM) was created and used to search through the intergenic regions of the V. fischeri ES114 genome. Some potential LuxR and LitR-regulated genes with high score were tested experimently to confirm direct activation. For the LuxR regulon, these possible LuxR-regulated promoters were cloned into a lacZ reporter and tested for their LuxR dependence. Beyond the genes found in microarray, the promoter of the intergenic region VFA0658-0659 was found to be activated by LuxR and 3-oxo-C6-HSL. For the LitR regulon, two LitR-regulated genes found in the microarray were also identified using PSWM and confirmed by real-time PCR to be dependent on LitR for expression. EMSA experiments showed that LitR can specifically bind to the litR boxes of LitR-regulated genes, litR and VF0170 which confirmed that the regulation is direct.
Ph. D.

The quorum-sensing response of Vibrio fischeri involves a complex network of genes (encoding regulatory proteins as well as sRNAs), that govern host-association and production of bioluminescence. A key regulator of this system is LuxR, which is the transcriptional activator of the lux operon as well as several other genes in. LuxR also autoregulates its own transcription, which we have shown causes bistability and hysteresis in the quorum-sensing response. This behavior allows the system to maintain a stable and robust response in the face of environmental fluctuation or decreases in external autoinducer concentration caused by other sources. There are many factors that are known to regulate luxR expression, including the ArcA redox-responsive regulator, the cAMP-CRP secondary metabolism regulator, and components of the quorum-sensing pathway like LitR. Because of this, LuxR levels are critical in both the timing of quorum-sensing induction, as well as the maintenance of the response over time. This makes it a potential target for multiple levels of regulation in response to factors such as environmental and metabolic conditions, as well as other components of the quorum-sensing network. Another important global regulatory protein in V. fischeri (and most other species of Gram-negative proteobacteria) is the post-transcriptional regulator CsrA. CsrA controls processes involved in carbon storage and utilization, as well as the transition from exponential to stationary phase growth. We have demonstrated that CsrA is regulated by two sRNAs (CsrB1 and CsrB2) in V. fischeri. Because CsrA regulates changes in cell behavior and is an important metabolic regulator, there is a good possibility that it has some interactions with the quorum-sensing regulon, whose endproduct, bioluminescence, creates a large metabolic demand from the cell. In an effort to determine at which point in the quorum-sensing regulatory network CsrA regulation is important, epistasis experiments were designed using factorial design, which is a subset of statistical analysis of variance (ANOVA). This method was used to generate a high degree of confidence in the data, so that even minor interactions in the regulatory networks could be established. By altering the levels of CsrA expression in various mutant strains of V. fischeri, we have demonstrated that CsrA acts by an unknown mechanism to increase the transcription of luxR when the quorum-sensing regulator LitR is absent. Our results also demonstrated that CsrA mediates this effect through repression of ArcA activity, which is known to act directly on the luxR and luxI intergenic region as a repressor. This indicates that CsrA may bypass the upstream parts of the quorum-sensing regulatory cascade that lead to litR activation, so that LitR and LuxR may be regulated differently in response to certain conditions. This work has shown that the interactions between global regulons can coordinately control the amount of quorum-sensing induction by affecting the level of LuxR in the cell. The balance of these regulatory networks allows the cell to tightly regulate the quorum-sensing response. Thus, LuxR serves as a critical regulatory hub in the cell, at which multiple signals can be integrated in order to generate the appropriate cellular response.
Ph. D.

Luminescence is a secondary metabolic process that can be detected and assayed in a non-disruptive, in vivo, and real-time manner. As such the Vibrio harveyi luminescence (lux) system constitutes a model system that can be used to delineate the relatively complex transcriptional control mechanisms that are inherent to secondary metabolic processes. Molecular insights into the control of luminescence induction in V. harveyi is limited to the two-component quorum-sensing system and LuxR. While the quorum-sensing system is thought to facilitate luminescence induction through a mechanism of derepression, LuxR has been shown to be an essential activator of the process by virtue of the absence of luminescence in a luxR null mutant. Additionally, it has been suggested that LuxR activates its own expression. Data presented here demonstrates that LuxR is involved in autorepression. Moreover, it is argued that LuxR may not be a typical activator in that it may mediate activation of lux gene expression through a mechanism of repression. An implication of this proposal is that a second unidentified activator is required for luminescence induction to occur. The search of candidates to fill this proposed activation role focused on two highly conserved and extensively studied activators, MetR and CRP. Both are involved in nutrient dependent pathways as MetR is an activator of methionine biosynthesis genes and CRP is implicated in the regulation of metabolic pathways in response to the presence of preferred carbohydrates. In the specific case of the V. harveyi lux system, MetR is shown to act as a repressor, while CRP is shown to be a critical activator of luminescence induction. The evolutionary and ecological implications of these findings are discussed.

Vibrio fischeri, a symbiotic bioluminescent bacterium, serves as one of the best understood model systems for a mechanism of cell-density dependent bacterial gene regulation known as quorum sensing. During quorum sensing in V. fischeri, an acylated homoserine chemical signal (autoinducer) is synthesized by the bacteria and used to sense their own species in a given environment. As the autoinducer levels rise, complexes form between the autoinducer and the N-terminal domain of a regulatory protein, LuxR. In response to autoinducer binding, LuxR is believed to undergo a conformational change that allows the C-terminal domain to activate transcription of the luminescence or lux operon. To further understand the mechanism of LuxR-dependent transcriptional activation of the lux operon, PCR-based site-directed mutagenesis procedures have been used to generate alanine-substitution mutants in the C-terminal forty-one amino acid residues of LuxR, a region that has been hypothesized to play a critical role in the activation process. An in vivo luminescence assay was first used to test the effects of the mutations on LuxR-dependent activation of the lux operon in recombinant Escherichia coli. Luciferase levels present in cell extracts obtained from these strains were also quantified and found to correlate with the luminescence results. Eight strains encoding altered forms of LuxR exhibited a "dark" phenotype with luminescence output less than 50% and luciferase levels less than 50% of the wildtype control strain. Western immunoblotting analysis with cell extracts from the luminescence and luciferase assays verified that the altered forms of LuxR were expressed at levels approximately equal to wildtype. Therefor, Low luminescence and luciferase levels could be the result of a mutation that either affects the ability of LuxR to recognize and bind its DNA target (the lux box) or to establish associations with RNA polymerase (RNAP) at the lux operon promoter necessary for transcriptional initiation. A third in vivo assay was used to test the ability of the altered forms of LuxR to bind to the lux box (DNA binding assay/repression). All of the LuxR variants exhibiting the "dark" phenotype in the luminescence and luciferase assay were also found to be unable to bind to the lux box in the DNA binding assay. Therefore, it can be concluded that the alanine substitutions made at these positions affect the ability of LuxR to bind to the lux box in the presence and absence of RNA polymerase. Another class of mutants exhibited wildtype phenotypes in the luminescence and luciferase assays but were unable to bind to the lux box in the DNA binding assay. The alanine substitutions made at these amino acid residues may be making contacts with RNAP that are important for maintaining the stability of the DNA binding region of LuxR. Alanine substitutions made at these positions have a defect in DNA binding at the promoter of the lux operon only in the absence of RNAP. None of the alanine substitutions made in the C-terminal forty-one amino acids of LuxR were found to affect activation of transcription of the lux operon without also affecting DNA binding. Taken together, these results support the conclusion that the C-terminal forty-one amino acids of LuxR are important for DNA recognition and binding of the lux box rather than positive control of the process of transcription initiation.
Master of Science

Vibrio vulnificus is a model environmental organism exhibiting a classical starvation response during nutrient limitation as well as a non-culturable state when exposed to low temperatures. In addition to these classic global responses, this organism is an opportunistic pathogen that exhibits numerous virulence factors. This organism was chosen as the model organism for the identification of regulators of the viable but nonculturable response (VBNC) and the starvation-induced maintenance of culturability (SIMC) that occurs when cells are starved prior to low temperature incubation. In order to accomplish this, three indirect approaches were used; proteomics, investigation of intercellular signalling pathways and genetic analysis of regulators involved in these responses. Two-dimensional gel electrophoresis was used to identify proteins expressed under conditions that induced SIMC. It was determined that carbon and long-term phosphorus starvation were important in the SIMC response. V. vulnificus was shown to possess genes, luxS and smcR, that are homologues of genes involved in signalling system system 2 in Vibrio harveyi. Signal molecules were produced upon starvation and the entry to stationary phase in V. vulnificus. Furthermore, a null mutation in smcR, a transcriptional regulator was shown to have pleiotropic effects in V. vulnificus, including up-regulation of numerous virulence factors and a defect in starvation survival and development of the SIMC response. We propose that V. vulnificus possesses a signalling system analogous to that of system 2 in V. harveyi, and that this system is involved in the regulation of stationary phase and starvation adaptation in this organism.

The mechanism of gene regulation used by Gram-negative bacteria during quorum sensing is well understood in the bioluminescent marine bacterium Vibrio fischeri. The cell-density dependent activation of the luminescence (lux) genes of V. fischeri relies on the formation of a complex between the autoinducer molecule, N-(3-oxohexanoyl) homoserine lactone, and the autoinducer-dependent transcriptional activator LuxR. LuxR, a 250 amino acid polypeptide, binds to a site known as the lux box centered at position -42.5 relative to the luxI transcriptional start site. During transcriptional activation of the lux operon, LuxR is thought to function as an ambidextrous activator capable of making multiple contacts with RNA polymerase (RNAP). The specific role of region 4 of the Escherichia coli sigma 70 subunit of RNAP in LuxR-dependent transcriptional activation of the luxI promoter has been investigated. Rich in basic amino acids, this conserved portion of sigma 70 is likely to be surface-exposed and available to interact with transcription factors bound near the -35 element. The effect of 16 single and 2 triple alanine substitution variants of sigma 70 between amino acid residues 590 and 613, was determined in vivo by measuring the rate of transcription from a luxI-lacZ translational fusion via b-galactosidase assays in recombinant E. coli. In vitro work was performed with LuxRDN, the autoinducer-independent C-terminal domain (amino acids 157 to 250) of LuxR because purified, full length LuxR is unavailable. Single-round transcription assays were performed in the presence of LuxRDN and 19 variant RNAPs, one of which contained a C-terminally truncated sigma 70 subunit devoid of region 4. Results indicate that region 4 is essential for LuxRDN-dependent luxI transcription with two specific amino acid residues, E591 and K597, having negative effects on the rate of LuxRDN-dependent luxI transcription in vivo and in vitro. None of the residues tested were identified as having any effect on LuxR-dependent luxI transcription in vivo. These findings suggest that region 4.2 is most likely to be in close proximity to LuxR when bound to the luxI promoter. However, unlike the situation found for other ambidextrous activators, no single residue within region 4.2 of sigma 70 may be critical by itself for LuxR-dependent during transcriptional activation.
Master of Science

Quorum sensing in Gram-negative bacteria is best understood in the bioluminescent marine microorganism, Vibrio fischeri. In V. fischeri, the luminescence or lux genes are regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule (3-oxo-hexanoyl homoserine lactone). LuxR, which binds to the lux operon promoter at position -42.5, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP). The specific role of the aCTD of RNAP in LuxR-dependent transcriptional activation of the lux operon promoter has been investigated. The effect of seventy alanine substitution variants of the a subunit was determined in vivo by measuring the rate of transcription of the lux operon via luciferase assays in recombinant Escherichia coli. The mutant RNAPs from strains exhibiting at least two fold increased or decreased activity in comparison to the wild-type were further examined by in vitro assays. Since full-length LuxR has not been purified to date, an autoinducer-independent N-terminal truncated form of LuxR, LuxRDN, was used for in vitro studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxRDN, and fourteen residues in the aCTD were identified as having negative effects on the rate of transcription from the lux operon promoter. Five of these fourteen residues were also involved in the mechanism of both LuxR and LuxRDN-dependent activation in vivo and were chosen for further analysis by DNA mobility shift assays. Results from these assays indicate that while the wild-type aCTD is capable of interacting with the lux DNA fragment tested, all five of the variant forms of the aCTD tested appear to be deficient in their ability to recognize and bind the DNA. These findings suggest that aCTD-DNA interactions may play a role in LuxR-dependent transcriptional activation of the lux operon during quorum sensing.
Master of Science

A luxO DNA binding protein (LuxT) was purified to homogeneity from V. harveyi after five major chromatography steps including a highly effective DNA affinity chromatography step and reverse phase HPLC. The sequences of three tryptic peptides obtained on digestion of the purified protein did not match any sequences in the protein data bank indicating that LuxT is a new V. harveyi protein. Inverse PCR was conducted sequentially to obtain the complete gene (luxT) coding for a protein of 153 amino acids which shares homology with the AcrR/TetR family of transcriptional regulators. Gene disruption of luxT in V. harveyi increased luxO expression and affected the cell density dependent induction of luminescence showing that LuxT is a repressor of luxO. As LuxT also affected the survival of the V. harveyi cells at high salt concentration and close homologues were present in other bacterial species suggested that the LuxT regulatory protein appears to be a general rather than a lux-specific regulator.
The rpoS gene in V. harveyi has been cloned in this work and shown to code for a protein with high homology to the RpoS proteins in other species. The null mutant of RpoS has been constructed and the effect of rpoS deletion on stress resistance as well as the cell density dependent luminescence in V. harveyi were examined.

The individual behaviour of prokaryotic organisms such as bacteria often gives rise to complexity that is commonly associated with multicellular behaviour. The transition from unicellular to multicellular behaviour occurs in response to chemical signals, called autoinducers, which bacteria generate and receive internally within a given population. These autoinducers control the gene expression necessary for the emergence of group-behaviour-phenotype. This phenomenon is called quorum sensing (QS). An example of the quorum sensing control of gene regulation has been the luminescence (lux) operon in Vibrio fischeri. The luxI and ainS quorum signalling systems work in conjunction to regulate luminescence in V. fischeri. LuxI and AinS are acyl-synthases that catalyse the production of the autoinducers C6-HSL and C8-HSL respectively. These autoinducers bind to LuxR, a transcriptional activator of the lux operon, which activates expression of the lux genes causing an increase in luminescence. It was shown that quorum signalling also affects motility and biofilm formation in bacteria. However, the evidence with respect to these phenotypes is conflicting and inconclusive, the reason being the state of quorum is ambiguously defined. It is not properly known whether the observed collective behaviour is purely a result of physical crowding of bacteria, or that both chemical signalling and crowding contribute to this phenomenon. This work attempts to address these issues by studying luminescence, motility, and biofilm, a diverse set of behaviours, yet closely linked to each other in V. fischeri-squid symbiosis. We studied the luminescence response of V. fischeri to both endogenous and externally added signals at per-cell and population level. Experiments with ES114, a wild-type strain of V. fischeri, and ainS mutant showed that (i) luminescence per cell does not mutually correlate with the cell-density, indicating that bacteria do not show greater response to the signal at higher densities; (ii) the activity of the lux signalling circuit shows a strong dependence on the growth stage, (iii) the cells do not show enhanced growth, i.e., they do not derive fitness benefits at higher densities in response to the signal. We anticipated that the culture with a higher cell-density should exhibit greater per-cell-luminescence. However, we found that the luminescence curve of the culture with lower density crosses that of the cultures with higher densities during the exponential phase. Kinetic modelling of the luxI mRNA expression showed that the expression profile qualitatively agrees with the luminescence trend observed in the cultures, supporting the observation that growth-phase plays a major role in regulating the luminescence gene expression. We also studied the effect of autoinducers on motility of V. fischeri. V. fischeri uses flagella to move into the inner crypts of the light organ of the squid. The bacterium secretes autoinducers, encounters secretions of the light organ, and slows down during the final stage of colonization process. Studies have shown that flagellar elaboration is repressed as a consequence of ainS signalling. However, those studies were soft-agar migration assays and carried out with the mutant strain of ainS. We measured real-time planktonic motility of ES114 and the signalling mutant strains of V. fischeri in response to autoinducers added exogenously at different concentrations. We found that the autoinducers do not affect the motility of the strains. We also showed that reduction in motility is purely a consequence of physical crowding of bacteria, and chemical signalling may not be involved in the process. It was shown that reduction in motility leads to biofilm formation. Motile bacteria must lose flagella in order to form biofilm, and signalling controls biofilm formation in many species. Our study on motility showed that reduction in motility occurs because of physical crowding in V. fischeri. Hence, we explored the possibility that physical crowding might lead to formation of biofilm rather than signalling in this species. We quantified exopolysaccharide production by crystal violet assay, which revealed that planktonic cells produce exopolysaccharides, in addition to biofilm cells. The study revealed that V. fischeri cells always produce exopolysaccharides irrespective of their physiological state. We examined the effect of signalling on biofilm in ES114 and the mutant strains using gene-expression analysis. We quantified the expression of various genes involved in biofilm formation and found that both ES114 and the mutants expressed rscS and sypP indicating that exopolysaccharide production is not under the control of autoinducers. Therefore, we hypothesized that biofilm formation in V. fischeri may be a result of physical agglomeration of cells. Our observations indicate that the state of quorum is inadequately defined and there is no direct measure of the underlying process. Multicellular behaviour in V. fischeri is regulated by a complex interplay of cell-density, signalling, and other factors such as the growth phase of the culture, indicating that the state of quorum employs different mechanisms to regulate various phenotypes. Our study reveals that QS is an intricate process, and the accepted mechanisms for QS are incomplete at best.

碩士
輔英科技大學
醫學檢驗生物技術系碩士班
101
Vibrio vulnificus is a halophilic gram-negative bacterial species. In nature, V. vulnificus is present in marine environment and causes opportunistic infections in humans including septicemia and wound infection. The capsular polysaccharide is one of potential virulence factors and associated with formation of biofilm. To understand the effect of capsular polysaccharide on the formation of biofilm, this study uses the acapsular strain JF046 (ΔORFVV0364) with a mutation in capsular polysaccharide surface factor. The characteristics of bacterial growth and formation of biofilm were analyzed between strains. Furthermore, the mRNA expression of quorum-sensing regulators including LuxR and SmcR were determined and compared. First, the colony of YJ016 appeared to be opaque and JF046 was translucent. Second, the results of growth curve indicated that the bacterial density for growth (A600) were lower in JF046 than those in YJ016 both in seawater environment (cultured for 24 hours, 1.795 for YJ016, 1.486 for JF046; p=0.006) and fresh water environment (cultured for 24 hours, 1.519 for YJ016, 1.141 for JF046; p=0.005), respectively. Third, the bacterial aggregates were existed in JF046 and absent in YJ016. Fourth, the ability for formations of biofilm (A492/A620) in acapsular mutant JF046 were higher than those in wild type YJ016 both in seawater environment (0.91±0.15 for YJ016, 1.50±0.23 for JF046; p=0.047) and fresh water (0.50±0.05 for YJ016, 1.26±0.20 for JF046; p=0.013). Fifth, the expressions of quorum-sensing regulators, LuxR and SmcR, were compared between JF046 and YJ016. The results form regression analysis revealed that the correlation between mRNA expressions of LuxR and SmcR were r2=0.885 (y=1.045x+7.798) in YJ016 and r2=0.798 (y=0.928x+25.835) in JF046, respectively. In conclusion, the results demonstrated that the ORFVV0364 for capsular polysaccharide surface factor of Vibrio vulnificus is associated with formation of biofilm and quorum-sensing regulators expression.

碩士
國立屏東科技大學
生物科技研究所
100
Communication among bacteria with quorum-sensing system is accomplished through the exchange of signaling molecules called autoinducers. When the density of autoinducers accumulated to a certain concentration, bacteria responded by specialized gene expression such as genes involved in biofilm formation, bioluminescence, virulence and sporulation. Currently it still remains unclear about the light-emitting and regulatory mechanism of the marine luminous bacteria V. orientalis. It is speculated that the expression of bioluminescence genes in V. orientalis is also regulated by the quorum sensing system. In this study, the lux genes of V. orientalis are identified with the same five major structural genes luxCDABE in contrast to other luminous bacteria. The synthesis of luciferase is mediated by luxA and luxB genes, and that of fatty acyl reductase, acyl transferase, and acyl protein synthase, comprising the fatty acyl reductase complex for providing the substrate for luciferase, are mediated by luxC, luxD, and luxE genes, respectively. Moreover genes of lux operon are found varied in different luminous bacteria. Among these, the luxAB genes could be used as reporter genes for monitoring promoter and terminator activities after cloning into the vectors for the construction of reporter vectors. Upstream sequences of V. orientalis lux operon are analyzed to predict the potential promoter region and the regulatory elements involved in the activation and inhibition of gene expression. Serial deleted DNA fragments from upstream sequences are cloned and analyzed by the promoter reporter vector for this purpose. Possible DNA sequences of rho-independent terminators are also cloned and analyzed by the terminator reporter vector. The results of this study would provide both useful information for the investigation of lux genes regulation in V. orientalis and facile tools for the monitoring of potential promoter and terminator activities from genes of various organisms.

碩士
國立屏東科技大學
生物科技系所
106
The process of communication from one cell to another in bacteria called Quorum Sensing (QS). This process can regulate the genes that must be perform in a group of bacteria. In V. orientalis, the luminous bacteria from Yellow coast of China there are six genes that linked with QS. That genes were: luxP (VIA_001151), luxQ (VIA_001152), luxS (VIA_003963), luxO (VIA_003573) and (VIA_003054), luxU (VIA_003574) and hapR (VIA_000273). This research focused on hapR gene since it was LuxR homologues and previous report showed that LuxR is an activator of the lux operon in V. harveyi (Swartzman et al., 1992; Showalter et al.,1990). LuxR in V. harveyi is not homolog with the luxR in A. fischeri, since it was the QS master regulator that controls expression of the genes in the QS of V. harveyi. This present study observed the role of HapR in bioluminescence process of V. orientalis. For confirmation, the interaction between HapR protein and promoter lux operon, the in vitro, in vivo and in silico study were done. DNA size of hapR was 615 bp and the protein size was about 24 kDa in pET28a, and around 19 kDa in pET29a. But only the pET28a that harbouring HapR that was protein fusion. The best treatment of IPTG induction for HapR was 1 mM IPTG in 25oC for 3 hour, and the buffer for purification the protein was Tris-HCl pH 8. In vitro study proved that HapR has an interaction with the promoter lux operon no.1-4 but not with the promoter lux operon no.5 and 6. It means upstream of the promoter lux operon no. 5 (292 bp) are sufficient to bind with HapR protein. In vivo study result showed that clones that has the higest Relative Light Unit (RLUs) was in HapR-PM with IPTG induction group, which is HapR-PM2 with 24.563 x 106 RLUs at 5 hour after IPTG induction. This result suggest that HapR bind the promoter lux operon around 792 bp-581 bp. In silico study result suggest that V. orientalis CIP 102891 = ATCC 33934 strain CIP 102891 has seven lux operon genes, luxCDABEGH and the arrangement is similar to V. harveyi strain ATCC 33843 (392 [MAV]) with 97.8% similarities. In silico study also demonstrated that V. orientalis only use one type of QS system that was AI2 system to manage their light production and it predicted that the regulation of bioluminescence was depend on phosphorylation-dephosphorylation cascade similar with AI2 system in V. harveyi and A. fischeri. So, it’s can conclude that HapR has a role on the regulation of lux genes from V. orientalis.

碩士
國立成功大學
微生物及免疫學研究所
92
Vibrio vulnificus, a halophilic gram-negative marine bacterium, is an opportunistic pathogen causing septicemia and wound infection in humans. A few virulence factors, such as the capsule, a few extracellular products, and iron-acquisition ability, have been proposed; however, the pathogenesis mechanism of this organism remains unclear. It has been shown previously that the quorum-sensing system can regulate the virulence of pathogens, such as V. cholerae, Pseudomonas aeruginosa, and Staphylococcus aureus. In the whole genome sequence of V. vulnificus completed in 2002, most genes required for AI-2 quorum-sensing signaling were identified. To understand whether the quorum-sensing system is involved in the regulation of V. vulnificus, a mutant (LD mutant) deficient in LuxO, the quorum-sensing regulator, and a luxOD47E mutant expressing constitutively active LuxO were isolated and characterized. We found that LuxO regulates not only the extracellular protease activity negatively but also the cytolysin activity positively. The virulence of LD mutant in mice infected by subcutaneous injection was 100-fold less than that of the wild-type strain or the luxOD47E mutant, suggesting that quorum-sensing system may be involved in regulating the virulence of V. vulnificus. The cytotoxicity to HEp-2 cells was not affected in the luxOD47E mutant, but has diminished in the LD mutant. Furthermore, the spread of bacteria from subcutaneous tissue to bloodstream was remarkably slower in mice infected with the LD mutant than those infected with the wild-type strain or luxOD47E mutant. In the histological examination, we found abundant infiltrating cells but few bacteria in the infection site with the LD mutant. However, a large number of infiltrating cells and bacteria were found clustered together with the wild-type strain and the luxOD47E mutant. On the other hand, the amount of siderophore produced, growth in murine blood, and resistance to serum bactericidal effect were not affected in either the LD or luxOD47E mutant. In conclusion, these results suggest that LuxO may be involved in regulating the virulence of V. vulnificus, probably via activating the expression of an unknown cytotoxin important for escaping the attack from the phagocytes and/or invasion of this organism to the bloodstream, where the organism multiplies and causes septic shock and death.

碩士
國立中興大學
分子生物研究所
85
V. fishcheri lux regulon luxR gene 下游 3,565 bp 之 DNA 經定序分 析，得知此 DNA 片段，具有 RNA-like gene cluster、ufo1 gene、 origin-like sequence 與 cnf1 gene，與 lux regulon 之排列序列為 ----cnf1-ori-like sequence-ufo1-RNA-like gene cluster-R&R'-luxR- R&R-luxI-luxC-luxD-luxA-luxB-luxE----。其中 cnf1 為主導 cytotoxic necrotizing factor 1 基因；ufo1 為一未鑑定之基因；而 RNA-like gene cluster 包括一 rRNA-like gene 與數個 tRNA-like genes；另存在一 origin-like sequence。此等基因顯然受 lux regulon 主調節區域 R&R (regulatory region) 的調控；之外，其內含調節區域 R&R'，則具有 terminator-like block 之功能，可弱化轉錄，減少其後 續基因之表現，亦具有可能為輔助啟動子 (intern promoter) 之序列， 可能為次調節 (sub-regulation) 系統，以調控基因表現。有關 lux regulon 的調控，除了主調節區域 R&R 受 LuxR-AI、cAMP-CRP 的調控之 外，內含於 lux regulon 之次調節機制 (sub-regulation)，包括 R&R' 、terminator-like block、intern promoter、potential hairpin loops 等，皆參與 lux regulon 基因的調控。實驗結果肯定次調節機制 (sub-regulation) 存在於 lux regulon；此等 sub-regulaton signals ，包括 terminator-like block 與可能之輔助啟動子 (intern promoter) 存在於 luxC gene 內與 luxD gene 5' 端之前段。另外存在 於 luxB gene 之後、luxE gene 之前的 potential hairpin loop Wo'' 則不具明顯之次調節機制。但此類似之 potential hairpin loop 亦存在 於 V. harveyi 與 P. leiognathi，皆具有弱化轉錄以調控螢光表現的作 用。結果顯示 lux regulon/operon 中確實存在 sub-regulation 次調節 機制以調控螢光表現。 The 3,565 bp DNA sequence downstream of the luxR gene in V. fischeri lux regulon has been determined, which contains RNA- like gene cluster, ufo1 gene, origin-like sequence, and cnf1 gene. Sequence analysis elucidates that there are several genes resided in the DNA fragment: the cnf1 gene encodes cytotoxic necrotizing factor 1; the ufo1 gene is not identified yet; the RNA-like gene cluster contains a rRNA-like gene and several tRNA-like genes; and an origin-like sequence is illustrated. The gene order of these genes and the lux regulon is <--cnf1-ori- like sequence-ufo1-RNA-like gene cluster-R&R'-luxR-R&R-luxI- luxC-luxD-luxA-luxB-luxE----; whereas the regulatory region R&R is the domain regulation for the lux regulon, and the regulatory region R&R' contained terminator-like block is the sub- regulation for the lux regulon. The regulatory region R&R' contains an intern promoter, which enables to reduce transcription and repress the gene expression of the genes linked to the luxR gene, might function for sub-regulation. The regulation of the lux regulon is not only under regulation by the regulatory region R&R, which regulated by LuxR-AI and cAMP- CRP, but also under sub-regulation by the regulatory region R& R', terminator-like block, intern promoter, and potential hairpin loop resided in the lux regulon. The terminator-like block and intern promoter bet-ween the luxC gene and 5'-end of the luxD gene is identified as sub-regulation signals. The potential hairpin loop Wo'', which resided in the 3'-end of the luxB gene, does not function for sub-regulation; but the similar potential hairpin loops resided in the lux operons of V. harveyi and P. leiognathi enable to deduce transcription. The results elicits that the sub-regulation indeed is functioned for gene expression and regulation of bioluminescence.

碩士
慈濟大學
微生物學免疫學暨生物化學研究所
99
Quorum sensing system, a two-component system in bacteria, is a cell-cell communication process. Bacteria use the production and detection of the extracellular chemical signals called autoinducers (AI) to monitor cell density and coordinate gene expression. The LuxS enzyme (S-ribosylhomocysteinlyase), which is conserved in a wide variety of bacteria, is responsible for the production of autoinduer-2 (AI-2). It has been reported that LuxS/AI-2 system has multiple roles in interfering with physiological and virulence functions in many bacteria. To gain insight into LuxS function in seafood-borne pathogen Vibrio parahaemolyticus, we have constructed and characterized a luxS mutant in the tdh-and trh-negative strain named VP93. Using the V. harveyi bioassay, the highest level of AI-2 was detected in the supernatant of stationary-phase cells of wild type. As expected, mutation of luxS abolished their AI-2 production. Besides, protease secretion, swarming activity and colony morphology showed no significant difference between the wild type and its mutant. However, it was found that biofilm formation was diminished but swimming motility was increased in the luxS mutant. The global protein expression profiles of the wild type and the mutant were also compared by 2D proteome analysis. In the analysis of intracellular proteomes, proteins generated under stress conditions (GroEL) and proteins involved in cell division (FtsZ) were detected in wild type. In contrast, cell envelope proteins, OmpA and OmpU, were abundant in the luxS mutant. Additionally, enzymes involved in amino acid synthesis and purine ribonucleotide synthesis were also increased in the mutant strain. In extracellular proteome analysis, outer membrane protein (OmpU) and outer membrane channel protein (TolC) were only abundant in the wild type, but the polar flagellin protein (FlaA) was detected in the luxS mutant. Finally, we also examined the reported virulence factor genes in V. parahaemolyticus in the wild type and its mutant by RT-PCR. The result showed that mutation of luxS resulted in down-regulation of capsular polysaccharide gene (cpsA) expression and up-regulation of flagella gene (flaA) expression. In conclusion, mutation of luxS in VP93 might influence biofilm formation and swimming motility. Besides, several LuxS-regulated genes were also found in this study.

Mittels Quorum sensing können Bakterienzellen die Expression von Genen Zelldichte-abhängig steuern. Dies spielt eine besondere Rolle bei der Expression von Virulenzfaktoren oder Antibiotikaproduktion, der Biofilmentwicklung oder Phänomenen wie genetischer Kompetenz, Sporulation oder Biolumineszenz. Vibrio harveyi, ein Gram-negativer, mariner, frei lebender Organismus, reguliert die Expression von Biolumineszenz-Genen Zelldichte-abhängig nach dem Prinzip des Quorum sensing und sollte im Rahmen dieser Arbeit als Modell für die Mechanismen der Signaltransduktion untersucht werden. Dazu wurden die Proteine der Lux-Signaltransduktionskaskade heterolog in E. coli überexprimiert und teilweise gereinigt. Mittels in vitro Phosphorylierung konnten die enzymatischen Aktivitäten der Proteine erstmals biochemisch charakterisiert werden. Für die Hybridsensorkinase LuxN konnte neben einer Kinase-Aktivität ein Phosphotransfer auf das Histidin-Phosphotransferprotein LuxU gezeigt werden. Die Autophosphorylierungsaktivität ist dabei eindeutig von der Konzentration des Signalmoleküls, eines Acyl-Homoserinlaktons, abhängig. Eine ebenfalls eindeutig nachgewiesene Phosphatase-Aktivität von LuxN, die zur Dephosphorylierung von LuxU führt, war dagegen konstitutiv. Damit konnte ein auf biochemischen Daten basierendes Modell der Signaltransduktion von V. harveyi postuliert werden. Basierend auf den Ergebnissen von Topologieuntersuchungen mittels luxN-Reportergenfusionen und Protease-Zugänglichkeitsstudien konnten neue Hinweise auf die Membrantopologie der Hybridsensorkinase ermittelt werden. Diese lassen auf ein Modell mit neun Transmembranhelices schließen, bei der der N-terminus des Proteins im Periplasma der Zelle lokalisiert zu sein scheint.

碩士
國立中興大學
分子生物研究所
81
We use the gene order rearrangement to analyze the regulatory region (R&R) of the lux regulon from Vibrio fischeri reveals that the expression of the L-promoter is much stronger than that of the R-promoter. The results of glucose repression experiment illustrate that the expression of the L-operon is enhanced by cAMP-CRP. In trans complementation tests elucidat that the LuxR- AI complex could enhance the expression of the two promoters by binding on the motif binding site of the R&R. The evidences of site-directed mutagenesis reveal that the LuxR- AI could bind on the R1-palindrome to enhance the expression of the divergent pro- moters. In addition, site-directed mutagenesis method was used to modify the R2-reversed repeat,it shows no effect on the of the divergent promoters. So far,the function of the repeat on the R&R of the lux regulon is not clearly known yet.

碩士
國立中興大學
分子生物研究所
81
For the purpose of simplicity, only the regulatory genes, luxR and luxI, were used for studying in this work. A series of gene order rearrangement constructions and in trans comple- mentary tests were designed to investigate the requirement of the sequences of the luxR and luxI genes, and functional analy- sis of the LuxR protein and autoinducer (AI). The luciferase genes, luxA-luxB, from V. harveyi were used as reporter gene to monitor the expression of the R- and L-promoter. The results show that the sequences of the luxR and luxI genes were the ne- gative control elements for the expression of the R- and L-pro- moter. The LuxR-AI complex is the positive control element for both the R- and L-promoter, and directly bound to the R&R se- quence. The sequence of the luxR gene repressed the expression of the L-promoter much stronger than the effect of the sequence of the luxI gene to the R-promoter. Nucleotide sequence analysis showed that an reversed repeat sequence, termed R3- reversed re- peat, was found on the sequence of the luxR gene, and it might be the LuxR motif-binding locus. The site-directed mutagenesis was used to inspect the function of this site. The results of the luxR mutations show that the premature translation of the LuxR protein repressed the expression of the following reporter genes. It suggested that the sequence of the luxR gene might re- gulate the lux regulon by attenuation-like termination. Further modification in this site was be done to define the function of the R3-reversed repeat, but the result can't confirm R3-reversed repeat is LuxR protein motif-binding locus for negative feedback control. The LuxR protein might bind on one specific site of the luxR sequence to enhance the attenuation-like transcriptional termination and negative feedback control the expression of L- operon.