• Journal of Microbiology
• /
• v.38 no.2
• /
• pp.80-87
• /
• 2000

The nucleotide sequence of the luxC gene coding for lux-specific fatty acyl-CoA reductase and the upstream DNA (325bp)of the structural gene from bioluminescent bacterium, Photobacterium phosphoreum, has been deternubed. An open reading frame extending for more than 20 codons in 325 bp DNA upstream of luxC was not present in both directions. The lux gene can be translated into a polypeptide of 54 kDa and the amino acid sequences of lux specific reductases of P. phosphoreum shares 80, 65, 58, and 62% identity with those of the Photobacterium leiognathi, Vibrio fischeri, Vibrio harveyi, and Xehnorhabdus luminescenens reductases, respectively. Analyses of codon usage, showing that a high frequency (2.3%) of the isoleucine codon, AUA, in the luxC gene compared to that found in Escherichia coli genes (0.2%) and its absence in the luxA and B genes, suggested that the AUA codon may play a modulator role in the expression of lux gene in E. coli. The structural genes (luxC, D, A, B, E) of the P. phosphoreum coding for luciferase (${\alpha}$,${\beta}$) and fatty acid reductase (r, s, t) polypeptides can be expressed exclusively in E. coli under the T7 phage RNA polymerase/promoter system and identificationof the [35S]methionine labelled polypeptide products. The degree of expression of lux genes in analyses of codon usage. High expression of the luxC gene could only be accomplished in a mutant E. coli 43R. Even in crude extracts, the acylated acyl-CoA reductase intermediate as well as acyl-CoA reductrase activities could be readily detected.

• Korean Journal of Microbiology
• /
• v.36 no.1
• /
• pp.1-7
• /
• 2000

The genes involved in riboflavin synthesis (ribI, II, III, and IV) were found immediately downstream of luxG in the lux operon from Photobacterium species. The single stranded DNA containing the intergenic region of lux genes and rib genes from Photobacterium phosphoreum was fully protected by P. phosphoreum mRNA from the S1 nuclease mapping assay suggesting that a transcriptional terminator was not present in the region. In addition, the levels of riboflavin synthase activity in P. phosphoreum was increased during the development of bacterial bioluminescence in the same fashion as the luciferase and fatty acid reductase activities. Insertion of the Photobacterium leiognathi DNA extending from luxB to ribII, between a strong lux promoter and a reporter gene (chloramphenicol acetyltransferase, CAT) and transferred by conjugation into P. leiognathi, did not affect expression of reporter gene. Moreover the CAT gene was not expressed in an analogous construct missing the lux promoter indicating that a promoter was not present in this region. Based on the data here, it can be concluded that the lux genes and rib genes in Photobacterium species are under common regulation.

• Korean Journal of Microbiology
• /
• v.38 no.3
• /
• pp.173-179
• /
• 2002

The functions of riboflavin synthesis genes ( ribI,II,III and IV) found immediately downstream of luxG in the lux operon from Photobacterium species were identified using the biochemical and genetical analysis. The ribI-III gene codes for protein corresponding to that coded by the second (riboflavin synthase), third (3,4-dihydroxy 2-butanone 4-phosphate synthase/GTP cyclohydrolase II) and fourth (lumazine synthase) gene, respectively, of Bacillus subtilis rib operon with the respective gene procuct sharing 41-50% amino acid sequence identity. Unexpectedly, the sequence of the ribIV product of Photobacterium phosphoreum does not correspond in sequence to the protein encoded by the fifth rib gene of Bacillus subtilis. Instead the gene (ribIV) codes for a polypeptide similar in sequence to GTP cyclohydrolase II of Escherichia coli and the carboxy terminal domain of the third rib gene from Bacillus subtilis. Complementation of Escherichia coli riboflavin auxotrophs showed that the function of the gene products of ribII and ribIV are DHBP synthase and GTP cyclohydrolase II, respectively. In addition the experiment, showing that increase in thermal stability of riboflavin synthase coded by ribIon coexpression with ribIII, provided indirect evidence that the latter gene codes for lumazine synthase.

• Journal of Microbiology and Biotechnology
• /
• v.7 no.5
• /
• pp.352-355
• /
• 1997

The promoter region of the pupA gene of Pseudomonas putida WCS358 was fused with the structural genes for bioluminescence (luxCDABE) from Vibrio fischeri, and the resulting fusion plasmid harbored by the WCS358 host. The pup-lux fusion gene was then used for quantitative analysis of the iron-dependence of pupA promoter activity. Factors affecting bioluminescence produced by the pup-lux bioreporter were found to be cell activity, iron-chelator concentrations, Fe(III) concentrations, and nutrient components. Light production rates of the pup-lux bioreporter were inversely dependent upon iron molecules when $FeCl_3$ concentrations were between $10^{-2}$ and 1 ${\mu}M$ in nutrient-poor minimal media, and between 0.1 and 10 mM in nutrient-rich complex media.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.8
• /
• pp.765-773
• /
• 2009

Edwardsiella tarda is a pathogen with a broad host range that includes human and animals. The E. tarda hemolysin (Eth) system, which comprises EthA and EthB, is a noted virulence element that is widely distributed in pathogenic isolates of E. tarda. Previous study has shown that the expression of ethB is regulated by iron, which suggests the possibility that the ferric uptake regulator (Fur) is involved in the regulation of ethB. The work presented in this report supports the previous findings and demonstrates that ethB expression was decreased under conditions when the E. tarda Fur ($Fur_{Et}$) was overproduced, and enhanced when $Fur_{Et}$ was inactivated. We also identified a second ethB regulator, EthR, which is a transcription regulator of the GntR family. EthR represses ethB expression by direct interaction with the ethB promoter region. In addition to ethB, EthR also modulates, but positively, luxS expression and AI-2 production by binding to the luxS promoter region. The expression of ethR itself is subject to negative autoregulation; interference with this regulation by overexpressing ethR during the process of infection caused (i) drastic changes in ethB and luxS expressions, (ii) vitiation in the tissue dissemination and survival ability of the bacterium, and (iii) significant attenuation of the overall bacterial virulence. These results not only provide new insights into the regulation mechanisms of the Eth hemolysin and LuxS/AI-2 quorum sensing systems but also highlight the importance of these systems in bacterial virulence.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.3 no.1
• /
• pp.6-10
• /
• 1998

A plasmid vector containing two reporter genes, mer-lux and lac-GFP, was transformed to both Escherichia coli and Pseudomonas putida. Their cellular activities and biofilm characteristics were investigated in flow-cell units by measuring bioluminescent lights and fluorescent levels of GFP. Bioluminescence was effective to monitor temporal cell activities, whereas fluorescent level of GFP was useful to indicate the overall cell activities during biofilm development. The light production rates of E. coli and P. putida cultures were dependent upon concentrations of HgCl2. Mercury molecules entrapped in P. putida biofilms were hardly washed out in comparison with those in E. coli biofilms, indicating that P. putida biofilms may have higher affinity to mercury molecules than E. coli biofilms. It was observed that P. putida expressed GFP cDNA in biofilms but not in liquid cultures. This may indicate that the genetic mechanisms of P. putida were favorably altered in biofilm conditions to make a foreign gene expression possible.

• Journal of Microbiology
• /
• v.44 no.2
• /
• pp.226-232
• /
• 2006

Swarming has proven to be a good in vitro model for bacterial surface adherence and colonization, and the swarming differentiation of a bacterium has been shown to be coupled with changes in the expression of virulence factors associated with its invasiveness, particularly in the early stages of infection. In this study, we attempted to determine whether the expression of vvhA, which encodes for hemolysin/cytolysin (VvhA), is either upregulated or downregulated during the swarming differentiation of V. vulnificus. The insertional inactivation of vvhA itself exerted no detectable effect on the expression of V. vulnificus swarming motility. However, in our lacZ-fused vvhA transcriptional reporter assay, vvhA expression decreased in swarming V. vulnificus as compared to non-swarming or planktonic V. vulnificus. The reduced expression of vvhA in swarming V. vulnificus increased as a result of the deletional inactivation of luxS, a gene associated with quorum sensing. These results show that vvhA expression in swarming V. vulnificus is downregulated via the activity of the LuxS quorum-sensing system, suggesting that VvhA performs no essential role in the invasiveness of V. vulnificus via the adherence to and colonization on the body surfaces required in the early stages of the infection. However, VvhA may playa significant role in the pathophysiological deterioration occurring after swarming V. vulnificus is differentiated into planktonic V. vulnificus.

• Journal of Microbiology and Biotechnology
• /
• v.32 no.9
• /
• pp.1134-1145
• /
• 2022

SCO6993 (606 amino acids) in Streptomyces coelicolor belongs to the large ATP-binding regulators of the LuxR family regulators having one DNA-binding motif. Our previous findings predicted that SCO6993 may suppress the production of pigmented antibiotics, actinorhodin, and undecylprodigiosin, in S. coelicolor, resulting in the characterization of its properties at the molecular level. SCO6993-disruptant, S. coelicolor ΔSCO6993 produced excess pigments in R2YE plates as early as the third day of culture and showed 9.0-fold and 1.8-fold increased production of actinorhodin and undecylprodigiosin in R2YE broth, respectively, compared with that by the wild strain and S. coelicolor ΔSCO6993/SCO6993⁺. Real-time polymerase chain reaction analysis showed that the transcription of actA and actII-ORF4 in the actinorhodin biosynthetic gene cluster and that of redD and redQ in the undecylprodigiosin biosynthetic gene cluster were significantly increased by SCO6993-disruptant. Electrophoretic mobility shift assay and DNase footprinting analysis confirmed that SCO6993 protein could bind only to the promoters of pathway-specific transcriptional activator genes, actII-ORF4 and redD, and a specific palindromic sequence is essential for SCO6993 binding. Moreover, SCO6993 bound to two palindromic sequences on its promoter region. These results indicate that SCO6993 suppresses the expression of other biosynthetic genes in the cluster by repressing the transcription of actII-ORF4 and redD and consequently negatively regulating antibiotic production.

• Journal of Life Science
• /
• v.23 no.9
• /
• pp.1133-1139
• /
• 2013

During the collection of boar semen, bacterial contamination usually occurs. The contamination has deleterious effects both on semen quality and on sow fertility. The majority of contaminants are gram-negative bacteria, especially Serratia marcescens. In this study, we developed a PCR assay for the identification of S. marcescens targeting the luxS gene (GenBank no. EF164926). S. marcescens yielded a specific 306 bp PCR product. However, no amplification was observed in the other strains tested. The detection limit of PCR was $50pg/{\mu}l$ of template DNA of S. marcescens. The antimicrobial susceptibility patterns of S. marcescens isolated from boar semen were tested using the disk diffusion method. Gentamicin, ceftiofur, florfenicol, and neomycin showed high sensitivity in this test. The minimum inhibitory concentration (MIC) was also determined by the broth microdilution method. The $MIC_{90}$ values of ceftiofur, enrofloxacin, gentamicin, and neomycin were 8, 8, 8, and $16{\mu}g/ml$, respectively. These results indicate that PCR amplification of the luxS gene is a reliable and effective method for the identification of S. marcescens and that ceftiofur, enrofloxacin, gentamicin, and neomycin are effective semen extenders for controlling S. marcescens.

• Korean Journal of Microbiology
• /
• v.42 no.4
• /
• pp.277-285
• /
• 2006

The most biofilm forming bacteria in catheter, Esctherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were isolated and identified from a patient's catheter occuring catheter-associated urinary tract infection (CA-UTI). We examined mRNA expression and its quantification of AIs synthetic genes encoding signal substance of quorum sensing from each bacterial species in order to elucidated quorum sensing mechanism. Both pure cultures for each bacterial strains and a mixed cultures with three were grown for 24 hr and 30 days. Initial densities to be able to detect mRNA expression oil single strains culture were shown at $2.4{\times}10^5$ CFU/ml, $5.4{\times}10^6$ CFU/ml of E. coli for ygaG and S. aureus for luxS, and at $6.9{\times}10^4$ CFU/ml of P. aeruginosa for rhlI and lasI. Also, in mixed culture of three, initial cell densities of mRNA expression were appear to at $7.3{\times}10^5$ CFU/ml, $1.6{\times}10^7$ CFU/ml of E. coli for ygaG and S. aureus for luxS, and at $2.1{\times}10^5$ CFU/ml of P. aeruginosa for rhlI and lasI. Each AIs synthetic gene was expressed in initial cell density and the mRNA expression of the genes were detected continously during 30 days. And then, the quantification of mRNA expression level of ygaG, rhlI, last, and luxS which were related AIs synthesis was done each time point by real-time RT-PCR. Interestingly, the mRNA levels of ygaG, rhlI, lasI, and luxS from the mixed culture was higher than those from each single strain culture. In the case of E. coli ygaG, the amount of transcript from the mixed culture was at least 30 times for that from single culture. In the case of P. aeruginosa rhlI and lasI, the amount of transcript from the mixed culture was at least 40 times and 250 times for that from single strain culture. In the case of S. aureus luxS, the amount of transcript from the mixed culture was at least 5 times for that from single strain culture. And specially, the mRNA expression of rhlI and lasI of P. aeruginosa showed the highest efficency among four AIs synthetic genes.