Kyu-Ho Lee

Bacterial communities in the initial stage of marine biofilm formation on artificial surfaces

Succession of bacterial communities during the first 36 h of biofilm formation in coastal water was investigated at 3∼15 h intervals. Three kinds of surfaces (i.e., acryl, glass, and steel substratum) were submerged in situ at Sacheon harbor, Korea. Biofilms were harvested by scraping the surfaces, and the compositions of bacterial communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP), and cloning and sequencing of 16S rRNA genes. While community structure based on T-RFLP analysis showed slight differences by substratum, dramatic changes were commonly observed for all substrata between 9 and 24 h. Identification of major populations by 16S rRNA gene sequences indicated that γ-Proteobacteria (Pseudomonas, Acinetobacter, Alteromonas, and uncultured γ-Proteobacteria) were predominant in the community during 0∼9 h, while the ratio of α-Proteobacteria (Loktanella, Methylobacterium, Pelagibacter, and uncultured α-Proteobacteria) increased 2.6∼4.8 folds during 24∼36 h of the biofilm formation, emerging as the most predominant group. Previously, α-Proteobacteria were recognized as the pioneering organisms in marine biofilm formation. However, results of this study, which revealed the bacterial succession with finer temporal resolution, indicated some species of γ-Proteobacteria were more important as the pioneering population. Measures to control pioneering activities of these species can be useful in prevention of marine biofilm formation.

Differential Expression of Vibrio vulnificus Elastase Gene in a Growth Phase-dependent Manner by Two Different Types of Promoters

Elastase activity of Vibrio vulnificus was highly dependent on growth phase, reached a maximum during the stationary phase, and was regulated at the level of transcription. The stationary phase production of elastase incrp or rpoS mutants, which were constructed by allelic exchanges, decreased about 3- and 10-fold, respectively. However, the promoter activity of vvpE encoding elastase was unaffected by those mutations in the log phase when analyzed using a vvpE-lux fusion. A primer extension analysis revealed that the transcription of vvpE begins at two different sites, consisting of putative promoter L (PL) and promoter S (PS). The PL activity was constitutive through the log and stationary phases, lower than the PS activity, and unaffected by the crp orrpoS mutations. The transcription of PS, induced only in the stationary phase, was dependent on RpoS. The mutation incrp reduced the activity of PS; however, the additional inactivation of crp did not influence the PS activity in the rpoS mutant, indicating that CRP exerted its effects through PS requiring RpoS. These results demonstrate thatvvpE expression is differentially directed by PL and PS depending on the growth phase and elevated by RpoS and CRP in the stationary phase.

Complete Genome Sequence of Vibrio vulnificus MO6-24/O

Vibrio vulnificus is the causative agent of life-threatening septicemia and severe wound infections. Here, we announce the complete annotated genome sequence of V. vulnificus MO6-24/O, isolated from a patient with septicemia. When it is compared with previously known V. vulnificus genomes, the genome of this bacterium shows a unique genetic makeup, including phagelike elements, carbohydrate metabolism-related genes, and the superintegron.

Transcriptional Regulatory Cascade for Elastase Production in Vibrio vulnificus LuxO ACTIVATES luxT EXPRESSION AND LuxT REPRESSES smcR EXPRESSION

Vibrio vulnificus causes diseases through actions of various virulence factors, including the elastase encoded by the vvpE gene. Through transposon mutagenesis of V. vulnificus, vvpE expression was shown to be increased by luxO mutation. Since the vvpE gene is known to be positively regulated by SmcR via direct binding to the vvpE promoter, the role of LuxO in smcR expression was investigated. The luxAB-transcriptional fusions containing different lengths of the smcR promoter region indicated that the smcR transcription was negatively regulated by LuxO and that a specific upstream region of the smcR gene was required for this repression. Since LuxO is a known member of positive regulators, the negative regulation of smcR transcription by LuxO prompted us to identify the factor(s) linking LuxO and smcR transcription. LuxT was isolated in a ligand fishing experiment using the smcR upstream region as bait, and smcR expression was increased by luxT mutation. Recombinant LuxT bound to a specific upstream region of the smcR gene, –154 to –129 relative to the smcR transcription start site. The expression of luxT was positively regulated by LuxO, and the luxT promoter region contained a putative LuxO-binding site. Mutagenesis of the LuxO-binding site in the luxT promoter region resulted in a loss of transcriptional control by LuxO. Therefore, this study demonstrates a transcriptional regulatory cascade for elastase production, where LuxO activates luxT transcription and LuxT represses smcR transcription.

Role of NtrC-regulated exopolysaccharides in the biofilm formation and pathogenic interaction of Vibrio vulnificus

Vibrio vulnificus has been shown to require a global transcription factor, NtrC for mature biofilm development via controlling the biosyntheses of lipopolysaccharide and exopolysaccharide (EPS). Biofilm formation and EPS production were dramatically increased in a medium including a tricarboxylic acid cycle‐intermediate as a carbon source. These phenotypes required functional NtrC and were abolished by the addition of ammonium chloride. During the initial stage of biofilm formation, both expression of the ntrC gene and the cellular content of NtrC protein increased. Thus, the regulatory roles of NtrC in EPS biosynthesis were studied with three gene clusters for EPS biosyntheses. Transcriptions of the three clusters were positively controlled by NtrC and showed maximal expression at the early stage of biofilm development. Mutants deficient in one of the genes (VV1_2661, VV2_1579 and VV1_2305) in each cluster showed decreased production of EPS, attenuated ability to form biofilm and lowered cytoadherence to human epithelial cells. However, mutations in VV2_1579 and VV1_2305 resulted in lower cytotoxicity to human cells and mortality to mice than the mutation in VV1_2661. These results demonstrate that NtrC‐regulated EPS are crucial in biofilm formation of V.u2003vulnificus, and some EPS components play important roles in interacting with hosts.

Role of NtrC in biofilm formation via controlling expression of the gene encoding an ADP‐glycero‐manno‐heptose‐6‐epimerase in the pathogenic bacterium, Vibrio vulnificus

To identify the genetic elements required for biofilm formation, we screened a pool of random Vibrio vulnificus mutants for their ability to form biofilms. One mutant displaying significantly decreased biofilm‐forming activity was found to contain a transposon insertion in the ntrC gene. The ntrC gene encodes a well‐known transcriptional activator. We examined how this regulator modulates a biofilm‐forming process in V.u2003vulnificus by searching for NtrC target gene(s). Comparison of the proteomes of ntrC mutant and wild‐type strains grown under planktonic and biofilm stages revealed that synthesis of the protein homologous to GmhD (ADP‐glycero‐manno‐heptose‐6‐epimerase) was elevated during the growth period for biofilm formation and was strongly influenced by NtrC. A luxAB‐transcriptional fusion with the gmhD promoter region indicated that gmhD expression was positively regulated by both NtrC and RpoN. The function of the gmhD gene product in V.u2003vulnificus was assessed by constructing and phenotypic analyses of an isogenic mutant. The gmhD mutant was defective in production of mature lipopolysaccharide (LPS) and exopolysaccharides (EPS), and demonstrated an attenuated ability to form a biofilm. These results suggest that NtrC acts as a key regulator of both LPS and EPS biosyntheses and, thereby, modulates critical steps in biofilm development of V.u2003vulnificus.

Enhanced Rapidity for Qualitative Detection of Listeria monocytogenes Using an Enzyme-Linked Immunosorbent Assay and Immunochromatography Strip Test Combined with Immunomagnetic Bead Separation

An enzyme-linked immunosorbent assay (ELISA), immunochromatography (ICG) strip test, and immunomagnetic bead separation (IMBS) system based on a monoclonal antibody were individually developed for the detection and isolation of Listeria monocytogenes in meat samples. The three methods showed a strong reaction with Listeria species and a weak reaction with Staphylococcus aureus. To increase the rapidity of L. monocytogenes detection, combinations of the ELISA and ICG strip test with the IMBS system (ELISA-IMBS and ICG-IMBS) were investigated. In comparative analyses of artificially inoculated meat and samples of processed meat, the ELISA and ICG strip test required 24 h of enrichment time to detect the inoculated meat samples with > or =1 X 10(2) CFU/10 g, whereas the ELISA-IMBS and ICG-IMBS required only 14 h of enrichment. Analyses of naturally contaminated meat samples (30 pork samples, 20 beef samples, 26 chicken samples, 20 fish samples, and 20 processed meat samples) performed by ELISA-IMBS, ICG-IMBS, and API kit produced similar results. The ELISA-IMBS and ICG-IMBS provide a more rapid assay than the individual ELISA and the ICG strip test and are appropriate for rapid and qualitative detection of L. monocytogenes (or Listeria species) in meat samples. With the ICG-IMBS, L. monocytogenes could be detected in meat samples within 15 h and the method has potential as a rapid, cost-effective on-site screening tool for the detection of L. monocytogenes in food samples and agricultural products at a minimum detection level of approximately 100 CFU/10 g.

Comparative proteomic analysis of trophozoites versus cysts of Giardia lamblia.

The proteome of Giardia lamblia at its cyst stage was compared with that of trophozoites by using two-dimensional SDS-PAGE gel electrophoresis. Protein spots that increased in the extracts of cysts compared to trophozoites were identified by MALDI-TOF mass spectroscopy and categorized as cytoskeletal proteins, metabolic enzymes, a cell-cycle-specific kinase, stress resistance proteins, and a protein involved in translation. Expression patterns of five of the identified proteins were examined during encystation by real-time PCR. Expression of cwp1 (encoding cyst wall protein 1), a marker for encystation, was increased 11-fold. In contrast, tim (encoding triose-1-phosphate isomerase) was expressed constitutively during encystation, and its transcription level was therefore used as a mRNA loading control. Expression of three genes encoding β-tubulin, vacuolar ATPase, and never-in-mitosis-A-related protein kinase did not vary significantly during encystation. Interestingly, genes encoding two heat shock proteins (Hsp70 and Hsp90) showed increased expression during encystation, suggesting that this differentiation process accompanies a cellular response to stress in G. lamblia.

Expression of the cpdA gene, encoding a 3',5'-cyclic AMP (cAMP) phosphodiesterase, is positively regulated by the cAMP-cAMP receptor protein complex.

The intracellular level of cyclic 3,5-AMP (cAMP), a signaling molecule that mediates a variety of cellular processes, is finely modulated by the regulation of its synthesis, excretion, and degradation. In this study, cAMP phosphodiesterase (CpdA), an enzyme that catalyzes the conversion of cAMP to AMP, was characterized in a pathogenic bacterium, Vibrio vulnificus. The cpdA gene exists in an operon composed of mutT, yqiB, cpdA, and yqiA, the transcription of which was initiated at position -22 upstream of mutT. A cpdA-null mutant of V. vulnificus contained significantly higher levels of cAMP than the wild type but showed no detectable cAMP when a multicopy plasmid of the cpdA gene was provided in trans, suggesting that CpdA is responsible for cAMP degradation. Cellular contents of the CpdA protein decreased dramatically in both cya and crp mutants. In addition, levels of expression of the cpdA::luxAB transcription fusion decreased in cya and crp mutants. The level of expression of cpdA::luxAB in the cya mutant increased in a concentration-dependent manner upon the exogenous addition of cAMP. The cAMP-cAMP receptor protein (CRP) complex bound directly to the upstream region of mutT, which includes a putative CRP-binding sequence centered at position -95.5 relative to the transcription start site. Site-directed mutagenesis or the deletion of this sequence in the cpdA::luxAB transcription fusion resulted in the loss of regulation by cAMP and CRP. Thus, this study demonstrates that CpdA plays a crucial role in determining the intracellular cAMP level and shows for the first time that the expression of cpdA is activated by the cAMP-CRP complex via direct binding to the regulatory region.

FrsA functions as a cofactor-independent decarboxylase to control metabolic flux

The interaction between fermentation-respiration switch (FrsA) protein and glucose-specific enzyme IIA(Glc) increases glucose fermentation under oxygen-limited conditions. We show that FrsA converts pyruvate to acetaldehyde and carbon dioxide in a cofactor-independent manner and that its pyruvate decarboxylation activity is enhanced by the dephosphorylated form of IIA(Glc) (d-IIA(Glc)). Crystal structures of FrsA and its complex with d-IIA(Glc) revealed residues required for catalysis as well as the structural basis for the activation by d-IIA(Glc).