Characterization of development-related genes for the cestode Bothriocephalus acheilognathi

Differential gene expression of mature and immature Bothriocephalus acheilognathi cestodes was analyzed using the suppression subtractive hybridization technique. Five mature-associated cDNAs were isolated and characterized. Virtual Northern blot and RT-PCR analyses confirmed that four of the five genes were up-regulated in mature parasites. The sequence analysis revealed that one gene encoded the structural protein chorion precursor, and that three encoded functional proteins homologous to yolk ferritin, sodium/hydrogen exchanger and muscin-like protein. Another gene appeared to be specific to B. acheilognathi, encoding a putative metal-bound protein. Although results obtained in the present study are preliminary, the information about the five genes may provide clues for further investigation on the decline in parasite numbers during the maturation of B. acheilognathi.

Cloning and expression analysis in mature individuals of two chicken type-II GnRH (cGnRH-II) genes in common carp (Cyprinus carpio)

Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide family, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differing cGnRH-II cDNAs of common carp, namely cGnRH-II cDNA1 and cDNA2, were firstly cloned from the brain by rapid amplification of cDNA end (RACE) and reverse transcription- polymerase chain reaction (RT-PCR). The length of cGnRH-II cDNA1 and cDNA2 was 622 and 578 base pairs (bp), respectively. The cGnRH-II precursors encoded by two cDNAs consisted of 86 amino acids, including a signal peptide, cGnRH-II decapeptide and a GnRH-associated peptide (GAP) linked by a Gly-Lys-Arg proteolytic site. The results of intron trapping and Southern blot showed that two differing cGnRH-II genes in common carp genome were further identified, and that two genes might exist as a single copy. The multi-gene coding of common carp cGnRH-II gene offered novel evidence for gene duplication hypothesis. Using semi-quantitative RT-PCR, expression and relative expression levels of cGnRH-II genes were detected in five dissected brain regions, pituitary and gonad of common carp. With the exception of no mRNA2 in ovary, two cGnRH-II genes could be expressed in all the detected tissues. However, expression levels showed an apparent difference in different brain regions, pituitary and gonad. According to the expression characterization of cGnRH-II genes in brain areas, it was presumed that cGnRH-II might mainly work as the neurotransmitter and neuromodulator and also operate in the regulation for the GnRH releasing. Then, the expression of cGnRH-II genes in pituitary and gonad suggested that cGnRH-II might act as the autocrine or paracrine regulator.

Profiling of Complex Microbial Populations by Denaturing Gradient Gel Electrophoresis Analysis of Polymerase Chain Reaction-Amplified Genes Coding for 16S rRNA

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain

A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50 degrees C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.

Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain

Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (Al-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the Al-2 synthase was cloned from TX1 and designated luxS(Et). LuxS(Et) was able to complement the Al-2 mutant phenotype of Escherichia coli strain DH5 alpha. Expression Of luxS(Et) correlated with Al-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS(Et) expression. Overexpression of luxS(Et) enhanced Al-2 activity in TX1, whereas disruption of luxS(Et) expression by antisense RNA interference (i) reduced the level of Al-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous Al-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the Al-2 activity in TX1 is controlled at least in part at the level of luxS(Et) expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS(Et) is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/Al-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.

Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain

Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (Al-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the Al-2 synthase was cloned from TX1 and designated luxS(Et). LuxS(Et) was able to complement the Al-2 mutant phenotype of Escherichia coli strain DH5 alpha. Expression Of luxS(Et) correlated with Al-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS(Et) expression. Overexpression of luxS(Et) enhanced Al-2 activity in TX1, whereas disruption of luxS(Et) expression by antisense RNA interference (i) reduced the level of Al-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous Al-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the Al-2 activity in TX1 is controlled at least in part at the level of luxS(Et) expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS(Et) is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/Al-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.