Identification of the Genes Involved in Riemerella anatipestifer Biofilm Formation by Random Transposon Mutagenesis

Riemerella anatipestifer causes epizootics of infectious disease in poultry that result in serious economic losses to the duck industry. Our previous studies have shown that some strains of R. anatipestifer can form a biofilm, and this may explain the intriguing persistence of R. anatipestifer on duck farms post infection. In this study we used strain CH3, a strong producer of biofilm, to construct a library of random Tn4351 transposon mutants in order to investigate the genetic basis of biofilm formation by R. anatipestifer on abiotic surfaces. A total of 2,520 mutants were obtained and 39 of them showed a reduction in biofilm formation of 47%–98% using crystal violet staining. Genetic characterization of the mutants led to the identification of 33 genes. Of these, 29 genes are associated with information storage and processing, as well as basic cellular processes and metabolism; the function of the other four genes is currently unknown. In addition, a mutant strain BF19, in which biofilm formation was reduced by 98% following insertion of the Tn4351 transposon at the dihydrodipicolinate synthase (dhdps) gene, was complemented with a shuttle plasmid pCP-dhdps. The complemented mutant strain was restored to give 92.6% of the biofilm formation of the wild-type strain CH3, which indicates that the dhdp gene is associated with biofilm formation. It is inferred that such complementation applies also to other mutant strains. Furthermore, some biological characteristics of biofilm-defective mutants were investigated, indicating that the genes deleted in the mutant strains function in the biofilm formation of R. anatipestifer. Deletion of either gene will stall the biofilm formation at a specific stage thus preventing further biofilm development. In addition, the tested biofilm-defective mutants had different adherence capacity to Vero cells. This study will help us to understand the molecular mechanisms of biofilm development by R. anatipestifer and to study the pathogenesis of R. anatipestifer further

TiO₂‑Based Phosphoproteomic Analysis of Schistosomes: Characterization of Phosphorylated Proteins in the Different Stages and Sex of <i>Schistosoma japonicum</i>

Protein phosphorylation is an important posttranslational modification in many organisms that regulates numerous cellular processes. However, it remains poorly characterized in schistosomes, the causative agent of schistosomiasis in humans and related animals. In the present study, we characterized phosphorylated proteins in different stages and sex of <i>Schistosoma japonicum</i> (<i>S. japonicum</i>) including schistosomula (14 days), adult females (35 days), and adult males (35 days) by a titanium dioxide (TiO₂) based phosphoproteomic method. A total of 180 phosphopeptides were identified in 148 proteins. Our further studies revealed that heat shock protein 90 (Hsp90), one of the phosphoproteins codetected in the different stage and sex of schistosomes, may play an important role in the regulation of schistosome development by directly or indirectly interacting with other codetected signal molecules. Additionally, some phosphoproteins were shown to be detected in a gender-specific manner, and the expressions of these proteins were further validated either by immunohistochemistry or by real-time reverse transcription polymerase chain reaction (RT-PCR) at transcript levels between male and female schistosomes. In summary, these findings as well as the providing of an inventory of phosphoproteins are expected to provide new insights into schistosome development and sexual maturation and then may result in the development of novel interventions against schistosomiasis

TiO₂‑Based Phosphoproteomic Analysis of Schistosomes: Characterization of Phosphorylated Proteins in the Different Stages and Sex of <i>Schistosoma japonicum</i>

Protein phosphorylation is an important posttranslational modification in many organisms that regulates numerous cellular processes. However, it remains poorly characterized in schistosomes, the causative agent of schistosomiasis in humans and related animals. In the present study, we characterized phosphorylated proteins in different stages and sex of <i>Schistosoma japonicum</i> (<i>S. japonicum</i>) including schistosomula (14 days), adult females (35 days), and adult males (35 days) by a titanium dioxide (TiO₂) based phosphoproteomic method. A total of 180 phosphopeptides were identified in 148 proteins. Our further studies revealed that heat shock protein 90 (Hsp90), one of the phosphoproteins codetected in the different stage and sex of schistosomes, may play an important role in the regulation of schistosome development by directly or indirectly interacting with other codetected signal molecules. Additionally, some phosphoproteins were shown to be detected in a gender-specific manner, and the expressions of these proteins were further validated either by immunohistochemistry or by real-time reverse transcription polymerase chain reaction (RT-PCR) at transcript levels between male and female schistosomes. In summary, these findings as well as the providing of an inventory of phosphoproteins are expected to provide new insights into schistosome development and sexual maturation and then may result in the development of novel interventions against schistosomiasis

Adherence assays of selected biofilm-defective mutants to Vero cells.

<p>The data represent the number of bacteria bound to Vero cells in each well of a 24-well plate. The error bars represent means ± standard deviations from three independent experiments.</p

Fluorescence microscopic observations of selected biofilm-defective mutants.

<p>Biofilm images of various biofilm-defective mutants were obtained with fluorescence microscopy after staining with Live/dead BacLight Bacterial Viability staining reagent after 24 h of incubation (400×).</p

Description of biofilm-defective <i>Riemerella anatipestifer</i> mutants.

a<p>Subcellular locations were predicted by the PSORTb v.3.0 server. Available: <a href="http://www.psort.org/psortb/index.html" target="_blank">http://www.psort.org/psortb/index.html</a>. Accessed 10 June 2012.</p>b<p>Functional characterization of the proteins was predicted by the software COGnitor. Available: <a href="http://www.ncbi.nlm.nih.gov/COG/old/xognitor.html" target="_blank">http://www.ncbi.nlm.nih.gov/COG/old/xognitor.html</a>. Accessed 10 June 2012. <b>Functional categories: (1) Information storage and processing:</b> (J: Translation, ribosomal structure and biogenesis; K: Transcription; L: DNA replication, recombination and repair); <b>(2) Cellular processes:</b> (D: Cell division and chromosome partitioning; O: Posttranslational modification, protein turnover, chaperones; M: Cell envelope biogenesis, outer membrane; P: Inorganic ion transport and metabolism; T: Signal transduction mechanisms); <b>(3) Metabolism:</b> (C: Energy production and conversion; E: Amino acid transport and metabolism; F: Nucleotide transport and metabolism); <b>(4) Poorly characterized:</b> (R: General function prediction only; S: Function unknown).</p>c<p>–: No related COG.</p>d<p>Gene not found on the genome <i>R. anatipestifer</i> DSM15868 (accession number: CP002346), but on that of strain CH3 (accession number: JN986833-JN986836).</p

Mutant BF19 restored by the pCP29-dhdps shuttle plasmid recovered the ability to form biofilm.

<p>(A) Biofilm formation by wild-type CH3, and mutants BF19, BF19 (pCP-dhdps) and BF19 (pCP29), was measured using crystal violet staining in a 96-well microtiter plate. (B) The biofilm of complemented BF19 was observed with fluorescence microscopy after staining with Live/dead BacLight Bacterial Viability staining reagent after 24 h of incubation (400×).</p

Strains, plasmids and primers used in this study.

<p>Strains, plasmids and primers used in this study.</p