Methanococcus voltae is a flagellated member of the Domain Archaea that has four flagellin genes arranged in two transcriptional units. One transcriptional unit encodes only flaA while the second is a multi-cistronic unit encoding three flagellin genes (flaB1, flaB2, and flaB3) as well as at least seven other open reading frames downstream. The polymerase chain reaction was used to amplify an internal fragment of the flaA gene which was subsequently cloned into an insertion vector developed for M. voltae. Transformation of protoplasts with this vector led to the isolation of mutant strains that had insertions in flaA or flaB2. Mutant strains carrying insertions in flaA had flagelia that were similar to wild-type cells in both number and appearance when viewed using the electron microscope. In addition, some of these mutant strains had profiles identical to the wild type in immunoblots developed with antisera raised against the 31 kDa flagellin of M. voltae. All flaA mutant strains and the wild-type cells showed immuno-cross-reactive bands at 33 and 31 kDa (corresponding to purified flagellins) as well as at 18 kDa. Some flaA mutant strains also showed an immuno-cross-reactive band at 27 kDa which probably represents a truncated flagellin produced by the insertion vector. However, both types of flaA mutant strains were less motile than the wild type in semi-swarm plate experiments. The mutant strain with an insertion in flaB2 was non-flagellated when examined by electron microscopy and it was non-motile in semi-swarm plate experiments. It represents the first structural mutant strain isolated in a methanogen. This mutant strain lacked the 33, 31, and 18 kDa immuno-cross-reactive bands observed in the wild type and flaA mutant strains, and instead had a novel band at 20 kDa. This band may represent an unmodified flagellin which still has an attached leader peptide. If so, then one of the downstream genes in the multi-cistronic transcriptional unit may encode a leader peptidase for the flagellin system.
The highly conserved nature of the 5'-termini of all archaeal flagellin genes was exploited by polymerase chain reaction (PCR) techniques to amplify the sequence of a portion of a flagellin gene family from the archaeon Methanococcus vannielii. Subsequent inverse PCR experiments generated fragments that permitted the sequencing of a total of three flagellin genes, which, by comparison with flagellin genes that have been sequenced, from other archaea appear to be equivalent to flaB1, flaB2, and flaB3 of M. voltae. Analysis of purified M. vannielii flagellar filaments by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed two major flagellins (Mr = 30,800 and 28,600), whose N-terminal sequences identified them as the products of the flaB1 and flaB2 genes, respectively. The gene product of flaB3 could not be detected in flagellar filaments by SDS-PAGE. The protein sequence data, coupled with the DNA sequences, demonstrated that both FlaB1 and FlaB2 flagellins are translated with a 12-amino acid signal peptide which is absent from the mature protein incorporated into the flagellar filament. These data suggest that archaeal flagellin export differs significantly from that of bacterial flagellins.
A vaccine protecting against different Streptococcus suis serotypes is highly needed in porcine practice to improve animal welfare and reduce the use of antibiotics. We hypothesized that immunogens prominently recognized by convalescence sera but significantly less so by sera of susceptible piglets are putative protective antigens. Accordingly, we investigated immunogenicity and protective efficacy of a multicomponent vaccine including six main conserved immunogens, namely SSU0934, SSU1869, SSU0757, SSU1950, SSU1664 and SSU0187. Flow cytometry confirmed surface expression of all six immunogens in S. suis serotypes 2, 9 and 14. Although prime-booster vaccination after weaning resulted in significantly higher specific IgG levels against all six immunogens compared to the placebo-treated group, no significant differences between bacterial survival in blood from either vaccinated or control animals were recorded for serotype 2, 9 and 14 strains. Furthermore, vaccinated piglets were not protected against morbidity elicited through intranasal challenge with S. suis serotype 14. As ~50% of animals in both groups did not develop disease, we investigated putative other correlates of protection. Induction of reactive oxygen species (ROS) in blood granulocytes was not associated with vaccination but correlated with protection as all piglets with >5% ROS survived the challenge. Based on these findings we discuss that the main immunogens of S. suis might actually not be a priori good candidates for protective antigens. On the contrary, expression of immunogens that evoke antibodies that do not mediate killing of this pathogen might constitute an evolutionary advantage conserved in many different S. suis strains.
The gene sequences of the second largest subunits of RNA polymerases I and II of Euplotes octocarinatus, RPA2 and RPB2, were determined and compared to the respective known sequences of Saccharomyces cerevisiae. The similarity of the derived polypeptide sequences permitted their assignment to the respective polymerases and allowed the comparison of the zinc binding regions. In frame TGA codons were detected, which are likely to encode conserved cysteinyl residues in the putative zinc-finger region of the RPA2 gene. They were also found in other positions in both the RPA2 and RPB2 genes. The RPB2 gene contains a 30 bp intron close to the 5'-end of its coding region. The 5'-ends of the coding regions of all three genes encoding the largest subunits of the three different polymerases were also analyzed. The zinc finger structures again show the use of TGA codons for conserved cysteinyl residues in two of the genes. An N-terminal intron is located in the RPB1 gene at a conserved position as compared to the respective genes of several other eucarya.
Streptococcus suis is an important pathogen of pigs that, as a zoonotic agent, can also cause severe disease in humans, including meningitis, endocarditis, and septicemia. We report complete and annotated genomes of S. suis strains 10, 13-00283-02, and 16085/3b, which represent the highly prevalent serotypes cps2, cps7, and cps9, respectively.
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