Protection Efficacy of Oral Bait Probiotic Vaccine Constitutively Expressing Tetravalent Toxoids against Clostridium perfringens Exotoxins in Livestock (Rabbits)

Clostridium perfringens is an opportunistic pathogen. Its main virulence factors are exotoxins, which are the etiological agents of enteritis necroticans and enterotoxemia caused in livestock (cattle, sheep, and rabbits). Here, we demonstrated effective immune protection for rabbits against α, β, and ε exotoxins of C. perfringens provided by an oral tetravalent bait probiotic vaccine delivering α, ε, β1, and β2 toxoids of C. perfringens. Results showed that the recombinant probiotic had good segregational stability and good colonization ability in the rabbit intestinal tract. Oral administration of the probiotic vaccine can effectively elicit significant levels of antigen-specific mucosa sIgA and sera IgG antibodies with exotoxin-neutralizing activity. Additionally, oral immunization with the probiotic vaccine effectively promoted lymphoproliferation and Th1/Th2-associated cytokine production. The protection rate of immunized rabbits with the probiotic vaccine was 80% after challenging rabbits with a combination of C. perfringens (toxinotypes A, C, and D) and exotoxin mixture, which was better than the 60% provided by a commercial inactivated C. perfringens A, C, and D trivalent vaccine. Moreover, obvious histopathological changes were observed in the intestinal tissues of rabbits in the commercial vaccine and PBS groups. The bait probiotic vaccine can provide effective protection against C. perfringens exotoxins, suggesting a promising C. perfringens vaccination strategy.

Effects of Three Different Nucleoid-Associated Proteins Encoded on IncP-7 Plasmid pCAR1 on Host Pseudomonas putida KT2440

ABSTRACTNucleoid-associated proteins (NAPs), which fold bacterial DNA and influence gene transcription, are considered to be global transcriptional regulators of genes on both plasmids and the host chromosome. Incompatibility P-7 group plasmid pCAR1 carries genes encoding three NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. In this study, the effects of single or double disruption ofpmr,pnd, andphuwere assessed in hostPseudomonas putidaKT2440. Whenpmrandpndorpmrandphuwere simultaneously disrupted, both the segregational stability and the structural stability of pCAR1 were markedly decreased, suggesting that Pmr, Pnd, and Phu act as plasmid-stabilizing factors in addition to their established roles in replication and partition systems. The transfer frequency of pCAR1 was significantly decreased in these double mutants. The segregational and structural instability of pCAR1 in the double mutants was recovered by complementation ofpmr, whereas no recovery of transfer deficiency was observed. Comprehensive phenotype comparisons showed that the host metabolism of carbon compounds, which was reduced by pCAR1 carriage, was restored by disruption of the NAP gene(s). Transcriptome analyses of mutants indicated that transcription of genes for energy production, conversion, inorganic ion transport, and metabolism were commonly affected; however, how their products altered the phenotypes of mutants was not clear. The findings of this study indicated that Pmr, Pnd, and Phu act synergistically to affect pCAR1 replication, maintenance, and transfer, as well as to alter the host metabolic phenotype.

Functional Analysis of the Stability Determinant AlfB of pBET131, a Miniplasmid Derivative of Bacillus subtilis (natto) Plasmid pLS32

ABSTRACT Bacillus subtilis plasmid pBET131 is a derivative of pLS32, which was isolated from a natto strain of Bacillus subtilis. The DNA region in pBET131 that confers segregational stability contains an operon consisting of three genes, of which alfA, encoding an actin-like ATPase, and alfB are essential for plasmid stability. In this work, the alfB gene product and its target DNA region were studied in detail. Transcription of the alf operon initiated from a σA-type promoter was repressed by the alfB gene product. Overproduction of AlfA was inhibitory to cell growth, suggesting that the repression of the alf operon by AlfB is important for maintaining appropriate levels of AlfA. An electrophoretic mobility shift assay and footprinting analysis with purified His-tagged AlfB showed that it bound to a DNA region containing three tandem repeats of 8-bp AT-rich sequence (here designated parN), which partially overlaps the −35 sequence of the promoter. A sequence alteration in the first or third repeat did not affect the AlfB binding and plasmid stability, whereas that in the second repeat resulted in inhibition of these phenomena. The repression of alfA-lacZ expression was observed in the constructs carrying a mutation in either the first or third repeat, but not in the second repeat, indicating a correlation between plasmid stability, AlfB binding, and repression. It was also demonstrated by the yeast two-hybrid system that AlfA and AlfB interact with each other and among themselves. From these results, it was concluded that AlfB participates in partitioning pBET131 by forming a complex with AlfA and parN, the mode of which is typified by the type II partition mechanism.

Improved Cloning Vectors for Bifidobacteria, Based on the Bifidobacterium catenulatum pBC1 Replicon

ABSTRACT This study reports the development of several cloning vectors for bifidobacteria based on the replicon of pBC1, a cryptic plasmid from Bifidobacterium catenulatum L48 thought to replicate via the theta mode. These vectors, in which antibiotic resistance genes encoding either erythromycin or tetracycline resistance acted as selection markers, were able to replicate in a series of eight Bifidobacterium species at frequencies ranging from 4.0 × 101 to 1.0 × 105 transformants μg−1 but not in Lactococcus lactis or Lactobacillus casei. They showed a relative copy number of around 30 molecules per chromosome equivalent and a good segregational stability, with more than 95% of the cells retaining the vectors after 80 to 100 generations in the absence of selection. Vectors contain multiple cloning sites of different lengths, and the lacZα peptide gene was introduced into one of the molecules, thus allowing the easy selection of colonies harboring recombinant plasmids in Escherichia coli. The functionality of the vectors for engineering Bifidobacterium strains was assessed by cloning and examining the expression of an α-l-arabinofuranosidase gene belonging to Bifidobacterium longum. E. coli and Bifidobacterium pseudocatenulatum recombinant clones were stable and showed an increase in α-arabinofuranosidase activity of over 100-fold compared to that of the untransformed hosts.

Localization of the Naturally Occurring Plasmid ColE1 at the Cell Pole

ABSTRACT The naturally occurring plasmid ColE1 was found to localize as a cluster in one or both of the cell poles of Escherichia coli. In addition to the polar localization of ColE1 in most cells, movement of the plasmid to the midcell position was observed in time-lapse studies. ColE1 could be displaced from its polar location by the p15A replicon, pBAD33, but not by plasmid RK2. The displacement of ColE1 by pBAD33 resulted in an almost random positioning of ColE1 foci in the cell and also in a loss of segregational stability, as evidenced by the large number of cells carrying pBAD33 with no visible ColE1 focus and as confirmed by ColE1 stability studies. The addition of the active partitioning systems of the F plasmid (sopABC) or RK2 (OB1 incC korB) resulted in movement of the ColE1 replicon from the cell pole to within the nucleoid region. This repositioning did not result in destabilization but did result in an increase in the number of plasmid foci, most likely due to partial declustering. These results are consistent with the importance of par regions to the localization of plasmids to specific regions of the cell and demonstrate both localization and dynamic movement for a naturally occurring plasmid that does not encode a replication initiation protein or a partitioning system that is required for plasmid stability.