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The success of the pathogenic bacteria is partly attributable to their ability to thwart host innate immune responses, which includes resisting the antimicrobial functions of macrophages. And reactive oxygen species (ROS) is one of the most effective antimicrobial components of macrophages to kill invading bacteria. Our previous studies found that Aeromonas hydrophila can survive in fish macrophages, which suggested that this bacterium might take fish macrophages as their shelters to resist drug killings and other immune damage. But how A. hydrophila survive in host macrophages remains unknown. Since KatG has been reported to have not only catalase activity but also peroxidase and peroxynitritase activity, the amino acid sequence and protein structure of KatG was analyzed in this study, the function of KatG in A. hydrophila survival in and escape from host macrophages was also carried out. The bioinformatics analysis displayed that KatG of A. hydrophila B11 showed >93% homologous to that of KatG in other Aeromonas. KatG of A. hydrophila was stable silenced by shRNA and RT-qPCR confirmed the expression of KatG in KatG-RNAi was significantly reduced. The survival rate of intracellular KatG-RNAi decreased by 80% compared to that of the wild type strain B11, while the intracellular ROS level of the macrophages that phagocytosed KatG-RNAi increased 65.9% when compared to that of the macrophages phagocytosed wild-type strain. The immune escape rate of A. hydrophila decreased by 85% when the expression of KatG was inhibited. These results indicated that (1) The amino acid sequence and protein structure of KatG of A. hydrophila is conserved; (2) KatG helped A. hydrophila to survive in fish macrophages by eliminating the harm of intracellular H 2 O 2 and inhibiting intracellular ROS levels increased; (3) A small portion of intracellular A. hydrophila could escape from host macrophages for further infection, in this process KatG also played important role., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A gene (eprA1) encoding the extracellular protease of Aeromonas hydrophila AH1 has been cloned and sequenced. Nucleotide sequence analysis of eprA1 predicted a single open reading frame (ORF) of 1038 bp encoding a 346 amino acid (aa) polypeptide, with a potential 21-aa signal peptide. When the eprA1 gene was expressed in minicells, one major band of approx. 37 kDa was identified, while protease activity staining experiments identified a caseinolytic band of approx. 29 kDa determined by SDS-PAGE analysis of the minicells. The deduced C-terminal aa region (Arg-290 to Gly-313) showed sequence homology to partial C-terminal sequences of other zinc metalloproteases including Penicillium citrinum metalloprotease (PlnC), Aspergillus oryzae metalloprotease (NpII), Aspergillus flavus metalloprotease (MepA), and Aspergillus fumigatus metalloprotease (Mep20), particularly with respect to zinc-binding residues.

Fifty isolates of chitinase (Cts)-producing bacteria were collected from soil samples and tested for their ability to degrade chitin using colloidal chitin agar as the primary plating medium. The results indicated that three isolates could degrade chitin at high pH. Further studies also demonstrated that crude Cts preparations from Bacillus circulans (Bc) No. 4.1 could enhance the toxicity of Bacillus thuringiensis subsp. kurstaki (Bt-k) toward diamondback moth larvae. Thus, it might be useful to increase the toxicity of B. thuringiensis (Bt) toward target insects by introducing a Cts-encoding gene (cts) into Bt. To investigate the expression of cts in Bt, cloned cts from Aeromonas hydrophila (pHYA1) and Pseudomonas maltophilia (pHYB1, pHYB2 and pHYB3) were cloned into the shuttle vector pHY300PLK and transformed into Escherichia coli DH5 alpha using 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside (4-MUF GlcNAc) as the detecting substrate. The four plasmids were then introduced into B. thuringiensis subsp. israelensis (Bt-i) strain c4Q272 by electroporation. Various transformants harboring cloned cts were selected, and expression and stability of the plasmids in Bt were studied.

The gene endA, encoding the periplasmic endonuclease I (EndoI) of Escherichia coli, was identified on a cloned chromosomal 1.5-kb HindIII fragment. The nucleotide sequence of the fragment revealed an open reading frame (ORF) coding for a polypeptide of 235 amino acids (aa). The ORF preceeded by a region with two possible promoter sites displays promoter activity when cloned into an expression vector. On the C-terminal side, two sequences with putative transcription termination function are present. The predicted aa sequence suggests the presence of a signal peptide of 22 aa and a signal peptide cleavage site. A cold-shock supernatant from cells harbouring a multicopy endA+ plasmid contained an approx. tenfold higher amount than wild-type cells of the DNA double-strand- and single-strand-cleavage activities characteristic of EndoI. The growth rate and viability of the cells was not affected. The predicted aa sequence of the ORF is 60 and 54% identical to the sequence of extracellular DNases from Vibrio cholerae and Aeromonas hydrophila, respectively.

The gene encoding an extracellular DNase from Aeromonas hydrophila CHC-1 has been cloned and sequenced. Following expression of the dns in Escherichia coli, it was revealed that some of the cloned enzyme was present in the cell-free extracellular supernatant fluid, and there was no cell lysis and concurrent release of cytoplasmic or periplasmic proteins. Therefore, results suggest that E. coli cells were capable of secreting the DNase extracellularly, albeit very inefficiently. The dns is transcribed from its own promoter in E. coli, and expressed as a 25-kDa product, as determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of the culture supernatant preparations followed by a DNA-hydrolysis assay. Nucleotide sequence analysis predicted a single open reading frame of 690 bp encoding a 230-amino acid (aa) polypeptide, with a potential 20-aa signal peptide located at the N terminus of the predicted protein. The deduced aa sequence of the entire protein is highly homologous with that of the DNase of Vibrio cholerae.