Revisiting host identification of bacteriophage Enc34: from biochemical to molecular.

In our 2012 genome announcement (J Virol 86:11403-11404, 2012, https://doi.org/10.1128/JVI.01954-12), we initially identified the host bacterium of bacteriophage Enc34 as Enterobacter cancerogenus using biochemical tests. However, later in-house DNA sequencing revealed that the true host is a strain of Hafnia alvei. Capitalizing on our new DNA-sequencing capabilities, we also refined the genomic termini of Enc34, confirming a 60,496-bp genome with 12-nucleotide 5' cohesive ends. IMPORTANCE: Our correction reflects the evolving landscape of bacterial identification, where molecular methods have supplanted traditional biochemical tests. This case underscores the significance of revisiting past identifications, as seemingly known bacterial strains may yield unexpected discoveries, necessitating essential updates to the scientific record. Despite the host identity correction, our genome announcement retains importance as the first complete genome sequence of a Hafnia alvei bacteriophage.

Degradation of phytate by the 6-phytase from Hafnia alvei: a combined structural and solution study.

Phytases hydrolyse phytate (myo-inositol hexakisphosphate), the principal form of phosphate stored in plant seeds to produce phosphate and lower phosphorylated myo-inositols. They are used extensively in the feed industry, and have been characterised biochemically and structurally with a number of structures in the PDB. They are divided into four distinct families: histidine acid phosphatases (HAP), ß-propeller phytases, cysteine phosphatases and purple acid phosphatases and also split into three enzyme classes, the 3-, 5- and 6-phytases, depending on the position of the first phosphate in the inositol ring to be removed. We report identification, cloning, purification and 3D structures of 6-phytases from two bacteria, Hafnia alvei and Yersinia kristensenii, together with their pH optima, thermal stability, and degradation profiles for phytate. An important result is the structure of the H. alvei enzyme in complex with the substrate analogue myo-inositol hexakissulphate. In contrast to the only previous structure of a ligand-bound 6-phytase, where the 3-phosphate was unexpectedly in the catalytic site, in the H. alvei complex the expected scissile 6-phosphate (sulphate in the inhibitor) is placed in the catalytic site.

Identification of a non-pathogenic surrogate organism for chlorine dioxide (ClO2) gas treatment.

The identification of non-pathogenic surrogate microorganisms is beneficial for determining and validating the efficacy of antimicrobial treatments in food manufacturing environments. A surrogate organism was identified to aid in the decontamination process of fresh produce when treated with chlorine dioxide (ClO(2)) gas. Thirty-two known strains of pathogenic and non-pathogenic microorganisms and seven unknown microbial isolates from mushroom, tomatoes, and strawberries were evaluated. The primary goal was to find alternative non-pathogenic organisms that had an equal or higher resistance compared to Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes. Among the strains tested, MR1 (mushroom isolate), E. coli O157:H7 C7927, E. coli O157:H7 204P, STB2 (strawberry isolate), and vegetative cells of Bacillus cereus 232 in wet inoculum were found to be the most resistant to gaseous ClO(2) treatment at 0.3 mg/l for 1 min and D-values at 0.3 mg/l ClO(2) were 3.53, 1.95, 1.72, 1.68, and 1.57 min, respectively. For identification, the MR1 and STB2 strains were identified using a Ribotyper with the EcoRI restriction enzyme of 16S rDNA sequence. MR1 was identified as Hafnia alvei with a similarity value of 94% using the ribotype pattern and with a 93.6% similarity using an API 20E strip, and with a 99% similarity using 16S rDNA analysis. The Ped-2E9-based cytotoxicity assay was conducted for the MRI strain extracellular toxin and whole cell toxicity and did not show cytotoxicity. Analysis, using multiplex PCR, was performed to verify absence of the eaeA gene. H. alvei is a suitable non-pathogenic surrogate, with higher resistance to ClO(2) gas compared to pathogens studied, that may be useful to establish optimum conditions of ClO(2) gas decontamination systems.

Conjugative plasmid mediated inducible nickel resistance in Hafnia alvei 5-5.

Hafnia alvei 5-5, isolated from a soil-litter mixture underneath the canopy of the nickel-hyperaccumulating tree Sebertia acuminata (Sapotaceae) in New Caledonia, was found to be resistant to 30 mM Ni(2+) or 2 mM Co(2+). The 70-kb plasmid, pEJH 501, was transferred by conjugation to Escherichia coli, Serratia marcescens, and Klebsiella oxytoca. Transconjugant strains expressed inducible nickel resistance to between 5 and 17 mM Ni(2+), and cobalt resistance to 2 mM Co(2+). A 4.8-kb Sal- EcoRI fragment containing the nickel resistance determinant was subcloned, and the hybrid plasmid was found to confer a moderate level of resistance to nickel (7 mM Ni(2+)) even to E. coli. The expression of nickel resistance was inducible by exposure to nickel chloride at a concentration as low as 0.5 mM Ni(2+). By random Tn phoA'-1 insertion mutagenesis, the fragment was shown to have structural genes as well as regulatory regions for nickel resistance. Southern hybridization studies showed that the nickel-resistance determinant from pEJH501 of H. alvei 5-5 was homologous to that of pTOM9 from Alcaligenes xylosoxydans 31A.