ABSTRACT
It has been shown previously, based largely on DNA-DNA hybridizations and partial 16S rRNA gene sequencing, that Hafnia alvei is genotypically heterogeneous and consists of at least two DNA hybridization groups (HGs). In the present study, the taxonomic status of H. alvei HGs 1 and 2 was reassessed. A panel of 24 reference strains and isolates previously assigned to one of the two HGs in H. alvei was subjected to (GTG)5-PCR fingerprinting; this resulted in the delineation of two (GTG)5-PCR clusters in perfect accordance with the respective HG designations. Based on full 16S rRNA gene sequencing of a selection of reference strains, H. alvei HGs 1 and 2 showed internal sequence similarities of 99.8 and 99.5%, respectively. Between the two groups, sequence similarities ranged from 98.8 to 99.1%. Mean DNA-DNA hybridization values of 74.7-99.9% were obtained within each of the two HGs, whereas cross-hybridizations between members of H. alvei HG 1 (including ATCC 13337T) and HG 2 revealed only 32.7-48.7 % DNA-DNA hybridization. Previously published and new phenotypic data revealed that a combination of malonate assimilation and beta-glucosidase activity enabled correct assignment of Hafnia isolates to one of the two HGs. Collectively, taxonomic data from this study confirm that H. alvei comprises at least two taxa at the species level, of which HG 1 corresponds to H. alvei sensu stricto because it includes the type strain ATCC 13337T. Strains formerly classified as members of H. alvei HG 2 represent a novel species, for which the name Hafnia paralvei sp. nov. is proposed; ATCC 29927T (=CDC 4510-73T =LMG 24706T), the former reference strain of H. alvei HG 2, is designated the type strain.
Subject(s)
Hafnia/classification , Hafnia/isolation & purification , Bacterial Proteins/metabolism , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , Enterobacteriaceae Infections/microbiology , Feces/microbiology , Hafnia/enzymology , Hafnia/genetics , Humans , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/genetics , beta-Glucosidase/metabolismABSTRACT
A gene appA encoding a novel phytase was firstly cloned from Hafnia alvei by PCR and sequenced. The gene was consisted of 1335 bp, encoding 444 amino acids. The calculated molecular weight of the mature APPA was about 45.2 kD. The gene appA was expressed in E. coli BL21 (DE3). Recombinant APPA was purified and its enzymatic properties were determined. The optimum pH for the enzyme was 4.5 and the optimum temperature was 60 degrees C. The pH stability of r-APPA is good, the relative phytase activity was above 80% after treated in buffers of pH 2.0-10.0. The specific activity of r-APPA is 356.7 U/mg, and the Km value was 0.49 mmol/L and Vmax of 238 U/mg. The enzyme showed resistance to pepsin and trypsin treatment.
