Characterization of the zinc-containing metalloprotease encoded by zpx and development of a species-specific detection method for Enterobacter sakazakii.

Enterobacter sakazakii causes a severe form of neonatal meningitis that occurs as sporadic cases as well as outbreaks. The disease has been epidemiologically associated with consumption of reconstituted, dried infant formulas. Very little information is available regarding pathogenicity of the organism and production of virulence factors. Clinical and environmental strains were screened for production of factors which have activity against Chinese hamster ovary (CHO) cells in tissue culture. Polymyxin B lysate and sonicate preparations but not culture supernatants from the strains caused "rounding" of CHO cells. Subsequent studies showed that the CHO cell-rounding factor is a proteolytic enzyme that has activity against azocasein. The cell-bound protease was isolated by using a combination of polymyxin B lysis, followed by sonication of cells harvested from tryptone broth. The protease was purified to homogeneity by sequential ammonium sulfate precipitation, gel filtration chromatography with Sephadex G-100, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, and a second gel filtration with Sephadex G-100. In addition to activity against azocasein, the purified protease also exhibits activity against azocoll and insoluble casein but not elastin. The protease has a molecular weight of 38,000 and an isoelectric point of 4.4. It is heat labile and for maximal activity against azocasein has an optimum temperature of 37 degrees C and a pH range of 5 to 7. Proteolytic activity is inhibited by ortho-phenanthroline and Zincov but is not affected by phenylmethylsulfonyl fluoride, N-ethylmaleimide, and trypsin inhibitors, which demonstrates that the protease is a zinc-containing metalloprotease. The metalloprotease does not hemagglutinate chicken or sheep erythrocytes. Twenty-three to 27 of the first 42 N-terminal amino acid residues of the metalloprotease are identical to proteases produced by Serratia proteamaculans, Pectobacterium carotovorum, and Anabaena sp. PCR analysis using primers designed from a consensus nucleotide sequence showed that 135 E. sakazakii strains possessed the metalloprotease gene, zpx, and 25 non-E. sakazakii strains did not. The cloned zpx gene of strain 29544 consists of 1,026 nucleotides, and the deduced amino acid sequence of the metalloprotease has 341 amino acid residues, which corresponds to a theoretical protein size of 37,782 with a theoretical pI of 5.23. The sequence possesses three well-characterized zinc-binding and active-site motifs present in other bacterial zinc metalloproteases.

Structure and seasonal dynamics of hyporheic zone microbial communities in free-stone rivers of the western United States.

The hyporheic zone of a river is characterized by being nonphotic, exhibiting chemical/redox gradients, and having a heterotrophic food web based on the consumption of organic carbon entrained from surface waters. Hyporheic microbial communities constitute the base of food webs in these environments and are important for maintaining a functioning lotic ecosystem. While microbial communities of rivers dominated by fine-grained sediments are relatively well studied, little is known about the structure and seasonal dynamics of microbial communities inhabiting the predominantly gravel and cobble hyporheic zones of rivers of the western United States. Here, we present the first molecular analysis of hyporheic microbial communities of three different stream types (based on mean base discharge, substratum type, and drainage area), in Montana. Utilizing 16S rDNA phylogeny, DGGE pattern analysis, and qPCR, we have analyzed the prokaryotic communities living on the 1.7 to 2.36 mm grain-size fraction of hyporheic sediments from three separate riffles in each stream. DGGE analysis showed clear seasonal community patterns, indicated similar community composition between different riffles within a stream (95.6-96.6% similarity), and allowed differentiation between communities in different streams. Each river supported a unique complement of species; however, several phylogenetic groups were conserved between all three streams including Pseudomonads and members of the genera Aquabacterium, Rhodoferax, Hyphomicrobium, and Pirellula. Each group showed pronounced seasonal trends in abundance, with peaks during the Fall. The Hyphomicrobium group was numerically dominant throughout the year in all three streams. This work provides a framework for investigating the effects of various environmental factors and anthropogenic effects on microbial communities inhabiting the hyporheic zone.