Characterization of extracellular RNases of host dependent Bdellovibrio bacteriovorus 109 D Public Deposited

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  • The growth of bdellovibrios at the expense of the host organism involves the catabolism of host cell macromolecular components and the subsequent anabolistic incorporation into bdellovibrio progeny. Extracellular enzymes specific for the various substrates provided by the host cell are present. In this study the extracellular RNase of Bdellovibrio bacteriovorus 109 D has been partially characterized. After several initial experiments, it was found that two extracellular RNA degrading enzymes are produced by this species. Initial studies involved the growth of H-D Bdellovibrio bacteriovorus 109D in cell-free host extract. An assay was developed to follow the digestion of radioactively labeled substrate nucleic acids by the RNase, utilizing a trichloroacetic acid precipitation of the substrate on membrane filters monitored by a scintillation counting device. A growth cycle of six hours was established, and the production and accumulation of RNase in the spent culture medium was found to be maximum at between four and five hours. The enzyme was stable in storage at -10° C. for up to one month. The optimum temperature for the activity of the mixture of the enzymes was between 30° C. and 37° C. The pH optimum for the crude enzyme mixture demonstrated a peak at pH 9. 5, with a shallow rise and a shoulder at pH 7. 5. The temperature profile and the pH optimum study suggested a two enzyme system. The crude RNase was shown to have activity with Spirillum serpens VHL RNA, as well as the ability to degrade host cell ribosomes in vitro. The crude enzyme did not degrade DNA, either double stranded or denatured. The reaction of the crude enzyme with the substrate RNA was shown to exhibit first order kinetics. The Km of the mixture of enzymes was investigated, and utilizing a Lineweaver-Burk plot, the Km was found to be 6. 74 μg /ml. , while the Vmax was established at 1,327 cpm/hr/μg. Separation of two enzymes was accomplished using column chromatography, with Sephadex G-50 dextran. The Bd. RNase I, so named because it was eluted first from the column, was determined to have a molecular weight of 18, 000. The second enzyme to be eluted from the column, called Bd. RNase II, has a molecular weight of 10, 000. Temperature has a markedly different effect on the two enzymes. Bd. RNase I has a temperature optimum of 37° C. , and is inactivated by heat at between 75° -80° C. Bd. RNase II was shown to have a temperature optimum of 31 ° C. and to be heat inactivated between 45° C. and 60° C. The separated R.Nase enzymes were found to have different pH profiles. Bd. R.Nase I has a sharp optimum pH at 9. 5, while Bd. R.Nase II exhibits a broad range with slight peaks at pH 7. 5 and pH 9. 5. A two dimensional high voltage paper electrophoretic "fingerprint" of the end products from an RNA digest by Bd. RNase I and Bd. R.Nase II revealed large oligonucleotide fragments. A further one dimensional electrophoretogram of a secondary digest of the Bd. RNase I produced RNA fragments, by various nucleases, gave evidence that the enzyme is most likely an exonuclease, cleaving from the 5' end of the ribonucleotide chain leaving a 5' phosphate group and a 3' hydroxyl group.
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