POWERFUL METHODS TO ESTABLISH CHROMOSOMAL MARKERS IN LACTOCOCCUS-LACTIS - AN ANALYSIS OF PYRIMIDINE SALVAGE PATHWAY MUTANTS OBTAINED BY POSITIVE SELECTIONS
POWERFUL METHODS TO ESTABLISH CHROMOSOMAL MARKERS IN LACTOCOCCUS-LACTIS - AN ANALYSIS OF PYRIMIDINE SALVAGE PATHWAY MUTANTS OBTAINED BY POSITIVE SELECTIONS
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DOI：
10.1099/13500872-141-8-1883
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发表时间：
1995-08-01
期刊：
影响因子：
--
通讯作者：
HAMMER, K
中科院分区：
文献类型：
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作者：
MARTINUSSEN, J;HAMMER, K
利用不同的5-氟嘧啶类似物，建立了乳酸菌嘧啶和嘌呤回收基因阻断突变体的阳性选择程序。由于相应基因突变，分离到的菌株缺乏以下酶活性：尿嘧啶磷酸核糖基转移酶（upp）、尿苷/胞苷激酶（udk）、嘧啶核苷磷酸化酶（pdp）、胞苷/脱氧胞苷脱氨酶（cdd）、胸苷激酶（tdk）和嘌呤核苷磷酸化酶（pup）。基于对获得的突变体的分析。验证了乳杆菌代谢尿嘧啶和不同嘧啶核苷的途径。测定了不同酶的底物特异性。结果表明，单嘌呤核苷磷酸化酶可进行尿苷、脱氧尿苷和胸腺嘧啶的磷酸化裂解，单嘌呤核苷磷酸化酶对腺嘌呤、鸟嘌呤和次黄嘌呤的核糖核苷和脱氧核糖核苷衍生物均有活性。没有针对黄嘌呤的磷酸化酶活性。在这项工作中开发的选择程序可用于在许多相关乳酸菌的染色体上建立标记。
Using different 5-fluoropyrimidine analogues, positive selection procedures for obtaining mutants blocked in pyrimidine and purine salvage genes of Lactococcus lactis were established. Strains lacking the following enzyme activities due to mutations in the corresponding genes were isolated: uracil phosphoribosyltransferase (upp), uridine/cytidine kinase (udk), pyrimidine nucleoside phosphorylase (pdp), cytidine/deoxycytidine deaminase (cdd), thymidine kinase (tdk) and purine nucleoside phosphorylase (pup). Based on an analysis of the mutants obtained. the pathways by which L. lactis metabolizes uracil and the different pyrimidine nucleosides were verified. The substrate specificities of the different enzymes were determined. It was demonstrated that a single pyrimidine nucleoside phosphorylase accounts for the phosphorolytical cleavage of uridine, deoxyuridine and thymidine, and a single purine nucleoside phosphorylase has activity towards both the ribonucleoside and deoxyribonucleoside derivatives of adenine, guanine and hypoxanthine. No phosphorylase activity towards xanthosine appeared to be present. The selection procedures developed during this work may be employed in establishing markers on the chromosome of many related lactic acid bacteria.