Somerville J E, Kahn M L
J Bacteriol. 1983 Oct;156(1):168-76. doi: 10.1128/jb.156.1.168-176.1983.
Glutamine synthetase is a major enzyme in the assimilation of ammonia by members of the genus Rhizobium. Two forms of glutamine synthetase are found in members of the genus Rhizobium, a heat-stable glutamine synthetase I (GSI) and a heat-labile GSII. As a step toward clarifying the role of these enzymes in symbiotic nitrogen fixation, we have cloned the structural gene for GSI from Rhizobium meliloti 104A14. A gene bank of R. meliloti was constructed by using the bacteriophage P4 cosmid pMK318. Cosmids that contain the structural gene for GSI were isolated by selecting for plasmids that permit ET8051, an Escherichia coli glutamine autotroph, to grow with ammonia as the sole nitrogen source. One of the cosmids, pJS36, contains an insert of 11.9 kilobases. ET8051(pJS36) grows slowly on minimal media. When a 3.7-kilobase HindIII fragment derived from this DNA is cloned into the HindIII site of pACYC177 and the plasmids are transformed into ET8051, rapid growth is observed when the insert is in one orientation (pJS44) but not the other (pJS45). Glutamine synthetase activity can be detected in ET8051(pJS44); most of this activity is heat stable. pJS36 hybridizes with the glnA structural gene from Escherichia coli. Insertion of a 2.7-kilobase Tetr determinant into a BglII site located within pJS44 abolishes all glutamine synthetase activity. This interrupted version of a glutamine synthetase gene was substituted for the normal R. meliloti sequence by homologous recombination in R. meliloti. Recombinants lose GSI activity, but retain GSII activity and grow well with ammonia as the sole nitrogen source. These mutants are unaffected in nodulation and nitrogen fixation.
谷氨酰胺合成酶是根瘤菌属成员同化氨的一种主要酶。在根瘤菌属成员中发现了两种形式的谷氨酰胺合成酶，一种是热稳定的谷氨酰胺合成酶I（GSI），另一种是热不稳定的GSII。作为阐明这些酶在共生固氮中作用的一个步骤，我们从苜蓿根瘤菌104A14中克隆了GSI的结构基因。利用噬菌体P4粘粒pMK318构建了苜蓿根瘤菌的基因文库。通过选择允许大肠杆菌谷氨酰胺自养菌ET8051以氨作为唯一氮源生长的质粒，分离出含有GSI结构基因的粘粒。其中一个粘粒pJS36含有一个11.9千碱基的插入片段。ET8051(pJS36)在基本培养基上生长缓慢。当从该DNA衍生的一个3.7千碱基的HindIII片段克隆到pACYC177的HindIII位点，并且将这些质粒转化到ET8051中时，当插入片段处于一种方向（pJS44）时观察到快速生长，而在另一种方向（pJS45）时则没有。在ET8051(pJS44)中可以检测到谷氨酰胺合成酶活性；这种活性的大部分是热稳定的。pJS36与大肠杆菌的glnA结构基因杂交。将一个2.7千碱基的四环素抗性决定子插入位于pJS44内的一个BglII位点，消除了所有谷氨酰胺合成酶活性。通过苜蓿根瘤菌中的同源重组，用这个谷氨酰胺合成酶基因的中断版本替代了苜蓿根瘤菌的正常序列。重组体失去了GSI活性，但保留了GSII活性，并且能够以氨作为唯一氮源良好生长。这些突变体在结瘤和固氮方面没有受到影响。
