WILD-TYPE GAS VESICLE FORMATION REQUIRES AT LEAST 10 GENES IN THE GVP GENE-CLUSTER OF HALOBACTERIUM-HALOBIUM PLASMID-PNRC100
WILD-TYPE GAS VESICLE FORMATION REQUIRES AT LEAST 10 GENES IN THE GVP GENE-CLUSTER OF HALOBACTERIUM-HALOBIUM PLASMID-PNRC100
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DOI：
10.1128/jb.176.24.7646-7652.1994
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发表时间：
1994-12-01
影响因子：
3.2
通讯作者：
YIN, LRS
中科院分区：
文献类型：
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作者：
DASSARMA, S;ARORA, P;YIN, LRS
为了研究盐生盐杆菌质粒pNRC 100上13个gvp基因gvpMLKJIHGFEDACN在气泡形成中的功能，我们对基因簇进行了连接子扫描突变。我们构建了一个24.5kb的大肠杆菌H.盐菌穿梭质粒pFL 2，其含有grp基因簇，并将卡那霉素抗性（kappa）盒引入每个基因（除了gvpA）。H.具有pFL 2和突变的pFL 2衍生物的缺失了整个gvp基因簇的盐生菌SD 109显示，虽然未突变的基因簇成功地编程了气泡形成，但在除gvpM之外的任何gvp基因中插入κ盒的衍生物都不导致正常气泡的产生。在gvpL、-K、-J、-I和-F中的插入导致在气体囊泡合成中的完全阻断，而在gvpH、-G、-E、-D、-C和-N中的插入导致大大减少的气体囊泡合成。在大多数情况下，气体囊泡合成中的阻断并不是由极性效应引起的，因为对于插入突变体的衍生物获得了类似的结果，其中大部分kappa盒的内部部分被删除，仅保留小的（15至54-bp）插入。唯一的例外是gvpH和gvpD，其中kappa插入的内部部分的缺失导致表型逆转。κ突变体的电子显微镜分析显示，中断gvpC和gvpN的结果在形成较小的气体囊泡比野生型，而中断gvpF，-G，-H，-I，-J，-K，和-L产生没有可辨别的囊泡中间体。这些结果表明，gvpA，-C，和-N，！其具有α转录方向，编码结构蛋白，其中gvpC和gvpN是囊泡形成的晚期所必需的，而gvpL、-K、-J、-I、-H、-G和-F具有α转录方向，编码参与气体囊泡组装的早期步骤的蛋白。
To study the functions of the 13 gvp genes, gvpMLKJIHGFEDACN, on plasmid pNRC100 of Halobacterium halobium in gas vesicle formation, we carried out linker scanning mutagenesis of the gene cluster. We constructed a 24.5-kb Escherichia coli-H. halobium shuttle plasmid, pFL2, containing the grp gene cluster and introduced a kanamycin resistance (kappa) cassette into each gene (except for gvpA). Transformation of H. halobium SD109, which had the entire gvp gene cluster deleted, with pFL2 and mutated pFL2 derivatives showed that while the unmutated gene cluster successfully programmed gas vesicle formation, derivatives with insertion of the kappa cassette in any of the gvp genes, except gvpM, did not lead to production of normal gas vesicles. Insertions in gvpL, -K, -J, -I, and -F resulted in a complete block in gas vesicle synthesis, while insertions in gvpH, -G, -E, -D, -C, and -N resulted in greatly reduced gas vesicle synthesis. In most cases, the block in gas vesicle synthesis did not result from polar effects, since similar results were obtained for derivatives of the insertion mutants in which most of the internal portion of the kappa cassette was deleted and only small (15 to 54-bp) insertions remained. The only exceptions were for gvpH and gvpD, where deletion of the internal portion of the kappa insertions resulted in phenotypic reversion. Electron microscopic analysis of the kappa mutants revealed that interruptions of gvpC and gvpN result in the formation of smaller gas vesicles than in the wild type, while interruptions of gvpF, -G, -H, -I, -J, -K, and -L produce no discernible vesicle intermediates. These results indicate that gvpA, -C, and -N,! which have the rightward transcriptional orientation, encode structural proteins, with gvpC and gvpN necessary for late stages of vesicle formation, and gvpL, -K, -J, -I, -H, -G, and -F, which have the leftward transcriptional orientation, encode proteins involved in early steps in the assembly of gas vesicles.