Of the citrate pathway in glutamate biosynthesis by Streptococcus mutans
Of the citrate pathway in glutamate biosynthesis by Streptococcus mutans
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DOI：
10.1128/jb.179.3.650-655.1997
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发表时间：
1997-02-01
影响因子：
3.2
通讯作者：
Bleiweis, AS
中科院分区：
文献类型：
--
作者：
Cvitkovitch, DG;Gutierrez, JA;Bleiweis, AS
在先前报道的工作中（J. a . Gutierrez， P. J. Crowley， D. P. Brown， J. D. Hillman， P. Youngman, and a . S. Bleiweis， J. Bacteriol. 178:4166-4175, 1996），分离出了一种Tn917转座子产生的不能厌氧合成谷氨酸的突变体JH1005，并确定该转座子的插入点位于编码异柠檬酸脱氢酶（ICDH）的icd基因中。通过与大肠杆菌宿主菌株EB106的icd突变互补，从突变链球菌基因组质粒文库中分离到完整的突变链球菌icd基因。对该互补质粒pJG400进行遗传分析，发现其开放阅读框（ORF）全长1182个核苷酸，编码393个氨基酸的酶，预测分子量为43 kDa，该核苷酸序列与其他3种细菌的icd基因具有高度同源性（60 ~ 72%）。在icd基因的5'处，我们发现了一个长度为1119个核苷酸的ORF，命名为citZ，编码来自其他细菌的已知柠檬酸合成酶基因的同源物，该ORF编码372个氨基酸的预测蛋白，分子量为43 kDa。此外，质粒pJG400还能够补充大肠杆菌W620的柠檬酸合成酶（gltA）突变。在携带质粒pJG400的野生型变形链球菌和大肠杆菌突变体的细胞提取物中，测定了ICDH （NAD（+）依赖性）和柠檬酸合成酶的活性。在citZ基因的5'区也发现了编码264个推定的乌头酶基因的羧基末端氨基酸的部分ORF。遗传和生化证据表明，变形链球菌具有将乙酰辅酶A和草酸酯转化为乙醇酮戊二酸所需的酶，这是合成谷氨酸所必需的。事实上，变形链球菌JH1005被证明在缺乏有机氮源的最小培养基中吸收氨作为唯一的氮源。
In work previously reported (J. A. Gutierrez, P. J. Crowley, D. P. Brown, J. D. Hillman, P. Youngman, and A. S. Bleiweis, J. Bacteriol. 178:4166-4175, 1996), a Tn917 transposon-generated mutant of Streptococcus mutans JH1005 unable to synthesize glutamate anaerobically was isolated and the insertion point of the transposon was determined to be in the icd gene encoding isocitrate dehydrogenase (ICDH). The intact icd gene of S. mutans has now been isolated from an S. mutans genomic plasmid library by complementation of an icd mutation in Escherichia coli host strain EB106. Genetic analysis of the complementing plasmid pJG400 revealed an open reading frame (ORF) of 1,182 nucleotides which encoded an enzyme of 393 amino acids with a predicted molecular mass of 43 kDa, The nucleotide sequence contained regions of high (60 to 72%) homology with icd genes from three other bacterial species. Immediately 5' of the icd gene, we discovered an ORF of 1,119 nucleotides in length, designated citZ, encoding a homolog of known citrate synthase genes from other bacteria, This ORF encoded a predicted protein of 372 amino acids with a molecular mass of 43 kDa, Furthermore, plasmid pJG400 was also able to complement a citrate synthase (gltA) mutation of E. coli W620. The enzyme activities of both ICDH, found to be NAD(+) dependent, and citrate synthase were measured in cell extracts of wild-type S. mutans and E. coli mutants harboring plasmid pJG400. The region 5' from the citZ gene also revealed a partial ORF encoding 264 carboxy-terminal amino acids of a putative aconitase gene. The genetic and biochemical evidence indicates that S. mutans possesses the enzymes required to convert acetyl coenzyme A and oxalacetate to ol-ketoglutarate, which is necessary for the synthesis of glutamic acid. Indeed, S. mutans JH1005 was shown to assimilate ammonia as a sole source of nitrogen in minimal medium devoid of organic nitrogen sources.