Singh Birendra, Röhm Klaus-Heinrich
Philipps University, Institute of Physiological Chemistry, D-35043 Marburg, Germany.
Microbiology (Reading). 2008 Mar;154(Pt 3):797-809. doi: 10.1099/mic.0.2007/013185-0.
We describe an ATP-binding cassette (ABC) transporter in Pseudomonas putida KT2440 that mediates the uptake of glutamate and aspartate. The system (AatJMQP, for acidic amino acid transport) is encoded by an operon involving genes PP1071-PP1068. A deletion mutant with inactivated solute-binding protein (KTaatJ) failed to grow on Glu and Gln as sole sources of carbon and nitrogen, while a mutant lacking a functional nucleotide-binding domain (KTaatP) was able to adapt to growth on Glu after an extended lag phase. Uptake of Glu and Asp by either mutant was greatly impaired at both low and high amino acid concentrations. The purified solute-binding protein AatJ exhibited high affinity towards Glu and Asp (K(d)=0.4 and 1.3 muM, respectively), while Gln and Asn as well as dicarboxylates (succinate and fumarate) were bound with much lower affinity. We further show that the expression of AatJMQP is controlled by the sigma(54)-dependent two-component system AauRS. Binding of the response regulator AauR to the aat promoter was examined by gel mobility shift assays and DNase I footprinting. By in silico screening, the AauR-binding motif (the inverted repeat TTCGGNNNNCCGAA) was detected in further P. putida KT2440 genes with established or putative functions in acidic amino acid utilization, and also occurred in other pseudomonads. The products of these AauR-responsive genes include the H(+)/Glu symporter GltP, a periplasmic glutaminase/asparaginase, AnsB, and phosphoenolpyruvate synthase (PpsA), a key enzyme of gluconeogenesis in Gram-negative bacteria. Based on these findings, we propose that AauR is a central regulator of acidic amino acid uptake and metabolism in pseudomonads.
我们描述了恶臭假单胞菌KT2440中一种介导谷氨酸和天冬氨酸摄取的ATP结合盒（ABC）转运蛋白。该系统（AatJMQP，用于酸性氨基酸转运）由一个包含基因PP1071 - PP1068的操纵子编码。溶质结合蛋白失活的缺失突变体（KTaatJ）无法以Glu和Gln作为唯一碳源和氮源生长，而缺乏功能性核苷酸结合结构域的突变体（KTaatP）在延长的延迟期后能够适应在Glu上生长。在低氨基酸浓度和高氨基酸浓度下，这两种突变体对Glu和Asp的摄取都受到极大损害。纯化的溶质结合蛋白AatJ对Glu和Asp表现出高亲和力（K(d)分别为0.4和1.3 μM），而Gln和Asn以及二羧酸盐（琥珀酸盐和富马酸盐）的结合亲和力则低得多。我们进一步表明，AatJMQP的表达受σ(54)依赖的双组分系统AauRS控制。通过凝胶迁移率变动分析和DNase I足迹法检测了响应调节因子AauR与aat启动子的结合。通过计算机筛选，在恶臭假单胞菌KT2440其他已确定或推测在酸性氨基酸利用中具有功能的基因中检测到了AauR结合基序（反向重复序列TTCGGNNNNCCGAA），并且在其他假单胞菌中也存在。这些AauR响应基因的产物包括H(+)/Glu同向转运体GltP、一种周质谷氨酰胺酶/天冬酰胺酶AnsB以及磷酸烯醇丙酮酸合酶（PpsA），后者是革兰氏阴性菌糖异生的关键酶。基于这些发现，我们提出AauR是假单胞菌中酸性氨基酸摄取和代谢的核心调节因子。
