Djordjevic G M, Tchieu J H, Saier M H
Department of Biology, University of California at San Diego, La Jolla, CA 92093, USA.
J Bacteriol. 2001 May;183(10):3224-36. doi: 10.1128/JB.183.10.3224-3236.2001.
The heterofermentative lactic acid bacterium Lactobacillus brevis transports galactose and the nonmetabolizable galactose analogue thiomethyl-beta-galactoside (TMG) by a permease-catalyzed sugar:H(+) symport mechanism. Addition of glucose to L. brevis cells loaded with [(14)C]TMG promotes efflux and prevents accumulation of the galactoside, probably by converting the proton symporter into a uniporter. Such a process manifests itself physiologically in phenomena termed inducer expulsion and exclusion. Previous evidence suggested a direct allosteric mechanism whereby the phosphocarrier protein, HPr, phosphorylated at serine-46 [HPr(Ser-P)], binds to the galactose:H(+) symporter to uncouple sugar transport from proton symport. To elucidate the molecular mechanism of inducer control in L. brevis, we have cloned the genes encoding the HPr(Ser) kinase, HPr, enzyme I, and the galactose:H(+) symporter. The sequences of these genes were determined, and the relevant phylogenetic trees are presented. Mutant HPr derivatives in which the regulatory serine was changed to either alanine or aspartate were constructed. The cloned galP gene was integrated into the chromosome of Bacillus subtilis, and synthesis of the mutant HPr proteins in this organism was shown to promote regulation of GalP, as expected for a direct allosteric mechanism. We have thus reconstituted inducer control in an organism that does not otherwise exhibit this phenomenon. These results are consistent with the conclusion that inducer exclusion and expulsion in L. brevis operates via a multicomponent signal transduction mechanism wherein the presence of glycolytic intermediates such as fructose 1,6-bisphosphate (the intracellular effector), derived from exogenous glucose (the extracellular effector), activates HPr(Ser) kinase (the sensor) to phosphorylate HPr on Ser-46 (the messenger), which binds to the galactose:H(+) symporter (the target), resulting in uncoupling of sugar transport from proton symport (the response). This cascade allows bacteria to quickly respond to changes in external sugar concentrations. Understanding the molecular mechanism of inducer control advances our knowledge of the link between metabolic and transport processes in bacteria.
H⁺同向转运机制转运半乳糖和不可代谢的半乳糖类似物硫代甲基-β-半乳糖苷（TMG）。向加载了[¹⁴C]TMG的短乳杆菌细胞中添加葡萄糖会促进外流并阻止半乳糖苷的积累，这可能是通过将质子同向转运体转变为单向转运体实现的。这样一个过程在生理上表现为诱导物排出和排除现象。先前的证据表明存在一种直接变构机制，即丝氨酸-46位点磷酸化的磷酸载体蛋白HPr[HPr(Ser-P)]与半乳糖：H⁺同向转运体结合，从而使糖转运与质子同向转运解偶联。为了阐明短乳杆菌中诱导物控制的分子机制，我们克隆了编码HPr(Ser)激酶、HPr、酶I和半乳糖：H⁺同向转运体的基因。测定了这些基因的序列，并给出了相关的系统发育树。构建了将调节性丝氨酸分别替换为丙氨酸或天冬氨酸的突变型HPr衍生物。将克隆的galP基因整合到枯草芽孢杆菌的染色体中，结果表明在该生物体中合成突变型HPr蛋白可促进对GalP的调节，这与直接变构机制的预期相符。因此，我们在原本不表现出这种现象的生物体中重建了诱导物控制。这些结果与以下结论一致：短乳杆菌中的诱导物排除和排出通过多组分信号转导机制起作用，其中源自外源葡萄糖（细胞外效应物）的糖酵解中间产物如1,6-二磷酸果糖（细胞内效应物）的存在激活HPr(Ser)激酶（传感器），使其将HPr的丝氨酸-46位点磷酸化（信使），磷酸化的HPr与半乳糖：H⁺同向转运体（靶标）结合，导致糖转运与质子同向转运解偶联（响应）。这种级联反应使细菌能够快速响应外部糖浓度的变化。了解诱导物控制的分子机制增进了我们对细菌代谢与转运过程之间联系的认识。
