Cabrera-Martinez Rosa-Martha, Tovar-Rojo Federico, Vepachedu Venkata Ramana, Setlow Peter
Department of Biochemistry University of Connecticut Health Center, Farmington, Connecticut 06032, USA.
J Bacteriol. 2003 Apr;185(8):2457-64. doi: 10.1128/JB.185.8.2457-2464.2003.
The rates of germination of Bacillus subtilis spores with L-alanine were increased markedly, in particular at low L-alanine concentrations, by overexpression of the tricistronic gerA operon that encodes the spore's germinant receptor for L-alanine but not by overexpression of gerA operon homologs encoding receptors for other germinants. However, spores with elevated levels of the GerA proteins did not germinate more rapidly in a mixture of asparagine, glucose, fructose, and K(+) (AGFK), a germinant combination that requires the participation of at least the germinant receptors encoded by the tricistronic gerB and gerK operons. Overexpression of the gerB or gerK operon or both the gerB and gerK operons also did not stimulate spore germination in AGFK. Overexpression of a mutant gerB operon, termed gerB*, that encodes a receptor allowing spore germination in response to either D-alanine or L-asparagine also caused faster spore germination with these germinants, again with the largest enhancement of spore germination rates at lower germinant concentrations. However, the magnitudes of the increases in the germination rates with D-alanine or L-asparagine in spores overexpressing gerB* were well below the increases in the spore's levels of the GerBA protein. Germination of gerB* spores with D-alanine or L-asparagine did not require participation of the products of the gerK operon, but germination with these agents was decreased markedly in spores also overexpressing gerA. These findings suggest that (i) increases in the levels of germinant receptors that respond to single germinants can increase spore germination rates significantly; (ii) there is some maximum rate of spore germination above which stimulation of GerA operon receptors alone will not further increase the rate of spore germination, as action of some protein other than the germinant receptors can become rate limiting; (iii) while previous work has shown that the wild-type GerB and GerK receptors interact in some fashion to cause spore germination in AGFK, there also appears to be an additional component required for AGFK-triggered spore germination; (iv) activation of the GerB receptor with D-alanine or L-asparagine can trigger spore germination independently of the GerK receptor; and (v) it is likely that the different germinant receptors interact directly and/or compete with each other for some additional component needed for initiation of spore germination. We also found that very high levels of overexpression of the gerA or gerK operon (but not the gerB or gerB* operon) in the forespore blocked sporulation shortly after the engulfment stage, although sporulation appeared normal with the lower levels of gerA or gerK overexpression that were used to generate spores for analysis of rates of germination.
编码芽孢杆菌对L-丙氨酸萌发受体的三顺反子gerA操纵子的过表达，显著提高了枯草芽孢杆菌芽孢与L-丙氨酸的萌发率，尤其是在低L-丙氨酸浓度下，但编码其他萌发剂受体的gerA操纵子同源物的过表达则没有这种效果。然而，GerA蛋白水平升高的芽孢在天冬酰胺、葡萄糖、果糖和K⁺（AGFK）的混合物中萌发并不更快，AGFK是一种萌发剂组合，至少需要三顺反子gerB和gerK操纵子编码的萌发受体参与。gerB或gerK操纵子或gerB和gerK操纵子两者的过表达也不会刺激芽孢在AGFK中的萌发。一种称为gerB的突变gerB操纵子的过表达，其编码一种允许芽孢响应D-丙氨酸或L-天冬酰胺而萌发的受体，也会使芽孢与这些萌发剂的萌发更快，同样在较低萌发剂浓度下芽孢萌发率提高最大。然而，过表达gerB的芽孢对D-丙氨酸或L-天冬酰胺的萌发率增加幅度远低于芽孢中GerBA蛋白水平的增加。gerB芽孢与D-丙氨酸或L-天冬酰胺的萌发不需要gerK操纵子产物的参与，但在也过表达gerA的芽孢中，这些试剂的萌发显著降低。这些发现表明：（i）对单一萌发剂有反应的萌发受体水平的增加可显著提高芽孢萌发率；（ii）存在某种芽孢萌发的最大速率，超过该速率，仅刺激GerA操纵子受体不会进一步提高芽孢萌发率，因为除萌发受体外的某些蛋白质的作用可能成为限速因素；（iii）虽然先前的研究表明野生型GerB和GerK受体以某种方式相互作用导致芽孢在AGFK中萌发，但AGFK触发的芽孢萌发似乎还需要一个额外的成分；（iv）用D-丙氨酸或L-天冬酰胺激活GerB受体可独立于GerK受体触发芽孢萌发；（v）不同的萌发受体可能直接相互作用和/或相互竞争芽孢萌发起始所需的某些额外成分。我们还发现，前芽孢中gerA或gerK操纵子（但不是gerB或gerB操纵子）的非常高水平的过表达在吞噬阶段后不久就会阻止芽孢形成，尽管用于分析萌发率的芽孢产生时gerA或gerK的过表达水平较低，芽孢形成看起来正常。
