Buxton R S
J Gen Microbiol. 1978 Apr;105(2):175-85. doi: 10.1099/00221287-105-2-175.
A mutant of Bacillus subtilis which grew in complex medium at 30 degrees C but lysed at 45 degrees C has been isolated. It could only grow on minimal medium at 45 degrees C with added aspartate (20 microgram ml-1) but lysed if lysine (20 microgram ml-1) was also present. The requirement for aspartate was due to a low activity of pyruvate carboxylase; the site of the mutation (pyc) was linked (16% cotransducible using phage PBSI) to the pyrD locus, and the order of markers deduced was: pyrD-cysC-pyc. This defect appeared to lead to decreased synthesis of mesodiaminopimelic acid (mesoA2pm), an amino acid unique to peptidoglycan and its precursors. At the restrictive temperature the mutant accumulated uridine-5'-diphosphate N-acetylmuramyl-L-alanyl-D-glutamate, since meso A2pm is the next amino acid to be added to the growing peptide chain of peptidoglycan. This resulted in an inhibition of peptidoglycan synthesis, determined as a reduced incorporation of N-acetyl[14C]glucosamine. Peptidoglycan synthesis was not decreased if the mutant was grown in media containing aspartate but lacking lysine. The sensitivity to lysine may arise because (i) at 45 degrees C the mutant was starved for aspartate and hence mesoA2pm even when aspartate was present, since aspartate utilization, as estimated by the incorporation of [3H]aspartate into trichloroacetic acid precipitable material, was relatively inefficient; and (ii) this diminished level of mesoA2pm synthesis from aspartate was further curtailed since lysine inhibits one of the aspartokinases in B. subtilis. Thus, addition of lysine allowed protein synthesis and hence autolysin production to proceed whilst peptidoglycan synthesis remained inhibited. When autolysis was blocked, either indirectly by stopping protein synthesis through starvation of aspartate and lysine, or directly by introducing a lyt mutation, then shifting the mutant to 45 degrees C did not result in lysis but growth still ceased.
已分离出一种枯草芽孢杆菌突变体，它在30℃的复合培养基中生长，但在45℃时裂解。它只能在添加了天冬氨酸（20微克/毫升）的45℃基本培养基上生长，但如果同时存在赖氨酸（20微克/毫升）则会裂解。对天冬氨酸的需求是由于丙酮酸羧化酶活性较低；突变位点（pyc）与pyrD基因座连锁（使用噬菌体PBSI共转导率为16%），推导的标记顺序为：pyrD-cysC-pyc。这种缺陷似乎导致中-二氨基庚二酸（mesoA2pm）合成减少，mesoA2pm是肽聚糖及其前体特有的一种氨基酸。在限制温度下，突变体积聚尿苷-5'-二磷酸N-乙酰胞壁酰-L-丙氨酰-D-谷氨酸，因为meso A2pm是要添加到肽聚糖不断增长的肽链上的下一个氨基酸。这导致肽聚糖合成受到抑制，表现为N-乙酰[14C]葡萄糖胺掺入减少。如果突变体在含有天冬氨酸但缺乏赖氨酸的培养基中生长，肽聚糖合成不会减少。对赖氨酸的敏感性可能源于：（i）在45℃时，即使存在天冬氨酸，突变体也缺乏天冬氨酸，因此缺乏mesoA2pm，因为从天冬氨酸掺入三氯乙酸可沉淀物质估计，天冬氨酸利用效率相对较低；（ii）由于赖氨酸抑制枯草芽孢杆菌中的一种天冬氨酸激酶之一，从天冬氨酸合成的mesoA2pm水平进一步降低。因此，添加赖氨酸允许蛋白质合成，从而使自溶素产生继续进行，而肽聚糖合成仍然受到抑制。当自溶被阻断时，要么通过天冬氨酸和赖氨酸饥饿间接停止蛋白质合成，要么直接引入lyt突变，然后将突变体转移到45℃不会导致裂解，但生长仍然停止。
