Ewann Fanny, Hoffman Paul S
Division of Infectious Diseases, University of Virginia Health Systems, MR-4 Building, Room 2146, 409 Lane Road, Charlottesville, VA 22908, USA.
Appl Environ Microbiol. 2006 Jun;72(6):3993-4000. doi: 10.1128/AEM.00684-06.
Growth of Legionella pneumophila on buffered charcoal-yeast extract (BCYE) medium is dependent on L-cysteine (but not L-cystine), which is added in excess over what is required for nutrition. We investigated the biochemical and genetic bases for this unusual requirement and determined that much of the L-cysteine in BCYE medium is rapidly oxidized to L-cystine and is unavailable to the bacteria. Analysis of cysteine consumption during bacterial growth indicated that of the 11% consumed, 3.85% (approximately 0.1 mM) was incorporated into biomass. The activities of two key cysteine biosynthetic enzymes (serine acetyltransferase and cysteine synthase) were not detected in cell extracts of L. pneumophila, and the respective genes were not present in the genome sequences, confirming cysteine auxotrophy. Kinetic studies identified two energy-dependent cysteine transporters, one with high affinity (apparent Km, 3.29 microM) and the other with low affinity (apparent Km, 93 microM), each of which was inhibited by the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone. Cystine was not transported by L. pneumophila; however, a mutant strain capable of growth on L-cystine (CYS1 mutant) transported L-cystine with similar kinetics (Km, 4.4 microM and 90 microM). Based on the bipartite kinetics, requirement for proton motive force, and inhibitor studies, we suggest that a high-affinity periplasmic binding protein and a major facilitator/symporter (low affinity) mediate uptake. The latter most likely is functional at high cysteine concentrations and most likely displays altered substrate specificity in the CYS-1 mutant. Our studies provide biochemical evidence to support a general view that L. pneumophila is restricted to an intracellular lifestyle in natural environments by an inability to utilize cystine, which most likely ensures that the dormant cyst-like transmissible forms do not germinate outside suitable protozoan hosts.
嗜肺军团菌在缓冲活性炭酵母提取物（BCYE）培养基上的生长依赖于L-半胱氨酸（而非L-胱氨酸），其添加量超过营养所需量。我们研究了这种异常需求的生化和遗传基础，确定BCYE培养基中的大部分L-半胱氨酸会迅速氧化为L-胱氨酸，细菌无法利用。对细菌生长过程中半胱氨酸消耗情况的分析表明，消耗的11%中，有3.85%（约0.1 mM）被整合到生物量中。在嗜肺军团菌的细胞提取物中未检测到两种关键半胱氨酸生物合成酶（丝氨酸乙酰转移酶和半胱氨酸合酶）的活性，并且在基因组序列中也不存在相应基因，证实了半胱氨酸营养缺陷型。动力学研究确定了两种能量依赖性半胱氨酸转运体，一种具有高亲和力（表观Km为3.29 microM），另一种具有低亲和力（表观Km为93 microM），每种转运体都受到解偶联剂羰基氰化物间氯苯腙的抑制。嗜肺军团菌不转运胱氨酸；然而，一株能够在L-胱氨酸上生长的突变菌株（CYS1突变体）以相似的动力学（Km分别为4.4 microM和90 microM）转运L-胱氨酸。基于双重动力学、对质子动力势的需求以及抑制剂研究，我们认为一种高亲和力的周质结合蛋白和一种主要转运体/同向转运体（低亲和力）介导摄取。后者最有可能在高半胱氨酸浓度下起作用，并且在CYS-1突变体中最有可能表现出改变的底物特异性。我们的研究提供了生化证据，支持这样一种普遍观点，即嗜肺军团菌在自然环境中因无法利用胱氨酸而局限于细胞内生活方式，这很可能确保了休眠的类似囊肿的可传播形式不会在合适的原生动物宿主之外萌发。
