Sage A E, Vasil A I, Vasil M L
Department of Microbiology, University of Colorado Health Sciences Centre, Denver 80262, USA.
Mol Microbiol. 1997 Jan;23(1):43-56. doi: 10.1046/j.1365-2958.1997.1681542.x.
Production of the two phospholipases C (PLCs) in Pseudomonas aeruginosa PAO1 is induced under conditions of phosphate limitation, or by the osmoprotectants choline or glycine betaine. Tn5 mutagenesis was performed on strain PAO1 to isolate mutants deficient in choline-dependent induction of PLC. Two mutants, Tn5T1 and Tn5G19, were identified which produce decreased levels of PLC in phosphate-replete media supplemented with choline. A total of 136 and 496 bp of flanking DNA from Tn5G19 and Tn5T1 was cloned by an inverse polymerase chain reaction (PCR) and sequenced. The DNA flanking the Tn5T1 insertion contains an open reading frame predicted to encode a peptide that is approx. 60% identical to the N-terminus of a previously identified protein (P35) of unknown function from Escherichia coli. The P35 gene, which is located in the nusA-infB operon in E. coll, was designated orp (osmoprotectant regulator of PLC). Haemolytic titres, total PlcH protein and beta-galactosidase activity expressed from a chromosomally inserted plcH-lacZ operon fusion were reduced in strain Tn5T1 in comparison with the parental strain (PAO1) carrying the same fusion. However, this mutant expressed several-fold higher levels of plcH message than strain PAO1 in the presence of choline, while the phosphate-starvation-dependent transcript of plcH could not be detected in this mutant. The defects in Tn5T1 are complemented by a DNA fragment, isolated from a genomic library of PAO1, that carries the orp gene. The deduced amino acid sequence of the DNA fragment cloned from Tn5G19 exhibits 84% identity with the betB gene product of E. coli that has betaine aldehyde dehydrogenase activity. This enzyme catalyses the conversion of betaine aldehyde to glycine-betaine. Unlike the parental strain, the Tn5G19 mutant could not utilize choline as a sole carbon, nitrogen and energy source, and it was deficient in betaine aldehyde dehydrogenase activity. Also, consistent with a disruption of betB in Tn5G19, choline inhibited growth of this strain in media containing 0.7 M NaCl, while glycine-betaine restores growth to wild-type levels. The defects in Tn5G19 are complemented by a DNA fragment from PAO1 that carries the betB gene. The orp gene is located between 0.6 to 6.6 min while betB is located between 10.5 to 12.5 min on the chromosome of PAO1.
铜绿假单胞菌PAO1中两种磷脂酶C（PLCs）的产生在磷酸盐限制条件下，或由渗透保护剂胆碱或甘氨酸甜菜碱诱导。对PAO1菌株进行Tn5诱变，以分离出胆碱依赖性PLC诱导缺陷的突变体。鉴定出两个突变体Tn5T1和Tn5G19，它们在补充有胆碱的磷酸盐充足培养基中产生的PLC水平降低。通过反向聚合酶链反应（PCR）克隆了来自Tn5G19和Tn5T1的总共136和496 bp的侧翼DNA并进行了测序。Tn5T1插入侧翼的DNA包含一个开放阅读框，预测编码一种肽，该肽与先前鉴定的来自大肠杆菌的功能未知的蛋白质（P35）的N端约60%相同。位于大肠杆菌nusA-infB操纵子中的P35基因被命名为orp（PLC的渗透保护剂调节因子）。与携带相同融合的亲本菌株（PAO1）相比，Tn5T1菌株中从染色体插入的plcH-lacZ操纵子融合表达的溶血滴度、总PlcH蛋白和β-半乳糖苷酶活性降低。然而，在存在胆碱的情况下，该突变体表达的plcH信息水平比PAO1菌株高几倍，而在该突变体中无法检测到plcH的磷酸盐饥饿依赖性转录本。Tn5T1中的缺陷由从PAO1基因组文库中分离的携带orp基因的DNA片段互补。从Tn5G19克隆的DNA片段的推导氨基酸序列与具有甜菜碱醛脱氢酶活性的大肠杆菌betB基因产物具有84%的同一性。这种酶催化甜菜碱醛转化为甘氨酸甜菜碱。与亲本菌株不同，Tn5G19突变体不能利用胆碱作为唯一的碳、氮和能源，并且它缺乏甜菜碱醛脱氢酶活性。此外，与Tn5G19中betB的破坏一致，胆碱抑制该菌株在含有0.7 M NaCl的培养基中的生长，而甘氨酸甜菜碱将生长恢复到野生型水平。Tn5G19中的缺陷由来自PAO1的携带betB基因的DNA片段互补。orp基因位于PAO1染色体上0.6至6.6分钟之间，而betB位于10.5至12.5分钟之间。
