Panas Panayiotis, Ternan Nigel G, Dooley James S G, McMullan Geoff
School of Biomedical Sciences, University of Ulster, Coleraine, County Londonderry, Northern Ireland BT52 1SA, UK.
Environ Microbiol. 2006 May;8(5):939-45. doi: 10.1111/j.1462-2920.2005.00974.x.
Phosphonoacetate is regarded as an antiviral xenobiotic whose mineralization can be catalysed by an enzyme, phosphonoacetate hydrolase, encoded by the phnA gene. To date the enzyme's activity has been detected in only a limited number of bacteria. Its expression has been shown to occur in a manner independent of the phosphate status of the cell, in direct contrast to the general rule of organophosphonate metabolism being under the control of the pho regulon. In this study the environmental occurrence of the phnA gene was evaluated by polymerase chain reaction amplification of DNA extracts obtained directly from various soil environments. Sensitivity of this method was improved such that a positive result was routinely obtained with soil spiked with as few as 6 colony-forming units (cfu) per gram of soil of Pseudomonas fluorescens 23F (phnA(+)). When total DNA from a variety of Northern Irish, Greek and Bolivian soils was tested, all were positive for phnA. Bacteria capable of utilizing phosphonoacetate as sole carbon, energy and phosphorus source, with the release of essentially equimolar concentrations of phosphate to the culture supernatant, were isolated from all soil samples tested. Analysis of three such isolates revealed all to be species of Pseudomonas sensu stricto, possessing phosphonoacetate hydrolase activity in cell-free extracts. Sequence determination of the phnA gene revealed a similarity of the putative protein sequences at levels of 98.3-99.3% between the Pseudomonas strains. This is the first study to use molecular methods to investigate the distribution of a gene encoding organophosphonate metabolism, and indicates that the phnA gene is ubiquitous within soils from geographically distinct regions. Such an observation supports the proposition that phosphonoacetate is a compound that may also have a biogenic origin.
膦乙酸被视为一种抗病毒的外源性物质，其矿化作用可由一种由phnA基因编码的酶——膦乙酸水解酶催化。迄今为止，仅在有限数量的细菌中检测到该酶的活性。研究表明，其表达的发生方式与细胞的磷酸盐状态无关，这与有机膦酸盐代谢受pho调节子控制的一般规律形成直接对比。在本研究中，通过对直接从各种土壤环境中获得的DNA提取物进行聚合酶链反应扩增，评估了phnA基因在环境中的存在情况。该方法的灵敏度得到了提高，以至于对于每克土壤中仅添加低至6个荧光假单胞菌23F（phnA(+)）菌落形成单位（cfu）的加标土壤，常规情况下都能获得阳性结果。当检测来自北爱尔兰、希腊和玻利维亚各种土壤的总DNA时，所有样本的phnA均呈阳性。从所有测试的土壤样本中分离出了能够利用膦乙酸作为唯一碳源、能源和磷源，并向培养上清液中释放基本等摩尔浓度磷酸盐的细菌。对其中三个这样的分离株进行分析发现，它们均为狭义假单胞菌属的物种，其无细胞提取物中具有膦乙酸水解酶活性。phnA基因的序列测定表明，假单胞菌菌株之间推定的蛋白质序列相似度在98.3%至99.3%之间。这是第一项使用分子方法研究编码有机膦酸盐代谢基因分布的研究，表明phnA基因在地理上不同区域的土壤中普遍存在。这一观察结果支持了膦乙酸可能也有生物源的观点。
