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首页> 外文期刊>Journal of bacteriology >Redundancy of Enzymes for Formaldehyde Detoxification in Pseudomonas putida
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Redundancy of Enzymes for Formaldehyde Detoxification in Pseudomonas putida

机译：恶臭假单胞菌中用于甲醛解毒的酶的冗余

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Pseudomonas putida KT2440 exhibits redundant formaldehyde dehydrogenases and formate dehydrogenases that contribute to the detoxification of formaldehyde, a highly toxic compound. Physical and transcriptional analyses showed that the open reading frame (ORF) PP0328, encoding one of the formaldehyde dehydrogenases, is self-sufficient, whereas the other functional formaldehyde dehydrogenase gene (ORF PP3970) forms an operon with another gene of unknown function. Two formate dehydrogenase gene clusters (PP0489 to PP0492 and PP2183 to PP2186) were identified, and genes in these clusters were found to form operons. All four transcriptional promoters were mapped by primer extension and revealed the presence of noncanonical promoters expressed at basal level in the exponential growth phase and at a higher level in the stationary phase regardless of the presence of extracellular formaldehyde or formate. These promoters were characterized by a 5′-AG-CCA-C/A-CT-3′ conserved region between ?7 and ?16. To determine the contribution of the different gene products to formaldehyde and formate mineralization, mutants with single and double mutations of formaldehyde dehydrogenases were generated, and the effect of the mutations on formaldehyde catabolism was tested by measuring 14CO2 evolution from 14C-labeled formaldehyde. The results showed that both enzymes contributed to formaldehyde catabolism. A double mutant lacking these two enzymes still evolved CO2 from formaldehyde, suggesting the presence of one or more still-unidentified formaldehyde dehydrogenases. Mutants with single and double mutations in the clusters for formate dehydrogenases were also generated, and all of them were able to metabolize [14C]formate to 14CO2, suggesting a redundancy of functions that was not limited to only the annotated genes. Single and double mutants deficient in formaldehyde dehydrogenases and formate dehydrogenases exhibited longer lag phases than did the parental strain when confronted with concentrations of formaldehyde close to the MICs. This suggests a role for the detoxification system in tolerance to sublethal concentrations of formaldehyde.
机译：恶臭假单胞菌 KT2440表现出多余的甲醛脱氢酶和甲酸脱氢酶，有助于高毒性化合物甲醛的解毒。物理和转录分析表明，编码其中一种甲醛脱氢酶的开放阅读框（ORF）PP0328是自给自足的，而另一个功能性甲醛脱氢酶基因（ORF PP3970）与另一个功能未知的基因形成操纵子。鉴定了两个甲酸脱氢酶基因簇（PP0489至PP0492和PP2183至PP2186），发现这些簇中的基因形成操纵子。所有四个转录启动子均通过引物延伸进行定位，并揭示了在指数生长期以基础水平表达而在固定相以较高水平表达的非经典启动子的存在，无论是否存在细胞外甲醛或甲酸盐。这些启动子的特征是在α7和β16之间的5′-AG-CCA-C / A-CT-3′保守区。为了确定不同基因产物对甲醛和甲酸矿化的贡献，生成了具有甲醛脱氢酶单突变和双突变的突变体，并通过测量 14 CO < sub> 2 从 14 C标记的甲醛演变而来。结果表明，两种酶均有助于甲醛分解代谢。缺少这两种酶的双突变体仍从甲醛释放出CO 2 ，表明存在一种或多种尚未鉴定的甲醛脱氢酶。还生成了甲酸脱氢酶簇中具有单突变和双突变的突变体，并且所有突变体均能够将[ 14 C]甲酸酯代谢为 14 CO 2 ，表明功能的冗余不仅限于注释的基因。甲醛脱氢酶和甲酸酯脱氢酶缺乏的单突变体和双突变体在面对接近MIC的甲醛浓度时，比亲本菌株表现出更长的滞后期。这表明了排毒系统在耐受亚致死浓度的甲醛中的作用。

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