Geszvain Kati, Smesrud Logan, Tebo Bradley M
Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health & Science University, Portland, Oregon, USA
Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health & Science University, Portland, Oregon, USA.
Appl Environ Microbiol. 2016 Jun 13;82(13):3774-3782. doi: 10.1128/AEM.00046-16. Print 2016 Jul 1.
The oxidation of soluble Mn(II) to insoluble Mn(IV) is a widespread bacterial activity found in a diverse array of microbes. In the Mn(II)-oxidizing bacterium Pseudomonas putida GB-1, two Mn(II) oxidase genes, named mnxG and mcoA, were previously identified; each encodes a multicopper oxidase (MCO)-type enzyme. Expression of these two genes is positively regulated by the response regulator MnxR. Preliminary investigation into putative additional regulatory pathways suggested that the flagellar regulators FleN and FleQ also regulate Mn(II) oxidase activity; however, it also revealed the presence of a third, previously uncharacterized Mn(II) oxidase activity in P. putida GB-1. A strain from which both of the Mn(II) oxidase genes and fleQ were deleted exhibited low levels of Mn(II) oxidase activity. The enzyme responsible was genetically and biochemically identified as an animal heme peroxidase (AHP) with domain and sequence similarity to the previously identified Mn(II) oxidase MopA. In the ΔfleQ strain, P. putida GB-1 MopA is overexpressed and secreted from the cell, where it actively oxidizes Mn. Thus, deletion of fleQ unmasked a third Mn(II) oxidase activity in this strain. These results provide an example of an Mn(II)-oxidizing bacterium utilizing both MCO and AHP enzymes.
The identity of the Mn(II) oxidase enzyme in Pseudomonas putida GB-1 has been a long-standing question in the field of bacterial Mn(II) oxidation. In the current work, we demonstrate that P. putida GB-1 employs both the multicopper oxidase- and animal heme peroxidase-mediated pathways for the oxidation of Mn(II), rendering this model organism relevant to the study of both types of Mn(II) oxidase enzymes. The presence of three oxidase enzymes in P. putida GB-1 deepens the mystery of why microorganisms oxidize Mn(II) while providing the field with the tools necessary to address this question. The initial identification of MopA as a Mn(II) oxidase in this strain required the deletion of FleQ, a regulator involved in both flagellum synthesis and biofilm synthesis in Pseudomonas aeruginosa Therefore, these results are also an important step toward understanding the regulation of Mn(II) oxidation.
将可溶性二价锰（Mn(II)）氧化为不溶性四价锰（Mn(IV)）是一种在多种微生物中广泛存在的细菌活性。在二价锰氧化细菌恶臭假单胞菌GB-1中，先前已鉴定出两个二价锰氧化酶基因，分别命名为mnxG和mcoA；每个基因编码一种多铜氧化酶（MCO）类型的酶。这两个基因的表达受应答调节因子MnxR的正调控。对假定的其他调控途径的初步研究表明，鞭毛调节因子FleN和FleQ也调节二价锰氧化酶活性；然而，研究还揭示了恶臭假单胞菌GB-1中存在第三种先前未表征的二价锰氧化酶活性。一个缺失了二价锰氧化酶基因和fleQ的菌株表现出低水平的二价锰氧化酶活性。通过遗传和生化方法鉴定出负责该活性的酶是一种动物血红素过氧化物酶（AHP），其结构域和序列与先前鉴定的二价锰氧化酶MopA相似。在ΔfleQ菌株中，恶臭假单胞菌GB-1的MopA过表达并从细胞中分泌出来，在细胞外它能积极地氧化锰。因此，fleQ的缺失揭示了该菌株中第三种二价锰氧化酶活性。这些结果提供了一个二价锰氧化细菌同时利用MCO和AHP酶的例子。
恶臭假单胞菌GB-1中二价锰氧化酶的身份一直是细菌二价锰氧化领域长期存在的问题。在当前的研究中，我们证明恶臭假单胞菌GB-1同时利用多铜氧化酶和动物血红素过氧化物酶介导的途径来氧化二价锰，使这种模式生物与这两种类型的二价锰氧化酶的研究相关。恶臭假单胞菌GB-1中存在三种氧化酶加深了微生物为何氧化二价锰这一谜团，同时为该领域提供了解决这个问题所需的工具。在该菌株中最初将MopA鉴定为二价锰氧化酶需要缺失FleQ，FleQ是一种参与铜绿假单胞菌鞭毛合成和生物膜合成的调节因子。因此，这些结果也是理解二价锰氧化调控的重要一步。
