Krzyżanowska Dorota M, Ossowicki Adam, Rajewska Magdalena, Maciąg Tomasz, Jabłońska Magdalena, Obuchowski Michał, Heeb Stephan, Jafra Sylwia
Laboratory of Biological Plant Protection, Department of Biotechnology, Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk Gdansk, Poland.
Laboratory of Molecular Bacteriology, Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk, Medical University of Gdansk Gdansk, Poland.
Front Microbiol. 2016 May 26;7:782. doi: 10.3389/fmicb.2016.00782. eCollection 2016.
Dickeya solani and Pectobacterium carotovorum subsp. brasiliense are recently established species of bacterial plant pathogens causing black leg and soft rot of many vegetables and ornamental plants. Pseudomonas sp. strain P482 inhibits the growth of these pathogens, a desired trait considering the limited measures to combat these diseases. In this study, we determined the genetic background of the antibacterial activity of P482, and established the phylogenetic position of this strain. Pseudomonas sp. P482 was classified as Pseudomonas donghuensis. Genome mining revealed that the P482 genome does not contain genes determining the synthesis of known antimicrobials. However, the ClusterFinder algorithm, designed to detect atypical or novel classes of secondary metabolite gene clusters, predicted 18 such clusters in the genome. Screening of a Tn5 mutant library yielded an antimicrobial negative transposon mutant. The transposon insertion was located in a gene encoding an HpcH/HpaI aldolase/citrate lyase family protein. This gene is located in a hypothetical cluster predicted by the ClusterFinder, together with the downstream homologs of four nfs genes, that confer production of a non-fluorescent siderophore by P. donghuensis HYS(T). Site-directed inactivation of the HpcH/HpaI aldolase gene, the adjacent short chain dehydrogenase gene, as well as a homolog of an essential nfs cluster gene, all abolished the antimicrobial activity of the P482, suggesting their involvement in a common biosynthesis pathway. However, none of the mutants showed a decreased siderophore yield, neither was the antimicrobial activity of the wild type P482 compromised by high iron bioavailability. A genomic region comprising the nfs cluster and three upstream genes is involved in the antibacterial activity of P. donghuensis P482 against D. solani and P. carotovorum subsp. brasiliense. The genes studied are unique to the two known P. donghuensis strains. This study illustrates that mining of microbial genomes is a powerful approach for predictingthe presence of novel secondary-metabolite encoding genes especially when coupled with transposon mutagenesis.
茄科果胶杆菌和胡萝卜软腐果胶杆菌巴西亚种是最近确定的细菌性植物病原体物种，可导致多种蔬菜和观赏植物发生黑胫病和软腐病。假单胞菌属菌株P482可抑制这些病原体的生长，鉴于对抗这些疾病的措施有限，这是一个理想的特性。在本研究中，我们确定了P482抗菌活性的遗传背景，并确定了该菌株的系统发育位置。假单胞菌属P482被归类为东湖假单胞菌。基因组挖掘表明，P482基因组不包含决定已知抗菌物质合成的基因。然而，旨在检测非典型或新型次生代谢物基因簇的ClusterFinder算法在该基因组中预测了18个此类基因簇。对一个Tn5突变体文库进行筛选，得到了一个抗菌阴性转座子突变体。转座子插入位于一个编码HpcH/HpaI醛缩酶/柠檬酸裂合酶家族蛋白的基因中。该基因位于ClusterFinder预测的一个假定基因簇中，与四个nfs基因的下游同源物在一起，这些同源物赋予东湖假单胞菌HYS(T)产生一种非荧光铁载体的能力。对HpcH/HpaI醛缩酶基因、相邻的短链脱氢酶基因以及一个必需的nfs基因簇基因的同源物进行定点失活，均消除了P482的抗菌活性，表明它们参与了一个共同的生物合成途径。然而，没有一个突变体的铁载体产量降低，高铁生物利用度也没有损害野生型P482的抗菌活性。一个包含nfs基因簇和三个上游基因的基因组区域参与了东湖假单胞菌P482对茄科果胶杆菌和胡萝卜软腐果胶杆菌巴西亚种的抗菌活性。所研究的基因是两个已知东湖假单胞菌菌株所特有的。本研究表明，微生物基因组挖掘是预测新型次生代谢物编码基因存在的一种强大方法, 特别是与转座子诱变结合时。
