Biosynthesis of the Nematode Attractant 2-Heptanone and Its Co-evolution Between the Pathogenic Bacterium Bacillus nematocida and Non-pathogenic Bacterium Bacillus subtilis.

Man Zhu,Xiaowei Huang,Pengfei Wang,Shanzhuang Niu,Yuhong Li,Keqin Zhang,Xiao’E Xu

doi:10.3389/fmicb.2019.01489

Abstract

Methylketones are broadly distributed in nature and perform a variety of functions. Most microorganisms are thought to produce methylketone by abortive β-oxidation of fatty acid catalytic metabolism. However, two methylketone synthetase genes in wild tomatoes are reported to synthesize methylketone using intermediates of the fatty acids biosynthetic pathway. In our previous study on Trojan horse-like interactions between the bacterium Bacillus nematocida B16 and its host worm, the chemical 2-heptanone was found to be an important attractant for the hosts. So here we used this model to investigate the genes involved in synthesizing 2-heptanone in microorganisms. We identified a novel methylketone synthase gene yneP in B. nematocida B16 and found enhancement of de novo fatty acid synthesis during 2-heptanone production. Interestingly, a homolog of yneP’ existed in the non-pathogenic species Bacillus subtilis 168, a close relative of B. nematocida B16 that was unable to lure worms, but GC-MS assay showed no 2-heptanone production. However, overexpression of yneP’ from B. subtilis in both heterologous and homologous systems demonstrated that it was not a pseudogene. The transcriptional analysis between those two genes had few differences under the same conditions. It was further shown that the failure to detect 2-heptanone in B. subtilis 168 was at least partly due to its conversion into 6-methyl-2-heptanone by methylation. Our study revealed methylketone biosynthesis of Bacillus species, and provided a co-evolution paradigm of second metabolites during the interactions between pathogenic/non-pathogenic bacteria and host.

Highlights

Methylketones are broadly distributed in natural environments and can be produced by a variety of bacteria, fungi, plants, insects, and even mammals
To investigate that the potential methylketone synthetase existed in our bacterial strain, we aligned shMKSI (AY701574) and shMKSII (EU883793) from wild tomatoes with the annotated genome of B. nematocida B16 at amino acid level, and two genes of ytpA and yneP were identified as having the highest sequence identity to shMKSI and shMKSII
Wild tomatoes produce methylketones as repellants against its pests (Yu, 2013), in which two genes shMKSI and shMKSII are identified to be responsible for the production of methylketones during fatty acid biosynthesis (Yu et al, 2010)

Summary

Introduction

Methylketones are broadly distributed in natural environments and can be produced by a variety of bacteria, fungi, plants, insects, and even mammals. In bacteria, methylketones, such as 2heptanone, 2-butanone, 2-petanone, and 3-methyl-1-butanone, are detected in the volatile organic compounds (VOCs) of Lactobacillus casei (Gallegos et al, 2017). 2-pentanone and a highly autolytic strain of Lactobacillus helveticus is reported to enhance the levels of methylketones during cheese ripening (Fadda et al, 2002; Hannon et al, 2006) In fungi, both 2-heptanone and 2-petanone are detected from a range of species, from unicellular yeast to filamentous fungi (Sunesson et al, 1996). A higher concentration of 2heptanone in the urine of male Rattus norvegicus was found to be more attractive to the females (Zhang et al, 2008)

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Conclusion