Bonatelli, M. L. | bioRxiv Science

bioRxiv2022

Recently evolved combination of unique sulfatase and amidase genes enables bacterial degradation of the wastewater micropollutant acesulfame worldwide

Xenobiotics often challenge the principle of microbial infallibility. One example is acesulfame introduced in the 1980s as zero-calorie sweetener, which was recalcitrant in wastewater treatment plants until the early 2010s. Then, efficient removal has been reported with increasing frequency. By studying acesulfame metabolism in alphaproteobacterial degraders of the genera Bosea and Chelatococcus, we experimentally confirmed the previously postulated route of two subsequent hydrolysis steps via acetoacetamide-N-sulfonate (ANSA) to acetoacetate and sulfamate. Genome comparison of wildtype Bosea sp. 100-5 and a spontaneous acesulfame degradation-defective mutant revealed the involvement of two plasmid-borne gene clusters. The acesulfame-hydrolyzing sulfatase is strictly manganese-dependent and belongs to the metallo beta-lactamase family. In all degraders analyzed, it is encoded on a highly conserved gene cluster embedded in a composite transposon. The ANSA hydrolase, on the other hand, is an amidase signature domain enzyme encoded in another gene cluster showing variable length among degrading strains. Transposition of the sulfatase gene cluster between chromosome and plasmid explains how the two catabolic gene clusters recently combined for the degradation of acesulfame. Searching available genomes and metagenomes for the two hydrolases and associated genes indicates that the acesulfame plasmid evolved and spread worldwide in short time. While the sulfatase is unprecedented and unique for acesulfame degraders, the amidase occurs in different genetic environments and might have evolved for the degradation of other substrates. Evolution of the acesulfame degradation pathway might have been supported by the presence of structurally related natural and anthropogenic compounds, such as aminoacyl sulfamate ribonucleotide or sulfonamide antibiotics.

microbiology↗

bioRxiv2021

Bacillus sp. RZ2MS9, a tropical PGPR, colonizes maize endophytically and alters the plant's production of volatile organic compounds both independently and when co-inoculated with Azospirillum brasilense Ab-V5

Bacillus spp. are among the most efficient known plant growth-promoting rhizobacteria (PGPR). The PGPR Bacillus sp. strain RZ2MS9 is a multi-trait maize growth promoter previously isolated from guarana plants cultivated in the Amazon rainforest. However, there are several aspects of its interaction with the host that need further investigation. To achieve effective performance of microbial inoculants in crop production, it is necessary to monitor the plants colonization by a PGPR and to assess the potential synergy among beneficial strains. Here, we obtained a stable mutant of RZ2MS9 labelled with green fluorescent protein (RZ2MS9-GFP). We verified that the insertion of the plasmid did not affect either bacterial growth nor its ability to promote maize growth in vitro. Using fluorescent microscopy and qPCR, we demonstrated that RZ2MS9-GFP successfully colonizes maizes roots and leaves endophytically. Subsequently, we evaluated whether RZ2MS9 has a synergistic effect on plant growth promotion when co-inoculated with Azospirillum brasilense Ab-V5, a commercial inoculant for maize. The two strains combined enhanced maizes roots and shoots dry weight by 50.8% and 79.6%, respectively, when compared to the non-inoculated control. In addition, we used co-inoculation experiments in glass chambers to analyze the plants Volatile Organic Compounds (VOCs) production during the maize-RZ2MS9 and maize-RZ2MS9-Ab-V5 interaction. We found that the single and co-inoculation altered maizes VOCs emission profile, with an increase in the production of indoles in the co-inoculation. Collectively, these results increase our knowledge about the interaction between the tropical PGPR Bacillus sp. RZ2MS9 and maize, and provide a new possibility of combined application with the commercial inoculant A. brasilense Ab-V5. ImportanceBacillus sp. RZ2MS9 is a PGPR, previously isolated from guarana plants cultivated in the Brazilian Amazon, which successfully promotes the growth of maize and soybean plants. To improve our knowledge about the interaction between this very promising PGPR and maize, we labelled RZ2MS9 with gfp and monitored its maize colonization. The transformation did not affect either RZ2MS9 growth nor its ability to promote maize growth in vitro. We demonstrated that RZ2MS9 colonizes endophytically maizes roots and leaves. We also verified that the co-inoculation of RZ2MS9 and Azospirillum brasilense Ab-V5, a known commercial maize inoculant enhanced maizes roots and shoots growth. Moreover, the co-inoculation altered the maizes volatile organic compounds, increasing the production of indoles, that is related with decreased upon the reduction of fertilization. Certainly, our research contributed with better Bacillus sp. RZ2MS9 - maize interaction understanding and also provided new information concerning RZ2MS9 activity when applied with A. brasilense Ab-V5.

molecular biology↗