Geert Cremers (Radboud University)

Summary of presentation:
Introduction
Bradyrhizobium is a genus of bacteria commonly found in soil. They are important for the nitrogen cycle, since many of them are capable of nitrogen fixation. Although Bradyrhizobium are metabolically diverse, they are not considered a methanotroph, nor is ammonia oxidation generally considered an associated trait. However, we isolated a Bradyrhizobium species which contains a particulate ammonium or methane monooxygenase (amo/pmo) encoding operon without the presence of a hydroxylamine dehydrogenase (hao). Interestingly, it is located before a polyhydroxyalkanoate (PHA) depolymerase (PHAD) on the genome.

Methods
Bradyrhizobium sp. was isolated as a contaminant from a DSMZ Terriglobus strain and its full genome was sequenced using Nanopore and Illumina technology. Growth experiments in presence or absence of alcohols, glucose, methane and ammonium were performed. In case of growth, RNA-seq was performed to compare expression levels of the genes. PHA was extracted from two week old cultures and analyzed by GC-MS. Phylogenetic trees of genes amoA, amoC and PHAD were constructed using the neighbor joining algorithm.

Results
Growth was observed when growing Bradyrhizobium on glucose or mannitol. When inoculated with a two week old glucose culture, poor growth was observed on CH4 or alcohols but disappeared completely after a new transfer. Growth was postponed or negative when supplying 4.6 mM NH4+ to the medium. Phylogenetically, amoA, amoC and PHAD form unique clades within their respective trees. GC-MS shows that levulinic acid is the most common monomer, hinting towards a polyketid as polymer. RNA-seq showed highly expressed PHA related phasins (5535-15257 RPKM), amoC (2789-6325 RPKM) and PHAD (145-353 RPKM) when grown on butanol, glucose or ethanol. Read mapping to the genome shows a continues transcription of the monooxygenase and PHAD operon.

Conclusion
Bradyrhizobium is capable of producing PHA and it contains an unique amo/pmo operon associated with the depolymerization of PHA.