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Abstract Aerobic methanotrophic bacteria are the primary organisms that consume atmospheric methane (CH₄) and have potential to mitigate the climate-active gas. However, a limited understanding of the genetic determinants of methanotrophy hinders the development of biotechnologies leveraging these unique microbes. Here, we developed and optimized a methanotroph CRISPR interference (CRISPRi) system to enable functional genomic screening. We built a genome-wide single guide RNA (sgRNA) library in the industrial methanotroph,Methylococcus capsulatus, consisting of ∼45,000 unique sgRNAs mediating inducible, CRISPRi-dependent transcriptional repression. A selective screen during growth on CH₄identified 233 genes whose transcription repression resulted in a fitness defect and repression of 13 genes associated with a fitness advantage. Enrichment analysis of the 233 putative essential genes linked many of the encoded proteins with critical cellular processes like ribosome biosynthesis, translation, transcription, and other central biosynthetic metabolism, highlighting the utility of CRISPRi for functional genetic screening in methanotrophs, including the identification of novel essential genes.M. capsulatusgrowth was inhibited when the CRISPRi system was used to individually target genes identified in the screen, validating their essentiality for methanotrophic growth. Collectively, our results show that the CRISPRi system and sgRNA library developed here can be used for facile gene-function analyses and genomic screening to identify novel genetic determinants of methanotrophy. These CRISPRi screening methodologies can also be applied to high-throughput engineering approaches for isolation of improved methanotroph biocatalysts. 

Symbiotic nitrogen fixation is a complex and regulated process that takes place in root nodules of legumes and allows legumes to grow in soils that lack nitrogen. Nitrogen is mostly acquired from the soil as nitrate and its level in the soil affects nodulation and nitrogen fixation. The mechanism(s) by which legumes modulate nitrate uptake to regulate nodule symbiosis remain unclear. In Medicago truncatula , the MtNPF1.7 transporter has been shown to control nodulation, symbiosis, and root architecture. MtNPF1.7 belongs to the nitrate/peptide transporter family and is a symporter with nitrate transport driven by proton(s). In this study we combined in silico structural predictions with in planta complementation of the severely defective mtnip-1 mutant plants to understand the role of a series of distinct amino acids in the transporter’s function. Our results support hypotheses about the functional importance of the ExxE(R/K) motif including an essential role for the first glutamic acid of the motif in proton(s) and possibly substrate transport. Results reveal that Motif A, a motif conserved among major facilitator transport (MFS) proteins, is essential for function. We hypothesize that it participates in intradomain packing of transmembrane helices and stabilizing one conformation during transport. Our results also question the existence of a putative TMH4-TMH10 salt bridge. These results are discussed in the context of potential nutrient transport functions for MtNPF1.7. Our findings add to the knowledge of the mechanism of alternative conformational changes as well as symport transport in NPFs and enhance our knowledge of the mechanisms for nitrate signaling.