Developing sustainable agricultural practices will require increasing our understanding of plant-microbe interactions. To study these interactions, new genetic tools for manipulating nonmodel microbes will be needed. To help meet this need, we recently reported development of chassis-independent recombinase-assisted genome engineering (CRAGE). CRAGE relies on cassette exchange between two pairs of mutually exclusive lox sites and allows direct, single-step chromosomal integration of large, complex gene constructs into diverse bacterial species. We then extended CRAGE by introducing a third mutually exclusive lox site, creating CRAGE-Duet, which allows modular integration of two constructs. CRAGE-Duet offers advantages over CRAGE, especially when a cumbersome recloning step is required to build single-integration constructs. To demonstrate the utility of CRAGE-Duet, we created a set of strains from the plant-growth-promoting rhizobacterium Pseudomonas simiae WCS417r that expressed various fluorescence marker genes. We visualized these strains simultaneously under a confocal microscope, demonstrating the usefulness of CRAGE-Duet for creating biological systems to study plant-microbe interactions.

Chemical signalling in the plant microbiome can have drastic effects on microbial community structure, and on host growth and development. Previously, we demonstrated that the auxin metabolic signal interference performed by the bacterial genus Variovorax via an auxin degradation locus was essential for maintaining stereotypic root development in an ecologically relevant bacterial synthetic community. Here, we dissect the Variovorax auxin degradation locus to define the genes iadDE as necessary and sufficient for indole-3-acetic acid (IAA) degradation and signal interference. We determine the crystal structures and binding properties of the operon's MarR-family repressor with IAA and other auxins. Auxin degradation operons were identified across the bacterial tree of life and we define two distinct types on the basis of gene content and metabolic products: iac-like and iad-like. The structures of MarRs from representatives of each auxin degradation operon type establish that each has distinct IAA-binding pockets. Comparison of representative IAA-degrading strains from diverse bacterial genera colonizing Arabidopsis plants show that while all degrade IAA, only strains containing iad-like auxin-degrading operons interfere with auxin signalling in a complex synthetic community context. This suggests that iad-like operon-containing bacterial strains, including Variovorax species, play a key ecological role in modulating auxins in the plant microbiome.

The genus Caldicellulosiruptor contains extremely thermophilic, cellulolytic bacteria capable of lignocellulose deconstruction. Currently, complete genome sequences for eleven Caldicellulosiruptor species are available. Here, we report genome sequences for three additional Caldicellulosiruptor species: Rt8.B8 DSM 8990 (New Zealand), Wai35.B1 DSM 8977 (New Zealand), and "Thermoanaerobacter cellulolyticus" strain NA10 DSM 8991 (Japan).