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ABSTRACT Mechanistic understanding of interactions in many host-microbe systems, including the honey bee microbiome, is limited by a lack of easy-to-use genome engineering approaches. To this end, we demonstrate a one-step genome engineering approach for making gene deletions and insertions in the chromosomes of honey bee gut bacterial symbionts. Electroporation of linear or non-replicating plasmid DNA containing an antibiotic resistance cassette flanked by regions with homology to a symbiont genome reliably results in chromosomal integration. This lightweight approach does not require expressing any exogenous recombination machinery. The high concentrations of large DNAs with long homology regions needed to make the process efficient can be readily produced using modern DNA synthesis and assembly methods. We use this approach to knock out genes, including genes involved in biofilm formation, and insert fluorescent protein genes into the chromosome of the betaproteobacterial bee gut symbiontSnodgrassella alvi. We are also able to engineer the genomes of multiple strains ofS. alviand another species,Snodgrassella communis, which is found in the bumble bee gut microbiome. Finally, we use the same method to engineer the chromosome of another bee symbiont,Bartonella apis, which is an alphaproteobacterium. As expected, gene knockout inS. alviusing this approach isrecA-dependent, suggesting that this straightforward procedure can be applied to other microbes that lack convenient genome engineering methods. IMPORTANCEHoney bees are ecologically and economically important crop pollinators with bacterial gut symbionts that influence their health. Microbiome-based strategies for studying or improving bee health have utilized wild-type or plasmid-engineered bacteria. We demonstrate that a straightforward, single-step method can be used to insert cassettes and replace genes in the chromosomes of multiple bee gut bacteria. This method can be used for investigating the mechanisms of host-microbe interactions in the bee gut community and stably engineering symbionts that benefit pollinator health.