A Metagenome-Wide Association Study and Arrayed Mutant Library Confirm Acetobacter Lipopolysaccharide Genes Are Necessary for Association with Drosophila melanogaster

G3 : Genes - Genomes - Genetics
K Makay WhiteJohn M Chaston

Abstract

A metagenome wide association (MGWA) study of bacterial host association determinants in Drosophila predicted that LPS biosynthesis genes are significantly associated with host colonization. We were unable to create site-directed mutants for each of the predicted genes in Acetobacter, so we created an arrayed transposon insertion library using Acetobacter fabarum DsW_054 isolated from Drosophila Creation of the A. fabarum DsW_054 gene knock-out library was performed by combinatorial mapping and Illumina sequencing of random transposon insertion mutants. Transposon insertion locations for 6,418 mutants were successfully mapped, including hits within 63% of annotated genes in the A. fabarum DsW_054 genome. For 45/45 members of the library, insertion sites were verified by arbitrary PCR and Sanger sequencing. Mutants with insertions in four different LPS biosynthesis genes were selected from the library to validate the MGWA predictions. Insertion mutations in two genes biosynthetically upstream of Lipid-A formation, lpxC and lpxB, show significant differences in host association, whereas mutations in two genes encoding LPS biosynthesis functions downstream of Lipid-A biosynthesis had no effect. These results suggest an impact of b...Continue Reading

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Citations

Nov 6, 2018·Applied and Environmental Microbiology·Andrew J Sommer, Peter D Newell
Mar 8, 2020·Applied and Environmental Microbiology·Melinda K MatthewsJohn M Chaston
Feb 23, 2020·Frontiers in Microbiology·James Emmanuel SanTulio de Oliveira
Oct 15, 2020·The Journal of Experimental Biology·John G McMullenAngela E Douglas
Sep 30, 2021·Microbiology Spectrum·Melinda K MatthewsJohn M Chaston

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Datasets Mentioned

BETA
PRJNA422683

Methods Mentioned

BETA
gene knock-out
PCR
Illumina sequencing
gene knockouts
genetic modification

Software Mentioned

TnSeq
BLASTKOALA
R package MAGNAMWAR
MGWA
RAST
KEGG Mapper – Search Pathway
KEGG pathway mapper
OrthoMCL

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