Proteomic and transcriptomic characterization of a virulence-deficient phosphatidylcholine-negative Agrobacterium tumefaciens mutant

Abstract

Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic membranes, whereas only a limited number of bacteria are able to synthesize PC. Intriguingly, many of the bacteria with PC-containing membranes interact with eukaryotic hosts. PC is one of the major membrane lipids in the phytopathogenic bacterium Agrobacterium tumefaciens. The presence of PC is critical for diverse cellular processes like motility, biofilm formation, stress resistance, and virulence. The exact role of PC in these processes is unknown. Here, we examined the global consequences of the complete loss of PC at the proteomic and transcriptomic levels. Both strategies validated the impaired virulence gene induction responsible for the virulence defect of the PC-deficient mutant. In addition, the proteomic approach revealed a limited subset of proteins with altered abundance including the reduced flagellar proteins FlaA and FlaB, which explains the motility defect of the PC mutant. At the whole-genome level, the loss of PC was correlated with altered expression of up to 13% of all genes, most encoding membrane or membrane-associated proteins and proteins with functions in the extracytoplasmic stress response. Our integrated analysis revealed that A. tumefaciens dynamically remodels its membrane protein composition in order to sustain normal growth in the absence of PC.

A Genome-Wide Survey of Highly Expressed Non-Coding RNAs and Biological Validation of Selected Candidates in Agrobacterium tumefaciens

“…Other vir operons, such as virB , virC and virD were only expressed under induction condition. These vir genes expression patterns were consistent with previous microarray and RNA-seq studies (Table S1 in File S1) [38], [51].…”

“…Several transcriptome analyses using microarrays have identified differentially expressed genes in A. tumefaciens in response to various conditions, such as low pH [36], plant-derived signaling molecules [37], and the loss of phosphatidylcholine biosynthesis [38]. A few recent studies showed that A. tumefaciens uses ncRNAs to regulate important biological processes, such as Ti plasmid replication [39] and the uptake of a plant-derived signaling molecule, γ-amynobutyric acid (GABA) by an ABC transporter [40].…”

Loss of PodJ in Agrobacterium tumefaciens Leads to Ectopic Polar Growth, Branching, and Reduced Cell Division

“…⌬podJ At was created through allelic exchange resulting in a markerless deletion of the Atu0499 open reading frame and the short hypothetical Atu0500 open reading frame that overlaps the 5= end of Atu0499. Transcription of Atu0500, however, was not detected in microarray data, indicating that it is not likely expressed (25). Deletion of podJ At was verified by PCR amplification of the Atu0499 locus and flanking regions from genomic DNA and sequencing of the PCR product.…”

Peptidoglycan Synthesis Machinery in Agrobacterium tumefaciens During Unipolar Growth and Cell Division

“…A subset of the A. tumefaciens LDTs were selected for localization studies to sample the various phylogenetic branches and to test the LDTs that were most highly expressed in transcriptome studies (28). A C-terminal superfolding GFP (sfGFP) (29, 30) fusion to Atu0845, one of the Rhizobiales / Rhodobacterales -specific LDTs, exhibited a striking localization to the growth pole (Fig.…”