Agrobacterium tumefaciens Growth Pole Ring Protein: C Terminus and Internal Apolipoprotein Homologous Domains Are Essential for Function and Subcellular Localization

Abstract

The Agrobacterium growth pole ring (GPR) protein forms a hexameric ring at the growth pole (GP) that is essential for polar growth. GPR is large (2,115 amino acids) and contains 1,700 amino acids of continuous α-helices. To dissect potential GPR functional domains, we created deletions of regions with similarity to human apolipoprotein A-IV (396 amino acids), itself composed of α-helical domains. We also tested deletions of the GPR C terminus. Deletions were inducibly expressed as green fluorescent protein (GFP) fusion proteins and tested for merodiploid interference with wild-type (WT) GPR function, for partial function in cells lacking GPR, and for formation of paired fluorescent foci (indicative of hexameric rings) at the GP. Deletion of domains similar to human apolipoprotein A-IV in GPR caused defects in cell morphology when expressed in trans to WT GPR and provided only partial complementation to cells lacking GPR. Agrobacterium-specific domains A-IV-1 and A-IV-4 contain predicted coiled coil (CC) regions of 21 amino acids; deletion of CC regions produced severe defects in cell morphology in the interference assay. Mutants that produced the most severe effects on cell shape also failed to form paired polar foci. Modeling of A-IV-1 and A-IV-4 reveals significant similarity to the solved structure of human apolipoprotein A-IV. GPR C-terminal deletions profoundly blocked complementation. Finally, peptidoglycan (PG) synthesis is abnormally localized circumferentially in cells lacking GPR. The results support the hypothesis that GPR plays essential roles as an organizing center for membrane and PG synthesis during polar growth. IMPORTANCE Bacterial growth and division are extensively studied in model systems (Escherichia coli, Bacillus subtilis, and Caulobacter crescentus) that grow by dispersed insertion of new cell wall material along the length of the cell. An alternative growth mode—polar growth—is used by some Actinomycetales and Proteobacteria species. The latter phylum includes the family Rhizobiaceae, in which many species, including Agrobacterium tumefaciens, exhibit polar growth. Current research aims to identify growth pole (GP) factors. The Agrobacterium growth pole ring (GPR) protein is essential for polar growth and forms a striking hexameric ring structure at the GP. GPR is long (2,115 amino acids), and little is known about regions essential for structure or function. Genetic analyses demonstrate that the C terminus of GPR, and two internal regions with homology to human apolipoproteins (that sequester lipids), are essential for GPR function and localization to the GP. We hypothesize that GPR is an organizing center for membrane and cell wall synthesis during polar growth.

RdsA Is a Global Regulator That Controls Cell Shape and Division in Rhizobium etli

“…Interestingly, the master cell division regulator CtrA ( Mann et al, 2016 ) was not downregulated ( Figure 6 and Supplementary Figure 4 ). On the other hand, genes such as pleC ( Kim et al, 2013 ), podZ ( Grangeon et al, 2017 ; Howell et al, 2017 ), popJ ( Fields et al, 2012 ; Anderson-Furgeson et al, 2016 ), divK, divJ ( Pini et al, 2013 ), and GPR ( Zupan et al, 2019 , 2021 ), participating in determination of asymmetric and unipolar growth, displayed normal expression levels in the cKD24 mutant. The downregulation in many genes for cell division suggests a major perturbation in cell division, which is consistent with the longer cell division times and cell division problems exhibited by the cKD24 mutant ( Figures 2 , 3 and Supplementary Movies 2 – 4 ).…”

“…Despite this caveat, the combination of changes observed is unusual among mutants affecting cell division in Rhizobiales. Regarding cell shape, round cells were detected upon depletion of GPR in A. tumefaciens ( Zupan et al, 2019 , 2021 ) or its ortholog ( rgsE ) in S. meliloti 1021 ( Krol et al, 2020 ), as well as upon elimination of cpdR in S. meliloti 1021 ( Kobayashi et al, 2009 ) or ntrY in S. meliloti GR4 ( Calatrava-Morales et al, 2017 ). A round cell phenotype has been described also in cells that overproduce RgsM in S. meliloti 1021 ( Schäper et al, 2018 ).…”

Centromere Interactions Promote the Maintenance of the Multipartite Genome in Agrobacterium tumefaciens

“…Since all the replicons have the origins localized to the cell pole, one possibility is that polar organizer proteins interact with each origin separately and indirectly cluster the origins together. In A. tumefaciens , three such proteins are known: PodJ, PopZ, and GPR ( 13 – 18 , 20 ). We found that these polar organizers are not required for the colocalization or clustering of the origins, suggesting that ParB1-RepBs directly interact with each other and cluster together.…”

“…There are three proteins (GPR, PopZ, and PodJ) that are thought to serve as polar organizing proteins. At the growth pole, GPR and PopZ are both required for unipolar growth, cell shape, and normal cell division ( 13 – 18 ). Moreover, in the absence of PopZ, the origin of Ch1 (or ori1 ) disassociates from the new pole, indicating that PopZ anchors the origin to the new pole ( 14 ), likely through interaction with ParB1 protein ( 19 ).…”

Outside-in: intracellular vesicles in giant sulfur bacteria contain peptidoglycan

“…Homologues were not detected in Ca. T. nelsonii's genomes nor did we find homologues to other known bacterial IFs [101][102][103][104][105][106][107][108][109][110][111][112][113][114][115]. The homopolymer bactofilin, CcmA (pfam04519), an alternative to IFs in bacteria [116], has been found broadly across bacteria and is present in Ca.…”