Topographical and mechanical properties of liposome surfaces harboring Na,K-ATPase by means of atomic force microscopy.

In this study, we obtained unprecedented AFM images of the Na,K-ATPase (NKA) pump after being reconstituted into DPPC and DPPC:DPPE liposomes. The mechanical properties observed in the phase images were associated with protrusions correlated to NKA microdomains, which are the darker areas seen in the AFM phase images. Protrusions in the DPPC-NKA proteoliposomes ranged from 38 to 115 nm, with 74 ± 21 nm diameter and 2.1 ± 1.4 nm height. DPPC:DPPE-NKA proteoliposomes showed protrusions from 21 to 78 nm, with 38 ± 16 nm diameter and 0.7 ± 0.5 nm height. We have estimated the presence of annular lipids in the microdomains considering that the areas of the protrusions should contain αß oligomers and annular phospholipids. For DPPC-NKA proteoliposomes, we hypothesize that 40 phospholipids surround an (αß)2 dimer and 46 phospholipids are present for the DPPC:DPPE-NKA proteoliposomes in an αß monomer. Catalytic activity measurements of both lipid compositions of proteoliposomes harboring NKA provide strong evidence regarding the protein orientation in the biomembrane. AFM data suggest that DPPC-NKA proteoliposomes are also rightside-out protein orientated, where the protrusions have an average height of 2.1 nm, while for DPPC:DPPE-NKA proteoliposomes, the majority of the protein reconstituted should be inside-out orientated, where the protrusions' average height is 0.5 nm. This result corroborates with the enzymatic analysis, where 61% and 91% of the enzymatic activity was recovered, respectively. Thus, a new application of AFM as a tool for the determination of topological features of protrusions in proteoliposomes has been brought to the scientific community, in addition to revealing the distinct catalytic orientation of enzymes present in the biomembranes model.

A Xanthomonas citri subsp citri hypothetical protein related to virulence contains a non-functional HD domain and is implicated in flagellar motility.

Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp citri (Xac), severely affects most economically important citrus varieties worldwide. A previous study showed that disruption of the ORF XAC1201 from the Xac 306 strain by transposon Tn5 decreased bacterium virulence in the Rangpur lime host (Citrus limonia L. Osbeck). However, little is known regarding the possible function of the hypothetical protein XAC1201 and how it affects the virulence of Xac 306. Here, we confirmed that disruption of ORF XAC1201 reduces Xac 306 virulence in two different hosts, delaying the onset of typical symptoms. In silico analysis suggested that XAC1201 interacts with the flagellar proteins FliM and FliL, known to be an important factor for virulence. In fact, motility assays revealed that the XAC1201 mutant has a significant difference in motility compared to the wild-type Xac 306. Also, a 3-D structure model revealed modified cofactor binding sites and suggested that XAC1201 has a non-functional HD domain. This hypothesis was confirmed by enzymatic assays performed in purified, XAC1201 recombinant protein expressed in Escherichia coli, which revealed no significant activities previously associated with HD domains for the tested substrates. Thus, the role of the XAC1201 protein in Xac 306 virulence seems to be related to flagellar motility, although a non-classic role for the HD domain cannot be dismissed.