Role of a high centrality residue in protein dynamics and thermal stability.

Centralities determined from Residue Interaction Networks (RIN) in proteins have been used to predict aspects of their structure and dynamics. Here, we correlate the Eigenvector Centrality (Ec) with the rate constant for thermal denaturation (kden) of the HisF protein from Thermotoga maritima based on 12 single alanine substitution mutants. The molecular basis for this correlation was further explored by studying a mutant containing a replacement of a high Ec residue, Y182A, which displayed increased kden at 80 °C. The crystallographic structure of this mutant showed few changes, mostly in two flexible loops. The 1H-15N -HSQC showed only subtle changes of cross peak positions for residues located near the mutation site and scattered throughout the structure. However, the comparison of the RIN showed that Y182 is the vertex of a set of high centrality residues that spreads throughout the HisF structure, which is lacking in the mutant. Cross-correlation displacements of Cα calculated from a molecular dynamics simulation at different temperatures showed that the Y182A mutation reduced the correlated movements in the HisF structure above 70 °C. 1H-15N NMR chemical shift covariance using temperature as perturbation were consistent with these results. In conclusion the increase in temperature drives the structure of the mutant HisF-Y182A into a less connected state, richer in non-concerted motions, located predominantly in the C-terminal half of the protein where Y182 is placed. Conversely, wild-type HisF responds to increased temperature as a single unit. Hence the replacement of a high Ec residue alters the distribution of thermal energy through HisF structure.

The opportunistic pathogen Stenotrophomonas maltophilia utilizes a type IV secretion system for interbacterial killing.

Bacterial type IV secretion systems (T4SS) are a highly diversified but evolutionarily related family of macromolecule transporters that can secrete proteins and DNA into the extracellular medium or into target cells. It was recently shown that a subtype of T4SS harboured by the plant pathogen Xanthomonas citri transfers toxins into target cells. Here, we show that a similar T4SS from the multi-drug-resistant opportunistic pathogen Stenotrophomonas maltophilia is proficient in killing competitor bacterial species. T4SS-dependent duelling between S. maltophilia and X. citri was observed by time-lapse fluorescence microscopy. A bioinformatic search of the S. maltophilia K279a genome for proteins containing a C-terminal domain conserved in X. citri T4SS effectors (XVIPCD) identified twelve putative effectors and their cognate immunity proteins. We selected a putative S. maltophilia effector with unknown function (Smlt3024) for further characterization and confirmed that it is indeed secreted in a T4SS-dependent manner. Expression of Smlt3024 in the periplasm of E. coli or its contact-dependent delivery via T4SS into E. coli by X. citri resulted in reduced growth rates, which could be counteracted by expression of its cognate inhibitor Smlt3025 in the target cell. Furthermore, expression of the VirD4 coupling protein of X. citri can restore the function of S. maltophilia ΔvirD4, demonstrating that effectors from one species can be recognized for transfer by T4SSs from another species. Interestingly, Smlt3024 is homologous to the N-terminal domain of large Ca2+-binding RTX proteins and the crystal structure of Smlt3025 revealed a topology similar to the iron-regulated protein FrpD from Neisseria meningitidis which has been shown to interact with the RTX protein FrpC. This work expands our current knowledge about the function of bacteria-killing T4SSs and increases the panel of effectors known to be involved in T4SS-mediated interbacterial competition, which possibly contribute to the establishment of S. maltophilia in clinical and environmental settings.