Impact of TamB in Rhizobium leguminosarum cell envelope stability

RUSSO DANIELA M.; BIALER MAGALÍ G.; ZIMMERMAN LUCIANA M.; DOMINGUEZ MALDONADO CAROLINA; KWEE PHOEBE; ZORREGUIETA ANGELES

Chapadmalal

Congreso; XVIII Congreso de la Sociedad Argentina de Microbiologia General SAMIGE 2023; 2023

SAMIGE

Our recent studies in Brucella spp., a facultative intracellular animal pathogen thatcauses brucellosis, suggest that the TAM system (Traslocation and Assembly module) isrequired for cell envelope integrity, cell division and full virulence. The TAM complexconsists of the outer membrane protein TamA and the inner membrane protein TamB. Theprecise contribution of this system to the biogenesis of the envelope is yet to be elucidatedin Gram-negative bacteria. Rhizobium leguminosarum is a soil free-living species, closelyrelated to Brucella spp., although they could adopt an endosymbiont lifestyle establishingN2 fixing-nodules in legume roots. Both lifestyles imply survival and adaptation toenvironmental fluctuations in which the bacterial envelope plays significant roles. To studythe role of the TAM system in rhizobia, we identify the TamB/MapB homologue fromRhizobium leguminosarum bv. viciae 3841 (Rlv 3841) by in silico analysis. The RhizobiumTamB orthologue (RL4382) encodes a 2033 amino acid protein that shares 27% amino acidsequence identity with MapB of B. suis (1579 aa). The protein domain prediction showedthat the Rhizobium MapB protein contained two COG2911 conserved domains, whichbelong to TamB superfamily at regions 13-1014 and 932-2033. Prediction of secondarystructure by Phyre2 suggests that beta strand secondary structure is present in most of theprotein (60%) except for an alpha helical (3%) and trans-membrane helix (1%) regions. Agene encoding a TamA homologue is found upstream and in the same orientation ofRL4382 tamB gene, comprising the homonymous operon in Rlv 3841. To study the role ofthis complex in Rhizobium, a deletion mutant was generated by double homologous recombination of tamB (delta tamB) and several envelope-associated phenotypes were studied. No significant differences in bacterial growth were found between wild type and tamB mutant in TY rich medium cultures. The mutant did not exhibit differences in the sensitivity to polymyxin B in comparison to the wild type strain, while in the presence of lysozyme (0.01mg/ml), the optical density of the mutant decreased up to 60% of the initial value after 15 minutes of incubation, indicating bacterial lysis. Besides, increased sensibility to 0.5% TRITON X-100 and 10 mM EDTA were observed in delta tamB, suggesting an alteration in the stability of the envelope. Taken together, these results suggest that absence of TamB affects the tolerance of Rhizobium cell envelope to membrane disrupting agents. Further investigations are required to understand the TAM system function in cell envelopebiogenesis in Rhizobium leguminosarum.