Genetic and Biochemical Characterization of the MinC-FtsZ Interaction in Bacillus subtilis

Abstract

Cell division in bacteria is regulated by proteins that interact with FtsZ and modulate its ability to polymerize into the Z ring structure. The best studied of these regulators is MinC, an inhibitor of FtsZ polymerization that plays a crucial role in the spatial control of Z ring formation. Recent work established that E. coli MinC interacts with two regions of FtsZ, the bottom face of the H10 helix and the extreme C-terminal peptide (CTP). Here we determined the binding site for MinC on Bacillus subtilis FtsZ. Selection of a library of FtsZ mutants for survival in the presence of Min overexpression resulted in the isolation of 13 Min-resistant mutants. Most of the substitutions that gave rise to Min resistance clustered around the H9 and H10 helices in the C-terminal domain of FtsZ. In addition, a mutation in the CTP of B. subtilis FtsZ also produced MinC resistance. Biochemical characterization of some of the mutant proteins showed that they exhibited normal polymerization properties but reduced interaction with MinC, as expected for binding site mutations. Thus, our study shows that the overall architecture of the MinC-FtsZ interaction is conserved in E. coli and B. subtilis. Nevertheless, there was a clear difference in the mutations that conferred Min resistance, with those in B. subtilis FtsZ pointing to the side of the molecule rather than to its polymerization interface. This observation suggests that the mechanism of Z ring inhibition by MinC differs in both species.

MinC Protein Shortens FtsZ Protofilaments by Preferentially Interacting with GDP-bound Subunits

“…In this study, it was proposed that MinC severs FtsZ polymers by specifically breaking the interaction between FtsZ and GDP present in polymers and the adjacent FtsZ molecule through this ␣-helix. A more recent mutational study on Bacillus subtilis FtsZ found mutations that conferred resistance to MinC in a different, more lateral region of FtsZ than in E. coli, suggesting that the MinC inhibition mechanism may be different in this organism (27).…”

Dynamics of theBacillus subtilisMin system

“…The Min system in E. coli consists of the three proteins MinC, MinD and MinE [15, 16], and has been studied extensively both experimentally [17-29] and theoretically [29-35]. MinC is the inhibitor of Z-ring formation, inhibiting the bundling of FtsZ filaments [22, 36-39]. MinC is localized through MinD, a protein that belongs to the WACA (Walker A cytomotive ATPase) family [40, 41].…”

Estudo do papel de MinD na ativação de MinC, um regulador chave na divisão bacteriana em Bacillus subtilis

“…FtsZ Blasios V., et al, 2013); além disso, estudos mais recentes sugerem que MinC também afeta as interações longitudinais que formam o protofilamento (Shen B. e Lutkenhaus J., 2010;Hernández V. et al, 2013, Arumugam S. et al, 2014. MinC é um dímero que consiste em dois domínios ligados por uma alça flexível, ambos os quais têm atividade inibitória de divisão no contexto correto (Hu Z. et al, 1999;Shen B. e Lutkenhaus J., 2009;Shen B. e Lutkenhaus J., 2010).…”

“…Sabe-se também que o verdadeiro inibidor é a proteína MinC (de Boer P. et al, 1990), e que ela só inibe a divisão na ausência de MinD quando é superexpressa ~50 vezes acima dos níveis endógenos (de Boer P. et al, 1992) (Johnson J. etal., 2002) e a proteína ZipA é requerida para esse recrutamento (Johnson J., et al, 2004 (Blasios V. et al 2013) …”

“…O primeiro, postula que o complexo MinCD atuaria encurtando os protofilamentos, ou seja, inibindo a polimerização longitudinalmente Shen B. e Lutkenhaus J., 2010;Hernández-Rocamora V. et al, 2013;Arumugam S. et al, 2014). O segundo, postula que o mecanismo de ação de MinCD se dá na inibição das interações laterais entre protofilamentos, inibindo a formação de feixes (Scheffers D., 2008;Blasios V. et al, 2013, Ghosal D. et al, 2014 (Johnson J. et al, 2002). Apesar disso, a estrutura resolvida do complexo MinCD de A. aeolicus não mostrou alguma mudança conformacional que indicasse um efeito alostérico em MinC por parte de MinD, apesar do fato de que a região N-terminal não está presente na estrutura.…”