Dörr Tobias, Lam Hubert, Alvarez Laura, Cava Felipe, Davis Brigid M, Waldor Matthew K
Division of Infectious Diseases, Brigham & Women's Hospital and Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, United States of America; Howard Hughes Medical Institute, Chevy Chase, Maryland, United States of America.
Division of Infectious Diseases, Brigham & Women's Hospital and Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, United States of America.
PLoS Genet. 2014 Jun 19;10(6):e1004433. doi: 10.1371/journal.pgen.1004433. eCollection 2014 Jun.
The bacterial cell wall, which is comprised of a mesh of polysaccharide strands crosslinked via peptide bridges (peptidoglycan, PG), is critical for maintenance of cell shape and survival. PG assembly is mediated by a variety of Penicillin Binding Proteins (PBP) whose fundamental activities have been characterized in great detail; however, there is limited knowledge of the factors that modulate their activities in different environments or growth phases. In Vibrio cholerae, the cause of cholera, PG synthesis during the transition into stationary phase is primarily mediated by the bifunctional enzyme PBP1A. Here, we screened an ordered V. cholerae transposon library for mutants that are sensitive to growth inhibition by non-canonical D-amino acids (DAA), which prevent growth and maintenance of cell shape in PBP1A-deficient V. cholerae. In addition to PBP1A and its lipoprotein activator LpoA, we found that CsiV, a small periplasmic protein with no previously described function, is essential for growth in the presence of DAA. Deletion of csiV, like deletion of lpoA or the PBP1A-encoding gene mrcA, causes cells to lose their rod shape in the presence of DAA or the beta-lactam antibiotic cefsulodin, and all three mutations are synthetically lethal with deletion of mrcB, which encodes PBP1B, V. cholerae's second key bifunctional PBP. CsiV interacts with LpoA and PG but apparently not with PBP1A, supporting the hypothesis that CsiV promotes LpoA's role as an activator of PBP1A, and thereby modulates V. cholerae PG biogenesis. Finally, the requirement for CsiV in PBP1A-mediated growth of V. cholerae can be overcome either by augmenting PG synthesis or by reducing PG degradation, thereby highlighting the importance of balancing these two processes for bacterial survival.
细菌细胞壁由通过肽桥交联的多糖链网组成（肽聚糖，PG），对维持细胞形状和存活至关重要。PG组装由多种青霉素结合蛋白（PBP）介导，其基本活性已得到详细表征；然而，对于在不同环境或生长阶段调节其活性的因素了解有限。在霍乱弧菌（霍乱的病原体）中，进入稳定期时的PG合成主要由双功能酶PBP1A介导。在这里，我们筛选了一个有序的霍乱弧菌转座子文库，寻找对非经典D-氨基酸（DAA）生长抑制敏感的突变体，DAA会阻止PBP1A缺陷型霍乱弧菌的生长和细胞形状维持。除了PBP1A及其脂蛋白激活剂LpoA，我们发现CsiV（一种以前没有描述过功能的小周质蛋白）在有DAA存在时对生长至关重要。删除csiV，就像删除lpoA或编码PBP1A的基因mrcA一样，会导致细胞在有DAA或β-内酰胺抗生素头孢磺啶存在时失去杆状形状，并且所有这三个突变与删除编码PBP1B（霍乱弧菌的第二个关键双功能PBP）的mrcB都是合成致死的。CsiV与LpoA和PG相互作用，但显然不与PBP1A相互作用，支持了CsiV促进LpoA作为PBP1A激活剂的作用，从而调节霍乱弧菌PG生物合成的假设。最后，通过增加PG合成或减少PG降解可以克服霍乱弧菌在PBP1A介导的生长中对CsiV的需求，从而突出了平衡这两个过程对细菌存活的重要性。
