Dual function of LapB (YciM) in regulating Escherichia coli lipopolysaccharide synthesis.

Levels of lipopolysaccharide (LPS), an essential glycolipid on the surface of most gram-negative bacteria, are tightly controlled-making LPS synthesis a promising target for developing new antibiotics. Escherichia coli adaptor protein LapB (YciM) plays an important role in regulating LPS synthesis by promoting degradation of LpxC, a deacetylase that catalyzes the first committed step in LPS synthesis. Under conditions where LPS is abundant, LapB recruits LpxC to the AAA+ protease FtsH for degradation. LapB achieves this by simultaneously interacting with FtsH through its transmembrane helix and LpxC through its cytoplasmic domain. Here, we describe a cryo-EM structure of the complex formed between LpxC and the cytoplasmic domain of LapB (LapBcyto). The structure reveals how LapB exploits both its tetratricopeptide repeat (TPR) motifs and rubredoxin domain to interact with LpxC. Through both in vitro and in vivo analysis, we show that mutations at the LapBcyto/LpxC interface prevent LpxC degradation. Unexpectedly, binding to LapBcyto also inhibits the enzymatic activity of LpxC through allosteric effects reminiscent of LpxC activation by MurA in Pseudomonas aeruginosa. Our findings argue that LapB regulates LPS synthesis in two steps: In the first step, LapB inhibits the activity of LpxC, and in the second step, it commits LpxC to degradation by FtsH.

Mutational analysis of predicted active site residues of an exoglucanase secreted by Xanthomonas oryzae pv. oryzae to determine their role in catalysis and in virulence on rice.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight disease in rice. As a part of its virulence repertoire, Xoo secretes a cell wall degrading enzyme Cellobiosidase (CbsA), which is a critical virulence factor and also a determinant of tissue specificity. CbsA protein is made up of an N-terminal catalytic domain and a C-terminal fibronectin type III domain. According to the CAZy classification, the catalytic domain of CbsA protein belongs to the glycosyl hydrolase-6 (GH6) family that performs acid-base catalysis. However, the identity of the catalytic acid and the catalytic base of CbsA is not known. Based on the available structural and biochemical data, we identified putative catalytic residues and probed them by site-directed mutagenesis. Intriguingly, the biochemical analysis showed that none of the mutations abolishes the catalytic activity of CbsA, an observation that is contrary to other GH6 family members. All the mutants exhibited altered enzymatic activity and caused significant virulence deficiency in Xoo emphasising the requirement of specific exoglucanase activity of wild-type CbsA for virulence on rice. Our study highlights the need for further studies and the detailed characterisation of bacterial exoglucanases.