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.

Role of the FnIII domain associated with a cell wall-degrading enzyme cellobiosidase of Xanthomonas oryzae pv. oryzae.

Cellobiosidase (CbsA) is an important secreted virulence factor of Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight of rice. CbsA is one of several cell wall-degrading enzymes secreted by Xoo via the type II secretion system (T2SS). CbsA is considered a fundamental virulence factor for vascular pathogenesis. CbsA has an N-terminal glycosyl hydrolase domain and a C-terminal fibronectin type III (FnIII) domain. Interestingly, the secreted form of CbsA lacks the FnIII domain during in planta growth. Here we show that the presence of the FnIII domain inhibits the enzyme activity of CbsA on polysaccharide substrates like carboxymethylcellulose. The FnIII domain is required for the interaction of CbsA with SecB chaperone, and this interaction is crucial for the stability and efficient transport of CbsA across the inner membrane. Deletion of the FnIII domain reduced virulence similar to ΔcbsA Xoo, which corroborates the importance of the FnIII domain in CbsA. Our work elucidates a hitherto unknown function of the FnIII domain in enabling the virulence-promoting activity of CbsA.

Suppression of XopQ-XopX-induced immune responses of rice by the type III effector XopG.

Effectors that suppress effector-triggered immunity (ETI) are an essential part of the arms race in the co-evolution of bacterial pathogens and their host plants. Xanthomonas oryzae pv. oryzae uses multiple type III secretion system (T3SS) secreted effectors such as XopU, XopV, XopP, XopG, and AvrBs2 to suppress rice immune responses that are induced by the interaction of two other effectors, XopQ and XopX. Here we show that each of these five suppressors can interact individually with both XopQ and XopX. One of the suppressors, XopG, is a predicted metallopeptidase that appears to have been introduced into X. oryzae pv. oryzae by horizontal gene transfer. XopQ and XopX interact with each other in the nucleus while interaction with XopG sequesters them in the cytoplasm. The XopG E76A and XopG E85A mutants are defective in interaction with XopQ and XopX, and are also defective in suppression of XopQ-XopX-mediated immune responses. Both mutations individually affect the virulence-promoting ability of XopG. These results indicate that XopG is important for X. oryzae pv. oryzae virulence and provide insights into the mechanisms by which this protein suppresses ETI in rice.

A mutation in an exoglucanase of Xanthomonas oryzae pv. oryzae, which confers an endo mode of activity, affects bacterial virulence, but not the induction of immune responses, in rice.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, a serious disease of rice. Xoo secretes a repertoire of cell wall-degrading enzymes, including cellulases, xylanases and pectinases, to degrade various polysaccharide components of the rice cell wall. A secreted Xoo cellulase, CbsA, is not only a key virulence factor of Xoo, but is also a potent inducer of innate immune responses of rice. In this study, we solved the crystal structure of the catalytic domain of the CbsA protein to a resolution of 1.86 Å. The core structure of CbsA shows a central distorted TIM barrel made up of eight ß strands with N- and C-terminal loops enclosing the active site, which is a characteristic structural feature of an exoglucanase. The aspartic acid at the 131st position of CbsA was predicted to be important for catalysis and was therefore mutated to alanine to study its role in the catalysis and biological functions of CbsA. Intriguingly, the D131A CbsA mutant protein displayed the enzymatic activity of a typical endoglucanase. D131A CbsA was as proficient as wild-type (Wt) CbsA in inducing rice immune responses, but was deficient in virulence-promoting activity. This indicates that the specific exoglucanase activity of the Wt CbsA protein is required for this protein to promote the growth of Xoo in rice.

Mutations in the Predicted Active Site of Xanthomonas oryzae pv. oryzae XopQ Differentially Affect Virulence, Suppression of Host Innate Immunity, and Induction of the HR in a Nonhost Plant.

Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, secretes a number of effectors through a type 3 secretion system. One of these effectors, called XopQ, is required for virulence and suppression of rice innate immune responses induced by the plant cell-wall-degrading enzyme lipase/esterase A (LipA). Bioinformatic analysis suggested that XopQ is homologous to inosine-uridine nucleoside hydrolases (NH). A structural model of XopQ with the protozoan Crithidia fasciculata purine NH suggested that D116 and Y279 are potential active site residues. X. oryzae pv. oryzae xopQ mutants (xopQ-/pHM1::xopQD116A and xopQ-/pHM1::xopQY279A) show reduced virulence on rice compared with xopQ-/pHM1::xopQ. The two predicted XopQ active site mutants (xopQ-/pHM1::xopQD116A and xopQ-/pHM1::xopQY279A) exhibit a reduced hypersensitive response (HR) on Nicotiana benthamiana, a nonhost. However, Arabidopsis lines expressing either xopQ or xopQY279A are equally proficient at suppression of LipA-induced callose deposition. Purified XopQ does not show NH activity on standard nucleoside substrates but exhibits ribose hydrolase activity on the nucleoside substrate analogue 4-nitrophenyl ß-D-ribofuranoside. The D116A and Y279A mutations cause a reduction in biochemical activity. These results indicate that mutations in the predicted active site of XopQ affect virulence and induction of the HR but do not affect suppression of innate immunity.