Regulation of cysteine residues in LsrB proteins from Sinorhizobium meliloti under free-living and symbiotic oxidative stress.

The development of legume nitrogen-fixing nodules is regulated by reactive oxygen species (ROS) produced by symbionts. Several regulators from Rhizobium are involved in ROS sensing. In a previous study, we found that Sinorhizobium meliloti LsrB regulates lipopolysaccharide production and is associated with H2 O2 accumulation in alfalfa (Medicago sativa) nodules. However, its underlying regulatory mechanism remains unclear. Here, we report that the cysteine residues in LsrB are required for adaptation to oxidative stress, gene expression, alfalfa nodulation and nitrogen fixation. Moreover, LsrB directly activated the transcription of lrp3 and gshA (encoding γ-glutamylcysteine synthetase, responsible for glutathione synthesis) and this regulation required the cysteine (Cys) residues in the LsrB substrate-binding domain. The Cys residues could sense oxidative stress via the formation of intermolecular disulfide bonds, generating LsrB dimers and LsrB-DNA complexes. Among the Cys residues, C238 is a positive regulatory site for the induction of downstream genes, whereas C146 and C275 play negative roles in the process. The lsrB mutants with Cys-to-Ser substitutions displayed altered phenotypes in respect to their adaptation to oxidative stress, nodulation and nitrogen fixation-related plant growth. Our findings demonstrate that S. meliloti LsrB modulates alfalfa nodule development by directly regulating downstream gene expression via a post-translational strategy.

Structural Model of a Porphyromonas gingivalis type IX Secretion System Shuttle Complex.

Porphyromonas gingivalis is a gram-negative oral anaerobic pathogen and is one of the key causative agents of periodontitis. P. gingivalis utilises a range of virulence factors, including the cysteine protease RgpB, to drive pathogenesis and these are exported and attached to the cell surface via the type IX secretion system (T9SS). All cargo proteins possess a conserved C-terminal signal domain (CTD) which is recognised by the T9SS, and the outer membrane ß-barrel protein PorV (PG0027/LptO) can interact with cargo proteins as they are exported to the bacterial surface. Using a combination of solution nuclear magnetic resonance (NMR) spectroscopy, biochemical analyses, machine-learning-based modelling and molecular dynamics (MD) simulations, we present a structural model of a PorV:RgpB-CTD complex from P. gingivalis. This is the first structural insight into CTD recognition by the T9SS and shows how the conserved motifs in the CTD are the primary sites that mediate binding. In PorV, interactions with extracellular surface loops are important for binding the CTD, and together these appear to cradle and lock RgpB-CTD in place. This work provides insight into cargo recognition by PorV but may also have important implications for understanding other aspects of type-IX dependent secretion.

Molecular and cellular insight into Escherichia coli SslE and its role during biofilm maturation.

Escherichia coli is a Gram-negative bacterium that colonises the human intestine and virulent strains can cause severe diarrhoeal and extraintestinal diseases. The protein SslE is secreted by a range of pathogenic and commensal E. coli strains. It can degrade mucins in the intestine, promotes biofilm maturation and it is a major determinant of infection in virulent strains, although how it carries out these functions is not well understood. Here, we examine SslE from the commensal E. coli Waksman and BL21 (DE3) strains and the enterotoxigenic H10407 and enteropathogenic E2348/69 strains. We reveal that SslE has a unique and dynamic structure in solution and in response to acidification within mature biofilms it can form a unique aggregate with amyloid-like properties. Furthermore, we show that both SslE monomers and aggregates bind DNA in vitro and co-localise with extracellular DNA (eDNA) in mature biofilms, and SslE aggregates may also associate with cellulose under certain conditions. Our results suggest that interactions between SslE and eDNA are important for biofilm maturation in many E. coli strains and SslE may also be a factor that drives biofilm formation in other SslE-secreting bacteria.

Two-component regulatory system ActS/ActR is required for Sinorhizobium meliloti adaptation to oxidative stress.

The two-component system ActS/ActR plays important roles in bacterial adaptation to abiotic stress, including acid tolerance and oxidant resistance. However, the underlying regulatory mechanism is not clear. In this study, we found that the ActS/ActR system is required for adaptation to oxidative stress by regulating the transcription of the genes actR, katB, gshA and gshB1. The actS and actR mutants were sensitive to low pH and oxidants such as H2O2, oxidized glutathione (GSSG) and sodium nitroprusside (SNP). The expression of actR by using a plasmid rescued the defect of SNP sensitivity for all actS and actR mutants. The expression of actS and actR were suppressed by treatment with H2O2. The expression of actS, actR, oxyR, katA and katB was required for ActS and ActR under normal conditions. The induction of katB, gshA and gshB1 depended on ActS and ActR during treatment with H2O2 and SNP. Our findings revealed that the ActS/ActR system is a key redox regulator in S. meliltoi and provides a new cue to understanding Rhizobium-legume symbiosis.