Significance Bacteria must respond quickly to environmental changes to survive. One way bacteria can respond to environmental stress is by undergoing a lifestyle transition from individual, free-swimming cells to a surface-associated community called a biofilm characterized by aggregative growth. The opportunistic pathogen Pseudomonas aeruginosa uses the Wsp chemosensory system to sense an unknown surface-associated cue. Here we show that the Wsp system senses cell envelope stress, specifically conditions that promote unfolded or misregulated periplasmic and inner membrane proteins. This work provides direct evidence that cell envelope stress is an important feature of surface sensing in P. aeruginosa.

Summary Envelope‐localized proteins, such as adhesins and secretion systems, play critical roles in host infection by Gram‐negative pathogens. As such, their folding is monitored by envelope stress response systems. Previous studies demonstrated that the Cpx envelope stress response is required for virulence of Citrobacter rodentium , a murine pathogen used to model infections by the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli ; however, the mechanisms by which the Cpx response promotes host infection were previously unknown. Here, we characterized the C. rodentium Cpx regulon in order to identify genes required for host infection. Using transcriptomic and proteomic approaches, we found that the Cpx response upregulates envelope‐localized protein folding and degrading factors but downregulates pilus genes and type III secretion effectors. Mouse infections with C. rodentium strains lacking individual Cpx‐regulated genes showed that the chaperone/protease DegP and the disulfide bond oxidoreductase DsbA were essential for infection, but Cpx regulation of these genes did not fully account for attenuation of C. rodentium ΔcpxRA . Both deletion of dsbA and treatment with the reducing agent dithiothreitol activated the C. rodentium Cpx response, suggesting that it may sense disruption of disulfide bonding. Our results highlight the importance of envelope protein folding in host infection by Gram‐negative pathogens.

Conjugation is a major mechanism for dissemination of antibiotic resistance and virulence among bacterial populations. The tra operon in the F family of conjugative plasmids encodes most of the proteins involved in bacterial conjugation. This work reveals that activation of tra operon transcription requires two proteins, TraJ and ArcA, to bind cooperatively to adjacent sites immediately upstream of the major tra promoter P Y . The interaction of TraJ and ArcA with the tra operon not only relieves P Y from silencers but also directly activates it. These findings provide insights into the regulatory circuit of the F-family plasmid-mediated bacterial conjugation.

SUMMARY Schistosoma japonicum , a human blood fluke, causes a parasitic disease affecting millions of people in Asia. Thioredoxin–glutathione system of S. japonicum plays a critical role in maintaining the redox balance in parasite, which is a potential target for development of novel antischistosomal agents. Here we cloned the gene of S. japonicum thioredoxin (SjTrx), expressed and purified the recombinant SjTrx in Escherichia coli . Functional assay shows that SjTrx catalyses the dithiothreitol (DTT) reduction of insulin disulphide bonds. The coupling assay of SjTrx with its endogenous reductase, thioredoxin glutathione reductase from S. japonicum (SjTGR), supports its biological function to maintain the redox homeostasis in the cell. Furthermore, the crystal structure of SjTrx in the oxidized state was determined at 2·0 Å resolution, revealing a typical architecture of thioredoxin fold. The structural information of SjTrx provides us important clues for understanding the maintenance function of redox homeostasis in S. japonicum and pathogenesis of this chronic disease.

The conjugative transfer of bacterial F plasmids relies on TraM, a plasmid-encoded protein that recognizes multiple DNA sites to recruit the plasmid to the conjugative pore. In spite of the high degree of amino acid sequence conservation between TraM proteins, many of these proteins have markedly different DNA binding specificities that ensure the selective recruitment of a plasmid to its cognate pore. Here we present the structure of F TraM RHH (ribbon–helix–helix) domain bound to its sbmA site. The structure indicates that a pair of TraM tetramers cooperatively binds an underwound sbmA site containing 12 base pairs per turn. The sbmA is composed of 4 copies of a 5-base-pair motif, each of which is recognized by an RHH domain. The structure reveals that a single conservative amino acid difference in the RHH β-ribbon between F and pED208 TraM changes its specificity for its cognate 5-base-pair sequence motif. Specificity is also dictated by the positioning of 2-base-pair spacer elements within sbmA; in F sbmA, the spacers are positioned between motifs 1 and 2 and between motifs 3 and 4, whereas in pED208 sbmA, there is a single spacer between motifs 2 and 3. We also demonstrate that a pair of F TraM tetramers can cooperatively bind its sbmC site with an affinity similar to that of sbmA in spite of a lack of sequence similarity between these DNA elements. These results provide a basis for the prediction of the DNA binding properties of the family of TraM proteins.

Thioredoxin glutathione reductase from Schistosoma japonicum (SjTGR), a multifunctional enzyme, plays a vital role in antioxidant pathways and is considered to be a potential drug target for the development of antischistosomal chemotherapy. In this study, two constructs of a truncated form of SjTGR without the last two residues (Sec597-Gly598) were cloned, overexpressed and purified using wild-type and codon-optimized genes. Only SjTGR from the wild-type gene was found to form a complex with flavin adenine dinucleotide (FAD), which could be crystallized in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 84.185, b = 86.47, c = 183.164 Å, at 295 K using the hanging-drop vapour-diffusion method. One dimer was present in the crystallographic asymmetric unit and the calculated Matthews coefficient (VM) and solvent content were 2.6 Å(3) Da(-1) and 52.8%, respectively. Structural determination of SjTGR is in progress using the molecular-replacement method.

TraJ is the essential activator of P-Y, the promoter of the F and F-like plasmid tra operon that encodes the majority of the proteins for bacterial conjugation. By combining error-prone PCR mutagenesis with a two-plasmid screen, we isolated 55 missense mutations in traJ, each affecting the ability of TraJ to activate P-Y. These mutations define two distinct functional clusters (amino acids [aa] 21 to 117 and aa 150 to 219). Limited proteolytic analysis of TraJ suggested that the N- and C-terminal functional clusters are two structurally distinct domains. Most TraJ mutants exhibited decreased intracellular protein levels, and the HslVU protease-chaperone pair was found to be responsible for degrading those mutants without extracytoplasmic stress-induced overexpression. In vivo cross-linking analysis of TraJ mutants indicated that the N-terminal domain is responsible for dimerization. This was confirmed by the finding that the purified N-terminal region of TraJ forms dimers in solution. The levels of dimerization and in vivo activities of TraJ mutants are well correlated, suggesting that dimerization of TraJ is required for its biological function. We propose that the regulation of TraJ dimerization and/or its susceptibility to HslVU could be a key mechanism in various signaling processes for controlling bacterial conjugation in response to physiological or environmental stimuli.