Structure of a translocation signal domain mediating conjugative transfer by Type IV secretion systems

Relaxases are proteins responsible for the transfer of plasmid and chromosomal DNA from one bacterium to another during conjugation. They covalently react with a specific phosphodiester bond within DNA origin of transfer sequences, forming a nucleo-protein complex which is subsequently recruited for transport by a plasmid-encoded type IV secretion system. In previous work we identified the targeting translocation signals presented by the conjugative relaxase TraI of plasmid R1. Here we report the structure of TraI translocation signal TSA. In contrast to known translocation signals we show that TSA is an independent folding unit and thus forms a bona fide structural domain. This domain can be further divided into three sub-domains with striking structural homology with helicase sub-domains of the SF1B family. We also show that TSA is part of a larger vestigial helicase domain which has lost its helicase activity but not its single-stranded DNA binding capability. Finally, we further delineate the binding site responsible for translocation activity of TSA by targeting single residues for mutations. Overall, this study provides the first evidence that translocation signals can be part of larger structural scaffolds, overlapping with translocation-independent activities

Structure of the Bacterial Sex F Pilus reveals an assembly of a Stoichiometric Protein-Phospholipid Complex

Conjugative pili are widespread bacterial appendages that play important roles in horizontal gene transfer, in spread of antibiotic resistance genes, and as sites of phage attachment. Among conjugative pili, the F “sex” pilus encoded by the F plasmid is the best functionally characterized, and it is also historically the most important, as the discovery of F-plasmid-mediated conjugation ushered in the era of molecular biology and genetics. Yet, its structure is unknown. Here, we present atomic models of two F family pili, the F and pED208 pili, generated from cryoelectron microscopy reconstructions at 5.0 and 3.6 Å resolution, respectively. These structures reveal that conjugative pili are assemblies of stoichiometric protein-phospholipid units. We further demonstrate that each pilus type binds preferentially to particular phospholipids. These structures provide the molecular basis for F pilus assembly and also shed light on the remarkable properties of conjugative pili in bacterial secretion and phage infection

Cryo-EM Structure of a Relaxase Reveals the Molecular Basis of DNA Unwinding during Bacterial Conjugation

Relaxases play essential roles in conjugation, the main process by which bacteria exchange genetic material, notably antibiotic resistance genes. They are bifunctional enzymes containing a trans-esterase activity, which is responsible for nicking the DNA strand to be transferred and for covalent attachment to the resulting 5′-phosphate end, and a helicase activity, which is responsible for unwinding the DNA while it is being transported to a recipient cell. Here we show that these two activities are carried out by two conformers that can both load simultaneously on the origin of transfer DNA. We solve the structure of one of these conformers by cryo electron microscopy to near-atomic resolution, elucidating the molecular basis of helicase function by relaxases and revealing insights into the mechanistic events taking place in the cell prior to substrate transport during conjugation

Structural basis for native agonist and synthetic inhibitor recognition by the Pseudomonas aeruginosa quorum sensing regulator PqsR (MvfR)

Bacterial populations co-ordinate gene expression collectively through quorum sensing (QS), a cell-to-cell communication mechanism employing diffusible signal molecules. The LysR-type transcriptional regulator (LTTR) protein PqsR (MvfR) is a key component of alkyl-quinolone (AQ)-dependent QS in Pseudomonas aeruginosa. PqsR is activated by 2-alkyl-4-quinolones including the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone), its precursor 2-heptyl-4- hydroxyquinoline (HHQ) and their C9 congeners, 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS) and 2-nonyl-4-hydroxyquinoline (NHQ). These drive the autoinduction of AQ biosynthesis and the up-regulation of key virulence determinants as a function of bacterial population density. Consequently, PqsR constitutes a potential target for novel antibacterial agents which attenuate infection through the blockade of virulence. Here we present the crystal structures of the PqsR co-inducer binding domain (CBD) and a complex with the native agonist NHQ. We show that the structure of the PqsR CBD has an unusually large ligand-binding pocket in which a native AQ agonist is stabilized entirely by hydrophobic interactions. Through a ligand-based design strategy we synthesized and evaluated a series of 50 AQ and novel quinazolinone (QZN) analogues and measured the impact on AQ biosynthesis, virulence gene expression and biofilm development. The simple exchange of two isosteres (OH for NH2) switches a QZN agonist to an antagonist with a concomitant impact on the induction of bacterial virulence factor production. We also determined the complex crystal structure of a QZN antagonist bound to PqsR revealing a similar orientation in the ligand binding pocket to the native agonist NHQ. This structure represents the first description of an LTTR-antagonist complex. Overall these studies present novel insights into LTTR ligand binding and ligand-based drug design and provide a chemical scaffold for further anti-P. aeruginosa virulence drug development by targeting the AQ receptor PqsR

Structural studies of Pseudomonas quinolone signal regulator PqsR

Pseudomonas aeruginosa is an opportunistic pathogen with a wide host range. It causes serious infection in cyctic fibrosis patients and patients with suppressed immunity. Most of the virulence and adaptational characters of this pathogen are controlled by quorum sensing. Quorum sensing in P. aeruginosa has two types of system, the first quorum sensing system is facilitated by N-acyl homoserine lactones which is further constituted by two system las and rhl system. The second type of quorum sensing system is facilitated by alkyl quinolones (AQ) and thus called the AQ signalling system. The AQ quorum sensing system plays a vital role in P. aeruginosa as it holds regulatory control over the rhl system and also is associated with the virulence factors such as pyocyanin, lectin and biofilm maturation. This system includes the phnA/B genes which produce the precursor molecule anthranilate and the pqsABCDE operon, which produces the first signal molecule 2-heptyl-4-quinolone (HHQ). HHQ gets converted to 2-heptyl-3hydroxy-4-quinolone also called Pseudomonas quinolone signal (PQS) which is the second signal molecule. The conversion of HHQ to PQS is facilitated by PqsH, an oxygen dependent monooxygenase which is not part of the pqsABCDE operon. PqsR, a transcriptional regulator of the LysR family of transcriptional regulator, holds transcriptional control over the phnA/B genes and pqsABCDDE operon. Avirulent phenotypes were observed with P. aeruginosa strains lacking PqsR, suggesting its important role in virulence. The cloning, expression, purification and crystal structure solution of PqsR coinducer binding domain has been described in this thesis. The structure of PqsR CBO shows a typical LTIR fold with 6 helices and 8 ~-strands. The structure also shows a unique dimer interface, unique hydrophobic pocket. In the apo-structure, two MPO molecules stabilised the hydrophobic pocket. The PqsR CBO - NHQ complex structure shows the ligand bound to the hydrophobic pocket with the quinolone head group buried deep within the protein and the alkyl chain bent and extending parallel to the dimer interface. PqsR is a potential anti-quorum sensingEThOS - Electronic Theses Online ServiceGBUnited Kingdo

Structural biology of the Gram-negative bacterial conjugation systems

Conjugation, the process by which plasmid DNA is transferred from one bacterium to another, is mediated by type IV secretion systems (T4SSs). T4SSs are versatile systems that can transport not only DNA, but also toxins and effector proteins. Conjugative T4SSs comprise 12 proteins named VirB1–11 and VirD4 that assemble into a large membrane-spanning exporting machine. Before being transported, the DNA substrate is first processed on the cytoplasmic side by a complex called the relaxosome. The substrate is then targeted to the T4SS for export into a recipient cell. In this review, we describe the recent progress made in the structural biology of both the relaxosome and the T4SS

SirA Inhibits the essential DnaA : DnaD Interaction to block helicase recruitment during Bacillus subtilis sporulation

Bidirectional DNA replication from a chromosome origin requires the asymmetric loading of two helicases, one for each replisome. Our understanding of the molecular mechanisms underpinning helicase loading at bacterial chromosome origins is incomplete. Here we report both positive and negative mechanisms for directing helicase recruitment in the model organism Bacillus subtilis. Systematic characterization of the essential initiation protein DnaD revealed distinct protein interfaces required for homo-oligomerization, interaction with the master initiator protein DnaA, and interaction with the helicase co-loader protein DnaB. Informed by these properties of DnaD, we went on to find that the developmentally expressed repressor of DNA replication initiation, SirA, blocks the interaction between DnaD with DnaA, thereby inhibiting helicase recruitment to the origin during sporulation. These results advance our understanding of the mechanisms underpinning DNA replication initiation in B. subtilis, as well as guiding the search for essential cellular activities to target for antimicrobial drug design

The DNA replication initiation protein DnaD recognises a specific strand of the Bacillus subtilis chromosome origin

Genome replication is a fundamental biological activity shared by all organisms. Chromosomal replication proceeds bidirectionally from origins, requiring the loading of two helicases, one for each replisome. However, the molecular mechanisms underpinning helicase loading at bacterial chromosome origins (oriC) are unclear. Here we investigated the essential DNA replication initiation protein DnaD in the model organism Bacillus subtilis. A set of DnaD residues required for ssDNA binding was identified, and photo-crosslinking revealed that this ssDNA binding region interacts preferentially with one strand of oriC. Biochemical and genetic data support the model that DnaD recognizes a new single-stranded DNA (ssDNA) motif located in oriC, the DnaD Recognition Element (DRE). Considered with single particle cryo-electron microscopy (cryo-EM) imaging of DnaD, we propose that the location of the DRE within oriC orchestrates strand-specific recruitment of helicase during DNA replication initiation. These findings significantly advance our mechanistic understanding of bidirectional replication from a bacterial chromosome origin. [Abstract copyright: © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.