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

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

The bacterial replication origin BUS promotes nucleobase capture

Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is initiated by the ubiquitous DnaA protein, which assembles into an oligomeric complex at the chromosome origin (oriC) that engages both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) to promote DNA duplex opening. However, the mechanism of DnaA specifically opening a replication origin was unknown. Here we show that Bacillus subtilis DnaAATP assembles into a continuous oligomer at the site of DNA melting, extending from a dsDNA anchor to engage a single DNA strand. Within this complex, two nucleobases of each ssDNA binding motif (DnaA-trio) are captured within a dinucleotide binding pocket created by adjacent DnaA proteins. These results provide a molecular basis for DnaA specifically engaging the conserved sequence elements within the bacterial chromosome origin basal unwinding system (BUS)

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 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

Architecture of the outer-membrane core complex from a conjugative type IV secretion system

AbstractConjugation is one of the most important processes that bacteria utilize to spread antibiotic resistance genes among bacterial populations. Interbacterial DNA transfer requires a large double membrane-spanning nanomachine called the type 4 secretion system (T4SS) made up of the inner-membrane complex (IMC), the outer-membrane core complex (OMCC) and the conjugative pilus. The iconic F plasmid-encoded T4SS has been central in understanding conjugation for several decades, however atomic details of its structure are not known. Here, we report the structure of a complete conjugative OMCC encoded by the pED208 plasmid from E. coli, solved by cryo-electron microscopy at 3.3 Å resolution. This 2.1 MDa complex has a unique arrangement with two radial concentric rings, each having a different symmetry eventually contributing to remarkable differences in protein stoichiometry and flexibility in comparison to other OMCCs. Our structure suggests that F-OMCC is a highly dynamic complex, with implications for pilus extension and retraction during conjugation.</jats:p