Bacterial type IV secretion systems (T4SSs) are a functionally diverse translocation superfamily. They consist mainly of two large subfamilies: (i) conjugation systems that mediate interbacterial DNA ...transfer and (ii) effector translocators that deliver effector macromolecules into prokaryotic or eukaryotic cells. A few other T4SSs export DNA or proteins to the milieu, or import exogenous DNA. The T4SSs are defined by 6 or 12 conserved “core” subunits that respectively elaborate “minimized” systems in Gram‐positive or ‐negative bacteria. However, many “expanded” T4SSs are built from “core” subunits plus numerous others that are system‐specific, which presumptively broadens functional capabilities. Recently, there has been exciting progress in defining T4SS assembly pathways and architectures using a combination of fluorescence and cryoelectron microscopy. This review will highlight advances in our knowledge of structure–function relationships for model Gram‐negative bacterial T4SSs, including “minimized” systems resembling the Agrobacterium tumefaciens VirB/VirD4 T4SS and “expanded” systems represented by the Helicobacter pylori Cag, Legionella pneumophila Dot/Icm, and F plasmid‐encoded Tra T4SSs. Detailed studies of these model systems are generating new insights, some at atomic resolution, to long‐standing questions concerning mechanisms of substrate recruitment, T4SS channel architecture, conjugative pilus assembly, and machine adaptations contributing to T4SS functional versatility.
Type IV secretion systems (T4SSs) are a functionally diverse superfamily of translocation nanomachines deployed by most species of bacteria. Collectively, T4SSs mediate the transfer of mobile genetic elements or protein toxins to other bacteria, or effector proteins or other macromolecules to eukaryotic cells to aid in infection. Recent structural and fluorescence approaches are generating exciting new insights into how T4SSs assemble in the cell envelope and recruit and translocate substrates to other cells.
Bacterial type IV secretion systems (T4SSs) are largely responsible for the proliferation of multi-drug resistance. We solved the structure of the outer-membrane core complex (OMCC
) of a T4SS ...encoded by a conjugative F plasmid at <3.0 Å resolution by cryoelectron microscopy. The OMCC
consists of a 13-fold symmetrical outer ring complex (ORC) built from 26 copies of TraK and TraV C-terminal domains, and a 17-fold symmetrical central cone (CC) composed of 17 copies of TraB β-barrels. Domains of TraV and TraB also bind the CC and ORC substructures, establishing that these proteins undergo an intraprotein symmetry alteration to accommodate the C13:C17 symmetry mismatch. We present evidence that other pED208-encoded factors stabilize the C13:C17 architecture and define the importance of TraK, TraV and TraB domains to T4SS
function. This work identifies OMCC
structural motifs of proposed importance for structural transitions associated with F plasmid dissemination and F pilus biogenesis.
F plasmids circulate widely among the Enterobacteriaceae through encoded type IV secretion systems (T4SSFs). Assembly of T4SSFs and associated F pili requires 10 VirB/VirD4‐like Tra subunits and ...eight or more F‐specific subunits. Recently, we presented evidence using in situ cryoelectron tomography (cryoET) that T4SSFs undergo structural transitions when activated for pilus production, and that assembled pili are deposited onto alternative basal platforms at the cell surface. Here, we deleted eight conserved F‐specific genes from the MOBF12C plasmid pED208 and quantitated effects on plasmid transfer, pilus production by fluorescence microscopy, and elaboration of T4SSF structures by in situ cryoET. Mutant phenotypes supported the assignment of F‐specific subunits into three functional Classes: (i) TraF, TraH, and TraW are required for all T4SSF‐associated activities, (ii) TraU, TraN, and TrbC are nonessential but contribute significantly to distinct T4SSF functions, and (iii) TrbB is essential for F pilus production but not for plasmid transfer. Equivalent mutations in a phylogenetically distantly related MOB12A F plasmid conferred similar phenotypes and generally supported these Class assignments. We present a new structure‐driven model in which F‐specific subunits contribute to distinct steps of T4SSF assembly or activation to regulate DNA transfer and F pilus dynamics and deposition onto alternative platforms.
Bacteria disseminate mobile genetic elements such as F plasmids, which often code for resistance to antibiotics, to other bacteria by a process termed conjugation. Here, we characterize the contributions of a set of highly conserved proteins to the conjugative transfer of F plasmids. We show that these proteins play distinct roles in assembly and activation of the F‐encoded conjugation machinery.
Streptococcus mutans, the primary aetiological agent of dental caries, is one of the major bacteria of the human oral cavity. The pathogenicity of this bacterium is attributed not only to the ...expression of virulence factors, but also to its ability to respond and adapt rapidly to the ever-changing conditions of the oral cavity. The two-component signal transduction system (TCS) CovR/S plays a crucial role in virulence and stress response in many streptococci. Surprisingly, in S. mutans the response regulator CovR appears to be an orphan, as the cognate sensor kinase, CovS, is absent in all the strains. We found that acetyl phosphate, an intracellular phosphodonor molecule known to act in signalling, might play a role in CovR phosphorylation in vivo. We also found that in vitro, upon phosphorylation by potassium phosphoramide (a high-energy phophodonor) CovR formed a dimer and showed altered electrophoretic mobility. As expected, we found that the conserved aspartic acid residue at position 53 (D53) was the site of phosphorylation, since neither phosphorylation nor dimerization was seen when an alanine-substituted CovR mutant (D53A) was used. Surprisingly, we found that the ability of CovR to act as a transcriptional regulator does not depend upon its phosphorylation status, since the D53A mutant behaved similarly to the wild-type protein in both in vivo and in vitro DNA-binding assays. This unique phosphorylation-mediated inhibition of CovR function in S. mutans sheds light on an unconventional mechanism of the signal transduction pathway.
Bacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as "minimized" or "expanded" based on whether they are composed of a core set of ...signature subunits or additional system-specific components. Prototypical minimized systems mediating Agrobacterium tumefaciens transfer DNA (T-DNA) and pKM101 and R388 plasmid transfer are built from subunits generically named VirB1 to VirB11 and VirD4. We visualized the pKM101-encoded T4SS in its native cellular context by
cryo-electron tomography (CryoET). The T4SS
is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCC exhibits 14-fold symmetry and resembles that of the T4SS
analyzed previously by single-particle electron microscopy. The IMC is highly symmetrical and exhibits 6-fold symmetry. It is dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMC closely resembles equivalent regions of three expanded T4SSs previously visualized by
CryoET but differs strikingly from the IMC of the purified T4SS
, whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers. Analyses of mutant machines lacking each of the three ATPases required for T4SS
function supplied evidence that TraB
as well as VirB11-like TraG contribute to distinct stages of machine assembly. We propose that the VirB4-like ATPases, configured as hexamers of dimers at the T4SS entrance, orchestrate IMC assembly and recruitment of the spatially dynamic VirB11 and VirD4 ATPases to activate the T4SS for substrate transfer.
Bacterial type IV secretion systems (T4SSs) play central roles in antibiotic resistance spread and virulence. By cryo-electron tomography (CryoET), we solved the structure of the plasmid pKM101-encoded T4SS in the native context of the bacterial cell envelope. The inner membrane complex (IMC) of the
T4SS differs remarkably from that of a closely related T4SS analyzed
by single-particle electron microscopy. Our findings underscore the importance of comparative
and
analyses of the T4SS nanomachines and support a unified model in which the signature VirB4 ATPases of the T4SS superfamily function as a central hexamer of dimers to regulate early-stage machine biogenesis and substrate entry passage through the T4SS. The VirB4 ATPases are therefore excellent targets for the development of intervention strategies aimed at suppressing the action of T4SS nanomachines.
Bacterial type IV secretion systems (T4SSs) are a versatile family of macromolecular translocators, collectively able to recruit diverse DNA and protein substrates and deliver them to a wide range of ...cell types. Presently, there is little understanding of how T4SSs recognize substrate repertoires and form productive contacts with specific target cells. Although T4SSs are composed of a number of conserved subunits and adopt certain conserved structural features, they also display considerable compositional and structural diversity. Here, we explored the structural bases underlying the functional versatility of T4SSs through systematic deletion and subunit swapping between two conjugation systems encoded by the distantly-related IncF plasmids, pED208 and F. We identified several regions of intrinsic flexibility among the encoded T4SSs, as evidenced by partial or complete functionality of chimeric machines. Swapping of VirD4-like TraD type IV coupling proteins (T4CPs) yielded functional chimeras, indicative of relaxed specificity at the substrate-TraD and TraD-T4SS interfaces. Through mutational analyses, we further delineated domains of the TraD T4CPs contributing to recruitment of cognate vs heterologous DNA substrates. Remarkably, swaps of components comprising the outer membrane core complexes, a few F-specific subunits, or the TraA pilins supported DNA transfer in the absence of detectable pilus production. Among sequenced enterobacterial species in the NCBI database, we identified many strains that harbor two or more F-like plasmids and many F plasmids lacking one or more T4SS components required for self-transfer. We confirmed that host cells carrying co-resident, non-selftransmissible variants of pED208 and F elaborate chimeric T4SSs, as evidenced by transmission of both plasmids. We propose that T4SS plasticity enables the facile assembly of functional chimeras, and this intrinsic flexibility at the structural level can account for functional diversification of this superfamily over evolutionary time and, on a more immediate time-scale, to proliferation of transfer-defective MGEs in nature.
Summary
Mobile genetic elements (MGEs) encode type IV secretion systems (T4SSs) known as conjugation machines for their transmission between bacterial cells. Conjugation machines are composed of an ...envelope‐spanning translocation channel, and those functioning in Gram‐negative species additionally elaborate an extracellular pilus to initiate donor‐recipient cell contacts. We report that pKM101, a self‐transmissible MGE functioning in the Enterobacteriaceae, has evolved a second target cell attachment mechanism. Two pKM101‐encoded proteins, the pilus‐tip adhesin TraC and a protein termed Pep, are exported to the cell surface where they interact and also form higher order complexes appearing as distinct foci or patches around the cell envelope. Surface‐displayed TraC and Pep are required for an efficient conjugative transfer, ‘extracellular complementation’ potentially involving intercellular protein transfer, and activation of a Pseudomonas aeruginosa type VI secretion system. Both proteins are also required for bacteriophage PRD1 infection. TraC and Pep are exported across the outer membrane by a mechanism potentially involving the β‐barrel assembly machinery. The pKM101 T4SS, thus, deploys alternative routing pathways for the delivery of TraC to the pilus tip or both TraC and Pep to the cell surface. We propose that T4SS‐encoded, pilus‐independent attachment mechanisms maximize the probability of MGE propagation and might be widespread among this translocation superfamily.
The tip‐adhesin TraC nucleates assembly of the Escherichia coli pKM101‐encoded conjugative pilus on a type IV secretion system (T4SS) scaffold. We show that TraC, and another pKM101‐encoded protein termed Pep, are alternatively routed via contributions of the BAM complex to the cell surface where they interact with each other and other surface proteins to form adhesin complexes. T4SSs may widely deploy surface adhesins as opposed to conjugative pili to stimulate intercellular interactions for substrate translocation.
•Characterization of polycyclic aromatic hydrocarbon dioxygenase in Sphingobium sp.•In silico analysis of large catalytic pocket and unique substrate access channel.•Cloning and expression of ...multi-component dioxygenase.•Evaluation of broad substrate specificity, guided by molecular docking analysis.
A Rieske non-heme iron ring-hydroxylating oxygenase (RHO) from Sphingobium sp. PNB involved in the initial oxidation of a wide range of low and high molecular weight polycyclic aromatic hydrocarbons (PAHs) was investigated. The RHO was shown to comprise of the gene products of distantly located ahdA1f-ahdA2f, ahdA3 and ahdA4 genes, which encoded the oxygenase α- and β-subunits, ferredoxin and reductase, respectively. In silico structural analysis of AhdA1f revealed a very large substrate-binding pocket, satisfying the spatial requirements to accommodate high molecular weight substrates. In addition, an atypical substrate access channel was noticed from the topology analysis of the oxygenase. Guided by molecular docking studies, dioxygenation of several PAHs as well as alkyl- and aryl benzenes was examined with the recombinant AhdA1fA2f expressed in Escherichia coli. Chromatographic and mass spectrometric analyses confirmed that AhdA1fA2f displays broad substrate specificity towards a wide range of aromatic hydrocarbons including potential xenobiotics, demonstrating metabolic robustness of strain PNB.
Polycyclic aromatic hydrocarbons (PAHs) comprise a group of priority organic pollutants that are toxic and/or carcinogenic. Phenanthrene, the simplest PAH among recognized priority pollutants, is ...commonly used as a model compound for the study of PAH biodegradation. Sphingobium sp. strain PNB, capable of degrading phenanthrene as a sole carbon and energy source, was isolated from a municipal waste-contaminated soil sample. A combination of chromatographic and spectrometric analyses, together with oxygen uptake and enzyme activity studies, suggested the presence of phenanthrene degradation pathways in this strain. Identification of metabolites suggested that initial dioxygenation of phenanthrene took place at both 3,4- and 1,2-carbon positions; meta-cleavage of resultant diols led to the formation of 1-hydroxy-2-naphthoic acid and 2-hydroxy-1-naphthoic acid, respectively. The hydroxynaphthoic acids, in turn, were metabolized by a meta-cleavage pathway(s), leading to the formation of 2,2-dicarboxychromene and 2-hydroxychromene-2-glyoxylic acid, respectively. These metabolites were subsequently transformed to catechol via salicylic acid, which further proceeds towards the tricarboxylic acid cycle leading to complete mineralization of the compound phenanthrene. The present study establishes the metabolism of hydroxynaphthoic acids by a meta-cleavage pathway in the degradation of phenanthrene, expanding our current understanding of microbial degradation of PAHs.
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