Chimeric systems composed of swapped Tra subunits between distantly-related F plasmids reveal striking plasticity among type IV secretion machines.

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.

IncFV plasmid pED208: Sequence analysis and evidence for translocation of maintenance/leading region proteins through diverse type IV secretion systems.

Two phylogenetically distantly-related IncF plasmids, F and pED208, serve as important models for mechanistic and structural studies of F-like type IV secretion systems (T4SSFs) and F pili. Here, we present the pED208 sequence and compare it to F and pUMNF18, the closest match to pED208 in the NCBI database. As expected, gene content of the three cargo regions varies extensively, although the maintenance/leading regions (MLRs) and transfer (Tra) regions also carry novel genes or motifs with predicted modulatory effects on plasmid stability, dissemination and host range. By use of a Cre recombinase assay for translocation (CRAfT), we recently reported that pED208-carrying donors translocate several products of the MLR (ParA, ParB1, ParB2, SSB, PsiB, PsiA) intercellularly through the T4SSF. Here, we extend these findings by reporting that pED208-carrying donors translocate 10 additional MLR proteins during conjugation. In contrast, two F plasmid-encoded toxin components of toxin-antitoxin (TA) modules, CcdB and SrnB, were not translocated at detectable levels through the T4SSF. Remarkably, most or all of the pED208-encoded MLR proteins and CcdB and SrnB were translocated through heterologous T4SSs encoded by IncN and IncP plasmids pKM101 and RP4, respectively. Together, our sequence analyses underscore the genomic diversity of the F plasmid superfamily, and our experimental data demonstrate the promiscuous nature of conjugation machines for protein translocation. Our findings raise intriguing questions about the nature of T4SS translocation signals and of the biological and evolutionary consequences of conjugative protein transfer.

Comprehensive Molecular Characterization of Papillary Renal-Cell Carcinoma.

BACKGROUND: Papillary renal-cell carcinoma, which accounts for 15 to 20% of renal-cell carcinomas, is a heterogeneous disease that consists of various types of renal cancer, including tumors with indolent, multifocal presentation and solitary tumors with an aggressive, highly lethal phenotype. Little is known about the genetic basis of sporadic papillary renal-cell carcinoma, and no effective forms of therapy for advanced disease exist. METHODS: We performed comprehensive molecular characterization of 161 primary papillary renal-cell carcinomas, using whole-exome sequencing, copy-number analysis, messenger RNA and microRNA sequencing, DNA-methylation analysis, and proteomic analysis. RESULTS: Type 1 and type 2 papillary renal-cell carcinomas were shown to be different types of renal cancer characterized by specific genetic alterations, with type 2 further classified into three individual subgroups on the basis of molecular differences associated with patient survival. Type 1 tumors were associated with MET alterations, whereas type 2 tumors were characterized by CDKN2A silencing, SETD2 mutations, TFE3 fusions, and increased expression of the NRF2-antioxidant response element (ARE) pathway. A CpG island methylator phenotype (CIMP) was observed in a distinct subgroup of type 2 papillary renal-cell carcinomas that was characterized by poor survival and mutation of the gene encoding fumarate hydratase (FH). CONCLUSIONS: Type 1 and type 2 papillary renal-cell carcinomas were shown to be clinically and biologically distinct. Alterations in the MET pathway were associated with type 1, and activation of the NRF2-ARE pathway was associated with type 2; CDKN2A loss and CIMP in type 2 conveyed a poor prognosis. Furthermore, type 2 papillary renal-cell carcinoma consisted of at least three subtypes based on molecular and phenotypic features. (Funded by the National Institutes of Health.).

The F plasmid conjutome: the repertoire of E. coli proteins translocated through an F-encoded type IV secretion system.

Bacterial conjugation systems pose a major threat to human health through their widespread dissemination of mobile genetic elements (MGEs) carrying cargoes of antibiotic resistance genes. Using the Cre Recombinase Assay for Translocation (CRAfT), we recently reported that the IncFV pED208 conjugation system also translocates at least 16 plasmid-encoded proteins to recipient bacteria. Here, we deployed a high-throughput CRAfT screen to identify the repertoire of chromosomally encoded protein substrates of the pED208 system. We identified 32 substrates encoded by the Escherichia coli W3110 genome with functions associated with (i) DNA/nucleotide metabolism, (ii) stress tolerance/physiology, (iii) transcriptional regulation, or (iv) toxin inhibition. The respective gene deletions did not impact pED208 transfer proficiencies, nor did Group 1 (DNA/nucleotide metabolism) mutations detectably alter the SOS response elicited in new transconjugants upon acquisition of pED208. However, MC4100(pED208) donor cells intrinsically exhibit significantly higher SOS activation than plasmid-free MC4100 cells, and this plasmid carriage-induced stress response is further elevated in donor cells deleted of several Group 1 genes. Among 10 characterized substrates, we gained evidence of C-terminal or internal translocation signals that could function independently or synergistically for optimal protein transfer. Remarkably, nearly all tested proteins were also translocated through the IncN pKM101 and IncP RP4 conjugation systems. This repertoire of E. coli protein substrates, here termed the F plasmid "conjutome," is thus characterized by functions of potential benefit to new transconjugants, diverse TSs, and the capacity for promiscuous transfer through heterologous conjugation systems. IMPORTANCE: Conjugation systems comprise a major subfamily of the type IV secretion systems (T4SSs) and are the progenitors of a second large T4SS subfamily dedicated to translocation of protein effectors. This study examined the capacity of conjugation machines to function as protein translocators. Using a high-throughput reporter screen, we determined that 32 chromosomally encoded proteins are delivered through an F plasmid conjugation system. The translocated proteins potentially enhance the establishment of the co-transferred F plasmid or mitigate mating-induced stresses. Translocation signals located C-terminally or internally conferred substrate recognition by the F system and, remarkably, many substrates also were translocated through heterologous conjugation systems. Our findings highlight the plasticity of conjugation systems in their capacities to co-translocate DNA and many protein substrates.

Chimeric systems composed of swapped Tra subunits between distantly-related F plasmids reveal striking plasticity among type IV secretion machines.

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.

A cell wall-anchored glycoprotein confers resistance to cation stress in Actinomyces oris biofilms.

Actinomyces oris plays an important role in oral biofilm development. Like many gram-positive bacteria, A. oris produces a sizable number of surface proteins that are anchored to bacterial peptidoglycan by a conserved transpeptidase named the housekeeping sortase SrtA; however, the biological role of many A. oris surface proteins in biofilm formation is largely unknown. Here, we report that the glycoprotein GspA-a genetic suppressor of srtA deletion lethality-not only promotes biofilm formation but also maintains cell membrane integrity under cation stress. In comparison to wild-type cells, under elevated concentrations of mono- and divalent cations the formation of mono- and multi-species biofilms by mutant cells devoid of gspA was significantly diminished, although planktonic growth of both cell types in the presence of cations was indistinguishable. Because gspA overexpression is lethal to cells lacking gspA and srtA, we performed a genetic screen to identify GspA determinants involving cell viability. DNA sequencing and biochemical characterizations of viable clones revealed that mutations of two critical cysteine residues and a serine residue severely affected GspA glycosylation and biofilm formation. Furthermore, mutant cells lacking gspA were markedly sensitive to sodium dodecyl sulfate, a detergent that solubilizes the cytoplasmic membranes, suggesting the cell envelope of the gspA mutant was altered. Consistent with this observation, the gspA mutant exhibited increased membrane permeability, independent of GspA glycosylation, compared to the wild-type strain. Altogether, the results support the notion that the cell wall-anchored glycoprotein GspA provides a defense mechanism against cation stress in biofilm development promoted by A. oris.

Protein Transfer through an F Plasmid-Encoded Type IV Secretion System Suppresses the Mating-Induced SOS Response.

Bacterial type IV secretion systems (T4SSs) mediate the conjugative transfer of mobile genetic elements (MGEs) and their cargoes of antibiotic resistance and virulence genes. Here, we report that the pED208-encoded T4SS (TrapED208) translocates not only this F plasmid but several plasmid-encoded proteins, including ParA, ParB1, single-stranded DNA-binding protein SSB, ParB2, PsiB, and PsiA, to recipient cells. Conjugative protein translocation through the TrapED208 T4SS required engagement of the pED208 relaxosome with the TraD substrate receptor or coupling protein. T4SSs translocate MGEs as single-stranded DNA intermediates (T-strands), which triggers the SOS response in recipient cells. Transfer of pED208 deleted of psiB or ssb, which, respectively, encode the SOS inhibitor protein PsiB and single-stranded DNA-binding protein SSB, elicited a significantly stronger SOS response than pED208 or mutant plasmids deleted of psiA, parA, parB1, or parB2. Conversely, translocation of PsiB or SSB, but not PsiA, through the TrapED208 T4SS suppressed the mating-induced SOS response. Our findings expand the repertoire of known substrates of conjugation systems to include proteins with functions associated with plasmid maintenance. Furthermore, for this and other F-encoded Tra systems, docking of the DNA substrate with the TraD receptor appears to serve as a critical activating signal for protein translocation. Finally, the observed effects of PsiB and SSB on suppression of the mating-induced SOS response establishes a novel biological function for conjugative protein translocation and suggests the potential for interbacterial protein translocation to manifest in diverse outcomes influencing bacterial communication, physiology, and evolution. IMPORTANCE Many bacteria carry plasmids and other mobile genetic elements (MGEs) whose conjugative transfer through encoded type IV secretion systems (T4SSs), or "mating" channels, can lead to a rapid intra- and interspecies proliferation of genes encoding resistance to antibiotics or heavy metals or virulence traits. Here, we show that a model IncF plasmid-encoded T4SS translocates not only DNA but also several proteins intercellularly. The repertoire of translocated proteins includes the plasmidic SOS inhibitor protein PsiB, single-stranded DNA-binding protein SSB, and several partitioning proteins. We demonstrate that intercellular transmission of PsiB and SSB suppresses the SOS response, which is triggered in recipient cells upon acquisition of the single-stranded DNA transfer intermediate during mating. Our findings identify a new biological function for conjugative protein translocation in mitigating potentially deleterious consequences to plasmid and genome integrity resulting from SOS-induced recombination and mutation events.

Spontaneous mutagenesis is elevated in protease-defective cells.

As a first step towards describing the role of proteolysis in maintaining genomic integrity, we have determined the effect of the loss of ClpXP, a major energy-dependent cytoplasmic protease that degrades truncated proteins as well as a number of regulatory proteins, on spontaneous mutagenesis. In a rifampicin-sensitive to rifampicin-resistance assay that detects base substitution mutations in the essential rpoB gene, there is a modest, but appreciable increase in mutagenesis in Delta(clpP-clpX) cells relative to wild-type cells. A colony papillation analysis using a set of lacZ strains revealed that genetic -1 frameshift mutations are strongly elevated in Clp-defective cells. A quantitative analysis using a valine-sensitive to valine-resistance assay that detects frameshift mutations showed that mutagenesis is elevated 50-fold in Clp-defective cells. Elevated frameshift mutagenesis observed in Clp-deficient cells is essentially abolished in lexA1[Ind(-)] (SOS-uninducible) cells, and in cells deleted for the SOS gene dinB, which codes for DNA polymerase IV. In contrast, mutagenesis is unaffected or stimulated in cells deleted for umuC or umuD, which code for critical components of DNA polymerase V. Loss of rpoS, which codes for a stress-response sigma factor known to upregulate dinB expression in stationary phase, does not affect mutagenesis. We propose that elevated DinB expression, as well as stabilization of UmuD/UmuD' heterodimers in Delta(clpP-clpX) cells, contributes to elevated mutagenesis. These findings suggest that in normal cells, Clp-mediated proteolysis plays an important role in preventing gratuitous mutagenesis.

Elevated expression of DNA polymerase II increases spontaneous mutagenesis in Escherichia coli.

Escherichia coli DNA polymerase II (Pol-II), encoded by the SOS-regulated polB gene, belongs to the highly conserved group B (alpha-like) family of "high-fidelity" DNA polymerases. Elevated expression of polB gene was recently shown to result in a significant elevation of translesion DNA synthesis at 3, N(4)-ethenocytosine lesion with concomitant increase in mutagenesis. Here, I show that elevated expression of Pol-II leads to an approximately 100-fold increase in spontaneous mutagenesis in a manner that is independent of SOS, umuDC, dinB, recA, uvrA and mutS functions. Cells grow slowly and filament with elevated expression of Pol-II. Introduction of carboxy terminus ("beta interaction domain") mutations in polB eliminates elevated spontaneous mutagenesis, as well as defects in cell growth and morphology, suggesting that these abilities require the interaction of Pol-II with the beta processivity subunit of DNA polymerase III. Introduction of a mutation in the proofreading exo motif of polB elevates mutagenesis by a further 180-fold, suggesting that Pol-II can effectively compete with DNA polymerase III for DNA synthesis. Thus, Pol-II can contribute to spontaneous mutagenesis when its expression is elevated.

Escherichia coli DNA polymerase II can efficiently bypass 3,N(4)-ethenocytosine lesions in vitro and in vivo.

Escherichia coli DNA polymerase II (pol-II) is a highly conserved protein that appears to have a role in replication restart, as well as in translesion synthesis across specific DNA adducts under some conditions. Here, we have investigated the effects of elevated expression of pol-II (without concomitant SOS induction) on translesion DNA synthesis and mutagenesis at 3,N(4)-ethenocytosine (varepsilonC), a highly mutagenic DNA lesion induced by oxidative stress as well as by exposure to industrial chemicals such as vinyl chloride. In normal cells, survival of transfected M13 single-stranded DNA bearing a single varepsilonC residue (varepsilonC-ssDNA) is about 20% of that of control DNA, with about 5% of the progeny phage bearing a mutation at the lesion site. Most mutations are C-->A and C-->T, with a slight predominance of transversions over transitions. In contrast, in cells expressing elevated levels of pol-II, survival of varepsilonC-ssDNA is close to 100%, with a concomitant mutation frequency of almost 99% suggesting highly efficient translesion DNA synthesis. Furthermore, an overwhelming majority of mutations at varepsilonC are C-->T transitions. Purified pol-II efficiently catalyzes translesion synthesis at varepsilonC in vitro, accompanied by high levels of mutagenesis with the same specificity. These results suggest that the observed in vivo effects in pol-II over-expressing cells are due to pol-II-mediated DNA synthesis. Introduction of mutations in the carboxy terminus region (beta interaction domain) of polB eliminates in vivo translesion synthesis at varepsilonC, suggesting that the ability of pol-II to compete with pol-III requires interaction with the beta processivity subunit of pol-III. Thus, pol-II can compete with pol-III for translesion synthesis.

Identity and function of a large gene network underlying mutagenic repair of DNA breaks.

Mechanisms of DNA repair and mutagenesis are defined on the basis of relatively few proteins acting on DNA, yet the identities and functions of all proteins required are unknown. Here, we identify the network that underlies mutagenic repair of DNA breaks in stressed Escherichia coli and define functions for much of it. Using a comprehensive screen, we identified a network of ≥93 genes that function in mutation. Most operate upstream of activation of three required stress responses (RpoS, RpoE, and SOS, key network hubs), apparently sensing stress. The results reveal how a network integrates mutagenic repair into the biology of the cell, show specific pathways of environmental sensing, demonstrate the centrality of stress responses, and imply that these responses are attractive as potential drug targets for blocking the evolution of pathogens.

Hypermutagenesis in mutA cells is mediated by mistranslational corruption of polymerase, and is accompanied by replication fork collapse.

Elevated mistranslation induces a mutator response termed translational stress-induced mutagenesis (TSM) that is mediated by an unidentified modification of DNA polymerase III. Here we address two questions: (i) does TSM result from direct polymerase corruption, or from an indirect pathway triggered by increased protein turnover? (ii) Why are homologous recombination functions required for the expression of TSM under certain conditions, but not others? We show that replication of bacteriophage T4 in cells expressing the mutA allele of the glyVtRNA gene (Asp-Gly mistranslation), leads to both increased mutagenesis, and to an altered mutational specificity, results that strongly support mistranslational corruption of DNA polymerase. We also show that expression of mutA, which confers a recA-dependent mutator phenotype, leads to increased lambdoid prophage induction (selectable in vivo expression technology assay), suggesting that replication fork collapse occurs more frequently in mutA cells relative to control cells. No such increase in prophage induction is seen in cells expressing alaVGlu tRNA (Glu-->Ala mistranslation), in which the mutator phenotype is recA-independent. We propose that replication fork collapse accompanies episodic hypermutagenic replication cycles in mutA cells, requiring homologous recombination functions for fork recovery, and therefore, for mutation recovery. These findings highlight hitherto under-appreciated links among translation, replication and recombination, and suggest that translational fidelity, which is affected by genetic and environmental signals, is a key modulator of replication fidelity.

DNA polymerase III from Escherichia coli cells expressing mutA mistranslator tRNA is error-prone.

Translational stress-induced mutagenesis (TSM) refers to the elevated mutagenesis observed in Escherichia coli cells in which mistranslation has been increased as a result of mutations in tRNA genes (such as mutA) or by exposure to streptomycin. TSM does not require lexA-regulated SOS functions but is suppressed in cells defective for homologous recombination genes. Crude cell-free extracts from TSM-induced E. coli strains express an error-prone DNA polymerase. To determine whether DNA polymerase III is involved in the TSM phenotype, we first asked if the phenotype is expressed in cells defective for all four of the non-replicative DNA polymerases, namely polymerase I, II, IV, and V. By using a colony papillation assay based on the reversion of a lacZ mutant, we show that the TSM phenotype is expressed in such cells. Second, we asked if pol III from TSM-induced cells is error-prone. By purifying DNA polymerase III* from TSM-induced and control cells, and by testing its fidelity on templates bearing 3,N(4)-ethenocytosine (a mutagenic DNA lesion), as well as on undamaged DNA templates, we show here that polymerase III* purified from mutA cells is error-prone as compared with that from control cells. These findings suggest that DNA polymerase III is modified in TSM-induced cells.