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.

Structural Biology of Bacterial Type IV Secretion Systems.

Type IV secretion systems (T4SSs) are large multisubunit translocons, found in both gram-negative and gram-positive bacteria and in some archaea. These systems transport a diverse array of substrates from DNA and protein-DNA complexes to proteins, and play fundamental roles in both bacterial pathogenesis and bacterial adaptation to the cellular milieu in which bacteria live. This review describes the various biochemical and structural advances made toward understanding the biogenesis, architecture, and function of T4SSs.

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.

Plasmids pick a bacterial partner before committing to conjugation.

Bacterial conjugation was first described by Lederberg and Tatum in the 1940s following the discovery of the F plasmid. During conjugation a plasmid is transferred unidirectionally from one bacterium (the donor) to another (the recipient), in a contact-dependent manner. Conjugation has been regarded as a promiscuous mechanism of DNA transfer, with host range determined by the recipient downstream of plasmid transfer. However, recent data have shown that F-like plasmids, akin to tailed Caudovirales bacteriophages, can pick their host bacteria prior to transfer by expressing one of at least four structurally distinct isoforms of the outer membrane protein TraN, which has evolved to function as a highly sensitive sensor on the donor cell surface. The TraN sensor appears to pick bacterial hosts by binding compatible outer membrane proteins in the recipient. The TraN variants can be divided into specialist and generalist sensors, conferring narrow and broad plasmid host range, respectively. In this review we discuss recent advances in our understanding of the function of the TraN sensor at the donor-recipient interface, used by F-like plasmids to select bacterial hosts within polymicrobial communities prior to DNA transfer.

In vitro dissolution profile comparison using bootstrap bias corrected similarity factor, f2.

In vitro dissolution profile has been shown to be correlated with the drug absorption and has often been considered as a metric for assessing in vitro bioequivalence between a test product and corresponding reference one. Various methods have been developed to assess the similarity between two dissolution profiles. In particular, similarity factor f2 has been reviewed and discussed extensively in many statistical articles. Although the f2 lacks inferential statistical properties, the estimation of f2 and its various modified versions were the most widely used metric for comparing dissolution profiles. In this paper, we investigated performances of the naive f2 estimate method, bootstrap f2 confidence interval method and bias corrected-accelerated (BCa) bootstrap f2 confidence interval method for comparing dissolution profiles. Our studies show that naive f2 estimate method and BCa bootstrap f2 confidence interval method are unable to control the type I error rate. The bootstrap f2 confidence interval method can control the type I error rate under a specific level. However, it will cause great conservatism on the power of the test. To solve the potential issues of the previous methods, we recommended a bootstrap bias corrected (BC) f2 confidence interval method in this paper. The type I error rate, power and sensitivity among different f2 methods were compared based on simulations. The recommended bootstrap BC f2 confidence interval method shows better control of type I error than the naive f2 estimate method and BCa bootstrap f2 confidence interval method. It also provides better power than the bootstrap f2 confidence interval method.

The Interaction of the F-Like Plasmid-Encoded TraN Isoforms with Their Cognate Outer Membrane Receptors.

Horizontal gene transfer via conjugation plays a major role in bacterial evolution. In F-like plasmids, efficient DNA transfer is mediated by close association between donor and recipient bacteria. This process, known as mating pair stabilization (MPS), is mediated by interactions between the plasmid-encoded outer membrane (OM) protein TraN in the donor and chromosomally-encoded OM proteins in the recipient. We have recently reported the existence of 7 TraN sequence types, which are grouped into 4 structural types, that we named TraNα, TraNß, TraNγ, and TraNδ. Moreover, we have shown specific pairing between TraNα and OmpW, TraNß and OmpK36 of Klebsiella pneumoniae, TraNγ and OmpA, and TraNδ and OmpF. In this study, we found that, although structurally similar, TraNα encoded by the Salmonella enterica pSLT plasmid (TraNα2) binds OmpW in both Escherichia coli and Citrobacter rodentium, while TraNα encoded by the R100-1 plasmid (TraNα1) only binds OmpW in E. coli. AlphaFold2 predictions suggested that this specificity is mediated by a single amino acid difference in loop 3 of OmpW, which we confirmed experimentally. Moreover, we show that single amino acids insertions into loop 3 of OmpK36 affect TraNß-mediated conjugation efficiency of the K. pneumoniae resistance plasmid pKpQIL. Lastly, we report that TraNß can also mediate MPS by binding OmpK35, making it the first TraN variant that can bind more than one OM protein in the recipient. Together, these data show that subtle sequence differences in the OM receptors can impact TraN-mediated conjugation efficiency. IMPORTANCE Conjugation plays a central role in the spread of antimicrobial resistance genes among bacterial pathogens. Efficient conjugation is mediated by formation of mating pairs via a pilus, followed by mating pair stabilization (MPS), mediated by tight interactions between the plasmid-encoded outer membrane protein (OMP) TraN in the donor (of which there are 7 sequence types grouped into the 4 structural isoforms α, ß, γ, and δ), and an OMP receptor in the recipient (OmpW, OmpK36, OmpA, and OmpF, respectively). In this study, we found that subtle differences in OmpW and OmpK36 have significant consequences on conjugation efficiency and specificity, highlighting the existence of selective pressure affecting plasmid-host compatibility and the flow of horizontal gene transfer in bacteria.

Contributions of F-specific subunits to the F plasmid-encoded type IV secretion system and F pilus.

F plasmids circulate widely among the Enterobacteriaceae through encoded type IV secretion systems (T4SSF s). Assembly of T4SSF s 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 T4SSF s 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.

Grand scale genome manipulation via chromosome swapping in Escherichia coli programmed by three one megabase chromosomes.

In bacterial synthetic biology, whole genome transplantation has been achieved only in mycoplasmas that contain a small genome and are competent for foreign genome uptake. In this study, we developed Escherichia coli strains programmed by three 1-megabase (Mb) chromosomes by splitting the 3-Mb chromosome of a genome-reduced strain. The first split-chromosome retains the original replication origin (oriC) and partitioning (par) system. The second one has an oriC and the par locus from the F plasmid, while the third one has the ori and par locus of the Vibrio tubiashii secondary chromosome. The tripartite-genome cells maintained the rod-shaped form and grew only twice as slowly as their parent, allowing their further genetic engineering. A proportion of these 1-Mb chromosomes were purified as covalently closed supercoiled molecules with a conventional alkaline lysis method and anion exchange columns. Furthermore, the second and third chromosomes could be individually electroporated into competent cells. In contrast, the first split-chromosome was not able to coexist with another chromosome carrying the same origin region. However, it was exchangeable via conjugation between tripartite-genome strains by using different selection markers. We believe that this E. coli-based technology has the potential to greatly accelerate synthetic biology and synthetic genomics.

Complete sequence of classic F-type plasmid pRK100 shows unique conservation over time and geographic location.

Plasmids exhibit great diversity of gene content and host ranges and are famous for quick adaptation to the genetic background of the bacterial host cell. In addition to observing ever evolving plasmids, some plasmids have conserved backbones: a stable core composition and arrangement of genes in addition to variable regions. There are a few reports of extremely conserved plasmids. Here we report the complete sequence of pRK100 plasmid - a large, well-characterized conjugative F-like plasmid found in an Escherichia coli strain isolated from a urinary tract infection patient in 1990. The sequence shows that the 142 kb-long pRK100 plasmid is nearly identical to plasmids circulating in distant geographical locations and found in different host E. coli strains between 2007 and 2017. We also performed additional functional characterization of pRK100. Our results showed that pRK100 does not have a strong pathogenicity phenotype in porcine primary bladder epithelial cell culture. Moreover, the conjugation of pRK100 seems to strongly depend on recipient characteristics. These observations and identification of the pRK100 plasmid in different strain genotypes leave the extreme sequence conservation and broad distribution of this plasmid unexplained.

A role for the last C-terminal helix of the F plasmid segregating protein SopA in nucleoid binding and plasmid maintenance.

The rapid emergence and spread of antibiotic resistance is a growing global burden. Antibiotic resistance is often associated with large single or low copy number plasmids, which rely upon cytoskeletal proteins for their stable maintenance. While the mechanism of plasmid partitioning has been well established for the R plasmids, the molecular details by which the F plasmid is maintained is only beginning to emerge. The partitioning function of the F plasmid depends upon a ParA/ MinD family of proteins known as SopA. SopA, by virtue of its ATP-dependent non-specific DNA binding activity and association with the bacterial nucleoid, drives the segregation of the F plasmid into the daughter cells. This function further depends upon the stimulation of the ATPase activity of SopA by the SopBC complex. Here, we report that several residues in the last C-terminal helix in SopA play a crucial but distinct role in SopA function and plasmid maintenance. While the deletion of the last five residues in SopA does not affect its ability to bind the nucleoid or SopB, they severely affect the plasmid partitioning function. Further, we show that while mutations in certain polar residues in the C-terminal helix only mildly affect its localisation to the nucleoid, others cause defects in nsDNA binding and disrupt plasmid maintenance functions.

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.

Structural bases for F plasmid conjugation and F pilus biogenesis in Escherichia coli.

Bacterial conjugation systems are members of the large type IV secretion system (T4SS) superfamily. Conjugative transfer of F plasmids residing in the Enterobacteriaceae was first reported in the 1940s, yet the architecture of F plasmid-encoded transfer channel and its physical relationship with the F pilus remain unknown. We visualized F-encoded structures in the native bacterial cell envelope by in situ cryoelectron tomography (CryoET). Remarkably, F plasmids encode four distinct structures, not just the translocation channel or channel-pilus complex predicted by prevailing models. The F1 structure is composed of distinct outer and inner membrane complexes and a connecting cylinder that together house the envelope-spanning translocation channel. The F2 structure is essentially the F1 complex with the F pilus attached at the outer membrane (OM). Remarkably, the F3 structure consists of the F pilus attached to a thin, cell envelope-spanning stalk, whereas the F4 structure consists of the pilus docked to the OM without an associated periplasmic density. The traffic ATPase TraC is configured as a hexamer of dimers at the cytoplasmic faces of the F1 and F2 structures, where it respectively regulates substrate transfer and F pilus biogenesis. Together, our findings present architectural renderings of the DNA conjugation or "mating" channel, the channel-pilus connection, and unprecedented pilus basal structures. These structural snapshots support a model for biogenesis of the F transfer system and allow for detailed comparisons with other structurally characterized T4SSs.

Identification of a Potential Membrane-Targeting Sequence in the C-Terminus of the F Plasmid Segregation Protein SopA.

Stable maintenance and partitioning of the 'Fertility' plasmid or the F plasmid in its host Escherichia coli require the function of a ParA superfamily of proteins known as SopA. The mechanism by which SopA mediates plasmid segregation is well studied. SopA is a nucleoid-binding protein and binds DNA in an ATP-dependent but sequence non-specific manner. ATP hydrolysis stimulated by the binding of the SopBC complex mediates the release of SopA from the nucleoid. Cycles of ATP-binding and hydrolysis generate an ATPase gradient that moves the plasmid through a chemophoresis force. Nucleoid binding of SopA thus assumes a central role in its plasmid-partitioning function. However, earlier work also suggests that the F plasmid can be partitioned into anucleate cells, thus implicating nucleoid independent partitioning. Interestingly, SopA is also reported to be associated with the inner membrane of the bacteria. Here, we report the identification of a possible membrane-targeting sequence, a predicted amphipathic helix, at the C-terminus of SopA. Molecular dynamics simulations indicate that the predicted amphipathic helical motif of SopA has weak affinity for membranes. Moreover, we experimentally show that SopA can associate with bacterial membranes, is detectable in the membrane fractions of bacterial lysates, and is sensitive to the membrane potential. Further, unlike the wild-type SopA, a deletion of the C-terminal 29 amino acids results in the loss of F plasmids from bacterial cells.

Antimicrobial antisense RNA delivery to F-pili producing multidrug-resistant bacteria via a genetically engineered bacteriophage.

Multidrug-resistant bacteria are a growing issue worldwide. This study developed a convenient and effective method to downregulate the expression of a specific gene to produce a novel antimicrobial tool using a small (140 nucleotide) RNA with a 24-nucleotide antisense (as) region from an arabinose-inducible expression phagemid vector in Escherichia coli. Knockdown effects of rpoS encoding RNA polymerase sigma factor were observed using this inducible artificial asRNA approach. asRNAs targeting several essential E. coli genes produced significant growth defects, especially when targeted to acpP and ribosomal protein coding genes rplN, rplL, and rpsM. Growth inhibited phenotypes were facilitated in hfq- conditions. Phage lysates were prepared from cells harboring phagemids as a lethal-agent delivery tool. Targeting the rpsM gene by phagemid-derived M13 phage infection of E. coli containing a carbapenem-producing F-plasmid and multidrug-resistant Klebsiella pneumoniae containing an F-plasmid resulted in the death of over 99.99% of infected bacteria. This study provides a possible strategy for treating bacterial infection and can be applied to any F-pilus producing bacterial species.

Initiation of homologous recombination at DNA nicks.

Discontinuities in only a single strand of the DNA duplex occur frequently, as a result of DNA damage or as intermediates in essential nuclear processes and DNA repair. Nicks are the simplest of these lesions: they carry clean ends bearing 3'-hydroxyl groups that can undergo ligation or prime new DNA synthesis. In contrast, single-strand breaks also interrupt only one DNA strand, but they carry damaged ends that require clean-up before subsequent steps in repair. Despite their apparent simplicity, nicks can have significant consequences for genome stability. The availability of enzymes that can introduce a nick almost anywhere in a large genome now makes it possible to systematically analyze repair of nicks. Recent experiments demonstrate that nicks can initiate recombination via pathways distinct from those active at double-strand breaks (DSBs). Recombination at targeted DNA nicks can be very efficient, and because nicks are intrinsically less mutagenic than DSBs, nick-initiated gene correction is useful for genome engineering and gene therapy. This review revisits some physiological examples of recombination at nicks, and outlines experiments that have demonstrated that nicks initiate homology-directed repair by distinctive pathways, emphasizing research that has contributed to our current mechanistic understanding of recombination at nicks in mammalian cells.

Cooperative Function of TraJ and ArcA in Regulating the F Plasmid tra Operon.

The F plasmid tra operon encodes most of the proteins required for bacterial conjugation. TraJ and ArcA are known activators of the tra operon promoter PY, which is subject to H-NS-mediated silencing. Donor ability and promoter activity assays indicated that PY is inactivated by silencers and requires both TraJ and ArcA for activation to support efficient F conjugation. The observed low-level, ArcA-independent F conjugation is caused by tra expression from upstream alternative promoters. Electrophoretic mobility shift assays showed that TraJ alone weakly binds to PY regulatory DNA; however, TraJ binding is significantly enhanced by ArcA binding to the same DNA, indicating cooperativity of the two proteins. Analysis of binding affinities between ArcA and various DNA fragments in the PY regulatory region defined a 22-bp tandem repeat sequence (from -76 to -55 of PY) sufficient for optimal ArcA binding, which is immediately upstream of the predicted TraJ-binding site (from -54 to -34). Deletion analysis of the PY promoter in strains deficient in TraJ, ArcA, and/or H-NS determined that sequences upstream of -103 are required by silencers including H-NS for PY silencing, whereas sequences downstream of -77 are targeted by TraJ and ArcA for activation. TraJ and ArcA appear not only to counteract PY silencers but also to directly activate PY in a cooperative manner. Our data reveal the cooperativity of TraJ and ArcA during PY activation and provide insights into the regulatory circuit controlling F-family plasmid-mediated bacterial conjugation.IMPORTANCE 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 PY The interaction of TraJ and ArcA with the tra operon not only relieves PY from silencers but also directly activates it. These findings provide insights into the regulatory circuit of the F-family plasmid-mediated bacterial conjugation.

CDI Systems Are Stably Maintained by a Cell-Contact Mediated Surveillance Mechanism.

Contact-dependent growth inhibition (CDI) systems are widespread amongst Gram-negative bacteria where they play important roles in inter-cellular competition and biofilm formation. CDI+ bacteria use cell-surface CdiA proteins to bind neighboring bacteria and deliver C-terminal toxin domains. CDI+ cells also express CdiI immunity proteins that specifically neutralize toxins delivered from adjacent siblings. Genomic analyses indicate that cdi loci are commonly found on plasmids and genomic islands, suggesting that these Type 5 secretion systems are spread through horizontal gene transfer. Here, we examine whether CDI toxin and immunity activities serve to stabilize mobile genetic elements using a minimal F plasmid that fails to partition properly during cell division. This F plasmid is lost from Escherichia coli populations within 50 cell generations, but is maintained in ~60% of the cells after 100 generations when the plasmid carries the cdi gene cluster from E. coli strain EC93. By contrast, the ccdAB "plasmid addiction" module normally found on F exerts only a modest stabilizing effect. cdi-dependent plasmid stabilization requires the BamA receptor for CdiA, suggesting that plasmid-free daughter cells are inhibited by siblings that retain the CDI+ plasmid. In support of this model, the CDI+ F plasmid is lost rapidly from cells that carry an additional cdiI immunity gene on a separate plasmid. These results indicate that plasmid stabilization occurs through elimination of non-immune cells arising in the population via plasmid loss. Thus, genetic stabilization reflects a strong selection for immunity to CDI. After long-term passage for more than 300 generations, CDI+ plasmids acquire mutations that increase copy number and result in 100% carriage in the population. Together, these results show that CDI stabilizes genetic elements through a toxin-mediated surveillance mechanism in which cells that lose the CDI system are detected and eliminated by their siblings.

ProQ/FinO-domain proteins: another ubiquitous family of RNA matchmakers?

Small RNAs (sRNAs), particularly those that act by limited base pairing with mRNAs, are part of most regulatory networks in bacteria. In many cases, the base-pairing interaction is facilitated by the RNA chaperone Hfq. However, not all bacteria encode Hfq and some base-pairing sRNAs do not require Hfq raising the possibility of other RNA chaperones. Candidates are proteins with homology to FinO, a factor that promotes base pairing between the FinP antisense sRNA and the traJ mRNA to control F plasmid transfer. Recent papers have shown that the Salmonella enterica FinO-domain protein ProQ binds a large suite of sRNAs, including the RaiZ sRNA, which represses translation of the hupA mRNA, and the Legionella pneumophila protein RocC binds the RocR sRNA, which blocks expression of competence genes. Here we discuss what is known about FinO-domain structures, including the recently solved Escherichia coli ProQ structure, as well as the RNA binding properties of this family of proteins and evidence they act as chaperones. We compare these properties with those of Hfq. We further summarize what is known about the physiological roles of FinO-domain proteins and enumerate outstanding questions whose answers will establish whether they constitute a second major class of RNA chaperones.

The interaction of TraW and TrbC is required to facilitate conjugation in F-like plasmids.

Bacterial conjugation, such as that mediated by the E. coli F plasmid, is a main mechanism driving bacterial evolution. Two important proteins required for F-pilus assembly and DNA transfer proficiency are TraW and TrbC. As members of a larger complex, these proteins assemble into a type IV secretion system and are essential components of pore formation and mating pair stabilization between the donor and the recipient cells. In the current report, we demonstrate the physical interaction of TraW and TrbC, show that TraW preferentially interacts with the N-terminal domain of TrbC, and that this interaction is important in restoring conjugation in traW/trbC knockouts.

Changes in Emotional and Behavioral Problems Between 2000 and 2011 Among 16-Year-Old Polish Children: A Cross-Sectional Study.

Since after the second world war there has been an increasing number of studies investigating secular changes in adolescent mental health. Although no general trends could be outlined, the majority of studies show at least partial deterioration of psychological wellbeing from year 2000 on. Our study adds to this knowledge by exploring changes in self-declared emotional and behavioral problems in Poland, which is a part of post-communist Europe. In this paper, we compared responses on the Youth Self-Report by Polish 16-year-olds from 2000 and those from 2011. Two independent samples consisted of 259 (year 2000) and 185 (year 2011) 16-year-olds of both genders, drawn from randomized, normative, school-based groups. We analyzed linear, ordinal and binary logistic regression models. The results revealed that teenagers from 2011 reported more self-rated internalizing and total problems. Social and thought problems also rose significantly. Gender related time trends hint at a male increase in externalizing, aggressive behaviors and anxiety/depression. Caseness rose significantly in most scales with female gender being an additional risk factor for internalizing and total problems. No reduction in self-reported emotional and behavioral problems was detected.