Conjugative DNA Transfer Is Enhanced by Plasmid R1 Partitioning Proteins.

Bacterial conjugation is a form of type IV secretion used to transport protein and DNA directly to recipient bacteria. The process is cell contact-dependent, yet the mechanisms enabling extracellular events to trigger plasmid transfer to begin inside the cell remain obscure. In this study of plasmid R1 we investigated the role of plasmid proteins in the initiation of gene transfer. We find that TraI, the central regulator of conjugative DNA processing, interacts physically, and functionally with the plasmid partitioning proteins ParM and ParR. These interactions stimulate TraI catalyzed relaxation of plasmid DNA in vivo and in vitro and increase ParM ATPase activity. ParM also binds the coupling protein TraD and VirB4-like channel ATPase TraC. Together, these protein-protein interactions probably act to co-localize the transfer components intracellularly and promote assembly of the conjugation machinery. Importantly these data also indicate that the continued association of ParM and ParR at the conjugative pore is necessary for plasmid transfer to start efficiently. Moreover, the conjugative pilus and underlying secretion machinery assembled in the absence of Par proteins mediate poor biofilm formation and are completely dysfunctional for pilus specific R17 bacteriophage uptake. Thus, functional integration of Par components at the interface of relaxosome, coupling protein, and channel ATPases appears important for an optimal conformation and effective activation of the transfer machinery. We conclude that low copy plasmid R1 has evolved an active segregation system that optimizes both its vertical and lateral modes of dissemination.

Common requirement for the relaxosome of plasmid R1 in multiple activities of the conjugative type IV secretion system.

Macromolecular transport by bacterial type IV secretion systems involves regulated uptake of (nucleo)protein complexes by the cell envelope-spanning transport channel. A coupling protein receptor is believed to recognize the specific proteins destined for transfer, but the steps initiating their translocation remain unknown. Here, we investigate the contribution of a complex of transfer initiation proteins, the relaxosome, of plasmid R1 to translocation of competing transferable substrates from mobilizable plasmids ColE1 and CloDF13 or the bacteriophage R17. We found that not only does the R1 translocation machinery engage the R1 relaxosome during conjugative self-transfer and during infection by R17 phage but it is also activated by its cognate relaxosome to mediate the export of an alternative plasmid. Transporter activity was optimized by the R1 relaxosome even when this complex itself could not be transferred, i.e., when the N-terminal activation domain (amino acids 1 to 992 [N1-992]) of TraI was present without the C-terminal conjugative helicase domain. We propose that the functional dependence of the transfer machinery on the R1 relaxosome for initiating translocation ensures that dissemination of heterologous plasmids does not occur at the expense of self-transfer.

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 subdomains with striking structural homology with helicase subdomains 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.

An activation domain of plasmid R1 TraI protein delineates stages of gene transfer initiation.

Bacterial conjugation is a form of type IV secretion that transports protein and DNA to recipient cells. Specific bacteriophage exploit the conjugative pili and cell envelope spanning protein machinery of these systems to invade bacterial cells. Infection by phage R17 requires F-like pili and coupling protein TraD, which gates the cytoplasmic entrance of the secretion channel. Here we investigate the role of TraD in R17 nucleoprotein uptake and find parallels to secretion mechanisms. The relaxosome of IncFII plasmid R1 is required. A ternary complex of plasmid oriT, TraD and a novel activation domain within the N-terminal 992 residues of TraI contributes a key mechanism involving relaxase-associated properties of TraI, protein interaction and the TraD ATPase. Helicase-associated activities of TraI are dispensable. These findings distinguish for the first time specific protein domains and complexes that process extracellular signals into distinct activation stages in the type IV initiation pathway. The study also provided insights into the evolutionary interplay of phage and the plasmids they exploit. Related plasmid F adapted to R17 independently of TraI. It follows that selection for phage resistance drives not only variation in TraA pilins but diversifies TraD and its binding partners in a plasmid-specific manner.

Molecular recognition determinants for type IV secretion of diverse families of conjugative relaxases.

In preparation for transfer conjugative type IV secretion systems (T4SS) produce a nucleoprotein adduct containing a relaxase enzyme covalently linked to the 5' end of single-stranded plasmid DNA. The bound relaxase is expected to present features necessary for selective recognition by the type IV coupling protein (T4CP), which controls substrate entry to the envelope spanning secretion machinery. We prove that the IncF plasmid R1 relaxase TraI is translocated to the recipient cells. Using a Cre recombinase assay (CRAfT) we mapped two internally positioned translocation signals (TS) on F-like TraI proteins that independently mediate efficient recognition and secretion. Tertiary structure predictions for the TS matched best helicase RecD2 from Deinococcus radiodurans. The TS is widely conserved in MOB(F) and MOB(Q) families of relaxases. Structure/function relationships within the TS were identified by mutation. A key residue in specific recognition by T4CP TraD was revealed by a fidelity switch phenotype for an F to plasmid R1 exchange L626H mutation. Finally, we show that physical linkage of the relaxase catalytic domain to a TraI TS is necessary for efficient conjugative transfer.

Protein and DNA effectors control the TraI conjugative helicase of plasmid R1.

The mechanisms controlling progression of conjugative DNA processing from a preinitiation stage of specific plasmid strand cleavage at the transfer origin to a stage competent for unwinding the DNA strand destined for transfer remain obscure. Linear heteroduplex substrates containing double-stranded DNA binding sites for plasmid R1 relaxosome proteins and various regions of open duplex for TraI helicase loading were constructed to model putative intermediate structures in the initiation pathway. The activity of TraI was compared in steady-state multiple turnover experiments that measured the net production of unwound DNA as well as transesterase-catalyzed cleavage at nic. Helicase efficiency was enhanced by the relaxosome components TraM and integration host factor. The magnitude of stimulation depended on the proximity of the specific protein binding sites to the position of open DNA. The cytoplasmic domain of the R1 coupling protein, TraDDeltaN130, stimulated helicase efficiency on all substrates in a manner consistent with cooperative interaction and sequence-independent DNA binding. Variation in the position of duplex opening also revealed an unsuspected autoinhibition of the unwinding reaction catalyzed by full-length TraI. The activity reduction was sequence dependent and was not observed with a truncated helicase, TraIDeltaN308, lacking the site-specific DNA binding transesterase domain. Given that transesterase and helicase domains are physically tethered in the wild-type protein, this observation suggests that an intramolecular switch controls helicase activation. The data support a model where protein-protein and DNA ligand interactions at the coupling protein interface coordinate the transition initiating production and uptake of the nucleoprotein secretion substrate.

Plasmid r1 conjugative DNA processing is regulated at the coupling protein interface.

Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. TraDDeltaN130 stimulated the TraI DNA transesterase activity apparently via interactions on both the protein and the DNA levels. TraM, a protein interaction partner of TraD, also increased DNA transesterase activity in vitro. The mechanism may involve altered DNA conformation as TraM induced underwinding of oriT plasmid DNA in vivo (DeltaL(k) = -4). Permanganate mapping of the positions of duplex melting due to relaxosome assembly with TraDDeltaN130 on supercoiled DNA in vitro confirmed localized unwinding at nic but ruled out formation of an open complex compatible with initiation of the TraI helicase activity. These data link relaxosome regulation to the T4CP and support the model that a committed step in the initiation of DNA export requires activation of TraI helicase loading or catalysis.

The combined contraceptive vaginal ring (NuvaRing): evaluation of the clinical and pharmacological evidence.

The NuvaRing((R)) is a vaginal ring contraceptive that releases a daily dose of 15 microg ethinylestradiol and 120 microg etonogestrel through the vaginal epithelium, thereby avoiding the daily fluctuations in serum levels typically observed with combined oral contraceptives. Each ring is designed for a single 3-week use followed by a 1-week ring-free period. The ring offers robust inhibition of ovulation, yielding a Pearl Index of 0.65 for European women in registration trials. The ring has the same contraindications as combined oral contraceptives. The vaginal ring is a highly effective, safe and well-tolerated method of hormonal contraception designed for reproductive-aged women who desire freedom from the daily pill.

Increased intrauterine frequency of Ureaplasma urealyticum in women with preterm labor and preterm premature rupture of the membranes and subsequent cesarean delivery.

OBJECTIVE: The purpose of this study was to evaluate the prevalence of microbial invasion of the amniotic cavity at the time of preterm cesarean delivery for therapy-resistant preterm labor or preterm premature rupture of membranes, which are events that commonly are induced by infection, and to compare this group of patients with a group of patients who underwent preterm cesarean delivery for indications other than preterm labor or preterm premature rupture of membranes. STUDY DESIGN: We studied 207 consecutive women between 23 and 34 weeks of gestation who underwent cesarean delivery. These patients were divided into 3 groups according to the indication for cesarean delivery: patients with preterm labor (group 1), patients with preterm premature rupture of membranes (group 2), and patients with other indications (group 3). In the course of the surgical procedure, amniotic fluid, amniotic membrane, and placental tissue specimens were collected for the detection of pathogens. RESULTS: Ureaplasma urealyticum was detected in 43.9% (58/132) of the patients of groups 1 and 2, with no significant difference between these 2 subgroups. In group 3, which served as the comparison group, Ureaplasma urealyticum was isolated in only 2.7% (2/75) of the patients. Ureaplasma urealyticum as a single pathogen was more frequent than all obligate pathogens together (43.9% vs 39.3%). CONCLUSION: Our results provide evidence for an association between intrauterine colonization with Ureaplasma urealyticum and both therapy-resistant preterm labor and preterm premature rupture of membranes.

The role of dydrogesterone in recurrent (habitual) abortion.

The published evidence regarding the administration of dydrogesterone in the treatment of habitual abortion is summarised in this review. Habitual abortion is defined as the loss of three or more consecutive pregnancies without known maternal or foetal pathology. The immunology of early pregnancy seems to determine the rejection or non-rejection of the allogenic embryo. When peripheral mononuclear cells from recurrent aborters are incubated with progesterone or dydrogesterone in vitro, T-helper (Th)2 cytokines such as interleukin (IL)-4 and IL-6 markedly increase whereas the Th1 cytokine interferon-gamma decreases. Additionally, both progesterone and dydrogesterone are thought to inhibit the activity of natural killer cells at the foeto-maternal interface in humans. Progesterone-induced blocking factor (PIBF) mediates the immunological effects of progesterone and dydrogesterone in pregnancy. It affects various phases of the maternal immune response involving both the cellular and humoral immune system, exerts anti-abortive effects and inhibits the release of arachidonic acid. It also favours the production of so-called asymmetric, pregnancy-protecting antibodies. In rodents, blockade of this factor results in the termination of pregnancy and in women considerably lower levels are found in those with threatened abortion or pre-term labour. In order to draw final conclusions as to the usefulness of dydrogesterone in women with a history of recurrent miscarriage, further controlled, blinded, randomised clinical trials are needed.

Large-for-gestational-age newborns in women with insulin-treated gestational diabetes under strict metabolic control.

OBJECTIVE: To assess the influence of strict metabolic control in women with insulin-treated gestational diabetes on the risk of large-for-gestational-age (LGA) newborns, the frequency of obstetrical complications and fetal outcome. METHODS: In this prospective cohort study, 875 women were screened for gestational diabetes mellitus with a 75 g oral glucose tolerance test (OGTT) between weeks 24 and 28 of gestation. The study group (n = 162) consisted of women with insulin-treated gestational diabetes mellitus (GDM) and the control group (n = 713) of women with normal glucose tolerance (NGT). In the women with diabetes, strict adjustments of fasting glucose levels to 90 mg/dl and 130 mg/dl postprandially were achieved with insulin administration. RESULTS: No increased risk for LGA newborns was observed in women with GDM and good metabolic control (16.7% vs. 12.3%; p = 0.1). In women with NGT, maternal prepregnancy BMI was significantly higher in those who delivered LGA newborns than in those who gave birth to newborns below the 90th percentile [27.2 kg/m(2) (5.0) vs. 24.4 kg/m(2) (5.6); p = 0.006], whereas there was no influence of maternal BMI on birth weight of newborns in women with GDM. There was no difference between the two groups with respect to maternal birth traumata and fetal outcome, except for plexus palsy which occurred in three GDM women with macrosomic newborns. CONCLUSION: Strict metabolic control and surveillance in women with insulin-treated GDM seems to attenuate the risk for LGA newborns, diabetic fetopathia, and the influence of maternal BMI on fetal growth.

IL-8 concentrations in maternal serum, amniotic fluid and cord blood in relation to different pathogens within the amniotic cavity.

OBJECTIVE: The association between elevated interleukin (IL)-8 concentrations in amniotic fluid and preterm delivery is well described. Little consideration has been given to the impact of different groups of microorganisms within the amniotic cavity on IL-8 concentration. METHODS: We collected amniotic fluid, placental tissue and amniotic membranes during preterm cesarean sections for bacterial culture. In addition, we determined IL-8 concentrations in maternal serum, amniotic fluid and cord blood and correlated them with the various intra-amniotic pathogens isolated by bacterial culture. RESULTS: IL-8 concentrations were determined in amniotic fluid in 107 cases, in cord blood in 185 cases and in maternal blood in 158 cases. Women with intra-amniotic Ureaplasma urealyticum infection had significantly higher amniotic fluid concentrations of IL-8 than those without (P< 0.001). In cord blood, we found significantly elevated IL-8 concentrations due to intra-amniotic infection with U. urealyticum (P=0.045) and other pathogens (P=0.04). In maternal sera, we found no significant elevation of maternal IL-8 in any of the groups. CONCLUSION: Intrauterine infection with U. urealyticum seems to play a profound role in the cascade of inflammation and increases IL-8 concentrations in amniotic fluid and cord blood.

Anatomy of the estrogen response element.

Estrogens exert their regulatory potential on gene expression through different nuclear and non-nuclear mechanisms. A direct nuclear approach is the interaction of estrogen with specific target sequences of DNA, estrogen response elements (ERE) or units. EREs can be grouped into perfect and imperfect palindromic sequences with the imperfect sequences differing from the consensus sequence in one or more nucleotides and being less responsive to the activated estrogen-estrogen receptor (ER) complex. Differences in the ERE sequence and the ER subtype involved can substantially alter ER-ERE interaction. In addition, cross-talk between ERs and other nuclear transcription factors profoundly influences gene expression. Here, we focus on the recent advances in the understanding of the structure of EREs and how ERs are recruited to these. Identifying known target genes for estrogen action could help us to understand the potential risks and benefits of the administration of this steroid to humans.

Body mass index, follicle-stimulating hormone and their predictive value in in vitro fertilization.

PURPOSE: The objective was to explore whether body mass and day 3 follicle-stimulating hormone have predictive value on odds of pregnancy after in vitro fertilisation. Few studies show that obesity produces a variety of alterations in the reproductive system, and that women with an elevation of day 3 FSH have declining ovarian function. METHODS: The data of one-hundred-seventy-one women who underwent a standard regime of controlled ovarian hyperstimulation was analyzed with particular reference to variations in body mass and hormone levels. RESULTS: By raising BMI and FSH (mIU/mL) by one unit, the odds for pregnancy were decreased by the respective factors 0.84 (95% confidence interval 0.73-0.97) and 0.77 (95% confidence interval 0.59-1.00). CONCLUSIONS: The results demonstrate that for the purpose of raising the odds of pregnancy BMI should be reduced. A low FSH value may cause the same effect. Nontheless, obesity and hormonal function may be independent risk factors for failure in assisted reproduction.

Differential effects of progestins on the brain.

Interactions exist between progestins and the gamma-aminobutyric acid (GABA) receptor subtype A where C(21)-steroids function as activators. Other interactions between progesterone and neurotransmitter systems include stimulation of dopamine release in striatal tissue, stimulation of GnRH release from hypothalamic neurons and inhibition of opioid receptor binding and activation. Cyproterone acetate increases dopaminergic responses and binds to opiate receptors independently of its classical effect on the androgen receptor. Progesterone substitution in perimenopausal women promotes length and quality of sleep. This effect seems most prominent for progesterone administered vaginally. Progestins also play a role in the pathogenesis of migraine. Migraine symptoms occur predominantly during the perimenstrual stage. Women who suffer from menstrual migraine triggered by premenstrual progesterone loss often benefit from cyclic progesterone administration. This may be because progesterone and allopregnenolone reduce meningeal release of substance P and inhibit the development of neurogenic oedema. Women whose migraine symptoms subside during pregnancy, however, benefit from intramuscular medroxyprogesterone acetate. Progesterone, generated from pregnenolone by Schwann cells, also enhances myelin synthesis. Myelination of axons is promoted when progesterone is added to cultures of rat dorsal root ganglia. No reliable data exist with respect to the effects of other progestins on demyelinating disease. Progestins promote the growth of meningioma as progesterone receptors predominate in meningioma tissue. Progesterone and synthetic progestins should therefore not be prescribed in these patients.

Physical characteristics of newborn babies after vaginal delivery: a 25-year study from Vienna.

This retrospective analysis evaluated whether the gestational age, mean birth weight, length and shoulder width of vaginally delivered newborns have increased during the last 25 years in Vienna, Austria. Women who have given birth at the University of Vienna Medical School Hospital were divided into four age groups: under 20 years, 20-24 years, 25-29 years, and 30 years of age or over. Patient data collected between 1976 to 2000 were grouped into five periods: 1976-1979, 1980-1984, 1985-1989, 1990-1994, and 1995-2000. A highly significant increase of all four parameters was observed for all age groups between 1976 and 2000. The changes were most marked in the 25-29 year age group.

Genetic factors in assisted reproduction.

It is still unclear whether the procedures of assisted reproduction increase the risk of congenital malformations. Thus, it remains to be clarified whether an increased risk, if any, of congenital malformations in these children is caused by the procedure of assisted reproduction itself or by the underlying maternal and paternal background. From the genetic point of view, infertility patients seeking assisted reproduction have to be classified as a high-risk group. The prevalence of numerical chromosomal abnormalities is around 10% in these patients, compared with 0.85% in the general population. The prevalence of structural chromosomal abnormalities is around 0.1% in the general population and is increased up to 1% in patients seeking assisted reproduction. In addition, patients with microdeletions of the Y-chromosome or mutations in the cystic fibrosis transmembrane-conductance regulator gene are likely to be encountered at the fertility clinic. Therefore, genetic screening and counselling should be routinely offered to infertility patients. They also need to understand that parental factors can be transferred to offspring that would most likely not have been conceived by natural means.

Absence of microdeletions in the azoospermia-factor region of the Y-chromosome in Viennese men seeking assisted reproduction.

PURPOSE: The azoospermia-factor region of the Y-chromosome is essential for spermatogenesis in humans. In the literature, a wide range is given for the frequency of microdeletions in this region. The purpose of this study was to evaluate our own population of patients. METHODS: During a two-year period at Vienna Medical School, all male patients (n = 383) seeking assisted reproduction were screened for microdeletions. Thirty-three men had azoospermia and 154 severe oligozoospermia. Genomic DNA was prepared from peripheral lymphocytes and polymerase chain reaction analysis of the azoospermia-factor region was performed using the Promega kit. RESULTS: No case tested positive for azoospermia-factor microdeletions. In all cases amplification of 18 non-polymorphic sequence tagged sites was obtained. CONCLUSIONS: Y-chromosome microdeletions do not seem to be an important factor for male infertility in our patients. This suggests that screening should be restricted to men with azoospermia or severe oligozoospermia only.