Functions of base flipping in E. coli nucleotide excision repair

UvrB is the main damage recognition protein in bacterial nucleotide excision repair and is capable of recognizing various structurally unrelated types of damage. Previously we have shown that upon binding of Escherichia coli UvrB to damaged DNA two nucleotides become extrahelical: the nucleotide directly 3' to the lesion and its base-pairing partner in the non-damaged strand. Here we demonstrate using a novel fluorescent 2-aminopurine-menthol modification that the position of the damaged nucleotide itself does not change upon UvrB binding. A co-crystal structure of B. caldotenax UvrB and DNA has revealed that one nucleotide is flipped out of the DNA helix into a pocket of the UvrB protein where it stacks on Phe249 [J.J. Truglio, E. Karakas, B. Hau, H. Wang, M.J. DellaVecchia, B. van Houten, C. Kisker, Structural basis for DNA recognition and processing by UvrB, Nat. Struct. Mol. Biol. 13 (2006) 360-364]. By mutating the equivalent of Phe249 (Tyr249) in the E. coli UvrB protein we show that on damaged DNA neither of the extrahelical nucleotides is inserted into this protein pocket. The mutant UvrB protein, however, resulted in an increased binding and incision of undamaged DNA showing that insertion of a base into the nucleotide-binding pocket is important for dissociation of UvrB from undamaged sites. Replacing the nucleotides in the non-damaged strand with a C3-linker revealed that the extruded base in the non-damaged strand is not directly involved in UvrB-binding or UvrC-mediated incision, but that its displacement is needed to allow access for residues of UvrB or UvrC to the neighboring base, which is directly opposite the DNA damage. This interaction is shown to be essential for optimal 3'-incision by UvrC. After 3'-incision base flipping in the non-damaged DNA strand is lost, indicative for a conformational change needed to prepare the UvrB-DNA complex for 5'-incision.     

Exo-inulinase of Aspergillus niger N402: A hydrolytic enzyme with significant transfructosylating activity

The purified exo-inulinase enzyme of Aspergillus niger N402 (AngInuE; heterologously expressed in Escherichia coli) displayed a sucrose:inulin (S/I) hydrolysis ratio of 2.3, characteristic for a typical exo-inulinase. The enzyme also had significant transfructosylating activity with increasing sucrose concentrations, producing various oligosaccharides. The AngInuE protein molecular mass was 57 kDa, close to the calculated value for the mature protein. AngInuE thus was active as a monomeric, non-glycosylated protein. Contradictory data on hydrolysis/transfructosylation activity ratios have been published for the (almost) identical (but monomeric or dimeric and glycosylated) exo-inulinases of other aspergilli. Our data clearly show that the AngInuE enzyme, produced in and purified from E. coli, is a broad specificity exo-inulinase that also has significant transfructosylating activity with sucrose. Analysis of site-directed mutants of AngInuE showed that the glycoside hydrolase family 32 conserved domain G is important for catalytic efficiency, with a clear role in hydrolysis of both sucrose and fructans.     

Architecture of nucleotide excision repair complexes: DNA is wrapped by UvrB before and after damage recognition

Nucleotide excision repair (NER) is a major DNA repair mechanism that recognizes a broad range of DNA damages. In Escherichia coli, damage recognition in NER is accomplished by the UvrA and UvrB proteins. We have analysed the structural properties of the different protein-DNA complexes formed by UvrA, UvrB and (damaged) DNA using atomic force microscopy. Analysis of the UvrA(2)B complex in search of damage revealed the DNA to be wrapped around the UvrB protein, comprising a region of about seven helical turns. In the UvrB-DNA pre-incision complex the DNA is wrapped in a similar way and this DNA configuration is dependent on ATP binding. Based on these results, a role for DNA wrapping in damage recognition is proposed. Evidence is presented that DNA wrapping in the pre-incision complex also stimulates the rate of incision by UvrC.     

Geographic diversity and temporal trends of antimicrobial resistance in Streptococcus pneumoniae in the United States

Resistance of Streptococcus pneumoniae to antibiotics is increasing throughout the United States, with substantial variation among geographic regions. We show that patterns of geographic variation are best explained by the intensity of selection for resistance, which is reflected by differences between the proportions of resistance within individual serotypes, rather than by differences between the frequencies of particular serotypes. Using a mathematical transmission model, we analyzed temporal trends in the proportions of singly and dually resistant organisms and found that pneumococcal strains resistant to both penicillin and erythromycin are increasing faster than strains singly resistant to either. Using the model, we predict that by 1 July 2004, in the absence of a vaccine, 41% of pneumococci at the Centers for Disease Control and Prevention (CDC)'s Active Bacterial Core surveillance (ABCs) sites, taken together, will be dually resistant, with 5% resistant to penicillin only and 5% to erythromycin only.     

H-NS mediated compaction of DNA visualised by atomic force microscopy

The Escherichia coli H-NS protein is a nucleoid-associated protein involved in gene regulation and DNA compaction. To get more insight into the mechanism of DNA compaction we applied atomic force microscopy (AFM) to study the structure of H-NS–DNA complexes. On circular DNA molecules two different levels of H-NS induced condensation were observed. H-NS induced lateral condensation of large regions of the plasmid. In addition, large globular structures were identified that incorporated a considerable amount of DNA. The formation of these globular structures appeared not to be dependent on any specific sequence. On the basis of the AFM images, a model for global condensation of the chromosomal DNA by H-NS is proposed.     

Two functionally divergent UDP-Gal nucleotide sugar transporters participate in phosphoglycan synthesis in Leishmania major

In the protozoan parasite Leishmania, abundant surface and secreted molecules, such as lipophosphoglycan (LPG) and proteophosphoglycans (PPGs), contain extensive galactose in the form of phosphoglycans (PGs) based on (Gal-Man-PO(4)) repeating units. PGs are synthesized in the parasite Golgi apparatus and require transport of cytoplasmic nucleotide sugar precursors to the Golgi lumen by nucleotide sugar transporters (NSTs). GDP-Man transport is mediated by the LPG2 gene product, and here we focused on transporters for UDP-Gal. Data base mining revealed 12 candidate NST genes in the L. major genome, including LPG2 as well as a candidate endoplasmic reticulum UDP-glucose transporter (HUT1L) and several pseudogenes. Gene knock-out studies established that two genes (LPG5A and LPG5B) encoded UDP-Gal NSTs. Although the single lpg5A(-) and lpg5B(-) mutants produced PGs, an lpg5A(-)/5B(-) double mutant was completely deficient. PG synthesis was restored in the lpg5A(-)/5B(-) mutant by heterologous expression of the human UDP-Gal transporter, and heterologous expression of LPG5A and LPG5B rescued the glycosylation defects of the mammalian Lec8 mutant, which is deficient in UDP-Gal uptake. Interestingly, the LPG5A and LPG5B functions overlap but are not equivalent, since the lpg5A(-) mutant showed a partial defect in LPG but not PPG phosphoglycosylation, whereas the lpg5B(-) mutant showed a partial defect in PPG but not LPG phosphoglycosylation. Identification of these key NSTs in Leishmania will facilitate the dissection of glycoconjugate synthesis and its role(s) in the parasite life cycle and further our understanding of NSTs generally.     

Determinants of Venous Thromboembolism among Hospitalizations of US Adults: A Multilevel Analysis

Background

Venous thromboembolism (VTE) is a significant clinical and public health concern. We evaluated a variety of multilevel factors—demographics, clinical and insurance status, preexisting comorbid conditions, and hospital characteristics—for VTE diagnosis among hospitalizations of US adults.

Methods

We generated adjusted odds ratios with 95% confidence intervals (CIs) and determined sources of outcome variation by conducting multilevel logistic regression analysis of data from the 2011 Nationwide Inpatient Sample that included 6,710,066 hospitalizations of US adults nested within 1,039 hospitals.

Results

Among hospitalizations of adults, age, sex, race or ethnicity, total days of hospital stay, status of health insurance, and operating room procedure were important determinants of VTE diagnosis; each of the following preexisting comorbid conditions—acquired immune deficiency syndrome, anemia, arthritis, congestive heart failure, coagulopathy, hypertension, lymphoma, metastatic cancer, other neurological disorders, obesity, paralysis, pulmonary circulation disorders, renal failure, solid tumor without metastasis, and weight loss—was associated independently with 1.04 (95% CI: 1.02−1.06) to 2.91 (95% CI: 2.81−3.00) times increased likelihood of VTE diagnosis than among hospitalizations of adults without any of these corresponding conditions. The presence of 2 or more of such conditions was associated a 180%−450% increased likelihood of a VTE diagnosis. Hospitalizations of adults who were treated in urban hospitals were associated with a 14%−15% increased likelihood of having a VTE diagnosis than those treated in rural hospitals. Approximately 7.4% of the total variation in VTE diagnosis occurred between hospitals.

Conclusion

The presence of certain comorbidities and hospital contextual factors is associated with significantly elevated likelihood of VTE diagnosis among hospitalizations of adults. The findings of this study underscore the importance of clinical risk assessment and adherence to evidence-based clinical practice guidelines in preventing VTE, as well as the need to evaluate potential contextual factors that might modify the risk of VTE among hospitalized patients.     

High HIV Prevalence among Asylum Seekers Who Gave Birth in the Netherlands: A Nationwide Study Based on Antenatal HIV Tests

Objectives

Asylum seekers are considered to be a particularly vulnerable group with respect to HIV. Data on the HIV prevalence among asylum seekers, however, are scarce. The aim of this study is to map the HIV prevalence among asylum seekers who gave birth in The Netherlands.

Methods

We used a nationwide electronic medical records database from the community health services for asylum seekers (MOA). The study population consisted of 4,854 women and girls who delivered in asylum reception between 2000 and 2008. A unique electronic health data base was used and case allocation was based on ICPC-codes.

Results

The number of women and girls that was HIV positive during their last pregnancy was 80, of which 79 originated from sub-Saharan Africa. The prevalence for women from this region of origin (3.4%) was high compared to women from all other regions of origin (0.04%; OR = 90.2; 95%CI 12.5–648.8). The highest HIV prevalence rates were found for women from Rwanda (17.0%) and Cameroon (13.2%). HIV prevalence rates were higher among women who arrived in reception without partner (OR = 1.82; 95%CI 0.75–4.44) and unaccompanied minors (OR = 2.59; 95%CI 0.79–8.49), compared to women who arrived in reception with partner.

Conclusions

We conclude that, among asylum-seeking women from sub-Saharan Africa giving birth in The Netherlands, the HIV prevalence is high compared to the host population. For women from other regions of origin, the prevalence is at the same level as in the host population. The high HIV prevalence underlines the importance of preventive interventions and voluntary HIV testing for sub-Saharan African asylum seekers as from shortly after arrival.     

Molecular and biochemical characterization of a novel intracellular invertase from Aspergillus niger with transfructosylating activity

A novel subfamily of putative intracellular invertase enzymes (glycoside hydrolase family 32) has previously been identified in fungal genomes. Here, we report phylogenetic, molecular, and biochemical characteristics of SucB, one of two novel intracellular invertases identified in Aspergillus niger. The sucB gene was expressed in Escherichia coli and an invertase-negative strain of Saccharomyces cerevisiae. Enzyme purified from E. coli lysate displayed a molecular mass of 75 kDa, judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Its optimum pH and temperature for sucrose hydrolysis were determined to be 5.0 and 37 to 40 degrees C, respectively. In addition to sucrose, the enzyme hydrolyzed 1-kestose, nystose, and raffinose but not inulin and levan. SucB produced 1-kestose and nystose from sucrose and 1-kestose, respectively. With nystose as a substrate, products up to a degree of polymerization of 4 were observed. SucB displayed typical Michaelis-Menten kinetics with substrate inhibition on sucrose (apparent K(m), K(i), and V(max) of 2.0 +/- 0.2 mM, 268.1 +/- 18.1 mM, and 6.6 +/- 0.2 mumol min(-1) mg(-1) of protein [total activity], respectively). At sucrose concentrations up to 400 mM, transfructosylation (FTF) activity contributed approximately 20 to 30% to total activity. At higher sucrose concentrations, FTF activity increased to up to 50% of total activity. Disruption of sucB in A. niger resulted in an earlier onset of sporulation on solid medium containing various carbon sources, whereas no alteration of growth in liquid culture medium was observed. SucB thus does not play an essential role in inulin or sucrose catabolism in A. niger but may be needed for the intracellular conversion of sucrose to fructose, glucose, and small oligosaccharides.     

Dynamics of the UvrABC nucleotide excision repair proteins analyzed by fluorescence resonance energy transfer

UvrB plays a key role in bacterial nucleotide excision repair. It is the ultimate damage-binding protein that interacts with both UvrA and UvrC. The oligomeric state of UvrB and the UvrAB complex have been subject of debate for a long time. Using fluorescence resonance energy transfer (FRET) between GFP and YFP fused to the C-terminal end of Escherichia coli UvrB, we unambiguously show that in solution two UvrB subunits bind to UvrA, most likely as part of a UvrA2B2 complex. This complex is most stable when both UvrA and UvrB are in the ATP-bound form. Analysis of a truncated form of UvrB shows that binding to UvrA promotes dimerization of the two C-terminal domain 4 regions of UvrB. The presence of undamaged DNA leads to dissociation of the UvrA2B2 complex, but when the ATPase site of UvrB is inactivated, the complex is trapped on the DNA. When the complex is bound to a damaged site, FRET between the two UvrB subunits could still be detected, but only as long as UvrA remains associated. Dissociation of UvrA from the damage-bound UvrB dimer leads to the reduction of the magnitude of the FRET signal, indicating that the domain 4 regions no longer interact. We propose that the UvrA-induced dimerization of the domain 4 regions serves to shield these domains from premature UvrC binding. Only after specific binding of the UvrB dimer to a damaged site and subsequent release of UvrA is the contact between the domain 4 regions broken, allowing recruitment of UvrC and subsequent incisions.