Purine Utilization by Klebsiella oxytoca M5al: Genes for Ring-Oxidizing and -Opening Enzymes

The enterobacterium Klebsiella oxytoca uses a variety of inorganic and organic nitrogen sources, including purines, nitrogen-rich compounds that are widespread in the biosphere. We have identified a 23-gene cluster that encodes the enzymes for utilizing purines as the sole nitrogen source. Growth and complementation tests with insertion mutants, combined with sequence comparisons, reveal functions for the products of these genes. Here, we report our characterization of 12 genes, one encoding guanine deaminase and the others encoding enzymes for converting (hypo)xanthine to allantoate. Conventionally, xanthine dehydrogenase, a broadly distributed molybdoflavoenzyme, catalyzes sequential hydroxylation reactions to convert hypoxanthine via xanthine to urate. Our results show that these reactions in K. oxytoca are catalyzed by a two-component oxygenase (HpxE-HpxD enzyme) homologous to Rieske nonheme iron aromatic-ring-hydroxylating systems, such as phthalate dioxygenase. Our results also reveal previously undescribed enzymes involved in urate oxidation to allantoin, catalyzed by a flavoprotein monooxygenase (HpxO enzyme), and in allantoin conversion to allantoate, which involves allantoin racemase (HpxA enzyme). The pathway also includes the recently described PuuE allantoinase (HpxB enzyme). The HpxE-HpxD and HpxO enzymes were discovered independently by de la Riva et al. (L. de la Riva, J. Badia, J. Aguilar, R. A. Bender, and L. Baldoma, J. Bacteriol. 190:7892-7903, 2008). Thus, several enzymes in this K. oxytoca purine utilization pathway differ from those in other microorganisms. Isofunctional homologs of these enzymes apparently are encoded by other species, including Acinetobacter, Burkholderia, Pseudomonas, Saccharomyces, and Xanthomonas.Purines and purine derivatives comprise a large portion of biomass and are involved in almost every step of life. Not only a major constituent of nucleic acids, they also are central to energy transfer and storage (ATP) as well as protein synthesis and signaling (GTP). Plants, animals, and many microorganisms use purines and purine derivatives to store and translocate nitrogen for assimilation or excretion (96).Salvage pathways operate to recycle purines, including hypoxanthine and xanthine, back into nucleoside pools (107). Additionally, some organisms can utilize purines as the sole source of nitrogen and carbon. Adenine and guanine are deaminated to form hypoxanthine and xanthine, respectively, which then are oxidized to form uric acid (urate at physiological pH) (Fig. ​(Fig.1).1). These oxidation steps are catalyzed by xanthine dehydrogenase, a well-studied molybdoflavoenzyme that is conserved from bacteria to humans (51). Two sequential ring-opening steps convert urate via allantoin to allantoate (Fig. ​(Fig.1).1). Subsequent steps, which comprise different pathways in different microorganisms (96), convert allantoate to ammonium, which is assimilated.Open in a separate windowFIG. 1.Purine ring oxidation and opening steps. The enzyme proposed to catalyze each step is shown. The K. oxytoca gene for adenine deaminase was not identified in this study. Dashed lines show reactions that can occur spontaneously.Some organisms express only the latter portion of the purine utilization pathway and cannot use purines or urate as sole sources of nitrogen. For example, Escherichia coli K-12 can use allantoin and its catabolites as the sole nitrogen source, albeit only under anaerobic conditions (21). Saccharomyces cerevisiae uses allantoin as a nitrogen storage compound (17). However, the complete pathway is present in other bacterial and fungal species, including Bacillus subtilis (84) and Aspergillus nidulans (83).Molybdoenzymes (excepting dinitrogenase) contain the molybdenum cofactor Mo-molybdopterin (42). Thus, mutations in genes for molybdenum cofactor biosynthetic enzymes (mol genes in bacteria and cnx in A. nidulans) confer pleiotropic phenotypes: these mutants can utilize neither nitrate nor purines, due to lack of the molybdoenzymes nitrate reductase and xanthine dehydrogenase (74). We previously reported that Klebsiella oxytoca mol mutants cannot assimilate nitrate but can utilize xanthine as the sole nitrogen source (32). This suggested, as one possibility, that K. oxytoca uses a molybdenum-independent enzyme in place of conventional xanthine dehydrogenase. Results reported here demonstrate that this is correct, as insertion mutants blocked specifically in xanthine and hypoxanthine utilization define the structural genes for an apparent two-component Reiske nonheme iron oxygenase.Here, we report analysis of 12 genes whose products catalyze conversion of purines to allantoate. Our investigation of the remaining genes, whose products catalyze allantoate utilization, is ongoing. Results show that several steps in the overall pathway are catalyzed by previously undescribed enzymes.While this paper was in review, the paper by de la Riva et al. (24), describing the hpxDE, hpxR, hpxO, and hpxPQT genes from Klebsiella pneumoniae W70, was posted in the “JB Accepts” section of the Journal of Bacteriology online edition. Results and conclusions concerning these seven genes are congruent between the two studies.(Some of the work presented here was submitted by Danielle Carl in 1994 as part of an undergraduate thesis to the Cornell University Division of Biological Sciences Honors Program.)     

Fathers have lower salivary testosterone levels than unmarried men and married non-fathers in Beijing, China

A growing body of evidence, almost entirely from North America, has found that male testosterone levels are positively associated with mating effort (male-male competition and mate-seeking behaviour), while lower testosterone levels have been associated with affiliative pair bonding and paternal care. To expand the cross-cultural scope of this research, here we investigate variation in salivary testosterone levels among Chinese men in relation to marital and parenting variables. One hundred and twenty-six men drawn from a Beijing university setting between the ages of 21 and 38 completed a questionnaire and provided both morning and late afternoon saliva samples from which testosterone levels were measured. The 66 unmarried men had slightly higher levels of testosterone than the 30 married non-fathers, but this difference was not statistically significant. However, the 30 fathers exhibited significantly lower testosterone levels than both unmarried men and married non-fathers. Among married non-fathers, marital relationship quality was not significantly related to testosterone levels. Among married fathers, men with children aged less than 4 years of age did not have lower testosterone levels than men with older children. These data are the first outside of North America to show lower testosterone levels among fathers, and lend support to the theoretical view that male testosterone levels differ according to mating and parenting effort.     

Development and optimisation of a sex pheromone lure for monitoring populations of saddle gall midge,Haplodiplosis marginata

Saddle gall midge, Haplodiplosis marginata (von Roser) (Diptera: Cecidomyiidae), is a sporadic pest of cereals in Northern and Central Europe and is of increasing importance in the UK. Recently, the major component of the sex pheromone produced by adult female H. marginata was reported to be 2‐nonyl butyrate. The importance of absolute configuration on attractiveness, the effects on trap catches of the addition of minor pheromone components, dispenser type, and pheromone loading are described in the development of an optimised pheromone lure with which to trap H. marginata males. In analyses of volatiles collected from virgin female H. marginata by gas chromatography (GC) coupled with electroantennographic recording (EAG) from the antenna of a male H. marginata, two EAG responses were observed. Analyses by coupled GC‐mass spectrometry (MS) indicated these were due to 2‐nonyl butyrate and a trace amount (1%) of 2‐heptyl butyrate. A similar trace amount of 2‐nonanol was detected in GC‐MS analyses but this compound did not elicit an EAG response when the synthetic compound was tested, whereas the other two compounds did. These three compounds were not observed in collections of volatiles made from male H. marginata. The 2‐nonyl butyrate was shown to be the (R)‐enantiomer. In field trapping tests (R)‐2‐nonyl butyrate was at least 10× more attractive to male H. marginata than the racemic compound, and the (S)‐enantiomer was unattractive. Addition of the potential minor components individually or together at the naturally occurring ratios did not increase or reduce the attractiveness of the lure. Polyethylene vials and rubber septa were equally effective as pheromone dispensers, lasting for at least 5 weeks in the field in the UK, although laboratory tests indicated release from the former was more uniform and more likely to last longer in the field. Increasing loading of pheromone in the dispenser increased attractiveness. Traps baited with polyethylene vials containing 0.5 mg of (R)‐2‐nonyl butyrate are recommended for monitoring H. marginata and these are far more sensitive than water or sticky traps currently used for monitoring this pest.     

Aspects of in vivo oocyte production,blastocyst development,and embryo transfer in the cat

A brief overview of the progress made during the past approximately 40 years on the development of methods for in vitro production of cat embryos and intra- and interspecies embryo transfer is described. The presentation is focused primarily on research done over the past 30 years at the Cincinnati Zoo (1980–1995) and at the Audubon Nature Institute, New Orleans (1996–present) beginning with original studies on determining optimal doses of porcine FSH for ovarian stimulation and uterine embryo recovery, cryopreservation, and transfer. A key early finding was the ability of cats to respond to multiple gonadotropin (porcine FSH) treatments by repeated stimulation of follicular development. With a ≥6-month interval between FSH treatments, over the past 15 years (1998–2013), we have done 1603 laparoscopic oocyte retrievals on 337 cats and recovered >38,000 mature oocytes (mean = 24.1 per laparoscopic oocyte retrieval). The limited information available on in vivo blastocyst development in the cat during the latter portion of the preimplantation period (approximately Days 8 to 12 after coitum or approximately Days 7 to 11 after ovulation) was assembled for the purpose of comparing and contrasting it with the growth, expansion, and zona functioning of in vitro-derived blastocysts. Also, results of transferring morulae and/or blastocysts into synchronous recipients are described to emphasize evidence that appears to allude to an essential role for an intact zona pellucida in successful implantation and subsequent development in the cat. Until 2003, our in vitro-derived embryos were transferred into the uterine horns of recipients to determine the feasibility of producing offspring from such primary methods as IVF, intracytoplasmic sperm injection, SCNT, and embryo cryopreservation. With the exception of SCNT embryos, pregnancy rates were satisfactory, but embryo survival rates were not. Subsequently, after finding that SCNT embryo survival rate could be improved using laparoscopic transfer of early cleavage stage embryos into the oviduct, we applied the technique to embryos derived using IVF with sex-sorted sperm, oocyte vitrification, and embryo cryopreservation. Overall, a pregnancy rate of 67% (14/21) has resulted. Most recently, with the oviductal embryo transfer technique, two litters of Black-Footed cat kittens have been born from intra- and interspecies transfer of cryopreserved embryos.     

High-Yield and Phylogenetically Robust Methods of DNA Recovery for Analysis of Microbial Biofilms Adherent to Plant Biomass in the Herbivore Gut

Recent studies have shown the microbial biofilms adherent to plant biomass in the gastrointestinal tracts of humans and other herbivores are quite different to planktonic populations. If these biofilm communities are to be properly characterized by metagenomics methods, then the microbial desorption methods used must ensure the phylogenetic diversity and genetic potential recovered is biologically valid. To that end, we describe here two different methods for desorbing microbes tightly adherent to plant biomass; and used PCR-DGGE analyses of the Bacteria and Archaea rrs genes to show both these desorption methods were effective in recovering the adherent microbial biofilm with no apparent biases in microbe recovery. We also present a derivation of the ??repeated bead beating and column (RBB+C) purification?? method of DNA extraction that results in the recovery of high molecular weight DNA. These DNA samples can be fragmented and size fractionated by sucrose density gradient centrifugation, bypassing the use of gel-plug lysis and pulsed-field gel electrophoresis separation of DNA for metagenomic library constructions.     

Thermotolerance and heat acclimation may share a common mechanism in humans

Thermotolerance and heat acclimation are key adaptation processes that have been hitherto viewed as separate phenomena. Here, we provide evidence that these processes may share a common basis, as both may potentially be governed by the heat shock response. We evaluated the effects of a heat shock response-inhibitor (quercetin; 2,000 mg/day) on established markers of thermotolerance [gastrointestinal barrier permeability, plasma TNF-α, IL-6, and IL-10 concentrations, and leukocyte heat shock protein 70 (HSP70) content]. Heat acclimation reduced body temperatures, heart rate, and physiological strain during exercise/heat stress) in male subjects (n = 8) completing a 7-day heat acclimation protocol. These same subjects completed an identical protocol under placebo supplementation (placebo). Gastrointestinal barrier permeability and TNF-α were increased on the 1st day of exercise/heat stress in quercetin; no differences in these variables were reported in placebo. Exercise HSP70 responses were increased, and plasma cytokines (IL-6, IL-10) were decreased on the 7th day of heat acclimation in placebo; with concomitant reductions in exercise body temperatures, heart rate, and physiological strain. In contrast, gastrointestinal barrier permeability remained elevated, HSP70 was not increased, and IL-6, IL-10, and exercise body temperatures were not reduced on the 7th day of heat acclimation in quercetin. While exercise heart rate and physiological strain were reduced in quercetin, this occurred later in exercise than with placebo. Consistent with the concept that thermotolerance and heat acclimation are related through the heat shock response, repeated exercise/heat stress increases cytoprotective HSP70 and reduces circulating cytokines, contributing to reductions in cellular and systemic markers of heat strain. Exercising under a heat shock response-inhibitor prevents both cellular and systemic heat adaptations.     

Borderline pulmonary arterial pressure in systemic sclerosis patients: a post-hoc analysis of the DETECT study

Introduction

Patients with mean pulmonary artery pressures (mPAP) of 21 to 24 mm Hg have a so-called borderline elevation of mPAP (BoPAP)—a condition thought to represent early-stage pulmonary arterial vasculopathy. Based on the DETECT study, this post-hoc analysis examined patient characteristics of systemic sclerosis (SSc) patients with normal mPAP, BoPAP and elevated mPAP, fulfilling pulmonary arterial hypertension (PAH) criteria.

Methods

Adult patients with a duration of SSc more than 3 years, a diffusing capacity of the lung for carbon monoxide less than 60% predicted, and no previous diagnosis of any form of pulmonary hypertension (PH) underwent screening tests followed by right heart catheterization. Subjects were divided into three groups: normal mPAP, BoPAP, and PAH. Exploratory comparative and binary logistic regression analyses were performed for the BoPAP versus normal mPAP and PAH versus BoPAP groups.

Results

Of 244 patients evaluated, 148 (60%) had normal mPAP, 36 (15%) had BoPAP, and 60 (25%) had definite PAH. Univariable logistic regression (ULR) showed the mean tricuspid regurgitation velocity in patients with BoPAP to be intermediate between normal mPAP and PAH. In the ULR analyses BoPAP versus normal mPAP and PAH versus BoPAP, the statistically significant predictors were, amongst others: demographic, clinical, pulmonary function, echocardiographic and hemodynamic variables.

Conclusions

In this exploratory post-hoc analysis of the DETECT study population patients with BoPAP could be distinguished from patients with normal mPAP and PAH, and it appears that BoPAP may be an intermediate stage on the continuum between normal PA pressures and PAH.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0493-1) contains supplementary material, which is available to authorized users.     

A meta-analysis of the risk of venous thromboembolism in inflammatory rheumatic diseases

Introduction

We performed a meta-analysis to investigate the risk of deep vein thrombosis (DVT) and/or pulmonary embolisms (PEs) in patients with inflammatory arthritis, vasculitis and connective tissue diseases (CTDs) (systemic lupus erythematosus (SLE), Sjögren’s syndrome, inflammatory myositis and systemic sclerosis (SSc)).

Methods

PubMed, Embase, the Cochrane databases and MEDLINE were searched to identify full-text English-language publications about adult patients with rheumatologic inflammatory diseases and venous thromboembolisms (VTEs). Data regarding rates of DVTs and PEs were extracted. Using random-effects models, pooled estimates for VTEs in individual and pooled diseases were compared with matched populations where possible. Studies were excluded if VTEs were described in the setting of pregnancy, postoperative outcomes or solely antiphospholipid antibody syndrome.

Results

Most of the 5,206 studies were excluded because they did not state the rate or incidence of VTEs. In total, 25 studies remained for analysis. Ten studies that included rheumatoid arthritis comprised an aggregate of 5,273,942 patients and 891,530,181 controls with a cumulative VTE incidence of 2.18% (95% confidence interval (CI): 1.82% to 2.54%) and an odds ratio of 2.23 (95% CI: 2.02 to 2.47) compared to age- and sex-matched populations. Ten studies comprised an aggregate of 54,697 SLE patients with a cumulative VTE incidence of 7.29% (95% CI: 5.82% to 8.75%). Four Sjögren’s syndrome studies comprising an aggregate of 25,100 patients demonstrated a cumulative VTE incidence of 2.18% (95% CI: 0.79% to 3.57%). Four studies of inflammatory myositis comprising an aggregate of 8,245 patients yielded a cumulative VTE incidence of 4.03% (95% CI: 2.38% to 5.67%). The SSc- and antineutrophil cytoplasmic antibody vasculitis–related cumulative VTE rates (four studies each) were 3.13% and 7.97%, respectively.

Conclusions

The inflammatory rheumatologic diseases studied were all associated with high rates of VTEs—more than three times higher than in the general population.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0435-y) contains supplementary material, which is available to authorized users.