Occurrence of Nitrate Reductase and Molybdopterin in Xanthomonas maltophilia

Fifteen of 23 ATCC strains and 2 of 9 clinical isolates of Xanthomonas maltophilia, all of which grew aerobically on ammonia, but not nitrate, as a sole nitrogen source, reduced nitrate to nitrite. X. maltophilia failed to grow anaerobically on complex medium with or without nitrate, so it is considered an obligate aerobe. Nitrate-reducing strains contained reduced methyl viologen nitrate reductase (MVH-NR) with specific activities ranging from 49.2 to 192 U mg of protein⁻¹. Strain ATCC 17666 doubled its cell mass after 3 h of growth on nitrate broth under low aeration, possessed maximal MVH-NR activity, and converted the added nitrate to nitrite, which accumulated. Dissolved oxygen above 15% saturation greatly suppressed nitrite formation. All strains, except ATCC 14535, possessed between 0.25 and 5.05 pmol of molybdopterin mg of protein⁻¹ as measured by the Neurospora crassa nit-1 assay. The molybdopterin activity in the soluble fraction sedimented as a single symmetrical peak with an s_20,w of 5.1. Studies identified MVH-NR in selected strains as a membrane-bound protein. The deoxycholate-solubilized MVH-NR sedimented as a single peak in sucrose density gradients with an s_20,w of 8.8. The MVH-NR of X. maltophilia has the physical characteristics of a respiratory nitrate reductase and may enable cells to use nitrate as an electron sink under semiaerobic conditions.     

Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype

The relationship between sequence variation and phenotype is poorly understood. Here, we use metabolomic analysis to elucidate the molecular mechanism underlying the filamentous phenotype of E. coli strains that carry destabilizing mutations in dihydrofolate reductase (DHFR). We find that partial loss of DHFR activity causes reversible filamentation despite SOS response indicative of DNA damage, in contrast to thymineless death (TLD) achieved by complete inhibition of DHFR activity by high concentrations of antibiotic trimethoprim. This phenotype is triggered by a disproportionate drop in intracellular dTTP, which could not be explained by drop in dTMP based on the Michaelis–Menten‐like in vitro activity curve of thymidylate kinase (Tmk), a downstream enzyme that phosphorylates dTMP to dTDP. Instead, we show that a highly cooperative (Hill coefficient 2.5) in vivo activity of Tmk is the cause of suboptimal dTTP levels. dTMP supplementation rescues filamentation and restores in vivo Tmk kinetics to Michaelis–Menten. Overall, this study highlights the important role of cellular environment in sculpting enzymatic kinetics with system‐level implications for bacterial phenotype.     

Biomass Production and Composition of Perennial Grasses Grown for Bioenergy in a Subtropical Climate Across Florida, USA

Carbohydrate and lignin composition of feedstock materials are major factors in determining their bioenergy potential. This study was conducted to quantify dry biomass yield and the carbohydrate and lignin composition of six potential biofuel grasses (elephantgrass, energycane, sweetcane, giant reed, giant miscanthus, and sugarcane) across three sites in Florida for plant (2009) and first ratoon (2010) crops. Dry biomass yields ranged from about 30 to 50 Mg ha^?1 and were generally greatest for elephantgrass, energycane, sweetcane, and sugarcane. Accordingly, total plant carbohydrate yields (20 to 25 Mg ha^?1) were comparable among sugarcane, energycane, sweetcane, and elephantgrass, but were generally less for giant reed and even less for giant miscanthus. However, the contribution of total extractable carbohydrates and total fiber carbohydrates to total plant carbohydrate yields differed among species. Sugarcane had the highest concentrations of extractable carbohydrates (219 to 356 mg g^?1), followed by energycane, then sweetcane, elephantgrass, and giant reed, with giant miscanthus having the lowest. Energycane and elephantgrass tended to have significantly more fiber glucose, and elephantgrass less xylose, than other species. Variability in total lignin concentrations on a fiber basis was relatively modest (250 to 285 mg g^?1) across species, but was generally highest in sweetcane and giant reed. Overall, elephantgrass and energycane were prime regional candidates for cellulosic conversion using fermentation processes due to high yields and favorable fiber characteristics, although energycane tended to have higher extractable carbohydrates.     

Aquifex aeolicus 3-Deoxy-d-manno-2-Octulosonic Acid 8-Phosphate Synthase: A New Class of KDO 8-P Synthase?

The relationship between 3-deoxy-D-manno-2-octulosonic acid 8-phosphate (KDO 8-P) synthase and 3-deoxy-D-arabino-2-heptulosonic acid 7-phosphate (DAH 7-P) synthase has not been adequately addressed in the literature. Based on recent reports of a metal requiring KDO 8-P synthase and the newly solved X-ray crystal structures of both Escherichia coli KDO 8-P synthase and DAH 7-P synthase, we begin to address the evolutionary kinship between these catalytically similar enzymes. Using a maximum likelihood-based grouping of 29 KDO 8-P synthase sequences, we demonstrate the existence of a new class of KDO 8-P synthase, the members of which we propose to require a metal cofactor for catalysis. Similarly, we hypothesize a class of DAH 7-P synthase that does not have the metal requirement of the heretofore model E. coli enzyme. Based on this information and a careful investigation of the reported X-ray crystal structures, we also propose that KDO 8-P synthase and DAH 7-P synthase are the product of a divergent evolutionary process from a common ancestor.     

Sex roles, parental experience and reproductive success of eastern kingbirds, Tyrannus tyrannus

We quantified parental behaviour of eastern kingbirds during the incubation and nestling periods to determine parental roles, and to examine the impact of previous breeding experience (defined as having bred on the territory in the past) on behaviour and reproductive success. Females performed all incubation, while males spent more than 60% of their time in vigilant or nest guarding behaviour during incubation. Parental roles were not defined as sharply during the nestling period. Females spent more time vigilant, but males provisioned young at only 54% of the rate of females. Vigilance and nest watching were still primarily male duties. Male and female behaviour did not vary with the pair's combination of experience (e.g. experienced-experienced versus inexperienced-inexperienced in previous-current breeding season, respectively) during either phase of reproduction, but experienced males were more vigilant during incubation and fed young relatively more than inexperienced males. Experienced females were also more efficient foragers. Although behaviour did not differ among the four combinations of pair experience, inexperienced pairs none the less lost the most young to starvation and predation. Consequently, inexperienced pairs fledged one less nestling per nesting attempt than did pairs with at least one experienced breeder. Our results suggest that having at least one experienced breeder substantially improved a pair's reproductive success. We propose that female site fidelity is a safeguard to avoid the lower breeding success a female would incur if she were to move to a new territory and breed with an inexperienced male. Copyright 1999 The Association for the Study of Animal Behaviour.     

Small proline-rich protein 1A is a gp130 pathway- and stress-inducible cardioprotective protein

The interleukin-6 cytokines, acting via gp130 receptor pathways, play a pivotal role in the reduction of cardiac injury induced by mechanical stress or ischemia and in promoting subsequent adaptive remodeling of the heart. We have now identified the small proline-rich repeat proteins (SPRR) 1A and 2A as downstream targets of gp130 signaling that are strongly induced in cardiomyocytes responding to biomechanical/ischemic stress. Upregulation of SPRR1A and 2A was markedly reduced in the gp130 cardiomyocyte-restricted knockout mice. In cardiomyocytes, MEK1/2 inhibitors prevented SPRR1A upregulation by gp130 cytokines. Furthermore, binding of NF-IL6 (C/EBPbeta) and c-Jun to the SPRR1A promoter was observed after CT-1 stimulation. Histological analysis revealed that SPRR1A induction after mechanical stress of pressure overload was restricted to myocytes surrounding piecemeal necrotic lesions. A similar expression pattern was found in postinfarcted rat hearts. Both in vitro and in vivo ectopic overexpression of SPRR1A protected cardiomyocytes against ischemic injury. Thus, this study identifies SPRR1A as a novel stress-inducible downstream mediator of gp130 cytokines in cardiomyocytes and documents its cardioprotective effect against ischemic stress.     

Escherichia coli YrbI is 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase

3-Deoxy-d-manno-octulosonate 8-phosphate (KDO 8-P) phosphatase, which catalyzes the hydrolysis of KDO 8-P to KDO and inorganic phosphate, is the last enzyme in the KDO biosynthetic pathway for which the gene has not been identified. Wild-type KDO 8-P phosphatase was purified from Escherichia coli B, and the N-terminal amino acid sequence matched a hypothetical protein encoded by the E. coli open reading frame, yrbI. The yrbI gene, which encodes for a protein of 188 amino acids, was cloned, and the gene product was overexpressed in E. coli. The recombinant enzyme is a tetramer and requires a divalent metal cofactor for activity. Optimal enzymatic activity is observed at pH 5.5. The enzyme is highly specific for KDO 8-P with an apparent K(m) of 75 microm and a k(cat) of 175 s(-1) in the presence of 1 mm Mg(2+). Amino acid sequence analysis indicates that KDO 8-P phosphatase is a member of the haloacid dehalogenase hydrolase superfamily.     

Escherichia coli YrbH is a D-arabinose 5-phosphate isomerase

A gene encoding for arabinose 5-phosphate isomerase (API), which catalyzes the interconversion of d-ribulose 5-phosphate (Ru5P) and d-arabinose 5-phosphate (A5P), has been identified from the genome of Escherichia coli K-12. API is the first enzyme in the biosynthesis of 3-deoxy-d-manno-octulosonate (KDO), a sugar moiety located in the lipopolysaccharide layer of most Gram-negative bacteria. The API gene yrbH is located next to the recently identified specific KDO 8-P phosphatase gene, yrbI. The 328-amino acid open reading frame yrbH was cloned, overexpressed, and characterized. The purified recombinant enzyme is a tetramer and is sensitive to inhibition by zinc cations. API has optimal activity at pH 8.4 and catalytic residues with estimated pKa values of 6.55 +/- 0.04 and 10.34 +/- 0.07. The enzyme is specific for A5P and Ru5P, with apparent Km values of 0.61 +/- 0.06 mm for A5P and 0.35 +/- 0.08 mm for Ru5P. The apparent kcat in the A5P to Ru5P direction is 157 +/- 4 s-1, and in the Ru5P to A5P direction it is 255 +/- 16 s-1. The value of Keq (Ru5P/A5P) is 0.50 +/- 0.06. Homology searches of the E. coli genome suggest yrbH may be one of multiple genes that encode proteins with API activity.