Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy. In order to identify the genes involved in this utilization, we cloned and sequenced a 12.7-kb fragment containing a conserved region of NAD(P)H:quinone oxidoreductase genes. Of the products of the 13 open reading frames deduced from this fragment, PnpA shares 24% identity to the large component of a 3-hydroxyphenylacetate hydroxylase from Pseudomonas putida U and PnpB is 58% identical to an NAD(P)H:quinone oxidoreductase from Escherichia coli. Both PnpA and PnpB were purified to homogeneity as His-tagged proteins, and they were considered to be a monomer and a dimer, respectively, as determined by gel filtration. PnpA is a flavin adenine dinucleotide-dependent single-component PNP 4-monooxygenase that converts PNP to para-benzoquinone in the presence of NADPH. PnpB is a flavin mononucleotide-and NADPH-dependent p-benzoquinone reductase that catalyzes the reduction of p-benzoquinone to hydroquinone. PnpB could enhance PnpA activity, and genetic analyses indicated that both pnpA and pnpB play essential roles in PNP mineralization in strain WBC-3. Furthermore, the pnpCDEF gene cluster next to pnpAB shares significant similarities with and has the same organization as a gene cluster responsible for hydroquinone degradation (hapCDEF) in Pseudomonas fluorescens ACB (M. J. Moonen, N.

The RNA polymerase inhibitor tiacumicin B is currently undergoing phase III clinical trial for treatment of Clostridium difficile associated diarrhea with great promise. To understand the biosynthetic logic and to lay a foundation for generating structural analogues via pathway engineering, the tiacumicin B biosynthetic gene cluster was identified and characterized from the producer Dactylosporangium aurantiacum subsp. hamdenensis NRRL 18085. Sequence analysis of a 110,633 bp DNA region revealed the presence of 50 open reading frames (orfs). Functional investigations of 11 orfs by in vivo inactivation experiments, preliminarily outlined the boundaries of the tia-gene cluster and suggested that 31 orfs were putatively involved in tiacumicin B biosynthesis. Functions of a halogenase (TiaM), two glycosyltransferases (TiaG1 and TiaG2), a sugar C-methyltransferase (TiaS2), an acyltransferase (TiaS6), and two cytochrome P450s (TiaP1 and TiaP2) were elucidated by isolation and structural characterization of the metabolites from the corresponding gene-inactivation mutants. Accumulation of 18 tiacumicin B analogues from 7 mutants not only provided experimental evidence to confirm the proposed functions of individual biosynthetic enzymes, but also set an example of accessing microbial natural product diversity via genetic approach. More importantly, biochemical characterization of the FAD-dependent halogenase TiaM reveals a sequentially acting dihalogenation step tailoring tiacumicin B biosynthesis.

In eukaryotes, aberrant expression of transposable elements (TEs) is detrimental to the host genome. Piwi-interacting RNAs (piRNAs) of ∼23 to 30 nucleotides bound to PIWI clade Argonaute proteins silence transposons in a manner that is strictly dependent on their sequence complementarity. Hence, a key goal in understanding piRNA pathways is to determine mechanisms that modulate piRNA sequences. Here, we identify a protein-protein interaction between the 3′-to-5′ exoribonuclease Nibbler (Nbr) and Piwi that links Nbr activity with piRNA pathways. We show that there is a delicate balance in the interplay between Nbr and Hen1, a methyltransferase involved in 2′-O-methylation at the 3′ terminal nucleotides of piRNAs, thus connecting two genes with opposing activities in the biogenesis of piRNA 3′ ends. With age, piRNAs become shorter and fewer in number, which is coupled with the derepression of select TEs. We demonstrate that activities of Nbr and Hen1 inherently contribute to TE silencing and age-dependent profiles of piRNAs. We propose that antagonistic roles of Nbr and Hen1 define a mechanism to modulate piRNA 3′ ends.

HighlightA new model of the dicot leaf shows that different chloroplasts can experience drastically different light conditions and that bundle sheath extensions and chloroplast positioning influence photosynthetic light use efficiency.

Mesophyll resistance (r ), stomatal resistance, and biochemical limitations are recognized as three critical factors limiting leaf photosynthesis. Contrary to the expectation of being a constant, r not only varies with light and CO conditions but also shows different responses among species. To elucidate the mechanistic basis of these responses, we derived an analytical model of r , which incorporates various anatomical and biochemical factors including permeabilities of cell wall and chloroplast envelope to CO and HCO , carbonic anhydrase activities in cytosol and stroma, Rubisco activities, and relative location of mitochondria and chloroplast. The robustness of this model was confirmed by comparing the predicted r and its components to numerical models developed at cell and leaf levels, which incorporate detailed 3-dimensional cell and leaf anatomies, CO hydration and diffusion processes from intercellular air space to stroma, and CO fixation by Rubisco. A combination of these model analyses shows that the varying r is influenced by four biochemical factors: (a) nonuniform photosynthesis status across the leaf, (b) photorespiration and respiration, (c) bicarbonate leakage on the chloroplast envelope, and (d) hydration activity in cytosol and stroma. This study provides a theoretical framework to study components of r and their responses to environmental perturbations.

Alcaligenes sp. strain NyZ215 was isolated for its ability to grow on ortho-nitrophenol (ONP) as the sole source of carbon, nitrogen, and energy and was shown to degrade ONP via a catechol ortho-cleavage pathway. A 10,152-bp DNA fragment extending from a conserved region of the catechol 1,2-dioxygenase gene was obtained by genome walking. Of seven complete open reading frames deduced from this fragment, three (onpABC) have been shown to encode the enzymes involved in the initial reactions of ONP catabolism in this strain. OnpA, which shares 26% identity with salicylate 1-monooxygenase of Pseudomonas stutzeri AN10, is an ONP 2-monooxygenase (EC 1.14.13.31) which converts ONP to catechol in the presence of NADPH, with concomitant nitrite release. OnpC is a catechol 1,2-dioxygenase catalyzing the oxidation of catechol to cis,cismuconic acid. OnpB exhibits 54% identity with the reductase subunit of vanillate O-demethylase in Pseudomonas fluorescens BF13. OnpAB (but not OnpA alone) conferred on the catechol utilizer Pseudomonas putida PaW340 the ability to grow on ONP. This suggests that OnpB may also be involved in ONP degradation in vivo as an o-benzoquinone reductase converting o-benzoquinone to catechol. This is analogous to the reduction of tetrachlorobenzoquinone to tetrachlorohydroquinone by a tetrachlorobenzoquinone reductase (PcpD, 38% identity with OnpB) in the pentachlorophenol degrader Sphingobium chlorophenolicum ATCC 39723.

Cassava is an important staple crop in tropical and sub-tropical areas. As a common farming practice, cassava is usually cultivated intercropping with other crops and subjected to various degrees of shading, which causes reduced productivity. Herein, a comparative transcriptomic analysis was performed on a series of developmental cassava leaves under both full sunlight and natural shade conditions. Gene expression profiles of these two conditions exhibited similar developmental transitions, e.g. genes related to cell wall and basic cellular metabolism were highly expressed in immature leaves, genes involved in lipid metabolism and tetrapyrrole synthesis were highly expressed during the transition stages, and genes related to photosynthesis and carbohydrates metabolism were highly expressed in mature leaves. Compared with the control, shade significantly induced the expression of genes involved in light reaction of photosynthesis, light signaling and DNA synthesis/chromatin structure; however, the genes related to anthocyanins biosynthesis, heat shock, calvin cycle, glycolysis, TCA cycle, mitochondrial electron transport, and starch and sucrose metabolisms were dramatically depressed. Moreover, the shade also influenced the expression of hormone-related genes and transcriptional factors. The findings would improve our understanding of molecular mechanisms of shade response, and shed light on pathways associated with shade-avoidance syndrome for cassava improvement.

We previously identified a subcortical maternal complex (SCMC) that is essential for early embryogenesis and female fertility in mice. However, the molecular mechanism by which the SCMC affects female fertility remains largely uncharacterized. Here, we report that a novel maternal protein, zinc finger BED-type containing 3 (Zbed3), participates in the SCMC. Depletion of maternal Zbed3 results in reduced fecundity of females, because of the impaired and delayed development in a proportion of mutant embryos. The loss of maternal Zbed3 results in asymmetric zygotic division and abnormal distributions of organelles in the affected oocytes and zygotes, similar to the phenotypes observed in females with disrupted core SCMC genes. Further investigation revealed that these phenotypes are associated with disrupted dynamics of microtubules and/or formation of cytoplasmic lattices (CPLs). The stability and localization of Zbed3 depend on, but are not required for, the formation of the SCMC. Thus, our data suggest Zbed3 as one of downstream proteins mediating SCMC functions and provide further insights into the roles of the SCMC and CPLs in female fertility.