Improving heterologous polyketide production in <Emphasis Type="Italic">Escherichia coli</Emphasis> by overexpression of an <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase gene

An S-adenosylmethionine synthetase gene (metK) from Streptomyces spectabilis was cloned into an expression plasmid under the control of an inducible T7 promoter and introduced into a strain of Escherichia coli (BAP1(pBP130/pBP144)) capable of producing the polyketide product 6-deoxyerythronolide B (6-dEB). The metK coexpression in BAP1(pBP130/pBP144) improved the specific production of 6-dEB from 10.86 to 20.08 mg l⁻¹ . In an effort to probe the reason for this improvement, a series of gene deletion and expression experiments were conducted based on a metK metabolic pathway that branches between propionyl-CoA (a 6-dEB precursor) and autoinducer compounds. The deletion and expression studies suggested that the autoinducer pathway had a larger impact on improved 6-dEB biosynthesis. Supporting these results were experiments demonstrating the positive effect conditioned media (the suspected location of the autoinducer compounds) had on 6-dEB production. Taken together, the results of this study show an increase in heterologous 6-dEB production concomitant with heterologous metK gene expression and suggest that the mechanism for this improvement is linked to native autoinducer compounds.     

Monomer composition and sequence of sodium alginate extracted at pilot plant scale from three commercially important seaweeds from Mexico

The marine waters of the Baja California peninsula (Mexico) are a rich source of brown seaweeds with a great potential for exploitation. For that reason, Sargassum sinicola, Eisenia arborea, and Macrocystis pyrifera collected from different locations were subjected to extraction of sodium alginate using a pilot-plant scale process developed in our facilities. The composition and sequence parameters of the recovered alginate were studied by infrared and nuclear magnetic resonance spectroscopy. The spectral analysis of the products revealed that sodium alginate from S. sinicola contains a greater proportion of guluronate monomers (64%) than that from E. arborea (48%), and M. pyrifera (38%). Computation of the frequencies of diads and triads indicated that the alginate from S. sinicola was constructed by intercalated guluronate-blocks of 14 residues in length. In contrast, the length of the G-block in the alginates from E. arborea and M. pyrifera were 7 and 4 residues, respectively. The results show that S. sinicola, E. arborea, and M. pyrifera are sources of sodium alginate with different mannuronate/guluronate ratios, as well as a varied building-block length. In consequence, aqueous dispersions of sodium alginate from the three studied species are expected to exhibit different physical properties.     

The Streptococcus mutans vicX gene product modulates gtfB/C expression, biofilm formation, genetic competence, and oxidative stress tolerance

Streptococcus mutans is considered one of the primary etiologic agents of dental caries. Previously, we characterized the VicRK two-component signal transduction system, which regulates multiple virulence factors of S. mutans. In this study, we focused on the vicX gene of the vicRKX tricistronic operon. To characterize vicX, we constructed a nonpolar deletion mutation in the vicX coding region in S. mutans UA159. The growth kinetics of the mutant (designated SmuvicX) showed that the doubling time was longer and that there was considerable sensitivity to paraquat-induced oxidative stress. Supplementing a culture of the wild-type UA159 strain with paraquat significantly increased the expression of vicX (P < 0.05, as determined by analysis of variance [ANOVA]), confirming the role of this gene in oxidative stress tolerance in S. mutans. Examination of mutant biofilms revealed architecturally altered cell clusters that were seemingly denser than the wild-type cell clusters. Interestingly, vicX-deficient cells grown in a glucose-supplemented medium exhibited significantly increased glucosyltransferase B/C (gtfB/C) expression compared with the expression in the wild type (P < 0.05, as determined by ANOVA). Moreover, a sucrose-dependent adhesion assay performed using an S. mutans GS5-derived vicX null mutant demonstrated that the adhesiveness of this mutant was enhanced compared with that of the parent strain and isogenic mutants of the parent strain lacking gtfB and/or gtfC. Also, disruption of vicX reduced the genetic transformability of the mutant approximately 10-fold compared with that of the parent strain (P < 0.05, as determined by ANOVA). Collectively, these findings provide insight into important phenotypes controlled by the vicX gene product that can impact S. mutans pathogenicity.     

Morphology and Taxonomy of <Emphasis Type="Italic">Apristurus longicephahis</Emphasis> (Lamniformes,Seyliorhinidae)

Data on the individual variation and changes with growth in proportions and morphology are presented for the poorly known Apristurus longicephalus, and compared with those of other species. A. longicephalus is concluded to be a distinct species without synonyms, characterized by its long snout, widely separate nostrils, long caudal fin, short abdomen, very sparse teeth, and low number of monospondylous vertebrae. It is a species of small size, maturing at about 42 cm in total length.     

Inflorescence architecture: A developmental genetics approach

We are characterizing a suiteof Pisum sativum mutants that alter inflorescence architecture to construct a model for the genetic regulation of inflorescence development in a plant with a compound raceme. Such a model, when compared with those created forAntirrhinum majus andArabidopsis thaliana, both of which have simple racemes, should provide insight into the evolution of the development of inflorescence architecture. The highly conserved nature of cloned genes that regulate reproductive development in plants and the morphological similarities among our mutants and those identified inA. majus andA. thaliana enhance the probability that a developmental genetics approach will be fruitful. Here we describe sixP. sativum mutants that affect morphologically and architecturally distinct aspects of the inflorescence, and we analyze interactions among these genes. Both vegetative and inflorescence growth of the primary axis is affected byUNIFOLIA TA, which is necessary for the function ofDETERMINATE (DET).DET maintains indeterminacy in the first-order axis. In its absence, the meristem differentiates as a stub covered with epidermal hairs.DET interacts withVEGETATIVE1 (VEG1).VEG1 appears essential for second-order inflorescence (I₂) development.veg1 mutants fail to flower or differentiate the I₂ meristem into a rudimentary stub,det veg1 double mutants produce true terminal flowers with no stubs, indicating that two genes must be eliminated for terminal flower formation inP. sativum, whereas elimination of a single gene accomplishes this inA. thaliana andA. majus. NEPTUNE also affects I₂ development by limiting to two the number of flowers produced prior to stub formation. Its role is independent ofDET, as indicated by the additive nature of the double mutantdet nep. UNI, BROC, and PIM all play roles in assigning floral meristem identity to the third-order branch.pim mutants continue to produce inflorescence branches, resulting in a highly complex architecture and aberrant flowers.uni mutants initiate a whorl of sepals, but floral organogenesis is aberrant beyond that developmental point, and the double mutantuni pim lacks identifiable floral organs. A wild-type phenotype is observed inbroc plants, butbroc enhancesthe pim phenotype in the double mutant, producing inflorescences that resemble broccoli. Collectively these genes ensure that only the third-order meristem, not higher- or lower-order meristems, generates floral organs, thus precisely regulating the overall architecture of the plant. Gene symbols used in this article: For clarity a common symbolization is used for genes of all species discussed in this article. Genes are symbolized with italicized capital letters. Mutant alleles are represented by lowercase, italicized letters. In both cases, the number immediately following the gene symbol differentiates among genes with the same symbol. If there are multiple alleles, a hyphen followed by a number is used to distinguish alleles. Protein products are represented by capital letters without italics.     

Protection by pyruvate against glutamate neurotoxicity is mediated by astrocytes through a glutathione-dependent mechanism

Pyruvate, an endogenous metabolite of glycolysis, is an anti-toxicity agent. Recent studies have suggested possible roles for pyruvate in protecting CNS neurons from excitotoxic and metabolic insults. Utilizing cultures derived from embryonic rat cortex, the studies presented in this paper indicate that an astroglia-mediated mechanism is involved in the neuroprotective effects of pyruvate against glutamate toxicity. Glutamate-induced toxicity could be reversed by pyruvate in a mixed culture of cortex cells. Importantly, in pure neuronal cultures from the same tissue, pyruvate failed to protect against glutamate toxicity. Addition of astroglia to the pure neuronal cultures restores the ability of pyruvate to protect neurons from glutamate-induced toxicity. Our results further suggest that pyruvate can induce glia to up-regulate the synthesis of glutathione (GSH), an antioxidant that protects cells from toxins such as free radicals. Taken together, our data suggest that astroglia in mixed cultures are essential for mediating the effects of pyruvate, revealing a novel mechanism by which pyruvate, an important intermediate of tricarboxylic acid cycle in the body, may act to protect neurons from damage during insults such as brain ischemia.     

Identification of Two Species of Yeast-like Symbiotes in the Brown Planthopper, <Emphasis Type="Italic">Nilaparvata lugens</Emphasis>

To determine the species of the yeast-like symbionts (YLS) in the brown planthoppers (BPH), Nilaparvata lugens, YLS were first isolated and purified by ultracentrifugation from the fat bodies of BPH, and then 18S rDNA and internal transcribed spacer (ITS)–5.8S rDNA sequences of YLS were amplified with the different general primers for fungi. The results showed that the two different 18S and ITS–5.8S rDNA sequences of YLS were obtained. One 2291-bp DNA sequence, which contained 18S and ITS–5.8S rDNA, showed the high similarity to Cryptococcus and was named Cryp-Like symbiotes. Another 1248-bp DNA sequence, which contained a part of 18S and ITS–5.8S rDNA, showed the high similarity to Pichia guilliermondii and was named Pichia-Like symbiotes. It was further proved that Cryp- and Pichia-Like symbiotes existed in BPH through nested PCR with specific primers for two symbiotes and in situ hybridization analysis using digoxigenin-labeled probes. Our results showed that BPH harbored more than one species of eukaryotic YLS, which suggested that diversity of fungal endosymbiotes may be occurred in planthoppers, just like bacterial endosymbiotes.     

Identifying habitat conservation priorities and gaps for migratory shorebirds and waterfowl in California

Conservation of migratory shorebirds and waterfowl presents unique challenges due to extensive historic loss of wetland habitats, and current reliance on managed landscapes for wintering and migratory passage. We developed a spatially-explicit approach to estimate potential shorebird and waterfowl densities in California by integrating mapped habitat layers and statewide bird survey data with expert-based habitat rankings. Using these density estimates as inputs, we used the Marxan site-selection program to identify priority shorebird and waterfowl areas at the ecoregional level. We identified 3.7 million ha of habitat for shorebirds and waterfowl, of which 1.4 million ha would be required to conserve 50% of wintering populations. To achieve a conservation goal of 75%, more than twice as much habitat (3.1 million ha) would be necessary. Agricultural habitats comprised a substantial portion of priority areas, especially at the 75% level, suggesting that under current management conditions, large areas of agricultural land, much of it formerly wetland, are needed to provide the habitat availability and landscape connectivity required by shorebird and waterfowl populations. These habitats were found to be largely lacking recognized conservation status in California (96% un-conserved), with only slightly higher levels of conservation for priority shorebird and waterfowl areas. Freshwater habitats, including wetlands and ponds, were also found to have low levels of conservation (67% un-conserved), although priority shorebird and waterfowl areas had somewhat higher levels of conservation than the state as a whole. Conserving migratory waterfowl and shorebirds will require a diversity of conservation strategies executed at a variety of scales. Our modeled results are complementary with other approaches and can help prioritize areas for protection, restoration and other actions. Traditional habitat protection strategies such as conservation easements and fee acquisitions may be of limited utility for protecting and managing significant areas of agricultural lands. Instead, conservation strategies focused on incentive-based programs to support wildlife friendly management practices in agricultural settings may have greater utility and conservation effectiveness.