In vivo study of fidelity of DNA double-strand break repair in bacteriophage T4

A method for in vivo studying the fidelity of DNA double-strand break (DSB) repair in bacteriophage T4 has been developed. The frequency of reversion of rII mutations to the wild phenotype was measured in i segC ⁺ × i ets1 segCΔ crosses, where ets1 is an insertion in the initial part of the rIB gene carrying a sequence recognized by SegC endonuclease; i designates a rIIB or rIIA mutation located at some distance from ets1, and segCΔ is a deletion in the segC gene. In such cross, a DSB occurs in the site of ets1. Their repair involves genetic recombination and DNA replication in the neighborhood of ets1. In parallel, the frequency of reversion of the same i mutant in the absence of DSBs is measured in i × i self-crosses. Reversions of different types (base substitutions, deletions, insertions) can be studied with the use of structurally different i mutations located at varying distances from ets1. The reversion frequencies were determined for three rIIB mutations and one rIIA mutation. The results obtained suggest that DSB repair in bacteriophage T4 is a process of high fidelity with the rate of errors that does not essentially exceed that in the case of usual phage multiplication.     

Genetic and biochemical evidences reveal novel insights into the mechanism underlying <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Sae2-mediated abrogation of DNA replication stress

In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) protein complex plays pivotal roles in double-strand break (DSB) repair, replication stress and telomere length maintenance. Another protein linked to DSB repair is Sae2, which regulates MRX persistence at DSBs. However, very little is known about its role in DNA replication stress and repair. Here, we reveal a crucial role for Sae2 in DNA replication stress. We show that different mutant alleles of SAE2 cause hypersensitivity to genotoxic agents, and when combined with Δmre11 or nuclease-defective mre11 mutant alleles, the double mutants are considerably more sensitive suggesting that the sae2 mutations synergize with mre11 mutations. Biochemical studies demonstrate that Sae2 exists as a dimer in solution, associates preferentially with single-stranded and branched DNA structures, exhibits structure-specific endonuclease activity and cleaves these substrates from the 5′ end. Furthermore, we show that the nuclease activity is indeed intrinsic to Sae2. Interestingly, sae2^G270D protein possesses DNA-binding activity, but lacks detectable nuclease activity. Altogether, our data suggest a direct role for Sae2 nuclease activity in processing of the DNA structures that arise during replication and DNA damage and provide insights into the mechanism underlying Mre11-Sae2-mediated abrogation of replication stress-related defects in S. cerevisiae.     

Comparison of microbial communities during the anaerobic digestion of <Emphasis Type="Italic">Gracilaria</Emphasis> under mesophilic and thermophilic conditions

Mesophilic and thermophilic anaerobic digesters (MD and TD, respectively) utilizing Gracilaria and marine sediment as the substrate and inoculum, respectively, were compared by analyzing their performances and microbial community changes. During three successive transfers, the average cumulative methane yields in the MD and TD were 222.6 ± 17.3 mL CH₄/g volatile solids (VS) and 246.1 ± 11 mL CH₄/g VS, respectively. The higher hydrolysis rate and acidogenesis in the TD resulted in a several fold greater accumulation of volatile fatty acids (acetate, propionate, and butyrate) followed by a larger pH drop with a prolonged recovery than in the MD. However, the operational stability between both digesters remained comparable. Pyrosequencing analyses revealed that the MD had more complex microbial diversity indices and microbial community changes than the TD. Interestingly, Methanomassiliicoccales, the seventh methanogen order was the predominant archaeal order in the MD along with bacterial orders of Clostridiales, Bacteriodales, and Synergistales. Meanwhile, Coprothermobacter and Methanobacteriales dominated the bacterial and archaeal community in the TD, respectively. Although the methane yield is comparable, both MD and TD show a different profile of pH, VFA and the microbial communities.     

Mammalian meiosis involves DNA double-strand breaks with 3′ overhangs

Meiotic recombination in yeast is initiated at DNA double-strand breaks (DSBs), processed into 3′ single-strand overhangs that are active in homology search, repair and formation of recombinant molecules. Are 3′ overhangs recombination intermediaries in mouse germ cells too? To answer this question we developed a novel approach based on the properties of the Klenow enzyme. We carried out two different, successive in situ Klenow enzyme-based reactions on sectioned preparations of testicular tubules. Signals showing 3′ overhangs were observed during wild-type mouse spermatogenesis, but not in Spo11 ^?/? males, which lack meiotic DSBs. In Atm ^?/? mice, abundant positively stained spermatocytes were present, indicating an accumulation of non-repaired DSBs, suggesting the involvement of ATM in repair of meiotic DSBs. Thus the processing of DSBs into 3′ overhangs is common to meiotic cells in mammals and yeast, and probably in all eukaryotes.     

Comparison of molecular genetic utilities of TD,AFLP, and MSAP among the accessions of <Emphasis Type="Italic">japonica,indica</Emphasis>, and <Emphasis Type="Italic">Tongil</Emphasis> of <Emphasis Type="Italic">Oryza sativa</Emphasis> L.

Rice is one of the most important food crops in the world. Genetic diversity is essential for cultivar improvement programs. We compared genetic diversity derived from insertion–deletion (in–del) or base substitutions by amplified fragment length polymorphism (AFLP), from transposon transposition mutations by transposon display (TD), and from cytosine methylation by methylation-sensitive amplified polymorphism (MSAP) in japonica, indica, and Tongil type varieties of Oryza sativa L. Polymorphic profiles from the three marker systems allowed us to clearly distinguish the three types of varieties. The indica type varieties showed the highest genetic diversity followed by the Tongil and japonica type varieties. Of the three marker systems, TD produced the highest marker indices, and AFLP and MSAP produced similar marker indices. Pair-wise comparisons of the three marker systems showed that the correlation between the two genetic markers systems (AFLP and TD, r = 0.959) was higher than the correlations between the genetic and epigenetic marker systems (AFLP and MSAP, r = 0.52; TD and MSAP, r = 0.505). Both genetic marker systems had similar levels of gene differentiation (G _ST ) and gene flow (N _m ), which differed in the epigenetic marker system. Although the G _ST of the epigenetic marker system was lower than the genetic marker systems, the N _m of the epigenetic marker system was higher than in the genetic marker systems, indicating that epigenetic variations have a greater influence than genetic variations among the O. sativa L. types.     

Interspecific variation in extracellular polysaccharide content and colony formation of <Emphasis Type="Italic">Microcystis</Emphasis> spp. cultured under different light intensities and temperatures

Single cells of five different Microcystis species (M. ichthyoblabe, M. viridis, M. flos-aquae, M. wesenbergii, and M. aeruginosa) were batch-cultured at different temperatures and light intensities: (a) 25 °C and 50 μmol photons m^?2 s^?1 (control culture); (b) 25 °C and 10 μmol photons m^?2 s^?1; and (c) 15 °C and 50 μmol photons m^?2 s^?1. The extracellular polysaccharide content was significantly higher in treatments b and c than in the control treatment. All Microcystis species existed as single cells under the control treatment but formed colonies in treatments b and c. All of the colonies were irregular with indistinct margins. M. ichthyoblabe, M. viridis, M. flos-aquae, and M. wesenbergii formed colonies with similar morphologies and their cells were loosely aggregated. In contrast, M. aeruginosa formed denser colonies with no distinct holes. The colony morphologies differed from the classic morphology of M. ichthyoblabe field-grown colonies but resembled that of small colonies found in Lake Taihu (Yangtze Delta Plain, China) during early spring. This indicates that field- and laboratory-grown colonies are governed by similar formation processes. We suggest that in laboratory and field environments, M. ichthyoblabe (or M. flos-aquae) colonies are representative of small colonies formed from single Microcystis cells, whereas the morphology of older colonies evolves to resemble M. wesenbergii and M. aeruginosa colonies.     

Production of <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12-conjugated linoleic acid using permeabilized whole-cell biocatalyst of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objective

To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.

Results

Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l^?1, 100 g wet cells l^?1, and 25 g LA l^?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l^?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l^?1.

Conclusion

Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.

     

Artemis and Nonhomologous End Joining-Independent Influence of DNA-Dependent Protein Kinase Catalytic Subunit on Chromosome Stability

Deficiency in both ATM and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is synthetically lethal in developing mouse embryos. Using mice that phenocopy diverse aspects of Atm deficiency, we have analyzed the genetic requirements for embryonic lethality in the absence of functional DNA-PKcs. Similar to the loss of ATM, hypomorphic mutations of Mre11 (Mre11^ATLD1) led to synthetic lethality when juxtaposed with DNA-PKcs deficiency (Prkdc^scid). In contrast, the more moderate DNA double-strand break response defects associated with the Nbs1^ΔB allele permitted viability of some Nbs1^ΔB/ΔB Prkdc^scid/scid embryos. Cell cultures from Nbs1^ΔB/ΔB Prkdc^scid/scid embryos displayed severe defects, including premature senescence, mitotic aberrations, sensitivity to ionizing radiation, altered checkpoint responses, and increased chromosome instability. The known functions of DNA-PKcs in the regulation of Artemis nuclease activity or nonhomologous end joining-mediated repair do not appear to underlie the severe genetic interaction. Our results reveal a role for DNA-PKcs in the maintenance of S/G₂-phase chromosome stability and in the induction of cell cycle checkpoint responses.The Mre11 complex, consisting of Mre11, Rad50, and Nbs1 (Xrs2 in Saccharomyces cerevisiae), is involved in diverse aspects of DNA double-strand break (DSB) metabolism. The Mre11 complex acts as a DSB sensor, mediates cell cycle checkpoint arrest and apoptosis, and promotes DSB repair (47, 48). The influence of the Mre11 complex on DSB responses is attributable partly to its influence on ataxia-telangiectasia mutated (ATM) kinase activity (29). ATM is a central signal transducer in the response to DSBs and is required for arrest throughout the cell cycle, as well as the efficient execution of apoptosis in response to many types of genotoxic stress (43).The Mre11 complex is required for ATM activation and governs the phosphorylation of ATM substrates such as SMC1, Chk2, and BID (4, 6, 26, 47, 49, 51). The C terminus of Nbs1 interacts with ATM and plays an important role in facilitating a subset of these events, particularly those important for apoptosis (11, 14, 47, 58). However, ATM makes multiple functional contacts with members of the Mre11 complex. Nbs1, Mre11, and Rad50 are all ATM substrates, and many aspects of ATM checkpoint signaling are impaired by hypomorphic Mre11 and Nbs1 mutations that do not affect the ATM binding domain in the C terminus of Nbs1 (32, 36, 52, 54).Several molecular and genetic observations support the view that the Mre11 complex''s role in preserving genome stability is particularly relevant to the S and G₂ phases of the cell cycle (3, 56). The complex, predominantly nucleoplasmic in G₁ cells, becomes predominantly chromatin associated and colocalizes with PCNA throughout S phase (35, 38). This association is a likely prerequisite for the complex''s influence on DNA damage signaling as well as DNA repair.Cell cultures established with samples from patients with Nijmegen breakage syndrome (NBS1 hypomorphism) and ataxia-telangiectasia-like disorder (MRE11 hypomorphism) exhibit checkpoint defects in S phase and at the G₂/M transition, while the G₁/S transition is relatively unaffected. These checkpoint defects are correlated with reduced Mre11 complex chromatin association both in human cells and in mouse models of Nijmegen breakage syndrome and ataxia-telangiectasia-like disorder (5, 45, 49, 52). Chromosomal aberrations arising in these cells are predominantly chromatid type breaks, consistent with impaired metabolism of DNA replication-associated DNA breaks (49, 52).Further supporting a predominant role for the Mre11 complex in S phase is the observation that its primary role in DSB repair is the promotion of recombination between sister chromatids (3, 24). Structural and genetic evidence that the Mre11 complex effects molecular bridging between DNA duplexes offers a mechanistic basis for this observation (10, 23, 53). Molecular bridging by the Mre11 complex may also contribute to its influence on nonhomologous end joining (NHEJ) (12, 34, 57). Collectively, these data strongly support the view that the Mre11 complex''s checkpoint and DSB repair functions are manifested predominantly in the S and G₂ phases of the cell cycle.Although the Mre11 complex and ATM function in the same arm of the DNA damage response, ATM deficiency is lethal in hypomorphic Mre11 and Nbs1 mutants (Mre11^ATLD1/ATLD1 and Nbs1^ΔB/ΔB mice, respectively) (49, 52), suggesting that aspects of ATM function are Mre11 complex independent. ATM deficiency is also synthetically lethal with mutations in Prkdc, the gene encoding the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is mutated in mice with severe combined immunodeficiency (Prkdc^scid mice) (22, 42). DNA-PKcs is an ATM paralog required for NHEJ, which appears to be the predominant mode of DSB repair in G₁ cells (16).Defective NHEJ is unlikely to be the basis for the embryonic lethality of Prkdc^−/− Atm^−/− or Prkdc^scid/scid Atm^−/− mice, as loss of ATM rescues the late embryonic lethality of both DNA ligase IV (Lig4) and XRCC4 null embryos, which have more severe NHEJ defects than Prkdc^scid mice abolished by the Atm^−/− genotype (31, 42). These observations argue that the DNA-PKcs functions required for viability in the absence of ATM do not include NHEJ.To address this issue, we crossed Mre11^ATLD1/ATLD1 and Nbs1^ΔB/ΔB mice with Prkdc^scid/scid mice. As these Mre11 complex hypomorphs do not completely phenocopy ATM deficiency, we reasoned that double-mutant animals would be viable and thus provide a venue in which to examine the functional relationship between the Mre11 complex/ATM arm of the DNA damage response and DNA-PKcs. Whereas the Mre11^ATLD1/ATLD1 mutation was synthetically lethal with the Prkdc^scid/scid genotype, some Nbs1^ΔB/ΔB Prkdc^scid/scid mice were born, consistent with the more moderate DNA damage response defects associated with the Nbs1^ΔB allele than with the Mre11^ATLD1 allele (48). Nbs1^ΔB/ΔB Prkdc^scid/scid embryos were born at drastically reduced Mendelian ratios, displayed gross developmental defects, and were severely runted. Nbs1^ΔB/ΔB Prkdc^scid/scid cell cultures exhibited profound chromosome instability, growth defects, and increased sensitivity to ionizing radiation (IR). DNA repair defects associated with DNA-PKcs deficiency did not appear to underlie the observed phenotypic synergy. Rather, the data suggest a novel regulatory function of DNA-PKcs in the maintenance of chromosomal stability during the S and G₂ phases of the cell cycle.     

Comparative analysis of prokaryotic diversity in solar salterns in eastern Anatolia (Turkey)

The prokaryotic communities of four salterns (Bingöl, Fadlum, Kemah, and Tuzlagözü) in Turkey were examined and compared using the cultivation and cultivation-independent methods [fluorescence in situ hybridization (FISH) and 454 pyrosequencing]. FISH analysis with universal probes revealed that feeding waters carried 1.6 × 10²–1.7 × 10³ cells mL^?1, while crystallization ponds carried 3.8 × 10⁶–2.0 × 10⁷ cells mL^?1 that were mostly haloarchaea, including square cells (except for Kemah). High-throughput 16S rRNA-based gene sequencing showed that the most frequent archaeal OTUs in Bingöl, Fadlum, Tuzlagözü, and Kemah samples were affiliated with Haloquadratum (76.8 %), Haloarcula (27.8 %), Halorubrum (49.6 %), and Halonotius (59.8 %), respectively. Bacteroidetes was the dominant bacterial phylum in Bingöl and Fadlum, representing 71.5 and 79.5 % of the bacterial OTUs (respectively), while the most abundant bacterial phylum found in the Kemah saltern was Proteobacteria (79.6 %). The majority of the bacterial OTUs recovered from Tuzlagözü belonged to the Cyanobacteria (35.7 %), Bacteroidetes (35.0 %), and Proteobacteria (25.5 %) phyla. Cultivation studies revealed that the archaeal isolates were closely related to the genera Halobacterium, Haloarcula, and Halorubrum. Bacterial isolates were confined to two phyla, Proteobacteria (Alphaproteobacteria and Gammaproteobacteria classes) and Bacteroidetes. Comparative analysis showed that members of the Euryarchaeota, Bacteroidetes, Proteobacteria, and Cyanobacteria phyla were major inhabitants of the solar salterns.     

Growth and phosphorus removal by <Emphasis Type="Italic">Synechococcus elongatus</Emphasis> co-immobilized in alginate beads with <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>

This study examined the co-immobilization of the cyanobacterium Synechococcus elongatus with the plant growth-promoting bacterium Azospirillum brasilense in alginate beads and its potential application for the removal of phosphorus from aquaculture wastewater. Co-immobilization of both microorganisms significantly increased the cell density of S. elongatus (2852.5?×?10⁴ cells mL^?1) compared with that of immobilization of cyanobacteria alone (1325.2?×?10⁴ cells mL^?1). Chlorophyll a content was similar in co-immobilized (11.1?±?3.5 pg cell^?1) and immobilized S. elongatus (14.5?±?4.9 pg cell^?1). Azospirillum brasilense showed continuous growth until day 2, after which its cell concentration declined until the end of the assay. Co-immobilized S. elongatus removed more phosphorus (44.8 %) than immobilized cyanobacteria cells alone (32.0 %). In conclusion, phosphate removal was greater with free cells of S. elongatus but overlapped with the values that were obtained with the treatment of co-immobilization of cells. Our results demonstrate that A. brasilense enhances the growth of S. elongatus and improves its removal of phosphorus when they are co-immobilized in alginate beads compared with only immobilization of cyanobacteria cells alone.     

Measurement of individual cell strength of <Emphasis Type="Italic">Botryococcus braunii</Emphasis> in cell culture

Botryococcus braunii is a microalga considered for biofuel production and may require physical disruption of cells/colonies for efficient hydrocarbon extraction. In this study, the strength of individual cells of B. braunii was measured using a nanoindenter. From the load and cell size, the pressure for bursting the cell was calculated to be 56.9 MPa. This value is 2.3–10 times those of Saccharomyces cerevisiae and Chlorella vulgaris found in another research, because B. braunii has two types of cell walls with different thicknesses. The energy required to disrupt 1 g of dry B. braunii cells, estimated by load-displacement curves, is 3.19 J g^?1 which is 0.19–1.2 times higher than those of S. cerevisiae and C. vulgaris. When using a high-pressure homogenizer for disrupting B. braunii cells, the cell disruption degree increased with the treatment pressure at above 30 MPa, and 70% of cells were disrupted at 80 MPa.     

Laboratory and simulated-field bioassays for assessing mixed cultures of <Emphasis Type="Italic">Lysinibacillus sphaericus</Emphasis> against <Emphasis Type="Italic">Aedes aegypti</Emphasis> (Diptera: Culicidae) larvae resistant to temephos

Aedes aegypti (L.) is the main vector of tropical diseases such as dengue, chikungunya and Zika. Due to the overuse of insecticides, Ae. aegypti resistant populations have increased. Biological control with Lysinibacillus sphaericus (Ahmed) has been used against Culex sp. and Anopheles sp. Although Ae. aegypti is refractory to the binary toxin of L. sphaericus spores, vegetative cells have been shown to be effective against Ae. aegypti larvae. In this work, the effect of L. sphaericus vegetative cells on Ae. aegypti temephos-resistant larvae was assessed under lab and simulated field conditions. L. sphaericus caused about 90% mortality of insecticide-resistant Ae. aegypti larvae under simulated field conditions. Likewise, Ae. aegypti larvae were more sensitive to mixed cultures of L. sphaericus than to individual strains; then, the most effective mixed culture exhibited an LC₅₀ of 1.21 × 10⁵ CFU/mL with Rockefeller larvae and 8.04 × 10⁴ CFU/mL with field-collected larvae. Additionally, we found that mixed cultures composed of two L. sphaericus strains were more effective than a culture formed by the three strains. Our results suggest that mixed cultures comprising L. sphaericus vegetative cells could be useful for controlling temephos-resistant populations of Ae. aegypti, as evidenced by the effectiveness demonstrated under laboratory and simulated field conditions.     

Detection and Quantification of <Emphasis Type="Italic">Vibrio cholerae,Vibrio parahaemolyticus</Emphasis>, <Emphasis Type="Italic">and Vibrio vulnificus</Emphasis> in Coastal Waters of Guinea-Bissau (West Africa)

V. cholerae, V. parahaemolyticus, and V. vulnificus are recognized human pathogens. Although several studies are available worldwide, both on environmental and clinical contexts, little is known about the ecology of these vibrios in African coastal waters. In this study, their co-occurrence and relationships to key environmental constraints in the coastal waters of Guinea-Bissau were examined using the most probable number-polymerase chain reaction (MPN-PCR) approach. All Vibrio species were universally detected showing higher concentrations by the end of the wet season. The abundance of V. cholerae (ISR 16S-23S rRNA) ranged 0–1.2 × 10⁴ MPN/L, whereas V. parahaemolyticus (toxR) varied from 47.9 to 1.2 × 10⁵ MPN/L. Although the presence of genotypes associated with virulence was found in environmental V. cholerae isolates, ctxA+ V. cholerae was detected, by MPN-PCR, only on two occasions. Enteropathogenic (tdh+ and trh+) V. parahaemolyticus were detected at concentrations up to 1.2 × 10³ MPN/L. V. vulnificus (vvhA) was detected simultaneously in all surveyed sites only at the end of the wet season, with maximum concentrations of 1.2 × 10⁵ MPN/L. Our results suggest that sea surface water temperature and salinity were the major environmental controls to all Vibrio species. This study represents the first detection and quantification of co-occurring Vibrio species in West African coastal waters, highlighting the potential health risk associated with the persistence of human pathogenic Vibrio species.     

Feed-backs between genetic structure and perturbation-driven decline in seagrass (<Emphasis Type="Italic">Posidonia oceanica</Emphasis>) meadows

We explored the relationships between perturbation-driven population decline and genetic/genotypic structure in the clonal seagrass Posidonia oceanica, subject to intensive meadow regression around four Mediterranean fish-farms, using seven specific microsatellites. Two meadows were randomly sampled (40 shoots) within 1,600 m² at each site: the “impacted” station, 5–200 m from fish cages, and the “control” station, around 1,000 m downstream further away (considered a proxy of the pre-impact genetic structure at the site). Clonal richness (R), Simpson genotypic diversity (D*) and clonal sub-range (CR) were highly variable among sites. Nevertheless, the maximum distance at which clonal dispersal was detected, indicated by CR, was higher at impacted stations than at the respective control station (paired t-test: P < 0.05, N = 4). The mean number of alleles (Â) and the presence of rare alleles ( _r) decreased at impacted stations (paired t-test: P < 0.05, and P < 0.02, respectively, N = 4). At a given perturbation level (quantified by the organic and nutrient loads), shoot mortality at the impacted stations significantly decreased with CR at control stations (R ² = 0.86, P < 0.05). Seagrass mortality also increased with  (R ² = 0.81, P < 0.10), R (R ² = 0.96, P < 0.05) and D* (R ² = 0.99, P < 0.01) at the control stations, probably because of the negative correlation between those parameters and CR. Therefore, the effects of clonal size structure on meadow resistance could play an important role on meadow survival. Large genotypes of P. oceanica meadows thus seem to resist better to fish farm-derived impacts than little ones. Clonal integration, foraging advantage or other size-related fitness traits could account for this effect.     

Exploiting Phosphate-Starved cells of <Emphasis Type="Italic">Scenedesmus</Emphasis> sp. for the Treatment of Raw Sewage

Phosphate depletion is one of the favorable ways to enhance the sewage water treatment with the algae, however, detailed information is essential with respect to internal phosphate concentration and physiology of the algae. The growth rate of the phosphate-starved Scenedesmus cells was reduced drastically after 48 h. Indicating cells entered in the stationary phase of the growth cycle. Fourier Transform Infrared analysis of phosphate-starved Scenedesmus cells showed the reduction in internal phosphate concentration and an increase in carbohydrate/phosphate and carbohydrate/lipid ratio. The phosphate-starved Scenedesmus cells, with an initial cell density of, 1 × 10⁶ cells mL^?1 shows 87% phosphate and 100 % nitrogen removal in 24 h. The normal Scenedesmus cells need approximately 48 h to trim down the nutrients from wastewater up to this extent. Other microalgae, Ankistrodesmus, growth pattern was not affected due to phosphate starvation. The cells of Ankistrodesmus was able to reduce 71% phosphate and 73% nitrogen within 24 h, with an initial cell density of, 1 × 10⁶ cells mL^?1.     

Toxin Gene Contents and Activity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strains Against Two Sugarcane Borer Species, <Emphasis Type="Italic">Diatraea saccharalis</Emphasis> (F.) and <Emphasis Type="Italic">D. flavipennella</Emphasis> (Box)

Bacillus thuringiensis (Berliner) bears essential characteristics in the control of insect pests, such as its unique mode of action, which confers specificity and selectivity. This study assessed cry gene contents from Bt strains and their entomotoxicity against Diatraea saccharalis (F.) and Diatraea flavipennella (Box) (Lepidoptera: Crambidae). Bioassays with Bt strains were performed against neonates to evaluate their lethal and sublethal activities and were further analyzed by PCR, using primers to identify toxin genes. For D. saccharalis and D. flavipennella, 16 and 18 strains showed over 30% larval mortality in the 7th day, respectively. The LC₅₀ values of strains for D. saccharalis varied from 0.08 × 10⁵ (LIIT-0105) to 4104 × 10⁵ (LIIT-2707) spores + crystals mL^?1. For D. flavipennella, the LC₅₀ values of strains varied from 0.40 × 10⁵ (LIIT-2707) to 542 × 10⁵ (LIIT-2109) spores + crystals mL^?1. For the LIIT-0105 strain, which was the most toxic to D. saccharalis, the genes cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C, cry1D, cry1F, cry1I, cry2Aa, cry2Ab, cry8, and cry9C were detected, whereas for the strain LIIT-2707, which was the most toxic to D. flavipennella, detected genes were cry1Aa, cry1Ab, cry1Ac, cry1B, cry1D, cry1F, cry1I, cry2Aa, cry2Ab, and cry9. The toxicity data and toxin gene content in these strains of Bt suggest a great variability of activity with potential to be used in the development of novel biopesticides or as source of resistance genes that can be expressed in plants to control pests.     

Parasitism Rate of <Emphasis Type="Italic">Habrobracon hebetor</Emphasis> to <Emphasis Type="Italic">Ephestia kuehniella</Emphasis> Larvae: Residual Effect of Deltamethrin,Fenvalerate and Azadirachtin

Sublethal concentrations of chemical insecticides may cause changes in some behavioral characteristics of natural enemies such as functional responses. The residual effect of three synthetic insecticides including deltamethrin, fenvalerate and azadirachtin were studied on functional response of Habrobracon hebetor Say to Ephestia kuehniella Zeller larvae. Seven host densities (2, 4, 8, 16, 32, 64 and 96) were used during a 24 h period. The resulting data were appropriately fit to Type II functional response models in all treatments: (1) control (0.0916 h^?1; and T _h  = 0.2011 h); (2) deltamethrin (a = 0.0839 h^?1; and T _h  = 0.3560 h); (3) fenvalerate (a = 0.0808 h^?1 and T _h  = 0.3623 h); and (4) azadirachtin (a = 0.0900 h^?1 and T _h  = 0.2042 h). Maximum theoretical parasitism rate (T/T _h ) was 119.34 estimated for control wasps. There was no significant difference between the values of attack rates (a and a + D _a ) in all treatments while the handling time was statistically affected in female wasps treated with fenvalerate. Our findings will be useful in safe application of these insecticides in pest management programmes.     

Enantioconvergent hydrolysis of racemic styrene oxide at high concentration by a pair of novel epoxide hydrolases into (<Emphasis Type="Italic">R</Emphasis>)-phenyl-1,2-ethanediol

Objectives

To prepare (R)-phenyl-1,2-ethanediol ((R)-PED) with high enantiomeric excess (ee _p) and yield from racemic styrene oxide (rac-SO) at high concentration by bi-enzymatic catalysis.

Results

The bi-enzymatic catalysis was designed for enantioconvergent hydrolysis of rac-SO by a pair of novel epoxide hydrolases (EHs), a Vigna radiata EH3 (VrEH3) and a variant (AuEH2^A250I) of Aspergillus usamii EH2. The simultaneous addition mode of VrEH3 and AuEH2^A250I, exhibiting the highest average turnover frequency (aTOF) of 0.12 g h^?1 g^?1, was selected, by which rac-SO (10 mM) was converted into (R)-PED with 92.6% ee _p and 96.3% yield. Under the optimized reaction conditions: dry weight ratio 14:1 of VrEH3-expressing E. coli/vreh3 to AuEH2^A250I-expressing E. coli/Aueh2 ^A250I and reaction at 20 °C, rac-SO (10 mM) was completely hydrolyzed in 2.3 h, affording (R)-PED with 98% ee _p. At the weight ratio 0.8:1 of rac-SO to two mixed dry cells, (R)-PED with 97.4% ee _p and 98.7% yield was produced from 200 mM (24 mg/ml) rac-SO in 10.5 h.

Conclusions

Enantioconvergent hydrolysis of rac-SO at high concentration catalyzed by both VrEH3 and AuEH2^A250I is an effective method for preparing (R)-PED with high ee _p and yield.

     

Occurrence and distribution of multiple antibiotic-resistant <Emphasis Type="Italic">Enterococcus</Emphasis> and <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. from Indian poultry: in vivo transferability of their erythromycin,tetracycline and vancomycin resistance

The objective of this study was to determine the occurrence and distribution of antibiotic resistant (AR) lactic acid bacteria (LAB) in Indian poultry. LAB from poultry farm feces (n = 21) and samples from slaughter houses comprising chicken intestine (n = 46), raw meat (n = 23), and sanitary water (n = 4) were evaluated and compared with those from organic chicken (OC) collected from nearby villages. Screening studies showed 5–7 log units higher erythromycin (ER), tetracycline (TC) and vancomycin (VAN) resistant LAB from conventional poultry chicken (CC) compared to OC. Molecular characterization of isolated cultures (n = 32) with repetitive-PCR profiling and 16S rRNA gene sequencing revealed their taxonomical status as Enterococcus faecium (n = 16), Enterococcus durans (n = 2), Lactobacillus plantarum (n = 10), Lactobacillus pentosus (n = 1) and Lactobacillus salivarius (n = 3). The isolates were found to harbor erm(B), msr(C), msr(A/B), tet(M), tet(L) and tet(K) genes associated with Tn916 and Tn917 family transposons. Expression studies through real-time PCR revealed antibiotic-induced expression of the identified AR genes. In vitro and in vivo conjugational studies revealed transfer of ER and TC resistant (ER^R and TC^R) genes with transfer frequencies of 10^?7 and 10^?4 transconjugants recipient^?1, respectively. Although no known VAN resistance (VAN^R) genes were detected, high phenotypic resistance was observed and was transferable to the recipient. From a public health point of view, this study reports Indian poultry as a major source of high levels of AR bacteria contaminating the food chain and the environment. Thus, urgent and determined strategies are needed to control the spread of multiple AR bacteria.     

A saponin isolated from <Emphasis Type="Italic">Agapanthus africanus</Emphasis> differentially induces apoplastic peroxidase activity in wheat and displays fungicidal properties

A spirostane with an attached trisaccharide, (25R)-5α-spirostane-2α,3β,5α-triol 3-O-(O-α-l-rhamnopyranosyl-(1 → 2)-O-(β-d-galactopyranosyl-(1 → 3))-β-d-glucopyranoside), was isolated and identified from the aerial parts of Agapanthus africanus by activity-guided fractionation. Fungicidal properties of the crude extract, semi-purified fractions as well as the purified active saponin from A. africanus were screened in vitro against Fusarium oxysporum. At a concentration of 1 mg mL^?1, the crude extract and semi-purified ethyl acetate and dichloromethane fractions showed significant antifungal activity. The purified saponin inhibited the in vitro mycelial growth of F. oxysporum completely (100 %) at a concentration of 125 µg mL^?1. Furthermore, to verify previously observed induced resistance by crude extracts of A. africanus towards leaf rust, intercellular PR-protein activity was determined in wheat seedlings following foliar application of the purified saponin at 100 µg mL^?1. In vitro peroxidase enzyme activity increased significantly (60 %) in wheat seedlings 48 h after treatment with the purified saponin, demonstrating its role as an elicitor to activate a defence reaction in wheat.