Purification of high molecular-weight antibacterial proteins of insect pathogenic Brevibacillus laterosporus isolates

Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium belonging to the Brevibacillus brevis phylogenetic cluster. Globally, insect pathogenic strains of the bacterium have been isolated, characterised, and some activities patented. Two isolates, Bl 1821L and Bl 1951, exhibiting pathogenicity against the diamondback moth and mosquitoes, are under development as a biopesticide in New Zealand. However, due to the suspected activity of putative antibacterial proteins (ABPs), the endemic isolates often grow erratically. Various purification methods including size exclusion chromatography, sucrose density gradient centrifugation, polyethylene glycol precipitation, and ammonium sulphate precipitation employed in this study enabled the isolation of two putative antibacterial proteins of ~30 kD and ~48 kD from Bl 1821L and one putative antibacterial protein of ~30 kD from Bl 1951. Purification of the uninduced cultures of Bl 1821L and Bl 1951 also yielded the protein bands of ~30 kD and ~48 kD on SDS-PAGE which indicated their spontaneous induction. Disc diffusion assay was used to determine the antagonistic activities of the putative ABPs. Subsequent transmission electron microscope (TEM) examination of purified putative antibacterial protein-containing solution showed the presence of encapsulin (~30 kD) and polysheath (~48 kD) like structures. Although only the ~30 kD protein was purified from Bl 1951, both structures were seen in this strain under TEM. Furthermore, while assessing the antibacterial activity of some fractions of Bl 1951 against Bl 1821L in size exclusion chromatography method, population of Bl 1821L persister cells was noted. Overall, this work added a wealth of knowledge for the purification of the HMW proteins (bacteriocins) of the Gram-positive bacteria including Bl

Isolation, purification, and characterisation of a phage tail-like bacteriocin from the insect pathogenic bacterium Brevibacillus laterosporus

The Gram-positive and spore-forming bacterium Brevibacillus laterosporus (Bl) belongs to the Brevibacillus brevis phylogenetic cluster. Isolates of the species have demonstrated pesticidal potency against a wide range of invertebrate pests and plant diseases. Two New Zealand isolates, Bl 1821L and Bl 1951, are under development as biopesticides for control of diamondback moth and other pests. However, due to the often-restricted growth of these endemic isolates, production can be an issue. Based on the previous work, it was hypothesised that the putative phages might be involved. During investigations of the cause of the disrupted growth, electron micrographs of crude lysate of Bl 1821L showed the presence of phages’ tail-like structures. A soft agar overlay method with PEG 8000 precipitation was used to differentiate between the antagonistic activity of the putative phage and phage tail-like structures (bacteriocins). Assay tests authenticated the absence of putative phage activity. Using the same method, broad-spectrum antibacterial activity of Bl 1821L lysate against several Gram-positive bacteria was found. SDS-PAGE of sucrose density gradient purified and 10 kD MWCO concentrated lysate showed a prominent protein band of ∼48 kD, and transmission electron microscopy revealed the presence of polysheath-like structures. N-terminal sequencing of the ∼48 kD protein mapped to a gene with weak predicted amino acid homology to a Bacillus PBSX phage-like element xkdK, the translated product of which shared >90% amino acid similarity to the phage tail-sheath protein of another Bl published genome, LMG15441. Bioinformatic analysis also identified an xkdK homolog in the Bl 1951 genome. However, genome comparison of the region around the xkdK gene between Bl 1821L and Bl 1951 found differences including two glycine rich protein encoding genes which contain imperfect repeats (1700 bp) in Bl 1951, while a putative phage region resides in the analogous Bl 1821L region. Although comparative analysis of the genomic organisation of Bl 1821L and Bl 1951 PBSX-like region with the defective phages PBSX, PBSZ, and PBP 180 of Bacillus subtilis isolates 168 and W23, and Bacillus phage PBP180 revealed low amino acids similarity, the genes encode similar functional proteins in similar arrangements, including phage tail-sheath (XkdK), tail (XkdO), holin (XhlB), and N-acetylmuramoyl-L-alanine (XlyA). AMPA analysis identified a bactericidal stretch of 13 amino acids in the ∼48 kD sequenced protein of Bl 1821L. Antagonistic activity of the purified ∼48 kD phage tail-like protein in the assays differed remarkably from the crude lysate by causing a decrease of 34.2% in the number of viable cells of Bl 1951, 18 h after treatment as compared to the control. Overall, the identified inducible phage tail-like particle is likely to have implications for the in vitro growth of the insect pathogenic isolate Bl 1821L

You are what you eat: Fungal metabolites and host plant affect the susceptibility of diamondback moth to entomopathogenic fungi

Background: Beauveria are entomopathogenic fungi of a broad range of arthropod pests. Many strains of Beauveria have been developed and marketed as biopesticides. Beauveria species are well-suited as the active ingredient within biopesticides because of their ease of mass production, ability to kill a wide range of pest species, consistency in different conditions, and safety with respect to human health. However, the efficacy of these biopesticides can be variable under field conditions. Two under-researched areas, which may limit the deployment of Beauveria-based biopesticides, are the type and amount of insecticidal compounds produced by these fungi and the influence of diet on the susceptibility of specific insect pests to these entomopathogens. Methods: To understand and remedy this weakness, we investigated the effect of insect diet and Beauveria-derived toxins on the susceptibility of diamondback moth larvae to Beauveria infection. Two New Zealand-derived fungal isolates, B. pseudobassiana I12 Damo and B. bassiana CTL20, previously identified with high virulence towards diamondback moth larvae, were selected for this study. Larvae of diamondback moth were fed on four different plant diets, based on different types of Brassicaceae, namely broccoli, cabbage, cauliflower, and radish, before their susceptibility to the two isolates of Beauveria was assessed. A second experiment assessed secondary metabolites produced from three genetically diverse isolates of Beauveria for their virulence towards diamondback moth larvae. Results: Diamondback moth larvae fed on broccoli were more susceptible to infection by B. pseudobassiana while larvae fed on radish were more susceptible to infection by B. bassiana. Furthermore, the supernatant from an isolate of B. pseudobassiana resulted in 55% and 65% mortality for half and full-strength culture filtrates, respectively, while the filtrates from two other Beauveria isolates, including a B. bassiana isolate, killed less than 50% of larvae. This study demonstrated different levels of susceptibility of the insects raised on different plant diets and the potential use of metabolites produced by Beauveria isolates in addition to their conidia

Purification of high-molecular-weight antibacterial proteins of insect pathogenic Brevibacillus laterosporus isolates

Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium belonging to the Brevibacillus brevis phylogenetic cluster. Globally, insect pathogenic strains of the bacterium have been isolated, characterised, and some activities have been patented. Two isolates, Bl 1821L and Bl 1951, exhibiting pathogenicity against the diamondback moth and mosquitoes, are under development as a biopesticide in New Zealand. However, due to the suspected activity of putative antibacterial proteins (ABPs), the endemic isolates often grow erratically. Various purification methods, including size exclusion chromatography, sucrose density gradient centrifugation, polyethylene glycol precipitation, and ammonium sulphate precipitation employed in this study, enabled the isolation of two putative antibacterial proteins of ∼30 and ∼48 kD from Bl 1821L and one putative antibacterial protein of ∼30 kD from Bl 1951. Purification of the uninduced cultures of Bl 1821L and Bl 1951 also yielded protein bands of ∼30 and ∼48 kD on SDS-PAGE, which indicated their spontaneous induction. A disc diffusion assay was used to determine the antagonistic activities of the putative ABPs. Subsequent transmission electron microscope (TEM) examination of a purified putative antibacterial protein-containing solution showed the presence of encapsulin (∼30 kD) and polysheath (∼48 kD)-like structures. Although only the ∼30 kD protein was purified from Bl 1951, both structures were seen in this strain under TEM. Furthermore, while assessing the antibacterial activity of some fractions of Bl 1951 against Bl 1821L in the size exclusion chromatography method, the population of Bl 1821L persister cells was noted. Overall, this work added a wealth of knowledge about the purification of the high-molecular-weight (HMW) proteins (bacteriocins) of Gram-positive bacteria including Bl

Photometric Redshifts of Quasars

We demonstrate that the design of the Sloan Digital Sky Survey (SDSS) filter system and the quality of the SDSS imaging data are sufficient for determining accurate and precise photometric redshifts (``photo-z''s) of quasars. Using a sample of 2625 quasars, we show that photo-z determination is even possible for z<=2.2 despite the lack of a strong continuum break that robust photo-z techniques normally require. We find that, using our empirical method on our sample of objects known to be quasars, approximately 70% of the photometric redshifts are correct to within delta z = 0.2; the fraction of correct photometric redshifts is even better for z>3. The accuracy of quasar photometric redshifts does not appear to be dependent upon magnitude to nearly 21st magnitude in i'. Careful calibration of the color-redshift relation to 21st magnitude may allow for the discovery of on the order of 10^6 quasars candidates in addition to the 10^5 quasars that the SDSS will confirm spectroscopically. We discuss the efficient selection of quasar candidates from imaging data for use with the photometric redshift technique and the potential scientific uses of a large sample of quasar candidates with photometric redshifts.Comment: 29 pages, 8 figures, submitted to A

Whey and pea protein fortification of rice starches: Effects on protein and starch digestibility and starch pasting properties

Rice is rich in starch but low in protein. In countries where rice is the staple food, people are at high risk of protein deficiency. Legumes (such as pea) and milk are an important part of the diet in many developing and developed countries, respectively. For this reason, pea protein isolate (PPI) and whey protein concentrate (WPC) are incorporated into basmati (≈20–25% amylose) and glutinous (≈0–3% amylose) starches to test the effect of protein incorporation on the pasting properties of rice starch, and on starch and protein digestibility. Increasing protein incorporation reduced the peak, breakdown, and final viscosities. The effect is more pronounced for basmati mixtures compared to glutinous mixtures. For starch digestibility, basmati starch mixtures exhibited an increasing trend in the amount of glucose released over 120 min, whereas glutinous starch mixtures show a decrease in the amount of glucose released after 60 min. In addition, samples with the same % protein incorporation (PPI and WPC) shows similar trend in terms of amount of glucose released at each time point. The amylose content appears to have an effect on starch digestibility over time. For pea and whey protein digestibility, basmati starch samples exhibited lower digestion after 120 min compared with glutinous starch samples. The starch amylose content appears to have an impact on protein digestibility. This provides a way of fortifying rice starch with proteins, in which the proteins are effectively digested without significantly increasing blood glucose levels

Physical, predictive glycaemic response and antioxidative properties of black ear mushroom (Auricularia auricula) extrudates

Black ear mushroom (Auricularia auricula) is an important genus of cultivated mushroom, which contains health benefits. Incorporating black ear (BE) mushroom into brown rice by extrusion changed the physicochemical, and more importantly, the nutritional characteristics of the extrudates. With increased incorporation of BE mushroom in the extrudates in vitro starch digestion of the different extrudates revealed significantly reduced starch digestion, suggesting a lower glycaemic index. In addition, incorporation of BE in brown rice extrudates increased the total phenolic concentration of the samples, which led to higher % scavenging effect against free-radicals in DPPH assay. In the ORAC assay for anti-oxidant activity, BE powder exhibited the highest anti-oxidant activity, followed by 10% BE and 15% BE, and 5% BE extruded products. The extruded brown rice control exhibited the lowest antioxidant activity. Inclusion of black ear mushroom was shown to improve the nutritional qualities of the food product illustrating the connection between plant bioactive ingredients and human health

The effect of selected bacteria on the virulence of Metarhizium novozealandicum C14 to Costelytra giveni larvae (Scarabaeidae)

The ability of disease-causing bacteria to synergise infection by the entomopathogen Metarhizium of larvae of Costelytra giveni, a New Zealand native scarab, has been previously demonstrated, but the role of soil bacteria in infections is unknown. Bacteria isolated from both dead C. giveni larvae and soil were assessed for their effect on the virulence of Metarhizium novozealandicum C14 towards C. giveni larvae. Seven bacterial isolates showed synergism with M. novozealandicum C14 against second instar larvae, but no synergism was found against third instar larvae. No apparent disease was noted with C. giveni larvae independently challenged with the bacteria isolates. An isolate of Yersinia enterocolitica (isolate 6-1) in combination with M. novozealandicum C14 caused higher larval mortality than the other bacterial isolate-entomopathogen combinations against second instar larvae. Using plate assays, Y. enterocolitica 6-1 was found to produce more chitinase, but not protease, compared with the other bacterial strains suggesting a role of chitinase in synergism. This study suggests that the combination of Y. enterocolitica (isolate 6-1) and M. novozealandicum C14 could provide effective biocontrol of second instar grass grub larvae

Correlations between the phenolic and fibre composition of mushrooms and the glycaemic and textural characteristics of mushroom enriched extruded products

In this study, semolina extruded snack products were supplemented with white button mushroom (Agaricus bisporus), shiitake (Lentinula edodes) and porcini mushrooms (Boletus edulis). The functional properties of the products were analysed, including the nutritional profile, starch characteristics (content, gelatinisation and digestibility) and antioxidant activities (total phenolic content, DPPH and ORAC), colour, and textural properties. Compared to the control samples, extrudates supplemented with mushroom powder showed a reduced product expansion, decreased water absorption index, increased water solubility index and altered microstructure characteristics. Differences in starch gelatinisation and starch hydrolysis resulted in a reduced predictive glycaemic response value for mushroom supplemented extrudates. Inclusion of mushroom material resulted in higher levels of phenolic compounds and antioxidants