Ecology and thermal inactivation of microbes in and on interplanetary space vehicle components

The experiments conducted to determine the heat resistance of Bacillus megaterium ATCC 6458 at 90 and 100 C were completed. Estimates from replicate experiments at eight percent relative humidities (less than 0.001 to 100% RH) for each temperature were computed. A Bacillus cereus strain with high heat resistance was cultured and the resistance determined in phosphate buffer (D sub 121.1 = 2.16 min and z = 8.7 C). The profile of the dry heat resistance of B. megaterium is summarized and the most resistant condition to the three spores (Bacillus subtilis var. niger, ATCC 29669, and Bacillus stearothermophilus, strain 1518) is compared

Engineering Bacillus megaterium for production of functional intracellular materials

Background: Over the last 10-15 years, a technology has been developed to engineer bacterial polyhydroxybutyrate (PHB) inclusions as functionalized beads, for applications such as vaccines, diagnostics and enzyme immobilization. This has been achieved by translational fusion of foreign proteins to the PHB synthase (PhaC). The respective fusion protein mediates self-assembly of PHB inclusions displaying the desired protein function. So far, beads have mainly been produced in recombinant Escherichia coli which is problematic for some applications as the lipopolysaccharides (LPS) co-purified with such inclusions are toxic to humans and animals. Results: In this study, we have engineered the formation of functional PHB inclusions in the Gram-positive bacterium Bacillus megaterium, an LPS-free and established industrial production host. As B. megaterium is a natural PHB producer, the PHB-negative strain PHA05 was used to avoid any background PHB production. Plasmid-mediated T7 promoter-driven expression of the genes encoding β-ketothiolase (phaA), acetoacetyl-CoA-reductase (phaB) and PHB synthase (phaC) enabled/effected PHB production by B. megaterium PHA05. To produce functionalized PHB inclusions, the N- and C-terminus of PhaC was fused to four and two IgG binding Z-domains from Staphylococcus aureus, respectively. The ZZ-domain PhaC fusion protein was strongly overproduced at the surface of the PHB inclusions and the corresponding isolated ZZ-domain displaying PHB beads were found to purify IgG with a binding capacity of 40-50 mg IgG/g beads. As B. megaterium has the ability to sporulate and respective endospores could co-purify with cellular inclusions, a sporulation negative production strain was generated by disrupting the spoIIE gene in PHA05. This strain did not produce spores when tested under sporulation inducing conditions and it was still able to synthesize ZZ-domain displaying PHB beads. Conclusions: This study provides proof of concept for the successful genetic engineering of B. megaterium as a host for the production of functionalized PHB beads. Disruption of the spoIIE gene rendered B. megaterium incapable of sporulation but particularly suitable for production of functionalized PHB beads. This sporulation-negative mutant represents an improved industrial production strain for biotechnological processes otherwise impaired by the possibility of endospore formation.fals

Effect of inoculation of Bacillus megaterium isolates on growth, biomass and nutrient content of Peppermint

The investigation was carried out to study the effect of Bacillus megaterium isolates on growth, biomass and nutrient content of Pepper mint. Pepper mint was used as a host plant to study the growth response, biomass and nutrient content. Bacillus megaterium strains from different soil types of various agroclimatic zones were isolated, identified and confirmed using standard synaptic keys. A pot culture experiment was carried out to find out the influence of Bacillus megaterium isolates on seedlings of Pepper mint under greenhouse conditions. Pepper mint plants inoculated with Bacillus megaterium isolates manifested increase in plant height, number of leaves, number of branches, biomass and nutrient content compared to uninoculated plants. Among the ten isolates inoculated, the Bacillus megaterium isolates from zone 3 showed significantly high values in almost all growth parameters chosen for the study. Similarly biochemical parameters of the Pepper mint inoculated with ten isolates of Bacillus megaterium was studied. In the inoculated plants the biochemical parameters like chlorophyll content, nitrogen content and phosphorus content was higher as compared to uninoculated plants. The results suggests that plants inoculated with Bacillus megaterium isolates showed better growth response, biomass yield and nutrient content when compared to uninoculated plants. Also the studies showed that Zone 3 isolate was more effective among other isolates

Diversity of culturable moderately halophilic and halotolerant bacteria in a marsh and two salterns a protected ecosystem of Lower Loukkos (Morocco)

To study the biodiversity of halophilic bacteria in a protected wetland located in Loukkos (Northwest, Morocco), a total of 124 strains were recovered from sediment samples from a marsh and salterns. 120 isolates (98%) were found to be moderately halophilic bacteria; growing in salt ranges of 0.5 to 20%. Of 124 isolates, 102 were Gram-positive while 22 were Gram negative. All isolates were identified based on 16S rRNA gene phylogenetic analysis and characterized phenotypically and by screening for extracellular hydrolytic enzymes. The Gram-positive isolates were dominated by the genus Bacillus (89%) and the others were assigned to Jeotgalibacillus, Planococcus, Staphylococcus and Thalassobacillus. The Gram negative isolates were dominated by the genus Vibrio (41%) and the others were assigned to Halomonas, Psychrobacter, Marinobacterium, Pseudoalteromonas, Salinivibrio and Photobacterium. The growth of strains obtained under different physico-chemical conditions and the screening for hydrolytic enzymes showed a high diversity even within the same species

Growth Response of Ayapana on inoculation with Bacillus megaterium isolated from different soil types of various agroclimatic zones of Karnataka

A study was undertaken to find out the growth response of Ayapana on inoculation with Bacillus megaterium isolated from different soil types of various agroclimatic zones of Karnataka. Bacillus megaterium strains from different soil types of various agroclimatic zones of Karnataka were isolated, identified and confirmed using standard synaptic keys. Ayapana was used as a host plant to study the growth response, biomass and nutrient content. Treatments of Bacillus megaterium isolates from ten different soil types of various agroclimatic zones of Karnataka were given to seedlings of Ayapana. There were significant changes in the plant growth, biomass and nutrient uptake in plants inoculated with Bacillus megaterium when compared to control plants. The parameters such as plant height, number of leaves, shoot and root fresh weight, shoot and root dry weight and nutrient uptake were studied. Plants inoculated with Bacillus megaterium isolates performed well when compared to uninoculated plants. The heights of plants inoculated with Bacillus megaterium isolates were found to be more than uninoculated plants. In plants inoculated with B. megaterium isolates, the height, number of leaves, fresh and dry weight of roots and shoots, nitrogen content, P content and chlorophyll content remained higher than the uninoculated plants. Among ten isolates inoculated, Zone 7 isolate recorded significantly high values in almost all growth parameters chosen for the study. The results suggests that plants inoculated with Bacillus megaterium isolates showed better growth response, biomass yield and nutrient content when compared to uninoculated plants.Â&nbsp

Recombinant protein immobilisation and display by alginate : a thesis presented in partial fulfilment of the requirements of the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand.

Biopolymers are a diverse group of organic materials with important applications in a number of industries. Their ability to adsorb and encapsulate compounds has been widely utilised in both biotechnologies and pharmaceuticals. In the last decade, biopolymers have been given new and enhanced functionality, including the separation and purification of compounds. This field is of increasing relevance as advances in the bacterial cell culture process have improved productivity in the biomanufacturing industry, with the establishment of several bacterial host cell lines and optimised protein production systems. This increase in upstream productivity is leading to bottlenecks in downstream processing as current technology platforms reach their limits of throughput and scalability. While previous studies have generated functionalised protein biopolymers using polyhydroxyalkanoate (PHA) biopolyester beads, very few studies have examined the commercially significant biopolymer alginate. Alginate is an exopolysaccharide produced by algae and some bacteria, and is widely utilised in food, pharmaceutical, and biomedical industries because of its stabilising, haemostatic, biocompatible properties and its modifiable structure. In this study, a partially functional alginate-binding recombinant protein was produced, which contained an α-amylase domain from Bacillus licheniformis (BLA) translationally fused to the alginate-binding domain of Pseudomonas aeruginosa AlgX – an alginate acetyltransferase. An Ssp DnaB mini-intein was included between BLA and AlgX to facilitate recovery of BLA, following immobilisation and display on the surface of alginate. However, aberrant activity of the intein caused total cleavage of the recombinant protein between its BLA and AlgX domains before it could be recovered from the protein production system. Additionally, the absence of a key cysteine residue in the alginate-binding domain prevented the formation of a disulfide bond, which is an essential structural element for the folding and functionality of this region. While this study was unable to overcome intein hyperactivity, functional analysis of the BLA domain showed consistent and significant levels of α-amylase activity, leading to a positive outlook for the functionality of a full-length recombinant protein if proper intein activity can be restored and the necessary cysteine included. In this way, alginate could be specifically functionalised with a desired protein, and in turn, alginate beads could be used for the separation and enrichment of target proteins

Effect of vitamins and bivalent metals on lysine yield in Bacillus megaterium

The effects of vitamins and bivalent metals on lysine accumulation in Bacillus strains were investigated. Biotin enhanced lysine production in all the Bacillus strains, while folic acid and riboflavin stimulated lysine yields in Bacillus megaterium SP 86 only. All bivalent metals stimulated lysineaccumulation in B. megaterium SP 86 and B. megaterium SP 14, while Co2+ and Zn2+ improved lysine levels in all the strains

Growth inhibition of Beauveria bassiana by bacteria isolated from the cuticular surface of the corn leafhopper, Dalbulus maidis and the planthopper, Delphacodes kuscheli, two important vectors of maize pathogens

The phytosanitary importance of the corn leafhopper, Dalbulus maidis (De Long and Wolcott) (Hemiptera: Cicadellidae) and the planthopper, Delphacodes kuscheli Fennah (Hemiptera: Delphacidae) lies in their ability to transmit phloem-associated plant pathogens, mainly viruses and mollicutes, and to cause considerable mechanical damage to corn plants during feeding and oviposition. Fungi, particularly some members of the Ascomycota, are likely candidates for biocontrol agents against these insect pests, but several studies revealed their failure to invade the insect cuticle possibly because of the presence of inhibitory compounds such as phenols, quinones, and lipids and also by the antibiosis effect of the microbiota living on the cuticular surface of the host. The present work aims to understand interactions between the entomopathogenic fungus Beauveria bassiana (Balsamao-Crivelli) Vuillemin (Hypocreales: Cordycipitaceae) and bacterial antagonists isolated from the cuticular surface of D. maidis and D. kuscheli. A total of 155 bacterial isolates were recovered from the insect's cuticle and tested against B. bassiana. Ninety-one out of 155 strains inhibited the growth of B. bassiana. Bacterial strains isolated from D. maidis were significantly more antagonistic against B. bassiana than those isolates from D. kuscheli. Among the most effective antagonistic strains, six isolates of Bacillus thuringiensis Berliner (Bacillales: Bacillaeae (after B. subtilis)), one isolate of B. mycoides Flügge, eight isolates of B. megaterium de Bary, five isolates of B. pumilus Meyer and Gottheil, one isolate of B. licheniformis (Weigmann) Chester, and four isolates of B. subtilis (Ehrenberg) Cohn were identified.Fil: Toledo, Andrea Vanesa. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Centro de Investigaciones de Fitopatología. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigaciones de Fitopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Alippi, Adriana Mónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Centro de Investigaciones de Fitopatología. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigaciones de Fitopatología; ArgentinaFil: de Remes Lenicov, A.M.M.. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. División Entomología; Argentin

Diversity of plant growth-promoting bacteria associated with sugarcane

The sugarcane (Saccharum spp) presents economic importance, mainly for tropical regions, being an important Brazilian commodity. However, this crop is strongly dependent on fertilizers, mainly nitrogen (N). This study assessed the plant growth-promoting bacteria (PGPB) associated with sugarcane that could be used as a potential inoculant to the crop. We evaluated the genetic diversity of PGPB in the plant tissue of sugarcane varieties (RB 867515, RB 1011, and RB 92579). The primer BOX-A1R was used to differentiate the similar isolated and further sequencing 16S rRNA ribosomal gene. The 16S rRNA gene showed the presence of seven different genera distributed into four groups, the genus Bacillus, followed by Paenibacillus (20%), Burkholderia (14%), Herbaspirillum (6%), Pseudomonas (6%), Methylobacterium (6%), and Brevibacillus (3%). The molecular characterization of endophytic isolates from sugarcane revealed a diversity of bacteria colonizing this plant, with a possible biotechnological potential to be used as inoculant and biofertilizers