Pumilus Isolates Research Articles

Spore UV and Acceleration Resistance of Endolithic Bacillus pumilus and Bacillus subtilis Isolates Obtained from Sonoran Desert Basalt: Implications for Lithopanspermia

Bacterial spores have been used as model systems for studying the theory of interplanetary transport of life by natural processes such as asteroidal or cometary impacts (i.e., lithopanspermia). Because current spallation theory predicts that near-surface rocks are ideal candidates for planetary ejection and surface basalts are widely distributed throughout the rocky planets, we isolated spore-forming bacteria from the interior of near-subsurface basalt rocks collected in the Sonoran desert near Tucson, Arizona. Spores were found to inhabit basalt at very low concentrations (</=28 colony-forming units/g) in these samples. Six isolates identified as being most closely related to Bacillus pumilus and one Bacillus subtilis isolate were recovered from near-subsurface basalt samples. Populations of purified spores prepared from the isolated strains were subjected to 254-nm UV and ballistics tests in order to assess their resistance to UV radiation and to extreme acceleration shock, two proposed lethal factors for spores during interplanetary transfer. Specific natural isolates of B. pumilus were found to be substantially more resistant to UV and extreme acceleration than were reference laboratory strains of B. subtilis, the benchmark organism, suggesting that spores of environmental B. pumilus isolates may be more likely to survive the rigors of interplanetary transfer.

Variability and interactions between endophytic bacteria and fungi isolated from leaf tissues of citrus rootstocks.

Fungi and bacteria were isolated from surface disinfected leaf tissues of several citrus rootstocks. The principal bacterial species isolated were Alcaligenes sp., Bacillus spp. (including B. cereus, B. lentus, B. megaterium, B. pumilus, and B. subtilis), Burkholderia cepacia, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium extorquens, and Pantoea agglomerans, with P. agglomerans and B. pumilus being the most frequently isolated species. The most abundant fungal species were Colletotrichum gloeosporioides, Guignardia citricarpa, and Cladosporium sp. Genetic variability between 36 endophytic bacterial isolates was analysed by the random amplified polymorphic DNA (RAPD) technique, which indicated that B. pumilus isolates were more diverse than P. agglomerans isolates, although genetic diversity was not related to the host plants. In vitro interaction studies between G. citricarpa isolates and the most frequently isolated endophytic bacteria showed that metabolites secreted by G. citricarpa have an inhibitory growth effect on some Bacillus species, and a stimulatory growth effect on P. agglomerans.

Toxic Bacillus pumilus from indoor air, Recycled Paper Pulp,Norway Spruce, Food Poisoning Outbreaks and Clinical Samples

Forty-four B. pumilus isolates of food poisoning, clinical, environmental and industrial origins were investigated for toxin production using the boar spermatozoan motility assay, previously shown to be a sensitive method for detecting non-protein toxins from B. cereus and B. licheniformis. The three toxic isolates originated from live tree, indoor air and recycled paper pulp and were more toxic than the previously described food poisoning isolates of B. licheniformis, whereas the B. pumilus food poisoning and clinical isolates were lower in toxicity. The type strain also produced inhibitory substances. The toxic substances were insensitive to heat (100 degrees C, 20 min), to pH 2 or pH 10 and to digestion with pronase. The substances were readily soluble in methanol and chloroform, but less soluble in toluene. Exposure of boar spermatozoa to 1-10 microg ml(-1) (EC50) of methanol soluble substance from the four strains disrupted the plasma membrane permeability barrier, induced abnormalities in the postacrosomal sheath, collapsed the mitochondrial and suppressed cytoplasmic NAD reduction. No change was observed in human peripheral blood lymphocytes exposed to concentrations of B. pumilus extract that affected spermatozoa. The toxin producing isolates were 99.4 to 99.6% similar in 16SrDNA (500 bp) to the type strain and could not be distinguished from the 41 non-toxic isolates by biochemical properties or whole cell fatty acid composition.

Expression of a Bacillus thuringiensis delta-endotoxin gene by Bacillus pumilus.

The delta-endotoxin genes from Bacillus thuringiensis were introduced into a rhizosphere-inhabiting Bacillus pumilus isolate to create a delta-endotoxin expression and delivery system for subterranean feeding insects such as the larvae of pale western cutworm (Agrotis orthogonia Morrison (Lepidoptera: Noctuidae)). Preliminary experiments indicated that Bacillus thuringiensis subsp. kurstaki cultures were toxic to pale western cutworm larvae. Three different cry genes from Bacillus thuringiensis subsp. kurstaki were cloned into high and low copy number vectors and mated into Bacillus pumilus RB8. When carried on high copy number vectors, cry genes appeared to inhibit sporulation and delta-endotoxin production in Bacillus pumilus RB8 cultures, since microscopic examination of these cultures revealed that < 0.1% of the cells of late stationary phase cultures had sporulated and produced parasporal inclusions. On low copy number vectors, the cry genes did not inhibit sporulation; however, production of delta-endotoxins was undetectable. Using a heat shock regime for enrichment of sporogenous crystalliferous variants, a Bacillus pumilus isolate, carrying cryIA(c) on a high copy number plasmid, was obtained in which high level delta-endotoxin production occurred concomitant with sporulation. Synthesis of functional delta-endotoxin by this strain was confirmed by Western blot analysis and bioassay with pale western cutworm larvae. These results show that rhizosphere-inhabiting bacilli are indeed a potential route for introduction of delta-endotoxins to the root environment for biocontrol purposes.

Multi-target and medium-independent fungal antagonism by hydrolytic enzymes in Paenibacillus polymyxa and Bacillus pumilus strains from barley rhizosphere

Glucanolytic bacteria from barley rhizosphere soil were isolated by a procedure selecting for isolates with β-glucosidase activity. Almost all isolates were fast-growing, Gram-positive rods. Sixteen out of 100 isolates showed in vitro fungal antagonism against widely different plant-pathogenic microfungi ( Aphanomyces cochleoides, Pythium ultimum and Rhizoctonia solani). The 16 isolates shared a characteristic profile of cell-wall-degrading enzymes, comprising glucanolytic (cellulase, mannanase and xylanase) and proteolytic enzymes. This enzyme profile was not observed in any of the non-antagonistic isolates and may thus be useful in selection protocols for fungal antagonists producing fungal cell-wall-degrading enzymes. The antagonistic isolates were identified as Paenibacillus ( Bacillus) polymyxa (2 strains) and Bacillus pumilus (13 strains), Bacillus sp. (1 strain). Both P. polymyxa isolates and a subgroup of 4 B. pumilus isolates showed a medium-independent antagonism as tested against A. cochleoides in four different media. This was apparently related to constitutive production of cell-wall-degrading enzymes ( P. polymyxa) or induction of a repertoire of enzymes substituting for each other in different media ( B. pumilus). Despite different strategies of enzyme production in the media, both the P. polymyxa and B. pumilus strains demonstrate a multi-target and medium-independent type of fungal antagonism, which is promising for application in biological control.

The infuence of native rhizobacteria on European alder (Alnus glutinosa (L.) Gaertn.) growth

Metabolite production was investigated in four bacterial strains that promoted (plant growth promoting rhizobacteria -PGPR-, B. licheniformis, isolate B.12 and B. pumilus isolate B.3) or inhibited (deleterious rhizobacteria -DRB-, P. fluorescens bv II, isolates P.9 and P.20) growth of nodulated and non-nodulated Alnus glutinosa seedlings. These strains were isolated and characterized from the rhizosphere of a natural alder population, and their biological effects on plant growth determined on previous studies. Biological assays were performed to confirm the observed effects on aerial length (AL), aerial surface (AS), number of leaves (NL) and total nitrogen (TN). According to the high resolution gas chromatography (HRGC) results, PGPR strains produced auxin-like (IAA-1) compounds at levels of 1.736 and 1.790 mg IAA-1 L -1 culture growth medium ; however, they did not produce HCN. These compounds are derived from IAA and not from the Trp originated by peptide degradation in culture media. The promoting effect is evidenced when comparing the effects of IAA and the filtered bacterial growth culture medium to control (increases of 64% in aerial surface, 277% in total N content and 32% in aerial length). The deleterious strains produced HCN (1.6 and 2.4 mg kg -1 detected in growth culture medium) and they did not produce IAA-1 compounds. The bacterial culture's -free of bacteria-inhibiting effects were 7% in aerial surface, 240% in total nitrogen content and 15% in aerial length. The results reported here suggest that the interactions that take place in the alder rhizosphere are in a delicate equilibrium. In view of this, the coexistence of PGPR and DRB strains in this environment is unquestionable, and does affect alder health in field conditions.

The influence of native rhizobacteria on european alder (Alnus glutinosa (L.) Gaertn.) growth

Metabolite production was investigated in four bacterial strains that promoted (plant growth promoting rhizobacteria -PGPR-, B. licheniformis, isolate B.12 and B. pumilus isolate B.3) or inhibited (deleterious rhizobacteria-DRB-, P. fluorescens bv II, isolates P.9 and P.20) growth of nodulated and non-nodulated Alnus glutinosa seedlings. These strains were isolated and characterized from the rhizosphere of a natural alder population, and their biological effects on plant growth determined on previous studies. Biological assays were performed to confirm the observed effects on aerial length (AL), aerial surface (AS), number of leaves (NL) and total nitrogen (TN). According to the high resolution gas chromatography (HRGC) results, PGPR strains produced auxin-like (IAA-1) compounds at levels of 1.736 and 1.790 mg IAA-1 L-1 culture growth medium; however, they did not produce HCN. These compounds are derived from IAA and not from the Trp originated by peptide degradation in culture media. The promoting effect is evidenced when comparing the effects of IAA and the filtered bacterial growth culture medium to control (increases of 64% in aerial surface, 277% in total N content and 32% in aerial length). The deleterious strains produced HCN (1.6 and 2.4 mg kg-1 detected in growth culture medium) and they did not produce IAA-1 compounds. The bacterial culture's-free of bacteria-inhibiting effects were 7% in aerial surface, 240% in total nitrogen content and 15% in aerial length. The results reported here suggest that the interactions that take place in the alder rhizosphere are in a delicate equilibrium. In view of this, the coexistence of PGPR and DRB strains in this environment is unquestionable, and does affect alder health in field conditions.

Seasonal occurrence of psychrotrophic Bacillus species in raw milk, and studies on the interactions with mesophilic Bacillus sp.

Mesophilic and psychrotrophic isolates of Bacillus species displayed seasonal incidences in raw and pasteurised milk. The incidence of mesophilic isolates was highest in the winter and lowest in the summer/autumn while psychrotroph incidence was conversely lowest in the winter and highest in the late summer/autumn. Spores of Bacillus sp. were isolated from raw milk taken from farm milk machines and bulk tanks, milk tankers, dairy silos and pasteurised milk. A consistent seasonal fluctuation in incidence throughout these samples suggested that spores of Bacillus sp. derived from the farm environment survived as important contaminants right through the milk chain to the pasteurised product. Up to seven mesophilic Bacillus sp. were isolated from a single sample with three species commonly occurring in most samples. The predominant mesophilic species isolated were B. pumilus, B. licheniformis and B. subtilis. The dominant psychrotrophic isolate was B. cereus. Selected mesophilic isolates were examined for possible antagonistic effects on the growth of psychrotrophic B. cereus and B. pumilus isolates. Bacillus subtilis and B. licheniformis were found to produce antagonistic factors. It was considered that these factors may influence the incidence and growth of psychrotrophic isolates in the farm environment or in milk but the factors are not yet fully characterised or identified.

Lecithinase and Toxin Production in Bacillus Species

A total of 96 Bacillus species consisting of B. licheniformis, B. pumilus, B. subtilis, B. megaterium and B. polymyxa isolated from smoked meat were screened for both lecithinase and toxin production. Lecithinase production was found to be wide-spread among these species and to be strain specific. A high percentage of the tested species (66.7-100%) was strongly lecithinase positive. Like lecithinase, toxin production is wide-spread among the Bacillus species and is equally strain specific. With the exception of the B. pumilus isolates, an absolute correlation between lecithinase and toxin production could not be established among the tested isolates. In B. pumilus both lecithinase and toxin are detectable in the culture filtrate after three hours incubation but while maximal lecithinase synthesis occurs within three hours, maximal toxin production is not attained within 15 h incubation. B. pumilus toxin proved toxic for mice but not for rats.