Antibacterial Screening of Secreted Compounds Produced by the Phase I Variant of Photorhabdus luminescens

In this study, antibacterial activity of metabolites secreted by the phase I variant of Photorhabdus luminescens was investigated. Bioactivity of these metabolites was screened against 28 different bacterial species and strains. Bacterial sensitivity was determined by a modified-version of the Kirby–Bauer disk diffusion susceptibility method, whereas the phase I variant’s culture permeate was utilized as the “antibacterial” agent. This investigation demonstrates that 11 of the 28 bacterial species tested were sensitive to at least one of the secreted compounds or a combination thereof.     

Biocontrol and Plant Growth Promotion Characterization of Bacillus Species Isolated from Calendula officinalis Rhizosphere

The phenotypic and genotypic diversity of the plant growth promoting Bacillus genus have been widely investigated in the rhizosphere of various agricultural crops. However, to our knowledge this is the first report on the Bacillus species isolated from the rhizosphere of Calendula officinalis. 15 % of the isolated bacteria were screened for their important antifungal activity against Fusarium oxysporum, Botrytis cinerea, Aspergillus niger, Cladosporium cucumerinium and Alternaria alternata. The bacteria identification based on 16S r-RNA and gyrase-A genes analysis, revealed strains closely related to Bacillus amyloliquefaciens, B. velezensis, B. subtilis sub sp spizezenii and Paenibacillus polymyxa species. The electro-spray mass spectrometry coupled to liquid chromatography (ESI-LC MS) analysis showed that most of the Bacillus isolates produced the three lipopeptides families. However, the P. polymyxa (18SRTS) didn’t produce any type of lipopeptides. All the tested Bacillus isolates produced cellulase but the protease activity was observed only in the B. amyloliquefaciens species (9SRTS). The Salkowsky colorimetric test showed that the screened bacteria synthesized 6–52 μg/ml of indole 3 acetic acid. These bacteria produced siderophores with more than 10 mm wide orange zones on chromazurol S. The greenhouse experiment using a naturally infested soil with Sclerotonia sclerotiorum showed that the B. amyloliquefaciens (9SRTS) had no significant (P > 0.05) effect on the pre-germination of the chickpea seeds. However, it increased the size of the chickpea plants and reduced the stem rot disease (P < 0.05).These results suggested that the Bacillus strains isolated in this work may be further used as bioinoculants to improve the production of C. officinalis and other crop systems.     

Antagonism of Bacillus spp. Isolated from Marine Biofilms Against Terrestrial Phytopathogenic Fungi

We aimed at determining the antagonistic behavior of bacteria derived from marine biofilms against terrestrial phytopathogenic fungi. Some bacteria closely related to Bacillus mojavensis (three isolates) and Bacillus firmus (one isolate) displayed antagonistic activity against Colletotrichum gloeosporioides ATCC 42374, selected as first screen organism. The four isolates were further quantitatively tested against C. gloeosporioides, Colletotrichum fragariae, and Fusarium oxysporum on two culture media, potato dextrose agar (PDA) and a marine medium-based agar [yeast extract agar (YEA)] at different times of growth of the antagonists (early, co-inoculation with the pathogen and late). Overall antagonistic assays showed differential susceptibility among the pathogens as a function of the type of culture media and time of colonization (P < 0.05). In general, higher suppressive activities were recorded for assays performed on YEA than on PDA; and also when the antagonists were allowed to grow 24 h earlier than the pathogen. F. oxysporum was the most resistant fungus while the most sensitive was C. gloeosporioides ATCC 42374. Significant differences in antagonistic activity (P < 0.05) were found between the different isolates. In general, Bacillus sp. MC3B-22 displayed a greater antagonistic effect than the commercial biocontrol strain Bacillus subtilis G03 (Kodiak). Further incubation studies and scanning electronic microscopy revealed that Bacillus sp. MC3B-22 was able to colonize, multiply, and inhibit C. gloeosporioides ATCC 42374 when tested in a mango leaf assay, showing its potential for fungal biocontrol. Additional studies are required to definitively identify the active isolates and to determine their mode of antifungal action, safety, and biocompatibility.     

The Determination of Assay for Laccase of Bacillus subtilis WPI with Two Classes of Chemical Compounds as Substrates

Ligninolytic enzyme complexes are involved in lignin degradation. Among them laccases are outstanding because they use molecular oxygen as a co-substrate instead of hydrogen peroxide as used by peroxidases. Bacterial laccase of Bacillus genus was first reported in Claus and Filip (Microbiol Res 152:209–216, 1997), since then more bacterial laccases have been found. In this research, laccase-producing bacteria were screened from pulp and paper industry wastewater, bagass and sugarcane rhizosphere. Nutrient agar medium containing 0.5 mM of guaiacol was used. It was observed that the laccase-producing strains developed brown colour from which 16 strains of Bacillus were identified. One of the isolated strains was identified as Bacillus subtilis WPI based on the results of biochemical tests and 16S rDNA sequence analysis. This strain showed laccase-like activity towards the oxidizing substrates ABTS and guaiacol. In this study guaiacol was used as the substrate of laccase activity assay. For determination of laccase activity of this isolate guaiacol was used as a substrate of assay for the first time in this study. SDS-PAGE and Native-PAGE confirmed the presence of laccase.     

Comparison of the bacterial Enhancin-like proteins from Yersinia and Bacillus spp. with a baculovirus Enhancin

Enhancins are a class of metalloproteases found in some baculoviruses that enhance viral infection by degrading the peritrophic membrane (PM) of the insect midgut. However, sequencing has revealed enhancin-like genes with 24-25% homology to viral enhancins, in the genomes of Yersinia pestis and Bacillus anthracis. AcMNPV does not encode enhancin therefore recombinant AcMNPV budded viruses (BVs) and polyhedra inclusion bodies (PIBs) were generated expressing the bacterial Enhancins. Bacterial Enhancins were found to be cytotoxic when compared to viral enhancin, however, larval bioassays suggested that the bacterial Enhancins did not enhance infection in the same way as viral Enhancin. This suggests that the bacterial Enhancins may have evolved a distinct biochemical function.     

A Novel Technique for Infesting Field Sites with Encapsulated Eggs of Meloidogyne spp.

Eggs of Meloidogyne arenaria race 1 were encapsulated in calcium alginate for use as inoculum to infest peanut field plots. Some eggs within the capsules remained viable up to 10 weeks after preparation. A field site was successfully infested at peanut planting and (or) 6 weeks later. Dual applications of nematode inoculum (at planting and 6 weeks later) were superior to single applications (at planting or 6 weeks after planting). Field-site infestation levels at the end of the first year were related to the amount of inoculum dispersed and timing of the infestation (P = 0.001). Peanut yield was only slightly affected in the first year, but significant (P = 0.02) yield suppression occurred during the second year after field infestations. The negative relationship between the numbers of M. arenaria eggs and juveniles per 500 cm³ soil in the fall and the percentage of peanut hull galled the second year was described by a quadratic model (P = 0.002, R² = 0.41).     

Two acidothermotolerant lipases from new variants of Bacillus spp.

Two acidothermotolerant lipases from new isolates of Bacillus stearothermophilus SB-1 and Bacillus licheniformis SB-3 are reported. In addition, a thermotolerant, neutral lipase from Bacillus atrophaeus SB-2 that hydrolyses castor oil is also reported. The lipase from B. stearothermophilus SB-1 retained 70% activity and that from B. licheniformis SB-3 retained 50% activity at pH 3.0 at 50 °C. In addition, at 100 °C B. stearothermophilus SB-1 lipase had a half life of 25 min at pH 3.0 and 15 min at pH 6.0. Lipase activity was markedly stimulated by glycerol in case of B. stearothermophilus SB-1 and by diethylether in cases of B. atrophaeus SB-2 and B. licheniformis SB-3. The lipases varied in their substrate specificity towards triacylglycerols. The rate of hydrolysis of neem oil with B. stearothermophilus SB-1 and B. atrophaeus SB-2 lipases was, respectively, nearly 4-fold and 2-fold more than with olive oil.     

Genomic comparison of 93 Bacillus phages reveals 12 clusters, 14 singletons and remarkable diversity

Background

The Bacillus genus of Firmicutes bacteria is ubiquitous in nature and includes one of the best characterized model organisms, B. subtilis, as well as medically significant human pathogens, the most notorious being B. anthracis and B. cereus. As the most abundant living entities on the planet, bacteriophages are known to heavily influence the ecology and evolution of their hosts, including providing virulence factors. Thus, the identification and analysis of Bacillus phages is critical to understanding the evolution of Bacillus species, including pathogenic strains.

Results

Whole genome nucleotide and proteome comparison of the 93 extant Bacillus phages revealed 12 distinct clusters, 28 subclusters and 14 singleton phages. Host analysis of these clusters supports host boundaries at the subcluster level and suggests phages as vectors for genetic transfer within the Bacillus cereus group, with B. anthracis as a distant member of the group. Analysis of the proteins conserved among these phages reveals enormous diversity and the uncharacterized nature of these phages, with a total of 4,922 protein families (phams) of which only 951 (19%) had a predicted function. In addition, 3,058 (62%) of phams were orphams (phams containing a gene product from a single phage). The most populated phams were those encoding proteins involved in DNA metabolism, virion structure and assembly, cell lysis, or host function. These included several genes that may contribute to the pathogenicity of Bacillus strains.

Conclusions

This analysis provides a basis for understanding and characterizing Bacillus phages and other related phages as well as their contributions to the evolution and pathogenicity of Bacillus cereus group bacteria. The presence of sparsely populated clusters, the high ratio of singletons to clusters, and the large number of uncharacterized, conserved proteins confirms the need for more Bacillus phage isolation in order to understand the full extent of their diversity as well as their impact on host evolution.     

Biodegradation of microcystin-RR by Bacillus flexus isolated from a Saudi freshwater lake

A bacterium capable of degrading microcystin-RR (MC-RR) was isolated from a Saudi eutrophic lake which was previously reported to have microcystin-producing cyanobacteria. Based on the analysis of the 16S rRNA gene sequence, the isolated strain SSZ01, most likely belong to the genus Bacillus with a highest sequence similarity (99%) with Bacillus flexus strain EMGA5. It was found that B. flexus strain SSZ01, possesses an mlrA gene encoding the most important enzyme for MC degradation. This strain was capable of degrading MC-RR, at a concentration of 10 mg l⁻¹, in batch experiments under environmentally relevant conditions. The degradation of MC-RR was completely removed within 4 d. The degradation of MC-RR by this strain occurred in a rich medium nutrient broth (NB), indicating that this could likely occur along with other organic compounds found in the environment. Therefore, the coexistence of such bacteria with MCs in the same environment can contribute to the self-purification of the ecosystem from such potent toxins. This is the first study to report that B. flexus can degrade MCs.     

Histological Comparisons of Parasitism by Schistonchus spp. (Nemata: Aphelenchoididae) in Neotropical Ficus spp.

Syconia (enclosed infructescences) infested with host-specific species of Schistonchus (Aphelenchoididae) were collected from six species of Ficus (Moraceae) native to Florida or Panama. They were sectioned and histologically examined to assess the effects of parasitism. Parasitism by Schistonchus spp. was associated with hypertrophied cells, tissue necrosis, and the presence of an exudate in all species. Occasional hypertrophy of the outer epidermal cells occurred on seed florets, wasp florets, and on the endothecial cells of male florets in F. aurea (subgenus Urostigma) from Florida. Aberrations of the inner mesocarp occurred under the hypertrophied cells on seed florets. In F. laevigata (subgenus Urostigma) from Florida, Schistonchus sp. infested immature male florets and was associated with hypertrophy of endothecial cells, epidermal cells of the anther filaments, and anthers. Schistonchus sp. also caused aberrations of the anther filament, anthers, and pollen. Ficus poponoei (subgenus Urostigma) and F. glabrata (subgenus Pharmacosycea), both from Panama, had hypertrophied outer epidermal cells on seed florets. Ficus poponoei also had Schistonchus sp. within the pedicel of an aborted floret, with hypertrophy of the cortical parenchyma. Ficus trigonata (subgenus Urostigma) from Panama had hypertrophy of the outer epidermis of seed florets. When the outer epidermis on these florets was missing, the inner mesocarp was hypertrophied. Ficus maxima (subgenus Pharmacosycea) from Panama had hypertrophy on the outer epidermis of seed and aborted florets. Schistonchus spp. were not found in wasp larvae or pupae in any of the Ficus spp. examined. Hypertrophy was never observed in the absence of Schistonchus spp.     

Erratum to: genomic comparison of 93 Bacillus phages reveals 12 clusters, 14 singletons and remarkable diversity

Background

The Bacillus genus of Firmicutes bacteria is ubiquitous in nature and includes one of the best characterized model organisms, B. subtilis, as well as medically significant human pathogens, the most notorious being B. anthracis and B. cereus. As the most abundant living entities on the planet, bacteriophages are known to heavily influence the ecology and evolution of their hosts, including providing virulence factors. Thus, the identification and analysis of Bacillus phages is critical to understanding the evolution of Bacillus species, including pathogenic strains.

Results

Whole genome nucleotide and proteome comparison of the 83 extant, fully sequenced Bacillus phages revealed 10 distinct clusters, 24 subclusters and 15 singleton phages. Host analysis of these clusters supports host boundaries at the subcluster level and suggests phages as vectors for genetic transfer within the Bacillus cereus group, with B. anthracis as a distant member. Analysis of the proteins conserved among these phages reveals enormous diversity and the uncharacterized nature of these phages, with a total of 4,442 protein families (phams) of which only 894 (20%) had a predicted function. In addition, 2,583 (58%) of phams were orphams (phams containing a single member). The most populated phams were those encoding proteins involved in DNA metabolism, virion structure and assembly, cell lysis, or host function. These included several genes that may contribute to the pathogenicity of Bacillus strains.

Conclusions

This analysis provides a basis for understanding and characterizing Bacillus and other related phages as well as their contributions to the evolution and pathogenicity of Bacillus cereus group bacteria. The presence of sparsely populated clusters, the high ratio of singletons to clusters, and the large number of uncharacterized, conserved proteins confirms the need for more Bacillus phage isolation in order to understand the full extent of their diversity as well as their impact on host evolution.     

Characterization of Insecticidal Genes of Bacillus thuringiensis Strains Isolated from Arid Environments

This study aimed at characterizing the insecticidal genes of eight Bacillus thuringiensis isolates that were recovered from the local environment of western Saudi Arabia. The screening for the presence of lepidopteran-specific cry1A family and vip3A genes, dipteran-specific cry4 family and coleopteran-specific cry3A, vip1A and vip2A genes, was carried out by PCR. All eight isolates produced PCR products that confirmed the presence of cry1Aa, cry1Ab, cry1Ac, cry4A, cry4B genes, but not cry3A, vip1A and vip2A genes. However, three isolates only were found to carry vip3A genes as revealed by PCR. The observation of cry1 and cry4 genes suggests that these eight isolates may have dual activity against Lepidoptera and Diptera species, while three isolates possessed vip3 genes in addition to cry1 and cry4 which suggests that these three isolates have toxic crystals and vegetative proteins. The results of this study are interesting in the sense that they may help developing new strategies for controlling insects of economic and medical importance in Saudi Arabia, using B. thuringiensis strains that naturally exist in the local environment instead of the current control strategies that are based solely on chemical insecticides.     

In Vitro Antibacterial Activity of 4-Phenyl-1-(2-phenyl-allyl)pyridinium bromide: A Novel Class of Pyridinium Based Antibacterial Compounds

The continuing increase in the incidence of multi drug resistant pathogenic bacteria and shortage of new antimicrobial agents are the prime driver in efforts to identify the novel antimicrobial classes. In vitro antibacterial activity of 4-phenyl-1-(2-phenylallyl) pyridinium bromide was tested against Gram positive Staphylococcus aureus, Streptococcus species, Bacillus subtilis, and Gram negative Klebsiella aerogenes and Escherichia coli using disk diffusion method. Among them S. aureus showed strong antibacterial activity (21.99 ± 0.03 mm) while E. coli showed very little activity (8.97 ± 0.06 mm) towards the compound. The MIC of 4-phenyl-1-(2-phenyl-allyl)-pyridinium bromide for 90% S. aureus was ≤20 μg/ml and was compared with phenoxymethylpenicillin, cloxacillin, erythromycin and vancomycin. When 4-phenyl-1-(2-phenyl-allyl)pyridinium bromide showed MIC at ≤20 μg/ml, all others showed MIC at ≤100 μg/ml. Strong antibacterial activity of 4-phenyl-1-(2-phenyl-allyl)pyridinium bromide against S. aureus indicates that there is a possibility to use it as an effective antibacterial agent.     

Ecobiotechnological Approach for Exploiting the Abilities of Bacillus to Produce Co-polymer of Polyhydroxyalkanoate

Ecobiotechnological approach is an attractive and economical strategy to enrich beneficial microbes on waste biomass for production of Polyhydroxyalkanoate (PHA). Here, six strains of Bacillus spp. were used to produce co-polymers of PHA from pea-shells. Of the 57 mixed bacterial cultures (BCs) screened, two of the BCs, designated as 5BC1 and 5BC2, each containing 5 strains could produce PHA co-polymer at the rate of 505–560 mg/l from feed consisting of pea-shell slurry (PSS, 2 % total solids) and 1 % glucose (w/v). Co-polymer production was enhanced from 65–560 mg/l on untreated PSS to 1,610–1,645 mg/l from PSS treated with defined hydrolytic bacteria and 1 % glucose. Supplementation of the PSS hydrolysate with sodium propionate enabled 5BC1 to produce co-polymer P(3HB-co-3HV) with a 3HV content up to 13 % and a concomitant 1.46-fold enhancement in PHA yield. Using the principles of ecobiotechnology, this is the first demonstration of PHA co-polymer production by defined co-cultures of Bacillus from biowaste as feed under non-axenic conditions.     

Biochemical characterization of ribosome assembly GTPase RbgA in Bacillus subtilis

The ribosome biogenesis GTPase A protein RbgA is involved in the assembly of the large ribosomal subunit in Bacillus subtilis, and homologs of RbgA are implicated in the biogenesis of mitochondrial, chloroplast, and cytoplasmic ribosomes in archaea and eukaryotes. The precise function of how RbgA contributes to ribosome assembly is not understood. Defects in RbgA give rise to a large ribosomal subunit that is immature and migrates at 45 S in sucrose density gradients. Here, we report a detailed biochemical analysis of RbgA and its interaction with the ribosome. We found that RbgA, like most other GTPases, exhibits a very slow k(cat) (14 h(-1)) and has a high K(m) (90 μM). Homology modeling of the RbgA switch I region using the K-loop GTPase MnmE as a template suggested that RbgA requires K(+) ions for GTPase activity, which was confirmed experimentally. Interaction with 50 S subunits, but not 45 S intermediates, increased GTPase activity by ～55-fold. Stable association with 50 S subunits and 45 S intermediates was nucleotide-dependent, and GDP did not support strong interaction with either of the subunits. GTP and guanosine 5'-(β,γ-imido)triphosphate (GMPPNP) were sufficient to promote association with the 45 S intermediate, whereas only GMPPNP was able to support binding to the 50 S subunit, presumably due to the stimulation of GTP hydrolysis. These results support a model in which RbgA promotes a late step in ribosome biogenesis and that one role of GTP hydrolysis is to stimulate dissociation of RbgA from the ribosome.     

Rhizosphere Colonization and Control of Meloidogyne spp. by Nematode-trapping Fungi

The ability of nematode-trapping fungi to colonize the rhizosphere of crop plants has been suggested to be an important factor in biological control of root-infecting nematodes. In this study, rhizosphere colonization was evaluated for 38 isolates of nematode-trapping fungi representing 11 species. In an initial screen, Arthrobotrys dactyloides, A. superba, and Monacrosporium ellipsosporum were most frequently detected in the tomato rhizosphere. In subsequent pot experiments these fungi and the non-root colonizing M. geophyropagum were introduced to soil in a sodium alginate matrix, and further tested both for establishment in the tomato rhizosphere and suppression of root-knot nematodes. The knob-forming M. ellipsosporum showed a high capacity to colonize the rhizosphere both in the initial screen and the pot experiments, with more than twice as many fungal propagules in the rhizosphere as in the root-free soil. However, neither this fungus nor the other nematode-trapping fungi tested reduced nematode damage to tomato plants.     

Ethylene Production by Meloidogyne spp.-Infected Plants

Gall size and rates of ethylene production by various hosts infected with Meloidogyne javanica and by excised tomato root cultures infected with M. javanica or M. hapla were measured. Infection with M. javanica increased the rate of ethylene production in dicotyledonous plants (cabbage, pea, carrot, cucumber, carnation, and tomato), but not in infected monocotyledonous plants (corn, wheat, and onion). Nematode infection induced large galls on roots of dicotyledonous, but not monocotyledonous, plants. Excised tomato roots in culture infected with M. javanica produced ethylene at high rates and formed large galls, whereas roots infected with M. hapla produced ethylene at low rates and induced smaller galls.     

Antibacterial activity of a bacteriocin-like substance produced by Bacillus sp. P34 that targets the bacterial cell envelope

The objective of this study was to investigate the mode of action of BLS P34, a bacteriocin-like substance (BLS) produced by a novel Bacillus sp. strain P34 isolated from the Amazon basin. The effect of the BLS was tested against Listeria monocytogenes, showing a bactericidal effect at 200 AU (activity units) ml⁻¹, while no inhibition of spore outgrowth of Bacillus cereus was observed with a dose of 1,600 AU ml⁻¹. Growth of Escherichia coli and Salmonella Enteritidis was inhibited, but only when the chelating agent EDTA was co-added with the BLS. The effect of BLS P34 on L. monocytogenes was also investigated by Fourier transform infrared spectroscopy. Treated cells showed an important frequency increase in 1,452 and 1,397 cm⁻¹ and decrease in 1,217 and 1,058 cm⁻¹, corresponding assignments of fatty acids and phospholipids. Transmission electron microscopy showed damaged cell envelope and loss of protoplasmic material. BLS P34 was bactericidal to Gram-positive, and also showed inhibitory effect against Gram-negative bacteria. There is evidence that its mode of action corresponds to that of a membrane-active substance. The knowledge about the mode of action of this BLS is essential to determine its effective application as an antimicrobial agent.     

Hydrogen and Polyhydroxybutyrate Producing Abilities of <Emphasis Type=Italic>Bacillus</Emphasis> spp. From Glucose in Two Stage System

Metabolic activities of four Bacillus strains to transform glucose into hydrogen (H₂) and polyhydroxybutyrate (PHB) in two stages were investigated in this study. Under batch culture conditions, Bacillus thuringiensis EGU45 and Bacillus cereus EGU44 evolved 1.67–1.92 mol H₂/mol glucose, respectively during the initial 3 days of incubation at 37°C. In the next 2 days, the residual glucose solutions along with B. thuringiensis EGU45 shaken at 200 rpm was found to produce PHB yield of 11.3% of dry cell mass. This is the first report among the non-photosynthetic microbes, where the Bacillus spp.—B. thuringiensis and B. cereus strains have been shown to produce H₂ and PHB in same medium under different conditions.