16S rDNA sequence analysis of culturable marine biofilm forming bacteria from a ship's hull

Marine bacteria from the hull of a ship in the form of biofilms or microfouling were isolated, cultured, and identified by phylogenetic analysis using 16S rDNA sequences. With an average length of 946 bp, all the 16 sequences were classified using the Ribosomal database project (RDP) and were submitted to the National Center for Biotechnology Information. Phylogenetic analysis using 16S rDNA sequences indicated that the 16 strains belonged to the Firmicutes (IK-MB6 Exiguobacterium aurantiacum, IK-MB7 Exiguobacterium arabatum, IK-MB8 Exiguobacterium arabatum, IK-MB9 Jeotgalibacillus alimentarius, IK-MB10 Bacillus megaterium, IK-MB11 Bacillus pumilus, IK-MB12 Bacillus pumilus, IK-MB13 Bacillus pumilus, IK-MB14 Bacillus megaterium), High GC, Gram-positive bacteria (IK-MB2 Micrococcus luteus, IK-MB5 Micrococcus luteus, IK-MB16 Arthrobacter mysorens), G-Proteobacteria (IK-MB3 Halomonas aquamarina, IK-MB15 Halotalea alkalilenta), CFB group bacteria (IK-MB1 Myroides odoratimimus), and Enterobacteria (IK-MB4 Proteus mirabilis). Among the 16 strains, representatives of the Firmicutes were dominant (56.25%) compared to the high GC, Gram-positive bacteria (18.75%), G-Proteobacteria (12.5%), CFB group bacteria (6.25%), and Enterobacteria (6.25%). Analysis revealed that majority of marine species found in marine biofilm are of anthropogenic origin.     

Potential metabolites of dimethoate produced by bacterial degradation

Microbial degradation of pesticide has been recognized as a potential solution for the disposal of pesticide. Two bacterial strains namely Bacillus licheniformis and Pseudomonas aeruginosa, which were isolated from water and fish intestine, respectively, were allowed to grow in mineral salt solution. The acetonitrile extracts of the bacterial cultures were subjected to thin layer chromatography using two different solvent systems: hexane–chloroform–methanol and cyclohexane–acetone–chloroform. The chromatogram revealed the presence of four metabolites of dimethoate with different R_f values, in the original P. aeruginosa strain using both hexane–chloroform–methanol and cyclohexane–acetone–chloroform solvent systems. Total disappearance of dimethoate spot occurred in the culture of B. licheniformis strain at day 3. Thus, the present study establishes the bacterial degradation of dimethoate and also suggests the role of bacteria in the bioremediation of pesticides.     

Cold-adapted Bacilli isolated from the Qinghai–Tibetan Plateau are able to promote plant growth in extreme environments

Nearly 1400 Bacillus strains growing in the plant rhizosphere were sampled from different sites on the Qinghai–Tibetan Plateau. Forty-five of the isolates, selected due to their biocontrol activity, were genome-sequenced and their taxonomic identification revealed that they were representatives of the Bacillus subtilis species complex (20) and the Bacillus cereus group (9). Majority of the remaining strains were found closely related to Bacillus pumilus, but their average nucleotide identity based on BLAST and electronic DNA/DNA hybridization values excluded closer taxonomic identification. A total of 45 different gene clusters involved in synthesis of secondary metabolites were detected by mining the genomes of the 45 selected strains. Except eight mesophilic strains, the 37 remaining strains were found either cold-adapted or psychrophilic, able to propagate at 10°C and below (Bacillus wiedmannii NMSL88 and Bacillus sp. RJGP41). Pot experiments performed at 10°C with winter wheat seedlings revealed that cold-adapted representatives of B. pumilus, B. safensis and B. atrophaeus promoted growth of the seedlings under cold conditions, suggesting that these bacilli isolated from a cold environment are promising candidates for developing of bioformulations useful for application in sustainable agriculture under environmental conditions unfavourable for the mesophilic bacteria presently in use.     

Riboflavin Production by Roseoflavin-resistant Strains of Some Bacteria

Riboflavin(RF)-productivity of reseoflavin(RoF)-resistant strains of Staphylococcus aureus, Bacillus pumilus and Bacillus subtilis was studied. Two of three types of resistant strains of S. aureus were shown to produce more RF than the parent strains. Resistant strains of B. pumilus were also RF-producing, and one type (fp) of the resistant strains from B. subtilis HW produced a large amount of RF in the culture medium. The hereditary stability of RF-producing properties of the fp type was also shown.     

Characterization of Bacillus Probiotics Available for Human Use

Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.     

Screening and characterization of antifungal clusterbean (Cyamopsis tetragonoloba) rhizobacteria

About 377 guar (Cyamopsis tetragonoloba) rhizobacteria were isolated from cultivated soils of north-west India (Thar Desert) and their antifungal activity against Macrophomina phaseolina (strains of groundnut, mungbean and guar) and Fusarium oxysporum (strains of chickpea and cumin) was examined. Isolates were characterised for generic types and physiological/functional diversity. About 19% isolates representing 24% locations were inhibitory to fungal growth. Isolates 009071, 009073, 009078 and 102354 recorded maximum inhibition of pathogenic fungi on plates. Isolate 034206 gave highest %RI, 009073 showed maximum protease activity and 102354 gave highest salt tolerance. Net house and field screening results revealed that isolates 004052, 009071, 009073, 001001, 094340 and 102354 had potential for biocontrol of disease. Partial sequencing of 16S rRNA gene of 61 isolates showed that 85% of isolates belonged to genus Bacillus. Phylogenetically, however, there were four clusters in the Bacillus group comprising of Bacillus subtilis, B. cereus, B. pumilus and B. sphaericus. One isolate was identified as B. flexus, while six isolates were Bacillus spp. Four isolates were identified as Achromobacter xylosoxidans, two as Bacterium (unclassified bacteria), and one each as Ochrobactrum intermedium, Pseudomonas aeruginosa and Ralstonia sp.     

Antibacterial activity of different degree of hydrolysis of palm kernel expeller peptides against spore-forming and non-spore-forming bacteria

Aims: The goal of this study was to determine inhibitory effect of palm kernel expeller (PKE) peptides of different degree of hydrolysis (DH %) against spore‐forming bacteria Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophillus, Bacillus subtilis, Bacillus thuringiensis, Clostridium perfringens; and non‐spore‐forming bacteria Escherichia coli, Lisinibacillus sphaericus, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Typhimurium and Staphylococcus aureus. Methods and Results: A range of DH % (50–100) of PKE peptides was prepared using alcalase, and hydrolysis conditions were determined using response surface methodology (RSM). The influence of pH (6·5–10·5), temperature (35–65°C), enzyme/substrate ratio (1–5%) and substrate concentration (1–2%) were studied on the response of the DH. The antibacterial activity of different DH % of PKE peptides was tested by using disc diffusion assay and micro‐broth dilution assay. According to the minimum inhibitory concentration (MIC) test on each of the PKE peptides of different DH %, the 70 DH % PKE peptide showed greater inhibitory effect compared to the 100 DH % PKE peptide against B. cereus, B. coagulans, B. megaterium, B. pumilus, B. stearothermophillus, B. subtilis, B. thuringiensis, Cl. perfringens, Lisinibacillus sphaericus and L. monocytogenes. Conclusions: The 70 DH % PKE peptides exhibited greatest overall antibacterial effect of the various peptides of PKE evaluated. Further research is needed to determine the mode of action of PKE peptides. Significance and Impact of the Study: Palm kernel expeller peptides, a natural plant product, effectively inhibited the growth of spore‐forming and non‐spore‐forming Gram‐positive bacteria. Potentially, PKE peptides could be used in food preservation and developed as antibacterial agent in the pharmaceutical industry.     

Comparative assessment of selenite (SeIV) detoxification to elemental selenium (Se0) by <Emphasis Type="Italic">Bacillus</Emphasis> sp.

Bacillus licheniformis, B. subtilis, B. cereus, Bacillus pumilus and Exiguobacterium sp., which were resistant up to 20 mg Na₂SeO₃/ml in nutrient broth and 40 mg/ml on nutrient agar plates, were isolated from contaminated soil and water. They grew from 25 to 45°C and pH 5 to 9. They had multiple metal and antibiotic resistances. All strains reduced selenite (SeIV) to elemental selenium (Se0) aerobically with a maximum reduction of 97% by B. pumilus after 144 h with Na₂SeO₃ at 500 μg/ml.     

Biochemical characterisation of grain mould resistant and susceptible genotypes and PGPR-induced resistance in the host to Curvularia lunata and Fusarium proliferatum

Resistance to biotic stresses in plants is either due to the presence of preformed biochemical compounds or induced in response to external stimulus. In this study, 13 grain mould resistant and seven susceptible lines of sorghum were analysed for biochemical defence mechanism. The levels of total phenols and phenylalanine ammonia lyase were almost same in the resistant and susceptible genotypes. However, two additional isoforms of peroxidase were found in the three of the 13 resistant genotypes. The isoform peroxidase corresponding to the R _f value of 0.25 was found in the resistant genotypes IS 13969, ICSB 377 and IS 8219-1, and two genotypes IS 13969 and ICSB 377 had an additional isoform corresponding to the R _f value of 0.32. The results indicated the genotype specific association of peroxidases with grain mould resistance in sorghum. Nine bacterial strains (Bacillus pumilus SB 21, Bacillus megaterium HiB 9, Bacillus subtilis BCB 19, Pseudomonas plecoglossicida SRI 156, Brevibacterium antiquum SRI 158, B. pumilus INR 7, P. fluorescens UOM SAR 80, P. fluorescens UOM SAR 14, B. pumilus SE 34) were tested to induce systemic resistance in sorghum cultivars 296B and Bulk Y against the highly pathogenic grain mould pathogens Curvularia lunata and Fusarium proliferatum, respectively. The bacterial isolates were effective in inducing resistance in sorghum. Among the strains tested, SRI 158 was found highly effective in reducing grain mould severity in both the genotypes.     

Marine bacterial isolates inhibit biofilm formation and disrupt mature biofilms of Pseudomonas aeruginosa PAO1

According to the Centers for Disease Control and Prevention, biofilms cause 65% of infections in developed countries. Pseudomonas aeruginosa biofilm cause life threatening infections in cystic fibrosis infection and they are 1,000 times more tolerant to antibiotic than the planktonic cells. As quorum sensing, hydrophobicity index and extracellular polysaccharide play a crucial role in biofilm formation, extracts from 46 marine bacterial isolates were screened against these factors in P. aeruginosa. Eleven extracts showed antibiofilm activity. Extracts of S6-01 (Bacillus indicus = MTCC 5559) and S6-15 (Bacillus pumilus = MTCC 5560) inhibited the formation of PAO1 biofilm up to 95% in their Biofilm Inhibitory Concentration(BIC) of 50 and 60 μg/ml and 85% and 64% in the subinhibitory concentrations (1/4 and 1/8 of the BIC, respectively). Furthermore, the mature biofilm was disrupted to 70–74% in their BIC. The antibiofilm compound from S6-15 was partially purified using solvent extraction followed by TLC and silica column and further characterized by IR analysis. Current study for the first time reveals the antibiofilm and antiquorum-sensing activity of B. pumilus, B. indicus, Bacillus arsenicus, Halobacillus trueperi, Ferrimonas balearica, and Marinobacter hydrocarbonoclasticus from marine habitat.     

Extreme Spore UV Resistance of <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Isolates Obtained from an Ultraclean Spacecraft Assembly Facility

Recent environmental microbial sampling of the ultraclean Spacecraft Assembly Facility at NASA Jet Propulsion Laboratory (JPL-SAF) identified spores of Bacillus pumilus as major culturable bacterial contaminants found on and around spacecraft. As part of an effort to assess the efficacy of various spacecraft sterilants, purified spores of 10 JPL-SAF B. pumilus isolates were subjected to 254-nm UV and their UV resistance was compared to spores of standard B. subtilis biodosimetry strains. Spores of six of the 10 JPL-SAF isolates were significantly more resistant to UV than the B. subtilis biodosimetry strain, and one of the JPL-SAF isolates, B. pumilus SAFR-032, exhibited the highest degree of spore UV resistance observed by any Bacillus spp. encountered to date.     

Genotyping and Toxigenic Potential of Bacillus subtilis and Bacillus pumilus Strains Occurring in Industrial and Artisanal Cured Sausages

Artisanal and industrial sausages were analyzed for their aerobic, heat-resistant microflora to assess whether new emerging pathogens could be present among Bacillus strains naturally contaminating cured meat products. Sixty-four isolates were characterized by randomly amplified polymorphic DNA (RAPD)-PCR and fluorescent amplified fragment length polymorphism (fAFLP). The biotypes, identified by partial 16S rRNA gene sequence analysis, belonged to Bacillus subtilis, Bacillus pumilus, and Bacillus amyloliquefaciens species. Both RAPD-PCR and fAFLP analyses demonstrated that a high genetic heterogeneity is present in the B. subtilis group even in strains harvested from the same source, making it possible to isolate 56 different biotypes. Moreover, fAFLP analysis made it possible to distinguish B. subtilis from B. pumilus strains. The strains were characterized for their toxigenic potential by molecular, physiological, and immunological techniques. Specific PCR analyses revealed the absence of DNA sequences related to HBL, BcET, NHE, and entFM Bacillus cereus enterotoxins and the enzymes sphingomyelinase Sph and phospholipase PI-PLC in all strains; also, the immunological analyses showed that Bacillus strains did not react with NHE- and HBL-specific antibodies. However, some isolates were found to be positive for hemolytic and lecithinase activity. The absence of toxigenic potential in Bacillus strains from the sausages analyzed indicates that these products can be considered safe under the processing conditions they were produced; however, great care should be taken when the ripening time is shortened, particularly in the case of traditional sausages, which could contain high amounts of Bacillus strains and possibly some B. cereus cells.     

Lytic Action of B-enzyme on Pseudomonas aeruginosa

B-enzyme was produced by Bacillus subtilis YT–25 and lysed the native cells of Pseudomonas aeruginosa extensively in the presence of NLF (Native Cell-Lytic Factor). NLF was a peptide which was also produced by B. subtilis YT–25. It was found that B-enzyme hydrolyzed the peptidoglycan of P. aeruginosa eventually to disaccharide units. Because the reducing end of the enzymatic digest was muramic acid, B-enzyme seemed to be an endo-N-acetyl-muramidase. Whereas, egg white iysozyme which was an endo-N-acetylmuramidase hardly lysed the native cells of P. aeruginosa. Specific activity of B-enzyme for the murein of P. aeruginosa was higher than that of egg white Iysozyme in the buffer of low ionic strength and the surface components of P. aeruginosa did not affect the activity of B-enzyme but strongly inhibited the activity of egg white Iysozyme. These facts seemed to explain the superiority of B-enzyme to egg white Iysozyme in the lysis of the native cells of P. aeruginosa.     

The parasporal crystals of Bacillus pumilus strain 15.1: a potential virulence factor?

Bacillus pumilus strain 15.1 was previously found to cause larval mortality in the Med‐fly Ceratitis capitata and was shown to produce crystals in association with the spore. As parasporal crystals are well‐known as invertebrate‐active toxins in entomopathogenic bacteria such as Bacillus thuringiensis (Cry and Cyt toxins) and Lysinibacillus sphaericus (Bin and Cry toxins), the B. pumilus crystals were characterized. The crystals were composed of a 45 kDa protein that was identified as an oxalate decarboxylase by peptide mass fingerprinting, N‐terminal sequencing and by comparison with the genome sequence of strain 15.1. Synthesis of crystals by a plasmid‐cured derivative of strain 15.1 (produced using a novel curing strategy), demonstrated that the oxalate decarboxylase was encoded chromosomally. Crystals spontaneously solubilized when kept at low temperatures, and the protein produced was resistant to trypsin treatment. The insoluble crystals produced by B. pumilus 15.1 did not show significant toxicity when bioassayed against C. capitata larvae, but once the OxdD protein was solubilized, an increase of toxicity was observed. We also demonstrate that the OxdD present in the crystals has oxalate decarboxylate activity as the formation of formate was detected, which suggests a possible mechanism for B. pumilus 15.1 activity. To our knowledge, the characterization of the B. pumilus crystals as oxalate decarboxylase is the first report of the natural production of parasporal inclusions of an enzyme.     

Bio-degradation and utilization of silica and quartz

Summary A mesophilic bacterium,Proteus mirabilis, which was known to be able to accumulate monomer silicate ions, a thermophilic bacterium,Bacillus caldolyticus, originating from a habitat with high silica concentrations, and a silicautilizing plant,Equisetum arvense, were all found to produce monomer silica from its polymer. The monomer silica, resulting from the mineralysis of either experimentally polymerized silica, or from quartz, is taken up byP. mirabilis cells, and also byEquisetum, which then deposits the silica again as a polymer in its stem and leaves. WithB. caldolyticus, which does not utilize the depolymerized product under the given conditions, we found that the intensity of the mineralysis depends on the growth rate of the organism.     

Effect of plant growth promoting rhizobacteria on bacterial canker of tomato

Use of plant growth promoting rhizobacteria in managing bacterial canker disease of tomato was studied in the present work. Tomato seeds were treated with PGPR strains viz., Bacillus pumilus INR7, Bacillus pumilus SE34, Bacillus pumilus T4, Bacillus subtilis GBO3, Bacillus amyloliquefaciens IN937a and Brevibacillus brevis IPC11 were subjected for seed germination and seedling vigor. Among the PGPR strains tested, only three strains (IN937a, GBO3 and IPC11) which showed enhancement in the seed quality parameters like seed germination and seedling vigor, were further subjected for estimation of one of the defence-related enzymes, Phenylalanine Ammonia Lyase (PAL) with total phenol contents. The same three strains were recorded for maximum disease protection under greenhouse conditions. The level of PAL and total phenol contents increased significantly upon the PGPR treatment. The rate of reduction in the bacterial canker disease incidence was directly proportional to the amount of increased level of PAL and total phenol content. The possible uses of these PGPR strains in effective management of bacterial canker of tomato were discussed in the present work.     

Identification of some Bacteria from Paddy Antagonistic to Several Rice Fungal Pathogens

The effect of 23 bacterial strains from ricefields in the tropics on rice seed germination and on radicle and hypocotyl development of four rice cultivars was determined. There was a varietal difference in response to seed bacterization with the different bacterial strains. Germination of cv. IR58 increased from 78 to 93 %, that of cv. IR64, from 89 to 97 %. Less effects on germination of cvs IR42 and IR36 were observed. All strains inhibited the mycelial growth of Rhizoctonia solani in vitro. The three strains, identified as Bacillus subtilis, inhibited the mycelial growth of eight fungal pathogens whereas the other strains were pathogen-specific. Seed bacterization with these bacterial strains provided a sheath blight protection of 4. 5 to 73 % in the glasshouse trial. These 23 bacterial strains were identified by phenotypic tests using the API systems, morphological and biochemical features, and by comparison of electrophoretic patterns after sodium dodecyl sulphate polyacrylamide gel electrophoresis. Bacterial strains were identified (number of strains in brackets) as: Bacillus subtilis (3), Bacillus laterosporus (1), Bacillus pumilus (1), Pseudomonas aeruginosa (7), Pseudomonas belonging to section 1 (5), Erwina herbicola-like (1), and Serratia marcescens (1). The features of the other four strains were similar to Serratia except for the DNAase and lipase activities.     

Induction of resistance in Camellia sinensis against Sclerotium rolfsii by dual application of Rhizophagus fasciculatus and Bacillus pumilus

Rhizophagus fasciculatus and Bacillus pumilus, isolated from rhizosphere of tea (Camellia sinensis (L.) O.Kuntze) bushes, were selected for the present study. Inoculation of tea bushes with any of the two micro-organisms increased growth of plants but significant increase was obtained in case of dual application. B. pumilus exhibited plant growth promoting rhizobacterial traits and antagonistic activity against Sclerotium rolfsii in vitro. Disease reduction following application of R. fasciculatus and B. pumilus was obtained when applied individually but joint application gave more significant results. A sharp increase was found in polyphenolic accumulation and activities of four defence enzymes which play a key role in disease suppression. Immunodetection of S. rolfsii in soil was done after treatments with bioinoculants using PAb of S. rolfsii and its population was significantly reduced owing to application of B. pumilus and R. fasciculatus.     

Antifungal chitinases from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> SG2: preliminary report

A halotolerant bacterium capable of chitinase production was previously isolated from saline soils of Gavkhooni marsh, Iran. The strain was identified as Bacillus pumilus strain SG2. This strain secretes two chitinases into the medium in response to the presence of chitin in the medium. The two chitinases, ChiS and ChiL, were active on both polymeric as well as oligosaccharide substrates. In this study, these two extracellular chitinases were purified from the supernatant of the Bacillus culture by ammonium sulfate precipitation. The optimum pH and temperature for both of these chitinases were pH 6 and 37°C, respectively. According to the conserved domain analysis, ChiS and ChiL were categorized in family 18 of the glycosylhydrolases. Three essential conserved amino acid residues (Asp-194, Asp-196 and Glu-198) in ChiS and (Asp-228, Asp-230 and Glu-232) in ChiL were found within the active site. Antifungal activities of the two purified chitinases were assessed on Rhizoctonia solani, Verticillium sp., Nigrospora sp., Stemphyllium botryosum and Bipolaris sp. demonstrating inhibition of all the tested strains. However, these chitinases did not inhibit cell wall formation of the non-chitin-containing fungi (oomycetes) Phytophthora citricola and Phytophthora capsici. This is the first investigation demonstrating antifungal activity of chitinases purified from B. pumilus SG2.     

Rhamnolipid–Biosurfactant Permeabilizing Effects on Gram-Positive and Gram-Negative Bacterial Strains

The potential of biosurfactant PS to permeabilize bacterial cells of Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis on growing (in vivo) and resting (in vitro) cells was studied. Biosurfactant was shown to have a neutral or detrimental effect on the growth of Gram-positive strains, and this was dependent on the surfactant concentration. The growth of Gram-negative strains was not influenced by the presence of biosurfactant in the media. Cell permeabilization with biosurfactant PS was shown to be more effective with B. subtilis resting cells than with Pseudomonas aeruginosa. Scanning-electron microscopy observations showed that the biosurfactant PS did not exert a disruptive action on resting cells such that it was detrimental to the effect on growing cells of B. subtilis. Low critical micelle concentrations, tender action on nongrowing cells, and neutral effects on the growth of microbial strains at low surfactant concentrations make biosurfactant PS a potential candidate for application in different industrial fields, in environmental bioremediation, and in biomedicine.