Deciphering Diversity of Salt-Tolerant Bacilli from Saline Soils of Eastern Indo-gangetic Plains of India

The intensive use of chemical fertilizers, monoculture and irrigation with surface saline water has resulted in the deterioration of soil health by enhancing the level of salinity in the Eastern Indo-Gangetic Plains of India. Therefore, diversity of halotolerant bacteria adapted to that environment and possessed the ability to produce plant growth hormones was explored, that could be used for salt stress amelioration. The 16S rRNA gene sequencing and fatty acid methyl ester (FAME) were used for diversity analysis of salt-tolerant bacilli. Among the 95 isolates, 55 strains showed plant growth promotion traits, production of industrially important enzymes (amylase, protease and cellulase) and tolerance to more than 4% NaCl. Using partial 16S rRNA sequences and FAME comparisons, 21 different species of Bacillus and Bacillus-derived genera were identified, viz. Bacillus megaterium, B. subtilis, B. licheniformis, B. firmus, B. horikoshii, B. pumilus, Bacillus sp., B. safensis, B. thuringiensis, B. simplex, B. agri, B. flexus, B. oceanisediminis, B. cereus, B. arsenicus, Paenibacillus dendritiformis, Lysinibacillus sp., L. sphaericus, B. marisflavi, Terribacillus sp., and B. mycoides. These isolates possess the ability to tolerate high salt, form endospores, withstand harsh environments, and also have the potential for plant growth promotion, which could be useful in formulation of new inoculants to enhance the availability of nutrients for crop growth under saline conditions.     

High-level expression of an endoxylanase gene from Bacillus sp. in Bacillus subtilis DB104 for the production of xylobiose from xylan

To produce xylobiose from xylan, high-level expression of an endoxylanase gene from Bacillus sp. was carried out in Bacillus subtilis DB104. A 1.62-kb SmaI DNA fragment, coding for an endoxylanase of Bacillus sp., was ligated into the Escherichia coli/B. subtilis shuttle vector pJH27Δ88, producing pJHKJ4, which was subsequently transformed into B. subtilis DB104. A maximum endoxylanase activity of 105 U/ml was obtained from the supernatant of B. subtilis DB104 harboring pJHKJ4. The endoxylanase was purified to homogeneity by ion-exchange chromatography and the production profile of xylooligosaccharides from xylan by the endoxylanase was examined by HPLC with a carbohydrate analysis column. Xylobiose was the major product from xylan at 40 °C and its proportion in the xylan hydrolyzates increased with the reaction time; at 12 h, over 60% of the reaction products was xylobiose. These results suggest that xylobiose, which has a stimulatory effect on the selective growth of the intestinal bacterium Bifidobacterium, can be mass-produced effectively by the endoxylanase of Bacillus sp. cloned in B. subtilis. Received: 2 January 1998 / Received revision: 4 March 1998 / Accepted: 4 March 1998     

Antifungal Effects of Bacilysin and Fengymycin from Bacillus subtilis F-29-3 A Comparison with Activities of Other Bacillus Antibiotics

Of the two antifungal antibiotics produced by Bacillus subtilis F-29-3, the dipeptide compound bacilysin inhibits yeasts (and bacteria), whereas the formerly unknown fengymycin, a complex of closely related lipopeptide components, shows antibiotic activity against filamentous fungi. Bacilysin production, formerly known for a few strains only, could be demonstrated for all 12 wild-type cultures of Bacillus subtilis tested during this study. The antibiotic also occurs in some strains of three other Bacillus species considered as closely realted to B. subtilis. Members of the lipopeptide class of antifungal Bacillus metabolites were formed by 8 of 12 Bacillus subtilis-isolates and several other Bacillus strains. The antibiotics of F-29-3 were compared with antifungal metabolites of other Bacillus isolates using TLC, agar-diffusion techniques and tests demonstrating the capacity of six lipopeptide and peptide preparations to protect rice seedlings from phytomycosis due to Rhizoctonia solani. Fengymycin proved to be different from the other compounds tested. It was less toxic to the test plants and protected them better from Rhizoctonia disease than the other antibiotics of the study did.     

Cloning of a gene for maltohexaose producing amylase of an alkalophilic Bacillus and hyper-production of the enzyme in Bacillus subtilis cells

Summary The gene for maltohexaose producing amylase from an alkalophilic bacterium, Bacillus sp. # 707, was cloned in an Escherichia coli phage D69 and recloned in an E. coli plasmid pBR322 and a Bacillus subtilis plasmid pUB110, designated the resulting plasmids as pTUE306 and pTUB812, respectively. A common DNA region of approximately 2.5 kb was defined among the inserted DNAs. The enzymatic activity was lost when a part of the common region was deleted. The plasmids were stably maintained and the gene was well expressed in the bacterium, B. subtilis[pTUB812] which produced more than 70 times higher activity in the culture medium than did Bacillus sp. # 707. The major product of hydrolysis of starch by the enzymes of B. subtilis[pTUB812] and E. coli[pTUE306] was maltohexaose. The cloned gene corresponded to one of the genes for five components of malto-oligosaccharide-producing amylases of Bacillus sp. # 707.Abbreviations G1, G2, G3, G4, G5, and G6 glucose, maltose, maltotriose, maltotetraose, maltopentaose, and maltohexaose, respectively - kb kilobase pairs - kdal kilodalton - [] designated plasmid-carrier state     

Cloning and Characterization of Flagellin Genes and Identification of Flagellin Glycosylation from Thermophilic Bacillus Species

Flagellin glycosylation was identified in Bacillus sp. PS3 and Geobacillus stearothermophilus. In vivo complementation showed that these flagellin genes did not restore the motility of a Bacillus subtilis flagellin mutant, whereas the genes encoding non-glycosylated flagellin from Geobacillus kaustophilus and Bacillus sp. Kps3 restored motility. Moreover, four types of flagellins expressed in B. subtilis were not glycosylated. We speculate that glycosylation is required for flagellar filament assembly of these bacilli.     

Alkaline protease from <Emphasis Type="Italic">Bacillus sp.</Emphasis> isolated from coffee bean grown on cheese whey

Two strains of Bacillus, one from a culture collection (B. subtilis ATCC 6633) and a wild type (Bacillus sp. UFLA 817CF) isolated during coffee fermentation in the south of Minas Gerais, Brazil, were evaluated in relation to secretion of alkaline proteases. The strains were grown on nutrient broth, nutrient broth with sodium caseinate and nutrient broth with three different concentrations of cheese whey powder for 72 h. Samples were collected at 24-h intervals to evaluate the proteolytic activity, protein content and cell population. Maximum protease activity was observed after 24-h growth for both the microorganisms, a period that coincided with the end of the exponential phase. The specific activity values were, respectively, 839.8 U/mg for B. subtilis ATCC 6633 and 975.9 U/mg for Bacillus sp. UFLA 817CF. The 60% saturation presented the best results for specific protease activity in all the growth culture media tested with B. sp. UFLA 817CF. Bacillus sp. UFLA 817CF showed highest enzymatic activity at pH 9.0 and 40°C in the three culture media tested. The protease obtained from culture of the wild Bacillus strain presented stability at pH 7.0 and considerable heat stability at 40°C and 50°C, and could be an alternative for the industry to utilize cheese whey to produce proteolytic enzymes.     

Screening of marine actinobacteria for amylase enzymes inhibitors

Amylase inhibitor producing actinobacteria were isolated and characterized from terrestrial environment and there is no much report found from marine environment, hence in the present study, 17 strains isolated from the rhizosphere sediments of mangroves were tested for their amylase inhibition ability. Seawater requirement test for the growth of actinobacteria found that the strains SSR-3, SSR-12 and SSR-16 requires at least 50% and SSR-6 requires at least 25% seawater for their growth. The inhibition activity of both prokaryotic and eukaryotic amylase was tested by using Bacillus subtilis and Aspergillus niger. The maximum amylase activity (40mm) produced by the A. niger was taken as positive control, when the test actinobacteria strains grown in the medium they inhibited amylase activity and was evidenced by the reduction in inhibition zone (14–37 mm) similarly the amylase produced by the Bacillus subtilis was also recorded maximum (35 mm) amylase activity and was taken as positive control, and the test atinobacterial strains reduced enzyme action(12–33 mm) it varied levals. This indicates that the actinobacteria strains were controlled amylase enzyme activity in both the cases. The strain SSR-10 was highly effective and SSR-8 was less effective in inhibiting eukaryotic amylase produced by A. niger. The strain SSR-2 was effective and SSR-6 showed very less effect in inhibiting the prokaryotic amylase produced by the B subtilis.     

Rapid Detection and Characterization of Surfactin-Producing <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and Closely Related Species Based on PCR

The surfactin production genetic locus (sfp) is responsible for the ability of Bacillus subtilis to produce the lipopeptide biosurfactant, surfactin. This report demonstrates the utility of using PCR of the sfp gene as a means of identifying Bacillus species that produce surfactin. We carried out a hemolysis zone assay, quantitative HPLC and NMR in parallel to ensure that the PCR provided correct results. PCR analyses were performed for the sfp gene on 15 standard strains and 20 field-collected Bacillus spp. isolates native to Taiwan. Among the 15 standard strains, surfactin was produced by seven strains of B. subtilis and two closely related species, B. amyloliquefaciens B128 and B. circulans ATCC 4513. Of the 20 field-collected Bacillus spp. isolates; 16 strains yielded surfactin- positive results with PCR and HPLC. A good correlation was observed. Within the 16 field isolates, B. amyloliquefaciens S13 (452.5 mg/L) and B. subtilis S15 (125.6 mg/L) had high productivity of surfactin. The technique is valuable for finding out potential good yields of surfactin-producing strains. The PCR method we used could also be used to find different species or genera containing homologous genes. This is the first report of the detection of surfactin production by B. amyloliquefaciens and B. circulans based on PCR screening.     

Application of the Upstream Region of a Bacillus Endoglucanase Gene to High-level Expression of Foreign Genes in Bacillus subtilis

A 0.4-kb ScaI-HpaI fragment, 199bp upstream of the structural gene for alkaline endoglucanase, from the alkalophilic Bacillus sp. KSM-64, was found to be essential for the extracellular production of the enzyme by recombinant Bacillus subtilis cells. We constructed a new vector, pHSP64 (5.5 kb), using pHY300PLK and part of the 5′ region of the endoglucanase that contained a possible promoter region. Using recombinant B. subtilis cells that carried this vector, very high production of two endoglucanases and of chloramphenicol acetyltransferase was done.     

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.     

Extracellular production of cycloisomaltooligosaccharide glucanotransferase and cyclodextran by a protease-deficient <Emphasis Type="Italic">Bacillus subtilis</Emphasis> host–vector system

A cycloisomaltooligosaccharide (CI; cyclodextran) production system was developed using a Bacillus subtilis expression system for the cycloisomaltooligosaccharide glucanotransferase (CITase) gene. The CITase gene of Bacillus circulans T-3040, along with the α-amylase promoter (PamyQ) and amyQ signal sequence of Bacillus amyloliquefaciens, was cloned into the Bacillus expression vector pUB110 and subsequently expressed in B. subtilis strain 168 and its alkaline (aprE) and neutral (nprE) protease-deficient strains. The recombinant CITase produced by the protease-deficient strains reached 1 U/mL in the culture supernatant within 48 h of cultivation, which was approximately 7.5 times more than that produced by the industrial CITase-producing strain B. circulans G22-10 derived from B. circulans T-3040. When aprE- and nprE-deficient B. subtilis 168 harboring the CITase gene was cultured with 10% dextran 40 for 48 h, 17% of the dextran in the culture was converted to CIs (CI-7 to CI-12), which was approximately three times more than that converted by B. circulans G22-10 under the same dextran concentration. The B. subtilis host–vector system enabled us to produce CIs by direct fermentation of dextran along with high CITase production, which was not possible in B. circulans G22-10 due to growth inhibition by dextran at high concentrations and limited production of CITase.     

Note on the bacterial flora of the Egyptian desert in summer

Summary The response of bacterial population to edaphic drought of the Egyptian desert in summer has been investigated. The sporeformers Bacillus subtilis, Bacillus licheniformis, and Bacillus megaterium have predominated in the soil. B. subtilis and B. licheniformis predominated in the rhizosphere of the studied chamaephytes. The total bacterial counts were found to be much higher in the rhizospheres than in the soils apart. Azotobacter sp. and Clostridium sp. have been isolated from rhizospheres but not from soils. The significance of the rhizosphere as constituting a microhabitat in the desert community, has been discussed. The probable contribution of the peritrophic mantle of bacteria for protecting root cells against edaphic drought has also been notified.     

Molecular phylogenetic diversity of Bacillus community and its temporal–spatial distribution during the swine manure of composting

In order to obtain the diversity and temporal–spatial distribution of Bacillus community during the swine manure composting, we utilized traditional culture methods and the modern molecular biology techniques of polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) and –denaturing gradient gel electrophoresis (PCR-DGGE). Bacillus species were firstly isolated from the composting. Based on temperature changes, the temporal–spatial characteristics of total culturable Bacillus were remarkable that the number of the culturable Bacillus detected at the high-temperature stage was the highest in each layer of the pile and that detected in the middle layer was the lowest at each stage of composting respectively. The diversity of cultivated Bacillus species isolated from different composting stages was low. A total of 540 isolates were classified by the RFLP method and partial 16S rDNA sequences. They affiliated to eight species including Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Bacillus anthracis, Bacillus megaterium, Bacillus licheniformis, Bacillus pumilus, and Bacillus circulans. The predominant species was B. subtilis, and the diversity of culturable Bacillus isolated in the middle-level samples at temperature rising and cooling stages was the highest. The DGGE profile and clone library analysis revealed that the temporal–spatial distribution of Bacillus community was not obvious, species belonging to the Bacillus were dominant (67%) with unculturable bacteria and B. cereus was the second major culturable Bacillus species. This study indicated that a combination of culture and culture-independent approaches could be very useful for monitoring the diversity and temporal–spatial distribution of Bacillus community during the composting process.     

Rope-Producing Strains of Bacillus spp. from Wheat Bread and Strategy for Their Control by Lactic Acid Bacteria

Two types of white wheat bread (high- and low-type loaves) were investigated for rope spoilage. Thirty of the 56 breads tested developed rope spoilage within 5 days; the high-type loaves were affected by rope spoilage more than the low-type loaves. Sixty-one Bacillus strains were isolated from ropy breads and were characterized on the basis of their phenotypic and genotypic traits. All of the isolates were identified as Bacillus subtilis by biochemical tests, but molecular assays (randomly amplified polymorphic DNA PCR assay, denaturing gradient gel electrophoresis analysis, and sequencing of the V3 region of 16S ribosomal DNA) revealed greater Bacillus species variety in ropy breads. In fact, besides strains of B. subtilis, Bacillus licheniformis, Bacillus cereus, and isolates of Bacillus clausii and Bacillus firmus were also identified. All of the ropy Bacillus isolates exhibited amylase activity, whereas only 32.4% of these isolates were able to produce ropiness in bread slices after treatment at 96°C for 10 min. Strains of lactic acid bacteria previously isolated from sourdough were first selected for antirope activity on bread slices and then used as starters for bread-making experiments. Prevention of growth of approximately 10⁴ rope-producing B. subtilis G1 spores per cm² on bread slices for more than 15 days was observed when heat-treated cultures of Lactobacillus plantarum E5 and Leuconostoc mesenteroides A27 were added. Growth of B. subtilis G1 occurred after 7 days in breads started with Saccharomyces cerevisiae T22, L. plantarum E5, and L. mesenteroides A27.     

Sequence Analysis of a 32-kb Region Including the Major Ribosomal Protein Gene Clusters from Alkaliphilic Bacillus sp. Strain C-125

Forty-one open reading frames (ORFs) were identified in a 32-kb DNA fragment of alkaliphilic Bacillus sp. C-125. A similarity search using the BSORF database found 37 ORFs with significant sequence similarity to B. subtilis RNA polymerase subunits, elongation factor G, elongation factor Tu, and ribosomal proteins. Each ORF product showed more than 70% identity to those of B. subtilis. Gene organization in the region of str, S10, spc, and the α cluster was highly conserved among three strains, C-125, B. subtilis, and B. stearothermophilus.     

New approach for the detection of non-ribosomal peptide synthetase genes in Bacillus strains by polymerase chain reaction

Bacillus strains produce non-ribosomal lipopeptides that can be grouped into three families: surfactins or lichenysins, iturins and fengycins or plispastatins. These biosurfactants show a broad spectrum of biological activities. To detect strains able to produce these lipopeptides, a new polymerase chain reaction screening approach was developed using degenerated primers based on the intraoperon alignment of adenylation and thiolation nucleic acid domains of all enzymes implicated in the biosynthesis of each lipopeptide family. The comparative bioinformatics analyses of each operon led to the design of four primer pairs for the three families taking into account the differences between open reading frames of each synthetase gene. Tested on different Bacillus sp. strains, this technique was used successfully to detect not only the expected genes in the lipopeptide producing strains but also the presence of a plispastatin gene in Bacillus subtilis ATCC 21332 and a gene showing a high similarity with the polyketide synthase type I gene in the B. subtilis ATCC 6633 genome. It also led to the discovery of the presence of non-ribosomal peptide synthetase genes in Bacillus thuringiensis serovar berliner 1915 and in Bacillus cereus LMG 2098. In addition, this work highlighted the differences between the fengycin and plipastatin operon at DNA level.     

Isolation,evaluation and characterization of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> from cotton rhizospheric soil with biocontrol activity against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

Present investigation is based on the isolation of Bacillus subtilis from cotton rhizosphere and their evaluation as biocontrol agent against Fusarium oxysporum. The production of extracellular hydrolytic enzyme was studied for determining the antagonism. 43% of 21 isolates were identified under the B. subtilis group on the basis of biochemical characterization. 38% isolates showed competitive activity against Fusarium oxysporum and exhibit more than 50% mycelial inhibition in dual culture bioassay. The pot assay of cotton by seed treatment and soil amendment technique under green house condition showed the competent activity of the isolates in preventing the wilting of cotton seedlings due to F. oxysporum infection. SVI values of 30 day old seedlings indicated that the soil inoculation with B. subtilis BP-2 and seed treatment with B. subtilis BP-9 significantly promoted the growth of cotton seedlings. RAPD profiling revealed the diversity in the Bacillus subtilis group, ranging from 10 to 32%. The discriminative pattern among the isolates belonging to the same species was validated by 16S rDNA partial sequencing which identified them into four different strains of B. subtilis.     

Production studies on the alkaline amylases of three alkalophilic Bacillus spp.

Summary Bacillus alcalophilus subsp. halodurans ATCC 21591, Bacillus sp. NCIB 11203 and Bacillus sp. IMD370 are obligate alkalophiles and produce alkaline -amylases with maxima for activity at pH 10.0. All three organisms yielded maximum amylase activity in a medium containing starch as a carbon source and yeatex as a nitrogen source with an initial pH 10.0.     

Thermostable,salt-tolerant amylase fromBacillus sp. 64

A thermostable, salt-tolerant amylase was produced byBacillus sp. 64, with maximum amylase production (8.0 U/ml culture filtrate) after 24-h growth. Partially purified amylase was stable at 60°C for 30 min and 80% of the original activity was retained when incubated in 5m NaCl over 24 h. Starch or dextrin was the best carbon source and peptone the best nitrogen source for the production of the enzyme. Amylase was secreted over a wide pH range (5 to 11) with the maximum activity between pH 7 and 8. Ca²⁺ and Mg²⁺ stimulated growth and enzyme production.NCL Communication No. 5209.     

Isolation,screening, and optimization of bacterial strains for novel transglutaminase production

Transglutaminases are a class of transferases known to form isopeptide bond between glutamine and lysine residues in a protein molecule. Increasing demand for transglutaminase in food and other industries and its low productivity have compelled researchers to isolate and screen micro-organisms with potential to produce it. In the present investigation around 200 isolates were screened for extracellular secretion of microbial transglutaminase (MTGase). Isolate B4 showed enzyme activity of 1.71?±?0.2?U/mL followed by isolate C2 which showed 1.61?±?0.17?U/mL activity, comparable with the activity of industrially used microbial strains. Biochemical analysis along with 16S r-RNA sequencing revealed these isolates (B4 and C2) to be Bacillus nakamurai and a variant of Bacillus subtilis, respectively. Amongst the various production media screened, a medium containing starch and peptone was found best for MTGase production. Correlation between growth, enzyme production, and sugar utilization was also studied and maximum enzyme production was obtained after 48 to 60?hr. Highest MTGase titer (3.95?±?0.03?U/mL for B4 and 2.65?±?0.17?U/mL for C2) was obtained by optimization of parameters. The enzyme was characterized for temperature and pH optima, pH and thermal stability, and effect of metal ions, suggesting its potential use in future applications.