Isolation and study of azobenzene-transforming soil bacteria

Heterotrophic bacteria were isolated from soil and glass slides and classified as Bacillus cereus SNK12, Paenibacillus polymyxa SNK2, Azotobacter chroococcum ANKII, and Ochrobactrum intermedium ANKI. Their cultures could degrade azobenzene under the conditions of co-metabolism. A rapid test for the ability of bacteria to transform azobenzenes is proposed.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 185–188.Original Russian Text Copyright © 2005 by Wackerow-Kouzova.     

Copper-dependent DNA damage induced by hydrazobenzene,an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5′-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.     

Effect of Carbon Source on the Antimicrobial Activity of the Air Flora

Summary In an attempt to screen for air flora producing new potent antimicrobial substances, Bacillus megaterium NB-3, Bacillus cereus NB-4, Bacillus cereus NB-5, Bacillus subtilis NB-6 and Bacillus circulans NB-7, were isolated and were found to be antagonistic to bacteria and/or fungi. Production of antimicrobial substances by the bacterial strains was greatly influenced by variation of carbon sources. Glycerol strongly enhanced the antimicrobial activity of strains NB-3 and NB-6, whereas glucose increased the antimicrobial activity of strains NB-4 and NB-5. The maximum antibiotic yield of NB-7 was achieved with fructose as a carbon source. Starch (Bacillus megaterium NB-3), maltose (Bacillus cereus NB-5), glycerol (Bacillus circulans NB-7), arabinose, ribose (Bacillus cereus NB-4) and arabinose, fructose, glucose, ribose and sucrose (Bacillus subtilis NB-6) repressed the production of antimicrobial substances by the respective bacterial strains.     

Monte Carlo simulation of photoselective cyclization of azobenzene-containing oligosarcosines

Conformations of (Sar)_n? Azo? (Sar)_n oligomers [Sar = sarcosine (N-methylglycine) unit. Azo = p-aminoazobenzene unit] were computer-simulated for trans and cis states of the azobenzene group. Each part of the oligosarcosine chain was assumed to behave independently and the ring-closure probability, i.e., the probability that the end-to-end distance of the above oligomer is shorter than 4 Å, was evaluated from an overlap integral of the two three-dimensional distribution functions for the left and right halves of the oligosarcosine chain. The ring-closure probability for the cis azobenzene unit was larger than that for trans, indicating the preference of cyclization reaction to the corresponding intermolecular reactions. The prediction agrees qualitatively with the experimental observation reported by us in our companion paper, but the calculated ring-closure probability for the cis state was substantially larger than the experimental value. The difference was explained as a result of intramolecular overlappings of the two halves of the oligosarcosine chain, which is facilitated when the azobenzene group is in the cis state.     

Conformational Changes of DNA by Photoirradiation of DNA-Bis(ZnII-Cyclen)-Azobenzene Complex

Bis(Zn^II-cyclen)-azobenzene derivative, which has two Zn^II-macrocyclic tetraamine complexes connected through azobenzene spacer, has been synthesized as a cross-linking agent for double stranded DNA in aqueous solution. The Zn^II-cyclen derivative selectively binds to A-T base pairs producing complexes between the Zn^II-cyclen moiety and the imide-deprotonated thymine with breaking A-T base pairs. The azobenzene spacer undergoes cis/trans photoisomerization in the complex between the Zn^II-cyclen derivative and the DNA duplex. The conformation of the DNA remarkably changed by photoisomerization of the azobenzene linker, when the Zn^II-cyclen derivative binds to the DNA duplex with an interstrand cross-linking manner.     

Cloning and expression of the PHA synthase genes phaC1 and phaC1AB into Bacillus subtilis

Summary Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this work, we constructed two recombinant plasmids, pBE2C1 and pBE2C1AB. The two plasmids were inserted into Bacillus subtilis DB104 and generated Bacillus subtilis/pBE2C1 and Bacillus subtilis/pBE2C1AB. The two recombinant strains were subjected to fermentation and showed PHA accumulation, the first reported example of medium-chain-length-PHA production in Bacillus subtilis. GC analysis identified the compound produced by Bacillus subtilis/pBE2C1 was a hydroxydecanoate-co-hydroxydodecanoate (HD-co-HDD) polymer while that produced by Bacillus subtilis/pBE2C1AB was a hydroxybutyrate-co-hydroxydecanoate-co-hydroxydodecanoate (HB-HD-HDD) polymer. The results also showed that the recombinant B. subtilis could utilize the malt waste in the medium as a carbon source better than that of glucose and thus could substantially lower the cost of production of PHA.     

Dynamic of the volume and structure of the soil bacterial complex in the presence of azobenzene

Azobenzene exerted no significant effect on the dynamics and the species composition of the saprophytic soil bacterial complex, which remained almost the same as in the control and was characterized by the predominance of Curtobacterium sp., Arthrobacter globiformis, and Bacillus megaterium in all stages of succession. Some heterotrophic bacteria were found to be able to accumulate azobenzene. Bac. cereus and Bac. polymyxa degraded azobenzene during their cultivation in nutrient media.     

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.     

The effect of Spo0A and AbrB proteins on expression of the genes of guanyl-specific ribonucleases from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis recombinant strains

Guanyl-specific ribonucleases from Bacillus intermedius and Bacillus pumilus are actively secreted under phosphate starvation by recombinant strains of Bacillus subtilis with native regulatory systems and by strains defective in some proteins of the Spo0A phosphorylation pathway. The level of expression of ribonuclease genes has been shown to increase approximately sixfold in recombinant strains with mutation in the spo0A gene and threefold in the spo0A/abrB mutants, as compared with native strains. These results demonstrate that the Spo0A protein regulates the production of ribonucleases and thus acts as a repressor, while the AbrB protein is an activator of expression of the genes encoding ribonucleases from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis cells. Original Russian Text ? V.V. Ul’yanova, V.I. Vershinina, M.A. Kharitonova, M.R. Sharipova, 2007, published in Mikrobiologiya, 2007, Vol. 76, No. 5, pp. 639–644.     

Bacillus gaemokensis sp. nov., isolated from foreshore tidal flat sediment from the Yellow Sea

A Gram-positive, rod-shaped, endospore-forming organism, strain BL3-6^T, was isolated from tidal flat sediments of the Yellow Sea in the region of Tae-An. A 16S rRNA gene sequence analysis demonstrated that this isolate belongs to the Bacillus cereus group, and is closely related to Bacillus mycoides (99.0% similarity), Bacillus thuringiensis (99.0%), Bacillus weihenstephanensis (99.0%), Bacillus cereus (98.9%), Bacillus anthracis (98.8%), and Bacillus pseudomycoides (98.1%). The phylogenetic distance from any validly described Bacillus species outside the Bacillus cereus group was less than 95.6%. The DNA G+C content of the strain was 39.4 mol% and the major respiratory quinone was menaquinone-7. The major cellular fatty acids were iso-C_14:0 (17.8%), iso-C_16:0 (15.8%), and iso-C_12:0 (11.3%). The diagnostic amino acid of the cell wall was meso-diaminopimelic acid and the major cell wall sugar was galactose. The results of DNA-DNA hybridization (<55.6%) and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain BL3-6^T from the published Bacillus species. BL3-6^T therefore represents a new species, for which the name Bacillus gaemokensis sp. nov. is proposed, with the type strain BL3-6^T (=KCTC 13318^T =JCM 15801^T).     

Mutually exclusive distribution of the sap and eag S-layer genes and the lytB/lytA cell wall hydrolase genes in Bacillus thuringiensis

Recently, two Bacillus thuringiensis strains were reported to synthesize parasporal inclusion bodies made not of the expected crystal (Cry) proteins but rather of the surface layer proteins (SLP) Sap (encoded by sap) and EA1 (encoded by eag), respectively. Whether the presence of the sap and eag genes is restricted to these two B. thuringiensis strains or ubiquitous in B. thuringiensis is unknown. We report here the distribution of the sap and eag genes in B. thuringiensis. Strains in the Bacillus cereus group were added for comparison purposes. We show that sap and eag are either present in tandem in 35% of the B. thuringiensis strains analysed and absent in 65% of the strains. When absent, a different tandem, the lytB/lytA cell wall hydrolase genes, is present. The distribution of the sap and eag S-layer and the lytB/lytA cell wall hydrolase genes is not species-specific in B. thuringiensis, B. cereus and Bacillus weihenstephanensis. Bacillus anthracis and Bacillus mycoides harbor sap and eag but not lytB/lytA. The sap, eag and lytB/lytA genes were absent in Bacillus pseudomycoides. Clearly, the distribution of the sap and eag S-layer and the lytB/lytA cell wall hydrolase genes in B. thuringiensis and in the Bacillus cereus group is mutually exclusive. We also showed that two genes involved in cell wall metabolism, csaA and csaB, are present not only upstream of the sap and eag S-layer genes, but also upstream of the lytB/lytA tandem in strains where sap and eag are absent. Bootstrapped neighbor-joining trees were inferred from the translated amino acid sequences of sap, eag and the tandem lytB/lytA, respectively.     

Effects of four Bacillus spp. of soil origin on the Colorado potato beetle Leptinotarsa decemlineata (Say)

Four species of Bacillus were isolated from soil in an effort to find safe, effective and alternative biological control agents against plant pests. These bacteria were identified as Bacillus pumilus, Bacillus sphaericus, Bacillus megaterium and Bacillus cereus on the basis of fatty acid methyl ester analysis and carbon utilization profiles by using Microbial Identification and Biolog Microplate Systems. Laboratory experiments carried out to determine the insecticidal activities of these isolates showed that B. pumilus caused 95.7 and 26.7% mortality and B. sphaericus caused 74.5 and 23.3% mortality of Leptinotarsa decemlineata larvae and adults, respectively. B. cereus and B. megaterium showed 51.1 and 29.7%, respectively, of L. decemlineata larvae. This study presents at least two Turkish isolates from the genus Bacillus showing high insecticidal activity against L. decemlineata.     

Proteolysis, histopathological effects, and immunohistopathological localization of delta-endotoxins of Bacillus thuringiensis subsp. kurstaki in the midgut of lepidopteran olive tree pathogenic insect Prays oleae

Considering the fact that Prays oleae is one of the most pathogenic insects to the olive tree in the Mediterranean particularly in Tunisia, the mode of action of Cry insecticidal toxins of Bacillus thuringiensis kurstaki in Prays oleae midgut was investigated. The proteolysis of Bacillus thuringiensis δ-endotoxins in the midgut was a key step in determining their potency against Prays oleae. The latter's proteases activated the δ-endotoxins early, yielding stable toxins. The in vitro and in vivo binding of these toxins to Prays oleae larvae midgut was studied immunohistochemically, evidencing a midgut columnar cell vacuolization, microvilli damage, and then a pass of epithelium cell content into the larvae midgut. Moreover, Bacillus thuringiensis toxins were shown to bind to the apical microvilli of the midgut epithelial cells. The in vitro study of the interaction of Prays oleae midgut proteins with biotinylated Bacillus thuringiensis toxins allowed the prediction of four suitable receptor proteins in Prays oleae.     

Bacillus manliponensis sp. nov., a new member of the Bacillus cereus group isolated from foreshore tidal flat sediment

A Gram-positive, endospore-forming, new Bacillus species, strain BL4-6^T, was isolated from tidal flat sediment of the Yellow Sea. Strain BL4-6^T is a straight rod, with motility by peritrichate flagella. The cell wall contains meso-diaminopimelic acid, and the major respiratory quinone is menaquinone-7. The major fatty acids are iso-C_15:0 and summed feature 3 (containing C_16:1 ω7c/iso-C_15:0 2OH, and/or iso-C_15:0 2OH/C_16:1 ω7c). Cells are catalase-positive and oxidase-negative. The G+C content of the genomic DNA is 38.0 mol%. Based on a comparative 16S rRNA gene sequence analysis, the isolate belongs to the genus Bacillus, forms a clade with the Bacillus cereus group, and is closely related to Bacillus mycoides (98.5%), Bacillus cereus (98.5%), Bacillus anthracis (98.4%), Bacillus thuringiensis (98.4%), Bacillus weihenstephanensis (98.1%), and Bacillus pseudomycoides (97.5%). The isolate showed less than 85% similarity of the gyrA gene sequence and below 95% similarity of the rpoB gene sequence to the members of this group. DNA-DNA relatedness between strain BL4-6^T and B. cereus group was found to be in a range of 22.8–42.3%, and thus BL4-6^T represents a unique species. On the basis of these studies, strain BL4-6^T (=KCTC 13319^T =JCM 15802^T) is proposed to represent the type strain of a novel species, Bacillus manliponensis sp. nov.     

Flexible Solar Thermal Fuel Devices: Composites of Fabric and a Photoliquefiable Azobenzene Derivative

Controllable storage and release of solar energy has always been a highlighted scientific issue for its benefit of mankind. Solar thermal fuels (STFs) supply a closed cycle and renewable energy‐storage strategy by transforming solar energy into chemical energy stored in the conformation of molecular isomers, such as cis/trans‐azobenzene, and releasing it as heat under various stimuli. Although the potential high energy density of the STFs which are based on the hybrids of azobenzene derivatives and carbon nanomaterials has been reported the solvent‐assistant charging hinders their practicability. In this study, a solid‐state STF device is designed and fabricated by compositing one photoliquefiable azobenzene (PLAZ) derivative with a flexible fabric template. The photoinduced phase transition of the PLAZ derivative enables the charging of the flexible STFs to be totally solvent‐free. Interestingly, the energy‐storage capacity (energy density ≈201 J g^?1) of flexible PLAZ STFs has been improved by the soft fabric template. The exothermic situation is monitored with one infrared camera, which shows 4 °C temperature difference between charged and discharged samples under blue light stimulus. The flexible STFs are may be used in practice as heating equipment.     

Analysis of the genome of an alkaliphilic Bacillus strain from an industrial point of view

Bacillus species and other microbes with pH optima for growth higher than pH 9 are defined as alkaliphiles. A large number of alkaliphilic Bacillus strains producing useful enzymes, have been isolated from various environments. Some of these enzymes, such as proteases and cellulases from alkaliphilic Bacillus strains, have been commercialized and have brought great advantages to industry and domestic life. To support further development of the enzyme industry, we initiated analysis of the genome of Bacillus halodurans C-125, which is 4.25 Mb in size, and constructed a physical and genetic map for comparison with the Bacillus subtilis chromosome. Systematic sequencing of the whole genome of Bacillus halodurans C-125 has been automated since the beginning of May 1998, and sequencing of 98% of the whole genome has been done so far. Through genome analysis, it became apparent that the genome organization of alkaliphilic Bacillus halodurans C-125 is totally different from that of B. subtilis orthologues. Received: July 11, 1999 / Accepted: December 27, 1999     

The Bioremediation Potential of Hydrocarbonoclastic Bacteria Isolated From a Mangrove Contaminated by Petroleum Hydrocarbons on the Cilacap Coast,Indonesia

The main purpose of the study was to isolate strains of bacteria capable of degrading hydrocarbons from contaminated mangroves and to investigate the ability of the isolated bacteria to degrade total petroleum hydrocarbons (TPH) in a microcosm model of an oily sludge. The potential use of these bacteria strains as environmental clean-up agents was tested by culturing them with six different polyaromatic hydrocarbon (PAH) compounds (phenothiazine, fluorene, fluoranthene, dibenzothiophene, phenanthrene, and pyrene). Six viable and culturable bacteria were isolated, and the 16S rDNA sequence for each was amplified using the primers 9F and 1510R. Sequence results were compared using the National Center for Biotechnology Information (NCBI) BLAST program and, combined with phenotypic and phylogenetic data, were used to identify three strains that belonged to the Bacillus genus and were most closely related (98–99%) to Bacillus aquimaris, Bacillus megaterium, and Bacillus pumilus. The other three strains were closely related (98–100%) to Flexibacteraceae bacterium, Halobacilus trueperi, and Rhodobacteraceae bacterium. Two isolates, BA-PZN and BM-PFFP, which were related to Bacillus aquimaris and Bacillus megaterium, respectively, were further characterized and showed great potential for the removal of more complex hydrocarbon compounds in the oily microcosm model.     

Survey of bacterial populations present in US‐produced linerboard with high recycle content

Aims: To survey paperboard products from 17 US mills for bacterial populations and for bacteria potentially harmful to human health. Methods and Results: Culturable aerobic bacteria were isolated from paperboard products using selective and nonselective medium. Resulting colonies from samples from three regions of the United States were identified using fatty acid methyl ester analysis. Percentages of bacteria species found were Bacillus megaterium (47), Bacillus licheniformis (15), Bacillus pumilus (12), Paenibacillus macerans (5), Paenibacillus pabuli (3), Bacillus subtilis (2), Bacillus cereus (2), Bacillus coagulans (1), Bacillus circulans (1), Bacillus brevis (1), Bacillus thuringiensis (1), Paenibacillus polymyxa (1), Cellulomonas turbata (1), Cellulomonas flavigena (1), unidentified Bacillus sp. (3) and unidentified bacteria (1). Conclusions: Recycled paperboard contained high populations of bacteria, and a positive correlation was found between recycle content and bacterial populations. Escherichia coli, Salmonella, Shigella or confirmed coliform bacteria were not found in any product. Significance and Impact of the Study: Populations of bacteria did not differ significantly from original counts over a 4‐month period of dry storage, indicating that bacteria persist in paperboard over long periods and may re‐enter the recycling process. The predominance of heat‐tolerant endospore‐forming bacteria explains the high bacteria counts found in paperboard made from recycled materials .     

Bacillus subtilis secretes a foreign protein by the signal sequence of Bacillus amyloliquefaciens neutral protease

Summary We constructed a secretion plasmid in which a truncated penicillinase gene of Bacillus licheniformis was introduced at the end of the signal peptide coding region of a Bacillus amyloliquefaciens neutral protease gene. A Bacillus subtilis recombinant secreted about 140 mg/liter of the penicillinase into the medium. Analysis of the purified product revealed that it was a mixture of two penicillinases containing one or two additional amino acids at the NH₂-terminus of B. licheniformis exo-small penicillinase.     

Recoverability of heat-injured Bacillus spores by lysozyme and EDTA or alkaline thioglycollate

The D_95°C value of Bacillus thuringiensis spores plated in the presence of lysozyme increased from 3.0 min to 3.6 min by post-treatment of heat-injured spores with 50mm EDTA. In the case of Bacillus alvei and Bacillus polymyxa spores D-values decreased from 4.9 to 4.3 min and from 4.7 to 4.1 min respectively. Post-treatment of heat-injured spores treated with alkaline thioglycollate increased D_95°C values of Bacillus alvei from 4.2 to 5.3 min, B. thuringiensis 3.6 to 4.7 min, and Bacillus polymyxa from 4.2 to 5.0 min when spores were plated in the presence of lysozyme. Electron micrographs of heat-injured B. alvei spores treated with sodium thioglycollate indicated that the coat layers of the treated spores were granulated and less intact than the control spores.