Bacillus Azotoformans Research Articles

In vitro Antibacterial and in vivo Antinociceptive Activities of Ethyl Acetate Fraction of Dipterocarpus turbinatus Leaves

The study was aimed to assess the antibacterial and analgesic properties of the ethyl acetate extract from the leaves of Dipterocarpus turbinatus. The antibacterial effectiveness of the extract obtained from the leaves of D. turbinatus was evaluated using the disc diffusion method against both positive and negative Gram strains. Furthermore, the analgesic qualities were assessed by doing the formalin-incited paw licking test in mice model. The ethyl acetate fraction demonstrated significant antibacterial activity against all tested microorganisms (Lactobacillus casei, Corynebacterium sp., Bacillus cereus, Staphylococcus aureus, Escherichia coli, Bacillus azotoformans, and Salmonella typhi) at concentrations of 0.1 mg/disc, 0.3 mg/disc, and 0.5 mg/disc using the disc diffusion method. The extract's zone of inhibition measured between 12 and 25 mm was less than the Kanamycin's diameter of 32–36 mm. The analgesic activity of the substance was evaluated in Swiss Albino mice using the tail immersion and formalin test procedures, revealing a modest level of analgesic activity. In the formalin test, the extract had a strong dose-dependent impact at dosages of 200 mg/kg and 400 mg/kg, as shown by a larger inhibition percentage compared to the standard. This result was statistically significant (***p<0.001) when compared to the control group. During the tail immersion test, the extract showed a significant impact at dose 400 mg/kg (**p<0.01). The plant extract has a modest level of antibacterial and analgesic properties. Bangladesh Pharmaceutical Journal 27(2): 207-214, 2024 (July)

Physicochemical and Characterization of Nitrogen Fixing Bacteria from Soil Samples Within the Vicinity of Telecommunication Mast (Site No: 000148) Located at Karfi Town Kura Local Government, Kano State

The telecommunication mast associated-radiation is one of the primary factors influencing the way in which microorganisms interact with ecosystem. This study aims to assess the physicochemical and non-symbiotic nitrogen fixing bacteria (NNFB) from soil samples within the vicinity of telecommunication mast located at Karfi town Kura local government Kano state. Soil samples A, B, and C were collected within the vicinity of the mast at an interval of 10 meters, 20 meters and 30 meters from the mast respectively and control sample (D) was collected from location *(outside the vicinity of the mast)*. Physicochemical parameters of the soil samples were analyzed, isolation and identification of non-symbiotic nitrogen fixing bacteria were carried out using standard procedures. Samples B showed higher values of pH (8.02), phosphorus (23.95mg/kg), organic carbon (1.45%), nitrogen (0.28%) and organic matter content (2.50%) while control sample (D) showed lower values with 6.24, 2.77mg/kg, 0.41%, 0.07% and 0.71% of pH, phosphorus, organic carbon, nitrogen and organic matter content respectively. However, the moisture content(0.21%) of control sample is higher than that of sample A and B with 0.12% and 0.11% respectively The mean count of NNFB of the soil samples were 3.20 ± 0.06, 1.80± 0.12, 1.40±0.23, 1.20±0.20 for sample B, C, A and D respectively. Total of 14 isolates of the species Azomonas agilis 1(7.14%), Azotomonas insolita 1(7.14%), Bacillus megaterium 2(14.28 %), Bacillus azotoformans 1(7.14%), Bacillus mycoides 3(21.42%), Enterobacter cloacae 3(21.42%), and Klebsiella pneumonia 3(21.42%) were obtained. This indicates that the electromagnetic radiation from the mast has no effect on soil physicochemical parameters as well as non symbiotic nitrogen fixing bacteria proliferation.

Induction of moisture stress tolerance by Bacillus and Paenibacillus in pigeon pea (Cajanus cajan. L).

Drought stress is the main growth-limiting factor in pigeon pea production. Plant growth-promoting bacteria (PGPB) induce abiotic stress tolerance in several plants. However, the physiological and molecular changes with PGPB priming are not well understood in pigeon pea. The present study explored the potential of Firmibacteria (Bacillus azotoformans MTCC2953, Bacillus aryabhattai KSBN2K7, and Paenibacillus stellifer M3T4B6) to induce stress tolerance in pigeon pea under pot culture condition. Different physiological and biochemical parameters, including osmolytes, stress enzymes, and antioxidants, were evaluated under two stress conditions (50% and 25% field capacity) and an unstressed condition in pigeon pea. Under moisture stress conditions significant differences were observed in physiological and biochemical parameters between firmibacteria inoculated and control plants.The quantitative real-time polymerase chain reaction was performed to study the bacterial inoculation mediated expression of proline and drought-responsive genes in enhancing the drought tolerance in pigeon pea. Results showed that the inoculation of Bacillus aryabhattai upregulated the expression of drought-responsive genes (C. cajan_29830 and C. cajan_33874) and downregulated the expression of the proline gene by inducing the drought stress tolerance in inoculated plants compared with the uninoculated control plants. Therefore, Bacillus aryabhattai may be recommended for inducing drought stress tolerance and increasing the growth of pigeon pea under moisture stress conditions after field evaluation.

Isolation and identification of amylolytic bacteria from garbage and garden soil

An analysis for the abundance and diversity of amylolytic bacteria of two different soil types viz. garbage and garden soil was carried out. pH of the garbage and garden soil samples ranged between 7.73 and 9.84, 6.88 and 7.93, respectively. Average bacterial load on both NA and PYG agar media was found to be higher in garbage than garden soils. Bacterial load of garbage soil samples ranged from 2.08 × 108 to 3.79 ×108 cfu/g and 1.45 × 108 to 2.74 × 108 cfu/g on NA and PYG agar, respectively. On the other hand, bacterial load of the garden soil samples ranged from 3.3×106 to 9.7 ×106 cfu/g on NA and 2.9 × 106 to 9.35 × 106 cfu/g on PYG agar. A total of 200 bacterial isolates (100 from each soil type) were primarily selected for their amylolytic potential. Among them, the percentage of amylolytic bacteria was higher in garbage soil (46) than garden soil (38). Finally, a total of 8 (4 from each soil type) amylolytic potential isolates were selected for detailed study and identification. All 4 isolates from garbage soil and 3 from garden soil were found to be Gram positive and by conventional identification belonged to the genus Bacillus with six different species viz. Bacillus azotoformans (2), B. stearothermophilus (1), B. acidocaldarius (1), B. subtilis (2) and B. megaterium (1) and the only Gram negative isolate was identified as Acetobacter liquefaciens. The conventional identification was further confirmed by molecular technique and isolates were identified as Bacillus sp. T5-12, B. cereus MSW, Bacillus sp. FJAT-14266, B. toyonensis KK25A, B. cereus T10, Stenotrophomonas sp. ZJZG10, B. subtilis XF-1 and Pseudomonas sp. NCCP-1179. As significance of amylase enzyme in various industries and biotechnological processes are on the rise, it is important to find better and cheaper source for it. This piece of work focuses on finding out which can be a better source for amylolytic bacteria between two different soil types.

The phenomenon of UV-Vis spectroscopic changing due the binding of CO to the nitric oxide reductase from Bacillus Azotoformans

Gram-positive bacterium, Bacillus azotoformans, possess a nitric oxide reductase named CuANOR (formerly qCuANOR). The purified enzyme consists of two subunits. On the basis of UV-Vis and EPR spectra, it was elucidated that its small subunit contains the mixed-valence dinuclear CuA center and its large subunit of CuANOR contains a low-spin heme b centre, a high-spin heme b and one nonheme iron centre. The latter two grouped is an EPR-silent binuclear iron centre where NO reduction occurred. The CuANOR revealed itself as a new class of nitric oxide reductase, a new member of cytochrome c oxidase superfamily. The structure of CuANOR is not available yet. Previous studies on Raman resonance and FTIR demonstrated the capability of both heme iron and non heme-iron to bind one CO molecule each in the highly free chloride of dithionite-reduced CuANOR, whereas at high chloride concentration the binding of CO at non-heme iron was lost. Whether the binding of CO to CuANOR is followed by the oxidation of CO to CO2, as found in cytochrome c oxidase, hemoglobin, myoglobin, and free heme, has never been reported. This paper reports the phenomenon of UV-Vis spectroscopic changing due the binding of CO to CuANOR which explain the enzyme reactivity toward CO molecule.

Antioxidant, antibacterial, antidiarrheal and antipyretic activities of organic crude fractions of Commelina paludosa

Background:The herb, Commelina paludosa (CP) Blume (Family-Commelinaceae) is medicinally used by the traditional practitioners in Bangladesh. However, there is a lack of scientific evidence on this medicinal herb.Aim and Objectives:This study aimed to evaluate the phytochemical and pharmacological activities of CP (ethanol [ECP], chloroform [CCP] and n-hexane [NHCP] of whole-plant extracts).Materials and Methods:For this antioxidant, antimicrobial, antidiarrheal and antipyretic activities of crude extracts of CP were conducted by 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging, disc diffusion and serial dilution, castor oil-induced diarrhea and yeast powder-induced pyrexia methods respectively.Results and Observation:The results suggest that all the fractions significantly scavenged DPPH radicals. In the disc diffusion test, the zones of inhibition were observed within the range of 7 mm to 30.67 mm at 500 mg/disc. The highest zones of inhibition were observed by ECP, CCP and NHCP against Bacillus azotoformans, Lactobacillus coryifomis and Salmonella typhi respectively. NHCP was found to exert stronger antibacterial effect than the ECP and CCP.Conclusion:Minimum inhibitory concentrations were detected within the range of 31.25 and 250 μg/ml. Moreover, the crude fractions also showed significant (P < 0.05) antidiarrheal and antipyretic activities in Swiss mice. CP may be a good source of therapeutic components.

An electrogenic nitric oxide reductase

Nitric oxide reductases (Nors) are members of the heme-copper oxidase superfamily that reduce nitric oxide (NO) to nitrous oxide (N2O). In contrast to the proton-pumping cytochrome oxidases, Nors studied so far have neither been implicated in proton pumping nor have they been experimentally established as electrogenic. The copper-A-dependent Nor from Bacillus azotoformans uses cytochrome c551 as electron donor but lacks menaquinol activity, in contrast to our earlier report (Suharti et al., 2001). Employing reduced phenazine ethosulfate (PESH) as electron donor, the main NO reduction pathway catalyzed by CuANor reconstituted in liposomes involves transmembrane cycling of the PES radical. We show that CuANor reconstituted in liposomes generates a proton electrochemical gradient across the membrane similar in magnitude to cytochrome aa3, highlighting that bacilli using CuANor can exploit NO reduction for increased cellular ATP production compared to organisms using cNor.

Draft genome sequence of Bacillus azotoformans MEV2011, a (Co-) denitrifying strain unable to grow with oxygen.

Bacillus azotoformans MEV2011, isolated from soil, is a microaerotolerant obligate denitrifier, which can also produce N2 by co-denitrification. Oxygen is consumed but not growth-supportive. The draft genome has a size of 4.7 Mb and contains key genes for both denitrification and dissimilatory nitrate reduction to ammonium.Electronic supplementary materialThe online version of this article (doi:10.1186/1944-3277-10-4) contains supplementary material, which is available to authorized users.

Study on Biosurfactant Production by Two Bacillus Species

With increasing demand of biosurfactants for environmental and industrial application, there is increased interest in prospecting of microorganisms and substrates for production of substantial quantities of biosurfactants. A study was carried out to investigate the ability of Bacillus sphaericus EN3 and Bacillus azotoformans EN16 to produce large quantities of biosurfactants and determine a suitable substrate for its production. The organisms were previously screened for biosurfactant production and found to be good candidates. Three carbon sources including glucose, diesel and crude oil were used and incorporated into modified Bushnell - Haas medium at 2% w/v concentration. Large quantities of biosurfactants were observed when glucose was used as substrate with a mean production of 1.01 ± 0.3 g/L and 0.58 ± 0.17 g/L for B. sphaericus EN3 and B. azotoformans EN16 respectively after 10 days. The surfactants were shown to be stable at various pH (2 - 12), temperature (50°C - 100°C) and salinity (2 - 10% NaCl) ranges. The biosurfactants were however more effective at pH of 8 to 10, 50 0 C and salinity of 2 to 4% NaCl. Chromatographic analysis revealed that the surfactants produced by both organisms were principally phospholipids. Therefore, the organisms were capable of producing substantial amounts of surfactants and harnessing their potentials would be essential.

Draft genome sequence of Bacillus azotoformans MEV2011, a (Co-) denitrifying strain unable to grow with oxygen.

Bacillus azotoformans MEV2011, isolated from soil, is a microaerotolerant obligate denitrifier, which can also produce N2 by co-denitrification. Oxygen is consumed but not growth-supportive. The draft genome has a size of 4.7 Mb and contains key genes for both denitrification and dissimilatory nitrate reduction to ammonium.

Experimental investigation of microbiologically influenced corrosion of selected steels in sugarcane juice environment

In the current study, ferritic stainless grades AISI 439 and AISI 444 were investigated as possible construction materials for machinery and equipment in the cane-sugar industry. Their performance in corrosive cane-sugar juice environment was compared with the presently used low carbon steel AISI 1010 and austenitic stainless steel AISI 304. The Tafel plot electrochemical technique was used to evaluate general corrosion performance. Microbiologically influenced corrosion (MIC) behaviour in sugarcane juice environment was studied. Four microbial colonies were isolated from the biofilms on the metal coupon surfaces on the basis of their different morphology. These were characterized as Brevibacillus parabrevis, Bacillus azotoformans, Paenibacillus lautus and Micrococcus sp. The results of SEM micrographs showed that AISI 439 and AISI 304 grades had suffered maximum localized corrosion. MIC investigations revealed that AISI 444 steel had the best corrosion resistance among the tested materials. However from the Tafel plots it was evident that AISI 1010 had the least corrosion resistance and AISI 439 the best corrosion resistance.

Structure of microbial communities performing the simultaneous reduction of Fe(II)EDTA.NO2−and Fe(III)EDTA−

BioDeNOx is a combined physicochemical and biological process for the removal of nitrogen oxides (NOx) from flue gas. In the present study, two anaerobic bioreactors performing BioDeNOx were run consecutively (RUN-1 and RUN-2) at a dilution rate of 0.01 h(-1) with Fe(II)EDTA.NO(2-) and Fe(III)EDTA(-) as electron acceptors and ethanol as electron donor. The measured protein concentration of the reactor biomass of both runs was 120 mg/l. Different molecular methods were used to determine the identity and abundance of the bacterial populations in both bioreactors. Bacillus azotoformans strain KT-1 was recognized as a key player in Fe(II)EDTA.NO(2-) reduction. PCR-denaturing gradient gel electrophoresis analysis of the reactor biomass showed a greater diversity in RUN-2 than in RUN-1. Enrichments of Fe(II)EDTA.NO(2-) and Fe(III)EDTA(-) reducers and activity assays were conducted using the biomass from RUN-2 as an inoculum. The results on substrate turnover, overall microbial diversity, and enrichments and finally activity assays confirmed that ethanol was used as electron donor for Fe(II)EDTA.NO(2-) reduction. In addition, the Fe(III)EDTA(-) reduction rate of the microbial community proved to be feasible enough to run the bioreactors, ruling out the chemical reduction of Fe(III)EDTA(-) with sulfide as was proposed by other researchers.

Identification of nif genes in N2‐fixing bacterial strains isolated from rice fields along the Yangtze River Plain

The aim of this research was to identify nifH and nifHDKYE ' genes in twenty strains of N2-fixing heterotrophic bacteria isolated from rice fields in the Yangtze River Plain. Southern hybridization of the total DNA from each strain was performed with the Klebsiella pneumoniae nifHDKYE ' gene probe (6.2 kb Eco RI fragment from pSA30) and the Azospirillum brasilense nifH gene probe (0.6 kb Eco RI-Hin dIII fragment from pHU8). We found that Eco RI fragments of total DNA from Aeromonas hydrophila HY2, Bacillus azotoformans FD, Bacillus licheniformis NCH1, NCH5, WH4, Bacillus brevis NC2, Bacillus pumilus NC12, Bacillus cereus NCH2, Citrobacter freundii HY5, HY9, Derxia gummosa HZ5, Pseudomonas mendocina HZ1 and Pseudomonas pseudoalcaligenes WH3 were positively hybridized with both of the probes. Agrobacterium radiobacter HY17, Corynebacterium sp. HY12, YZ and Pseudomonas sp. HY11 had Eco RI fragments hybridized with the K. pneumoniae nifHDKYE ' gene probe. An Eco RI fragment of total DNA from Bacillus megaterium YY4 was positively hybridized to the A. brasilense nifH gene probe. No hybridization sign was found in the total DNA fragments from Alcaligenes cupidus YY6 and Corynebacterium sp. NC11 hybridized with either of the gene probes. The data provide the number and size of EcoRI fragments of the total DNA hybridized with the nif gene probes for these strains of rarely studied species, suggesting additional evidence for N2 fixing and nif gene diversity of N2-fixing bacteria in rice fields along the Yangtze River Plain.

Membrane-bound denitrification in the Gram-positive bacteriumBacillus azotoformans

Gram+ bacteria are capable of complete denitrification just like Gram- (Gram-negative) bacteria. However, Gram+ (Gram-positive) bacteria have a very small periplasmic-like space. This leads to the question of whether those enzymes and electron carriers involved in denitrification, which are normally located in the periplasmic space in Gram- bacteria, are located in the periplasmic-like space in Gram+ bacteria or have been modified as membrane-bound proteins. Using Bacillus azotoformans as a Gram+ bacterial model, our study demonstrates that anaerobic denitrification is catalysed by four membrane-bound enzymes and that the electron carriers are membrane-bound c-type cytochromes and menaquinol. NADH dehydrogenase is coupled with the denitrification pathway providing menaquinol. In addition, the cytochrome b(6)f complex forms part of the denitrification pathway, oxidizing menaquinol and reducing at least three different membrane-bound c-type cytochromes. We determined that the NO reductase, qCu(A)NOR (where NOR stands for nitric oxide reductase), can accept electrons from two donors, a specific cytochrome c(551) and menaquinol. Similarly, nitrite reductase, a copper enzyme, and nitrous oxide reductase may be bifunctional enzymes. Regarding the bifunctionality of qCu(A)NOR, we propose that the menaquinol-linked pathway is involved in the detoxification of NO.

NO Reductase from Bacillus azotoformans Is a Bifunctional Enzyme Accepting Electrons from Menaquinol and a Specific Endogenous Membrane-Bound Cytochrome c551

Bacillus azotoformans is a Gram-positive denitrifying soil bacterium, which is capable of respiring nitrate, nitrite, nitric oxide, and nitrous oxide under anaerobic conditions. It contains a unique menaquinol-dependent nitric oxide reductase (qCu(A)NOR) with a Cu(A) center in its small subunit. The qCu(A)NOR exhibits menaquinol-dependent NO reductase activity, whereas reduced horse heart cytochrome c was inactive. Here we describe the purification of three membrane-bound c cytochromes from B. azotoformans. Their apparent molecular masses on SDS-PAGE are approximately 11 kDa. At neutral pH, these c cytochromes are negatively charged and the E(m) for all is close to 150 mV. Only one of these c cytochromes, which exhibits an alpha-band maximum at 551 nm, acts as a direct electron donor to qCu(A)NOR. Further investigation demonstrated that this cytochrome c(551) possesses two lipoyl moieties, which presumably function to anchor it to the membrane. Steady-state kinetic studies reveal that cytochrome c(551) is a noncompetitive inhibitor of NO reduction when menaquinol is used as an electron donor. This finding points to the presence of two different electron donation pathways in qCu(A)NOR. The ability of qCu(A)NOR to accept electrons from both menaquinol and cytochrome c(551) might be related to the regulation of the rate of NO reduction especially as a defense mechanism of B. azotoformans against the toxicity of NO. Growth experiments in batch culture indeed show that B. azotoformans is highly NO tolerant, in contrast to, for example, Paracoccus denitrificans that has a monofunctional cytochrome c-dependent NOR. We propose that the menaquinol pathway, which has a 4-fold greater maximal activity than the pathway via cytochrome c(551), is used for NO detoxification, whereas electron donation via the endogenous cytochrome c involves the cytochrome b(6)f complex serving the bioenergetic needs of the organism.

A novel copper A containing menaquinol NO reductase from Bacillus azotoformans.

The molecular biology and biochemistry of denitrification in gram-negative bacteria has been studied extensively. However, little is known about this process in gram-positive bacteria. We have purified the NO reductase from the cytoplasmic membrane of the gram-positive bacterium Bacillus azotoformans. The purified enzyme consists of two subunits with apparent molecular masses of 16 and 40 kDa based on SDS-PAGE. Analytical and spectroscopic determinations revealed the presence of one non-heme iron, two copper atoms and of two b-type hemes per enzyme complex. Heme c was absent. Using EPR and UV-visible spectroscopy, it was determined that one of the hemes is a low-spin heme b, in which the two axial histidine imidazole planes are positioned at an angle of 60-70 degrees. The second heme b is high-spin binding CO in the reduced state. The high-spin heme center and the non-heme iron are EPR silent. They are proposed to form a binuclear center where reduction of NO occurs. There are two novel features of this enzyme that distinguish it from other NO reductases. First, the enzyme contains copper in form of copper A, an electron carrier up to now only detected in cytochrome oxidases and nitrous oxide reductases. Second, the enzyme uses menaquinol as electron donor, whereas cytochrome c, which is the substrate of other NO reductases, is not used. Copper A and both hemes are reducible by menaquinol. This new NO reductase is thus a menaquinol:NO oxidoreductase. With respect to its prosthetic groups the B. azotoformans NO reductase is a true hybrid between copper A containing cytochrome oxidases and NO reductases present in gram-negative bacteria. It may represent the most ancient "omnipotent" progenitor of the family of heme-copper oxidases.

Criteria and methodology for identifying respiratory denitrifiers

Respiratory denitrification is not always adequately established when bacteria are characterized. We have tested a simple method that allows one to evaluate whether the two necessary criteria to claim denitrification have been met, namely, that N(inf2) or N(inf2)O is produced from nitrate or nitrite and that this reduction is coupled to a growth yield increase. Microorganisms were cultured in sealed tubes under a helium headspace and in the presence of 0, 2, 4, 7, and 10 mM nitrate or nitrite. After growth had ceased, N(inf2) and N(inf2)O were quantified by gas chromatography and the final protein concentration was measured. Net protein production was linearly related to nitrate concentration for all denitrifiers tested and ranged from 2 to 6 g of protein per mol of electron equivalent reduced. Nitrogen recovery as N(inf2) plus N(inf2)O from nitrate and nitrite transformed exceeded 80% for all denitrifiers. We also suggest that a rate of N gas production of >10 (mu)mol/min/g of protein can be used as an additional characteristic definitive of denitrification since this process produces gas more rapidly than other processes. These characteristics were established after evaluation of a variety of well-characterized respiratory denitrifiers and other N(inf2)O-producing nitrate reducers. Several poorly characterized denitrifiers were also tested and confirmed as respiratory denitrifiers, including Aquaspirillum itersonii, Aquaspirillum fasciculus, Bacillus azotoformans, and Corynebacterium nephridii. These criteria distinguished respiratory denitrifiers from other groups that reduce nitrate or produce N(inf2)O. Furthermore, they correctly identified respiratory denitrification in weak denitrifiers, a group in which the existence of this process may be overlooked.

Two structurally different cytochromes c from Bacillus azotoformans: on the evolution of Gram-positive bacteria

c-552 and split-alpha c-555 cytochromes from Bacillus azotoformans are classified on the basis of partial sequence information. The haem-containing polypeptides are postulated to be structurally equivalent to small IC and ID subclass cytochromes found in purple bacteria.

Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria.

Polyclonal antibodies were used to identify heme or copper nitrite reductases in the following groups: 23 taxonomically diverse denitrifiers from culture collections, 100 numerically dominant denitrifiers from geographically diverse environments, and 51 denitrifiers from a culture collection not selected for denitrification. Antisera were raised against heme nitrite reductases from Pseudomonas aeruginosa and Pseudomonas stutzeri and against copper nitrite reductase from Achromobacter cycloclastes. Nitrite reductases were identified by Western immunoblot. Diethyldithiocarbamate, which specifically inhibits copper nitrite reductases, was used to confirm the immunological characterization and determine which type was present in strains nonreactive with any antiserum. For groups in which the type of nitrite reductase has not been previously described, we found that Alcaligenes eutrophus, Bacillus azotoformans, Bradyrhizobium japonicum, Corynebacterium nephridii, and Rhizobium spp. contained copper nitrite reductase, while Aquaspirillum itersonii, Flavobacterium spp., and Pseudomonas fluorescens contained heme nitrite reductase. Heme nitrite reductases dominated, regardless of soil type or geographic origin. They occurred in 64 and 92%, respectively, of denitrifiers in the numerically dominant and nonselected collections. The two nitrite reductase types were mutually exclusive in individual bacteria, but both appeared in different strains from the Alcaligenes and Pseudomonas genera. The heme type predominated in Pseudomonas strains. The heme-type nitrite reductase appeared more conserved if judged by similarities in molecular weights and immunological reactions. The Cu type was found in more taxonomically unrelated strains and varied in molecular weight and antiserum recognition.

Taxonomic Study of Bacillus coagulans Hammer 1915 with a Proposal for Bacillus smithii sp. nov.

Guanine-plus-cytosine (G+C) content determinations, deoxyribonucleic acid (DNA) relatedness estimations, and phenotypic similarity analyses of 90 strains identified previously as Bacillus coagulans Hammer 1915 revealed that 52 (group 1) strains were Bacillus coagulans sensu stricto; 30 of the remaining organisms segregated into two distinct DNA relatedness groups, one consisting of 26 (group 2) strains and the other of 4 (group 4) organisms. Five (group 3) strains were Bacillus licheniformis, and three (group 5) strains were B. stearothermophilus. Because group 2 was a major cluster of strains which did not correspond in their characteristics to any previously known group, efforts were made to determine its taxonomic position. Group 2, with G+C contents ranging from 37 to 40 mol%, was compared with other species generally characterized as having G+C contents ranging from 37 to 44 mol% or capable of growing at 50°C or both (namely, Bacillus alcalophilus, Bacillus azotoformans, Bacillus badius, Bacillus firmus, Bacillus globiformis, Bacillus laterosporus, Bacillus macquariensis, Bacillus marinus, Bacillus megaterium, Bacillus pumilus, and Bacillus subtilis). Low DNA relatedness values and poor matching of phenotypic characteristics strongly indicated that group 2 organisms are strains of a new species, for which the name Bacillus smithii is proposed. The type strain of the new species is strain NRRL NRS-173.