Gram-positive Sporeforming Research Articles

Troubleshooting high laboratory pasteurization counts in organic raw milk requires characterization of dominant thermoduric bacteria, which includes nonsporeformers as well as sporeformers

Laboratory pasteurization count (LPC) enumerates thermoduric bacteria and is one parameter used to assess raw milk quality. No regulatory limit has presently been set for LPC, but LPC data are used by some dairy processors and cooperatives to designate raw milk quality premiums paid to farmers and may also be used for troubleshooting bacterial contamination issues. Although it is occasionally used as a proxy for levels of bacterial spores in raw milk, limited knowledge is available on the types of organisms that are enumerated by LPC in contemporary raw milk supplies. Although historical studies have reported that thermoduric bacteria quantified by LPC may predominantly represent gram-positive cocci, updated knowledge on microbial populations enumerated by LPC in contemporary organic raw milk supplies is needed. To address this gap, organic raw milk samples from across the United States (n = 94) were assessed using LPC, and bacterial isolates were characterized. LPC ranged from below detection (<0.70 log cfu/mL) to 4.07 log cfu/mL, with a geometric mean of 1.48 log cfu/mL. Among 380 isolates characterized by 16S rDNA sequencing, 52.6%, 44.5%, and 2.4% were identified as gram-positive sporeformers, gram-positive nonsporeformers, and gram-negatives, respectively; 0.5% could not be categorized into those groups because they could only be assigned a higher level of taxonomy. Isolates identified as gram-positive sporeformers were predominantly Bacillus (168/200), and gram-positive nonsporeformers were predominantly Brachybacterium (56/169) and Kocuria (47/169). To elucidate if the LPC level can be an indicator of the type of thermoduric (e.g., sporeforming bacteria) present in raw milk, we evaluated the proportion of sporeformers in raw milk samples with LPC of ≤100 cfu/mL, 100 to 200 cfu/mL, and ≥200 cfu/mL (51%, 67%, and 35%), showing a trend for sporeformers to represent a smaller proportion of the total thermoduric population when LPC increases, although overall linear regression showed no significant association between the proportion of sporeformers and the LPC concentration. Hence, LPC level alone provides no insight into the makeup of the thermoduric population in raw milk, and further characterization is needed to elucidate the bacterial drivers of elevated LPC in raw milk. We therefore further characterized the isolates from this study using MALDI-TOF mass spectrometry (MALDI-TOF MS), a rapid microbial identification tool that is more readily available to dairy producers than 16S rDNA PCR and sequencing. Although our data indicated agreement between 16S rDNA sequencing and MALDI-TOF MS for 66.6% of isolates at the genus level, 24.2% and 9.2% could not be reliably identified or were mischaracterized using MALDI-TOF MS, respectively. This suggests that further optimization of this method is needed to allow for accurate characterization of thermoduric organisms commonly found in raw milk. Ultimately, our study provides a contemporary perspective on thermoduric bacteria selected by the LPC method and establishes that the LPC alone is not sufficient for identifying the bacterial drivers of LPC levels. Further development of rapid characterization methods that are accessible to producers, cooperatives, and processors will support milk quality troubleshooting efforts and ultimately improve outcomes for dairy industry community members.

Role of the Spore Coat Proteins CotA and CotB, and the Spore Surface Protein CDIF630_02480, on the Surface Distribution of Exosporium Proteins in Clostridioides difficile 630 Spores.

Clostridioides difficile is Gram-positive spore-former bacterium and the leading cause of nosocomial antibiotic-associated diarrhea. During disease, C. difficile forms metabolically dormant spores that persist in the host and contribute to recurrence of the disease. The outermost surface of C. difficile spores, termed the exosporium, plays an essential role in interactions with host surfaces and the immune system. The main exosporium proteins identified to date include three orthologues of the BclA family of collagen-like proteins, and three cysteine-rich proteins. However, how the underlying spore coat influences exosporium assembly remains unclear. In this work, we explore the contribution of spore coat proteins cotA and cotB, and the spore surface protein, CDIF630_02480, to the exosporium ultrastructure, formation of the polar appendage and the surface accessibility of exosporium proteins. Transmission electron micrographs of spores of insertional inactivation mutants demonstrate that while cotB contributes to the formation of thick-exosporium spores, cotA and CDIF630_02480 contribute to maintain proper thickness of the spore coat and exosporium layers, respectively. The effect of the absence of cotA, cotB and CDIF630_02480 on the surface accessibility of the exosporium proteins CdeA, CdeC, CdeM, BclA2 and BclA3 to antibodies was affected by the presence of the spore appendage, suggesting that different mechanisms of assembly of the exosporium layer might be implicated in each spore phenotype. Collectively, this work contributes to our understanding of the associations between spore coat and exosporium proteins, and how these associations affect the assembly of the spore outer layers. These results have implications for the development of anti-infecting agents targeting C. difficile spores.

Awareness of Botox Injections in Facial Aesthetics among Dental Students

The aim of this study was to evaluate the awareness and knowledge on the use of Botox injections in facial aesthetics among undergraduate dental students. Botulinum toxin is considered as a quick and effective non-surgical solution for enhancing physical appearance. Botox is considered as the best choice, as it is affordable and less radical than surgery. It is derived from gram-positive spore forming, clostridium botulinum. A questionnaire was developed to evaluate the awareness of dentists towards using botulinum toxins in facial aesthetics. The survey was conducted online via survey planet. There were about 103 study population, participated in this study. Data is collected using an online survey planet link and statistically analysed. 95.1% of participants reported that they have general knowledge on the Botox (95.1%). 66 % of respondents agreed that clostridium botulinum bacterium is the source of Botox. 24.51% of respondents stated that Botox was commonly used for wrinkle reduction and 96.1% were aware of the toxicity effect of Botox when it is administered in a high dosage. Association between response of the participants and knowledge on Botox was found to be statistically significant (p&lt;0.05). According to our study, the majority of students showed an acceptable level of knowledge and awareness on Botox and its use in facial aesthetics.

Regulation and Anaerobic Function of the Clostridioides difficile β-Lactamase.

Clostridioides difficile causes severe antibiotic-associated diarrhea and colitis. C. difficile is an anaerobic, Gram-positive sporeformer that is highly resistant to β-lactams, the most commonly prescribed antibiotics. The resistance of C. difficile to β-lactam antibiotics allows the pathogen to replicate and cause disease in antibiotic-treated patients. However, the mechanisms of β-lactam resistance in C. difficile are not fully understood. Our data reinforce prior evidence that C. difficile produces a β-lactamase, which is a common β-lactam resistance mechanism found in other bacterial species. Here, we characterize the C. difficilebla operon that encodes a lipoprotein of unknown function and a β-lactamase that was greatly induced in response to several classes of β-lactam antibiotics. An in-frame deletion of the operon abolished β-lactamase activity in C. difficile strain 630Δerm and resulted in decreased resistance to the β-lactam ampicillin. We found that the activity of this β-lactamase, BlaCDD, is dependent upon the redox state of the enzyme. In addition, we observed that transport of BlaCDD out of the cytosol and to the cell surface is facilitated by an N-terminal signal sequence. Our data demonstrate that a cotranscribed lipoprotein, BlaX, aids in BlaCDD activity. Further, we identified a conserved BlaRI regulatory system and demonstrated via insertional disruption that BlaRI controls transcription of the blaXCDD genes in response to β-lactams. These results provide support for the function of a β-lactamase in C. difficile antibiotic resistance and reveal the unique roles of a coregulated lipoprotein and reducing environment in C. difficile β-lactamase activity.

Response of bentonite microbial communities to stresses relevant to geodisposal of radioactive waste

Microbes have been isolated previously from bentonite materials that may be used as barriers for the disposal of radioactive waste. Actively respiring microbes in such barrier materials, within a repository environment, have the potential to adversely affect waste container corrosion rates. Additionally, they could potentially alter the properties of the bentonite barrier itself. This is of significance, since the integrity of the waste container and properties of the bentonite barrier are required to fulfil defined safety functions. To help identify the critical factors that affect microbial activity in bentonite materials, this study examines the impact of a range of parameters that could affect microbial metabolism in a geodisposal environment. Several bentonites from different sources (bentonite mined from locations in Spain and the USA, along with commercially-sourced bentonite) were subjected to increased pressure (74 MPa, 30 s), heat (90 °C, 24 h), and irradiation (1000 Gy, 24.17 Gy min−1), before incubation in growth media selective for sulfate-reducing bacteria (SRB) or iron-reducing bacteria (IRB). The amount of SRB, and IRB were counted using the most probable number method and identified by 16S rRNA gene sequencing. The bentonites initially contained 660–6600 SRB cells g−1, and the number of SRB was correlated with the initial water content of the bentonite. A similar number of IRB was also present (400–4000 cells g−1), and the number of IRB was correlated with the ratio of bioavailable Fe(II)/Fe(III) present in the bentonite. The bentonites hosted sulfate-reducing species from two bacterial genera, with Desulfotomaculum dominating the SRB communities in the Spanish bentonite used in the Full-scale Engineered Barriers Experiment (FEBEX), while the other communities contained Desulfosporosinus species. The nature of the SRB community played a significant role in the microbial community response to different stresses, with the FEBEX material producing high SRB cell counts in response to pressure and irradiation but yielding low numbers in response to heat. Initially, the IRB communities contained a mixture of Gram-negative bacteria such as Geobacter, and Gram-positive spore-forming bacteria such as Bacillus and Desulfosporosinus, with an increase in the number of Gram-positive spore-formers in response to stress. The ability of Gram-positive spore-formers to grow, despite exposure to pressure, heat and irradiation, highlights the need to generate a swelling pressure sufficient to minimise microbial activity. In addition, we suggest that the microbial communities naturally present in the bentonite should be considered as part of the selection process for buffer materials in a geological disposal facility for radioactive waste.

Clostridium difficile exosporium cysteine-rich proteins are essential for the morphogenesis of the exosporium layer, spore resistance, and affect C. difficile pathogenesis.

Clostridium difficile is a Gram-positive spore-former bacterium and the leading cause of nosocomial antibiotic-associated diarrhea that can culminate in fatal colitis. During the infection, C. difficile produces metabolically dormant spores, which persist in the host and can cause recurrence of the infection. The surface of C. difficile spores seems to be the key in spore-host interactions and persistence. The proteome of the outermost exosporium layer of C. difficile spores has been determined, identifying two cysteine-rich exosporium proteins, CdeC and CdeM. In this work, we explore the contribution of both cysteine-rich proteins in exosporium integrity, spore biology and pathogenesis. Using targeted mutagenesis coupled with transmission electron microscopy we demonstrate that both cysteine rich proteins, CdeC and CdeM, are morphogenetic factors of the exosporium layer of C. difficile spores. Notably, cdeC, but not cdeM spores, exhibited defective spore coat, and were more sensitive to ethanol, heat and phagocytic cells. In a healthy colonic mucosa (mouse ileal loop assay), cdeC and cdeM spore adherence was lower than that of wild-type spores; while in a mouse model of recurrence of the disease, cdeC mutant exhibited an increased infection and persistence during recurrence. In a competitive infection mouse model, cdeC mutant had increased fitness over wild-type. Through complementation analysis with FLAG fusion of known exosporium and coat proteins, we demonstrate that CdeC and CdeM are required for the recruitment of several exosporium proteins to the surface of C. difficile spores. CdeC appears to be conserved exclusively in related Peptostreptococcaeace family members, while CdeM is unique to C. difficile. Our results sheds light on how CdeC and CdeM affect the biology of C. difficile spores and the assembly of the exosporium layer and, demonstrate that CdeC affect C. difficile pathogenesis.

Corrigendum: Bacteriophages and bacteriophage-derived endolysins as potential therapeutics to combat Gram-positive sporeforming bacteria.

Since their discovery in 1915, bacteriophages have been routinely used within Eastern Europe to treat a variety of bacterial infections. Although initially ignored by the West due to the success of antibiotics, increasing levels and diversity of antibiotic resistance is driving a renaissance for bacteriophage-derived therapy, which is in part due to the highly specific nature of bacteriophages as well as their relative abundance. This review focuses on the bacteriophages and derived lysins of relevant Gram-positive spore formers within the Bacillus cereus group and Clostridium genus that could have applications within the medical, food and environmental sectors.

Rhizosphere microorganisms affected by soil solarization and cover cropping in Capsicum annuum and Phaseolus lunatus agroecosystems

Field experiments were conducted to evaluate the effects of soil solarization or cover crop on bell pepper (Capsicum annuum) and lima bean (Phaseolus lunatus) rhizosphere microorganisms. In Experiment I, flat surface solarization (FSS), raised bed solarization (RBS), cowpea (Vigna unguiculata) cover crop alone (CP), and the combination of cowpea cover crop followed by raised bed soil solarization (CP+RBS) were compared with methyl bromide fumigation (MB) and an untreated control (UTC) for effects on rhizosphere bacteria and fungi on bell pepper. In Experiment II, effects of cover crop on rhizosphere microorganisms were examined on lima bean in a 3×3 split-plot experiment with three summer cover crop treatments: sunn hemp (SH) (Crotalaria juncea), cowpea (CP), and fallow (F) as main plots, and three organic amendments: sunn hemp hay (SHH), cowpea hay (CPH), and no hay (NH) as subplots. Rhizosphere bacteria including siderophore producers, gram-positive spore-formers, and fluorescent pseudomonads were assessed, as well as two fungi, Fusarium oxysporum and Rhizoctonia solani. In year 1 of Experiment I, CP+RBS reduced F. oxysporum and R. solani in the rhizosphere, resulting in levels similar to those in MB fumigation. This was not observed in year 2. Heavy rainfall in year 2 resulted in a severe outbreak of Pythium on bell pepper, which was most destructive in MB fumigated plots. Effects of solarization and cover crop on rhizosphere bacteria also differed between years. Fluorescent pseudomonads were abundant across treatments in year 1, but were greatly reduced in year 2, when the Pythium outbreak occurred. In Experiment II, populations of rhizobacteria on lima bean were not affected by cover crop in either year. At the end of year 1, isolation of R. solani from lima bean roots increased in SHH plots. SH cover crop reduced gram-positive spore forming bacteria and fluorescent pseudomonads. In year 2, CP followed by CPH had higher levels of fungi and bacteria than CP followed by either NH or SHH. Overall, cover crop amendments did not result in consistent enhancement of rhizobacteria, and some treatments resulted in increased abundance of pathogenic fungi. The most consistent treatment for reducing rhizosphere fungal populations was MB.

Taxonomy, virulence and life cycles of Bacillus cereus sensu lato

Abstract Bacillus cereus sensu lato is a group of several species of Gram-positive sporeformers ubiquitous in nature and showing huge impact on human activities. They are often found in soil, air, plant material, animal tissues and digestive tracts as well as in food products. Their genetic similarities and frequent horizontal gene transfer causes doubts regarding their taxonomy. In addition, their toxicity and psychrotolerance constitute serious problems in the dairy industry, being responsible for food-poisonings and spoilage of cold-stored products. Finally, recent finding indicate that B. cereus sensu lato toxicity plays an important role not only in their virulence, but also in social interactions with other bacteria. 1. Introduction. 2. The most important aspects of B. cereus sensu lato biology. 2.1. First challenge – coherent taxonomy. 2.2. Second challenge – life cycles and interactions with the environment. 2.3. Third challenge – adaptation to low temperatures. 2.4. Fourth challenge – toxins of B. cereus sensu lato. 3. Summary

Culturability and diversity of microorganisms recovered from an eight-year old highly-compacted, saturated MX-80 Wyoming bentonite plug

Highly-compacted bentonite (HCB)-based sealing systems are being developed for potential use in nuclear fuel waste repositories because of their inherent physicochemical and rheological properties. Results from previous short-duration (40–90days) studies with highly-compacted (HC), water infused, confined Wyoming MX-80 bentonite revealed that a uniform dry density≥ 1.6g/cm3 (resulting in a swelling pressure above 2000KPa, water activity below 0.96 and an average pore size of <0.02μm) suppressed microbial culturability. A longer-term (7-year, 264-day) experiment using the same material and conditions was therefore conducted to determine whether these conclusions held over extended periods. Viable cell counts in material from the ~8-yr clay plug approximated those in the original uncompacted bentonite, confirming suppression of microbial growth. Bacteria recovered from the ~8yr clay plug were comprised of primarily Gram-positive spore-formers. Together, these results confirmed that HCB suppressed microbial culturability (and presumably activity and growth) over extended time periods, and further revealed that the surviving culturable microorganisms were those adapted to life in low water activity soil environments.

Bacteriophages and bacteriophage-derived endolysins as potential therapeutics to combat Gram-positive spore forming bacteria.

Since their discovery in 1915, bacteriophages have been routinely used within Eastern Europe to treat a variety of bacterial infections. Although initially ignored by the West due to the success of antibiotics, increasing levels and diversity of antibiotic resistance is driving a renaissance for bacteriophage-derived therapy, which is in part due to the highly specific nature of bacteriophages as well as their relative abundance. This review focuses on the bacteriophages and derived lysins of relevant Gram-positive spore formers within the Bacillus cereus group and Clostridium genus that could have applications within the medical, food and environmental sectors.

The potential of flow cytometry in the study of Bacillus cereus

Flow cytometry (FCM) is a rapid method allowing the acquisition of multiparametric data from thousands of individual cells within a sample. As well as measuring the intrinsic light scattering properties of cells, a plethora of fluorescent dyes may be employed to yield information on macromolecule content, surface antigens present or physiological status. Despite FCM's indispensability within other fields e.g. immunology, it is underutilized within microbiological research. In this review, a strong case is presented for the potential of FCM in the study of Gram-positive spore-former, Bacillus cereus. Previous reports where FCM was successfully used in the study of B. cereus are reviewed along with relevant studies involving other members of the genus. Under headings reflecting common research themes associated with B. cereus, specific instances where FCM has generated novel data, providing a unique insight into the organism, are discussed. Further applications are posited, based on the authors' own research with FCM and B. cereus and work extant in the broader field of microbial cytometry. The authors conclude that, while the expense of equipment and reagents is an undeniable disadvantage, FCM is a technique capable of generating significantly novel data and allows the design and execution of experiments that are not possible with any other technique.

Microbial populations stimulated for hexavalent uranium reduction in uranium mine sediment.

Uranium-contaminated sediment and water collected from an inactive uranium mine were incubated anaerobically with organic substrates. Stimulated microbial populations removed U almost entirely from solution within 1 month. X-ray absorption near-edge structure analysis showed that U(VI) was reduced to U(IV) during the incubation. Observations by transmission electron microscopy, selected area diffraction pattern analysis, and energy-dispersive X-ray spectroscopic analysis showed two distinct types of prokaryotic cells that precipitated only a U(IV) mineral uraninite (UO(2)) or both uraninite and metal sulfides. Prokaryotic cells associated with uraninite and metal sulfides were inferred to be sulfate-reducing bacteria. Phylogenetic analysis of 16S ribosomal DNA obtained from the original and incubated sediments revealed that microbial populations were changed from microaerophilic Proteobacteria to anaerobic low-G+C gram-positive sporeforming bacteria by the incubation. Forty-two out of 94 clones from the incubated sediment were related to sulfate-reducing Desulfosporosinus spp., and 23 were related to fermentative Clostridium spp. The results suggest that, if in situ bioremediation were attempted in the uranium mine ponds, Desulfosporosinus spp. would be a major contributor to U(VI) and sulfate reduction and Clostridium spp. to U(VI) reduction.

Protease production by a thermophilic Bacillus species (P-001A) which degrades various kinds of fibrous proteins

An aerobic Gram-positive sporeforming bacterium was isolated from an alkaline hot spring at Wondo Genet, Ethiopia. In an optimized culture medium it produced maximum activity of protease at 55°C and pH 9.5. The protease activity against casein was 65 units/ml. Enzyme activity was detected between 30–70°C and pH of 4.5–11.5. The enzyme had a half-life of 55 and 30 min at 60° and 70°C, respectively. The isolate hydrolysed 90, 60 and 50% of the skin, feather and horn used in the optimized medium within 120 h.

Characteristics of a Rumen Clostridium Capable of Degrading Mimosine, 3(OH)-4-(1H)-Pyridone and 2,3 Dihydroxypyridine

Summary A Gram-positive sporeforming bacterium isolated from the rumen of sheep in Venezuela is described. The isolated strain (162) degraded the toxic amino acid mimosine, a component of the forage legume Leucaena. Strain 162 also degraded two products of mimosine metabolism, 3(hydroxy)-4-(1H)-pyridone and 2,3 dihydroxypyridine. Dihydroxyphenylalanine was also degraded, but 3 hydroxypyridine and catechol were not. Glucose, maltose and trehalose were fermented and supported growth. The major products were acetate, butyrate and ethanol, with traces of propionate, isobutyrate, isovalerate, valerate and hydrogen. Phytone stimulated growth, the optimal temperature for which was 38°C. The mol% G plus C in the DNA was 23. The strain appears to be a representative of a previously unrecognised species of the genus Clostridium.

Degradation of mimosine, 2,3-dihydroxy pyridine and 3-hydroxy-4(1H)-pyridine by bacteria from the rumen of sheep in Venezuela

The addition of Leucaena leucocephala herbage did not diet of sheep in Venezuela did not affect the concentration of volatile fatty acids (VFA) in the rumen, the degradation of rice straw incubated in sacco, or the numbers of rumen fungi or bacteria. However, feeding Leucaena increased the concentration of ammonia in the rumen. In addition, two products of the degradation of the toxic amino acid mimosine were detected in the rumen when Leucaena was fed. One of these products, 2,3-dihydroxy pyridine (2,3-DHP), was detected at concentrations of up to 1.1 μmol/ml. The other, 3-hydroxy-4(1H)-pyridone (3,4-DHP) was found at concentrations of up to 0.96 μmol/ml. The examination of bacterial cultures isolated from the rumen of the sheep under investigation showed that feeding Leucaena increased the relative proportions of short Gram-negative rods and decreased the proportion of long roads and coccobacilli present. Although the animals fed Leucaena showed a small loss in weight during the feeding trial, no evidence of Leucaena toxicity was seen. A total of 18 cultures capable of degrading 2,3-DHP or 3,4-DHP were isolated from the rumen of the sheep before Leucaena was fed. These included both Gram-positive and Gram-negative bacteria, and a Gram-positive sporeformer. It seems that 2,3-DHP and 3,4-DHP may be degraded by a much wider range of bacteria than has been recognised previously. The detection of these bacteria before Leucaena was fed suggests that they were indigenous members of the rumen microflora of sheep in Venezuela.

Degradation of mimosine, 2,3-dihydroxy pyridine and 3-hydroxy-4(1H)-pyridine by bacteria from the rumen of sheep in Venezuela

The addition of Leucaena leucocephala herbage did not diet of sheep in Venezuela did not affect the concentration of volatile fatty acids (VFA) in the rumen, the degradation of rice straw incubated in sacco, or the numbers of rumen fungi or bacteria. However, feeding Leucaena increased the concentration of ammonia in the rumen. In addition, two products of the degradation of the toxic amino acid mimosine were detected in the rumen when Leucaena was fed. One of these products, 2,3-dihydroxy pyridine (2,3-DHP), was detected at concentrations of up to 1.1 μmol/ml. The other, 3-hydroxy-4(1H)-pyridone (3,4-DHP) was found at concentrations of up to 0.96 μmol/ml. The examination of bacterial cultures isolated from the rumen of the sheep under investigation showed that feeding Leucaena increased the relative proportions of short Gram-negative rods and decreased the proportion of long roads and coccobacilli present. Although the animals fed Leucaena showed a small loss in weight during the feeding trial, no evidence of Leucaena toxicity was seen. A total of 18 cultures capable of degrading 2,3-DHP or 3,4-DHP were isolated from the rumen of the sheep before Leucaena was fed. These included both Gram-positive and Gram-negative bacteria, and a Gram-positive sporeformer. It seems that 2,3-DHP and 3,4-DHP may be degraded by a much wider range of bacteria than has been recognised previously. The detection of these bacteria before Leucaena was fed suggests that they were indigenous members of the rumen microflora of sheep in Venezuela.

Effect of delignification pretreatment and selective enrichment on methane production from cattle waste

Attempts were made to see whether delignification by pretreatment of cattle waste with alkaline peroxide or by selective enrichment of gram-negative and gram-positive spore formers in the digester with penicillin and heat treatment will lead to biogas improvement

14] Cytochrome oxidase from thermophilic bacterium PS3

This chapter focuses on the cytochrome oxidase obtained from thermophilic bacteria PS3. The thermophilic bacterium PS3 is a Gram-positive spore former. It produces a-, b-, and c-type cytochromes. Under highly aerobic conditions, the terminal oxidase is an aa3-type cytochrome oxidase, but under air-restricted conditions cytochrome o replaces cytochrome aa3. From cells grown under highly aerated conditions, aa3-type cytochrome oxidase can be prepared by following the α-band spectrum of the reduced form. The chapter describes a freeze-thaw method for reconstituting beef heart cytochrome oxidase and Thermus thermophilus cytochrome oxidase into vesicles. The cholate-deoxycholate dialysis method was not good for reconstitution of PS3 cytochrome oxidase. Two or three cycles of the freeze-thaw sonication gave the highest H+ pumping activity and a slow leakage. PS3 cytochrome oxidase vesicles thus prepared pump H+ upon addition of reduced cytochrome c under aerobic conditions.

Microbial ecology of the soils of Indian desert

A comprehensive study on the microbial ecology of Indian desert soils was carried out. Although different groups of microorganisms e.g., fungi, actinomycetes, bacteria, Azotobacter and nitrifying bacteria were present, but in relatively low numbers, there was considerable variation in numbers from one site to another within the desert as the physicochemical characters of the soils varied. Actinomycetes formed a high proportion of the total microflora. In the majority of the soils, 80% of the fungal population belonged to the genera Aspergillus and Penicillium, while Gram-positive spore formers were the dominant group among bacteria. No significant decline in the population of microorganisms was observed during summer, in spite of high surface soil temperatures. Microbiological properties of these soils also varied depending on the type of land use pattern: grasslands, in general, supported higher numbers of microorganisms than three plantations, cultivated fields or barren land. Stabilization of shifting and sand dunes introduction of vegetation has markedly increased the soil microflora. In general the low organic matter content and poor moisture availability of desert soils were the major factors limiting optimum microbial activity.