Subtilis Extracts Research Articles

Exploring the antibacterial efficacy of Satureja hortensis and Bacillus subtilis extracts in combating Xanthomonas citri

Biofilm plays a critical role in protecting the associated bacterial colonies. Citrus canker caused by Xanthomonas citri, resulted in remarkable yield reduction in citrus orchards. This study aims to evaluate the anti-biofilm properties of S. hortensis and B. subtilis extracts against X. citri. The antibacterial effects of both extracts were evaluated using the disk diffusion method and 96-well microdilution plates. Crystal violet and XTT procedures were employed to assess the inhibition of adhesion and antibiofilm effects of both extracts. The checkboard titration method was applied to determine the synergistic effects of the plant-bacterial extracts. The antibiofilm effects were confirmed by the light microscopy method. The results showed that the antibacterial and antibiofilm properties of both extracts varied. The MIC values of S. hortensis and B. subtilis extracts were 6.25 and 50 mg/ml, respectively. In these concentrations the inhibition of adhesion effects of S. hortensis and B. subtilis were 70% and 80%, whereas their antibiofilm effects were 60% and 72%. S. hortensis and B. subtilis extracts showed 63% and 76% antibiofilm activity, respectively, using the XTT test. The extracts showed synergistic effects, resulting in higher inhibition rates in combinatorial applications. Molecular docking results also confirmed that the secondary metabolites of S. hortensis could interact with different X. citri proteins differentially. Further large-scale studies on combinatorial antibiofilm effects of plant and bacterial extracts against citrus pathogens will provide new opportunities to develop safe and applicable reagents to control these destructive diseases.

Bacillus subtilis Modulated the Expression of Osteogenic Markers in a Human Osteoblast Cell Line.

Several in vivo trials have previously demonstrated the beneficial effects of the administration of various probiotic forms on bone health. In this study, we explored the potency of two probiotics, Bacillus subtilis and Lactococcus lactis, alone or in combination with vitamin D (VD), to modulate the transcription of genes involved in the ossification process in a human osteoblast cell line. Genes that mark the "osteoblast proliferation phase", such as RUNX2, TGFB1, and ALPL, "extracellular matrix (ECM) maturation", such as SPP1 and SPARC, as well as "ECM mineralization", such as BGN, BGLAP, and DCN, were all highly expressed in osteoblasts treated with B. subtilis extract. The observed increase in the transcription of the ALPL mRNA was further in agreement with its protein levels as observed by Western blot and immunofluorescence. Therefore, this higher transcription and translation of alkaline phosphatase in osteoblasts treated with the B. subtilis extract, indicated its substantial osteogenic impact on human osteoblasts. Although both the probiotic extracts showed no osteogenic synergy with VD, treatment with B. subtilis alone could increase the ECM mineralization, outperforming the effects of L. lactis and even VD. Furthermore, these results supported the validity of employing probiotic extracts rather than live cells to investigate the effects of probiotics in the in vitro systems.

Evaluation of chemical composition, trace element content, antioxidant and antimicrobial activities of Verbascum pseudoholotrichum

Given that some Verbascum species are effective against influenza viruses, an endemic species, Verbascum pseudoholotrichum is thought to have the potential to be used in the treatment of virucidal diseases. It was extensively investigated for its antioxidant and antimicrobial activities, chemical and trace element composition. The soxhlet extraction method was applied in the extraction process of the above-ground parts of the plant using three solvents (methanol, ethanol and water). The total phenolic content of extracts was between 175.7 and 186.1 mg GAE/100 g on the dried weight basis. DPPH radical scavenging activity analysis was performed and IC50 values were 0.539–1.254 mg mL−1. In total, 38 compounds were detected by GC–MS analysis. ICP-MS analysis was applied to identify the total contents of 24 elements in dried samples. The antimicrobial activity of the extracts was evaluated on six different pathogens using the spectrophotometric microdilution technique. The obtained minimum inhibitory concentration values were 17.37 µ–113.9 µg mL−1 at the end of the 24-h incubation. The highest and lowest inhibition zones were 5.0 and 0.3 mm for ethanol extract against Bacillus subtilis and water extract against Enterococcus fecalis, respectively, and no resistance was seen against Escherichia coli using disc diffusion method.

The Study of Bacillus Subtils Antimicrobial Activity on Some of the Pathological Isolates

The study involved the selection of two isolates from Bacillus subtilis to investigate their inhibitory activity against some bacterial pathogens. B sub-bacteria were found to have a broad spectrum against test bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa. They were about 23-30 mm and less against Klebsiella sp. The sensitivity of some antibodies was tested on the test samples. The results showed that the inhibitory ability of bacterial growth in the test samples using B. subtilis extract was more effective than the antibiotics used.

Isolation and Identification of Antibiotic-Producing Halophilic Bacteria from Dagh Biarjmand and Haj Aligholi Salt Deserts, Iran

Background: Halophilic bacteria are potent organisms in production of novel bioactive antimicrobial compounds which might be considered in drug innovation and control of plant pathogens. Salt deserts in Semnan province are of the most permanent hypersaline areas in the North of Iran. Despite the importance of these areas, there is no scientific report regarding the biodiversity and potency of their halophilic bacteria. Thus, aforementioned areas were selected to detect the halophilic bacteria. Methods: Here, seven strains were isolated and cultured on their molecular and biochemical properties were characterized. To determine the antibiotic potency of the isolates, agar well diffusion method was conducted. Phylogenetic analysis was done to reveal the isolates relationship with previously known strains. Results: As a result, growth of the strains in the medium containing 5 to 20% (w/v) NaCl determined that the majority of the isolates were moderately halophile. Catalase activity of all strains was positive. The results represented that D6A, Dar and D8B have antimicrobial effects against different plant and human pathogens. Phylogenic tree analysis also showed that two strains of D6A and Dar are belonged to Bacillus subtilis and D8B is belonged to Virgibacillus olivae. The bacteria extracts were evaluated for their antifungal and antibacterial activities on human and Plant pathogenic strains. The MIC of the extract B. subtilis against was found active against human pathogenic fungi and Plant pathogenic bacteria and fungi, ranging from 12.5 to 25 µg/mL. Conclusion: This study highlights the therapeutic and prophylactic potential of B. subtilis extracts as antibacterial and antifungal agents.

STUDY OF ANTIMICROBIAL ACTIVITY OF THE FOLKLORE MEDICINAL PLANT, ACALYPHA FRUTICOSA FORSSK.

The purpose of the study is to examine the antimicrobial efficacy of root extracts of the folklore medicinal plant species, Acalypha fruticosa Forssk by using three alcoholic solvents viz; petroleum ether, ethyl acetate and methanol were tested against ten human pathogenic bacteria viz., Pseudomonas aeruginosa, P. stutzeri, Escherichia coli, Micrococcus sp., Lactobacillus sp., Servatia sp., Moraxetta sp., Bacillus subtilis, B. thuriengensis, and Klebsiella pneumoniae and ten human pathogenic fungi viz., Aspergillus niger, A. flavus, A.baumannii, Fusarium oxysporum, F. solani, Mucor rouxii, Alternaria alternata, Candida albicans, Cladosporium sp. and Rhizopus sp. for assessing the antimicrobial properties by adapting disc diffusion method. The results of the study revealed that all extracts showed varied degree of antimicrobial activity against the tested pathogens. However, the methanol extracts exhibited higher inhibition zone (21.83 mm) against the bacterium, Bacillus subtilis and ethyl acetate extracts showed higher inhibiotion zone (24.83 mm) against the fungus, Rhizopus sp. Results concluded that this spices contain high amount of secondary metabolites due to these metabolites they have high antimicrobial activity and it can be used as good bio- preservater and it can also use for medicinal purpose.

Кариопатическое действие биопрепарата Bacillus subtilis 12В на состояние сперматогенного эпителия животных при воздействии гельминтов

Objective of research: to study the caryopathic effect of bio-preparation Bacillus subtilis
 12В on the status of spermatogenic epithelium of white mice after a single intra-abdominal
 administration of the Fasciola hepatica extract.
 Materials and methods: The investigations were conducted on white male mice after oral
 use of preparation Sporovite based on B. subtilis 12B; then the intra-abdominal injection of F.
 hepatica extracts at a dose of 100 mkg/head was applied. The protein extract from F. hepatica
 was administered to mice of the second group, and animals of the third group received only
 the probiotic Sporovite. Animals of the fourth group did not get the preparation and served as
 controls. 48 hours later the animals were killed; touch smears obtained from testis were stained
 by the Romanovsky method and examined under a microscope what enables to determine the mitotic index and the number of pathological meiosis forms. The experiments were conducted
 on lambs whose seminal vesicles were placed into a solution of 10% Formalin and examined
 histologically. 2-3μ-thick slices were stained with Haematoxylin and Eosin by Van Gieson method
 and examined under a microscope at 50, 400 and 1000 x magnifications.
 Results and discussion: During the pathomorphological and caryomitotic studies of testis
 of white mice and lambs after administration of Sporovite on the background of intra-abdominal
 injection of F. hepatica extract the reduction of negative effects on the status of spermatogenic
 epithelium of testis in animals wasn’t observed. A decrease in mitotic index by 2-3 times and a
 high amount of pathological forms were registered. The number of metaphases with preterm
 chromosome disjunction under the joint effect of F. hepatica and B. subtilis extracts has decreased
 by half.

In vitro Antibacterial and Antifungal Activity of Salvia multicaulis

Some aromatic plants such as Salvia are used for medical purposes. Compounds such as germacrene, linalool, 1,8-cineole (eucalyptol), borneol, ot-pinene, ß-pinene, camphor and camphene are found in the genus Salvia. Salvia multicaulis is one of 17 Salvia species endemic to Iran. In this study, antimicrobial activity of essential oil and ethyl acetate and ether extracts of S. multicaulis were examined against some species of bacteria and fungi. The essential oil of the aerial part of S. multicaulis was examined by GC and GC-MS. In the essential oil of S .multicaulis 27 Compounds have been identified. Benzyl benzoate (60.3 %), n-hexyl benzoate (16.7 %), Amyl benzoate (5.2 %) and 2- octyl benzoate (4.2 %) were the main components of the essential oil. The essential oil analysis showed greatest antimicrobial activity against Staphylococcus epidermidis (5.3 µg/ml) and S. cerevisiae (9.3 µg/ml). The ethyl acetate showed greatest antimicrobial activity against Bacillus subtilis (106.7 µg/ml), Candida albicans (5.3 µg/ml) and ether extract showed greatest antimicrobial activity against Klebseilla pneumoniae (10.7 µg/ml) and Saccharomyces cerevisiae (10.7 µg/ml). In conclusion, we suggest that the antimicrobial activity of S. multicaulis may be due to its content of germacrene and linalool.

Expression of, and in vivo stressosome formation by, single members of the RsbR protein family in Bacillus subtilis

The Bacillus subtilis stressosome is a 1.8 MDa complex that is the focal point for activating the bacterium's general response to physical stress. In vitro studies demonstrated that the stressosome's core element can be formed from one or more of a family of paralogous proteins (RsbRA, -RB, -RC and -RD) onto which the system's activator protein (RsbT) and its principal inhibitor (RsbS) are bound. The RsbR components of the stressosome are envisioned to be the initial receptors of stress signalling with the stressosome structure itself serving as a device to integrate multiple stress signals for a coordinated response. In the current work, we examine several of the in vivo characteristics of the RsbR family members, including their expression and ability to form stressosomes to regulate sigma(B). Translational fusions of lacZ to each rsbR paralogue revealed that rsbRA, -RB and -RC are expressed at similar levels, which remain relatively constant during growth, ethanol stress and entry into stationary phase. rsbRD, in contrast, is expressed at a level that is only slightly above background during growth, but is induced to 30 % of the rsbRA expression level following ethanol stress. Velocity sedimentation analyses of B. subtilis extracts from strains expressing single rsbR paralogues demonstrated that each incorporates RsbS into fast-sedimenting complexes. However, consistent with rsbRD's lower expression, the RsbRD-dependent RsbS complexes were present at only 20 % of the level of the complexes seen in a wild-type strain. The lower stressosome level in the RsbRD strain is still able to hold RsbT's activity in check, implying that the RsbR/S component of stressosomes is normally in excess for the control of RsbT. Consistent with such a notion, reporter gene and Western blot assays demonstrate that although RsbT is synthesized at the same rate as RsbRA and RsbS, RsbT's ultimate level in growing B. subtilis is only 10 % that of RsbRA. Apparently, RsbT's inherent structure and/or its passage between the stressosome and its activation target compromises its persistence.

Bacillus subtilis inhibits growth of Cladophialophora carrionii in vitro

The antifungal activity of Bacillus subtilis against a strain of Cladophialophora carrionii was evaluated using plate diffusion (PD) and sealed plate (SP) methods. For the PD test, the inoculum was obtained by homogenizing the log-phase fungus, adjusted to 0.5 McFarland and seeded (0.5 ml) on Sabouraud dextrose agar (SDA) plates; the B. subtilis extract was prepared from an 8-day culture, whose supernatant was lyophilized, concentrated 10 × and distributed (40 μl) in 4-mm holes previously punched in the seeded SDA plates. The plates were incubated at 30 °C for 72 hours. For the SP method, the inoculum was obtained from a 3-day fungal culture, seeded in the center of a SDA plate and placed over a SDA plate which had been inoculated with 0.2 ml of B. subtilis suspension. The two plates were sealed together and incubated for 7 days, either at room temperature or at 30 °C. With the PD method, a 20-mm diameter halo was observed after 72 h of incubation and the SP method showed 25% of fungal inhibition at both temperatures assayed. The results obtained with either method suggest the presence of metabolites from the bacterium that effectively inhibit the growth of C. carrionii. Although a single strain of C. carrionii was used here, these preliminary results provide grounds for further work to confirm the antifungal potential of B. subtilis against agents of chromoblastomycosis or to further characterize the chemical nature of the inhibitory substances involved.

Associations between Bacillus subtilis B regulators in cell extracts

The general stress regulon of Bacillus subtilis is induced by the activation of the sigma(B) transcription factor. Activation of sigma(B) occurs as a consequence of the dephosphorylation of its positive regulator RsbV by one of two phosphatases that respond to either physical or nutritional stress. The physical stress phosphatase (RsbU) requires a second protein (RsbT) for activity. Stress is thought to initiate a process that triggers the release of RsbT from a large inhibitory complex composed of multiple copies of two protein species, RsbR (and/or its paralogues) and RsbS. The stress-derived signal driving RsbT release is unknown, but it fails to develop in B. subtilis lacking either ribosome protein L11 or the ribosome-associated protein Obg. RsbR, RsbS, RsbT, Obg and ribosomes elute in common high-molecular-mass fractions during gel-filtration chromatography of crude B. subtilis extracts. This paper reports the investigation of the basis of this coelution by the examining of associations between these proteins in extracts prepared from wild-type and mutant B. subtilis, and Escherichia coli engineered to express RsbR, RsbS and RsbT. Large RsbR/RsbS complexes, distinct from ribosomes, were detected in extracts of both B. subtilis and E. coli. In E. coli, high-molecular-mass forms of RsbS were less abundant when RsbR was absent, but in B. subtilis, only when both RsbR and its principal paralogues were missing from the extract was this form less abundant. This finding is consistent with the notion that the RsbR paralogues, present in B. subtilis but not E. coli, can substitute for RsbR in such complexes. RsbT was not bound to RsbR/RsbS in any extract that was examined, including one prepared from a B. subtilis strain with an RsbS variant (RsbS59SA) that is believed to continuously associate with RsbT. The high-molecular-mass forms of RsbT were found to be Triton-sensitive and independent of any other B. subtilis protein for their formation. These probably represent RsbT aggregates. The data suggest that the contribution of ribosomes/Obg to sigma(B) activation does not involve formation of a stable association between these proteins and the Rsb complex. In addition, the binding of RsbT to RsbS/RsbR appears to be more labile than the binding between the previously analysed Rsb proteins which form inhibitory complexes. This, and the apparent proclivity of RsbT to aggregate, suggests an inherent instability in RsbT which may play a role in its regulation.

Guanine nucleotides stabilize the binding of Bacillus subtilis Obg to ribosomes

Obg is a GTP-binding protein of Bacillus subtilis with essential, but undefined roles in the bacterium’s growth, sporulation, and stress responses. Obg orthologs are widely conserved among both bacteria and eukaryotes. Gel filtration and affinity blot assays have suggested that Obg may be ribosome-associated. In the current work, we continue an examination of the putative Obg:ribosome interaction. Velocity centrifugation analyses of crude B. subtilis extracts or purified Obg:ribosome mixtures suggest that Obg is initially ribosome-bound, but can separate from ribosomes during sedimentation in the absence of added nucleotides. Addition of either GTP, GDP or ATP to the gradient prolonged the Obg:ribosome association, while inclusion of a nonhydrolyzable GTP analog (5-guanylyl-imidodiphosphate) preserved it. The data strengthen the notion that Obg is a ribosome-associated protein, demonstrate that Obg’s association with ribosomes is stabilized by GTP, and indicate that the ribosome-bound Obg can likely hydrolyze GTP and be released as a consequence.

Interactions between a Bacillus subtilis anti-sigma factor (RsbW) and its antagonist (RsbV)

The activity of sigma B, a secondary sigma factor of Bacillus subtilis, is primarily controlled by an anti-sigma factor protein (RsbW) that binds to sigma B and blocks its ability to form an RNA polymerase holoenzyme (E-sigma B). Inhibition of sigma B by RsbW is counteracted by RsbV, a protein that is essential for the activation of sigma B-dependent transcription. When crude B. subtilis extracts were fractionated by gel filtration chromatography or electrophoresis through nondenaturing polyacrylamide gels, a complex composed of RsbW and RsbV that is distinct from the previously observed RsbW-sigma B complex was detected. In analogous experiments, RsbX, an additional regulator of sigma B-dependent transcription that is thought to act independently of RsbV-RsbW, was not found to associate with any of the other sigB operon products. Two forms of RsbV were visualized when crude cell extracts of B. subtilis were subjected to isoelectric focusing (IEF), with the more negatively charged RsbV species absent from extracts prepared from RsbW- strains. In vitro, RsbV became phosphorylated when incubated with ATP and RsbW but not with ATP alone. The phosphorylated RsbV species comigrated during IEF with the RsbW-dependent form of RsbV found in crude cell extracts. These results suggest that the modified RsbV, present in crude cell extracts, is phosphorylated. When gel filtration fractions containing RsbV-RsbW complexes or RsbV alone were subjected to IEF, only the unmodified form of RsbV was found associated with RsbW. The presumed phosphorylated variant of RsbV was present only in fractions that did not contain RsbW. The data support a model whereby RsbV binds directly to RsbW and blocks its ability to form the RsbW-sigma B complex. This activity of RsbV appears to be inhibited by RsbW-dependent phosphorylation.

Molecular characterization and cloning of an esterase which inactivates the macrolide toxin brefeldin A.

The macrolide antibiotic brefeldin A (BFA) was described as a phytotoxin and pathogenicity factor from Alternaria carthami Chowdhury, the causal agent of a devastating blight disease in safflower (Carthamus tinctorius L.). The toxin is known to inhibit the endoplasmic reticulum-Golgi flux and processing. Conventional breeding of safflower for resistance to the Alternaria blight disease has failed, and in situ detoxification of brefeldin A is a novel approach for the protection of field-grown safflower plants from the blight disease. As a first step towards this goal, a strain of Bacillus subtilis has been isolated which is capable of hydrolyzing brefeldin A to a non-toxic metabolite, brefeldin A acid. The BFA esterase was purified to homogeneity from B. subtilis extracts and shown to consist of a monomeric peptide of approximately 40 kDa. Besides brefeldin A, the esterase hydrolyzed ethyl valerate, which structurally resembles the lactone portion in the brefeldin A molecule, but failed to accept other macrolides such as erythromycin or zearalenone. Roughly 12% of the esterase sequence was identified by microsequencing tryptic peptides and the N terminus, which lacked a methionine leader residue. Corresponding oligonucleotide probes were employed to clone the esterase gene in pUC18. One of seven clones was sequenced and shown to code for the full size esterase protein of 372 amino acid residues. The esterase gene was subcloned in pT7-7 and expressed in Escherichia coli yielding a fusion protein with a specific esterase activity 3-fold over that of the enzyme purified from B. subtilis. The cloned esterase provides the basis for the generation of transgenic safflower plants as a valuable asset in the research on nonspecific phytotoxins and in supporting breeding for Alternaria blight resistance.

Bacillus subtilis sigma B is regulated by a binding protein (RsbW) that blocks its association with core RNA polymerase.

sigma B is a secondary sigma factor of Bacillus subtilis. RNA polymerase containing sigma B transcribes a subset of genes that are expressed after heat shock or the onset of the stationary phase of growth. Three genes (rsbV, rsbW, and rsbX), cotranscribed with the sigma B structural gene (sigB), regulate sigma B-dependent gene expression. RsbW is the primary inhibitor of this system with the other gene products acting upstream of RsbW in the sigma B regulatory pathway. Evidence is now presented that RsbW inhibits sigma B-dependent transcription by binding to sigma B and blocking the formation of a sigma B-containing RNA polymerase holoenzyme. Antibodies specific for either RsbW or sigma B will coprecipitate both proteins from crude cell extracts. This is not due to the presence of both proteins on RNA polymerase. Western blot analysis of B. subtilis extracts that had been fractionated by gel-filtration chromatography revealed a single peak of RsbW that did not coelute with RNA polymerase and two peaks of sigma B protein: one that eluted with RNA polymerase and a second that overlapped the fractions that contained RsbW. Reconstitution experiments were performed in which partially purified sigma B and RsbW were added to core RNA polymerase and tested for their ability to influence the transcription of a sigma B-dependent promoter (ctc) in vitro. RsbW efficiently blocked sigma B-dependent transcription but only if it was incubated with sigma B prior to the addition of the core enzyme.

Synthesis and fractionation properties of SpoIIGA, a protein essential for pro-sigma E processing in Bacillus subtilis

sigma E, a major sporulation-specific sigma factor of Bacillus subtilis, is derived from an inactive precursor protein (pro-sigma E). The formation of sigma E from pro-sigma E requires the products of several stage II genes, including spoIIGA, a gene that is cotranscribed with the pro-sigma E coding region (spoIIGB, or sigE). SpoIIGA has been hypothesized to be both a membrane-bound protein and the protease which converts pro-sigma E into sigma E. to learn more of its properties, we joined the Escherichia coli lacZ gene to the 3' end of spoIIGA as a translational fusion, creating a gene whose product was found to contain both beta-galactosidase and SpoIIGA activities. Assaying for the beta-galactosidase activity of the chimeric protein as a measure of its abundance, we determined that the spoIIGA::lacZ product accumulated to approximately 10% the level of a spoIIGB::lacZ fusion protein. Using differential centrifugation to fractionate B. subtilis extracts that contained beta-galactosidase fusion proteins, we observed that the beta-galactosidase activity of the spoIIGA::lacZ fusion protein was preferentially associated with a Triton X-100-sensitive, fast-sedimenting portion of the extract, while the beta-galactosidase activity of the spoIIGB::lacZ fusion protein remained primarily in the supernatant fraction. If the properties of the fusion proteins are assumed to be representative of those of the products of the genes to which lacZ is joined, these results support the hypothesis that SpoIIGA is a membrane-bound protein that acts catalytically in the processing of pro-sigma E into sigma E.

Enzymatic basis for hydrolytic versus phosphorolytic mRNA degradation in Escherichia coli and Bacillus subtilis.

The rapid synthesis and breakdown of mRNA in prokaryotes can impose a significant energy drain on these cells. Previous in vivo studies [Duffy, J. J., Chaney, S. G. & Boyer, P. D. (1972) J. Mol. Biol. 64, 565-579; Chaney, S. G. & Boyer, P. D. (1972) J. Mol. Biol. 64, 581-591] indicated that while RNA turnover in Escherichia coli was hydrolytic, it was nonhydrolytic in Bacillus subtilis. Here we provide an explanation for these observations based on enzymatic analysis of extracts of these two organisms. RNA degradation to the mononucleotide level in E. coli extracts is due solely to two active ribonucleases, RNase II and polynucleotide phosphorylase, which act hydrolytically and phosphorolytically, respectively. RNase II activity represents close to 90% of the total activity of the extract, as expected for predominantly hydrolytic degradation in this organism. In contrast, RNase II is absent from B. subtilis extracts, and the primary mode of RNA degradation is phosphorolytic, employing the Bacillus equivalent of polynucleotide phosphorylase and releases nucleoside diphosphates as products. A low level of a Mn2(+)-stimulated, hydrolytic ribonuclease is also detectable in B. subtilis extracts. Overall, E. coli and B. subtilis extracts differ by about 20- to 100-fold, depending on the substrate, in their relative use of hydrolytic and phosphorolytic routes of RNA degradation. The relation of the mode of mRNA degradation to the environment of the cell is discussed.

Purification, characterization, and immunological properties of fumarase from Euglena gracilis var. bacillaris.

A rapid three-step procedure utilizing heat treatment, ammonium sulfate fractionation, and affinity chromatography on Matrex gel Orange A purified fumarase (EC 4.2.1.2) 632-fold with an 18% yield from crude extracts of Euglena gracilis var. bacillaris. The apparent molecular weight of the native enzyme was 120,000 as determined by gel filtration on Sephacryl S-300. The preparation was over 95% pure, and the subunit molecular weight was 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme is a dimer composed of two identical subunits. The pH optimum for E. gracilis fumarase was 8.4. The Km values for malate and fumarate were 1.4 and 0.031 mM, respectively. Preparative two-dimensional gel electrophoresis was used to further purify the enzyme for antibody production. On Ouchterlony double-immunodiffusion gels, the antifumarase serum gave a sharp precipitin line against total E. gracilis protein and purified E. gracilis fumarase. It did not cross-react with purified pig heart fumarase. On immunoblots of purified E. gracilis fumarase and crude cell extracts of E. gracilis, the antibody recognized a single polypeptide with a molecular weight of approximately 60,000, indicating that the antibody is monospecific. This polypeptide was found in E. gracilis mitochondria. The antibody cross-reacted with an Escherichia coli protein whose molecular weight was approximately 60,000, the reported molecular weight of the fumA gene product of E. coli, but it failed to cross-react with proteins found in crude mouse cell extracts, Bacillus subtilis extracts, or purified pig heart fumarase.

The monomeric glutamyl-tRNA synthetase from Bacillus subtilis 168 and its regulatory factor. Their purification, characterization, and the study of their interaction.

The glutamyl-tRNA synthetase from Bacillus subtilis has been purified to homogeneity. It is a monomer of Mr = 65,500 whose NH2-terminal sequence is Met-Asn-Glu-Val-Arg-Val-Arg-Tyr-Ser-Pro-Ser-Pro-Thr-Gly-His-Leu. The number of tryptic peptides indicates the absence of a significant amount of sequence duplication. Under certain conditions, this monomeric enzyme is co-purified with a polypeptide beta of Mr = 46,000, which increases the affinity of the enzyme about 10-fold for glutamate and for ATP, and stabilizes it against heat inactivation. gamma-Globulins prepared against the monomeric enzyme can inhibit completely the glutamyl-tRNA synthetase activity of a B. subtilis extract and precipitate from this extract both the monomeric enzyme and the regulatory factor beta. These anti-alpha immunoglobulins do nt precipitate pure beta. These results show that the glutamyl-tRNA synthetase of B. subtilis has a structure similar to that of the Escherichia coli enzyme (Lapointe, J., and Söll, D. (1972) J. Biol. Chem. 247, 4966-4974) and indicate that the beta factor has a function in the regulation of glutamyl-tRNA biosynthesis in vivo.

Specificity of the serine protease inhibitor, phenylmethylsulfonyl fluoride

Clarified cell-free extracts were prepared from rapidly dividing Bacillus subtilis cells and from rabbit liver cells. These extracts were treated with [ 3H]-phenylmethylsulfonyl fluoride (PMSF) and analyzed by electrophoresis in isoelectric focusing polyacrylamide gels or detergent gels. Not less than 14 proteins in the B. subtilis extracts and not less than 15 proteins in rabbit liver extracts reacted covalently with PMSF. These results suggest that PMSF is not as specific for serine proteases as sometimes supposed, and its effects in physiological experiments should be interpreted with caution.