High production of poly(3-hydroxybutyrate) from a wild Bacillus megaterium Bolivian strain.

AIM: Taking into account that a novel strain of Bacillus megaterium was isolated from Uyuni salt lake (Bolivia) in a previous work, the objectives of this new study were to determine the maximal Poly-3-hydroxybutyrate production potential of B. megaterium strain uyuni S29 in an industrial conventional media, the possibility that the strain accumulates different types of polyhydroxyalkanoates, the cellular morphology during the biosynthesis process and the characterization of the produced biopolymers. METHODS AND RESULTS: The micro-organism was first tested in a 3-L bioreactor obtaining a high specific growth rate of 1·64 h(-1). A second fed-batch experiment was carried out in shaking flasks, reaching up to 70% PHB of cell dry mass. The biosynthesized polymers were extracted by two different extraction procedures and characterized. The results showed that all of them were PHB with thermal properties different to the conventional PHB. The micrographs taken by TEM show the different cell morphology during the fermentation process. CONCLUSIONS: In this previous study, the strain not only grew properly in the industrial conditions proposed without spore formation, but also produced and accumulated a large content of PHB, never reached before for its genus. Therefore, if the culture conditions can be optimized, the biopolymer production could be increased. SIGNIFICANCE AND IMPACT OF THE STUDY: The impact of the study has related to the area of the biomaterials and their production. The study provides new data related to the high production of PHB from the wild novel strain B. megaterium uyuni S29, the highest polymer accumulation for the genus Bacillus without spores formation.

Alteration of the mode of antibacterial action of a defensin by the amino-terminal loop substitution.

Ancient invertebrate-type and classical insect-type defensins (AITDs and CITDs) are two groups of evolutionarily related antimicrobial peptides (AMPs) that adopt a conserved cysteine-stabilized α-helical and ß-sheet (CSαß) fold with a different amino-terminal loop (n-loop) size and diverse modes of antibacterial action. Although they both are identified as inhibitors of cell wall biosynthesis, only CITDs evolved membrane disruptive ability by peptide oligomerization to form pores. To understand how this occurred, we modified micasin, a fungus-derived AITDs with a non-membrane disruptive mechanism, by substituting its n-loop with that of an insect-derived CITDs. After air oxidization, the synthetic hybrid defensin (termed Al-M) was structurally identified by circular dichroism (CD) and functionally evaluated by antibacterial and membrane permeability assays and electronic microscopic observation. Results showed that Al-M folded into a native-like defensin structure, as determined by its CD spectrum that is similar to that of micasin. Al-M was highly efficacious against the Gram-positive bacterium Bacillus megaterium with a lethal concentration of 1.76µM. As expected, in contrast to micasin, Al-M killed the bacteria through a membrane disruptive mechanism of action. The alteration in modes of action supports a key role of the n-loop extension in assembling functional surface of CITDs for membrane disruption. Our work provides mechanical evidence for evolutionary relationship between AITDs and CITDs.

Identification and characterization of a novel intracellular poly(3-hydroxybutyrate) depolymerase from Bacillus megaterium.

A gene that codes for a novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase, designated PhaZ1, has been identified in the genome of Bacillus megaterium. A native PHB (nPHB) granule-binding assay showed that purified soluble PhaZ1 had strong affinity for nPHB granules. Turbidimetric analyses revealed that PhaZ1 could rapidly degrade nPHB granules in vitro without the need for protease pretreatment of the granules to remove surface proteins. Notably, almost all the final hydrolytic products produced from the in vitro degradation of nPHB granules by PhaZ1 were 3-hydroxybutyric acid (3HB) monomers. Unexpectedly, PhaZ1 could also hydrolyze denatured semicrystalline PHB, with the generation of 3HB monomers. The disruption of the phaZ1 gene significantly affected intracellular PHB mobilization during the PHB-degrading stage in B. megaterium, as demonstrated by transmission electron microscopy and the measurement of the PHB content. These results indicate that PhaZ1 is functional in intracellular PHB mobilization in vivo. Some of these features, which are in striking contrast with those of other known nPHB granule-degrading PhaZs, may provide an advantage for B. megaterium PhaZ1 in fermentative production of the biotechnologically valuable chiral compound (R)-3HB.

Membrane asymmetry.

The components of biological membranes are asymmetrically distributed between the membrane surfaces. Proteins are absolutely asymmetrical in that every copy of a polypeptide chain has the same orientation in the membrane, and lipids are nonabsolutely asymmetrical in that almost every type of lipid is present on both sides of the bilayer, but in different and highly variable amounts. Asymmetry is maintained by lack of transmembrane diffusion. Two types of membrane proteins, called ectoproteins and endoproteins, are distinguished. Biosynthetic pathways for both types of proteins and for membrane lipids are inferred from their topography and distribution in the formed cells. Note added in proof. A cell-free system has now been developed which permits the mechanisms of membrane protein assembly to be studied (108). The membrane glycoprotein of vesicular stomatitis virus has been synthesized by wheat germ ribosomes in the presence of rough endoplasmic reticulum from pancreas. The resulting polypeptide is incorporated into the membrane, spans the lipid bilayer asymmetrically, and is glycosylated (108). The amino terminal portion of this transmembrane protein is found inside the endoplasmic reticulum vesicle, while the carboxyl terminal portion is exposed on the outer surface of the vesicle. Furthermore, addition of the glycoprotein to membranes after protein synthesis does not result in incorporation of the protein into the membrane in the manner described above (108). Consequently, protein synthesis and incorporation into the membrane must be closely coupled. Indeed, using techniques to synchronize the growth of nascent polypeptides, it has been shown (109) that no more than one-fourth of the glycoprotein chain can be made in the absence of membranes and still cross the lipid bilayer when chains are subsequently completed in the presence of membranes. These findings demonstrate directly that the extracytoplasmic portion of an ectoprotein can cross the membrane only during biosynthesis, and not after.

Use of high-content analysis and machine learning to characterize complex microbial samples via morphological analysis.

High Content Analysis (HCA) has become a cornerstone of cellular analysis within the drug discovery industry. To expand the capabilities of HCA, we have applied the same analysis methods, validated in numerous mammalian cell models, to microbiology methodology. Image acquisition and analysis of various microbial samples, ranging from pure cultures to culture mixtures containing up to three different bacterial species, were quantified and identified using various machine learning processes. These HCA techniques allow for faster cell enumeration than standard agar-plating methods, identification of "viable but not plate culturable" microbe phenotype, classification of antibiotic treatment effects, and identification of individual microbial strains in mixed cultures. These methods greatly expand the utility of HCA methods and automate tedious and low-throughput standard microbiological methods.

Characterization of thermophilic bacteria using surface-enhanced Raman scattering.

Surface-enhanced Raman scattering (SERS) can provide molecular-level information about the molecules and molecular structures in the vicinity of nanostructured noble metal surfaces such as gold and silver. The three thermophilic bacteria Bacillus licheniformis, Geobacillus stearothermophilus, and Geobacillus pallidus, a Gram-negative bacterium E. coli, and a Gram-positive bacterium B. megaterium are comparatively characterized using SERS. The SERS spectra of thermophilic bacteria are similar, while they show significant differences compared to E. coli and B. megaterium. The findings indicate that a higher number of thiol residues and possible S-S bridges are present in the cell wall structure of thermophilic bacteria, providing their stability at elevated temperatures. Incubating the thermophilic bacteria with colloidal silver suspension at longer times improved the bacteria-silver nanoparticle interaction kinetics, while increased temperature does not have a pronounced effect on spectral features. A tentative assignment of the SERS bands was attempted for thermophilic bacteria. The results indicate that SERS can be a useful tool to study bacterial cell wall molecular differences.

Cell surface hydrophobicity of Bacillus spp. as a function of nutrient supply and lipopeptides biosynthesis and its role in adhesion.

Cell surface hydrophobicity (CSH) is recognised as a important factor in microbial adhesion to solid surfaces. Growth conditions have been found to determine the synthesis of extracellular molecules by microorganisms. It has major consequences in modification of bacterial surface properties and consequently, in bacterial adhesion to solid surfaces. In this paper, CSH properties of Bacillus spp. depending on the nutrient supply and lipopeptide biosynthesis and its role in bacterial adhesion to solid surfaces were investigated. The obtained results indicate that the examined factors (nitrogen and carbon availability) influence the CSH of Bacillus spp. cells. In most variants of the experiments the role of nutrient supply in adhesion process was characteristic for species. The strongest effect was observed for peptone concentration (P < 0.001). A decrease of CSH was noticed in optimal nitrogen availability (10 g/l) and it was connected with maximum yield of surfactin biosynthesis. The highest values of CSH of examined Bacillus spp. strains were observed under nitrogen starvation and in excess of carbon source. In these conditions the adhesion to stainless steel surface was more extensive.

Cardiolipin, a major phospholipid of Gram-positive bacteria that is not readily extractable.

Extraction of phospholipids from stationary phase grown cells of the Gram+ bacteria, Bacillus megaterium, Bacillus subtilis, Bacillus cereus and Micrococcus lysodeikticus was found to be incomplete with various commonly used extraction procedures. Phosphatidylglycerol and phosphatidylethanolamine were readily extracted but up to 95% of the cardiolipin appeared to be retained within the cell residue. Extraction of the cardiolipin could be slightly enhanced by increasing the temperature or the acidity of the extraction solutions but complete extraction was obtained only after lysozyme treatment of intact cells or cell residues remaining after extraction. In addition complete extraction could be observed in the case of cells harvested in the early logarithmic phase. Freeze-fracture electron microscopy was carried out on the cell residue remaining after extraction of all phospholipids except cardiolipin. A fracture plane through the plasma membrane could not be observed anymore. Instead fracture planes through lipid vesicles were observed. These vesicles reside within the remnants of the cytoplasm and consist most likely of the non-extracted cardiolipin.

Sites of interaction of streptogramin A and B antibiotics in the peptidyl transferase loop of 23 S rRNA and the synergism of their inhibitory mechanisms.

Streptogramin antibiotics contain two active A and B components that inhibit peptide elongation synergistically. Mutants resistant to the A component (virginiamycin M1 and pristinamycin IIA) were selected for the archaeon Halobacterium halobium. The mutations mapped to the universally conserved nucleotides A2059 and A2503 within the peptidyl transferase loop of 23 S rRNA (Escherichia coli numbering). When bound to wild-type and mutant haloarchaeal ribosomes, the A and B components (pristinamycins IIA and IA, respectively) produced partially overlapping rRNA footprints, involving six to eight nucleotides in the peptidyl transferase loop of 23 S rRNA, including the two mutated nucleotides. An rRNA footprinting study, performed both in vivo and in vitro, on the A and B components complexed to Bacillus megaterium ribosomes, indicated that similar drug-induced effects occur on free ribosomes and within the bacterial cells. It is inferred that position 2058 and the sites of mutation, A2059 and A2503, are involved in the synergistic inhibition by the two antibiotics. A structural model is presented which links A2059 and A2503 and provides a structural rationale for the rRNA footprints.

Structure-function analysis of the Bacillus megaterium GerUD spore germinant receptor protein.

Germination of Bacillus spores is triggered by the interaction of germinant molecules with specialized receptor proteins localized to the spore inner membrane. Germinant receptors (GRs) are comprised typically of three interacting protein subunits, each of which is essential for receptor function. At least some GRs appear to have a fourth component, referred to as a D-subunit protein. A number of D-subunit proteins were shown previously to be capable of modulating the activity of associated GRs. Here, we investigate the topology and structure-function relationships of the Bacillus megaterium QM B1551 GerUD protein, which is associated with the GerU GR. The presented data demonstrate that GerUD can be subjected to relatively extensive structural modifications while retaining function. Indeed, the presence of either of the two transmembrane spanning domains is sufficient to modulate an efficient GerU-mediated germinative response. The precise function of D-subunit proteins has yet to be established, although they may act as molecular chaperones within the spore inner-membrane environment.

Gene dosage effect on the expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis and Bacillus megaterium.

Significant differences in expression of the delta-endotoxin genes cryA1 and cryA2 of Bacillus thuringiensis subsp. kurstaki were observed in B. subtilis and B. megaterium. The cryA1 gene was expressed when present on a high-copy-number (hcn) vector in B. megaterium but not in B. subtilis. The cryA2 gene was expressed in both hosts, but at a higher level in B. megaterium. Expression of the cryA2 gene in B. megaterium was better from a hcn vector than from a low copy number vector. Inhibition of sporulation was observed when the toxin genes were present on hcn plasmids in B. subtilis while no such effect was evident in B. megaterium. In addition, there was a significant reduction in copy numbers in both B. subtilis and B. megaterium when delta-endotoxin genes or a spoVG promoter-containing fragment of DNA were cloned into hcn plasmids.

Protein catabolism in growing Bacillus megaterium during adaptation to salt stress.

Elevated concentration of NaCl in liquid medium caused a concentration-dependent growth delay (adaptation lag) and decrease in the maximal growth rate of Bacillus megaterium. The adaptation to salt stress was accompanied by transformation of some otherwise stable (long-lived; LLP) cell proteins into quickly degraded (short-lived; SLP) ones. Exposure to the strongly growth-reducing 1 M NaCl increased the size of the SLP 'pool' of intracellular proteins from about 5 to about 15% of total protein. The major intracellular proteolytic capacity of B. megaterium is represented by intracellular serine proteinases (ISP). Paradoxically, their specific activity was lowered or masked during the adaptation phase marked by increased catabolism of short-lived and/or destabilized proteins by the stress. This documents that intracellular proteolytic activity cannot be a key regulator of protein catabolism during adaptation to stress.

Near-field optical imaging of unstained bacteria: comparison with normal atomic force and far-field optical microscopy in air and aqueous media.

Simultaneous near-field scanning optical and atomic force imaging of bacteria is presented. The bacteria imaged in these studies were unstained. The near-field optical images had excellent signal-to-noise and showed excellent contrast even in these unstained specimens. The images obtained were interpreted in terms of the images that have been obtained by transmission electron microscopy and X-ray imaging. The results show that bacterial near-field optical imaging is going to be a very important tool in the arsenal of the bacteriologist both in terms of understanding the fundamental processes in the life cycle of bacteria with and without cytochemical staining and in terms of clinical diagnostic applications.

Production of PHB by a Bacillus megaterium strain using sugarcane molasses and corn steep liquor as sole carbon and nitrogen sources.

Poly(hydroxybutyric acid) (PHB) and other biodegradable polyesters are promising candidates for the development of environment-friendly, totally biodegradable plastics. The use of cane molasses and corn steep liquor, two of the cheapest substrates available in Egypt, may help to reduce the cost of producing such biopolyesters. In this work, the effect of different carbon sources was studied. Maximum production of PHB was obtained with cane molasses and glucose as sole carbon sources (40.8, 39.9 per mg cell dry matter, respectively). The best growth was obtained with 3% molasses, while maximum yield of PHB (46.2% per mg cell dry matter) was obtained with 2% molasses. Corn steep liquor was the best nitrogen source for PHB synthesis (32.7 mg per cell dry matter), on the other hand, best growth was observed when ammonium chloride, ammonium sulphate, ammonium oxalate or ammonium phosphate were used as nitrogen sources.

Cell viability and protein turnover in nongrowing Bacillus megaterium at sporulation suppressing temperature.

In Bacillus megaterium, a temperature that suppresses sporulation (43 degrees C) only slightly exceeds both the optimum growth temperature and the temperature still permitting sporulation (40-41 degrees C). Here we show that, when cells grown at 35 degrees C and transferred to a sporulation medium, were subjected to shifts between 35 degrees C and the sporulation suppressing temperature (SST, 43 degrees C), their development and proteolytic activities were deeply affected. During the reversible sporulation phase that took place at 35 degrees C for 2-3 h (T2-T3), the cells developed forespores and their protein turnover was characterized by degradation of short-lived proteins and proteins made accessible to the proteolytic attack because of starvation. During the following irreversible sporulation phase refractile heat-resistant spores appeared at T4-T5. Protein turnover rate increased again after T2 and up to T8 60-70% prelabelled proteins were degraded. The SST suppressed sporulation at its beginning; at T3 no asymmetric septa were observed and the amount of heat-resistant spores at T8 was by 4-5 orders lower than at 35 degrees C. However, the cells remained viable and were able to sporulate when transferred to a lower temperature. Protein degradation was increased up to T3 but then its velocity sharply dropped and the amount of degraded protein at T8 corresponded to slightly more than one-half of that found at 35 degrees C. The cytoplasmic proteolytic activity was enhanced but the activity in the membrane fraction was decreased. When a temperature shift to SST was applied at the beginning of the irreversible sporulation phase (T2.5), the sporulation process was impaired. A portion of forespores lyzed, the others were able to complete their development but most spores were not heat-resistant and their coats showed defects. Protein degradation increased again because an effective proteolytic system was developed during the reversible sporulation phase but the amount of degraded protein was slightly lower than at 35 degrees C. A later (T4) shift to SST had no effect on the sporulation process.

Growth of Bacillus megaterium in phosphate-limited medium.

Batch cultures of Bacillus megaterium grown in phosphate-limited media were compared with control cultures grown in phosphate-sufficient media. The effects of phosphate limitation on growth were determined by viable cells counts. Intracellular levels of protein, RNA, poly-3-hydroxybutyrate, carbohydrate and oxygen uptake were significantly affected by phosphate limitation. Electron micrographs of sectioned cells revealed differences in the structure; in particular the thick, rigid cell wall was absent from cells grown in phosphate-limited media, and such cells were larger, pleomorphic, and after 2 d were insensitive to lysozyme.

[Poly-(3-hydroxybutyrate) granules in Bacillus megaterium. Isolation and analysis of associated proteins]. / Gránulos de poli(3-hidroxibutirato) en Bacillus megaterium. Aislamiento y análisis de las proteínas asociadas.

Bacillus megaterium accumulates poly-(3-hydroxybutyrate) (PHB) as a reserve material in intracellular granules. In this work we described a method for the preparation of PHB granules from B. megaterium PV447 and the analysis by polyacrylamide gel electrophoresis of the associated proteins. By comparison with another species a function is proposed for some of these proteins.

[Experiments with Bacillus thuringiensis protoplasts. II. Study of the potential interspecies fusion of bacterial protoplasts: Bacillus thuringiensis and Bacillus megaterium]. / Eksperimenty s protoplastami Bacillus thuringiensis. Soobshchenie II. Issledovanie vozmozhnosti mezhvidovogo sliianiia bakterial'nykh protoplastov: Bacillus thuringiensis i Bacillus megaterium.

In the present study the possibility of obtaining interspecific bacterial hybrids by polyethylene glycol-assisted fusion of protoplasts from Bacillus thuringiensis and Bac. megaterium has been examined. Electron microscopic and genetic data allow to confirm with great probability that cytological fusion takes place. However, genetic analysis revealed that neither of methods applied for protoplast fusion gave stable recombinants. Apparently, it is due to the lack of recombination or the death of recombinants that follows the functioning of the cell correction system. The mechanism of protoplast fusion and its most important steps are also studied in the present work.

[Membranolytic activity of gradex]. / Membranoliticheskaia aktivnost' gradeksa.

The membranolytic activity of gradex towards bacterial protoplasts and human erythrocytes has been demonstrated. Dextran shows no such activity. The lytic activity of gradex towards protoplasts is much lower as compared to gramicidin S. The increase in gramicidin S content from 2 to 5% caused no significant changes in the lytic activity of gradex towards protoplasts. The reduced form of gradex nad no hemolytic effect on human erythrocytes up to a concentration of 300 micrograms/ml. However, the non-reduced form of gradex af a concentration of 500 micrograms/ml lysed from 31 to 48% of erythrocytes depending on the antibiotic content in the polymeric matrix.

BMQ_0737 encodes a novel protein crucial to the integrity of the outermost layers of Bacillus megaterium QM B1551 spores.

Bioinformatic and electron microscopy analyses indicate that the composition of the B. megaterium QM B1551 spore coat is likely to differ substantially from other Bacillus species. We report here on the identification and characterisation of novel B. megaterium proteins that appear to be abundant in the spore coat. All three proteins, encoded by loci BMQ_0737, BMQ_3035 and BMQ_4051, were identified by proteomic analysis of alkaline detergent extracts from mature spores. Putative spore coat proteins were characterised by transcriptional, reporter-fusion and mutagenesis analyses supported by fluorescence and transmission electron microscopy. These analyses revealed that BMQ_0737 is a novel morphogenetic protein that is required for the correct assembly of the B. megaterium outer spore coat and exosporium, both of which are structurally compromised or missing in BMQ_0737 null mutant spores.