Search

In the present study, the taxonomic position of Bacillus aryabhattai and Bacillus megaterium was evaluated using morphological, biochemical, phylogenomic and genome analysis. The morphological and biochemical of these two species were almost similar with few exceptions. The major fatty acids in B. megaterium DSM 32T and B. aryabhattai 21047T were anteiso-C15:0 and iso-C15:0. In the phylogenomic tree, both species clade together and shared high 16S rRNA gene sequence similarity (99.6%). The average nucleotide identity values between Bacillus aryabhattai and Bacillus megaterium were above the threshold values for bacterial species delineation. Based upon morphological, biochemical, chemotaxonomic and comparative genome analysis, we propose to reclassify Bacillus aryabhattai Shivaji et al. 2009 as a later heterotypic synonym of Bacillus megaterium de Bary 1884 (Approved Lists 1980)., (© FEMS 2020.)

The components of natural quorum-sensing (QS) systems can be used to engineer synthetic communication systems that regulate gene expression in response to chemical signals. We have used the machinery from the peptide-based agr QS system from Staphylococcus aureus to engineer a synthetic QS system in Bacillus megaterium to enable autoinduction of a target gene at high cell densities. Growth and gene expression from these synthetic QS cells were characterized in both complex and minimal media. We also split the signal production and sensing components between two strains of B. megaterium to produce sender and receiver cells and characterized the resulting communication in liquid media and on semisolid agar. The system described in this work represents the first synthetic QS and cell-cell communication system that has been engineered to function in a Gram-positive host, and it has the potential to enable the generation of dynamic gene regulatory networks in B. megaterium and other Gram-positive organisms.

During the past 2 decades, Bacillus megaterium has been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single-cell level using green fluorescent protein as a model product revealed cell culture heterogeneity characterized by a significant proportion of less productive bacteria. Due to the enormous size of B. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time lapse microscopy and flow cytometry. Cell culture heterogeneity was not caused simply by plasmid loss: instead, an asymmetric distribution of plasmids during cell division was detected during the exponential-growth phase. Multicopy plasmids are generally randomly distributed between daughter cells. However, in vivo and in vitro experiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was achieved mainly by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multicopy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in a Bacillus species., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The dynamics of bacterial spore germination were successfully observed using a fast Raman imaging system, in combination with real-time phase contrast microscopy. By using a multifocus scan scheme, the spontaneous Raman-scattering imaging acquisition speed was increased to ~30 s per frame while maintaining diffraction-limited resolution, which allowed monitoring of the dynamics of spore germination on a time scale of tens of seconds to a few minutes. This allowed simultaneous gathering of rich spatial distribution information on different cellular components including time-lapse molecular images of Ca-dipicolinic acid, protein, and nucleic acid during germination of single bacterial spores for the periods of 30 to 60 min.

Motivation: Time-lapse imaging in combination with fluorescence microscopy techniques enable the investigation of gene regulatory circuits and uncovered phenomena like culture heterogeneity. In this context, computational image processing for the analysis of single cell behaviour plays an increasing role in systems biology and mathematical modelling approaches. Consequently, we developed a software package with graphical user interface for the analysis of single bacterial cell behaviour., Results: A new software called TLM-Tracker allows for the flexible and user-friendly interpretation for the segmentation, tracking and lineage analysis of microbial cells in time-lapse movies., Availability: The software package, including manual, tutorial video and examples, is available as Matlab code or executable binaries at http://www.tlmtracker.tu-bs.de.

Single cell analysis is an important tool to gain deeper insights into microbial physiology for the characterization and optimization of bioprocesses. In this study a novel single cell analysis technique was applied for estimating viability and membrane potential (MP) of Bacillus megaterium cells cultured in minimal medium. Its measurement principle is based on the analysis of the electrical cell properties and is called impedance flow cytometry (IFC). Comparatively, state-of-the-art fluorescence-based flow cytometry (FCM) was used to verify the results obtained by IFC. Viability and MP analyses were performed with cells at different well-defined growth stages, focusing mainly on exponential and stationary phase cells, as well as on dead cells. This was done by PI and DiOC(2)(3) staining assays in FCM and by impedance measurements at 0.5 and 10 MHz in IFC. In addition, transition growth stages of long-term cultures and agar plate colonies were characterized with both methods. FCM and IFC analyses of all experiments gave comparable results, quantitatively and qualitatively, indicating that IFC is an equivalent technique to FCM for the study of physiological cell states of bacteria., (Copyright © 2011 Wiley Periodicals, Inc.)

The use of cytochromes P450 for the regio- and stereoselective hydroxylation of non-activated carbon atoms in biotechnological applications reflects an efficient and cost-effective alternative in comparison to classical organic chemistry. The prokaryotic cytochrome P450 CYP106A2 from Bacillus megaterium ATCC 13368 hydroxylates a variety of 3-oxo-Δ⁴ steroids and recently it was identified to carry out a one-step regioselective allylic hydroxylation of the diterpene abietic acid. The anti-inflammatory pentacyclic triterpene 11-keto-β-boswellic acid (KBA) was found to be a further substrate of CYP106A2, being the first report of a pentacyclic triterpene conversion by a prokaryotic P450. The reaction products were analyzed by HPLC and the corresponding kinetic parameters were investigated. Structure determination of the main product by NMR revealed a 15α-hydroxylation of this substrate. In order to overcome the inability of a recombinant P450 whole-cell system in E. coli for the uptake of acids with terpene structure, we developed for the first time an expression system for cytochromes P450 in B. megaterium (strains MS941 and ATCC 13368). Interestingly, CYP106A2 was only successfully expressed in the plasmid-less B. megaterium strain MS941 but not in ATCC13368. This recombinant system, with the co-expressed heterologous redox chain of the P450, bovine adrenodoxin reductase (AdR), and bovine adrenodoxin (Adx), was applied for the whole-cell conversion of KBA. The formation of 15α-hydroxy-KBA was increased 15-fold in comparison with the naturally CYP106A2-expressing B. megaterium strain ATCC 13368.

The antimicrobial activity of the peptide enantiomers cyclo[D-Tle-D-Lys-D-Tle-L-Ala-D-Tle-L-Ala-D-Tle-L-Ala] and cyclo[L-Tle-L-Lys-L-Tle-D-Ala-L-Tle-D-Ala-L-Tle-D-Ala] against Bacillus megaterium was investigated. Both these peptides showed very low activity in both an agar diffusion assay and a broth microdilution assay. However, when both peptides were present during the experiments a potent inhibition with an IC(50) value of 2 μM was observed. Furthermore, the peptides also showed low hemolytic activity. Neither peptide had any hemolytic activity in concentrations up to 1mM but when erythrocytes were exposed to both peptides a weak hemolytic activity could be observed with a HC(50) value of 316 μM., (Copyright © 2011. Published by Elsevier Ltd.)

Dynamic processes during wet-heat treatment of individual spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis at 80 to 90°C were investigated using dual-trap Raman spectroscopy, differential interference contrast (DIC) microscopy, and nucleic acid stain (SYTO 16) fluorescence microscopy. During spore wet-heat treatment, while the spores' 1:1 chelate of Ca(2+) with dipicolinic acid (CaDPA) was released rapidly at a highly variable time T(lag), the levels of spore nucleic acids remained nearly unchanged, and the T(lag) times for individual spores from the same preparation were increased somewhat as spore levels of CaDPA increased. The brightness of the spores' DIC image decreased by ~50% in parallel with CaDPA release, and there was no spore cortex hydrolysis observed. The lateral diameters of the spores' DIC image and SYTO 16 fluorescence image also decreased in parallel with CaDPA release. The SYTO 16 fluorescence intensity began to increase during wet-heat treatment at a time before T(lag) and reached maximum at a time slightly later than T(release). However, the fluorescence intensities of wet-heat-inactivated spores were ~15-fold lower than those of nutrient-germinated spores, and this low SYTO 16 fluorescence intensity may be due in part to the low permeability of the dormant spores' inner membranes to SYTO 16 and in part to nucleic acid denaturation during the wet-heat treatment.

Background: Single cell analysis for bioprocess monitoring is an important tool to gain deeper insights into particular cell behavior and population dynamics of production processes and can be very useful for discrimination of the real bottleneck between product biosynthesis and secretion, respectively., Results: Here different dyes for viability estimation considering membrane potential (DiOC2(3), DiBAC4(3), DiOC6(3)) and cell integrity (DiBAC4(3)/PI, Syto9/PI) were successfully evaluated for Bacillus megaterium cell characterization. It was possible to establish an appropriate assay to measure the production intensities of single cells revealing certain product secretion dynamics. Methods were tested regarding their sensitivity by evaluating fluorescence surface density and fluorescent specific concentration in relation to the electronic cell volume. The assays established were applied at different stages of a bioprocess where the antibody fragment D1.3 scFv production and secretion by B. megaterium was studied., Conclusions: It was possible to distinguish between live, metabolic active, depolarized, dormant, and dead cells and to discriminate between high and low productive cells. The methods were shown to be suitable tools for process monitoring at single cell level allowing a better process understanding, increasing robustness and forming a firm basis for physiology-based analysis and optimization with the general application for bioprocess development.

This study was conducted to investigate the effects of fertilizers and biosurfactants on biodegradation of crude oil by three marine bacterial isolates; Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa. Five sets of experiments were carried out in shake flask and microcosm conditions with crude oil as follows: Set 1-only bacterial cells added (no fertilizer and biosurfactant), Set 2-with additional fertilizer only, Set 3-with additional biosurfactant only, Set 4-with added biosurfactant+fertilizer, Set 5-with no bacterial cells added (control), all the above experimental sets were incubated for 168 h. The biosurfactant+fertilizer added Set 4, resulted in maximum crude oil degradation within shake flask and microcosm conditions. Among the three bacterial isolates, P. aeruginosa and biosurfactant produced by this strain resulted in maximum crude oil degradation compared to the other two bacterial strains investigated. Interestingly, when biosurfactant and bacterial cells were used (Set 3), significant oil biodegradation activity occurred and the difference between this treatment and that in Set 4 with added fertilizer+biosurfactant were only 4-5% higher degradation level in shake flask and 3.2-7% in microcosm experiments for all three bacterial strains used. It is concluded that, biosurfactants alone capable of promoting biodegradation to a large extent without added fertilizers, which will reduce the cost of bioremediation process and minimizes the dilution or wash away problems encountered when water soluble fertilizers used during bioremediation of aquatic environments., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The automated monitoring of cell variables in cultivation processes is a key technology in modern bioscience. In this article an innovative analytical tool for monitoring of cell densities in shaking-flask cultivations--consisting of a flow-through microscope with an automated image analysis software integrated in a FIA sampling system--is presented. This atline multitesting system was optimized by varying the height of the microscopes sampling zone. A calibration of the system was performed by correlating the FIA result peaks to known concentrations of Baker's yeast. It was successfully applied in cell density monitoring of cultivation processes of Saccharomyces cerevisiae with a good correlation with offline methods, and further used to monitor 3 parallel cultivations. This methodology was successfully transferred to Bacillus megaterium cultures and applied to measure the cell densities of parallel cultivation setups of B. megaterium and S. cerevisiae., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A novel small antifungal peptide-producing strain D4 was isolated from the dung of wild plateau yak and identified as Bacillus megaterium. The purification procedure of peptide consisted of acid precipitation, methanol extract and C(18) reverse-phase chromatography. The amino acid composition of peptide YP differed from those of antifungal peptides which have been reported to date. Peptide YP was proved to be a novel small antifungal cyclic peptide. It exhibited strong inhibitory activity against many phytopathogenic fungi and had a wild range of thermo stability and pH stability and was not susceptible to all proteases tested.

A bench study was carried out to characterize the kinetics of two plant growth promoting rhizobacteria (PGPR) Azotobacter chroococcum and Bacillus megaterium to adsorb heavy metals from solution. Adsorption of Pb(2+) and Cd(2+) by bacterial cells was processed quickly with an equilibration achieved within 5 min. The adsorptions were fitted well with Freundlich and Langmuir isotherm models. The comparison of isotherm parameters indicated that A. chroococcum had a stronger capacity to bind metal ions than B. megaterium, with an average increase of 59.8% for Pb(2+) and 75.6% for Cd(2+), respectively. Both bacteria had a stronger affinity to Pb(2+) than Cd(2+) since Pb(2+) was more easily bound with the phosphoryl groups on the cell surface than Cd(2+). This demonstrated that the presence of bacteria in the rhizosphere may result in the reduction of mobile ions in soil solution.

Interactions of cationic antimicrobial peptides with living bacterial and mammalian cells are little understood, although model membranes have been used extensively to elucidate how peptides permeabilize membranes. In this study, the interaction of F5W-magainin 2 (GIGKWLHSAKKFGKAFVGEIMNS), an equipotent analogue of magainin 2 isolated from the African clawed frog Xenopus laevis, with unfixed Bacillus megaterium and Chinese hamster ovary (CHO)-K1 cells was investigated, using confocal laser scanning microscopy. A small amount of tetramethylrhodamine-labeled F5W-magainin 2 was incorporated into the unlabeled peptide for imaging. The influx of fluorescent markers of various sizes into the cytosol revealed that magainin 2 permeabilized bacterial and mammalian membranes in significantly different ways. The peptide formed pores with a diameter of approximately 2.8 nm (< 6.6 nm) in B. megaterium, and translocated into the cytosol. In contrast, the peptide significantly perturbed the membrane of CHO-K1 cells, permitting the entry of a large molecule (diameter, >23 nm) into the cytosol, accompanied by membrane budding and lipid flip-flop, mainly accumulating in mitochondria and nuclei. Adenosine triphosphate and negatively charged glycosaminoglycans were little involved in the magainin-induced permeabilization of membranes in CHO-K1 cells. Furthermore, the susceptibility of CHO-K1 cells to magainin was found to be similar to that of erythrocytes. Thus, the distinct membrane-permeabilizing processes of magainin 2 in bacterial and mammalian cells were, to the best of our knowledge, visualized and characterized in detail for the first time.

By simultaneously scanning both the excitation and emission monochromators of a common spectrofluorometer with same starting excitation and emission wavelength (namely, Delta lambda = 0), we obtained synchronous light scattering (SLS) signals that related to Rayleigh and Mie scatterings. It was found that the SLS signals could be applied for quantitation and differentiation of model bioparticles such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Bacillus thuringiensis and Bacillus megaterium. In PBS buffer, these model bioparticles could form colloidal suspensions or dispersions of sizes ranging from hundreds of nanometers to tens of micrometers, giving SLS signals with the intensity being proportional to the amount of bioparticles in the range from 1.7 x 10 (5) to 1.7 x 10 (9) CFU/mL. A further finding is that polarized synchronous light scattering (PSLS) signals of I 0 degrees -30 degrees against I 0 degrees -0 degrees , which could be obtained by introducing polarizing sheets accessory of the spectrofluorometer, and the derivative synchronous light scattering (DrSLS) signals, which could be obtained directly with the extension function of the spectrofluorometer, offer differentiation information of bioparticles connected with their size, shape, refractive indexes, and inner structure. Refractive indexes of spherical bacteria were then calculated based on light scattering signals.

Suspended microbial aggregates, which are always in dynamic equilibrium with suspended cells and cells attached to surface, are very common structures in natural and engineering environmental systems. To study and design physiologically diverse suspended microbial aggregates the physiological classification of chemotrophic prokaryotes in 12 groups formed by four evolutionary periods (fermenting, anaerobic respiring, microaerophilic and facultative aerobic, aerobic prokaryotes) and three parallel lines (Gram-negative, Gram-positive Eubacteria, and Archaea) could be used. This type of physiological heterogeneity has been studied in microbial granules using fluorescence in situ hybridization, identification of 16S rRNA genes, and conferring the physiological properties from the description of the species. In spherical granules with diameter of 2.4 mm cells of aerobes were spread to the depth 0.55 mm below surface (85% of granule volume), facultative anaerobes dominated between the depths 0.55 mm and 0.85 mm (13% of granule volume), and anaerobes were concentrated at the depths from 0.85 to 1.0 mm (2% of granule volume). Percentages of aerobic, facultative anaerobic, and anaerobic species in granules, identified by 16S rRNA gene sequencing, were 69%, 9%, and 2% of total number of bacterial clones, respectively. Another type of physiological heterogeneity on the cellular level was due to the changes of cell physiological status during cell cycle. This type of heterogeneity has been studied in the populations of Escherichia coli, Bacillus megaterium, Saccharomyces cerevisiae, and Candida tropicalis. A significant proportion of cells from the exponential phase were killed after 10 min treatment with 1% solution of allyl alcohol, which specifically kills cells with high activity of alcohol dehydrogenase (ADH). However, there was no such effect in starved cell population. Percentage of cells with high activity ADH in microbial population can be used to monitor its physiological status. Physiological diversity of ecosystem may be due to mechanical mixing of cells from the different inflows. An example of such system is an ecosystem of aeration tank in municipal wastewater treatment plant. This ecosystem contains a mechanical mixture of dead anaerobic and live aerobic bacteria as well as attached and suspended cell aggregates supplied from anaerobic digester, raw sewage, and settling tank., (IWA Publishing 2008.)

Penicillin G acylase (PGA) is one of the most important enzymes for the pharmaceutical industry. Bacillus megaterium has the advantage of producing extra-cellular PGA. This work compares two neural networks (NNs) architectures for on-line inference of B. megaterium cell mass in an aerated stirred tank bioreactor, during the production of PGA. Nowadays, intelligent computing tools such as artificial NNs and fuzzy logic are commonly applied for state inference and modeling of bioreactors. Combining these two approaches in hybrid, neuro-fuzzy systems, may be advantageous. Our results indicate that a neuro-fuzzy inference system showed a better performance to infer cell concentrations, when compared to multilayer perceptrons networks.

Surface-enhanced Raman scattering (SERS) is a powerful technique for the analysis of a variety of molecules and molecular structures. Due to its great complexity, the acquisition of detailed molecular information from biological organizations such as bacteria is still a challenging task. SERS can provide valuable information once silver or gold surfaces can be brought in close contact with the biological organization. Because several experimental parameters can affect SERS spectra of bacteria, the experimental conditions must be well defined for comparable and reproducible results. The influence of experimental parameters, such as the type of noble metal, size, and aggregation properties of nanoparticles, and the wavelength of the laser light on the SERS of E. coli and B. megaterium are examined. It is demonstrated that the impact of these parameters could be enormous and a standard protocol must be developed depending on the goal of the study.

The peptidoglycan recognition protein (PGRP) family is conserved from insects to mammals and is involved in immune regulation and bacterial clearance. They form at least three functional classes; receptors required for immune gene expression; amidases that degrade peptidoglycan and scavenge the tissues from immune-stimulating peptidoglycan; and as proteins with antibacterial activity. We here report that PGRP-SB1 is an N-acetylmuramoyl l-alanine amidase, which (in contrast to the previously described PGRP-amidases) shows antibacterial activity. PGRP-SB1 is highly active against peptidoglycans that have a diaminopimelic acid (DAP) residue in the cross-linking peptide, but lack activity to most lysine-containing peptidoglycans. The antibacterial activity is pronounced against Bacillus megaterium with an LD(50) of 1.5microg ml(-1). The bactericidal effect of PGRP-SB1 is dependent on its enzymatic activity, as the zinc co-factor is essential. The bactericidal mode of action is thus different from non-enzymatic vertebrate PGRPs that have been reported to be antibacterial.

Floating pellets containing spores of bacterial biological control agent, Bacillus megaterium were prepared by extrusion-spheronization process. The formulations composed of hydrogenated vegetable oil (HVO), lactose, microcrystalline cellulose (Avicel(R) PH101), and a disintegrant; cross-linked sodium carboxymethylcellulose (Ac-Di-Sol(R)). The finishing pellets contained bacteria ranging from 10(7) to 10(8) CFU/g and the viability of bacteria in all formulations remained high after 6 months storage. The scanning electron microscope (SEM) was used to observe endospores of B. megaterium on both the surface and the inside of the pellets. The formulations were tested for their physical properties, floating ability and bacterial release. The level of disintegrant in the formulations influenced the floating ability and the liberation of antagonistic bacteria from pellets. The bacterial pellets showed promising result in suppression of the development of sheath blight lesions in greenhouse experiment.

This essay describes the author's studies with bacteria in post-war Hungary; the difficulties encountered, with funding, collaboration and publication; and how the Szeged Institute consolidated itself as the one outstanding scientific institute in Eastern Europe, with the author at the helm.

The results of the interspecific protoplast fusion between B. thuringensis sub. kurstaki Bt-3701 which has pesticide ability, and B. megaterium var. phosphaticum Bm-107 which has decomposing phosphate activity, were reported. High frequency of protoplast formation and regeneration was obtained with 4h activated Bm-107 treated by 100 micrograms/ml lysozyme, and with 2h activated Bt-3701 treated by 3% glycin and mild temperature. Using 40% PEG and 5% nascent Ca2+ to treat the parential protoplast mixture for 3 min at 37 degrees C, 4 stable fusants were obtained. Biological tests show that they have both pesticide ability and decomposing phosphate activity, but which are weaker than that of parential strains.

Study of synchronous and asynchronous cultures of Bacillus megaterium, Bacillus thuringiensis and Bacillus licheniformis has shown that the duration of chromosomal DNA replication (period C) is proportional to the generation time, and time between two cycles of the DNA replication (known as period I). The duration of period C is nearly constant and makes up from 0.5 to 1.0 hour at the variations of the generation time from 1.5 to 2.75 hours. The duration of period B (the time between the termination of the cell division and initiation of DNA replication), and period D (the time between the termination of DNA replication and initiation of cell division) were experimentally revealed as stochastic parameters. The theoretical model of the bacterial cell cycle and the age structure of bacterial population was suggested. The main points of this theory are that periods C and I may be stochastically disposed in the division cycle of individual cells and a sum of duration of C- and I-periods is equal to generation time. The data calculated from the theoretical model were confirmed by the experimental data of flow cytofluorometric analysis of the age structure of synchronous and asynchronous cultures of the bacilli.

A particular combination of elements of the Mueller matrix for scattering of polarized light given by (S34 + S14)/(S11 + S13) identical to (S34/S11)++ is measured vs angle at a wavelength of 633 nm for randomly oriented suspensions of several species of bacteria in different stages of growth. (This combination of elements is dominated in the present measurements by the behavior of the normalized S34 matrix element, as is indicated by the notation defined on the right side of the equation.) The resulting graph in each case shows an oscillating function of angle. This function is compressed toward smaller angles when the bacteria are in the exponential phase of growth in comparison with results for a suspension of the same bacteria in the stationary (starving-smaller cells) phase of growth. Microscopic measurements were made to determine, for each case, the average dimensions of the bacterial population. Graphs were then plotted of the peak positions from the Mueller matrix function plots vs either cell length or cell diameter. The function was shown to be strongly correlated with cell diameter under the conditions of this experiment and poorly correlated with cell length. The measurements were shown to have a sensitivity to changes in average diameter of about 20 nm.

It is shown that the Bacillus megaterium population has two types of qualitatively differing cells: exo- and endotrophic ones, i.e. the cells which assimilate in a given moment of time the source of carbon and energy from the medium or only intracellular sources of carbon and energy. Trophic structure of the population has been analyzed in different phases of the batch culture as well as in case of cell starvation. In all cases the content of exotrophic cells in the bacilli population considerably decreases with exhaustion of the nutrient medium. The obtained results are hypothetically explained by the alteration of the exo- and endotrophic processes in a cell cycle of bacteria.

Filaments of a thermosensitive Bacillus megaterium mutant showed an altered macromolecular composition compared with salt-cured mutant cells and parental cells. Filaments contained more peptidoglycan, polyglucose, poly-beta-hydroxy-butyrate, and deoxyribonucleic acid per unit of protein. The ribonucleic acid-to-protein ratio of filaments was similar to that of rods or salt-cured cells. Filament formation seemed to be due to defective protein or ribonucleic acid metabolism.

Protoplasts of Bacillus megaterium readily reverted to bacillary form in liquid media and when plated in a soft-agar layer onto the surface of appropriate agar media. Three phases of the reversion sequence could be differentiated by phase contrast microscopy: (i) increase in size of the individual protoplasts, (ii) non oriented division of the protoplasts and (iii) outgrowth of the bacillary forms. With time-lapse photomicrography, reversion sequences of single protoplasts were demonstrated.

The structure of the surface of spores of 10 strains of Bacillus megaterium was investigated by the method of carbon replicas. They were separated into three groups according to the peculiarities of the structural organization of the surface of the spores. The combination of other morphological and physiological-biochemical characteristics of these strains was characteristic of the species Bac. megaterium. Strains with differences in the structure of the spore surface also showed similarities in other peculiarities of carbohydrate and nitrogen metabolism studied in the work, as well as in the primary structure of DNA. The date obtained lead to the conclusion that structural differences in the surface of the spores cannot be considered a sufficient basis for isolating the bacteria studied into separate taxonomic groups.

Analysis of the penicillin-binding proteins in the membranes from germinated spores of Bacillus megaterium and a filamentous mutant indicated that (a) no specific synthesis of any penicillin-binding protein occurred before or during two relatively synchronous cell divisions, (b) penicillin-binding proteins 1, 3a and 3b may be involved in cell elongation and (c) filamentation of the mutant may be due to a decrease in the concentration of penicillin-binding protein 1.

A new system in which the in vivo and in vitro formation of cross-links in the peptidoglycan of Bacillus megaterium can be compared directly has been developed. The method for the determination of the in vivo cross-linking consists of lysozyme digestion of acetylated [(14)C]diaminopimelic acid-labeled cells and Bio-Gel P-6 gel filtration of the digest. The elution profile indicates the cell wall synthesized in vivo consists of highly cross-linked fractions (44%), bisdisaccharide peptide(s) (38%), and disaccharide peptide(s) (18%). The in vitro system showed a high synthetic activity of cross-linked peptidoglycan. The synthesis was inhibited completely by 83.3 mug of ristocetin or vancomycin per ml or 10(-4) M p-chloromercuribenzoate and inhibited only partially by penicillins. The polymerization was stimulated by high concentrations of sucrose, glycerol, amino acids, or dimethyl sulfoxide. The formation of cross-links was inhibited 50% at 0.3 mug of dicloxacillin per ml and 90% at 0.5 mug or more. It was also stimulated by high concentrations of sucrose, glycerol, or dimethyl sulfoxide. Effective concentrations of dicloxacillin on the growth, viability, and morphology of B. megaterium were determined. Sharp inhibition of cross-linking occurred in vivo and in vitro at these effective concentrations, whereas the incorporation of [(14)C]-diaminopimelate into bacterial cells was not affected at all. Cell-bound dicloxacillin reduced severely the degree of cross-linking in the cell wall synthesized after transfer to a dicloxacillin-free medium. Cell wall synthesized in the presence of dicloxacillin showed a higher rate of turnover than did the normal cell wall. Moreover, disaccharide peptide(s) was degraded faster than was bisdisaccharide peptide(s) in dicloxacillin-treated cells. From these observations, the primary target of penicillin action in B. megaterium is discussed in relation to the inhibition of cross-linking, penicillin-binding components, and cell lysis.

Several recent technological advances have increased the practicality and usefulness of the technique of electron microscopy of wet objects. (i) There have been gains in the effective penetration of high-voltage microscopes, scanning transmission microscopes, and high-voltage scanning microscopes. The extra effective penetration gives more scope for obtaining good images through film windows, gas, and liquid layers. (ii) Improved methods of obtaining contrast are available (especially dark field and inelastic filtering) that often make it possible to obtain sufficient contrast with wet unstained objects. (iii) Improved environmental chamber design makes it possible to insert and examine wet specimens as easily as dry specimens. The ultimate achievable resolution for wet objects in an environmental chamber will gradually become clear experimentally. Resolution is mainly a function of gas path, liquid and wet specimen thickness, specimen stage stability, acceleration voltage, and image mode (fixed or scanning beam) (13). Much depends on the development of the technique for controlling the thickness of extraneous water film around wet objects or the technique for depositing wet objects onto dry, hydrophobic support films. Although some loss of resolution due to water or gas scattering will always occur, an effective gain is anticipated in preserving the shape of individual molecules and preventing the partial collapse that usually occurs on drying or negative staining. The most basic question for biological electron microscopy is probably whether any living functions of cells can be observed so that the capabilities of the phase contrast and interference light microscopes can be extended. Investigators are now rapidly approaching a final answer to this question. The two limiting factors are (i) maintaining cell motility in spread cells immersed in thin layers of media and (ii) reducing beam radiation damage to an acceptable level. The use of sensitive emulsions and image intensifiers can bring the observation dose below that required to stop cell motility. Use of a timed, pulsed deflector system enables sufficiently short exposures to be obtained to eliminate blurring due to Brownian motion. Environmental chambers have enhanced the possibilities of electron diffraction analysis of minute crystals and ordered biological structures. High-resolution electron diffraction patterns (especially kinematic) of protein crystals can only be obtained in a wet environment. Hence, it may now be possible to obtain undistorted images of protein molecules. Moreover, by subjecting diffraction patterns to image-iterative techniques (56), it will be possible to phase the electron diffraction patterns to give a calculated image with a higher resolution than that which can be produced by electron microscope objective lenses. Environmental chambers offer exciting prospects for the determination of water structure and water and ice nucleation (atmospheric science). Nucleation data near the molecular level have been badly needed for some time. The application of environmental chambers in industrial chemistry, for example, in studies of polymerization, catalysis, and corrosion, are awaiting exploration. They offer an unusual approach to measurements of reaction kinetics through images that should be both sensitive and rapid.

B. megaterium NCTC 5637 has been shown to grow into long chains in 1% glucose and 1% suramin (w/v). This effect was not noticeable in lower concentration of the inhibitor. The effect diminished in suramin free medium, indicating the change to be phenotypic.

The behavior of a number of motile flagellated bacteria toward viscosity characteristics of their fluid environments was observed. All showed an increase in velocity (micrometers per second) in more viscous solutions. Velocity reached a maximum at a characteristic value, however, and thereafter decreased with higher viscosities. Peritrichously flagellated bacteria had maximum velocities at higher viscosities than polarly flagellated bacteria. Effects of temperature, and possible utilization of chemical constituents in the viscous solutions, were studied and found to be negligible factors under the experimental conditions used. Different agents produced the same phenomenon, thus indicating that there probably were no chemically induced metabolic effects. Loss of available water and the possibility of a variable energy supply to the flagellar propulsive system were considered but are believed minimal. Theoretically derived thermodynamic equations were utilized and suggest that the conformation of the flagellar helix affects efficiency of propulsion. Such a relationship between helix waveform and velocity was experimentally observed with Thiospirillum jenese.

Mutant TH14 of Bacillus megaterium ATCC 19213 is thermosensitive and defective in cell-division septation and spore formation at the restrictive temperature (39 C). As a consequence, the mutant forms multinucleate aseptate filaments and is asporogenic. The mutation does not result in any qualitative compositional changes in extractable membrane proteins. At the restrictive temperature, the mutant membrane has a reduced content of a small molecular weight protein(s). A membrane protein(s) with a molecular weight of nearly 80,000 appears to be partially derepressed in the mutant grown at the restrictive temperature. In addition, numerous unidentified spherical inclusions of fairly uniform size (diameter approximately 100 nm) are present in the cytoplasm at the restrictive temperature. They are especially concentrated at only one pole of each filament. Filamentous growth of the mutant is less sensitive to penicillin than growth in the rod form. Growth in either form is equally sensitive to d-cycloserine at the concentrations used for selection of the mutant. Temperature shift-up experiments suggest that one to two rounds of deoxyribonucleic acid (DNA) replication occur before the phenotypic expression of the mutation occurs. The septations after these replication events can be either two-division septations or a single-division septation plus a subsequent sporulation septation. This conclusion, coupled with previously reported work, supports the hypothesis that the early stages of sporulation represent a modified cell division.

A protein of apparent mol.wt. 35000 that is extractable from the purified coat fraction of Bacillus megaterium KM spores is synthesized during sporulation as a precursor protein from which a 12-13 amino acid peptide is removed. Cleavage of this small peptide is delayed until 60-90 min after precursor synthesis and is concomitant with the morphological appearance of stage VI. The addition of chloramphenicol, subsequent to precursor synthesis, prevents the appearance of this late processing event. Two-dimensional non-equilibrium pH-gradient gel electrophoresis of the integument extract of forespores isolated at stage V from sporangia pulse-labelled with L-[35S]methionine 1 h before isolation, revealed both unprocessed and processed components. Similar analysis of total protein from the corresponding mother cells revealed only the unprocessed component in relatively small amounts, suggesting that, although the protein may be synthesized in the mother-cell compartment, processing may be restricted to the forespore. Peptide analysis by limited proteolysis was used to examine the relationship between the 35000- and a 17500-mol.wt. coat protein. The possible implications of limited proteolytic processing to maturation of the spore coat are discussed.

The frequency of association of spore loci with the "old" and "new" ends of rod-shaped sporangia in batch cultures of Bacillus megaterium ATCC 19213 was estimated by phase contrast microscopy. The analysis was facilitated by (i) the association of most of the sporangia into chains of two to five sporangia and (ii) the occurrence of two types of cross wall distinguishable by their degree of splitting. It was concluded that a newly formed spore is located at the "old" end of a sporangium. By inference, the sporulation division septum locus is distal to the ultimate normal cell division septum, i.e., proximal to the "old" pole of the B. megaterium sporangium. This result is discussed in relation to deoxyribonucleic acid segregation during sporulation.

Spontaneous release of the temperate bacteriophage T (phiT), carried by Bacillus megaterium 899a, occurred during early growth of the host cells. Rejuvenated cells (accomplished by a 5x dilution in fresh medium) and unrejuvenated cells were induced by mitomycin C during the course of sporulation and subsequent phage phiT production measured by burst size. Induction of sequential samples of unrejuvenated cells resulted in burst sizes that fell to zero as T(0) sporulation time in the main culture was approached. This drop in burst size was not considered a sporulation event, as it also occurred during analogous stages of growth in an asporogenous mutant. Rejuvenated, induced portions of the culture of sporulating cells of B. megaterium 899a gave large burst sizes until T(+2), when the burst sizes fell to zero. The stage I asporogenous mutant, treated in a similar manner, gave lower, but still substantial, burst sizes; thus, the sharp decline in burst size of induced rejuvenated sporulating cells appeared to be a sporulation event. Sporulating cells induced at times shortly after T(1.5) formed spores in which the induced phage were trapped until germination of the spores, which formed infectious centers. This induced phage-trapping was maximal when the sporulating cultures were induced at T(2.25). Commitment to sporulation could be defined by our system as that point beyond which rejuvenated sporulating cells were unable to support the replication of the phage. This point also correlated with the increase in induced phage-trapping by spores. Two other methods gave a similar commitment time. Commitment to sporulation, in spite of added glucose or fresh complex medium, occurred at the same time. Electron micrography showed that the committed cell was still undergoing engulfment. The fate of induced phage phiT was determined at different points during growth and sporulation. Induction at times prior to T(0), which were longer than the eclipse period of the phage, resulted in a burst size of approximately 50. At times prior to T(0), shorter than the phage eclipse period, induction led to lysis with low burst sizes, approaching zero. The pattern of spontaneous phage release during growth was similar. From T(0) up to the point of commitment to sporulation, induction resulted in the blocking of spore formation without lysis. At the commitment point, induced phage were trapped and carried into spores which germinated to give infectious centers. The spontaneous derepression of phage at a time which blocked spore formation led to 7 x 10(4) infectious centers per ml and would not normally be noticed. Derepression at the time of phage entrapment was not observed to occur without induction with mitomycin C.

Photomicrographic evidence of reversion of Bacillus megaterium protoplasts to the bacillary form on soft agar plates hypertonic medium is demonstrated.