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.

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.

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.

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.

Spatial configuration of extracellular polymeric substances of Bacillus megaterium TF10 in aqueous solution.

Configuration of extracellular polymeric substances (EPS) excreted by microorganisms is related greatly to the inherent properties of EPS, and has a significant effect on the physicochemical characteristics of microbial aggregates, such as activated sludge for wastewater treatment, as well as their interaction with other substances in aqueous systems. In this work, the spatial configuration of microbial EPS is characterized using laser light scattering (LLS) technique, with EPS extracted from Bacillus megaterium TF10 as an example. The combined utilization of static light scanning (SLS) and dynamic light scanning (DLS) offers an effective avenue to explore the EPS configuration in aqueous solution, thus enables a better understanding about the physicochemical properties of EPS. The results show that EPS exist in the form of colloids in neutral aqueous solution (pH 7.0) and that their shape is random coil with incompletely extending chains. The attraction interaction between EPS colloids is related with the high flocculability of B. megaterium TF10. The cryo-electron microscopy image further confirms the spherical shape of EPS colloids. The LLS approach offers a powerful and convenient tool for characterizing microbial EPS configuration and understanding their behaviors in biological wastewater treatment systems.

Structural and metabolic correlation for Bacillus megaterium ACBT03 in response to colchicine biotransformation.

This study aims to evaluate the effects of colchicine on metabolic and structural changes in Bacillus megaterium ACBT03, enduring colchicine bioconversion. Electron microscopy examination of cells adapted to different concentrations of colchicine for its bioconversion to pharmacologically active 3-demethylated colchicine, endowed changes in cell shape, decreased cell wall and plasma membrane thickness. In line with microscopic studies, lipid and membrane protein contents were drastically reduced in bacterial cells adapted to higher concentrations of colchicine and resulting into decrease in cell membrane thickness. More numbers of polyhydroxybutyrate (PHB) rich inclusion bodies were found inside the colchicine adapted cells and presence of higher amount of PHB, a carbon source for generation of redox potential, indicates that it might be responsible for activation of P450 BM-3 enzyme and plays significant role in colchicine demethylation. The presence of dense ribosome like bodies in colchicine adapted cells showed higher biosynthesis of P450 BM-3. Reduction in cell wall and cell membrane thickness, presence of more inclusion bodies and ribosome like masses in colchicine adapted cells were some of the key interlinked phenomena responsible for colchicine bioconversion. This is the first study which reports that colchicine demethylation process severely affects the structural and metabolic functions of the bacteria.

Determination of the X-ray structure of the snake venom protein omwaprin by total chemical synthesis and racemic protein crystallography.

The 50-residue snake venom protein L-omwaprin and its enantiomer D-omwaprin were prepared by total chemical synthesis. Radial diffusion assays were performed against Bacillus megaterium and Bacillus anthracis; both L- and D-omwaprin showed antibacterial activity against B. megaterium. The native protein enantiomer, made of L-amino acids, failed to crystallize readily. However, when a racemic mixture containing equal amounts of L- and D-omwaprin was used, diffraction quality crystals were obtained. The racemic protein sample crystallized in the centrosymmetric space group P2(1)/c and its structure was determined at atomic resolution (1.33 A) by a combination of Patterson and direct methods based on the strong scattering from the sulfur atoms in the eight cysteine residues per protein. Racemic crystallography once again proved to be a valuable method for obtaining crystals of recalcitrant proteins and for determining high-resolution X-ray structures by direct methods.

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.

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.

An alternative bactericidal mechanism of action for lantibiotic peptides that target lipid II.

Lantibiotics are polycyclic peptides containing unusual amino acids, which have binding specificity for bacterial cells, targeting the bacterial cell wall component lipid II to form pores and thereby lyse the cells. Yet several members of these lipid II-targeted lantibiotics are too short to be able to span the lipid bilayer and cannot form pores, but somehow they maintain their antibacterial efficacy. We describe an alternative mechanism by which members of the lantibiotic family kill Gram-positive bacteria by removing lipid II from the cell division site (or septum) and thus block cell wall synthesis.

Interactions between penicillin-binding proteins (PBPs) and two novel classes of PBP inhibitors, arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-ones.

Several non-beta-lactam compounds were active against various gram-positive and gram-negative bacterial strains. The MICs of arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-ones were lower than those of ampicillin and cefotaxime for methicillin-resistant Staphylococcus aureus MI339 and vancomycin-resistant Enterococcus faecium EF12. Several compounds were found to inhibit the cell wall synthesis of S. aureus and the last two steps of peptidoglycan biosynthesis catalyzed by ether-treated cells of Escherichia coli or cell wall membrane preparations of Bacillus megaterium. The effects of the arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-one derivatives on E. coli PBP 3 and PBP 5, Streptococcus pneumoniae PBP 2xS (PBP 2x from a penicillin-sensitive strain) and PBP 2xR (PBP 2x from a penicillin-resistant strain), low-affinity PBP 2a of S. aureus, and the Actinomadura sp. strain R39 and Streptomyces sp. strain R61 DD-peptidases were studied. Some of the compounds exhibited inhibitory activities in the 10 to 100 microM concentration range. The inhibition of PBP 2xS by several of them appeared to be noncompetitive. The dissociation constant for the best inhibitor (Ki = 10 microM) was not influenced by the presence of the substrate.

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.

Antibacterial activity of human prostasomes.

BACKGROUND: Prostasomes are prostate-derived organelles in semen exhibiting pluripotent properties. The present study deals with their possible antibacterial effects. METHODS: Antibacterial activity was assessed by growth inhibition of bacteria in an incubation medium containing prostasomes, after which the incubate was inoculated on cystine lactose electrolyte deficient agar (CLED) plates. In cases involving Bacillus megaterium, the effects were also documented ultrastructurally with scanning electron microscopy and atomic force microscopy. RESULTS: A dose-dependent growth inhibition was apparent, and a complete inhibition of growth was seen at a prostasome protein concentration of 30 microg/ml with Bacillus megaterium. Ultrastructurally, increasingly irregular contours and a loosening of the smooth surface were observed, combined with a fragmentation of the bacteria. Among 9 other bacterial strains tested, a complete growth inhibition by prostasomes was attained in 3 strains, while the other 6 were unaffected. CONCLUSIONS: Our data suggest that prostasomes, or prostasome-derived proteins, are responsible for the antibacterial effects on Bacillus megaterium and some other bacterial strains. The results may serve as a basis of development of a new class of antibacterial drugs.

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.

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.

Polyhydroxyalkanoate inclusion body-associated proteins and coding region in Bacillus megaterium.

Polyhydroxyalkanoic acids (PHA) are carbon and energy storage polymers that accumulate in inclusion bodies in many bacteria and archaea in response to environmental conditions. This work presents the results of a study of PHA inclusion body-associated proteins and an analysis of their coding region in Bacillus megaterium 11561. A 7, 917-bp fragment of DNA was cloned and shown to carry a 4,104-bp cluster of 5 pha genes, phaP, -Q, -R, -B, and -C. The phaP and -Q genes were shown to be transcribed in one orientation, each from a separate promoter, while immediately upstream, phaR, -B, and -C were divergently transcribed as a tricistronic operon. Transfer of this gene cluster to Escherichia coli and to a PhaC- mutant of Pseudomonas putida gave a Pha+ phenotype in both strains. Translational fusions to the green fluorescent protein localized PhaP and PhaC to the PHA inclusion bodies in living cells. The data presented are consistent with the hypothesis that the extremely hydrophilic protein PhaP is a storage protein and suggests that PHA inclusion bodies are not only a source of carbon, energy, and reducing equivalents but are also a source of amino acids.

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.

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.