Structural Characterization of Native and Modified Encapsulins as Nanoplatforms for in Vitro Catalysis and Cellular Uptake.

Recent years have witnessed the emergence of bacterial semiorganelle encapsulins as promising platforms for bio-nanotechnology. To advance the development of encapsulins as nanoplatforms, a functional and structural basis of these assemblies is required. Encapsulin from Brevibacterium linens is known to be a protein-based vessel for an enzyme cargo in its cavity, which could be replaced with a foreign cargo, resulting in a modified encapsulin. Here, we characterize the native structure of B. linens encapsulins with both native and foreign cargo using cryo-electron microscopy (cryo-EM). Furthermore, by harnessing the confined enzyme (i.e., a peroxidase), we demonstrate the functionality of the encapsulin for an in vitro surface-immobilized catalysis in a cascade pathway with an additional enzyme, glucose oxidase. We also demonstrate the in vivo functionality of the encapsulin for cellular uptake using mammalian macrophages. Unraveling both the structure and functionality of the encapsulins allows transforming biological nanocompartments into functional systems.

Enhanced dibenzothiophene biodesulfurization by immobilized cells of Brevibacterium lutescens in n-octane-water biphasic system.

In this study, it was the first report that the Brevibacterium lutescens CCZU12-1 was employed as a sulfur removing bacteria. Using dibenzothiophene (DBT) as the sole sulfur source, B. lutescens could selectively degrade DBT into 2-hydroxybiphenyl (2-HBP) via the "4S" pathway. In the basal salt medium (BSM) supplemented with 0.25 mM DBT and 0.5 g/L Tween-80, high desulfurization rate (100 %) was obtained by growth cells after 60 h. Furthermore, the n-octane-water (10:90, v/v) biphasic system was built for the biodesulfurization by resting cells. Moreover, a combination of magnetic nano Fe3O4 particles with calcium alginate immobilization was used for enhancing biodesulfurization. In this n-octane-water biphasic system, immobilized B. lutescens cells could be reused for not less than four times. Therefore, B. lutescens CCZU12-1 shows high potential in the biodesulfurization.

Self-sorting of foreign proteins in a bacterial nanocompartment.

Nature uses bottom-up approaches for the controlled assembly of highly ordered hierarchical structures with defined functionality, such as organelles, molecular motors, and transmembrane pumps. The field of bionanotechnology draws inspiration from nature by utilizing biomolecular building blocks such as DNA, proteins, and lipids, for the (self-) assembly of new structures for applications in biomedicine, optics, or electronics. Among the toolbox of available building blocks, proteins that form cage-like structures, such as viruses and virus-like particles, have been of particular interest since they form highly symmetrical assemblies and can be readily modified genetically or chemically both on the outer or inner surface. Bacterial encapsulins are a class of nonviral protein cages that self-assemble in vivo into stable icosahedral structures. Using teal fluorescent proteins (TFP) engineered with a specific native C-terminal docking sequence, we report the molecular self-sorting and selective packaging of TFP cargo into bacterial encapsulins during in vivo assembly. Using native mass spectrometry techniques, we show that loading of either monomeric or dimeric TFP cargo occurs with unprecedented high fidelity and exceptional loading accuracy. Such self-assembling systems equipped with self-sorting capabilities would open up exciting opportunities in nanotechnology, for example, as artificial (molecular storage or detoxification) organelles or as artificial cell factories for in situ biocatalysis.

Synthesis of PHB nanoparticles from optimized medium utilizing dairy industrial waste using Brevibacterium casei SRKP2: a green chemistry approach.

Polyhydroxyalkanoates (PHAs) are natural, biodegradable polymers accumulated by bacteria under nutritional exhausted condition where carbon source is in excess. A gram positive bacterium (designated strain SRKP2) that potentially accumulated polyhydroxybutyrate (PHB) was isolated from dairy industrial waste. From its morphological and physiological properties and nucleotide sequence of its 16S rRNA, it was suggested that strain SRKP2 was similar to Brevibacterium casei. PHAs were synthesized from a medium containing dairy waste, yeast extract and sea water. The synthesized PHAs were characterized by FT-IR as Polyhydroxybutyrate (PHB). Response surface methodology was applied to optimize the production of PHB. From the optimized medium the yield of PHB was found to be 2.940 g/L. Here we report the direct use of dairy waste and sea water as potential sources for the production of PHB. Produced PHB was used to synthesize nanoparticles using solvent displacement technique.

Real-time nanomechanical and topographical mapping on live bacterial cells-Brevibacterium casei under stress due to their exposure to Co2+ ions during microbial synthesis of Co3O4 nanoparticles.

The study of elastic properties of microbial and mammalian cells using atomic force microscopy, with force-sensitivity as high as pico-Newtons and spatial resolution of a few nanometers, is proving to be a great tool for the real-time observation of the effects of drugs, biomolecules, metal ions, and nanoparticles on cell physiology in their natural environment. It has been shown that the Young's modulus of the cell surfaces is extremely sensitive to the surrounding environment. Recently, a broad array of microbes have been used successfully to synthesize nanocrystals of several metal and metal oxides in a controlled manner at room temperature after exposing them to various metal ion precursors. However, so far there is no report on the fate of their elastic properties and cell topography etc. during and after their exposure to the metal ions during the microbial synthesis of nanomaterials. Additionally, this information is also found to be extremely relevant to areas such as bioremediation, bioleaching, and biomineralization, where it is important to study the direct influence on the cell physiology in the presence of metal ions. Here, we report, for the first time, the use of AFM force-distance curves on live cells, to directly monitor (in real time) the changes in the surface-topography, surface-adhesion, indentation-depth, and Young's modulus of a metal-tolerant marine bacterium, Brevibacterium casei, isolated from the coast of the Arabian Sea, after its exposure to the Co2+ ions during the process of biosynthesis of nanoparticles. We earlier reported that this bacterium is capable of using the cobalt acetate as a precursor to synthesize protein-functionalized Co3O4 nanoparticles with very high crystallinity. Our study indicates a significant change in the morphology as well as elastic and adhesive properties of the Brevibacterium casei, where we found an increase in the adhesive properties and the indentation depth of the bacterial surfaces and a decrease in the cell stiffness after several hours of exposure to the cobalt acetate. We have discussed both qualitative and quantitative analysis of the force-spectroscopy data in detail.

Brevibacterium celere sp. nov., isolated from degraded thallus of a brown alga.

Two whitish yellow, Gram-positive, non-motile, aerobic bacteria were isolated from enrichment culture during degradation of the thallus of the brown alga Fucus evanescens. The bacteria studied were chemo-organotrophic, mesophilic and grew well on nutrient media containing up to 15 % (w/v) NaCl. The DNA G+C content was 61 mol%. The two isolates exhibited a conspecific DNA-DNA relatedness value of 98 %, indicating that they belong to the same species. A comparative analysis of 16S rRNA gene sequences revealed that strain KMM 3637(T) formed a distinct phyletic lineage in the genus Brevibacterium (family Brevibacteriaceae, class Actinobacteria) and showed the highest sequence similarity (about 97 %) to Brevibacterium casei. DNA-DNA hybridization experiments demonstrated 45 % binding with the DNA of B. casei DSM 20657(T). Physiological and chemotaxonomic characteristics (meso-diaminopimelic acid in the peptidoglycan, major cellular fatty acids 15 : 0ai and 17 : 0ai) of the bacteria studied were consistent with the genomic and phylogenetic data. On the basis of the results of this study, a novel species, Brevibacterium celere sp. nov., is proposed. The type strain is KMM 3637(T) (=DSM 15453(T)=ATCC BAA-809(T)).

Cell wall teichoic acids of two Brevibacterium strains.

Structurally identical teichoic acids were detected in cell walls of two soil isolates assigned to Brevibacterium linens based on phylogenetic data. Both cell walls contain unsubstituted 1,3-poly(glycerol phosphate) and poly(glycosylglycerol phosphate). Repeating units of the latter--alpha-D-GlcpNAc-(1-->4)-beta-D-Galp-(1-->1)-Gro--are bound by phosphodiester bonds including OH-3 of galactose and OH-3 of glycerol. Some of the N-acetylglucosamine residues have 4,6-pyruvic acid acetal, amounts of the latter in the two strains being unequal. Species-specificity of the structures of teichoic acids in the genus Brevibacterium is discussed.

The cell division genes ftsQ and ftsZ, but not the three downstream open reading frames YFIH, ORF5 and ORF6, are essential for growth and viability in Brevibacterium lactofermentum ATCC 13869.

The three ORFs (YFIH, ORF5 and ORF6) located downstream of the cell division genes ftsQ and ftsZ in Brevibacterium lactofermentum were disrupted by single homologous recombination events between internal fragments of the corresponding genes and the chromosomal sequences. The phenotypes of the disrupted mutants were similar to that of the wild type, suggesting that these genes are dispensable for growth and viability. However, using different plasmid constructs, it was not possible to obtain disrupted ftsZ or ftsQ mutants by single crossover events. When the ftsZ or ftsQ gene sequence was disrupted in vitro and used to replace the homologous chromosomal gene by double recombination, only single recombination events took place, and therefore no disruptants were obtained. It may be concluded therefore that, as in Escherichia coli, the cell division genes ftsQ and ftsZ are indispensable for growth and viability of B. lactofermentum. Northern hybridisation analyses performed using internal fragments of the genes coding for YFIH, ORF5 and ORF6 allowed us to dissect their transcriptional organization and to confirm the disruption of these genes.

Cloning, sequencing, and characterization of the ftsZ gene from coryneform bacteria.

Taking advantage of highly conserved domains present in the ftsZ genes from Escherichia coli, Rhizobium meliloti, and Bacillus subtilis, we designed degenerate oligonucleotides (oligos) corresponding to these regions. These oligos were used as primers in PCR in order to amplify DNA sequences from Brevibacterium flavum MJ233 chromosomal DNA. The PCR product was used as a probe to recover genomic fragments from a lambda library of Br. flavum MJ233. The complete nucleotide sequence (nt) of the cloned 4.2-kb EcoRI fragment containing the ftsZ homolog from Br. flavum MJ233 indicated that the deduced gene product of the Br. flavum ftsZ homolog is composed of 438 amino acids (aa) with a deduced molecular weight of 46.9 kDa. This size of molecular weight was also confirmed by the in vitro protein synthesis assay. Comparison of this aa sequence to the corresponding sequences from E. coli, Rh. meliloti, B. subtilis, and Streptomyces coelicolor revealed a high degree of conservation and suggested that the Br. flavum ftsZ homolog has a putative GTP binding motif and a GTP hydrolizing region. Expression of Br. flavum ftsZ gene in E. coli, JM109 inhibited its cell division, leading to filamentation. This suggested that the Br. flavum ftsZ product competed with the E. coli ftsZ product.

A new Brevibacterium sp. isolated from infected genital hair of patients with white piedra.

A new aerobic gram-positive non-sporeforming bacillus has been isolated from infected genital hair of patients with white piedra in association with Trichosporon beigelii. This species has been characterised morphologically, nutritionally, by DNA base composition, cell-wall analysis and cellular fatty-acid profile on the basis of 14 isolates. The G+C content of DNA is 63.05 mol%. Cell walls possess meso-diaminopimelic acid (Type IV) and the sugars glucose, galactose, xylose and ribose; mycolic acids are not present. The species has a distinct colonial and microscopic morphology, is strongly proteolytic and produces methanethiol. These findings and the cellular fatty-acid profile are compatible with the genus Brevibacterium. A new species is proposed based on the following characters: colonial and microscopic growth and morphology; conditions for rod-to-coccus cycle; ribose utilisation; and tellurite reduction. The type strain has been named Brevibacterium mcbrellneri E2cr (ATCC 49030). The strong proteolytic properties may be the mechanism of pathogenesis.

Characterization of 15 selected coccal bacteria isolated from Antarctic rock and soil samples from the McMurdo-Dry Valleys (South-Victoria Land).

Approximately 1500 cultures of microorganisms were isolated from rocks and soils of the Ross Desert (McMurdo-Dry Valleys). From these, 15 coccoid strains were chosen for more detailed investigation. They were characterized by morphological, physiological and chemotaxonomical properties. All isolates were Gram-positive, catalase-positive and nonmotile. Six strains showed red pigmentation and could be identified as members of the genera Micrococcus (M. roseus, M. agilis) or Deinococcus. In spite of their coccoid morphology, the remaining nine strains had to be associated with coryneform bacteria (Arthrobacter, Brevibacterium), because of their cell wall composition and G+C ratios. Most of the strains were psychrotrophic, but one strain was even obligately psychrophilic, with a temperature maximum below 20 degrees C. Red cocci had in vitro pH optima above 9.0 although they generally originated from acid samples. Most isolates showed a preference for sugar alcohols and organic acids, compounds which are commonly known to be released by lichens, molds and algae, the other components of the cryptoendolithic ecosystem. These properties indicate that our strains are autochthonous members of the natural Antarctic microbial population.

[Taxonomic position of the lysine producer Brevibacterium flavum]. / O sistematicheskom polozhenii produtsenta lizina Brevibacterium flavum.

Brevibacterium flavum 22 and 22L producing lysine and glutamic acid should be reclassified as Corynebacterium glutamicum on the basis of their chemotaxonomic characteristics: the IV type of the cell wall, corynomycolic acids C32--C34, 57.8% of GC in DNA.

Continuous production of NADP by immobilized Brevibacterium ammoniagenes cells.

Whole cells of Brevibacterium ammoniagenes IAM 1645 having the polyphosphate NAD-kinase were successfully immobilized in a polyacrylamide gel lattice. The immobilized cells were activated by treatment with organic solvents or detergents. The pH optimum of the immobilized cells for the production of NADP was 7.0, and divalent metal ions were required to maintain the elevated activity of polyphosphate NAD-kinase. Highly pure NADP was continuously produced in high yield by the immobilized cell column. The half-life of this column was about eight days.

Differentiation of genera of the coryneform bacteria.

A scheme of the generic structure of the group of coryneform bacteria, including the genera Arthrobacter, Brevibacterium, Cellulomonas, Corynebacterium, and Rhodococcus, is suggested. Morphological, chemotaxonomic (presence and stereochemical form of diaminopimelic acid, lipid A, and L-arabinose), and physiological features were used as diagnostic criteria. The position of Microbacterum and Mycococcus and of coryneforms with a nocardial wall but giving a positive Hugh-Leifson anaerobic test, and also of Arthrobacter pascens, which may be identical with Mycoplana sp., remains uncertain.

Unbalanced growth death due to depletion of Mn2+ in Brevibacterium ammoniagenes.

In the microbial conversion of added hypoxanthine to 5'-inosinic acid, Mn(2+) concentration in the growth medium is known to have a profound effect both on the yield of 5'-inosinic acid and the morphology of cells of Brevibacterium ammoniagenes. To elucidate the mechanism in which Mn(2+) was concerned with cell morphology and 5'-inosinic acid production, effects of Mn(2+) on the macromolecular synthesis were measured. It was found that Mn(2+) strongly governed deoxyribonucleic acid (DNA) synthesis and that, in the medium lacking Mn(2+), DNA synthesis was stopped at the level corresponding to one-fourth to one-third that in the medium supplemented with Mn(2+) (100 mug/liter). On the other hand, cellular ribonucleic acid and protein synthesis was quite indifferent to Mn(2+) concentration. Consequently, cells showed so-called "unbalanced growth death" after 10 hr of culture, losing the ability to form colonies while cell mass was increasing. The elongated cells turned into irregular forms (bulbous, club-shaped, etc.) which finally lysed. Two main reaction components in the conversion of hypoxanthine to 5'-inosinic acid, phosphoribosylpyrophosphate and hypoxanthine phosphoribosyltransferase, were liberated into the medium during lysis. The role of Mn(2+) in the synthesis of DNA and the role of the unbalanced growth death in the conversion of hypoxanthine to 5'-inosinic acid are discussed.