Strain Brevibacterium Research Articles

THREONINE MUTANT STRAIN-PRODUCER BREVIBACTERIUM FLAVUM ІМВ В-7446 CHARACTERISTICS AND OPTIMIZATION PROCESS OF ITS BIOSYNTHESIS

Aim. Identification and optimization process of biosynthesis threonine of mutant strain-producer Brevibacterium flavum ІМВ В-7446. Methods. The strain Brevibacterium flavum ІМВ В-7446 was studied using standard microbiological and biochemical methods. A fragment of genomic DNA isolated from agarose gel using a set of «Macherey-Nagel NucleoSpin Extract» according to the instructions of the manufacturer and sequenced. A comparative analysis was done by evaluation of statistical significance adjustment nucleotide sequences using the program ClustalW. Phylogenetic dendrogramm was created using a combination of techniques nearest neighbors and maximum likelihood. Results. The influence of different process parameters on the synthesis of cultivation threonine mutant strain and stability. Intensification synthesis was achieved by introducing growth factors in the culture medium. Powered dendrogramm of phylogenetic relationships investigated the strains and related the strains of Brevibacterium databases «GenBank». Conclusions. Comparative analysis of nucleotide sequences of gene 16S rRNA sequences included in the database GenBank, showed that the mutant and parent strains may belong to the genus Corynebacterium. Mutant strain had a high percentage of similarity (98%) with a species C. glutamicum. Optimization of the medium for culturing and producing the optimal cultivation parameters (temperature, pH, dissolved oxygen, the amount of inoculation, growth factors, various carbon sources) was established. Threonine strain was produced 11,9 g/dm3 by optimized cultivation conditions.

Biosynthesis, characterization, and antimicrobial applications of silver nanoparticles.

In the present study, the strain Brevibacterium frigoritolerans DC2 was explored for the efficient and extracellular synthesis of silver nanoparticles. These biosynthesized silver nanoparticles were characterized by ultraviolet-visible spectrophotometry, which detected the formation of silver nanoparticles in the reaction mixture and showed a maximum absorbance at 420 nm. In addition, field emission transmission electron microscopy revealed the spherical shape of the nanoparticles. The dynamic light scattering results indicated the average particle size of the product was 97 nm with a 0.191 polydispersity index. Furthermore, the product was analyzed by energy dispersive X-ray spectroscopy, X-ray diffraction, and elemental mapping, which displayed the presence of elemental silver in the product. Moreover, on a medical platform, the product was checked against pathogenic microorganisms including Vibrio parahaemolyticus, Salmonella enterica, Bacillus anthracis, Bacillus cereus, Escherichia coli, and Candida albicans. The nanoparticles demonstrated antimicrobial activity against all of these pathogenic microorganisms. Additionally, the silver nanoparticles were evaluated for their combined effects with the commercial antibiotics lincomycin, oleandomycin, vancomycin, novobiocin, penicillin G, and rifampicin against these pathogenic microorganisms. These results indicated that the combination of antibiotics with biosynthesized silver nanoparticles enhanced the antimicrobial effects of antibiotics. Therefore, the current study is a demonstration of an efficient biological synthesis of silver nanoparticles by B. frigoritolerans DC2 and its effect on the enhancement of the antmicrobial efficacy of well-known commercial antibiotics.

Poster #S28 IMMUNE RESPONSE TO STRESS IN POSTPARTUM PSYCHOSIS

Seawater, sediments and fuel samples extracted from a small area of the Prestige wreck (4000 m deep) were studied to check the microbial activity of the area, the occurrence of indigenous hydrocarbon-degrading bacteria and the presence of bacteria able to produce bioemulsifiers. Twenty-one strains with the capacity to degrade hydrocarbons and/or produce useful bioemulsifiers were selected. Phylogenetic affiliation of these isolates placed them in the genus Bacillus (8 strains), Pseudomonas (3 strains), Halomonas (4 strains), Pseudoalteromonas (1 strain), Brevibacterium (2 strains), and Marinobacter (1 strain) and 2 strains were identified as marine bacterium. Hydrocarbon degradation was established by determining the amount of alkanes and polycyclic aromatic hydrocarbons by GC/MS analyses. In this study, Pseudomonas, Bacillus and Brevibacterium strains were the most efficient hydrocarbon-degrading marine bacteria. In contrast, Halomonas strains produced the highest amount of efficient biopolymers with emulsifying activity. The yield, chemical composition and functional properties of these bioemulsifiers were affected by the incubation time required for production. In addition, supplementation of the hydrocarbon culture medium with bioemulsifiers (S22-BE, S24-BE and AD2-BE) clearly stimulated the growth of strains S25, S28 and S29 and enhanced the ability of these strains to biodegrade alkanes, alkenes and aromatic compounds.

Selection of new highly active L-alanine producer strains of Brevibacterium flavum and comparison of their activity in alanine synthesis

New mutants resistant to L-cycloserine and β-chloro-L-alanine and not described previously were obtained from a parental strain Brevibacterium flavum AA5. Their ability to produce alanine was studied. It was found that the resistance to L-cycloserine did not significantly affect the yield of L-alanine, whereas the resistance to β-chloro-L-alanine of B. flavum GL1 and B. flavum GL18 producers exceeded the initial level of the L-alanine synthesis by 23 and 38%, respectively.

Spelaeicoccus albus gen. nov., sp. nov., an actinobacterium isolated from a natural cave

A novel Gram-stain-positive, non-endospore-forming, coccoid actinobacterium, designated strain D3-40(T), was isolated from the soil of a natural cave and characterized by means of a polyphasic taxonomic analysis. 16S rRNA gene sequence analysis showed that strain D3-40(T) is a member of the suborder Micrococcineae and forms a distinct branch at the base of a Brevibacteriaceae cluster. Its closest relative is the type strain of Brevibacterium samyangense (95.7 % sequence similarity). The chemotaxonomic characteristics were as follows: the cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid; the major menaquinone was MK-9(H2); the polar lipids consisted of phosphatidylglycerol, phosphatidylinositol, an unknown glycolipid and an unknown phospholipid; the major fatty acids were anteiso-C15 : 0, anteiso-C17 : 0, iso-C15 : 0, C16 : 0 and cyclohexyl-C17 : 0; mycolic acids were absent. The G+C content of the DNA was 64.3 mol%. On the basis of morphological, chemotaxonomic and phylogenetic data, it is suggested that the organism represents a novel species of a new genus within the family Brevibacteriaceae, for which the name Spelaeicoccus albus gen. nov., sp. nov. is proposed. The type strain of the type species is D3-40(T) ( = KCTC 29141(T) = DSM 26341(T)).

Characterization of a novel cyfluthrin-degrading bacterial strain Brevibacterium aureum and its biochemical degradation pathway

Brevibacterium aureum DG-12, a new bacterial strain isolated from active sludge, was able to degrade and utilize cyfluthrin as a growth substrate in the mineral medium. Response surface methodology using central composite rotatable design of cultural conditions was successfully employed for optimization resulting in 88.6% degradation of cyfluthrin (50mgL−1) within 5days. The bacterium degraded cyfluthrin by cleavage of both the carboxylester linkage and diaryl bond to form 2,2,3,3-tetramethyl-cyclopropanemethanol, 4-fluoro-3-phenexy-benzoic acid, 3,5-dimethoxy phenol, and phenol, and subsequently transformed these compounds with a maximum specific degradation rate, half-saturation constant and inhibition constant of 1.0384day−1, 20.4967mgL−1, and 141.9013mgL−1, respectively. A novel degradation pathway for cyfluthrin was proposed based on analysis of these metabolites. In addition, this strain was found capable of degrading a wide range of synthetic pyrethroid insecticides. Our results suggest that B. aureum DG-12 may be an ideal microorganism for bioremediation of the pyrethroid-contaminated environments.

Study of strains brevibacterium as producer of lysine

Study of strains brevibacterium as producer of lysine

Effect of ilvBN Overexpression on the Metabolic Flux Distributions in Brevibacterium Flavum XVO5O5

In this stuty, overexpression of the ilvBN gene, enconding acetohydroxy acid synthase (AHAS), in the valine-prouducing strain Brevibacterium flavum XVO5O5 resulted in increased valine production by 9.85% and decreased alanine and acetic acid (the main by-products) by 30.49% and 20.89%, respectively. Metabolic flux analysis revealed a 19.9 % increase of valine synthase pathway in response to overexpression of the ilvBN. The benefit of overexpressing ilvBN was that it resulted in an intracellular pyruavte concentration decresed from 10.45mM to 4.42mM, which means that more carbon flux entering efficiently the pathway of synthesizing L-valine. It could be concluded from the results that ilvBN overexpression was essential achieve suffient carbon flux towards the desired product. Overexpression of ilvBN reduced pyruvate availability for L-alanine formation and therefor resulted in L-alanine reduction.

Computational approach for comparative phylogenetic analysis of isolated chromium resistant strain Brevibacterium casei

The isolated (Cr [VI]) resistant bacterial strain was identified as Brevibacterium caseiby 16S rRNA sequencing (Genbank Accession Number: EU781952). The generated sequenced data was used for construction of Phylogenetic tree (MEGA 3.1and clustalw2) and predict the two dimensional alignment of the highly conserved regions. Computational comparative 16S rRNA sequence analysis of the isolated strain with different chromium resistant strains was carried out .The result shows that the compared sequence differs from the other especially in the region of 210 to 242, 440 to 480 nucleotides and had higher resemblance from 245 to 440, 500 to 724 in nucleotide base pair (in the Escherichia coli numbering system). From the overall study it can be predicted that the conserved region in the nucleotide base pair in all Cr (VI) resistant species may play an important role in chromium resistance. Key words: Hexavalent chromium, 16S rRNA sequence, Brevibacterium casei, computational approach, phylogenetic analysis.

Biodegradative potential and characterization of bioemulsifiers of marine bacteria isolated from samples of seawater, sediment and fuel extracted at 4000 m of depth (Prestige wreck)

Seawater, sediments and fuel samples extracted from a small area of the Prestige wreck (4000 m deep) were studied to check the microbial activity of the area, the occurrence of indigenous hydrocarbon-degrading bacteria and the presence of bacteria able to produce bioemulsifiers. Twenty-one strains with the capacity to degrade hydrocarbons and/or produce useful bioemulsifiers were selected. Phylogenetic affiliation of these isolates placed them in the genus Bacillus (8 strains), Pseudomonas (3 strains), Halomonas (4 strains), Pseudoalteromonas (1 strain), Brevibacterium (2 strains), and Marinobacter (1 strain) and 2 strains were identified as marine bacterium. Hydrocarbon degradation was established by determining the amount of alkanes and polycyclic aromatic hydrocarbons by GC/MS analyses. In this study, Pseudomonas, Bacillus and Brevibacterium strains were the most efficient hydrocarbon-degrading marine bacteria. In contrast, Halomonas strains produced the highest amount of efficient biopolymers with emulsifying activity. The yield, chemical composition and functional properties of these bioemulsifiers were affected by the incubation time required for production. In addition, supplementation of the hydrocarbon culture medium with bioemulsifiers (S22-BE, S24-BE and AD2-BE) clearly stimulated the growth of strains S25, S28 and S29 and enhanced the ability of these strains to biodegrade alkanes, alkenes and aromatic compounds.

Brevibacterium sandarakinum sp. nov., isolated from a wall of an indoor environment

A Gram-stain-positive, rod-shaped, non-endospore-forming, orange-pigmented (coloured) actinobacterium (01-Je-003(T)) was isolated from the wall of an indoor environment primarily colonized with moulds. On the basis of 16S rRNA gene sequence similarity studies, strain 01-Je-003(T) was shown to belong to the genus Brevibacterium and was most similar to the type strains of Brevibacterium picturae (98.8 % similarity), Brevibacterium marinum (97.3 %) and Brevibacterium aurantiacum (97.2 %). Chemotaxonomic data [predominant quinone menaquinone MK-8(H2); polar lipid profile consisting of major compounds diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid; characteristic cell-wall diamino acid meso-diaminopimelic acid; polyamine pattern showing major compounds putrescine and cadaverine; major fatty acids anteiso-C(15 : 0) and anteiso-C(17 : 0)] supported the affiliation of strain 01-Je-003(T) to the genus Brevibacterium. The results of DNA-DNA hybridizations and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain 01-Je-003(T) from the two most closely related species, B. picturae and B. marinum. Strain 01-Je-003(T) therefore represents a novel species, for which the name Brevibacterium sandarakinum sp. nov. is proposed, with the type strain 01-Je-003(T) (=DSM 22082(T) =CCM 7649(T)).

Brevibacterium pityocampae sp. nov., isolated from caterpillars of Thaumetopoea pityocampa (Lepidoptera, Thaumetopoeidae)

This work deals with the taxonomic study of a bacterium, strain Tp12(T), isolated from caterpillars of the pine processionary moth (Thaumetopoea pityocampa Denis & Schiffermüller, 1775; Lepidoptera, Thaumetopoeidae). The isolate was assigned to the genus Brevibacterium on the basis of a polyphasic taxonomic study, including morphological and biochemical characteristics, 16S rRNA gene sequence analysis, fatty acid analysis and DNA G+C content. The highest 16S rRNA gene sequence similarity to this isolate was approximately 96 %, with the type strains of Brevibacterium album and Brevibacterium samyangense. Cellular fatty acids of the isolate are of the branched type, with the major components being anteiso-C(15 : 0) and anteiso-C(17 : 0). The DNA G+C content was 69.8 mol%. Although the strain was related to B. album and B. samyangense according to 16S rRNA gene sequence analysis, it differed from any known species of Brevibacterium. Based on this evidence, the novel species Brevibacterium pityocampae sp. nov. is proposed, with strain Tp12(T) (=DSM 21720(T) =NCCB 100255(T)) as the type strain.

Development of a large scale process for the conversion of polysialogangliosides to monosialotetrahexosylganglioside with a novel strain of Brevibacterium casei producing sialidase

A bioconversion process of producing GM1 (monosialotetrahexosylganglioside) on an industrial scale was developed with a novel sialidase-producing strain Brevibacterium casei. The sialidase hydrolyzed polysialogangliosides to produce GM1 but did not act on GM1. When Brevibacterium casei was cultured in a synthetic medium containing crude pig brain gangliosides (10% w/v) at 30 degrees C for 24 h in a 50 l fermenter, most of the polysialogangliosides were converted to GM1. The content of GM1 was increased from 9% in crude gangliosides to 45% with 70% (w/w) yield.

Host–parasite interactions in closed and open microbial cultivation system

The study addresses interaction of bacteria and phages in the host–parasite system in batch and continuous cultures. The study system consists of the auxotrophic strain of Brevibacterium – Brevibacterium sp. 22L – and the bacteriophage of Brevibacterium sp., isolated from the soil by the enrichment method. 1. Closed system. In the investigation of the relationship between the time of bacterial lysis and multiplicity of phage infection it has been found that at a lower phage amount per cell it takes a longer time for the lysis of the culture to become discernible. Another important factor determining cytolysis in liquid medium is the physiological state of bacterial population. Specific growth rate of bacteria at the moment of phage infection has been chosen as an indicator of the physiological state of bacteria. It has been shown that the shortest latent period and the largest output of the phage are observed during the logarithmic growth phase of bacteria grown under favorable nutrient conditions. In the stationary phase, bacterial cells become “a bad host” for the phage, whose reproduction rate decreases, and the lysis either slows down significantly or does not occur at all. 2. Open system. It has been found that in continuous culture, the components of the host–parasite system can coexist over a long period of time. After phage infection, the sizes of the both populations vary for some time and then the density of the host population reaches the level close to that of the uninfected culture. The phage population copies the variations in the density of the host population, but in antiphase. It has been proven that the bacterium becomes resistant to the phage rather soon. It has been supposed that primary resistance is of physiological origin, because the percentage of cells that have survived lysis – about 0.2% of the initial bacterial population – is too high for phage-resistant mutants. Bacteria and phages cultured over extended periods of time in the host–parasite system have been found to co-evolve. The stressful effect of the phage causes development of bacteria resistant to this phage, then a mutant phage capable of lysing these bacteria evolves, etc.

Growth stimulation of Brevibacterium sp. by siderophores

To assess which types of siderophores are typically produced by Brevibacterium and how siderophore production and utilization traits are distributed within this genus. During co-cultivation experiments it was found that growth of B. linens Br5 was stimulated by B. linens NIZO B1410 by two orders of magnitude. The stimulation was caused by the production of hydroxamate siderophores by B. linens NIZO B1410 that enabled the siderophore-auxotrophic strain Br5 to grow faster under the applied iron-limited growth conditions. Different patterns of siderophore production and utilization were observed within the genus Brevibacterium. These patterns did not reflect the phylogenetic relations within the group as determined by partial 16S rDNA sequencing. Most Brevibacterium strains were found to utilize hydroxamate siderophores. Brevibacteria can produce and utilize siderophores although certain strains within this genus are siderophore-auxotrophic. It is reported for the first time that brevibacteria produce and utilize siderophores. This knowledge can be utilized to stimulate growth of auxotrophic strains under certain conditions. Enhancing the growth rate of Brevibacterium is of importance for the application of this species, for example, for cheese manufacturing or for industrial production of enzymes or metabolites.

Molecular identification and differentiation of Brevibacterium species and strains

Amplified ribosomal DNA restriction enzyme analysis (ARDRA), pulsed field gel electrophoresis (PFGE) and ribotyping were used to differentiate among 24 strains of Brevibacterium linens, Brevibacterium casei and Brevibacterium epidermidis obtained from type culture collections or isolated from various smear ripened cheeses. ARDRA was applied to the 16S rDNA. B. linens was shown to be a quite heterogenic group with 2 to at least 4 copies of rrn operons per strain with aberrant nucleotide sequences. AccI gave genus specific restriction patterns and was used to separate Brevibacterium from Corynebacterium species. The expected species specificity of TaqI applied to B. linens type culture strains, but not to all strains isolated from cheese. By AvaI restriction, B. casei and B. linens were differentiated from B. epidermidis and the orange pigmented Arthrobacter casei, a new species of coryneform bacteria; by XmnI restriction, B. linens and B. epidermidis were differentiated from B. casei. One of 4 B. linens genotypes could not be distinguished from B. casei by this method. Here, the typical orange B. linens pigments were used for classification, which was confirmed by partial sequencing of the 16S rDNA.

Beneficial Role of Hydrophytes in Removing Cr(Vi) From Wastewater in Association With Chromate-Reducing Bacterial Strains Ochrobactrum Intermedium and Brevibacterium

This study deals with the use of three chromium-resistant bacterial strains (Ochrobactrum intermedium CrT-1, Brevibacterium CrT-13, and CrM-1) in conjunction with Eichornia crassipes for the removal of toxic chromium from wastewater. Bacterial strains resulted in reduced uptake of chromate into inoculated plants as compared to noninoculated control plants. In the presence of different heavy metals, chromium uptake into the plants was 28.7 and 7.15% less at an initial K 2 CrO 4 concentration of 100 and 500 μg ml−1 in comparison to a metal free chromium solution. K 2 CrO 4 uptake into the plant occurred at different pHs tested, but maximum uptake was observed at pH 5. Nevertheless, the bacterial strains caused some decrease in chromate uptake into the plants, but the combined effect of plants and bacterial strains conduce more removal of Cr(VI) from the solution.

Isolation and Identification of a Brevibacterium linens strain degrading p-nitrophenol

A bacterium was isolated from garden soil in basal salts medium containing p-nitrophenol (PNP). Subsequent subcultures in agar, nutrient agar plates and agar slants by streaking led to isolation of pure colonies. The pure culture could degrade up to 300 mg/L PNP in presence of yeast extract. It was Gram positive rods, mostly single, catalase-positive, hydrolyzing strach and casein but not urea. Gelatin liquefaction was positive whereas acid production from carbohydrates was negative. It showed tyrosine clearing and had meso-DAP as the characteristic cell wall amino acid. On the basis of the morphological, physiological, and biochemical tests the organism was identified as Brevibacterium linens . To our knowledge, this is the first report of any Brevibacterium strain able to degrade PNP. African Journal of Biotechnology Vol. 4 (3), pp. 256-257, 2005

Isolation and Identification of a Brevibacterium linens strain degrading p-nitrophenol

A bacterium was isolated from garden soil in basal salts medium containing p-nitrophenol (PNP). Subsequent subcultures in agar, nutrient agar plates and agar slants by streaking led to isolation of pure colonies. The pure culture could degrade up to 300 mg/L PNP in presence of yeast extract. It was Gram positive rods, mostly single, catalase-positive, hydrolyzing strach and casein but not urea. Gelatin liquefaction was positive whereas acid production from carbohydrates was negative. It showed tyrosine clearing and had meso-DAP as the characteristic cell wall amino acid. On the basis of the morphological, physiological, and biochemical tests the organism was identified as Brevibacterium linens. To our knowledge, this is the first report of any Brevibacterium strain able to degrade PNP. Key words: p-Nitrophenol, nitroaromatics, (bio) degradation, isolation, Brevibacterium.

A mannitol teichoic acid containing rhamnose and pyruvic acid acetal from the cell wall of Brevibacterium permense VKM Ac-2280

The cell wall of Brevibacterium permense VKM Ac-2280 contains two teichoic acids. The major polymer represents a 1,6-poly(mannitol phosphate) substituted wirh either l-rhamnose (∼70%, unit A) or ( S)-acetal of pyruvic acid (∼30%, unit B) with the overall chain length ∼10 mannitol phosphate units. The other polymer is an unsubstituted 1,3-poly(glycerol phosphate). The structures of the polymers were established using chemical degradations and NMR spectroscopy. The data obtained may be helpful in determination of the species-specific status of newly isolated Brevibacterium strains.