Functional genomics and taxonomic insights into heavy metal tolerant novel bacterium Brevibacterium metallidurans sp. nov. NCCP-602T isolated from tannery effluent in Pakistan.

The strain designated NCCP-602T was isolated from tannery effluent, and displayed aerobic, gram-positive, rod-shaped cells that were characterized by oxidase negative, catalase positive, and non-motile features. The most favourable growth conditions were observed at a temperature of 30°C, pH 7.0, and NaCl concentration of 1% (w/v). It tolerated heavy metals at high concentrations of chromium (3600 ppm), copper (3300 ppm), cadmium (3000 ppm), arsenic (1200 ppm) and lead (1500 ppm). The results of phylogenetic analysis, derived from sequences of the 16S rRNA gene, indicated the position of strain NCCP-602T within genus Brevibacterium and showed that it was closely related to Brevibacterium ammoniilyticum JCM 17537T. Strain NCCP-602 T formed a robust branch that was clearly separate from closely related taxa. A comparison of 16S rRNA gene sequence similarity and dDDH values between the closely related type strains and strain NCCP-602T provided additional evidence supporting the classification of strain NCCP-602T as a distinct novel genospecies. The polar lipid profile included diphosphatidylglycerol, glycolipid, phospholipids and amino lipids. MK-7 and MK-8 were found as the respiratory quinones, while anteiso-C15:0, iso-C15:0, iso-C16:0, iso-C17:0, and anteiso-C17:0 were identified as the predominant cellular fatty acids (> 10%). Considering the convergence of phylogenetic, phenotypic, chemotaxonomic, and genotypic traits, it is suggested that strain NCCP-602 T be classified as a distinct species Brevibacterium metallidurans sp. nov. within genus Brevibacterium with type strain NCCP-602T (JCM 18882T = CGMCC1.62055T).

Influence of Brevibacterium linens RS16 on foliage photosynthetic and volatile emission characteristics upon heat stress in Eucalyptus grandis.

Heat stress induces secondary metabolic changes in plants, channeling photosynthetic carbon and energy, away from primary metabolic processes, including, growth. Use of ACC (1-aminocyclopropane-1-carboxylate) deaminase containing plant growth promoting bacteria (PGPB) in conferring heat resistance in plants and the role of PGPB, in altering net carbon assimilation, constitutive and stress volatile emissions has not been studied yet. We exposed leaves of Eucalyptus grandis inoculated and non-inoculated with PGPB Brevibacterium linens RS16 to two levels of heat stress (37 °C and 41 °C for 5 min) and quantified temporal changes in foliage photosynthetic characteristics and volatile emission rates at 0.5 h, day 1 and day 5 after the stress application. Heat stress resulted in immediate reductions in dark-adapted photosystem II (PSII) quantum yield (Fv/Fm), net assimilation rate (A), stomatal conductance to water vapor (gs), and enhancement of stress volatile emissions, including enhanced emissions of green leaf volatiles (GLV), mono- and sesquiterpenes, light weight oxygenated volatile organic compounds (LOC), geranyl-geranyl diphosphate pathway volatiles (GGDP), saturated aldehydes, and benzenoids, with partial recovery by day 5. Changes in stress-induced volatiles were always less in leaves inoculated with B. linens RS16. However, net assimilation rate was enhanced by bacterial inoculation only in the 37 °C treatment and overall reduction of isoprene emissions was observed in bacterially-treated leaves. Principal component analysis (PCA), correlation analysis and partial least squares discriminant analysis (PLS-DA) indicated that different stress applications influenced specific volatile organic compounds. In addition, changes in the expression analysis of heat shock protein 70 gene (DnaK) gene in B. linens RS16 upon exposure to higher temperatures further indicated that B. linens RS16 has developed its own heat resistance mechanism to survive under higher temperature regimes. Taken together, this study demonstrates that foliar application of ACC deaminase containing PGPB can ameliorate heat stress effects in realistic biological settings.

Isolation, functional characterization and efficacy of biofilm-forming rhizobacteria under abiotic stress conditions.

Abiotic stresses such as salinity, drought and excessive heat are associated with significant loss of crop productivity globally, and require effective strategies for their reduction or tolerance. Biofilm-forming rhizobacteria, which harbor multifarious plant growth promoting traits and tolerance to abiotic stress, are believed to benefit plant health and production even under environmental stresses. The primary objective of this study was to investigate indigenous biofilm-forming rhizobacteria (Pseudomonas spp., Bacillus sp., Pantoea sp., Brevibacterium sp. and Acinetobacter sp.) for their functional diversity relevant to plant growth promoting activities, biofilm development and tolerance to abiotic stress conditions. The most promising isolates among FAP1, FAP2, FAP3, FAP4, FAP5, FAP10, FAB1, FAB3 and FAA1 were selected. Rhizobacteria exhibited high tolerance to salinity (1.5 M NaCl) and drought stress (up to 55% PEG-6000) conditions in vitro. The isolates demonstrated varying levels of PGP activities (IAA production and phosphate solubilization), biofilm development, and production of alginate and exopolysaccharides in the presence of salinity, drought stress and elevated temperature. Further efficacy of the isolates was demonstrated by inoculating to wheat (Triticum aestivum L.) plants in greenhouse conditions under both normal and drought stress for up to 30 days inoculation. The plant growth potential of the isolates was in the order of FAP3 > FAB3 > FAB1 > FAP10 > FAP5 > FAP4 > FAA1 > FAP2 > FAP1. The present study resulted in successful selection of promising PGPR as identified by 16S rRNA gene sequence analysis. Field study is needed to evaluate their relative performance in both 'normal' and stressed environments in order to be exploited for plant stress management.

Brevibacterium from Austrian hard cheese harbor a putative histamine catabolism pathway and a plasmid for adaptation to the cheese environment.

The genus Brevibacterium harbors many members important for cheese ripening. We performed real-time quantitative PCR (qPCR) to determine the abundance of Brevibacterium on rinds of Vorarlberger Bergkäse, an Austrian artisanal washed-rind hard cheese, over 160 days of ripening. Our results show that Brevibacterium are abundant on Vorarlberger Bergkäse rinds throughout the ripening time. To elucidate the impact of Brevibacterium on cheese production, we analysed the genomes of three cheese rind isolates, L261, S111, and S22. L261 belongs to Brevibacterium aurantiacum, whereas S111 and S22 represent novel species within the genus Brevibacterium based on 16S rRNA gene similarity and average nucleotide identity. Our comparative genomic analysis showed that important cheese ripening enzymes are conserved among the genus Brevibacterium. Strain S22 harbors a 22 kb circular plasmid which encodes putative iron and hydroxymethylpyrimidine/thiamine transporters. Histamine formation in fermented foods can cause histamine intoxication. We revealed the presence of a putative metabolic pathway for histamine degradation. Growth experiments showed that the three Brevibacterium strains can utilize histamine as the sole carbon source. The capability to utilize histamine, possibly encoded by the putative histamine degradation pathway, highlights the importance of Brevibacterium as key cheese ripening cultures beyond their contribution to cheese flavor production.

Corrosion behavior of X80 pipeline steel in the presence of Brevibacterium halotolerans in Beijing soil.

In this study, the corrosion behavior of X80 pipeline steel in the presence of Brevibacterium halotolerans (B. halotolerans) was investigated by surface analysis and electrochemical measurements. Results show that B. halotolerans can attach to the surface of X80 steel, and the entire surface is covered with B. halotolerans. The corrosion products induced by B. halotolerans were FeOOH, Fe2O3 and FeSO4. X80 steel corrosion was accelerated in the presence of B. halotolerans and was susceptible to pitting corrosion. The formation of pitting corrosion could be due that the B. halotolerans oxidization of elemental iron in X80 steel to either obtain electrons from the reduction of nitrate in the underlying biofilm or in the damaged corrosion product film.

Brevibacterium linens RS16 confers salt tolerance to Oryza sativa genotypes by regulating antioxidant defense and H+ ATPase activity.

Soil salinity is one of the major limitations that affects both plant and its soil environment, leading to reduced agricultural production. Evaluation of stress severity by plant physical and biochemical characteristics is an established way to study plant-salt stress interaction, but the halotolerant properties of plant growth promoting bacteria (PGPB) along with plant growth promotion is less studied till date. The aim of the present study was to elucidate the strategy, used by ACC deaminase-containing halotolerant Brevibacterium linens RS16 to confer salt stress tolerance in moderately salt-tolerant (FL478) and salt-sensitive (IR29) rice (Oryza sativa L.) cultivars. The plants were exposed to salt stress using 0, 50, and 100 mM of NaCl with and without bacteria. Plant physiological and biochemical characteristics were estimated after 1, 5, 10 days of stress application. H+ ATPase activity and the presence of hydroxyectoine gene (ectD) that is responsible for compatible solute accumulation were also analyzed in bacteria. The height and dry mass of bacteria inoculated plants significantly increased compared to salt-stressed plants, and the differences increased in time dependent manner. Bacteria priming reduced the plant antioxidant enzyme activity, lipid peroxidation and it also regulated the salt accumulation by modulating vacuolar H+ ATPase activity. ATPase activity and presence of hydroxyectoine gene in RS16 might have played a vital role in providing salt tolerance in bacteria inoculated rice cultivars. We conclude that dual benefits provided by the halotolerant plant growth promoting bacteria (PGPB) can provide a major way to improve rice yields in saline soil.

Inoculation of Brevibacterium linens RS16 in Oryza sativa genotypes enhanced salinity resistance: Impacts on photosynthetic traits and foliar volatile emissions.

The emission of volatiles in response to salt stress in rice cultivars has not been studied much to date. Studies addressing the regulation of stress induced volatile emission by halotolerant plant growth promoting bacteria containing ACC (1-aminocyclopropane-1-carboxylate) deaminase are also limited. The objective of the present study was to investigate the salt alleviation potential of bacteria by regulating photosynthetic characteristics and volatile emissions in rice cultivars, and to compare the effects of the bacteria inoculation and salt responses between two rice genotypes. The interactive effects of soil salinity (0, 50, and 100 mM NaCl) and inoculation with Brevibacterium linens RS16 on ACC accumulation, ACC oxidase activity, carbon assimilation and stress volatile emissions after stress application were studied in the moderately salt resistant (FL478) and the salt-sensitive (IR29) rice (Oryza sativa L.) cultivars. It was observed that salt stress reduced foliage photosynthetic rate, but induced foliage ACC accumulation, foliage ACC oxidase activity, and the emissions of all the major classes of volatile organic compounds (VOCs) including the lipoxygenase pathway volatiles, light-weight oxygenated volatiles, long-chained saturated aldehydes, benzenoids, geranylgeranyl diphosphate pathway products, and mono- and sesquiterpenes. All these characteristics scaled up quantitatively with increasing salt stress. The effects of salt stress were more pronounced in the salt-sensitive genotype IR29 compared to the moderately salt resistant FL478 genotype. However, the bacterial inoculation significantly enhanced photosynthesis, and decreased ACC accumulation and the ACC oxidase activity, and VOC emissions both in control and salt-treated plants. Taken together, these results suggested that the ACC deaminase-containing Brevibacterium linens RS16 reduces the temporal regulation of VOC emissions and increases the plant physiological activity by reducing the availability of ethylene precursor ACC and the ACC oxidase activity under salt stress.

Effect of dairy matrices on the survival of Streptococcus thermophilus, Brevibacterium aurantiacum and Hafnia alvei during digestion.

This study evaluated the ability of dairy matrices, different in composition (with and without fat) and structure (liquid and gel), to enhance microorganisms survival through digestion. The viability of three dairy microorganisms Streptococcus thermophilus, Brevibacterium aurantiacum and Hafnia alvei was measured during in vitro and in vivo digestion. S. thermophilus was highly sensitive to gastric stress, and was not found in the duodenal compartment. B. auranticum was moderately sensitive to gastric stress but resistant to duodenal stress. H. alvei was highly resistant to both stresses. LIVE/DEAD confocal microscopy's images, probed the effect of low pH on microorganisms survival. However, in vivo analyses (16S rRNA gene metabarcoding) failed to confirm in vitro observations since tested microorganisms were not detected. Despite of the different evolutions during digestion on buffer capacity, lipolysis, and rheological characteristics, we did not observe any protective effect of the dairy matrices on microorganisms survival.

Survival of cheese-ripening microorganisms in a dynamic simulator of the gastrointestinal tract.

A mixture of nine microorganisms (six bacteria and three yeasts) from the microflora of surface-ripened cheeses were subjected to in vitro digestive stress in a three-compartment "dynamic gastrointestinal digester" (DIDGI). We studied the microorganisms (i) grown separately in culture medium only (ii) grown separately in culture medium and then mixed, (iii) grown separately in culture medium and then included in a rennet gel and (iv) grown together in smear-ripened cheese. The yeasts Geotrichum candidum, Kluyveromyces lactis and Debaryomyces hansenii, were strongly resistant to the whole DIDGI process (with a drop in viable cell counts of less than <1 log CFU mL(-1)) and there were no significant differences between lab cultures and cheese-grown cultures. Ripening bacteria such as Hafnia alvei survived gastric stress less well when grown in cheese (with no viable cells after 90 min of exposure of the cheese matrix, compared with 6 CFU mL(-1) in lab cultures). The ability of Corynebacterium casei and Staphylococcus equorum to withstand digestive stress was similar for cheese and pure culture conditions. When grow in a cheese matrix, Brevibacterium aurantiacum and Arthrobacter arilaitensis were clearly more sensitive to the overall digestive process than when grown in pure cultures. Lactococcus lactis displayed poorer survival in gastric and duodenal compartments when it had been grown in cheese. In vivo experiments in BALB/c mice agreed with the DIDGI experiments and confirmed the latter's reliability.

Draft genome sequence of Brevibacterium massiliense strain 541308T.

A draft genome sequence of Brevibacterium massiliense, an aerobic bacterium isolated from a human ankle discharge, is described here. CRISPR-associated proteins were found to be encoded in the genome, and analysis of transport proteins was performed.

Brevibacillus expression system: host-vector system for efficient production of secretory proteins.

Brevibacillus expression system is an effective bacterial expression system for secretory proteins. The host bacterium, Brevibacillus choshinensis, a gram-positive bacterium, has strong capacity to secrete a large amount of proteins (approximately 30 g/L), which mostly consist of cell wall protein. A host-vector system that utilizes such high expression capacity has been constructed for the production of secretory proteins and tested for various heterologous proteins, including cytokines, enzymes, antigens, and adjuvants.

Optimization and characterization of a new lipopeptide biosurfactant produced by marine Brevibacterium aureum MSA13 in solid state culture.

The biosurfactant production of a marine actinobacterium Brevibacterium aureum MSA13 was optimized using industrial and agro-industrial solid waste residues as substrates in solid state culture (SSC). Based on the optimization experiments, the biosurfactant production by MSA13 was increased to threefold over the original isolate under SSC conditions with pre-treated molasses as substrate and olive oil, acrylamide, FeCl(3) and inoculums size as critical control factors. The strain B. aureum MSA13 produced a new lipopeptide biosurfactant with a hydrophobic moiety of octadecanoic acid methyl ester and a peptide part predicted as a short sequence of four amino acids including pro-leu-gly-gly. The biosurfactant produced by the marine actinobacterium MSA13 can be used for the microbially enhanced oil recovery processes in the marine environments.

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.

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 marinum sp. nov., isolated from seawater.

A novel yellow-pigmented actinobacterium was isolated from seawater collected from Hwasun Beach in Jeju, Republic of Korea. A comparative analysis of the 16S rRNA gene sequence indicated that the organism, designated HFW-26(T), was closely related to members of the genus Brevibacterium. As found for other species of the genus Brevibacterium, strain HFW-26(T) possessed meso-diaminopimelic acid as the diagnostic cell-wall diamino acid, contained MK-8(H(2)) as the major menaquinone, contained polar lipids that included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and an unknown phospholipid, and had anteiso-C(15 : 0) and anteiso-C(17 : 0) as the predominant fatty acids. The G+C content of the DNA was 71.4 mol%. The phylogenetically closest relative was Brevibacterium picturae DSM 16132(T) (99.0 % 16S rRNA gene sequence similarity). However, DNA-DNA hybridization of strain HFW-26(T) showed 35.1-43.7 % relatedness with respect to B. picturae DSM 16132(T). The novel isolate could be clearly distinguished from B. picturae DSM 16132(T) on the basis of some cultural, physiological and biochemical characteristics. A battery of phenotypic and genetic data obtained in this study suggest that strain HFW-26(T) represents a novel species of the genus Brevibacterium, for which the name Brevibacterium marinum sp. nov. is proposed. The type strain is HFW-26(T) (=JBRI 2001(T)=KCTC 19221(T)=DSM 18964(T)).

Brevibacterium oceani sp. nov., isolated from deep-sea sediment of the Chagos Trench, Indian Ocean.

Two bacterial strains, designated BBH5 and BBH7(T), were isolated from a deep-sea sediment sample collected from the Chagos Trench of the Indian Ocean (1 degrees 06' S 7 degrees 31' E). Based on their 16S rRNA gene sequence similarity (99.9%), level of DNA-DNA relatedness (93%) and a number of similar phenotypic characteristics, the two strains are identified as representing the same species. Their phylogenetically nearest neighbours, based on 16S rRNA gene sequence similarity values (97.9-98.4%), were identified as Brevibacterium iodinum, Brevibacterium epidermidis, Brevibacterium linens and Brevibacterium permense. However, strains BBH5 and BBH7(T) could be distinguished from the above four species by a number of phenotypic characteristics, and levels of DNA-DNA relatedness between the two new isolates and these Brevibacterium species were 35-42%. Therefore, strains BBH5 and BBH7(T) are considered to represent a novel species of the genus Brevibacterium, for which the name Brevibacterium oceani sp. nov. is proposed. The type strain is BBH7(T) (=LMG 23457(T) =IAM 15353(T)).

Efficient cyclic system to yield ectoine using Brevibacterium sp. JCM 6894 subjected to osmotic downshock.

Brevibacterium sp. JCM 6894 cells grown in the presence of 1.5-2.5 M NaCl for 24 h at 30 degrees C were subjected to the osmotic downshock. Downshocked cells after ectoine release were grown for further 24 h in the fresh medium with same salinity as before shock. When this cyclic system was applied to the strain JCM 6894, the amount of ectoine in the cells increased with an increase of incubation time, which indicates that the cells manipulated by the present conditions were enough active to survive and synthesize ectoine after several times of osmotic downshock. In the presence of 2 M NaCl, the highest yield of ectoine released was achieved in this cyclic system, more than 2.4 g/L during 7 days of incubation. (1)H and (13)C-NMR analyses of solutes released from the cells by the osmotic downshock showed the presence of only ectoine with high purity. Release of ectoine from the cells was carried out within 5 min and its rates were increased by the dilution in the downshock treatment. For the convenience of operations, non-sterilized medium containing 2 M NaCl was examined for the cell growth in the present system, in which almost same level of ectoine yield, release rates, and cell viability were observed as those of sterilized medium.

Supplementation effects of hydroxyectoine on proline uptake of downshocked Brevibacterium sp. JCM 6894.

Downshock treatment of the halotolerant Brevibacterium sp. JCM 6894 was a prerequisite for proline uptake which is a function for cell survival. Hydroxyectoine served as an effective stimulator for the proline uptake and cell survival of the downshocked cells of this strain. Duration of osmotic downshock, downshock strength, and the kinds of osmolyte affected the efficient rate of growth (ERG) and the uptake of proline. A shorter duration of osmotic downshock, that is

Effectiveness of autochthonous bacterium and mycorrhizal fungus on Trifolium growth, symbiotic development and soil enzymatic activities in Zn contaminated soil.

AIMS: This study investigates how autochthonous micro-organisms [bacterium and/or arbuscular mycorrhizal (AM) fungi] affected plant tolerance to Zn contamination. METHODS AND RESULTS: Zinc-adapted and -nonadapted Glomus mosseae strains protected the host plant against the detrimental effect of Zn (600 microg g(-1)). Zn-adapted bacteria increased root growth and N, P nutrition in plants colonized by adapted G. mosseae and decreased the specific absorption rate (SAR) of Cd, Cu, Mo or Fe in plants colonized by Zn-nonadapted G. mosseae. Symbiotic structures (nodule number and extraradical mycelium) were best developed in plants colonized by those Zn-adapted isolates that were the most effective in increasing plant Zn tolerance. The bacterium also increased the quantity and quality (metabolic characteristics) of mycorrhizal colonization, with the highest improvement for arbuscular vitality and activity. Inocula also enhanced soil enzymatic activities (dehydrogenase, beta-glucosidase and phosphatase) and indol acetic acid (IAA) accumulation, particularly in the rhizosphere of plants inoculated with Zn-adapted isolates. CONCLUSIONS: Glomus mosseae strains have a different inherent potential for improving plant growth and nutrition in Zn-contaminated soil. The bacterium increased the potential of mycorrhizal mycelium as inoculum. SIGNIFICANCE AND IMPACT OF THE STUDY: Mycorrhizal performance, particularly that of the autochthonous strain, was increased by the bacterium and both contributed to better plant growth and establishment in Zn-contaminated soils.