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).

Exploring the biosynthetic gene clusters in Brevibacterium: a comparative genomic analysis of diversity and distribution.

Exploring Brevibacterium strains from various ecosystems may lead to the discovery of new antibiotic-producing strains. Brevibacterium sp. H-BE7, a strain isolated from marine sediments from Northern Patagonia, Chile, had its genome sequenced to study the biosynthetic potential to produce novel natural products within the Brevibacterium genus. The genome sequences of 98 Brevibacterium strains, including strain H-BE7, were selected for a genomic analysis. A phylogenomic cladogram was generated, which divided the Brevibacterium strains into four major clades. A total of 25 strains are potentially unique new species according to Average Nucleotide Identity (ANIb) values. These strains were isolated from various environments, emphasizing the importance of exploring diverse ecosystems to discover the full diversity of Brevibacterium. Pangenome analysis of Brevibacterium strains revealed that only 2.5% of gene clusters are included within the core genome, and most gene clusters occur either as singletons or as cloud genes present in less than ten strains. Brevibacterium strains from various phylogenomic clades exhibit diverse BGCs. Specific groups of BGCs show clade-specific distribution patterns, such as siderophore BGCs and carotenoid-related BGCs. A group of clade IV-A Brevibacterium strains possess a clade-specific Polyketide synthase (PKS) BGCs that connects with phenazine-related BGCs. Within the PKS BGC, five genes, including the biosynthetic PKS gene, participate in the mevalonate pathway and exhibit similarities with the phenazine A BGC. However, additional core biosynthetic phenazine genes were exclusively discovered in nine Brevibacterium strains, primarily isolated from cheese. Evaluating the antibacterial activity of strain H-BE7, it exhibited antimicrobial activity against Salmonella enterica and Listeria monocytogenes. Chemical dereplication identified bioactive compounds, such as 1-methoxyphenazine in the crude extracts of strain H-BE7, which could be responsible of the observed antibacterial activity. While strain H-BE7 lacks the core phenazine biosynthetic genes, it produces 1-methoxyphenazine, indicating the presence of an unknown biosynthetic pathway for this compound. This suggests the existence of alternative biosynthetic pathways or promiscuous enzymes within H-BE7's genome.

Reclassification of Brevibacterium frigoritolerans as Peribacillus frigoritolerans comb. nov. based on phylogenomics and multiple molecular synapomorphies.

The taxonomic assignment of Brevibacterium frigoritolerans together with the in-house environmental isolate EB93 was reassessed in this study using phylogenetic and phylogenomic approaches, and the detection of multiple molecular synapomorphies. Results from the reconstructed phylogenetic trees based on the 16S rRNA gene sequences, the concatenated protein sequences of GyrA-GyrB-RpoB-RpoC, and the whole-genome sequences revealed the consistent exclusion of B. frigoritolerans and the environmental isolate EB93 from the cluster formed by the type strains of the genus Brevibacterium. In addition, B. frigoritolerans and the environmental isolate EB93 were both observed to form a clade together with the type strains of the genus Peribacillus. The results from the analysis of the digital DNA-DNA hybridization, average nucleotide identity, average amino acid identity and the difference in the G+C content also corroborated with the phylogenetic inference, and that B. frigoritolerans and the environmental isolate EB93 were of the same species. Furthermore, the presence of the molecular synapomorphies in the protein sequences noted in the description of the genus Peribacillus were also observed in B. frigoritolerans, further strengthening its taxonomic affiliation in the genus. Based on the evidence from the multiple lines of analyses, we propose the reclassification of Brevibacterium frigoritolerans as a member of the genus Peribacillus and assume the name Peribacillus frigoritolerans comb. nov. (type strain DSM 8801 T=ATCC 25097T=CCUG 43489T=CIP 67.20T=JCM 11681T).

Cervical Squamous Intraepithelial Lesions Are Associated with Differences in the Vaginal Microbiota of Mexican Women.

Cervical cancer is an important health concern worldwide and is one of the leading causes of death in Mexican women. Previous studies have shown changes in the female genital tract microbe community related to human papillomavirus (HPV) infection and cervical cancer; yet, this link remains unexplored in many human populations. This study evaluated the vaginal bacterial community among Mexican women with precancerous squamous intraepithelial lesions (SIL). We sequenced the V3 region of the 16S rRNA gene in cervical samples from 228 Mexican women, including 121 participants with SIL, most of which were HPV positive, and 107 healthy women without HPV infection or SIL. The presence of SIL was associated with changes in composition (beta diversity) and with a higher species richness (Chao1). A comparison of HPV-positive women with and without SIL showed that microbiota changes occurred even in the absence of SIL. Multivariate association with linear models (MaAsLin) analysis yielded independent associations between HPV infection and an increase in the relative abundance of Brachybacterium conglomeratum and Brevibacterium aureum as well as a decrease in two Lactobacillus iners operational taxonomic units (OTUs). We also identified a positive independent association between HPV-16, the most common HPV subtype linked to SIL, and Brachybacterium conglomeratum. Our work indicates that HPV infection leading to SIL is primarily associated with shifts in vaginal microbiota composition, some of which may be specific to this human population. IMPORTANCE Human papillomavirus (HPV) plays a critical role in cervical carcinogenesis but is not sufficient for cervical cancer development, indicating the involvement of other factors. The vaginal microbiota is an important factor in controlling infections caused by HPV, and, depending on its composition, it can modulate the microenvironment in vaginal mucosa against viral infections. Ethnic and sociodemographic factors influence differences in vaginal microbiome composition, which underlies the dysbiotic patterns linked to HPV infection and cervical cancer across different populations of women. Here, we provide evidence for associations between vaginal microbiota patterns and HPV infection linked to ethnic and sociodemographic factors. To our knowledge, this is the first report of the species Brevibacterium aureum and Brachybacterium conglomeratum linked to HPV infection or squamous intraepithelial lesions (SIL).

Brevibacterium limosum sp. nov., Brevibacterium pigmenatum sp. nov., and Brevibacterium atlanticum sp. nov., three novel dye decolorizing actinobacteria isolated from ocean sediments.

During a study of the marine actinobacterial biodiversity, a large number of Brevibacterium strains were isolated. Of these, five that have relatively low 16S rRNA gene similarity (98.5-99.3%) with validly published Brevibacterium species, were chosen to determine taxonomic positions. On the basis of 16S rRNA gene sequence analysis and BOX-PCR fingerprinting, strains o2T, YB235T, and WO024T were selected as representative strains. Genomic analyses, including average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH), clearly differentiated the three strains from each other and from their closest relatives, with values ranging from 82.8% to 91.5% for ANI and from 26.7% to 46.5% for dDDH that below the threshold for species delineation. Strains YB235T, WO024T, and o2T all exhibited strong and efficient decolorization activity in congo red (CR) dyes, moderate decolorization activity in toluidine blue (TB) dyes and poor decolorization in reactive blue (RB) dyes. Genes coding for peroxidases and laccases were identified and accounted for these strains' ability to effectively oxidize a variety of dyes with different chemical structures. Mining of the whole genome for secondary metabolite biosynthesis gene clusters revealed the presence of gene clusters encoding for bacteriocin, ectoine, NRPS, siderophore, T3PKS, terpene, and thiopeptide. Based on the phylogenetic, genotypic and phenotypic data, strains o2T, YB235T and WO024T clearly represent three novel taxa within the genus Brevibacterium, for which the names Brevibacterium limosum sp. nov. (type strain o2T = JCM 33844T = MCCC 1A09961T), Brevibacterium pigmenatum sp. nov. (type strain YB235T = JCM 33843T = MCCC 1A09842T) and Brevibacterium atlanticum sp. nov. (type strain WO024T = JCM 33846T = MCCC 1A16743T) are proposed.

Genome sequence analysis and bioactivity profiling of marine-derived actinobacteria, Brevibacterium luteolum, and Cellulosimicrobium funkei.

Genome analysis gives important insights into the biosynthetic potential of marine actinobacteria. The genomes of two marine actinomycetes Brevibacterium luteolum MOSEL-ME10a and Cellulosimicrobium funkei MOSEL-ME6 were sequenced to identify the biosynthetic gene clusters (BGCs). Additionally, anti-proliferative, antioxidant, and enzyme inhibitory activities were studied in vitro. We report a total genome size of 2.77 Mb with GC content of 67.8% and 6.81 Mb with GC content of 69% for Brevibacterium sp. MOSEL-ME10a and Cellulosimicrobium sp. MOSEL-ME6, respectively. Biosynthetic gene clusters (BGCs) encoding different classes of natural products were predicted including terpenes, peptides, siderophores, ectoines, and bacteriocins. The bioactivity potential of crude extracts derived from these strains was evaluated. Notable anti-proliferative activity was observed against HepG2 cell line (hepatocellular carcinoma) with an IC50 value of 182 µg/mL for Brevibacterium sp. MOSEL-ME10a. Furthermore, antioxidant activity was assessed with IC50 values of 48.91 µg/mL and 102.5 µg/mL for Brevibacterium sp. MOSEL-ME10a and Cellulosimicrobium sp. MOSEL-ME6, respectively. Protein kinase inhibition potential was observed only for Brevibacterium sp. MOSEL-ME10a. Our study also reports lower amylase enzyme inhibition potential for both strains. Moreover, both crude extracts showed only slight-to-no toxic effect on erythrocytes at 400 µg/mL and below, indicating erythrocyte membrane stability. Our data present the genomic features revealing biosynthetic potential of marine actinobacteria as well as biological activities found in vitro.

Brevibacterium renqingii sp. nov., isolated from the Daqu of Baijiu.

Two bacterial strains, designated REN4T and REN4-1, were isolated from daqu sample collected from baijiu factory located in Shanxi, China. The two strains shared highly similar 16S rRNA gene sequences (99.67% identities) and formed a monophyletic clade within the Brevibacterium 16S rRNA gene tree, showing 97.56-97.85% 16S rRNA gene sequence identities with type strains Brevibacterium permense VKM Ac-2280 T, Brevibacterium sediminis FXJ8.269 T, Brevibacterium oceani BBH7T and Brevibacterium epidermidis NCIMB 702286 T. They contained MK-8(H2) as the most predominant menaquinone, antesio-C15:0, antesio-C17:0, Iso-C16:0 and Iso-C17:0 as the major cellular fatty acids, DPG (diphosphatidylglycerol), PG (phosphatidylglycerol), PGL (phosphatidylglycerollipids), and PL (phospholipids) as the main polar lipids. The genomic DNA G + C content of strains REN4 and REN4-1 were 64.35, 65.82 mol%. Moreover, the low DNA-DNA relatedness values, physiological and biochemical characteristics, and taxonomic analysis allowed the differentiation of strains REN4T and REN4-1 from the other recognized species of the genus Brevibacterium. Therefore, strain REN4T represents a novel species of the genus Brevibacterium, for which the name Brevibacterium renqingii sp. nov. is proposed, with the type strain REN4T (= JCM 33953 T = KCTC 49366 T).

Draft whole-genome sequence of Brevibacterium casei strain isolated from a bloodstream infection.

Despite its low virulence potential and a commensal lifestyle as a member of the human skin microbiota, Brevibacterium casei has been increasingly reported as an opportunistic pathogen, especially in immunocompromised patients. Here, we present the draft genome sequence of the S51 strain isolated from a bloodstream infection. To the best of the authors' knowledge, this is the first report of the draft genome sequence of the B. casei strain isolated from the clinical infection. The strain was identified using phenotypic and molecular methods and subsequently sequenced using the next-generation sequencing. The draft whole genome was assembled de novo, automatically annotated by Rapid Annotations using Subsystems Technology (RAST) server and scrutinized to predict the presence of virulence, resistance, and stress response proteins. The genome size of the S51 strain was 3,743,532 bp and an average G+C content was 68.3%. The predicted genes included 48 genes involved in resistance to antibiotics (including vancomycin, fluoroquinolones, and beta-lactams) and toxic compounds (heavy metals), 16 genes involved in invasion and intracellular resistance (Mycobacterium virulence operons), and 94 genes involved in stress response (osmotic, oxidative stress, cold and heat shock). ResFinder has indicated the presence of a beta-lactamase, and a phenotypic analysis showed resistance to penicillin. This whole-genome NGS project for the S51strain has been deposited at EMBL/GenBank under the accession no. QNGF00000000.

Culturable endophytic bacteria of Camellia species endowed with plant growth promoting characteristics.

AIM: Tea (Camellia sinensis (L.) O. Kuntze) is an economically important caffeine-containing beverage crop with massive plantation in the Northeast corner of the agroclimatic belt of India. The main aim of the work was to isolate, identify and characterize the native plant growth promoting endophytes associated with tea for future microbe based bioformulation. METHODS AND RESULTS: A total of 129 endophytic bacteria were isolated and characterized for plant growth promoting traits such as indole-3-acetic acid (IAA), phosphate solubilization, ammonia production, biocontrol traits like siderophore and extracellular enzyme production. BOX-PCR fingerprinting was used to differentiate the various bacterial isolates obtained from six different tea species. 16S rRNA sequencing and blast analysis showed that these isolates belonged to different genera, that is, Bacillus, Brevibacterium, Paenibacillus and Lysinibacillus. Lysinibacillus sp. S24 showed the highest phosphate solubilization and IAA acid production efficiency of 268·4 ± 14·3 and 13·5 ± 0·5 µg ml-1 , respectively. Brevibacterium sp. S91 showed the highest ammonia production of 6·2 ± 0·5 µmol ml-1 . Chitinase, cellulase, protease and pectinase activities were shown by 4·6, 34·1, 27·13 and 13·14% of the total isolates, respectively. Similarly, 41% of the total isolates were positive for 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity. Further, the potent PGP isolates, S24 and S91 were able to enhance the vegetative parameters such as dry/fresh weight of root and shoot of tea plants in nursery conditions. CONCLUSION: Our findings corroborate that tea endophytic bacteria possess the potential to demonstrate multiple PGP traits both, in vivo and in vitro and have the potential for further large-scale trials. SIGNIFICANCE AND IMPACT OF THE STUDY: The exploration of tea endophytic bacterial community is suitable for the development of bioformulations for an integrated nutrient management and thus sustainable crop production and decreasing the hazardous effects of chemical fertilizers on the environment and human health.

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.

Brevibacterium paucivorans bacteremia: case report and review of the literature.

BACKGROUND: Brevibacteria are obligate aerobic gram-positive rods that are associated with milk products and are also found on human skin. Brevibacterium has been reported as a rare cause of catheter related blood steam infection mainly in immunocompromised hosts such as malignancies or AIDS patients. CASE PRESENTATION: A 94-year old woman, which had a past history of diabetes mellitus and chronic heart failure, presented with high fever associated with decreased oral intake and appetite loss and was admitted to our institute. A physical examination at the time of presentation was unremarkable. On day 2, both blood cultures collected on admission became positive with coryneform organism within 24 h without Staphylococci and Brevibacterium species were identified by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Subsequently, genetic investigation by 16S ribosomal RNA analysis was performed in order to identify the organism. Finally, the result identified this pathogen as Brevibacterium paucivorans with 99.5% homology on the Ez taxon database. The patient was started empirically on meropenem and teicoplanin for broad-spectrum antibiotic coverage. The patient's fever finally abated and labs were also improved. On day 14, the antibiotic therapy was discontinued. The site of infections was unknown. We hereby report a case of Brevibacterium paicivorans bacteremia in an immunocompetent patient and review cases of Brevibacterium specises bacteremia previously reported. This is the first case of B. paucivorans bacteremia as far as we could search. CONCLUSION: Physicians and microbiologists should be aware that Brevibacteria are uncommon but important agents which could cause opportunistic infections in immunocompetent.

Brevibacterium hankyongi sp. nov., isolated from compost.

A Gram-positive, strictly aerobic, non-motile, milky-white to creamy coloured and rod-shaped bacterium, designated BS05T, was isolated from compost. Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that the strain formed a distinct lineage within the genus Brevibacterium and was most closely related to Brevibacterium avium NCFB 3055T (96.3 %), Brevibacterium oceani BBH7T (96.2 %) and Brevibacterium epidermidis NBRC 14811T (96.1 %). The DNA G+C content was 62.3 mol%. The predominant quinone was MK-8(H2). The major fatty acids were anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 0. The cell-wall peptidoglycan of strain BS05T contained meso-diaminopimelic acid. The major polar lipid was phosphatidylglycerol. Moreover, the low sequence similarity of the 16S rRNA gene sequencing, physiological, biochemical and chemotaxonomic analyses allowed the phenotypic and genotypic differentiation of strain BS05T from the recognized species of the genus Brevibacterium. Therefore, strain BS05T represents a novel species of the genus Brevibacterium, for which the name Brevibacteriumhankyongi sp. nov. is proposed, with the type strain BS05T (=KACC 18875T=LMG 29562T).

Brevibacterium anseongense sp. nov., isolated from soil of ginseng field.

Gram-positive, aerobic, non-motile, pale-yellow, and rodshaped bacterium, designated as Gsoil 188T, was isolated from the soil of a ginseng field in Pocheon, South Korea. A phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that the strain formed a distinct lineage within the genus Brevibacterium and was most closely related to B. epidermidis NBRC 14811T (98.4%), B. sediminis FXJ8.269T (98.2%), B. avium NCFB 3055T (98.1%), and B. oceani BBH7T (98.1%), while it shared less than 98.1% identity with the other species of this genus. The DNA G + C content was 68.1 mol%. The predominant quinone was MK-8(H2). The major fatty acids were anteiso-C15:0 and anteiso-C17:0. The cell wall peptidoglycan of strain Gsoil 188T contained meso-diaminopimelic acid. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, and an unidentified aminolipid. The physiological and biochemical characteristics, low DNA-DNA relatedness values, and taxonomic analysis allowed the differentiation of strain Gsoil 188T from the other recognized species of the genus Brevibacterium. Therefore, strain Gsoil 188T represents a novel species of the genus Brevibacterium, for which the name Brevibacterium anseongense sp. nov. is proposed, with the type strain Gsoil 188T (= KACC 19439T = LMG 30331T).

Heterologous Expression of a VioA Variant Activates Cryptic Compounds in a Marine-Derived Brevibacterium Strain.

A new 14-membered homodimeric macrodiolide, brevidiolide (3), along with four known aromatic compounds (1, 2, 4 and 5) were obtained by heterologous expression of the recombinant plasmid pWLI823 expressing the G231L variant of VioA in the marine-derived Brevibacterium sp. 7002-073. The structures of 1⁻5 were elucidated on the basis of LC-MS and 2D NMR spectroscopic analyses. In the evaluation for the antibacterial activities of the compounds against multi-drug resistant (MDR) strains, 5 showed notable growth inhibition against Staphylococcus aureus CCARM 3090 and Klebsiella pneumoniae ATCC 13883, with a minimum inhibitory concentration (MIC) value of 3.12 µg/mL.

Comparative genomic analysis of Brevibacterium strains: insights into key genetic determinants involved in adaptation to the cheese habitat.

BACKGROUND: Brevibacterium strains are widely used for the manufacturing of surface-ripened cheeses, contributing to the breakdown of lipids and proteins and producing volatile sulfur compounds and red-orange pigments. The objective of the present study was to perform comparative genomic analyses in order to better understand the mechanisms involved in their ability to grow on the cheese surface and the differences between the strains. RESULTS: The genomes of 23 Brevibacterium strains, including twelve strains isolated from cheeses, were compared for their gene repertoire involved in salt tolerance, iron acquisition, bacteriocin production and the ability to use the energy compounds present in cheeses. All or almost all the genomes encode the enzymes involved in ethanol, acetate, lactate, 4-aminobutyrate and glycerol catabolism, and in the synthesis of the osmoprotectants ectoine, glycine-betaine and trehalose. Most of the genomes contain two contiguous genes encoding extracellular proteases, one of which was previously characterized for its activity on caseins. Genes encoding a secreted triacylglycerol lipase or involved in the catabolism of galactose and D-galactonate or in the synthesis of a hydroxamate-type siderophore are present in part of the genomes. Numerous Fe3+/siderophore ABC transport components are present, part of them resulting from horizontal gene transfers. Two cheese-associated strains have also acquired catecholate-type siderophore biosynthesis gene clusters by horizontal gene transfer. Predicted bacteriocin biosynthesis genes are present in most of the strains, and one of the corresponding gene clusters is located in a probable conjugative transposon that was only found in cheese-associated strains. CONCLUSIONS: Brevibacterium strains show differences in their gene repertoire potentially involved in the ability to grow on the cheese surface. Part of these differences can be explained by different phylogenetic positions or by horizontal gene transfer events. Some of the distinguishing features concern biotic interactions with other strains such as the secretion of proteases and triacylglycerol lipases, and competition for iron or bacteriocin production. In the future, it would be interesting to take the properties deduced from genomic analyses into account in order to improve the screening and selection of Brevibacterium strains, and their association with other ripening culture components.

Improved scFv Anti-LOX-1 Binding Activity by Fusion with LOX-1-Binding Peptides.

The oxidized low-density lipoprotein receptor-1 (LOX-1) targeted single-chain variable fragment (scFvs) is a promising molecule for the targeted delivery of imaging and therapeutic molecules of atherosclerotic diseases; however, its applications are limited by the inherent low antigen affinity. In this study, the three-dimensional (3D) model of the anti-LOX-1 scFv was constructed and its docking with the LOX-1 protein was developed. To improve the LOX-1-binding activity, the anti-LOX-1 scFv was designed to fuse with one of three LOX-1-binding heptapeptides, LTPATAI, FQTPPQL, and LSIPPKA, at its N-terminus and C-terminus and in the linker region, which have different LOX-1-binding interfaces with the anti-LOX-1 scFv analyzed by an array of computational approaches. These scFv/peptide fusions were constructed, successfully expressed in Brevibacillus choshinensis hosts, and purified by a two-step column purification process. The antigen binding activity, structural characteristics, thermal stability, and stability in serum of these fusion proteins were examined. Results showed that the scFv with N-terminal fusing peptides proteins demonstrated increased LOX-1-binding activity without decrease in stability. These findings will help increase the application efficacy of LOX-1 targeting scFv in LOX-1-based therapy.

Effects of transgenic expression of Brevibacterium linens methionine gamma lyase (MGL) on accumulation of Tylenchulus semipenetrans and key aminoacid contents in Carrizo citrange.

KEY MESSAGE: Carrizo transgenic plants overexpressing methionine-gamma-lyase produced dimethyl sulfide. The transgenic plants displayed more resistance to nematode attacks (Tylenculus semipenetrans) and may represent an innovative strategy for nematode control. Tylenchulus semipenetrans is a nematode pest of many citrus varieties that causes extensive damage to commercial crops worldwide. Carrizo citrange vr. (Citrus sinensis L. Usb × Poncirus trifoliate L. Raf) plants overexpressing Brevibacterium linens methionine-gamma-lyase (BlMGL) produced the sulfur volatile compound dimethyl sulfide (DMS). The aim of this work was to determine if transgenic citrus plants expressing BlMGL showed increased tolerance to T. semipenetrans infestation and to determine the effect on the content of key amino acids. While transgenic lines emitted dimethyl sulfide from leaves and roots, no sulfur-containing volatiles were detectable in wild-type Carrizo in the same tissues. Significant changes detected some key amino acids from leaves of transgenic plants such as aspartate, lysine, glycine, leucine and threonine with no changes in the amounts of methionine and α-ketobutyrate. In roots only glycine showed significant changes across all transgenic lines in comparison to wild-type plants. Transgenic plants expressing BlMGL and emitting DMS had less T. semipenetrans aggregation and more biomass than infected WT control plants, indicating that they may represent an innovative management alternative to pesticide/nematicide-based remedies.

Analysis of strain-specific genes in glutamic acid-producing Corynebacterium glutamicum ssp. lactofermentum AJ 1511.

Strains of the bacterium, Corynebacterium glutamicum, are widely used for the industrial production of L-glutamic acid and various other substances. C. glutamicum ssp. lactofermentum AJ 1511, formerly classified as Brevibacterium lactofermentum, and the closely related C. glutamicum ATCC 13032 have been used as industrial strains for more than 50 years. We determined the whole genome sequence of C. glutamicum AJ 1511 and performed genome-wide comparative analysis with C. glutamicum ATCC 13032 to determine strain-specific genetic differences. This analysis revealed that the genomes of the two industrial strains are highly similar despite the phenotypic differences between the two strains. Both strains harbored unique genes but gene transpositions or inversions were not observed. The largest unique region, a 220-kb AT-rich region located between 1.78 and 2.00 Mb position in C. glutamicum ATCC 13032 genome, was missing in the genome of C. glutamicum AJ 1511. The next two largest unique regions were present in C. glutamicum AJ 1511. The first region (413-484 kb position) contains several predicted transport proteins, enzymes involved in sugar metabolism, and transposases. The second region (1.47-1.50 Mb position) encodes restriction modification systems. A gene predicted to encode NADH-dependent glutamate dehydrogenase, which is involved in L-glutamate biosynthesis, is present in C. glutamicum AJ 1511. Strain-specific genes identified in this study are likely to govern phenotypes unique to each strain.

Reclassification of Brevibacterium halotolerans DSM8802 as Bacillus halotolerans comb. nov. Based on Microbial and Biochemical Characterization and Multiple Gene Sequence.

Brevibacterium halotolerans is currently classified as a member of the Brevibacterium genus, a genus that groups together many bacterial species of similar morphology but diverse biochemical and physiological features. Here we suggest, based on multiple gene sequencing and microbial and biochemical characterization of two environmental isolates and one type strain (DSM8802), that the B. halotolerans DSM8802 (and probably the other deposited under this species name) should be re-classified into the Bacillus genus, and offered the name B. halotolerans comb. nov.

Brevibacterium sediminis sp. nov., isolated from deep-sea sediments from the Carlsberg and Southwest Indian Ridges.

Three actinobacterial strains, FXJ8.128, FXJ8.269T and FXJ8.309, were isolated from deep-sea sediments collected from the Carlsberg Ridge and Southwest Indian Ridge at depths of 3690, 1800 and 2461 m, respectively. The three strains had highly similar 16S rRNA gene sequences (99.8-99.9 % identities) and formed a monophyletic clade within the Brevibacterium 16S rRNA gene tree, showing 98.2-98.9 % 16S rRNA gene sequence identities with type strains Brevibacterium epidermidis NCIMB 702286T, Brevibacterium iodinum DSM 20626T, Brevibacterium linens DSM 20425T, Brevibacterium oceani BBH7T and Brevibacterium permense VKM Ac-2280T. All three isolates showed activity towards the breakdown of pectin and fluoranthene. They contained MK-8(H2) as the most predominant menaquinone, diphosphatidylglycerol, phosphatidylglycerol and a glycolipd as the main polar lipids, and anteiso-C15 : 0 and anteiso-C17 : 0 as the major cellular fatty acids. Moreover, the three isolates were distinguished readily from the phylogenetically related type strains by DNA-DNA hybridization values, by random amplified polymorphic DNA fingerprint profiles and by a range of physiological and biochemical characteristics. On the basis of the above polyphasic taxonomic data, strains FXJ8.128, FXJ8.269T and FXJ8.309 represent a novel species of the genus Brevibacterium, for which the name Brevibacterium sediminis sp. nov. is proposed. The type strain is FXJ8.269T (=CGMCC 1.15472T=DSM 102229T).