Protein Pept Lett, 2005 Jan, 12(1), 95 - 8 Novel processing and localization of catA, ccdA associated thiol-disulfide oxidoreductase, in protein hyper-producing bacterium Brevibacillus choshinensis; Tanaka R et al.; Previously, we have cloned ccdA and its associated thiol-disulfide oxidoreductase gene, catA, in Brevibacillus choshinensis . CcdA is known to be an integral membrane protein and its associated oxidoreductase homologues are believed to be membrane anchoring proteins, both providing reducing equivalents across the membrane to control correct disulfide bond formation . Here, we found that CatA is first localized as a membrane bound form and then slowly released into the cellular periphery and culture medium with cleavage at a novel processing site. Biochemistry (Mosc), 2004 Dec, 69(12), 1353 - 9 Structures of Cell Wall Teichoic Acids of Brevibacterium iodinum VKM Ac-2106(T); Potekhina NV et al.; Structures of two cell wall teichoic acids of Brevibacterium iodinum VKM Ac-2106(T) were studied . The structure of mannitol teichoic acid described earlier was mainly confirmed . This polymer is 1,6-poly(mannitol phosphate) bearing beta-D-glucopyranosyl residues at the C-2 of mannitol and pyruvic acid residues at the C-4 and C-5 . The absolute configurations of D-mannitol and S-pyruvic acid were found . The following distinctions from the earlier described structure were found: unsubstituted 1,6-poly(mannitol phosphate) residues and residues substituted only by beta-D-glucopyranosyl at the C-2 of mannitol but unsubstituted by pyruvic acid are present in the chain . The structure of glycerol teichoic acid present in the cell wall as a minor component (~7%) is also described . This acid is identified as 1,3-poly(glycerol phosphate) substituted at the C-2 of glycerol by 2-acetamido-2-deoxy-alpha-D-galactopyranosyl residues bearing R-pyruvic acid residues at the C-4 and C-6 of galactose . This polymer is for the first time described in the cell wall of Gram-positive bacteria. Syst Appl Microbiol, 2004 Nov, 27(6), 746 - 54 Bacterial diversity in spent mushroom compost assessed by amplified rDNA restriction analysis and sequencing of cultivated isolates; Ntougias S et al.; Spent mushroom compost (SMC) is the residual by-product of commercial Agaricus spp . cultivation, and it is mainly composed of a thermally treated cereal straw/animal manure mixture colonized by the fungal biomass . Research on the valorization of this material is mainly focusing on its use as soil conditioner and plant fertilizer . An investigation of the bacterial diversity in SMC was performed using molecular techniques in order to reveal the origin of SMC microflora and its potential effect on soil microbial communities after incorporation into agricultural soils . The bacterial population was estimated by the plate count method to a mean of 2.7 10(9) colony forming units (cfu) per g of dry weight, while the numbers of Gram-positive and Gram-negative bacteria were 1.9 10(9) and 4.9 10(8) cfu per g dw respectively as estimated by enumeration on semi-selective media . Fifty bacterial isolates were classified into 14 operational taxonomic units (OTUs) following ARDRA-PCR of the 16S rDNA gene . Sequencing of the 16S rDNA amplicon assigned 12 of the 14 OTUs to Gram-positive bacteria, associated with the genera Bacillus, Paenibacillus, Exiguobacterium, Staphylococcus, Desemzia, Carnobacterium, Brevibacterium, Arthrobacter and Microbacterium of the bacterial divisions Firmicutes and Actinobacteria . Two bacterial groups have phylogenetic links with the genera Comamonas and Sphingobacterium, which belong to beta-Proteobacteria and Bacteroidetes respectively . Two potentially novel bacteria are reported, which are associated with the genera Bacillus and Microbacterium . Most of the bacteria identified are of environmental origin, while strains related to species usually isolated from insects, animal and clinical sources were also detected . It appears that bacterial diversity in SMC is greatly affected by the origin of the initial material, its thermal pasteurization treatment and the potential unintended colonization of the mushroom substrate during the cultivation process. J Appl Microbiol, 2005, 98(1), 184 - 92 Resistance of corynebacterial strains to infection and lysis by corynephage BFK 20; Halgasova N et al.; Abstract n . halgasova, t . majtan, j . ugorcakova, j . timko and g . bukovska . 2004.Aims: Defence mechanisms of the corynebacterial strains against corynephage BFK 20, which causes lysis of Brevibacterium flavum CCM 251 . Methods and Results: We tested adsorption of the phage BFK 20 to the corynebacterial cell surface . We observed strong adsorption ranging from ca 79 to 93% on the cells of B . flavum ATCC strains, but only ca 76% for B . flavum CCM 251 . Minor adsorption for Brevibacterium lactofermentum BLOB (ca 13%) and no adsorption for Corynebacterium glutamicum RM3 were determined . BFK 20 infection had no significant effect on growth and viability of C . glutamicum and B . lactofermentum, but significantly influenced growth and viability of B . flavum ATCC 21127, 21128 and 21474 . Cell growth stopped in short time after infection but with no lysis . Brevibacterium flavum CCM 251 cell growth was arrested too and lysis occurred . The Southern hybridization confirmed the presence of significant amount of BFK 20 DNA in samples from B . flavum CCM 251 and B . flavum ATCC strains after BFK 20 infection . Only weak hybridization signal was detected for DNA from infected cells of B . lactofermentum BLOB and no signal for C . glutamicum RM3 . Conclusions: Based on the above results we suggest presence of a mechanism leading to abortive infection in B . flavum ATCC 21127, 21128 and 21474 . In B . lactofermentum BLOB and C . glutamicum RM3 the adsorption barrier is more likely . Significance and Impact of the Study: This study increases the knowledge on defence mechanisms of corynebacteria against bacteriophages. Biotechnol Lett, 2004 Nov, 26(21), 1623 - 8 Comparative study of Cr(VI) uptake and reduction in industrial effluent by Ochrobactrum intermedium and Brevibacterium sp; Faisal M et al.; Two chromium-resistant bacterial strains, CrT-1 and CrT-13, tolerant up to 40mg K(2)CrO(4) ml(-1) on nutrient agar, 25mgml(-1) in nutrient broth, and up to 10mgml(-1) in acetate-minimal media, were identified as Ochrobactrum intermedium and Brevibacterium sp., respectively, on the basis of 16S rRNA gene sequencing . Uptake of chromate was greater in living cells than in heat-killed on dried cells . CrT-1 reduced 82%, 28% and 16% of Cr(VI) at 100, 500, and 1000mugml(-1) after 24h while CrT-13 reduced 41%, 14% and 9% . Other heavy metals at low concentrations did not affect these reductions . At 150 and 300mugml(-1) in an industrial effluent sample Cr(VI) was reduced by 87% and 71%, respectively, with CrT-1 and by 68% and 47% with CrT-13. Biotechnol Lett, 2004 Oct, 26(19), 1481 - 5 Ectoine accumulation in Brevibacterium epidermis; Onraedt A et al.; As a halotolerant bacterial species, Brevibacterium epidermis DSM 20659 can grow at relatively high salinity, tolerating up to 2 M NaCl . It synthesizes ectoine and the intracellular content increases with the medium salinity, with a maximum of 0.14 g ectoine/g CDW at 1 M NaCl . Sugar-stressed cells do not synthesize ectoine . Ectoine synthesis is also affected by the presence of external osmolytes . Added betaine is taken up and completely replaced ectoine, while L-proline is only temporarily accumulated after which ectoine is synthesized . The strain can metabolize ectoine; L-glutamate is a better carbon source for ectoine synthesis than L-aspartate. Biotechnol Lett, 2004 Sep, 26(17), 1385 - 8 Efficient oxidation of alcohols by a 2-phenylethanol-degrading Brevibacterium sp; Miyamoto K et al.; Microorganisms which can assimilate 2-phenylethanol were screened from soil for oxidation of various alcohols into carbonyl compounds . One strain, identified as Brevibacterium sp . KU1309, had high oxidative activity towards ArCH(CH(3))CH(2)OH and Ar(CH(2))(n)OH . When the substrate concentration was 0.1 approximately 0.4% (w/v), the oxidation reaction proceeded smoothly (6 approximately 96 h), and the corresponding carboxylates were obtained in good yields (68 approximately 87%). Environ Pollut, 2005 Mar, 134(2), 257 - 66 Interactive effect of Brevibacillus brevis and Glomus mosseae, both isolated from Cd contaminated soil, on plant growth, physiological mycorrhizal fungal characteristics and soil enzymatic activities in Cd polluted soil; Vivas A et al.; The interaction between two autochthonous microorganisms (Brevibacillus brevis and Glomus mosseae) isolated from Cd amended soil increased plant growth, arbuscular mycorrhizal (AM) colonization and physiological characteristics of the AM infection (measured as SDH or ALP activities) . The enhanced plant Cd tolerance after coinoculation with native microorganisms seemed to be a consequence of increased P and K acquisition and, simultaneously, of decreased concentration of Cd, Cr, Mn, Cu, Mo, Fe and Ni in plant tissue . Autochthonous microbial strains were more efficient for nutrient uptake, to immobilize metals and decrease their translocation to the shoot than reference G . mosseae (with or without bacteria) . Indole acetic acid produced by B . brevis may be related to its ability for improving root growth, nodule production and AM fungal intra and extraradical development . Dehydrogenase, phosphatase and beta-glucosidase activities, indicative of microbial metabolism and soil fertility, were maximized by the coinoculation of autochthonous microorganisms in cadmium polluted conditions . As a consequence, the use of native microorganisms may result very efficient in bioremediation. Appl Environ Microbiol, 2004 Dec, 70(12), 7348 - 54 Identification and functional analysis of the gene encoding methionine-gamma-lyase in Brevibacterium linens; Amarita F et al.; The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese . L-methionine-gamma-lyase (MGL) can convert L-methionine to methanethiol (MTL), alpha-ketobutyrate, and ammonia . The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs . The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain . Our work shows that L-methionine degradation via gamma-elimination is a key step in the formation of VSCs in B . linens. Biochemistry, 2004 Dec 7, 43(48), 15141 - 53 Single-turnover kinetics of homoprotocatechuate 2,3-dioxygenase; Groce SL et al.; Homoprotocatechuate 2,3-dioxygenase isolated from Brevibacterium fuscum utilizes an active site Fe(II) and O(2) to catalyze proximal extradiol cleavage of the substrate aromatic ring . In contrast to other members of the ring cleaving dioxygenase family, the transient kinetics of the extradiol dioxygenase catalytic cycle have been difficult to study because the iron is nearly colorless and EPR silent . Here, it is shown that the reaction cycle kinetics can be monitored by utilizing the alternative substrate 4-nitrocatechol (4NC), which is also cleaved in the proximal extradiol position . Changes in the optical spectrum of 4NC occurring as a result of ionization, environmental changes, and ring cleavage allow both the substrate binding and product formation phases of the reaction to be studied . It is shown that substrate binding occurs in a four-step process probably involving binding to two ionization states of the enzyme at different rates . Following an initial rapid binding of the monoanionic 4NC in the active site, slower binding to the Fe(II) and conversion to the dianionic form occur . The bound dianionic 4NC reacts rapidly with O(2) in four additional steps, apparently occurring in sequence . On the basis of the optical properties of the intermediates, these steps are hypothesized to be O(2) binding to the iron, isomerization of the resulting complex, ring opening, and product release . The natural substrate appears to form the same intermediates but with much larger rate constants . These are the first transient intermediates to be reported for an extradiol dioxygenase reaction. Lett Appl Microbiol, 2004, 39(6), 533 - 7 Production of L-2,3-butanediol by a new pathway constructed in Escherichia coli; Ui S et al.; AIMS: A metabolic pathway for L-2,3-butanediol (BD) as the main product has not yet been found . To rectify this situation, we attempted to produce L-BD from diacetyl (DA) by producing simultaneous expression of diacetyl reductase (DAR) and L-2,3-butanediol dehydrogenase (BDH) using transgenic bacteria, Escherichia coli JM109/pBUD-comb . METHODS AND RESULTS: The meso-BDH of Klebsiella pneumoniae was used for its DAR activity to convert DA to L-acetoin (AC) and the L-BDH of Brevibacterium saccharolyticum was used to reduce L-AC to L-BD . The respective gene coding each enzyme was connected in tandem to the MCS of pFLAG-CTC (pBUD-comb) . The divided addition of DA as a source, addition of 2% glucose, and the combination of static and shaking culture was effective for the production . CONCLUSIONS: L-BD (2200 mg l(-1)) was generated from 3000 mg l(-1) added of DA, which corresponded to a 73% conversion rate . Meso-BD as a by-product was mixed by 2% at most . SIGNIFICANCE AND IMPACT OF THE STUDY: An enzyme system for converting DA to L-BD was constructed with a view to using DA-producing bacteria in the future. Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2107 - 11 Brevibacterium celere sp . nov., isolated from degraded thallus of a brown alga; Ivanova EP et al.; 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)). Appl Environ Microbiol, 2004 Nov, 70(11), 6657 - 64 Molecular characterization of Brevibacillus laterosporus and its potential use in biological control; de Oliveira EJ et al.; Thirty-three strains of Brevibacillus laterosporus, including three novel strains isolated from Brazilian soil samples, were examined for genetic variability by the use of different PCR-based methods . Molecular markers that could characterize bacterial strains with regards to their pathogenic potential were investigated . In addition, toxicity was assessed by the use of insects belonging to the orders Lepidoptera and Coleoptera and the mollusk Biomphalaria glabrata . Among the targets tested, Biomphalaria glabrata demonstrated the highest degree of sensitivity to B . laterosporus, with some strains inducing 90 to 100% mortality in snails aged 3 and 12 days posteclosion . Larvae of the coleopteron Anthonomus grandis were also susceptible, presenting mortality levels of between 33 and 63% . Toxicity was also noted towards the lepidopteron Anticarsia gemmatalis . In contrast, no mortality was recorded among test populations of Tenebrio molitor or Spodoptera frugiperda . The application of intergenic transcribed spacer PCR and BOX-PCR generated 15 and 17 different genotypes, respectively . None of the molecular techniques allowed the identification of a convenient marker that was associated with any entomopathogenic phenotype . However, a 1,078-bp amplicon was detected for all strains of B . laterosporus when a primer for amplification of the BOXA1R region was used . Similarly, a 900-bp amplicon was generated from all isolates by use of the primer OPA-11 for randomly amplified polymorphic DNA analysis . These amplicons were not detected for other phenotypically related Brevibacillus species, indicating that they represent markers that are specific for B . laterosporus, which may prove useful for the isolation and identification of new strains of this species. Appl Environ Microbiol, 2004 Nov, 70(11), 6385 - 93 Fatty acid production from amino acids and alpha-keto acids by Brevibacterium linens BL2; Ganesan B et al.; Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile . Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and alpha-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of alpha-keto acids to flavor-associated compounds is controversial . The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and alpha-keto acids and determine the occurrence of the likely genes in the draft genome sequence . BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions . The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid . In contrast, logarithmic-phase cells of BL2 produced fatty acids from alpha-keto acids only . BL2 also converted alpha-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved . At least 100 genes are potentially involved in five different metabolic pathways . The genome of B . linens ATCC 9174 contained these genes for production and degradation of fatty acids . These data indicate that brevibacteria have the ability to produce fatty acids from amino and alpha-keto acids and that carbon metabolism is important in regulating this event. Syst Appl Microbiol, 2004 Sep, 27(5), 612 - 9 Molecular characterisation of bacterial contamination in semi-final gelatine extracts, using denaturing gradient gel electrophoresis; de Clerck E et al.; Contamination of gelatine may affect the safety and/or quality of its applications . Characterisation of bacterial isolates from semi-final gelatine batches revealed thermotolerant, aerobic, endosporeforming contaminants . In this paper, bacterial contamination in gelatine batches is analysed without previous isolation, by means of denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA sequences . V9 and V6-V8 regions of the 16S rDNA gene were found more suitable for this purpose than V1 or V3 regions . Bacillus fumarioli, Bacillus licheniformis, members of the 'Bacillus cereus group', Bacillus subtilis, Bacillus shackletonii, Brevibacillus borstelensis and Brevibacillus agri were detected. J Dairy Sci, 2004 Nov, 87(11), 3976 - 88 Deacidification by Debaryomyces hansenii of smear soft cheeses ripened under controlled conditions: relative humidity and temperature influences; Bonaiti C et al.; Model smear soft cheeses were prepared from pasteurized milk inoculated with Debaryomyces hansenii (304, GMPA) and Brevibacterium aurantiacum (ATCC 9175) under aseptic conditions . Debaryomyces hansenii growth and curd deacidification were studied in relation to ripening chamber temperature and relative humidity (RH) . A total of 9 descriptors, mainly based on kinetic data, were defined to represent D . hansenii growth (2 descriptors), cheese deacidification (5 descriptors), and cheese ripening (2 descriptors) . Regardless of the temperature, when the RH was 85%, D . hansenii growth was inhibited due to limitation of carbon substrate diffusions; consequently, cheese deacidification did not take place . Debaryomyces hansenii growth was most prolific when the temperature was 16 degrees C, and the RH was 95% . Kinetic descriptors of lactate consumption and pH increase were maximal at 16 degrees C and 100% RH . Under these 2 ripening conditions, on d 14 (packaging) the creamy underrind represented a third of the cheese; however, at the end of ripening (d 42), cheese was too liquid to be sold . Statistical analysis showed that the best ripening conditions to achieve an optimum between deacidification and appearance of cheeses (thickness of the creamy underrind) were 12 degrees C and 95 +/- 1% RH. J Nutr, 2004 Oct, 134(10 Suppl), 2854S - 2857S; discussion 2895S Production of arginine by fermentation; Utagawa T; Studies on the production of L-arginine by fermentation using mutants of Corynebacterium (Brevibacterium), Bacillus, and Serratia have been conducted since the 1960s . More recently, the breeding of L-arginine production strains by gene recombination techniques using Escherichia coli has been investigated . To produce L-arginine efficiently by fermentation, it is necessary to breed strains with a strong biosynthetic pathway to L-arginine . Because L-arginine is biosynthesized from the precursor L-glutamic acid through ornithine and citrulline, the use of strains with a high capability for producing L-glutamic acid is desirable . Corynebacterium (Brevibacterium), which is well known in the production of L-glutamic acid, was selected as a starting strain for the breeding of an L-arginine producer and has been used on a commercial scale . Regarding the fermentation conditions, as for other amino acids, L-arginine fermentation is controlled by regulating pH near the neutral point . Due to its high oxygen requirement, L-arginine production is seriously impaired without sufficient oxygen . Advanced purification methods are necessary to obtain highly pure L-arginine from the fermentation broth . After fermentation is complete, bacterial cells and proteins are removed by means of a membrane or centrifugation, and impurities are removed by means of an ion-exchange resin or activated carbon . Highly pure L-arginine crystals can be obtained through concentration at the end of the process. Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1537 - 41 Brevibacterium picturae sp . nov., isolated from a damaged mural painting at the Saint-Catherine chapel (Castle Herberstein, Austria); Heyrman J et al.; Three strains showing highly similar (GTG)5-PCR patterns were isolated from a heavily damaged mural painting at the Saint-Catherine chapel (Castle Herberstein, Austria) . On the basis of 16S rRNA gene sequence similarity, the strains were attributed to Brevibacterium, with Brevibacterium casei (96.7 %), Brevibacterium iodinum (96.7 %) and Brevibacterium linens (96.6 %) as the closest related species . Chemotaxonomic data {peptidoglycan contains meso-diaminopimelic acid; mycolic acids absent; MK-8(H2) as the major menaquinone; polar lipids phosphatidylglycerol and diphosphatidylglycerol present; anteiso-C(15 : 0) and anteiso-C(17 : 0) as major fatty acids} supported the affiliation of the strains to the genus Brevibacterium . Additional physiological and biochemical tests confirmed the taxonomic position of the strains and allowed phenotypic differentiation from Brevibacterium species with validly published names . The isolates from the mural painting, therefore, represent a novel species, for which the name Brevibacterium picturae sp . nov . is proposed, with LMG 22061T (= DSM 16132T) as the type strain. J Dairy Sci, 2004 Oct, 87(10), 3224 - 34 Influence of adjunct cultures on volatile free fatty acids in reduced-fat Edam cheeses; Tungjaroenchai W et al.; The effects of the adjunct cultures Lactococcus lactis ssp . diacetylactis, Brevibacterium linens BL2, Lactobacillus helveticus LH212, and Lactobacillus reuteri ATCC 23272 on volatile free fatty acid production in reduced-fat Edam cheese were studied . Lipase activity evaluation using p-nitrophenyl fatty acid ester substrates indicated that L . lactis ssp . diacetylactis showed the highest activity among the 4 adjunct cultures . Full-fat and 33% reduced-fat control cheeses (no adjunct) were made along with 5 treatments of reduced-fat cheeses, which included individual, and a mixture of the adjunct cultures . Volatile free fatty acids of cheeses were analyzed using static headspace analysis with 4-bromofluorobenzene as an internal standard . Changes in volatile free fatty acid concentrations were found in headspace gas of cheeses after 3-and 6-mo ripening . Acetic acid was the most abundant acid detected throughout ripening . Full-fat cheese had the highest relative amount of propionic acid among the cheeses . Certain adjunct cultures had a definite role in lipolysis at particular times . Reduced-fat cheese with L . lactis ssp . diacetylactis at 3-mo showed the highest levels of butyric, isovaleric, n-valeric, iso-caproic, and n-caproic acid . Reduced-fat cheese with Lactobacillus reuteri at 6 mo produced the highest relative concentration of isocaproic, n-caproic, and heptanoic, and the highest relative concentration of total acids. Protein Expr Purif, 2004 Oct, 37(2), 385 - 91 Expression and purification of thioredoxin (TrxA) and thioredoxin reductase (TrxB) from Brevibacillus choshinensis; Tanaka R et al.; Brevibacillus choshinensis (formerly Bacillus brevis) is a protein-hyperproducing bacterium and has been used for commercial protein production . Here, we cloned thioredoxin (trxA) and thioredoxin reductase (trxB) genes from B . choshinensis, and expressed the gene products in Escherichia coli with an amino-terminal hexa-His-tag for purification and characterization . His-TrxA and His-TrxB were purified to homogeneity with one-step Ni-NTA affinity column chromatography, and the two recombinant proteins showed identical specific activity with or without removal of the amino-terminal His-tag, indicating that the extrasequence containing the hexa-His-tag did not affect their enzymatic activities . The E . coli expression system used here resulted in a 40-fold increase in production of His-TrxB protein compared to the level of native TrxB produced in non-recombinant B . choshinensis cells . TrxA and TrxB proteins with carboxy-terminal His-tag (TrxA-His and TrxB-His) were successfully expressed in B . choshinensis and were purified by Ni-NTA column chromatography . Co-expression of TrxA-His with recombinant human epidermal growth factor (hEGF) in B . choshinensis promoted the extracellular production of hEGF by up to about 200%. J Dairy Res, 2004 Aug, 71(3), 355 - 66 Controlled production of Camembert-type cheeses . Part II . Changes in the concentration of the more volatile compounds; Leclercq-Perlat MN et al.; Flavour generation in cheese is a major aspect of ripening . In order to enhance aromatic qualities it is necessary to better understand the chemical and microbiological changes . Experimental Camembert-type cheeses were prepared in duplicate from pasteurized milk inoculated with Kluyveromyces lactis, Geotrichum candidum, Penicillium camemberti and Brevibacterium linens under aseptic conditions . Two replicates performed under controlled conditions of temperature (12 degrees C), relative humidity (95 +/- 2%), and atmosphere showed similar ripening characteristics . The evolutions of metabolite concentrations were studied during ripening . The volatile components were extracted by dynamic headspace extraction, separated and quantified by gas chromatography and identified by mass spectrometry . For each cheese the volatile concentrations varied with the part considered (rind or core) . Except for ethyl acetate and 2-pentanone, the volatile quantities observed were higher than their perception thresholds . The flavour component production was best correlated with the starter strains . During the first 10 days the ester formations (ethyl, butyl and isoamyl acetates) were associated with the concentrations of K . lactis and G . candidum . The rind quantity of esters was lower than that observed in core probably due to (1) a diffusion from the core to the surface and (2) evaporation from the surface to the chamber atmosphere . G . candidum and Brev . linens association produced 3 methyl butanol and methyl 3-butanal from leucine, respectively . DMDS came from the methionine catabolism due to Brev . linens . Styrene production was attributed to Pen . camemberti . 2-Pentanone evolution was associated with Pen . camemberti spores and G . candidum . 2-Heptanone changes were not directly related to flora activities while 2-octanone production was essentially due to G . candidum . This study also demonstrates the determining role of volatile component diffusion. J Dairy Res, 2004 Aug, 71(3), 346 - 54 Controlled production of Camembert-type cheeses . Part I: Microbiological and physicochemical evolutions; Leclercq-Perlat MN et al.; A holistic approach of a mould cheese ripening is presented . The objective was to establish relationships between the different microbiological and biochemical changes during cheese ripening . Model cheeses were prepared from pasteurized milk inoculated with Kluyveromyces lactis, Geotrichum candidum, Penicillium camemberti and Brevibacterium linens under aseptic conditions . Two cheese-making trials with efficient control of environmental parameters were carried out and showed similar ripening characteristics . K . lactis grew rapidly between days 1 and 6 (generation time around 48 h) . G . candidum grew exponentially between days 4 and 10 (generation time around 4.6 d) . Brevi . linens also grew exponentially but after day 6 when Pen . camemberti mycelium began developing and the pH of the rind was close to 7 . Its exponential growth presented 3 phases in relation to carbon and nitrogen substrate availability . Concentrations of Pen . camemberti mycelium were not followed by viable cell count but they were evaluated visually . The viable microorganism concentrations were well correlated with the carbon substrate concentrations in the core and in the rind . The lactose concentrations were negligible after 10 d ripening, and changes in lactate quantities were correlated with fungi flora . The pH of the inner part depended on NH3 . Surface pH was significantly related to NH3 concentration and to fungi growth . The acid-soluble nitrogen (ASN) and non-protein nitrogen (NPN) indexes and NH3 concentrations of the rind were low until day 6, and then increased rapidly to follow the fungi concentrations until day 45 . The ASN and NPN indexes and NH3 concentrations in the core were lower than in the rind and they showed the same evolution . G . candidum and Pen . camemberti populations have a major effect on proteolysis; nevertheless, K . lactis and Brevi . linens cell lysis also had an impact on proteolysis . Viable cell counts of K . lactis, G . candidum, Pen . camemberti and Brevi . linens were correlated with the environmental conditions, with proteolytic products and with carbon substrate assimilation . NH3 diffusion from surface to the cheese core during ripening was highly suspected . Interaction phenomena between microorganisms are discussed. Saudi Med J, 2004 Aug, 25(8), 1073 - 9 Isolation of coryneform bacteria from blood cultures of patients at a University Hospital in Saudi Arabia; Babay HA et al.; OBJECTIVE: Coryneform bacteria have been increasingly recognized as opportunistic pathogens in recent years . The aim of this study is to identify and determine the antimicrobial susceptibility of coryneform bacteria isolated from blood cultures of patients seen at King Khalid University Hospital (KKUH), Riyadh, Kingdom of Saudi Arabia and review the literature . METHODS: All coryneform bacteria isolated from blood culture specimens between January 2001 and March 2003 were prospectively identified by API Coryne System (BioMerieux, France) . Clinical data were collected from each patient's medical record . Antimicrobial susceptibility to 16 antimicrobial agents were determined by minimum inhibitory concentration (MIC) using E-test (AB Biodisk, Solna, Sweden) . RESULTS: Out of 50 coryneform bacteria isolated, 19 different species were identified . Corynebacterium propinquum was the most common species 6/50 (12%) followed by Corynebacterium auris 5/50 (10%), Corynebacterium afermentans, Corynebacterium striatum, Dermabacter hominis, Brevibacterium, and Arthrobacter species 4/50 (8%) each . Underlying chest diseases were common among the patients 11/50 (22%), followed by different surgeries 10/50 (20%) . Of all, 12/50 (24%) patients were from different intensive care units (ICUs), 36/50 (72%) had either vascular, urinary or respiratory intubation . Three patients in ICUs died, one was an elderly patient with gastrointestinal bleeding and 2 teenagers (one had tracheoesophageal fistula and the other was post-arrest road traffic accident patient) . Vancomycin was the most active antimicrobial agent against all coryneform species . The majority had MIC <1 ug/ml . For most isolates, the MIC90s of erythromycin, clindamycin, and ciprofloxacin were above the break points . Corynebacterium striatum was the only isolate susceptible to ampicillin . CONCLUSION: This study revealed that coryneform bacteria are increasingly being recognized as a cause of serious infections in immunocompromised patients . We recommend identification and susceptibility testing of predominant isolates of coryneform bacteria from different clinical sites of seriously ill patients to select the antimicrobial agent necessary for clinical intervention. Biosci Biotechnol Biochem, 2004 Aug, 68(8), 1801 - 4 Expression and purification of the Bacillus subtilis thioredoxin superfamily protein YkvV; Tanaka R et al.; Bacillus subtilis YkvV protein, an extracellular thioredoxin superfamily protein, was successfully expressed both in Brevibacillus choshinensis culture medium using an efficient promoter and the secretion signal of its surface layer protein, and in Escherichia coli cytoplasm with the amino-terminal His-tag (His-YkvV) . His-YkvV was purified to homogeneity by Ni-NTA column . Both secreted YkvV and purified His-YkvV exhibited thiol-disulfide oxidoreductase activity. Ann Univ Mariae Curie Sklodowska {Med}, 2003, 58(1), 385 - 91 Characteristics of opportunistic species of the Corynebacterium and related coryneforms isolated from different clinical materials; Chudnicka A et al.; Taking into account the increasing contribution of species, which enter into the composition of purely physiological flora of the organism, of the Corynebacterium type and related coryneforms in opportunistic infections in people, the analysis of strains was made from different clinical materials from patients . Their identification was made on the basis of biochemical properties and their antibiotic sensitivity was characterized . It was found that strains with similar biochemical properties (C.striatum, C.amycolatum ) should be identified by means of genetic methods, all the more that they were isolated from clinically important materials . Out of the examined strains the biggest number of infections were caused by C.pseudodiphtheriticum, next C . striatum/C . amycolatum, Brevibacterium sp., C.propinquum, one: C.afermentans, C.jeikeium, C.group G, C.group F1, C.accolens, C.macqinleyi . The highest sensitivity of isolated strains was to Vancomycin, Teicoplanin and Imipenem. Commun Agric Appl Biol Sci, 2003, 68(2 Pt A), 241 - 6 Dynamics and optimal conditions of intracellular ectoine accumulation in Brevibacterium sp; Onraedt A et al.; The optimal conditions for the intracellular synthesis of ectoine were determined in a halotolerant Brevibacterium sp . The size of the intracellular ectoine pool in the bacterial cells is shown to depend on the external salt concentrations, type of carbon source and aeration level . In erlenmeyer flasks a maximum concentration of intracellular ectoine of about 0.9 g/l was obtained . Under controlled aeration in a 1.5 l fermentor this level could be increased to 1.2 g/l . Consecutive cell transfers to media with increasingly higher salt concentrations enabled us to reach even higher levels, up to 1.6 g/l on erlenmeyer scale . The ectoine synthesis takes place immediately after the osmotic upshock . Within one generation time, the new corresponding specific intracellular ectoine concentration is reached. Environ Microbiol, 2004 Aug, 6(8), 820 - 30 Molecular detection and isolation of facultatively methylotrophic bacteria, including Methylobacterium podarium sp . nov., from the human foot microflora; Anesti V et al.; This is the first study to demonstrate that diverse methylotrophic bacteria occur in the human foot microflora . Polymerase chain reaction (PCR) amplification of DNA from the soles and toe clefts of feet of five subjects indicated Methylobacterium strains to be present in all cases . Polymerase chain reaction amplification also showed the gene for the alpha-subunit of methanol dehydrogenase (mxaF) to be present in all samples . Two types of mxaF were recovered, one closest to that of Methylobacterium extorquens and the other most similar to that of Hyphomicrobium methylovorum . Numerous methylotrophic strains able to grow on methylamine were isolated with ease from the feet of nine volunteers . These were found by 16S rRNA analysis to be most closely related to Methylobacterium species, Brevibacterium casei, Pseudomonas strain NZ099 and P . migulae . Three strains from two subjects were of a novel species, Methylobacterium podarium sp . nov . This facultatively methylotrophic, obligately aerobic, pink-pigmented, non-motile rod grew with a wide range of multicarbon and one-carbon compounds including citrate, xylose, mono-, di-, and trimethylamine, dimethylsulphide, methanethiol, dimethylsulphoxide, dimethylsulphone and methanol. Biochemistry (Mosc), 2004 Jun, 69(6), 658 - 64 Cell wall teichoic acids of two Brevibacterium strains; Shashkov AS et al.; 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. Biodegradation, 2004 Jun, 15(3), 145 - 51 A microbial consortium isolated from a crude oil sample that uses asphaltenes as a carbon and energy source; Pineda-Flores G et al.; A microbial consortium capable of mineralizing asphaltenes was obtained from the Maya crude oil . The enrichment system was built with a glass column reactor containing mineral medium supplied with asphaltenes as energy and carbon source . The consortium growth was evaluated in Casoy agar during 40 weeks . The steady-state phase of the enriched bacterial community was observed after 10 weeks when the culture reach 10(5) to 10(6) CFU ml(-1) . The isolates belong to bacterial genus reported for degradation of other hydrocarbons and they were identified as Corynebacterium sp., Bacillus sp., Brevibacillus sp . and Staphylococcus sp . The bacterial consortium growth was evaluated by a viable counts during 14 days exposed to different aeration, temperature, salinity, and pH conditions . The ability of the consortium to mineralize asphaltenes was evaluated using the method of ISO 9439 in glass column reactors of 20 x 3.2 cm during 13 days . Temperatures of 55 degrees C and salinity of 1.8% were growth limiting . The respiration of the microbial consortium using asphaltenes as a sole carbon source (800 micromoles CO2 in 13 days) was significantly higher than those of the samples containing only the microbial consortium (200 micromoles CO2) or only asphaltenes (300 micromoles CO2) . These results indicated the existence of asphaltenes-degradating microbes in the crude oil and confirmed that the consortium could mineralize asphaltenes in conditions of room temperature, salinity of 100 ppm, aeration of 1 l min(-1) and pH of 7.4. Mikrobiologiia, 2004 Mar-Apr, 73(2), 218 - 25 {Three new species of brevibacteria--Brevibacterium antiquum sp . nov., Brevibacterium aurantiacum sp . nov . and Brevibacterium permense sp . nov.}; Gavrish EIu et al.; This work deals with the taxonomic study of 12 orange-pigmented bacteria isolated from permafrost sediments, rice plots, and soils contaminated with wastes from the chemical and salt industries, which were assigned to the genus Brevibacterium on the basis of phenotypic characteristics, as well as of some strains described previously as Brevibacterium linens . The study revealed three genomic species, whose members and the type strains of the closest species of Brevibacterium had DNA similarity levels between 24 and 59% . The strains of the genomic species differed from each other and from the known species of Brevibacterium in some physiological and biochemical characteristics, as well as in the sugar and polyol composition of their teichoic acids . The 16S rDNA sequence analysis confirmed the assignment of the environmental isolates to the genus Brevibacterium and showed the phylogenetic distinction of the three genomic species . The results obtained in this study allow three new Brevibacterium species to be described: Brevibacterium antiquum (type strain VKM Ac-2118T = UCM Ac-411T), Brevibacterium aurantiacum (type strain VKM Ac-2111T = NCDO 739T = ATCC 9175T), and Brevibacterium permense (type strain VKM Ac-2280T = UCM Ac-413T). Mikrobiologiia, 2004 Mar-Apr, 73(2), 199 - 203 {Cobalt- and chromium-containing inclusions in bacterial cells}; Ariskina EV et al.; Bacteria belonging to different taxonomic and physiological groups (members of the genera Pseudomonas, Brevibacterium, Rhodopseudomonas, and Lactococcus) are able to form intracellular cobalt- and chromium-containing magnetic inclusions . The paper deals with the structure and the intracellular localization of these inclusions and their similarity to the known noncrystalline iron-containing magnetic inclusions . The possible biological role of the magnetic inclusions is discussed. J Clin Microbiol, 2004 Jun, 42(6), 2829 - 32 Identification of a novel Brevibacterium species isolated from humans and description of Brevibacterium sanguinis sp . nov; Wauters G et al.; Six coryneforms isolated from blood and dialysate fluid were phenotypically similar to Brevibacterium casei, but 16S rRNA gene sequencing and DNA-DNA hybridization indicate that they belong to a new species for which the name Brevibacterium sanguinis is proposed. Appl Environ Microbiol, 2004 Jun, 70(6), 3664 - 72 Isolation, characterization, and identification of bacterial contaminants in semifinal gelatin extracts; De Clerck E et al.; Bacterial contamination of gelatin is of great concern . Indeed, this animal colloid has many industrial applications, mainly in food and pharmaceutical products . In a previous study (E . De Clerck and P . De Vos, Syst . Appl . Microbiol . 25:611-618), contamination of a gelatin production process with a variety of gram-positive and gram-negative bacteria was demonstrated . In this study, bacterial contamination of semifinal gelatin extracts from several production plants was examined . Since these extracts are subjected to harsh conditions during production and a final ultrahigh-temperature treatment, the bacterial load at this stage is expected to be greatly reduced . In total, 1,129 isolates were obtained from a total of 73 gelatin batches originating from six different production plants . Each of these batches was suspected of having bacterial contamination based on quality control testing at the production plant from which it originated . For characterization and identification of the 1,129 bacterial isolates, repetitive-element PCR was used to obtain manageable groups . Representative strains were identified by means of 16S rRNA gene sequencing, species-specific gyrB PCR, and gyrA and rpoB sequencing and were tested for gelatinase activity . The majority of isolates belonged to members of Bacillus or related endospore-forming genera . Representative strains were identified as Bacillus cereus, Bacillus coagulans, Bacillus fumarioli, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus sonorensis, Bacillus subtilis, Bacillus gelatini, Bacillus thermoamylovorans, Anoxybacillus contaminans, Anoxybacillus flavithermus, Brevibacillus agri, Brevibacillus borstelensis, and Geobacillus stearothermophilus . The majority of these species include strains exhibiting gelatinase activity . Moreover, some of these species have known pathogenic properties . These findings are of great concern with regard to the safety and quality of gelatin and its applications. J Biol Chem, 2004 Aug 13, 279(33), 34903 - 12 Epub 2004 Jun 04. Functional domains of Brevibacillus thermoruber lon protease for oligomerization and DNA binding: role of N-terminal and sensor and substrate discrimination domains; Lee AY et al.; Lon protease is a multifunctional enzyme, and its functions include the degradation of damaged proteins and naturally short lived proteins, ATPase and chaperone-like activities, as well as DNA binding . A thermostable Lon protease from Brevibacillus thermoruber WR-249 (Bt-Lon) has been cloned and characterized with an N-terminal domain, a central ATPase domain that includes a sensor and substrate discrimination (SSD) domain, and a C-terminal protease domain . Here we present a detailed structure-function characterization of Bt-Lon, not only dissecting the individual roles of Bt-Lon domains in oligomerization, catalytic activities, chaperone-like activity, and DNA binding activity but also describing the nature of oligomerization . Seven truncated mutants of Bt-Lon were designed, expressed, and purified . Our results show that the N-terminal domain is essential for oligomerization . The truncation of the N-terminal domain resulted in the failure of oligomerization and led to the inactivation of proteolytic, ATPase, and chaperone-like activities but retained the DNA binding activity, suggesting that oligomerization of Bt-Lon is a prerequisite for its catalytic and chaperone-like activities . We further found that the SSD is involved in DNA binding based on gel mobility shift assays . On the other hand, the oligomerization of Bt-Lon proceeds through a dimer <--> tetramer <--> hexamer assembly model revealed by chemical cross-linking experiments . The results also showed that hydrophobic interactions may play important roles in the dimerization of Bt-Lon, and ionic interactions are mainly responsible for the assembly of hexamers. Appl Microbiol Biotechnol, 2004 Sep, 65(4), 401 - 6 Epub 2004 May 27. Streptomyces lividans and Brevibacterium lactofermentum as heterologous hosts for the production of X22 xylanase from Aspergillus nidulans; Diaz M et al.; The Aspergillus nidulans gene xlnA coding for the fungal xylanase X22 has been cloned and expressed in two heterologous bacterial hosts: Streptomyces lividans and Brevibacterium lactofermentum . Streptomyces strains yielded 10 units/ml of xylanase when the protein was produced with its own signal peptide, and 19 units/ml when its signal peptide was replaced by the one for xylanase Xys1 from Streptomyces halstedii . B . lactofermentum was also able to produce xylanase X22, affording 6 units/ml upon using either the Aspergillus xlnA signal peptide or Streptomyces xysA . These production values are higher than those previously reported for the heterologous expression of the A . nidulans xlnA gene in Saccharomyces cerevisiae (1 unit/ml) . Moreover, the X22 enzyme produced by Streptomyces lividans showed oenological properties, indicating that this Streptomyces recombinant strain is a good candidate for the production of this enzyme at the industrial scale. Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 783 - 9 Gulosibacter molinativorax gen . nov., sp . nov., a molinate-degrading bacterium, and classification of 'Brevibacterium helvolum' DSM 20419 as Pseudoclavibacter helvolus gen . nov., sp . nov; Manaia CM et al.; A Gram-positive, molinate-degrading bacterium, strain ON4(T) (=DSM 13485(T)=LMG 21909(T)), was isolated from a mixed bacterial culture able to mineralize the herbicide molinate . The strain was strictly aerobic, oxidase- and catalase-positive and non-acid-fast, with a growth temperature of 10-41 degrees C . It contained the major menaquinone MK-9 and a cell-wall peptidoglycan based on D-ornithine . 16S rDNA sequence analysis revealed that the strain formed a distinct line of descent in the family Microbacteriaceae, showing the highest 16S rDNA similarity ( approximately 95 %) to members of the genus Curtobacterium and 'Brevibacterium helvolum' DSM 20419 (=ATCC 13715) . The latter was reported to have the cell-wall peptidoglycan type B2gamma and the major menaquinone MK-9, which are typical of Clavibacter, but it is clearly separated from this genus at the phylogenetic level . Based on low values of 16S rDNA sequence similarity to previously described genera and their distinctive phenotypic characteristics, it is proposed that strains ON4(T) and 'B . helvolum' DSM 20419 be classified as two novel genera and species, with the respective names Gulosibacter molinativorax gen . nov., sp . nov . and Pseudoclavibater helvolus gen . nov., sp . nov. Lett Appl Microbiol, 2004, 38(6), 532 - 5 Development of a 16S rRNA primer for the detection of Brevibacterium spp; Gelsomino R et al.; AIM: To develop a PCR method for the rapid identification of the genus Brevibacterium . METHODS AND RESULTS: Genus-specific primers were designed by aligning and comparing the 16S sequence of all Brevibacterium spp . with closely related genera . The primer set was tested with all validly described Brevibacterium spp . and their closest neighbours . SIGNIFICANCE: Until today brevibacteria could only be identified with laborious and time-consuming phenotypic characterization . The primer from this study offers a rapid alternative to the detection of Brevibacterium spp . Brevibacteria have been isolated from food, blood, ear discharge, from a wound and from an intravascular catheter. J Biochem (Tokyo), 2004 Mar, 135(3), 347 - 54 Module-specific antibodies against human connective tissue growth factor: utility for structural and functional analysis of the factor as related to chondrocytes; Minato M et al.; Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24/CCN2) shows diverse functions in the process of endochondral ossification . It promotes not only the proliferation and differentiation of chondrocytes and osteoblasts in vitro, but also angiogenesis in vivo . The ctgf gene is a member of the gene family called CCN, and it encodes the characteristic 4-module structure of this family, with the protein containing IGFBP, VWC, TSP and CT modules . We raised several monoclonal antibodies and polyclonal antisera against CTGF, and located the epitopes in the modules by Western blotting . For mapping the epitopes, Brevibacillus-produced independent modules were utilized . As a result, at least 1 antibody or antiserum was prepared for the detection of each module in CTGF . Western blotting with these antibodies is expected to be useful for the analysis of CTGF fragmentation . Moreover, we examined the effects of these monoclonal antibodies on the biological functions of CTGF . One out of 3 humanized monoclonal antibodies was found to neutralize efficiently the stimulatory effect of CTGF on chondrocytic cell proliferation . This particular antibody bound to the CT module . In contrast, surprisingly, all of the 3 antibodies recognizing IGFBP, VWC and CT modules stimulated proteoglycan synthesis in chondrocytic cells . Together with previous findings, these results provide insight into the structural-functional relationships of CTGF in executing multiple functions. Clin Microbiol Infect, 2004 May, 10(5), 465 - 7 Management of long-term catheter-related Brevibacterium bacteraemia; Beukinga I et al.; Brevibacterium has been reported as a rare cause of implanted-device infection . In two cases of recurrent Brevibacterium casei bacteraemia associated with infection of surgically implanted intravascular devices, relapse occurred 2 and 5 months, respectively, after completion of therapy with vancomycin via the infected catheter . A second intravenous antibiotic therapy course by the antibiotic-lock technique led to bacteriological cure in one patient . Molecular typing results demonstrated that the recurrent bacteraemia was caused by the same strain . Implanted-device removal may be necessary, in addition to appropriate antibiotics, for successful management of such infections. Biotechnol Lett, 2004 Feb, 26(4), 265 - 8 Overproduction of thymidine by recombinant brevibacterium helvolum amplified with thymidine monophosphate phosphohydrolase gene from bacteriophage PBS2; Lee HC et al.; A microbial fermentation process could be used to produce thymidine biologically but many of the enzymes related to nucleotide biosynthesis are highly regulated . To overcome the complex regulation steps, an analogue mutant of Brevibacterium helvolum resistant to fluorouracil, hydroxyurea, and trimethoprim was constructed . This mutant accumulated 380 mg thymidine 1(-1) in 16 h in shake-flask culture . However, the accumulation of thymidine monophosphate (TMP) inside the cells suggested a low activity of nucleotidase which degrades TMP to thymidine . This limitation was overcome by cloning the TMP phosphohydrolase (TMPase) gene of the unusual bacteriophage, PBS2 . As a result, TMP in recombinant cells decreased from 230 micromol g(-1) cell to 20 micromol g(-1) cell with accumulation of 500 mg thymidine l(-1) in the medium. J Bacteriol, 2004 Apr, 186(7), 1945 - 58 Crystallographic comparison of manganese- and iron-dependent homoprotocatechuate 2,3-dioxygenases; Vetting MW et al.; The X-ray crystal structures of homoprotocatechuate 2,3-dioxygenases isolated from Arthrobacter globiformis and Brevibacterium fuscum have been determined to high resolution . These enzymes exhibit 83% sequence identity, yet their activities depend on different transition metals, Mn2+ and Fe2+, respectively . The structures allow the origins of metal ion selectivity and aspects of the molecular mechanism to be examined in detail . The homotetrameric enzymes belong to the type I family of extradiol dioxygenases (vicinal oxygen chelate superfamily); each monomer has four betaalphabetabetabeta modules forming two structurally homologous N-terminal and C-terminal barrel-shaped domains . The active-site metal is located in the C-terminal barrel and is ligated by two equatorial ligands, H214NE1 and E267OE1; one axial ligand, H155NE1; and two to three water molecules . The first and second coordination spheres of these enzymes are virtually identical (root mean square difference over all atoms, 0.19 A), suggesting that the metal selectivity must be due to changes at a significant distance from the metal and/or changes that occur during folding . The substrate (2,3-dihydroxyphenylacetate {HPCA}) chelates the metal asymmetrically at sites trans to the two imidazole ligands and interacts with a unique, mobile C-terminal loop . The loop closes over the bound substrate, presumably to seal the active site as the oxygen activation process commences . An "open" coordination site trans to E267 is the likely binding site for O2 . The geometry of the enzyme-substrate complexes suggests that if a transiently formed metal-superoxide complex attacks the substrate without dissociation from the metal, it must do so at the C-3 position . Second-sphere active-site residues that are positioned to interact with the HPCA and/or bound O2 during catalysis are identified and discussed in the context of current mechanistic hypotheses. Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 615 - 6 Reclassification of Brevibacterium liquefaciens Okabayashi and Masuo 1960 as Arthrobacter nicotianae Giovannozzi-Sermanni 1959; Gelsomino R et al.; Brevibacterium liquefaciens ATCC 14929(T) was reclassified as Corynebacterium liquefaciens by Laneelle et al . (1980) . A further study by Stackebrandt et al . (1983) described the same strain, indicating high genomic and chemotaxonomic relatedness to Arthrobacter nicotianae; however, reclassification was not formally proposed . Because of the discrepancies between their previous work and the data of Collins & Kroppenstedt (1983), Laneelle and colleagues re-examined B . liquefaciens and withdrew their recommendation for the assignment of B . liquefaciens to the genus Corynebacterium (Laneelle et al., 1984) . Formal reclassification of B . liquefaciens as A . nicotianae is now proposed. Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 419 - 27 Reclassification of Brevibacillus brevis strains NCIMB 13288 and DSM 6472 (=NRRL NRS-887) as Aneurinibacillus danicus sp . nov . and Brevibacillus limnophilus sp . nov; Goto K et al.; Comparison of the hypervariable region (269-279 bases in length) at the 5' end of the 16S rDNA sequences of 29 bacterial strains that were identified previously as Brevibacillus brevis showed that 13 strains clustered with Aneurinibacillus species, eight strains clustered with Bacillus species and eight strains clustered with Brevibacillus species . Based on DNA-DNA hybridization results, 27 strains, not including {Brevibacillus brevis} NCIMB 13288 and {Brevibacillus brevis} DSM 6472, were reidentified as Aneurinibacillus migulanus, Aneurinibacillus thermoaerophilus, Bacillus methanolicus, Bacillus oleronius, Brevibacillus agri, Brevibacillus brevis and Brevibacillus parabrevis . {Brevibacillus brevis} NCIMB 13288, which was located in the Aneurinibacillus cluster, showed low DNA-DNA relatedness (<14 %) and low 16S rDNA sequence similarity (96.8-97.9 %) to other Aneurinibacillus species . {Brevibacillus brevis} DSM 6472, which was located in the Brevibacillus cluster, also showed low DNA-DNA relatedness (<12 %) and low 16S rDNA sequence similarity (95.4-98.8 %) to other Brevibacillus species . These genotypic and phylogenetic data, plus phenotypic and chemotaxonomic characteristics, suggest that {Brevibacillus brevis} NCIMB 13288 (=IAM 15048) and {Brevibacillus brevis} DSM 6472 (=NRRL NRS-887) represent novel species of the genera Aneurinibacillus and Brevibacillus, respectively, for which the names Aneurinibacillus danicus sp . nov . and Brevibacillus limnophilus sp . nov . are proposed. Anal Chem, 2004 Feb 1, 76(3), 585 - 91 Characterization of microorganisms using UV resonance Raman spectroscopy and chemometrics; Lopez-Diez EC et al.; The past decade has seen an increased interest in the application of several physicochemical analytical techniques for the rapid detection and identification of microorganisms . We report the development of UV resonance Raman (UVRR) spectroscopy for the reproducible acquisition of information rich Raman fingerprints from endospore-forming bacteria belonging to the genera Bacillus and Brevibacillus . UVRR was conducted at 244 nm, and spectra were collected in typically 60 s . Cluster analyses of these spectra showed that UVRR spectroscopy could be used to discriminate between these microorganisms to species level, and the clustering pattern from this phenotypic classification was highly congruent with phylogenetic trees constructed from 16S rDNA sequence analysis . Therefore, we conclude that UVRR spectroscopy when coupled with chemometrics constitutes a powerful approach to the characterization and speciation of microorganisms. Clin Chim Acta, 2004 Jan, 339(1-2), 135 - 45 Performance of four sources of cholesterol oxidase for serum cholesterol determination by the enzymatic endpoint method; Lolekha PH et al.; BACKGROUND: Cholesterol oxidase is used for the determination of serum cholesterol . It can be derived from Streptomyces, Pseudomonas fluorescens, Cellulomonas, and Brevibacterium . This study compared the performance characteristics of four enzymes in the endpoint cholesterol determination . METHODS: Using the Mega analyzer, we studied assay optimization, linearity, precision, recovery, interference, stability, and compared 110 patient samples . RESULTS: The linearity for the four enzymes was up to 13.0 mmol/l at the optimal enzyme activity . The average within-run CVs ranged from 1.6% to 1.9% and between-day ranged from 2.8% to 3.0%, within the NCEP analytical criteria . The analytical recoveries obtained from four reagents ( approximately 96.5%) were excellent . The assays using these enzyme sources compared favorably with the commercial method and appeared accurate near the clinical decision cut-points . Hemoglobin concentration at 1.9 g/l interfered with the P . fluorescens cholesterol oxidase . Bilirubin caused a negative interference while lipemia generated a positive interference with all enzyme sources . Reagents were stable up to 6 weeks . CONCLUSIONS: Streptomyces, Cellulomonas, and Brevibacterium were essentially analytically equivalent . Streptomyces and Cellulomonas cholesterol oxidase are one-quarter as expensive Brevibacterium . Cellulomonas is a new source of cholesterol oxidase for determining serum cholesterol by the endpoint method. Carbohydr Res, 2003 Nov 14, 338(23), 2745 - 9 A mannitol teichoic acid containing rhamnose and pyruvic acid acetal from the cell wall of Brevibacterium permense VKM Ac-2280; Potekhina NV et al.; 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 (approximately 70%, unit A) or (S)-acetal of pyruvic acid (approximately 30%, unit B) with the overall chain length approximately 10 mannitol phosphate units . {carbohydrate structure: see text} 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. Can J Microbiol, 2003 Oct, 49(10), 577 - 88 Influence of bacterial strains isolated from lead-polluted soil and their interactions with arbuscular mycorrhizae on the growth of Trifolium pratense L . under lead toxicity; Vivas A et al.; We isolated two bacterial strains from an experimentally lead (Pb)-polluted soil in Hungary, 10 years after soil contamination . These strains represented the two most abundant cultivable bacterial groups in such soil, and we tested their influence on Trifolium pratense L . growth and on the functioning of native mycorrhizal fungi under Pb toxicity in a second Pb-spiked soil . Our results showed that bacterial strain A enhanced plant growth, nitrogen and phosphorus accumulations, nodule formation, and mycorrhizal infection, demonstrating its plant-growth-promoting activity . In addition, strain A decreased the amount of Pb absorbed by plants, when expressed on a root weight basis, because of increased root biomass due to the production of indoleacetic acid . The positive effect of strain A was not only evident after a single inoculation but also in dual inoculation with arbuscular mycorrhizal fungi . Strain A also exhibited higher tolerance than strain B when cultivated under increasing Pb levels in the spiked soil . Molecular identification unambiguously placed strain A within the genus Brevibacillus . We showed that it is important to select the most tolerant and efficient bacterial strain for co-inoculation with arbuscular mycorrhizal fungi to promote effective symbiosis and thus stimulate plant growth under adverse environmental conditions, such as heavy-metal contamination. Microbiology, 2003 Dec, 149(Pt 12), 3531 - 42 Involvement of DivIVA in the morphology of the rod-shaped actinomycete Brevibacterium lactofermentum; Ramos A et al.; In Brevibacterium lactofermentum, as in many Gram-positive bacteria, a divIVA gene is located downstream from the dcw cluster of cell-division- and cell-wall-related genes . This gene (divIVA(BL)) is mostly expressed during exponential growth, and the protein encoded, DivIVA(BL,) bears some sequence similarity to antigen 84 (Ag84) from mycobacteria and was detected with monoclonal antibodies against Ag84 . Disruption experiments using an internal fragment of the divIVA(BL) gene or a disrupted divIVA(BL) cloned in a suicide conjugative plasmid were unsuccessful, suggesting that the divIVA(BL) gene is needed for cell viability in BREV: lactofermentum . Transformation of BREV: lactofermentum with a multicopy plasmid containing divIVA(BL) drastically altered the morphology of the corynebacterial cells, which became larger and bulkier, and a GFP fusion to DivIVA(BL) mainly localized to the ends of corynebacterial cells . This localization pattern, together with the overproduction phenotype, suggests that DivIVA may be important in regulating the apical growth of daughter cells. Appl Environ Microbiol, 2003 Dec, 69(12), 7467 - 79 Enumeration and characterization of iron(III)-reducing microbial communities from acidic subsurface sediments contaminated with uranium(VI); Petrie L et al.; Iron(III)-reducing bacteria have been demonstrated to rapidly catalyze the reduction and immobilization of uranium(VI) from contaminated subsurface sediments . Thus, these organisms may aid in the development of bioremediation strategies for uranium contamination, which is prevalent in acidic subsurface sediments at U.S . government facilities . Iron(III)-reducing enrichment cultures were initiated from pristine and contaminated (high in uranium, nitrate; low pH) subsurface sediments at pH 7 and pH 4 to 5 . Enumeration of Fe(III)-reducing bacteria yielded cell counts of up to 240 cells ml(-1) for the contaminated and background sediments at both pHs with a range of different carbon sources (glycerol, acetate, lactate, and glucose) . In enrichments where nitrate contamination was removed from the sediment by washing, MPN counts of Fe(III)-reducing bacteria increased substantially . Sediments of lower pH typically yielded lower counts of Fe(III)-reducing bacteria in lactate- and acetate-amended enrichments, but higher counts were observed when glucose was used as an electron donor in acidic enrichments . Phylogenetic analysis of 16S rRNA gene sequences extracted from the highest positive MPN dilutions revealed that the predominant members of Fe(III)-reducing consortia from background sediments were closely related to members of the Geobacteraceae family, whereas a recently characterized Fe(III) reducer (Anaeromyxobacter sp.) and organisms not previously shown to reduce Fe(III) (Paenibacillus and Brevibacillus spp.) predominated in the Fe(III)-reducing consortia of contaminated sediments . Analysis of enrichment cultures by terminal restriction fragment length polymorphism (T-RFLP) strongly supported the cloning and sequencing results . Dominant members of the Fe(III)-reducing consortia were observed to be stable over several enrichment culture transfers by T-RFLP in conjunction with measurements of Fe(III) reduction activity and carbon substrate utilization . Enrichment cultures from contaminated sites were also shown to rapidly reduce millimolar amounts of U(VI) in comparison to killed controls . With DNA extracted directly from subsurface sediments, quantitative analysis of 16S rRNA gene sequences with MPN-PCR indicated that Geobacteraceae sequences were more abundant in pristine compared to contaminated environments,whereas Anaeromyxobacter sequences were more abundant in contaminated sediments . Thus, results from a combination of cultivation-based and cultivation-independent approaches indicate that the abundance/community composition of Fe(III)-reducing consortia in subsurface sediments is dependent upon geochemical parameters (pH, nitrate concentration) and that microorganisms capable of producing spores (gram positive) or spore-like bodies (Anaeromyxobacter) were representative of acidic subsurface environments. Eur J Biochem, 2003 Nov, 270(22), 4420 - 5 A novel mannitol teichoic acid with side phosphate groups of Brevibacterium sp . VKM Ac-2118; Potekhina NV et al.; The cell wall of Brevibacterium sp . VKM Ac-2118 isolated from a frozen (mean annual temperature -12 degrees C) late Pliocene layer, 1.8-3 Myr, Kolyma lowland, Russia, contains mannitol teichoic acid with a previously unknown structure . This is 1,6-poly(mannitol phosphate) with the majority of the mannitol residues bearing side phosphate groups at O-4(3) . The structure of the polymer was established by chemical methods, NMR spectroscopy, and MALDI-TOF mass spectrometry. J Biotechnol, 2003 Nov 6, 105(3), 245 - 53 Catabolism of volatile sulfur compounds precursors by Brevibacterium linens and Geotrichum candidum, two microorganisms of the cheese ecosystem; Arfi K et al.; Two Brevibacterium linens strains and the cheese-ripening yeast Geotrichum candidum were compared with regard to their ability to produce volatile sulfur compounds (VSCs) from three different precursors namely L-methionine, 4-methylthio-2-oxobutyric acid (KMBA) and 4-methylthio-2-hydroxybutyric acid (HMBA) . All microorganisms were able to convert these precursors to VSCs . However, although all were able to produce VSCs from L-methionine, only G . candidum accumulated KMBA when cultivated on this amino acid, contrary to B . linens suggesting that the transamination pathway is not active in this microorganism . Conversely, a L-methionine gamma-lyase activity--which catalyses the one step L-methionine to methanethiol (MTL) degradation route--was only found in B . linens strains . Several other enzymatic activities involved in the catabolism of the precursors tested were investigated . KMBA transiently accumulated in G . candidum cultures, and was then reduced to HMBA by a KMBA dehydrogenase (KDH) activity . This activity was not detected in B . linens . Despite no HMBA dehydrogenase (HDH) was found in G . candidum, a strong HMBA oxidase (HOX) activity was measured in this microorganism . This latter activity was weakly active in B . linens . KMBA and HMBA demethiolating activities were found in all the microorganisms . Our results illustrate the metabolic diversity between cheese-ripening microorganisms of the cheese ecosystem. Biotechnol Adv, 1990, 8(1), 97 - 103 Threonine production by dihydrodipicolinate synthase-defective mutants of Brevibacterium flavum; Shiio I; A novel type of threonine-producing strains, dihydrodipicolinate synthase (DPS)-defective mutants of Brevibacterium flavum, was isolated as alpha-amino-beta-hydroxyvaleric acid (AHV)-resistant producers . The third selection markers used were a strong lysine inhibition of threonine production and a lower production of lysine than that of threonine in those derived from strains with feedback-sensitive and-resistant aspartokinase (AK), respectively . The maximum threonine production by these DPS-defective mutants was 13.7 g/l at the optimum concentration of DL-diaminopimelic acid (DAP) in a medium containing 100 g/l of glucose, comparable to that by the previously reported conventional producers with feedback-resistant homoserine dehydrogenase (HD(R)) . The DPS-defective mutants with feedback-sensitive AK showed a slow but substantial growth in the absence of DAP and their growth was markedly stimulated by DAP, while those with feedback-resistant AK grew well in the absence of DAP and their growth was not promoted by DAP more than that of the parent strain . DPS-defective mutants with HD(R) were derived from an HD(R) mutant producing 10 g/l of L-threonine and selected as AHV-resistant mutants with a higher productivity . The maximum production was 16 g/l. Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1321 - 5 Brevibacterium lutescens sp . nov., from human and environmental samples; Wauters G et al.; Three strains of coryneform rods isolated from clinical samples and one of environmental origin exhibited phenotypic and chemotaxonomic properties characteristic of the genus Brevibacterium and their 16S rRNA gene sequences were closely related (98.5-99.0 %) to that of Brevibacterium otitidis . However, DNA-DNA hybridization of one strain (CF87(T)) showed only 59.6 % relatedness to the type strain of B . otitidis, DSM 10718(T), and 75-82 % relatedness to the three other strains . The four strains could be differentiated from B . otitidis by cellular fatty acid composition and some phenotypic characteristics . These findings suggest that the four strains belong to a novel species, for which the name Brevibacterium lutescens sp . nov . is proposed . The type strain of B . lutescens is CF87(T) (=DSM 15022(T)=CCUG 46604(T)). Appl Environ Microbiol, 2003 Sep, 69(9), 5128 - 37 Nature of polymorphisms in 16S-23S rRNA gene intergenic transcribed spacer fingerprinting of Bacillus and related genera; Daffonchio D et al.; The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels . We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-G+C-content spore-forming bacteria belonging to the genera Bacillus, Brevibacillus, Geobacillus, and Paenibacillus: We found that besides the polymorphisms in the homoduplex fragments amplified by PCR, heteroduplex products formed during PCR between amplicons from different ribosomal operons, with or without tRNA genes in the ITS, contribute to the interstrain variability in ITS-PCR fingerprinting patterns obtained in polyacrylamide-based gel matrices . The heteroduplex nature of the discriminating bands was demonstrated by fragment separation in denaturing polyacrylamide gels, by capillary electrophoresis, and by cloning, sequencing, and recombination of purified short and tRNA gene-containing long ITS . We also found that heteroduplex product formation is enhanced by increasing the number of PCR cycles . Homoduplex-heteroduplex polymorphisms (HHP) in a conserved region, such as the 16S and 23S rRNA gene ITS, allowed discrimination of closely related strains and species undistinguishable by other methods, indicating that ITS-HHP analysis is an easy and reproducible additional tool for strain typing. J Biotechnol, 2003 Sep 4, 104(1-3), 301 - 9 The mobile element IS1207 of Brevibacterium lactofermentum ATCC21086: isolation and use in the construction of Tn5531, a versatile transposon for insertional mutagenesis of Corynebacterium glutamicum; Bonamy C et al.; IS1207 is the insertion most frequently found among the spontaneous mutations that abolish the activity of an Escherichia coli phage lambda cI gene integrated in the Corynebacterium Brevibacterium lactofermentum ATCC21086 genome . We examined the transposition of transposon-like structures composed of a selective kanamycin resistance gene (aph3), and one or two IS1207 sequences . One of these, the Tn5531 transposon, transposed efficiently in Corynebacterium glutamicum . A replicative and a non-replicative Tn5531 delivery vector were used in Tn5531 mutagenesis . As IS1207, transposon Tn5531 shows a high frequency of transposition and mutagenesis, and a low target specificity . These features make of Tn5531 an adequate choice for gene identification and gene tagging experiments. J Biotechnol, 2003 Sep 4, 104(1-3), 41 - 53 Ribosomal RNA and ribosomal proteins in corynebacteria; Martin JF et al.; Ribosomal RNAs (rRNAs) (16S, 23S, 5S) encoded by the rrn operons and ribosomal proteins play a very important role in the formation of ribosomes and in the control of translation . Five copies of the rrn operon were reported by hybridization studies in Brevibacterium (Corynebacterium) lactofermentum but the genome sequence of Corynebacterium glutamicum provided evidence for six rrn copies . All six copies of the C . glutamicum 16S rRNA have a size of 1523 bp and each of the six copies of the 5S contain 120 bp whereas size differences are found between the six copies of the 23S rRNA . The anti-Shine-Dalgarno sequence at the 3'-end of the 16S rRNA was 5'-CCUCCUUUC-3' . Each rrn operon is transcribed as a large precursor rRNA (pre-rRNA) that is processed by RNaseIII and other RNases at specific cleavage boxes that have been identified in the C . glutamicum pre-rRNA . A secondary structure of the C . glutamicum 16S rRNA is proposed . The 16S rRNA sequence has been used as a molecular evolution clock allowing the deduction of a phylogenetic tree of all Corynebacterium species . In C . glutamicum, there are 11 ribosomal protein gene clusters encoding 42 ribosomal proteins . The organization of some of the ribosomal protein gene cluster is identical to that of Escherichia coli whereas in other clusters the organization of the genes is rather different . Some specific ribosomal protein genes are located in a different cluster in C . glutamicum when compared with E . coli, indicating that the control of expression of these genes is different in E . coli and C . glutamicum. Environ Pollut, 2003, 126(2), 179 - 89 Symbiotic efficiency of autochthonous arbuscular mycorrhizal fungus (G . mosseae) and Brevibacillus sp . isolated from cadmium polluted soil under increasing cadmium levels; Vivas A et al.; The effect of inoculation with indigenous naturally occurring microorganisms (an arbuscular mycorrhizal (AM) fungus and rhizosphere bacteria) isolated from a Cd polluted soil was assayed on Trifolium repens growing in soil contaminated with a range of Cd . One of the bacterial isolate showed a marked PGPR effect and was identified as a Brevibacillus sp . Mycorrhizal colonization also enhanced Trifolium growth and N, P, Zn and Ni content and the dually inoculated (AM fungus plus Brevibacillus sp.) plants achieved further growth and nutrition and less Cd concentration, particularly at the highest Cd level . Increasing Cd level in the soil decreased Zn and Pb shoot accumulation . Coinoculation of Brevibacillus sp . and AM fungus increased shoot biomass over single mycorrhizal plants by 18% (at 13.6 mg Cd kg(-1)), 26% (at 33.0 mg Cd kg(-1)) and 35% (at 85.1 mg Cd (kg(1)) . In contrast, Cd transfer from soil to plants was substantially reduced and at the highest Cd level Brevibacillus sp . lowered this value by 37.5% in AM plants . Increasing Cd level highly reduced plant mycorrhization and nodulation . Strong positive effect of the bacterium on inocula, are important in plant Cd tolerance and development in Cd polluted soils. FEMS Microbiol Lett, 2003 Aug 8, 225(1), 85 - 92 Cloning and expression of the inorganic pyrophosphatase gene from the amino acid producer Brevibacterium lactofermentum ATCC 13869; Ramos A et al.; A 20-kDa Brevibacterium lactofermentum protein was detected when purifying the His-tagged FtsZBL . The protein was identified by matrix-assisted laser desorption/ionisation time of flight as the inorganic pyrophosphatase encoded by the ppa gene, which is present as a single copy in the genome of Corynebacterium glutamicum . The ppa gene was cloned from B . lactofermentum chromosomal DNA by polymerase chain reaction; it seemed to be an essential gene and it might represent an attractive target for drug discovery . The cloned ppa gene complemented a ppa- Escherichia coli mutant and a ppa-gfp gene fusion revealed that the gene product mainly accumulated at the cell poles in both E . coli and B . lactofermentum. Biodegradation, 2003 Jun, 14(3), 183 - 8 Isolation and identification of thiocyanate utilizing chemolithotrophs from gold mine soils; Lee C et al.; A mixed bacterial culture capable of growing in potassium-thiocyanate containing medium (200 mg KSCN) has been isolated from bacterial suspensions of soil samples collected near gold mines in Kumjung (Korea) . The isolates were initially characterized by metabolic profile analysis and were identified as Bacillus thermoglucosidasius, Bacillus cereus, Bacillus licheniformis, Bacillus mycoides, Brevibacterium epidermidis, Brevibacterium otitidis, and Corynebacterium nitrilophilus . One of the seven isolates was initially characterized as Brevibacterium epidermidis, which is not known to degrade thiocyanate . However, using 16S rDNA sequencing, this strain was identified as a member of Klebsiella . The strain shows high similarity values (95.8 to 96.4%) with Klebsiella species, and the closest known relative was found to be K . ornithinolytica ATCC 31898 . The result indicates that species of the genus Klebsiella were the closest phylogenetic relatives of the investigated strain . This is the first known report of a member of Klebsiella that is capable of utilizing thiocyanate as sole source of carbon and nitrogen. Appl Microbiol Biotechnol, 2003 Aug, 62(2-3), 163 - 7 Epub 2003 Apr 02. Influence of growth conditions on bacteriocin production by Brevibacterium linens; Motta AS et al.; The influence of temperature, NaCl concentration and cheese whey media on growth of Brevibacterium linens ATCC 9175 and production of bacteriocin-like antimicrobial activity was studied . Bacteriocin production and activity were higher at 25 degrees C than at 30 degrees C . No significant growth or production of bacteriocins was observed at 37 degrees C . When bacteriocin production was investigated in media containing different concentrations of NaCl, increased activity was observed in media containing 40 or 80 g l(-1), but not 120 g l(-1) NaCl . The addition of NaCl resulted in a significant increase in specific production rates of bacteriocin-like activity . Antimicrobial activity was also observed by cultivation of B . linens at 25 degrees C in cheese whey media. Biotechnol Lett, 2003 Jan, 25(2), 143 - 7 Arrest of cell cycle by inhibition of ribonucleotide reductase induces accumulation of NAD+ by Mn2+-supplemented growth of Corynebacterium ammoniagenes; Abbouni B et al.; Cell division of the wild type strain Corynebacterium (formerly Brevibacterium) ammoniagenes ATCC 6872 which requires 1 microM Mn2+ for balanced growth was inhibited by addition of 20 mM hydroxyurea (HU) or 10 mM p-methoxyphenol (MP) to a Mn2+-supplemented fermentation medium at an appropriate time . Scanning electron microscopy (SEM) showed a restricted elongation characteristic of arrest of the cell cycle in coryneform bacteria . The cultures treated with HU or MP had, respectively, a fourfold or sixfold enhanced accumulation of NAD+ by a salvage biosynthetic pathway . An assay of nucleotide-permeable cells for ribonucleotide reductase activity using {3H-CDP} as substrate revealed a pre-early and complete decline of DNA precursor biosynthesis not found in the untreated control . Overproduction of NAD+ is an alternative to the conventional fermentation process using Mn2+ deficiency . A simple model is presented to discuss the metabolic regulation of the new process based on the presence of a manganese ribonucleotide reductase (Mn-RNR) in the producing strain. Biotechnol Lett, 2003 May, 25(9), 705 - 8 High cell density cultivation of Brevibacterium linens and formation of proteinases and lipase; Adamitsch BF et al.; Brevibacterium linens forms hydrolytic enzymes which can be used to accelerate the ripening of cheese without causing bitterness . B . linens ATCC 9172 was grown to a high cell density (50 g dry wt l-1 after 60 h) in a mineral medium containing lactic acid, soy-peptone and ammonium sulphate by applying a continuous feed of nutrients . The maximal activities of L-leucine aminopeptidase and cell-associated proteinase were 286 U l-1 and 202 U l-1, respectively . The cell-associated lipolytic activity exhibited a strong and sudden increase at 46 h, resulting in a maximum of 9.5 U g-1 dry wt; thus the volumetric productivity of proteolytic and lipolytic activity was 4220 U l-1 h-1 and 7.3 U l-1 h-1, respectively. J Clin Microbiol, 2003 Jul, 41(7), 3241 - 5 Rapid identification of Rhodococcus equi by a PCR assay targeting the choE gene; Ladron N et al.; The actinomycete Rhodococcus equi is an important pathogen of horses and an emerging opportunistic pathogen of humans . Identification of R . equi by classical bacteriological techniques is sometimes difficult, and misclassification of an isolate is not uncommon . We report here on a specific PCR assay for the rapid and reliable identification of R . equi . It is based on the amplification of a fragment of the choE gene encoding cholesterol oxidase . The choE-based PCR was assessed by using a panel of strains comprising 132 isolates from different sources and of different geographical origins, all initially identified biochemically as R . equi, and 30 isolates of representative non-R . equi actinomycete species, including cholesterol oxidase producers . The expected 959-bp amplicon was observed only with R . equi isolates, as confirmed by sequencing of a variable region of the 16S RNA gene from a random sample of 20 PCR-positive isolates . All R . equi isolates gave a positive choE-based PCR result, which correlated with a high degree of conservation of the choE gene . Three of the 132 strains originally identified as R . equi were negative for the choE gene, and subsequent analysis of their 16S RNA gene sequences confirmed that they belonged to other bacterial species (Dietzia maris, Mycobacterium peregrinum, and Staphylococcus epidermidis) . All non-R . equi isolates were negative by the choE-based PCR . ATCC 21387, the only known isolate of Brevibacterium sterolicum, gave a 959-bp amplicon whose DNA sequence was virtually identical to that of R . equi choE . Comparison of the 16S RNA genes indicated that ATCC 21387 should be considered an R . equi isolate. Bioorg Med Chem, 2003 Jul 17, 11(14), 3133 - 40 Modified glycopeptides related to cell wall peptidoglycan: conformational studies by NMR and molecular modelling; Feher K et al.; Polymeric peptidoglycans of bacterial cell walls, and smaller glycopeptides derived from them, exhibit versatile biological activities including immunomodulating properties . Peptidoglycan monomer (PGM) was isolated from Brevibacterium divaricatum and novel lipophilic derivatives of PGM bearing either (adamantyl-1-yl)-acetyl or Boc-Tyr substituents (Ad-PGM and BocTyr-PGM respectively) have recently been synthesized . We have obtained full assignments of the 1H and 13C spectra, using 2D NMR techniques, for all three compounds in DMSO solutions . NOESY/ROESY experiments have provided interproton distance restraints that were used in distance geometry modelling calculations to derive conformational preferences for each of these molecules . These data were supplemented with information available from chemical shifts, temperature dependence of amide proton shifts and proton-proton scalar couplings . Analysis of the results suggest that the lipophilic substituents attached to the Dap(3)- epsilon amino group of the parent PGM molecule introduce changes to the conformational preferences of the peptide moiety . In PGM electrostatic interactions between charged end groups apparently promote folded conformations with participation of the long Dap side chain . Derivatives wherein such interactions are suppressed by acylation of the Dap(3)- epsilon amino group are characterized by more extended conformations of the peptide chain . The new synthetic derivatives exhibit biological properties similar to those of the parent PGM . This may indicate that peripheral parts of the peptide chain such as the C-terminal and end groups of the long Dap side chain do not significantly contribute to the binding to receptors or enzymes participating in the biochemical interactions referred to above. Arch Microbiol, 2003 Jul, 180(1), 53 - 9 Epub 2003 Jun 07. Improved vectors for transcriptional/translational signal screening in corynebacteria using the melC operon from Streptomyces glaucescens as reporter; Adham SA et al.; The tyrosinase operon ( melC) from Streptomyces glaucescens was cloned and functionally expressed in Brevibacterium lactofermentum and Corynebacterium glutamicum under the control of the promoter of the kan gene from Tn 5 . Recombinant corynebacterial cells containing the tyrosinase operon produced melanin on agar plates and in liquid culture when supplemented with copper and tyrosine . A conjugative bifunctional replacement vector for transcriptional/translational signal screening (pEMel-1) was constructed using expression of the melC operon from S . glaucescens, which can be used for cloning promoter sequences as EcoRI- NdeI fragments . When the DNA fragments with promoter activity such as cspBp or trpp were inserted into pEMel-1, B . lactofermentum harboring the chimeric plasmids produced melanin at different stages of growth, allowing temporal detection of promoter activity . The vector was also used to detect the activity of a Streptomyces promoter ( xysAp), which was inactive in B . lactofermentum, after PCR mutagenesis . The melC operon can be used for the visual, inexpensive (compared to the high price of starch azure for amylase detection), and non-selective (in contrast to the kan or cat genes) screening of several thousand clones at high colony density without killing of the transformants due to the presence of iodine (as in the case of amylase assay). J Biotechnol, 2003 Jun 12, 103(1), 1 - 10 Cloning and expression of the ccdA-associated thiol-disulfide oxidoreductase (catA) gene from Brevibacillus choshinensis: stimulation of human epidermal growth factor production; Tanaka R et al.; Brevibacillus choshinensis (Bacillus brevis) is a protein-hyperproducing bacterium with a useful host-vector system for the production of recombinant proteins . Here, we cloned the ccdA-catA (cmacr;cdA associated thioredoxin-like tmacr;hiol-disulfide oxidoreductase) locus of B . choshinensis HPD31-S5 . CatA protein (molecular weight, 19664) contains a thioredoxin-like motif, Cys-Gly-Pro-Cys . It was successfully expressed in B . choshinensis extracellularly ( approximately 100 microg x ml(-1) culture) using the secretion vector pNCMO2, and in Escherichia coli intracellularly ( approximately 350 microg x ml(-1) culture) with an amino-terminal His-tag . Both recombinant proteins showed thiol-disulfide oxidoreductase activity . Incubation of non-native human epidermal growth factor (hEGF) containing incorrect disulfide bonds with B . choshinensis cells secreting CatA protein resulted in the stimulation of the conversion of non-native hEGF to the native form . Furthermore, co-expression of CatA protein with recombinant hEGF in the B . choshinensis production system increased the yield of native hEGF. Syst Appl Microbiol, 2003 Mar, 26(1), 30 - 7 Staphylococcus equorum subsp . linens, subsp . nov., a starter culture component for surface ripened semi-hard cheeses; Place RB et al.; Two staphylococcal strains, RP29T and RP33, were isolated from the main microflora of a surface ripened Swiss mountain cheese made from raw milk . These two strains were differentiated from the most closely related species Staphylococcus equorum on the basis of DNA-DNA hybridisation and phenotypic characteristics and are proposed as Staphylococcus equorum subsp . linens subsp . nov . They could be distinguished phenotypically from S . equorum by their sensitivity to all 14 tested antibiotics, especially to novobiocin, their incapability to ferment alpha-D-lactose, maltose, sucrose, D-trehalose, D-xylose, L-arabinose, salicin, D-ribose, D-raffinose, D-mannitol, and D-alanine . The GenBank accession numbers for the reference sequences of the 16S rDNA and the hsp60 gene used in this study are AF527483 and AF527484, respectively . 30 tons of a semi-hard Swiss cheese were produced with Staphylococcus equorum subsp . linens DSM 15097T as starter culture component in addition to Debaryomyces hansenii, Geotrichum candidum, Brevibacterium linens, Corynebacterium casei for surface ripened cheeses . The products were sensorically and hygienically perfect . Therefore, Staphylococcus equorum subsp . linens DSM 15097T can be proposed as starter culture component for surface ripened cheeses without any detected antibiotic resistances . The type strain of Staphylococcus equorum subsp . linens is DSM 15097T (CIP 107656T). Plasmid, 2003 Mar, 49(2), 160 - 8 Complete nucleotide sequence of a native plasmid from Brevibacterium linens; Moore M et al.; Brevibacterium linens has commercial significance in the dairy industry and potential application in the production of bacteriocins and carotenoids . Strain development of these industrially significant organisms would be facilitated by the use of vectors, yet few are available . In this study we report the isolation of four novel plasmids from the Gram-positive coryneform B . linens, and determine the first complete nucleotide sequence of a native plasmid of B . linens . The cryptic plasmid pLIM is 7610 bp in length, and belongs to a subfamily of theta replicating ColE2-related plasmids . Initial investigation suggests that replication in pLIM requires two replicases, a primase (RepA) and a DNA binding protein (RepB), encoded by a single operon repAB . The origin of replication is located upstream of repAB transcription. Appl Microbiol Biotechnol, 2003 May, 61(3), 252 - 6 Epub 2003 Feb 26. Hydrolysis of fenamiphos and its oxidation products by a soil bacterium in pure culture, soil and water; Megharaj M et al.; A bacterium, identified as Brevibacterium sp . MM1, readily hydrolysed fenamiphos, a widely used organophosphorus insecticide and its toxic oxides (fenamiphos sulfoxide, fenamiphos sulfone), which all contain a common P-O-C bond, in a mineral salts medium . The bacterium also hydrolysed fenamiphos and its oxides in soil and groundwater . Interestingly, fenamiphos phenol, fenamiphos sulfoxide phenol and fenamiphos sulfone phenol, formed during bacterial hydrolysis of fenamiphos and its oxides, persisted in the mineral salts medium, but were transitory in soil and groundwater due to their further metabolism by indigenous micro-organisms . The cell-free preparation (crude enzyme) of this bacterium was very effective in hydrolysing fenamiphos . This is the first report on exceptionally rapid hydrolysis of fenamiphos by a bacterium in pure cultures, soil and groundwater. J Agric Food Chem, 2003 Apr 23, 51(9), 2653 - 8 Bioavailability of an organophosphorus pesticide, fenamiphos, sorbed on an organo clay; Singh N et al.; Hydrolysis of an insecticide/nematicide, fenamiphos {ethyl-3-methyl-4-(methylthio)phenyl-(1-methylethyl)phosphoramidate}, immobilized through sorption by cetyltrimethylammonium-exchanged montmorillonite (CTMA-clay) by a soil bacterium, Brevibacterium sp., was examined . X-ray diffraction analysis, infrared spectra, and a negative electrophoretic mobility strongly indicated that fenamiphos was intercalated within the bacterially inaccessible interlayer spaces of CTMA-clay . The bacterium hydrolyzed, within 24 h, 82% of the fenamiphos sorbed by the CTMA-clay complex . There was a concomitant accumulation of hydrolysis product, fenamiphos phenol, in nearly stoichiometric amounts . During the same period, in abiotic (uninoculated) controls, 4.6% of the sorbed insecticide was released into the aqueous phase as compared to 6.0% of the sorbed fenamiphos in another abiotic control where activated carbon, a sink for desorbed fenamiphos, was present . Thus, within 24 h, the bacterium hydrolyzed 77% more fenamiphos sorbed by organo clay than the amounts desorbed in abiotic controls . Such rapid degradation of an intercalated pesticide by a bacterium has not been reported before . Evidence indicated that extracellular enzymes produced by the bacterium rapidly hydrolyzed the nondesorbable fenamiphos, even when the enzyme itself was sorbed . Fenamiphos strongly sorbed to an organo clay appears to be readily available for exceptionally rapid degradation by the bacterium. Bioorg Med Chem Lett, 2003 Apr 17, 13(8), 1479 - 82 First enantiodivergent Baeyer-Villiger oxidation by recombinant whole-cells expressing two monooxygenases from Brevibacterium; Mihovilovic MD et al.; Microbial Baeyer-Villiger oxidations of representative mesomeric ketones with recombinant Escherichia coli cells expressing two monooxygenases from Brevibacterium were investigated . The two enzymes displayed enantiodivergent biotransformations on an array of structurally diverse substrates, allowing access to some key lactone intermediates in natural compound synthesis. J Biol Inorg Chem, 2003 Feb, 8(3), 263 - 72 Epub 2002 Nov 09. 4-nitrocatechol as a probe of a Mn(II)-dependent extradiol-cleaving catechol dioxygenase (MndD): comparison with relevant Fe(II) and Mn(II) model complexes; Reynolds MF et al.; Mn(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase (MndD) is an extradiol-cleaving catechol dioxygenase from Arthrobacter globiformis that has 82% sequence identity to and cleaves the same substrate (3,4-dihydroxyphenylacetic acid) as Fe(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase (HPCD) from Brevibacterium fuscum . We have observed that MndD binds the chromophoric 4-nitrocatechol (4-NCH(2)) substrate as a dianion and cleaves it extremely slowly, in contrast to the Fe(II)-dependent enzymes which bind 4-NCH(2) mostly as a monoanion and cleave 4-NCH(2) 4-5 orders of magnitude faster . These results suggest that the monoanionic binding state of 4-NC is essential for extradiol cleavage . In order to address the differences in 4-NCH(2) binding to these enzymes, we synthesized and characterized the first mononuclear monoanionic and dianionic Mn(II)-(4-NC) model complexes as well as their Fe(II)-(4-NC) analogs . The structures of {(6-Me(2)-bpmcn)Fe(II)(4-NCH)}(+), {(6-Me(3)-TPA)Mn(II)(DBCH)}(+), and {(6-Me(2)-bpmcn)Mn(II)(4-NCH)}(+) reveal that the monoanionic catecholate is bound in an asymmetric fashion (Delta r(metal-O(catecholate))=0.25-0.35 A), as found in the crystal structures of the E(.)S complexes of extradiol-cleaving catechol dioxygenases . Acid-base titrations of {(L)M(II)(4-NCH)}(+) complexes in aprotic solvents show that the p K(a) of the second catecholate proton of 4-NCH bound to the metal center is half a p K(a) unit higher for the Mn(II) complexes than for the Fe(II) complexes . These results are in line with the Lewis acidities of the two divalent metal ions but are the opposite of the trend observed for 4-NCH(2) binding to the Mn(II)- and Fe(II)-catechol dioxygenases . These results suggest that the MndD active site decreases the second p K(a) of the bound 4-NCH(2) relative to the HPCD active site. Wei Sheng Wu Xue Bao, 1999 Oct, 39(5), 430 - 5 {Expression of genes aroG and pheA in phenylalanine biosynthesis}; Fan C et al.; aroG and pheA genes, encoding 3-Deoxy-D-arabinoheptulonate-7-phosphate synthase(DS) and Chorismate mutase (CM)-prephenate dehydratase(PD) in the pathway of phenylalanine biosynthesis respectively, were amplified by polymerase chain reaction(PCR) . The genes were assembled on the multicopy vectors and expressed in both Escherichia coli and Brevibacterium . The products of two gene were detected by SDS-PAGE . The activities of relevant enzymes were measured in the crude extract of the host strain . When aroG-pheA genes were introduced into E . coli p2392, the activities of DS, CM and PD were increased by 4.3-fold, 4.4-fold and 2.2-fold respectively . Whereas in the case of Brevibacterium flavum 2732, the activities of DS, CM and PD were increased by 12.3-fold, 2.3-fold and 5.6-fold, respectively . As the results, the overproduction of phenylalanine was brought about by using the genetic engineering strain of B . flavum. World J Gastroenterol, 2003 Feb, 9(2), 342 - 6 Co-expression of five genes in E coli for L-phenylalanine in Brevibacterium flavum; Wu YQ et al.; AIM: To study the effect of co-expression of ppsA, pckA, aroG, pheA and tyrB genes on the production of L-phenylalanine, and to construct a genetic engineering strain for L-phenylalanine . METHODS: ppsA and pckA genes were amplified from genomic DNA of E . coli by polymerase chain reaction, and then introduced into shuttle vectors between E coli and Brevibacterium flavum to generate constructs pJN2 and pJN5 . pJN2 was generated by inserting ppsA and pckA genes into vector pCZ; whereas pJN5 was obtained by introducing ppsA and pckA genes into pCZ-GAB, which was originally constructed for co-expression of aroG, pheA and tyrB genes . The recombinant plasmids were then introduced into B . flavum by electroporation and the transformants were used for L-phenylalanine fermentation . RESULTS: Compared with the original B . flavum cells, all the transformants were showed to have increased five enzyme activities specifically, and have enhanced L-phenylalanine biosynthesis ability variably . pJN5 transformant was observed to have the highest elevation of L-phenylalanine production by a 3.4-fold . Co-expression of ppsA and pckA increased activity of DAHP synthetase significantly . CONCLUSION: Co-expression of ppsA and pckA genes in B . flavum could remarkably increase the expression of DAHP synthetase; Co-expression of ppsA, pckA, aroG, pheA and tyrB of E . coli in B . flavum was a feasible approach to construct a strain for phenylalanine production. J Infect, 2003 Jan, 46(1), 61 - 4 Brevibacterium casei bacteremia and line sepsis in a patient with AIDS; Janda WM et al.; Brevibacteria are obligately aerobic gram-positive bacilli that are associated with milk products and are also found on human skin . Strains of Brevibacterium casei have been found to correspond to Centers for Disease Control coryneform groups B-1 and B-3 and have been isolated from a variety of human clinical specimens . In this report, we describe a case of B . casei bacteremia and sepsis in a patient with AIDS associated with a contaminated Hickmann catheter and review the microbiology and characteristics of these emerging opportunistic pathogens. J Gen Appl Microbiol, 2000 Aug, 46(4), 217 - 224 Screening of microbes, isolation, genetic manipulation, and physiological optimization of Brevibacterium helvolum to produce and excrete thymidine and deoxyuridine in high concentrations; Kalirai SK et al.; Analogues of deoxypyrimidines are used in the treatment of a variety of human ailments . Azidothymidine, or AZT, is one such analogue used to treat AIDS . Thymidine is the precursor of AZT, and its cost contributes to the high price of AZT . Attempts are being made to isolate and genetically manipulate microbes that can produce and excrete this compound in high concentrations . To this end, 145 different microbial species from Zeneca and the American Type Culture Collection were screened . Moreover, soil samples were collected from 36 different sites in England, and microbes from these samples were isolated and screened . >From approximately 25,000 isolates screened as single colonies and from 4,000 in liquid cultures, a strain of Brevibacterium helvolum showed the most promising results . Pyrimidine metabolic pathways of this bacterium were worked out, the isolate was genetically manipulated, and physiological conditions were optimized to increase the production of thymidine and deoxyuridine . These mutants of B . helvolum are considered to be of commercial importance. FEMS Microbiol Lett, 2002 Oct 29, 216(1), 77 - 84 The Brevibacterium flavum sigma factor SigB has a role in the environmental stress response; Halgasova N et al.; We have previously cloned a gene encoding a SigB, a principal-like sigma factor in Brevibacterium flavum, which was induced by several stress conditions . To clarify the in vivo function of this sigma factor, the sigB gene was disrupted by a homologous recombination, replacing the internal essential coding region in B . flavum chromosome by a kanamycin resistance marker gene . This mutation dramatically decreased vegetative growth rates of B . flavum . Studies of the effect of the sigB mutation on growth and viability of the cells under conditions of stress showed that the sigB mutant had increased susceptibility to acid, salt, alcohol, heat and cold stress . The plasmid-born wild-type sigB gene complemented the mutation . Based on the results, we propose that SigB has a role in vegetative growth and in response to various environmental stresses. Haematologia (Budap), 2002, 32(2), 151 - 3 Bacteremia caused by Brevibacterium species in a patient with chronic lymphocytic leukemia; Ogunc D et al.; We report a case of bacteremia caused by Brevibacterium species which is one of the coryneform bacteria, in a patient with chronic lymphocytic leukemia . We conclude that, if a coryneform bacteria is isolated from sterile body sites, it must be carefully evaluated, and especially in immunocompromised patients, Brevibacterium species should be considered as potential pathogens. J Infect Dis, 2002 Nov 1, 186(9), 1261 - 9 Epub 2002 Oct 03. A nontoxic chimeric enterotoxin adjuvant induces protective immunity in both mucosal and systemic compartments with reduced IgE antibodies; Kweon MN et al.; A novel nontoxic form of chimeric mucosal adjuvant that combines the A subunit of mutant cholera toxin E112K with the pentameric B subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli was constructed by use of the Brevibacillus choshinensis expression system (mCTA/LTB) . Nasal immunization of mice with tetanus toxoid (TT) plus mCTA/LTB elicited significant TT-specific immunoglobulin A responses in mucosal compartments and induced high serum immunoglobulin G and immunoglobulin A anti-TT antibody responses . Although TT plus native CT induced high total and TT-specific immunoglobulin E responses, use of the chimera molecule as mucosal adjuvant did not . Furthermore, all mice immunized with TT plus mCTA/LTB were protected from lethal systemic challenge with tetanus toxin . Importantly, the mice were completely protected from influenza virus infection after nasal immunization with inactivated influenza vaccine together with mCTA/LTB . These results show that B . choshinensis-derived mCTA/LTB is an effective and safe mucosal adjuvant for the induction of protective immunity against potent bacterial exotoxin and influenza virus infection. FEMS Microbiol Lett, 2002 Oct 8, 215(2), 243 - 8 Purification and properties of a new Brevibacterium sterolicum cholesterol oxidase produced by E . coli MM294/pnH10; Fujishiro K et al.; A gene encoding a cholesterol oxidase from Brevibacterium sterolicum nov . sp . ATCC21387 was isolated by an expression cloning method and highly expressed by a recombinant strain Escherichia coli MM294/pnH10 . The purified cholesterol oxidase was a typical flavoprotein with a molecular mass of 46.5 kDa, absorption peaks at 280, 360, and 450 nm . Optimum pH and temperature were found at pH 6.5 and 55 degrees C, respectively . The enzyme acted on 3beta-hydroxysteroids such as cholesterol, pregnenolone, and beta-sitosterol at high rates, but on dehydro-epi-androsterone to a lesser degree . The molecular and catalytic properties were different from those of cholesterol oxidase I, which was initially discovered in B . sterolicum nov . sp . ATCC21387 . The new enzyme, designated cholesterol oxidase II, was distinguished by its high affinity toward cholesterol (K(m)=30 microM). Sheng Wu Gong Cheng Xue Bao, 2002 Jul, 18(4), 438 - 41 {The construction of shuttle vectors of Brevibacillus brevis-Escherichia coli}; Peng QZ et al.; The 5' region of the cell wall protein(CWP) gene containing multiple tandem promoters and the signal peptide-coding sequence was isolated by PCR from Br . brevis 50, and used to construct the shuttle vector pBKE50, which included the replication origin of pUB110 and the erythromycin-resistance gene of pGK12 . The alpha-amylase gene of Bacillus subtilis 168 was ligated to pBKE50, producing plasmid pBKE50/alpha-amy . After the resulting plasmid was introduced into Br . brevis 50, soluble and biologically active alpha-amylase was secreted directly into the culture medium . The expression level of alpha-amylase in the recombinant Br . brevis 50 was twice higher than that of the donor strain. Vaccine, 2002 Oct 4, 20(29-30), 3543 - 50 Influence of adjuvant-active peptidoglycan monomer on specific T cell responses in mice; Halassy Spoljar B et al.; Peptidoglycan monomer (PGM) originating from Brevibacterium divaricatum is a non-toxic, non-pyrogenic, water-soluble immunostimulator . It potentiates humoral immune response to ovalbumin (OVA) in mice upregulating both immunoglobulin (IgG) 1 and IgG2a antibody subclasses . This study concerns the influence of PGM on T cell activation and cytokine networks in response to OVA . OVA-specific proliferative response as well as interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) secretion in lymph node cell cultures of immunised mice were studied . Due to pharmacokinetic properties of PGM, namely its fast metabolism and excretion, special emphasis was on choosing the appropriate time for lymph node removal and duration of cell cultivation for each cytokine . PGM treatment in addition to OVA resulted in an increase of lymph node cellularity, stimulation of OVA-specific IFN-gamma and IL-4 production as well as of OVA-specific proliferative response . Results demonstrate that PGM stimulated both Th1 and Th2 subpopulations. Bioorg Khim, 2002 Jul-Aug, 28(4), 298 - 302 {Isolation, biological properties, and spatial structure of an antibiotic loloatin A}; Krachkovskii SA et al.; Peptide antibiotic with cyanolytic activity was isolated from the IGM52 strain of the Brevibacillus laterosporus Gram-positive spore-forming bacteria . By 1H NMR spectroscopy, this antibiotic was identified as loloatin A, a cyclic decapeptide cyclo(-Asn-Asp-Tyr-Val-Orn-Leu-DTyr-Pro-Phe-DPhe-) . The spatial structure of loloatin A in solution was determined . The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol . 28, no . 4; see also http://www.maik.ru. J Food Prot, 2002 Aug, 65(8), 1309 - 16 Apparent antifungal activity of several lactic acid bacteria against Penicillium discolor is due to acetic acid in the medium; Cabo ML et al.; Fifty-six dairy bacteria belonging to the genera Lactococcus, Lactobacillus, Pediococcus, Propionibacterium, Streptococcus, Enterococcus, Leuconostoc, and Brevibacterium were screened for antifungal activity against four species of fungi relevant to the cheese industry (Penicillium discolor, Penicillium commune, Penicillium roqueforti, and Aspergillus vesicolor) . Most of the active strains belonged to the genus Lactobacillus, whereas Penicillium discolor was found to be the most sensitive of the four fungi investigated . Further studies on P . discolor showed antifungal activity only below pH 5 . This effect of pH suggests that organic acids present in the culture could be involved in the detected activity . Determination of acid composition revealed lactic acid production for active dairy strains and the presence of acetic acid in active as well as inactive strains . It was demonstrated that the undissociated acetic aci