Brevibacteria

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 acid originates from the bacterial growth medium . The synergistic effect of the acetic acid present and the lactic acid produced was likely the main factor responsible for the antifungal properties of the selected bacteria . These results could explain some discrepancies in reports of the antifungal properties of lactic acid bacteria, since the role of acetic acid has not been considered in previous studies.

FEMS Microbiol Lett, 2002 Aug 6, 213(2), 205 - 11
Evaluation of methods for molecular typing and identification of members of the genus Brevibacterium and other related species; Alves A et al.; The genus Brevibacterium includes pleomorphic Gram-positive bacteria with a high mol% G+C content . Species in the genus are difficult to identify by classical methods . The discriminatory power of DNA-based methods is assessed . Strains representing the four well established Brevibacterium species, and other related bacteria, were compared by amplified ribosomal DNA restriction analysis (ARDRA), repetitive-sequence-based PCR (rep-PCR) and ribotyping . Fingerprinting by rep-PCR and ribotyping provided complex genomic profiles with the highest discriminatory potential for molecular typing at the strain level, whereas ARDRA showed differentiation from the genus to the species levels . A high degree of heterogeneity within the genus Brevibacterium is apparent, thus indicating that the taxonomy of the genus should be further studied.

Int J Food Microbiol, 2002 Aug 25, 77(3), 175 - 86
Diversity and functionality of Bacillus and related genera isolated from spontaneously fermented soybeans (Indian Kinema) and locust beans (African Soumbala); Sarkar PK et al.; A total of 126 isolates of Bacillus and related genera from indigenous, spontaneously fermented soybeans (Kinema) and locust beans (Soumbala) were characterized with the purpose of defining interspecific, as well as intraspecific relationships among the components of their microflora . B . subtilis was the dominant species, and species diversity was more pronounced in Soumbala than in Kinema . While from Kinema, six species were isolated (B . subtilis, B . licheniformis, B . cereus, B . circulans, B . thuringiensis and B . sphaericus), in Soumbala, the species found were B . subtilis, B . thuringiensis, B . licheniformis, B . cereu, B . badius, Paenibacillus alvei, B . firmus, P . larvae, Brevibacillus laterosporus, B . megaterium, B . mycoides and B . sphaericus . Genomic diversity in the isolates of B . subtilis was investigated by random amplified polymorphic DNA (RAPD) analysis using the polymerase chain reaction (PCR) . The RAPD-PCR fingerprint analysis showed a high level of diversity . With more than 90% similarity, all 52 RAPD subdivisions were source and continent-wise homogeneous . Profiles of carbon source fermentation also showed a wide but corresponding phenotypic diversity, largely corresponding with RAPD subdivisions . The various strains were tested for several criteria for functionality in soybean fermentation, viz . protein degradation, pH increase, and development of desirable stickiness caused by viscous exopolymers . Profiles of functionality, based upon estimations of pH, free amino nitrogen and stickiness were associated with genotypic and phenotypic profiles . Notwithstanding the heterogenous fermentation results for some genotypic profiles, a ranking of RAPD groups is possible and can be useful in the further selection and study of B . subtilis strains.

Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(2), 235 - 8
{Study on the kinetics of immobilized cells of Brevibacterium ammoniagenes MA-2 and Brevibacterium flavum MA-3}; Hu YH et al.; The kinetics of immobilized cells of Brevibacterium ammoniagenes MA-2 and Brevibacterium flavum MA-3 cells were studied . By means of both a theoretical analysis of diffusion in the gel particles and an experimental determination of apparent kinetic parameters, the intrinsic kinetic parameters of immobilized cells of B . ammoniagenes MA-2 and B . flavum MA-3 cells were obtained.

Appl Environ Microbiol, 2002 Jul, 68(7), 3655 - 60
Differentiation of Paenibacillus larvae subsp . larvae, the cause of American foulbrood of honeybees, by using PCR and restriction fragment analysis of genes encoding 16S rRNA; Alippi AM et al.; A rapid procedure for the identification of Paenibacillus larvae subsp . larvae, the causal agent of American foulbrood (AFB) disease of honeybees (Apis mellifera L.), based on PCR and restriction fragment analysis of the 16S rRNA genes (rDNA) is described . Eighty-six bacterial strains belonging to 39 species of the genera Paenibacillus, Bacillus, Brevibacillus, and Virgibacillus were characterized . Amplified rDNA was digested with seven restriction endonucleases . The combined data from restriction analysis enabled us to distinguish 35 profiles . Cluster analysis revealed that P . larvae subsp . larvae and Paenibacillus larvae subsp . pulvifaciens formed a group with about 90% similarity; however, the P . larvae subsp . larvae restriction fragment length polymorphism pattern produced by endonuclease HaeIII was found to be unique and distinguishable among other closely related bacteria . This pattern was associated with DNA extracted directly from honeybee brood samples showing positive AFB clinical signs that yielded the restriction profile characteristic of P . larvae subsp . larvae, while no amplification product was obtained from healthy larvae . The method described here is particularly useful because of the short time required to carry it out and because it allows the differentiation of P . larvae subsp . larvae-infected larvae from all other species found in apiarian sources.

Ann Agric Environ Med, 2002, 9(1), 85 - 90
Exposure to airborne microorganisms in furniture factories; Krysinska-Traczyk E et al.; Microbiological air sampling was performed in 2 furniture factories located in eastern Poland . In one factory furniture were made from fibreboards and chipboards while in the other from beech wood . It was found that the concentration of total microorganisms (bacteria + fungi) in the air of the facility using beech wood for furniture production (mean 10.7 x (3) cfu/m(3), range 3.3 27.5 x (3) cfu/m(3)) was significantly higher (p < 0.01) compared to microbial concentration in the facility using fibre- and chipboards (mean 3.6 x (3) cfu/m(3), range 1.9-6.2 x (3) cfu/m(3)) . On average, the commonest microorganisms in the air of the furniture factories were corynebacteria (Corynebacterium spp., Arthrobacter spp., Brevibacterium spp.) which formed 18.1-50.0% of the total airborne microflora, and fungi (mostly Aspergillus spp., Penicillium spp., Absidia spp . and yeasts) which formed 6.2-54.4% of the total count . The values of the respirable fraction of airborne microflora in the furniture factories varied within fairly wide limits and were between 15.0-62.4% . Altogether, 28 species or genera of bacteria and 12 species or genera of fungi were identified in the air of examined factories, of which respectively 8 and 7 species or genera were reported as having allergenic and/or immunotoxic properties . In conclusion, the workers of furniture factories are exposed to relatively low concentrations of airborne microorganisms which do not exceed the suggested occupational exposure limits . Nevertheless, the presence of allergenic and/or immunotoxic microbial species in the air of factories poses a potential risk of respiratory disease, in particular in sensitive workers.

Clin Infect Dis, 2002 Jul 15, 35(2), e20 - 1 Epub 2002 Jun 14.
Brevibacterium endocarditis: a first report; Dass KN et al.; There are few case reports of infections caused by Brevibacterium species, and there have been no previously reported cases of endocarditis caused by any of the 6 known species of Brevibacterium . We report the first case of Brevibacterium endocarditis (caused by Brevibacterium otitidis) in a patient with prosthetic heart valves . The patient responded to 6 weeks of treatment with vancomycin and 2 weeks with gentamicin, and she has been receiving long-term maintenance therapy with oral azithromycin.

Protein Eng, 2002 Jun, 15(6), 477 - 84
Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp . by site-directed mutagenesis; Toyama M et al.; Despite the structural similarities between cholesterol oxidase from Streptomyces and that from Brevibacterium, both enzymes exhibit different characteristics, such as catalytic activity, optimum pH and temperature . In attempts to define the molecular basis of differences in catalytic activity or stability, substitutions at six amino acid residues were introduced into cholesterol oxidase using site-directed mutagenesis of its gene . The amino acid substitutions chosen were based on structural comparisons of cholesterol oxidases from Streptomyces and BREVIBACTERIUM: Seven mutant enzymes were constructed with the following amino acid substitutions: L117P, L119A, L119F, V145Q, Q286R, P357N and S379T . All the mutant enzymes exhibited activity with the exception of that with the L117P mutation . The resulting V145Q mutant enzyme has low activities for all substrates examined and the S379T mutant enzyme showed markedly altered substrate specificity compared with the wild-type enzyme . To evaluate the role of V145 and S379 residues in the reaction, mutants with two additional substitutions in V145 and four in S379 were constructed . The mutant enzymes created by the replacement of V145 by Asp and Glu had much lower catalytic efficiency for cholesterol and pregnenolone as substrates than the wild-type enzyme . From previous studies and this study, the V145 residue seems to be important for the stability and substrate binding of the cholesterol oxidase . In contrast, the catalytic efficiencies (k(cat)/K(m)) of the S379T mutant enzyme for cholesterol and pregnenolone were 1.8- and 6.0-fold higher, respectively, than those of the wild-type enzyme . The enhanced catalytic efficiency of the S379T mutant enzyme for pregnenolone was due to a slightly high k(cat) value and a low K(m) value . These findings will provide several ideas for the design of more powerful enzymes that can be applied to clinical determination of serum cholesterol levels and as sterol probes.

Biotechnol Appl Biochem, 2002 Jun, 35(Pt 3), 191 - 7
Determination of total cholesterol in serum by cholesterol esterase and cholesterol oxidase immobilized and co-immobilized on to arylamine glass; Malik V et al.; Commercial cholesterol esterase from bovine pancreas and cholesterol oxidase from Brevibacterium recombinant type have been immobilized individually and co-immobilized on to arylamine glass beads (pore diameter, 55 nm) through diazotization . A method for discrete analysis of total cholesterol in serum was developed employing individually immobilized cholesterol esterase (0.36 mg/50 mg of glass beads) and cholesterol oxidase (0.41 mg/50 mg of glass beads) or co-immobilized cholesterol esterase and cholesterol oxidase (0.56 mg/100 mg of glass beads) . Peroxidase from horseradish immobilized on to arylamine glass (0.9 mg/50 mg of glass beads) was common in both the cases . 4-Aminophenazone (0.25 mg/1.5 ml of reaction mixture) and phenol (0.5 mg/1.5 ml of reaction mixture) were used to form dye . In the method, cholesterol ester is hydrolysed by cholesterol esterase to free fatty acid and cholesterol, which is oxidized by cholesterol oxidase to cholestenone and H(2)O(2) . H(2)O(2) is determined enzymically with horseradish peroxidase by additive coupling of 4-aminophenazone with phenol, and the resulting quinoneimine dye is measured at 520 nm, (epsilon=4.0x10(-4)) . The lower detection limit of the method was 42.8 mg/l for individually immobilized enzymes and 21.4 mg/l for co-immobilized enzymes . Within-day and between-day coefficients of variation were <1.5% and <4.0% respectively for individually immobilized enzymes and <1.0% and <2.5% respectively for co-immobilized enzymes . A good correlation (r=0.99) was found between total serum cholesterol obtained by the present method and a commercial enzo-kit method employing free enzymes . The individually immobilized/co-immobilized enzymes did not show much loss of activity after their 300 uses, when stored at 4 degrees C in distilled water . The co-immobilized enzymes showed better efficiency in terms of sensitivity, linearity and precision compared with individually immobilized enzymes.

Mediators Inflamm, 2002 Apr, 11(2), 129 - 35
Augmentation of NKT and NK cell-mediated cytotoxicity by peptidoglycan monomer linked with zinc; Mrakovcic-Sutic I et al.; BACKGROUND: Peptidoglycan monomer (PGM), which was originally prepared by biosynthesis from culture fluids of penicillin-treated Brevibacterium divaricatum, is an immunostimulator, the activities of which might be improved by addition of zinc (Zn) to the basic molecule . METHODS: To test the possible cytotoxic effects of this new analogue, we analyzed the ability of PGM-Zn and PGM to change the phenotypic profile of hepatic and splenic mononuclear lymphatic cells and to affect the growth of malignant T-cell line YAC-1 and syngeneic thymocytes . RESULTS: Pretreatment of C57BL/6 mice primarily with PGM-Zn over 6 days (10/mg/kg intraperitoneally) significantly enhanced the proportions of NK1.1high+, CD4-CD8-, CD69+, and CD3intermediate/NK1.1+/IL2R-beta+ (NKT) cells in the liver, and major histocompatibility complex class II+, CD69+, and CD8+ cells in the spleen . Both types of cells were highly cytotoxic against YAC-1 and syngeneic thymocytes, increasing the destruction of YAC-1 by 70% on addition of hepatic cells and by 30% on addition of splenic cells . Destruction of thymocytes increased by 10 and 50%, respectively . CONCLUSION: The results point to PGM-Zn as a potent cytotoxicity-inducing agent, which also generates autoreactive NKT cells.

J Agric Food Chem, 2002 Jun 19, 50(13), 3810 - 7
Optimization of headspace solid-phase Microextraction (SPME) for the odor analysis of surface-ripened cheese; Lecanu L et al.; Fifty volatile compounds of surface smear-ripened cheese were detected and identified using headspace solid-phase microextraction (HS-SPME) and vacuum distillation coupled to gas chromatography-mass spectrometry . Changes in the headspace of aroma compounds were monitored over the whole packaging period (47 days) using the HS-SPME method . Initially, the concentration of methanethiol increased before reaching a plateau . This evolution could be linked to the growth of Brevibacterium linens . During the shelf life of cheese, levels of acetic acid and 3-methylbutanoic acid remained constant, whereas butane-2,3-dione, 3-hydroxybutan-2-one, and hydroxypropan-2-one levels gradually declined and acetone and 3-methylbutanol levels dropped sharply to a plateau . Changes in odor could be related to changes of the rind, which behaved as a barrier, strongly influencing the distribution of volatile compounds in the headspace . Using a gas chromatography-olfactometry technique without separation, it was shown that the SPME extract was representative of the cheese odor.

Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 953 - 66
Polyphasic identification of Bacillus and Brevibacillus strains from clinical, dairy and industrial specimens and proposal of Brevibacillus invocatus sp . nov.; Logan NA et al.; Thirty-three clinical, dairy and industrial isolates of aerobic endospore-forming bacteria which were unreactive in routine identification tests were characterized genotypically by using amplified rDNA restriction analysis (ARDRA), 16S rDNA sequencing and DNA-DNA reassociation, and phenotypically by using fatty acid methyl ester (FAME) analysis, SDS-PAGE of whole-cell proteins, API Biotype 100 assimilation tests and 16 other routine phenotypic tests . Three isolates were identified as strains of Bacillus badius, 12 as Brevibacillus agri, including 3 strains associated with an outbreak of waterborne illness, 4 as Brevibacillus centrosporus and 2 as Brevibacillus parabrevis; 12 strains contaminating an antibiotic production plant were recognized as members of a new species, for which the name Brevibacillus invocatus is proposed, with the type strain LMG 18962T (= B2156T = CIP 106911T = NCIMB 13772T).

Int J Food Microbiol, 2002 Jun 25, 76(3), 231 - 40
Peptidolytic, esterolytic and amino acid catabolic activities of selected bacterial strains from the surface of smear cheese; Curtin AC et al.; Enzymes produced by bacteria present on the surface of smear cheeses play essential roles in flavour development during cheese ripening . In this study, strains including brevibacteria, corynebacteria, staphylococci and brachybacteria, from the surface of two smear cheese (Tilsit and Gubeen) were screened for a range of enzyme activities including aminopeptidase (substrates: Leu-pNA and His-pNA), dipeptidase (Met-Ala, Ala-Met, Pro-Ala, His-Leu and Pro-Leu), tripeptidase (Phe-Gly-Gly, Gly-Gly-Gly and Leu-Ala- Pro), esterase (beta-naphthyl butyrate, beta-naphthyl caprate and beta-naphthyl palmitate) . L-methionine aminotransferase and cystathionine lyase activities . There were marked differences in the activities observed between different bacteria studied . Brachybacteria showed low activity on all substrates assayed . There was no consistency in activities within groups of related bacteria . For example, Staphylococcus equorum 14 showed higher activity than S . equorum 6 on all the substrates tested . Among the corynebacteria, Coryebacterium ammoniagenes CA8 had greatest aminopeptidase, esterase and cystathionine lyase activity while C . casei B showed more di- and tri-peptidase activity . It was noted that individual bacteria displayed similar activities on all three esterase substrates, i.e., the chain length of the fatty acid did not appear to affect activity . L-Methionine aminotransferase activity was observed in only one strain (S . equorm 14) whereas all strains had cystathionine lyase activity.

Microbiology, 2002 May, 148(Pt 5), 1523 - 32
Intraspecific diversity of Brevibacterium linens, Corynebacterium glutamicum and Rhodococcus erythropolis based on partial 16S rDNA sequence analysis and Fourier-transform infrared (FT-IR) spectroscopy; Oberreuter H et al.; The intraspecific diversity of 31 strains of Brevibacterium linens, 27 strains of Corynebacterium glutamicum and 29 strains of Rhodococcus erythropolis was determined by partial 16S rDNA sequence analysis and Fourier-transform infrared (FT-IR) spectroscopy . As a prerequisite for the analyses, 27 strains derived from culture collections which had carried invalid or wrong species designations were reclassified in accordance with polyphasic taxonomical data . FT-IR spectroscopy proved to be a rapid and reliable method for screening for similar isolates and for identifying these actinomycetes at the species level . Two main conclusions emerged from the analyses . (1) Comparison of intraspecific 16S rDNA similarities suggested that R . erythropolis strains have a very low diversity, B . linens displays high diversity and C . glutamicum occupies an intermediate position . (2) No correlation of FT-IR spectral similarity and 16S rDNA sequence similarity below the species level (i.e . between strains of one species) was observed . Therefore, diversification of 16S rDNA sequences and microevolutionary change of the cellular components detected by FT-IR spectroscopy appear to be de-coupled.

Mikrobiol Z, 2002 Jan-Feb, 64(1), 66 - 76
{Serological affinity of some species of nonpathogenic corynebacteria}; Furtat IM et al.; Serological peculiarities of the species strains Corynebacterium glutamicum, C . ammoniagenes, C . vitaeruminis, C . variabilis and strain of Corynebacterium sp . (Brevibacterium stationis) UCM Ac-719 have been investigated with the help of immunoenzyme analysis ELISA with the use of mice immune serum, specific to C . ammoniagenes UCM Ac-732T, C . vitaeruminis UCM Ac-718T, C . variabilis UCM Ac-717T, C . glutamicum UCM Ac-733 and Corynebacterium sp . UCM Ac-719 . It has been established that the species of nonpathogenic corynebacteria differ between themselves as to the degree of serological affinity . C . variabilis, C . ammoniagenes and C . glutamicum are the least similar as to this indication . Weak antigenic relations have been revealed in C . vitaeruminis and C . ammoniagenes . The latter displayed the higher, as compared with other strains, affinity for Corynebacterium sp . UCM Ac-719 . The highest degree of serological affinity within the species was registered in strains C . glutamicum and C . variabilis . Data obtained evidence that the ELISA method permits conducting the high-reliability species diagnosis of nonpathogenic corynebacteria on the basis of their antigenic characteristics.

J Dairy Res, 2001 Nov, 68(4), 663 - 74
L-methionine degradation potentialities of cheese-ripening microorganisms; Bonnarme P et al.; Volatile sulphur compounds are major flavouring compounds in many traditional fermented foods including cheeses . These compounds are products of the catabolism of L-methionine by cheese-ripening microorganisms . The diversity of L-methionine degradation by such microorganisms, however, remains to be characterized . The objective of this work was to compare the capacities to produce volatile sulphur compounds by five yeasts, Geotrichum candidum, Yarrowia lipolytica, Kluyveromyces lactis, Debaryomyces hansenii, Saccharomyces cerevisiae and five bacteria, Brevibacterium linens, Corynebacterium glutamicum, Arthrobacter sp., Micrococcus lutens and Staphylococcus equorum of technological interest for cheese-ripening . The ability of whole cells of these microorganisms to generate volatile sulphur compounds from L-methionine was compared . The microorganisms produced a wide spectrum of sulphur compounds including methanethiol, dimethylsulfide, dimethyldisulfide, dimethyltrisulfide and also S-methylthioesters, which varied in amount and type according to strain . Most of the yeasts produced methanethiol, dimethylsulfide, dimethyldisulfide and dimethyltrisulfide but did not produce S-methylthioesters, apart from G . candidum that produced S-methyl thioacetate . Bacteria, especially Arth . sp . and Brevi . linens, produced the highest amounts and the greatest variety of volatile sulphur compounds includling methanethiol, sulfides and S-methylthioesters, e.g . S-methyl thioacetate, S-methyl thiobutyrate, S-methyl thiopropionate and S-methyl thioisovalerate . Cell-free extracts of all the yeasts and bacteria were examined for the activity of enzymes possibly involved in L-methionine catabolism, i.e . L-methionine demethiolase, L-methionine aminotransferase and L-methionine deaminase . They all possessed L-methionine demethiolase activity, while some (K . lactis, Deb . hansenii, Arth . sp., Staph . equorum) were deficient in L-methionine aminotransferase, and none produced L-methionine deaminase . The catabolism of L-methionine in these microorganisms is discussed.

Huan Jing Ke Xue, 2001 Nov, 22(6), 83 - 5
{Biodegradation of polycyclic aromatic hydrocarbons by a preponderant brevibacterium}; Nie M et al.; A preponderant brevibacterium was isolated from the sludge contaminated by coke-plantwaste water for a long time . The bacterium was effective on the degradation and transformation of anthracene, phenanthrene and pyrene . The degradation process can be accelerated by ferric ions . 10 hours after aeration, the removing rates of anthracene, phenanthrene and pyrene by the brevibacterium were about 75%, 87% and 62% respectively . 106 hours after aeration, the removing rates of total organic carbon (TOC) caused by anthracene, phenanthrene and pyrene were 50%, 90%, and 50% respectively.

J Appl Microbiol, 2002, 92(1), 63 - 70
Characterization of an antibacterial peptide produced by Brevibacterium linens; Motta AS et al.; AIMS: The aim of this research was to investigate the antimicrobial activity produced by Brevibacterium linens ATCC 9175 . METHODS AND RESULTS: A bacteriocin produced by the red smear cheese bacterium B . linens ATCC 9175 was identified . The antimicrobial activity was first produced at the exponential growth phase . A crude bacteriocin obtained from the culture supernatant fluid was inhibitory to some indicator strains . It inhibited the growth of Listeria monocytogenes ATCC 7644, B . linens ATCC 9172 and Corynebacterium fimi NCTC 7547, but was inactive against the Gram-negative bacteria and yeast tested . The bacteriocin was stable at 30 degrees C but the activity was lost when the temperature reached 50 degrees C . It was sensitive to the proteolytic action of trypsin, papain and pronase E and was active between pH 6.0 and 9.0 . The bacteriocin was bactericidal to L . monocytogenes at 40 AU ml(-1) . Bacteriostasis was observed for a low dose of bacteriocin (20 AU ml(-1)) . CONCLUSIONS: An antibacterial peptide produced by B . linens was characterized, presenting potential for use as a biopreservative in food systems . SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of a novel bacteriocin active against L . monocytogenes addresses an important aspect of food protection against pathogens and spoilage micro-organisms.

J Am Soc Mass Spectrom, 2002 Feb, 13(2), 118 - 28
Flow-injection electrospray ionization mass spectrometry of crude cell extracts for high-throughput bacterial identification; Vaidyanathan S et al.; Flow-injection electrospray ionization mass spectrometry (FI-ESI-MS) of unfractionated cell-free extracts obtained from bacterial cells suspended in a solvent mixture was investigated as a rapid analytical method for reproducible, high-throughput bacterial identification . Five bacterial strains (two Escherichia coli, two Bacillus spp . and one Brevibacillus laterosporus) were studied in this investigation . Axenically grown bacterial cells were suspended in an acidic organic solvent and the cell-free extract was sequentially injected into a solvent flow stream that was sprayed into the ionization chamber of the ESI-MS . The spectra produced contained reproducible information, which was useful for discriminating between the bacteria . Tandem mass spectrometry was used to characterize further the peaks, and at least three classes of macromolecules, namely phospholipids, glycolipids, and proteins, were found to contribute most to the spectral information . Bacterial extracts stored under different conditions gave very similar mass spectra for each of the five bacterial strains, indicating that the extracts were stable even at room temperature for up to 24 h, with no loss of information content, which has obvious implications for automated high-throughput analysis . An analysis of the components of the extracting solvent mixture and their effects on the spectral information showed that acetonitrile contributes most significantly to the extraction process and hence to the information content of the spectra.

Appl Environ Microbiol, 2002 Feb, 68(2), 820 - 30
Biodiversity of the bacterial flora on the surface of a smear cheese; Brennan NM et al.; The bacteria on the surface of a farmhouse smear-ripened cheese at four stages of ripening (4, 16, 23, and 37 days) from inoculated (i.e., deliberately inoculated with Brevibacterium linens BL2) and noninoculated (not deliberately inoculated with B . linens BL2) cheese were investigated . The results show that, contrary to accepted belief, B . linens is not a significant member of the surface flora of smear cheese and no microbial succession of species occurred during the ripening of the cheeses . Of 400 isolates made, 390 were lactate-utilizing coryneforms and 10 were coagulase-negative Staphylococcus spp . A detailed analysis of the coryneforms was undertaken using phenotypic analysis, molecular fingerprinting, chemotaxonomic techniques, and 16S rRNA gene sequencing . DNA banding profiles (ramdom amplified polymorphic DNA {RAPD}-PCR) of all the coryneform isolates showed large numbers of clusters . However, pulsed-field gel electrophoresis (PFGE) of the isolates from the cheeses showed that all isolates within a cluster and in many contiguous clusters were the same . The inoculated and noninoculated cheeses were dominated by single clones of novel species of Corynebacterium casei (50.2% of isolates), Corynebacterium mooreparkense (26% of isolates), and Microbacterium gubbeenense (12.8% of isolates) . In addition, five of the isolates from the inoculated cheese were Corynebacterium flavescens . Thirty-seven strains were not identified but many had similar PFGE patterns, indicating that they were the same species . C . mooreparkense and C . casei grew at pH values below 4.9 in the presence of 8% NaCl, while M . gubbeenense did not grow below pH 5.8 in the presence of 5 to 10% NaCl . B . linens BL2 was not recovered from the inoculated cheese because it was inhibited by all the Staphylococcus isolates and many of the coryneforms . It was concluded that within a particular batch of cheese there was significant bacterial diversity in the microflora on the surface.

Arch Microbiol, 2001 Dec, 177(1), 91 - 7 Epub 2001 Oct 27.
Expression of the genes coding for the xylanase Xys1 and the cellulase Cel1 from the straw-decomposing Streptomyces halstedii JM8 cloned into the amino-acid producer Brevibacterium lactofermentum ATCC13869; Adham SA et al.; The xylanase ( xysA) and the cellulase ( celA1) genes from Streptomyces halstedii JM8 were cloned into Escherichia coli/ Brevibacterium lactofermentum shuttle vectors and successfully expressed in both hosts when placed downstream from the kanamycin resistance promoter (Pkan) from Tn 5 but not when under the control of their own promoters . Xylanase was secreted into the culture media of B . lactofermentum by removal of the same leader peptide as is removed in S . halstedii . The main difference between the production of xylanase by Streptomyces and corynebacteria was the low level of processing of the mature extracellular xylanase by B . lactofermentum, probably due to the lack of protease activity in this microorganism.

J Dairy Sci, 2001 Nov, 84(11), 2419 - 23
Phylogenetic studies on Corynebacterium bovis isolated from bovine mammary glands; Watts JL et al.; Coryneform bacteria are frequently isolated from bovine mastitis with the lipophilic species, and Corynebacterium bovis is the most frequently isolated organism of this group . However, previous studies on the phylogeny of corynebacteria have incorporated only a single reference strain . We examined the phylogeny of C . bovis using 47 strains isolated from bovine mammary glands . Phylogenetic studies were performed by direct sequencing of the 16S ribosomal RNA and comparison to sequences of reference strains . All strains identified as C . bovis demonstrated similarity of 98% or higher to the ribosomal RNA gene sequences of the type strain of C . bovis . Phylogenetic analyses indicated that all strains tested clustered with members of the Corynebacterium urealyticum group confirming that C . bovis is a legitmate member of the genus Corynebacterium . Further investigation into the diversity within the species using repetitive element palindrome PCR indicated only minor differences between the strains tested . Corynebacterium bovis ATCC 13722 demonstrated the highest similarity (95%) with Brevibacterium helvolum, indicating that this organism does not belong in the genus Corynebacterium.

Appl Microbiol Biotechnol, 2001 Nov, 57(4), 534 - 40
H+-ATPase defect in Corynebacterium glutamicum abolishes glutamic acid production with enhancement of glucose consumption rate; Sekine H et al.; A mutant of Corynebacterim glutamicum ('Brevibacterium flayum') ATCC14067 with a reduced H+-ATPase activity, F172-8, was obtained as a spontaneous neomycin-resistant mutant . The ATPase activity of strain F172-8 was reduced to about 25% of that of the parental strain . Strain F172-8 was cultured in a glutamic-acid fermentation medium containing 100 g/l of glucose using ajar fermentor . It was found that glucose consumption per cell during the exponential phase was higher by 70% in the mutant than in the parent . The respiration rate per cell of the mutant also increased to twice as much as that of the parent . However, the growth rate of the mutant was lower than that of the parent . Under those conditions, the parent produced more than 40 g/l glutamic acid, while the mutant hardly produced any glutamic acid . Instead the mutant produced 24.6 g/l lactic acid as the main metabolite of glucose . Remarkably, the accumulation of pyruvate and pyruvate-family amino acids, i.e., alanine and valine, was detected in the mutant . On the other hand, the parent accumulated alpha-ketoglutaric acid and a glutamate-family amino acid, proline, as major by-products . It was concluded that the decrease in the H+-ATPase activity caused the above-mentioned metabolic changes in strain F172-8, because a revertant of strain F172-8, R2-1, with a H+-ATPase activity of 70% of that of strain ATCC14067, showed a fermentation profile similar to that of the parent . Sequence analyses of the atp operon genes of these strains identified one point mutation in the gamma subunit in strain F172-8.

Appl Environ Microbiol, 2001 Dec, 67(12), 5425 - 30
Construction of a xylanase-producing strain of Brevibacterium lactofermentum by stable integration of an engineered xysA gene from Streptomyces halstedii JM8; Adham SA et al.; A xylanolytic strain of Brevibacterium lactofermentum containing the Streptomyces halstedii His-tagged xysA gene was generated . The new strain contains DNA derived from S . halstedii, expresses xylanolytic activity, and was obtained by an integrative process mediated by a conjugative plasmid targeted to a dispensable chromosomal region located downstream from the essential cell division gene ftsZ . The His-tagged Xys1 enzyme was constitutively expressed under the control of the kan promoter from Tn5 and was easily purified by use of Ni-nitrilotriacetic acid-agarose . The new strain is stable for more than 200 generations, lacks any known antibiotic resistance gene, and does not need any selective pressure to maintain the integrated gene . This strategy can be used to integrate any gene into the B . lactofermentum chromosome and to maintain it stably without the use of antibiotics for selection.

J Dairy Sci, 2001 Oct, 84(10), 2125 - 35
Aroma compound production in cheese curd by coculturing with selected yeast and bacteria; Martin N et al.; The microorganisms involved in cheese ripening produce various volatile compounds and induce typical flavors that contribute to cheese variety . To investigate aroma compound generation of cheese microflora, we used a dynamic headspace-gas chromatography-mass spectrometry analysis . To obtain good sensitivity and repeatability of quantification, dynamic headspace conditions and sample preparation were first optimized and led to an extraction set up in which samples were heated at 60 degrees C and diluted with water without pH adjustment . Then three different yeasts and three Geotrichum candidum commonly used in mold surface ripened cheeses were studied in pure culture in a cheese model medium . Thirty-nine cocultures of these three yeasts, the three G . candidum, and five bacteria were studied in the same medium to assess the interaction between microorganisms on aroma compound production . Twenty-four volatile compounds belonging to different chemical classes (alcohols, aldehydes, esters, sulfides, terpenes) were identified and quantified . Yeasts and especially Kluyveromyces lactis produced large amounts of alcohols, aldehydes, esters, and terpenes when cultured alone or in association . Geotrichum candidum and especially G . candidum strain G3 generated the largest amount of sulfides when cultured alone or in association . Finally, bacteria also produced aroma compounds but, except for Brevibacterium linens strain B5, which produced dimethyl trisulfide and ketones, no specific trend in the production of particular aroma compounds could be evidenced.

J Dairy Sci, 2001 Oct, 84(10), 2117 - 24
Influence of adjunct cultures on ripening of reduced fat Edam cheeses; Tungjaroenchai W et al.; The influence of four adjunct cultures {Brevibacterium linens (BL2), Lactococcus lactis ssp . diacetylactis, Lactobacillus helveticus (LH212), and Lactobacillus reuteri (ATCC 23272)} on chemical and sensory characteristics of reduced fat Edam cheese was studied . The aminopeptidase activity of Lactococcus lactis ssp . diacetylactis was higher than that of Lactobacillus helveticus, Lactobacillus reuteri, and Brevibacterium linens, respectively . Mean percent fat and moisture contents of reduced fat cheese were 20.85 +/- 0.69 and 42.95 +/- 0.43, respectively . Percentage of fat and moisture of full fat control cheese were 30.06 +/- 0.78 and 39.11 +/- 0.60 . Titratable acidity increased in all cheese with aging while pH initially decreased but increased in cheese after 6 mo aging at 7 degrees C . Lactic acid bacteria counts were on average one log higher for reduced fat cheeses than for full fat control cheese and counts decreasing with aging . Free amino acids (FAA) in cheeses increased with aging, and were higher in reduced fat cheeses than in the full fat control cheese . Reduced fat cheeses containing L . helveticus exhibited the highest FAA content . Descriptive sensory panelists (n = 9) did not detect differences among cheeses after 3 and 6 mo ripening, but aged/developed flavors (fruity, nutty, brothy, sulfur, free fatty acid) and sweetness increased between 3 and 6 mo . Expert panelists (n = 6) detected differences in texture quality among the cheeses . Reduced fat control cheeses and reduced fat cheeses with L . helveticus and L . reuteri received the highest texture quality scores . Addition of L . helveticus and Lc . lactis ssp . diacetylactis, as adjunct cultures to reduced fat Edam cheeses increased proteolysis, while the addition of L . helveticus and L . reuteri increased texture quality of cheeses.

Curr Microbiol, 2001 Oct, 43(4), 249 - 54
Cloning and transcriptional characterization of two sigma factor genes, sigA and sigB, from Brevibacterium flavum; Halgasova N et al.; Using a DNA fragment containing the principal sigma factor gene hrdB of Streptomyces aureofaciens, we identified two sigma70-like genes in a library of Brevibacterium flavum . Sequence analysis of the complete genes revealed two ORFs coding for gene products of 498 and 331 amino acid residues, which showed the greatest similarity to SigA and SigB sigma factors from Brevibacterium lactofermentum . We designated them similarly sigA and sigB . Transcription of B . flavum sigA and sigB has been investigated by S1-nuclease mapping by using RNA from different growth phases and after exposure to several stress conditions . Both genes are transcribed from a single promoter with transcription start points of 368 bp and 25 bp upstream from the proposed translation initiation codon of the sigA and sigB genes, respectively . Whereas sigA is transcribed almost constitutively during growth and after stress conditions, expression of sigB is significantly induced after several stress conditions, like acid stress, ethanol shock, and cold shock . Expression of both genes is significantly reduced after heat shock . Considering these transcriptional results, and also on the basis of the similarity to other principal sigma factor genes, sigA probably encodes the functional principal sigma factor, and sigB might have a function in stress response.

Inorg Chem, 1999 Aug 9, 38(16), 3676 - 3683
Spectroscopic Investigation of Reduced Protocatechuate 3,4-Dioxygenase: Charge-Induced Alterations in the Active Site Iron Coordination Environment; Davis MI et al.; Chemical reduction of the mononuclear ferric active site in the bacterial intradiol cleaving catecholic dioxygenase protocatechuate 3,4-dioxygenase (3,4-PCD, Brevibacterium fuscum) produces a high-spin ferrous center . We have applied circular dichroism (CD), magnetic circular dichroism (MCD), variable-temperature-variable-field (VTVH) MCD, X-ray absorption (XAS) pre-edge, and extended X-ray absorption fine structure (EXAFS) spectroscopies to investigate the geometric and electronic structure of the reduced iron center . Excited-state ligand field CD and MCD data indicate that the site is six-coordinate where the (5)E(g) excited-state splitting is 2033 cm(-)(1) . VTVH MCD analysis of the ground state indicates that the site has negative zero-field splitting with a small rhombic splitting of the lowest doublet (delta = 1.6 +/- 0.3 cm(-)(1)) . XAS pre-edge analysis also indicates a six-coordinate site while EXAFS analysis provides accurate bond lengths . Since previous spectroscopic analysis and the crystal structure of oxidized 3,4-PCD indicate a five-coordinate ferric active site, the results presented here show that the coordination number increases upon reduction . This is attributed to the coordination of a second solvent ligand . The coordination number increase relative to the oxidized site also appears to be associated with a large decrease in the ligand donor strength in the reduced enzyme due to protonation of the original hydroxide ligand.

Int J Syst Evol Microbiol, 2001 Sep, 51(Pt 5), 1703 - 7
Brevibacterium paucivorans sp . nov., from human clinical specimens; Wauters G et al.; Seven isolates from various human body sites displayed general chemotaxonomic and phenotypic characteristics of the genus Brevibacterium . This was corroborated by the 16S rRNA gene sequence analysis of strain CF62T, showing a sequence similarity of 99% to Brevibacterium mcbrellneri . However, DNA-DNA hybridization, a peculiar amino acid content of the cell wall and some phenotypic properties clearly suggested that these strains belong to a new species, for which the name Brevibacterium paucivorans sp . nov . is proposed . The type strain of B . paucivorans is CF62T (= DSM 13657T = LMG 19814T) . The DNA G+C content of the type strain is 55.8 mol%.

Int J Syst Evol Microbiol, 2001 Sep, 51(Pt 5), 1687 - 92
Paenibacillus jamilae sp . nov., an exopolysaccharide-producing bacterium able to grow in olive-mill wastewater; Aguilera M et al.; Endospore-forming strains were isolated from corn-compost treated with olive-mill wastewater ('alpechin') . The strains were taxonomically studied and proposed as a novel Paenibacillus species . These organisms (strains B.3T, B.7 and B.9) were particularly distinguishable from other aerobic spore-forming species by their ability to grow optimally in 100% (v/v) olive-mill wastewater at 30 degrees C and pH 7.0 and concomitant production of an interesting exopolysaccharide . Chemotaxonomic analysis revealed that MK-7 was the predominant menaquinone, the major fatty acid was anteiso C15:0 and the cell wall contained meso-diaminopimelic acid . The DNA G+C content was 40.7 mol% . Comparative sequence analysis of 16S rDNA with different reference species from the genera Bacillus, Paenibacillus, Brevibacillus, Aneurinibacillus, Alicyclobacillus, Halobacillus, Virgibacillus, Amphibacillus, Coprobacillus and Gracilibacillus indicated that the isolated strains were highly related to the genus Paenibacillus . Strain B.3T formed an evolutionary lineage distinct from other species within the evolutionary radiation encompassing the genus Paenibacillus . Strain B.3T was a close relative of Paenibacillus polymyxa, but DNA-DNA relatedness data with this species was very low (relative binding ratio < 16%) . Based on the morphological and physiological characteristics, as well as on the phylogenetic position determined by 16S rDNA analysis and DNA-DNA relatedness data, it is concluded that these strains should be designated a novel species, for which the name Paenibacillus jamilae sp . nov . is proposed . The type strain is B.3T (= CECT 5266T = DSM 13815T).

Int J Food Microbiol, 2001 Sep 19, 69(1-2), 1 - 10
Interactions between yeasts and bacteria in the smear surface-ripened cheeses; Corsetti A et al.; In the initial phase of ripening, the microflora of bacterial smear surface-ripened cheeses such as Limburger, Taleggio, Brick, Munster and Saint-Paulin and that of surface mould-ripened cheeses such as Camembert and Brie may be similar, but at the end of the ripening, bacteria such as Brevibacterium spp., Arthrobacter spp., Micrococcus spp., Corynebacterium spp . and moulds such as Penicillium camemberti are, respectively, the dominant microorganisms . Yeasts such as Candida spp., Cryptococcus spp., Debaryomyces spp., Geotrichum candidum, Pichia spp., Rhodotorula spp., Saccharomyces spp . and Yarrowia lipolytica are often and variably isolated from the smear surface-ripened cheeses . Although not dominant within the microorganisms of the smear surface-ripened cheeses, yeasts establish significant interactions with moulds and especially bacteria, including surface bacteria and lactic acid bacteria . Some aspects of the interactions between yeasts and bacteria in such type of cheeses are considered in this paper.

Biosci Biotechnol Biochem, 2001 Aug, 65(8), 1876 - 8
Purification and characterization of L-2,3-butanediol dehydrogenase of Brevibacterium saccharolyticum C-1012 expressed in Escherichia coli; Takusagawa Y et al.; The L-2,3-butanediol dehydrogenase produced in E . coli JM109/pLBD2-CTC was purified by 5 steps . The molecular mass of this enzyme was estimated at 110 kDa and the subunit was measured to be 30 kDa . The L-BDH had some differences from the BDHs from other sources in substrate specificity, pI value, pH stability, effects of divalent cations, and organic acids.

J Appl Microbiol, 2001 Oct, 91(4), 652 - 9
Mode of antagonism of Brevibacillus brevis against Botrytis cinerea in vitro; Edwards SG et al.; AIMS: To assess the activity of Brevibacillus brevis (formerly Bacillus brevis) Nagano and the antibiotic it produces, gramicidin S, against the plant pathogen Botrytis cinerea . METHODS AND RESULTS: Germination and growth of Bot . cinerea were assessed in the presence of B . brevis or gramicidin S in liquid media, on solid media and on leaf sections of Chinese cabbage . Germination was 10-fold more sensitive to gramicidin S than growth . Inhibition of Bot . cinerea was greater in liquid media compared with on solid media . Activity of gramicidin S against Bot . cinerea on leaf sections was much lower than in vitro . In vitro inhibition of Bot . cinerea by B . brevis Nagano was similar to equivalent levels of gramicidin . CONCLUSIONS: Antibiosis, via gramicidin S, is the mode of antagonism exhibited by B . brevis Nagano against Bot . cinerea in vitro . SIGNIFICANCE AND IMPACT OF THE STUDY: The mode of antagonism of B . brevis against Bot . cinerea was elucidated . The differing activity of gramicidin S against Bot . Cinerea in vitro and on leaf sections indicates one mechanism by which biocontrol activity may differ between laboratory and field conditions.

Appl Environ Microbiol, 2001 Oct, 67(10), 4520 - 30
Identification of bacteria in pasteurized zucchini purées stored at different temperatures and comparison with those found in other pasteurized vegetable purées; Guinebretiere MH et al.; One hundred nineteen isolates from a commercial zucchini puree stored at 4, 10, and 20 to 25 degrees C were fingerprinted using repetitive sequence-based PCR (REP-PCR) and classified into 35 REP types . One representative isolate of each REP type was subsequently identified by API50CHB/20E profile and partial rrs gene sequence analysis . Nine REP types were misidentified by the API system . Strains were misidentified as being in the Bacillus circulans (group 2) API taxon or in taxa with a low number of positive API characters such as Brevibacillus brevis . A phylogenetic analysis pointed to one new species of Bacillus and three new species of Paenibacillus among the misidentified REP types . Bacterial components in zucchini puree were compared phenotypically with those obtained in previous work on broccoli, carrot, leek, potato, and split pea purees, based on simple matching coefficient and unweighted pair group method with averages cluster analysis . Out of 254 strains, 69 strains previously identified as B . circulans (group 2) or B . circulans/B . macerans/B . polymyxa were assigned to a new Paenibacillus taxon phylogenetically related to P . azotofixans . Storage conditions at 4 degrees C favored the development of "B . macroides/B . maroccanus" and Paenibacillus spp . in zucchini purees and Paenibacillus spp . in other purees . Storage conditions at 20 to 25 degrees C favored the development of B . subtilis group (B . licheniformis and B . subtilis) and B . cereus group strains . At 10 degrees C, Paenibacillus spp . were always present at high frequencies, whereas the occurrence of B . macroides/B . maroccanus (in zucchini purees), B . cereus, and B . pumilus varied with the experiment.

Protein Expr Purif, 2001 Oct, 23(1), 113 - 20
High-level production of recombinant chicken interferon-gamma by Brevibacillus choshinensis; Yashiro K et al.; Cytokines, such as interferon-gamma have been shown to have adjuvant and growth promoting activity in poultry and livestock and have the potential to be used as alternatives to antibiotics . We have developed an efficient system for commercial-scale synthesis of recombinant chicken interferon-gamma (ChIFN-gamma) using Brevibacillus choshinensis as the host for protein production . The ChIFN-gamma expression vector, pNCIFN, was constructed using the novel Escherichia coli-B . choshinensis shuttle vector, pNCMO2 . ChIFN-gamma expression was optimized by investigating different culture conditions and different host B . choshinensis mutants . The highest level of production was observed using the B . choshinensis HPD31-MB2 strain grown at 30 degrees C, where ChIFN-gamma was produced at approximately 300-500 mg/L . ChIFN-gamma was also produced as a His-tagged fusion protein by using the pNCHis-IFN expression vector, a derivative of pNCMO2 . The protein was constitutively secreted into the culture supernatant and could be partially purified in a single step using a Ni-nitrilotriacetic acid column . This recombinant His-ChIFN-gamma was shown to have the same biological activity as native ChIFN-gamma .

Anal Chem, 2001 Sep 1, 73(17), 4134 - 44
Discrimination of aerobic endospore-forming bacteria via electrospray-lonization mass spectrometry of whole cell suspensions; Vaidyanathan S et al.; Direct injection electrospray ionization mass spectrometry (ESI-MS) without prior analyte separation was investigated for the analysis of whole cell suspensions of bacteria . Thirty-six strains of aerobic endospore-forming bacteria, consisting of six Bacillus species and one Brevibacillus species, were studied

Mediators Inflamm, 2001 Jun, 10(3), 155 - 62
The involvement of CD14 in the activation of human monocytes by peptidoglycan monomers; Muhvic D et al.; BACKGROUND: Cell-wall components of Gram-positive and Gram-negative bacteria induce the production of cytokines in human peripheral blood mononuclear cells . These cytokines are the main mediators of local or systemic inflammatory reaction that can contribute to the development of innate immunity . AIMS: This study was performed to analyze the involvement of CD14 molecule in the activation of human monocytes by peptidoglycan monomer (PGM) obtained by biosynthesis from culture fluid of penicillin-treated Brevibacterium divaricatum NRLL-2311 . METHODS: Cytokine release of interleukin (IL)-1, IL-6 and tumor necrosis factor-alpha from human monocytes via soluble CD14 (sCD14) or membrane-associated (mCD14) receptor using anti-CD14 monoclonal antibody (MEM-18) or lipid A structure (compound 406) was measured in bioassays . RESULTS: The results demonstrated that PGM in the presence of human serum might induce the monokine release in a dose-dependent manner . The addition of sCD14 at physiologic concentrations enhanced the PGM-induced monokine release, while the monokine inducing capacity of PGM in the presence of sCD14 was inhibited by MEM-18 . Effects of PGM were also blocked by glycolipid, compound 406, suggesting the involvement of binding structures similar to those for lipopolysaccharide . CONCLUSION: Activation of human monocytes by PGM involves both forms of CD14 molecule, sCD14 and mCD14.

Polar Biol, 1988, 9, 37 - 44
Characterization of 15 selected coccal bacteria isolated from Antarctic rock and soil samples from the McMurdo-Dry Valleys (South-Victoria Land); Siebert J et al.; Approximately 1500 cultures of microorganisms were isolated from rocks and soils of the Ross Desert (McMurdo-Dry Valleys) . From these, 15 coccoid strains were chosen for more detailed investigation . They were characterized by morphological, physiological and chemotaxonomical properties . All isolates were Gram-positive, catalase-positive and nonmotile . Six strains showed red pigmentation and could be identified as members of the genera Micrococcus (M . roseus, M . agilis) or Deinococcus . In spite of their coccoid morphology, the remaining nine strains had to be associated with coryneform bacteria (Arthrobacter, Brevibacterium), because of their cell wall composition and G+C ratios . Most of the strains were psychrotrophic, but one strain was even obligately psychrophilic, with a temperature maximum below 20 degrees C . Red cocci had in vitro pH optima above 9.0 although they generally originated from acid samples . Most isolates showed a preference for sugar alcohols and organic acids, compounds which are commonly known to be released by lichens, molds and algae, the other components of the cryptoendolithic ecosystem . These properties indicate that our strains are autochthonous members of the natural Antarctic microbial population.

Mol Genet Genomics, 2001 Aug, 265(6), 1022 - 30
The cell division genes ftsQ and ftsZ, but not the three downstream open reading frames YFIH, ORF5 and ORF6, are essential for growth and viability in Brevibacterium lactofermentum ATCC 13869; Honrubia MP et al.; The three ORFs (YFIH, ORF5 and ORF6) located downstream of the cell division genes ftsQ and ftsZ in Brevibacterium lactofermentum were disrupted by single homologous recombination events between internal fragments of the corresponding genes and the chromosomal sequences . The phenotypes of the disrupted mutants were similar to that of the wild type, suggesting that these genes are dispensable for growth and viability . However, using different plasmid constructs, it was not possible to obtain disrupted ftsZ or ftsQ mutants by single crossover events . When the ftsZ or ftsQ gene sequence was disrupted in vitro and used to replace the homologous chromosomal gene by double recombination, only single recombination events took place, and therefore no disruptants were obtained . It may be concluded therefore that, as in Escherichia coli, the cell division genes ftsQ and ftsZ are indispensable for growth and viability of B . lactofermentum . Northern hybridisation analyses performed using internal fragments of the genes coding for YFIH, ORF5 and ORF6 allowed us to dissect their transcriptional organization and to confirm the disruption of these genes.

Int J Syst Evol Microbiol, 2001 Jul, 51(Pt 4), 1361 - 71
Simplified technique for identification of the aerobic spore-forming bacteria by phenotype; Reva ON et al.; The use of modern research approaches of genetics, biochemistry and molecular biology has led to progress in bacterial taxonomy . Systematic study of the aerobic spore-forming bacteria has resulted in the realignment of the genus Bacillus into several new genera . In the meantime, the identification process has become more difficult for the non-specialist in Bacillus taxonomy . This paper presents a key for the simplified phenotypic identification of the mesophilic, aerobic, spore-forming bacteria belonging to the genera Bacillus, Paenibacillus, Brevibacillus, Aneurinibacillus, Geobacillus and Virgibacillus . A total of 81 species were included and 115 morphological and physiological tests were analysed for their discriminative efficiency . This key is practical for rough but quick identification of aerobic spore-forming bacteria isolated from nature . Such preliminary identification will be helpful for the selection of reference strains and methods for more precise identification using the newest techniques . The reliability of the proposed identification key was tested on 100 cultures from the Ukrainian Collection of Microorganisms . The developed identification key is represented in interactive mode on a website .

J Appl Microbiol, 2001 Aug, 91(2), 312 - 21
Amino acid catabolism in cheese-related bacteria: selection and study of the effects of pH, temperature and NaCl by quadratic response surface methodology; Curtin AC et al.; AIMS: To screen the cystathionine lyase and L-methionine aminotransferase activities of cheese-related bacteria (lactococci, non-starter lactobacilli and smear bacteria) and to determine the individual and interactive effects of temperature, pH and NaCl concentration on selected enzyme activities . METHODS AND RESULTS: A subcellular fractionation protocol and specific enzyme assays were used, and a quadratic response surface methodology was applied . The majority of the strains, 21 of 33, had detectable cystathionine lyase activity which differed in the specificity . Aminotransferase activity on L-methionine was observed in only three strains . The cystathionine lyase activities of Lactobacillus reuteri DSM20016, Lactococcus lactis subsp . cremoris MG1363, Brevibacterium linens 10 and Corynebacterium ammoniagenes 8 and the L-methionine aminotransferase activity of Lact . reuteri DSM20016 had temperature and pH optima of 30-45 degrees C, and 7.5-8.0, respectively . As shown by the quadratic response surface methodology these enzymes retained activities in the range of temperature, pH and NaCl concentration which characterized the cheeses from which the bacteria originated . CONCLUSION: The enzyme activities may have a role in flavour development during cheese ripening . SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge about the amino acid catabolic enzymes in order to improve cheese ripening.

J Bacteriol, 2001 Aug, 183(16), 4796 - 805
Identification and mutagenesis by allelic exchange of choE, encoding a cholesterol oxidase from the intracellular pathogen Rhodococcus equi; Navas J et al.; The virulence mechanisms of the facultative intracellular parasite Rhodococcus equi remain largely unknown . Among the candidate virulence factors of this pathogenic actinomycete is a secreted cholesterol oxidase, a putative membrane-damaging toxin . We identified and characterized the gene encoding this enzyme, the choE monocistron . Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified in Brevibacterium sterolicum and Streptomyces spp . ChoE also exhibits significant similarities to putative cholesterol oxidases encoded by Mycobacterium tuberculosis and Mycobacterium leprae . Genetic tools for use with R . equi are poorly developed . Here we describe the first targeted mutagenesis system available for this bacterium . It is based on a suicide plasmid, a selectable marker (the aacC4 apramycin resistance gene from Salmonella), and homologous recombination . The choE allele was disrupted by insertion of the aacC4 gene, cloned in pUC19 and introduced by electroporation in R . equi . choE recombinants were isolated at frequencies between 10(-2) and 10(-3) . Twelve percent of the recombinants were double-crossover choE mutants . The choE mutation was associated with loss of cooperative (CAMP-like) hemolysis with sphingomyelinase-producing bacteria (Listeria ivanovii) . Functional complementation was achieved by expression of choE from pVK173-T, a pAL5000 derivative conferring hygromycin resistance . Our data demonstrate that ChoE is an important cytolytic factor for R . equi . The highly efficient targeted mutagenesis procedure that we used to generate choE isogenic mutants will be a valuable tool for the molecular analysis of R . equi virulence.

Eur J Clin Microbiol Infect Dis, 2001 May, 20(5), 315 - 23
Natural antibiotic susceptibility of recently established coryneform bacteria; Troxler R et al.; The natural susceptibility of 20 strains each of Brevibacterium casei (formerly CDC coryneform groups B-1 and B-3), Dermabacter hominis (formerly CDC coryneform groups 3 and 5), and Turicella otitidis (formerly coryneform group ANF-1-like) isolated from clinical specimens to 71 antibiotics was investigated . Susceptibility testing was carried out with a microdilution procedure using H medium . All three species were naturally sensitive to tetracyclines, most aminoglycosides, carbapenems, macrolides, lincosamides, glycopeptides, and rifampin . Susceptibility patterns indicating natural resistance to pipemidic acid, sulfamethoxazole, and cotrimoxazole also were found for all three species . Species-dependent discrepancies in susceptibility leading to completely different categorizations (changing from sensitive to resistant or vice versa) were found for some penicillins (e.g., oxacillin and amoxicillin), a few cephalosporins (e.g., ceftibutene), aztreonam, tobramycin, norfloxacin, fleroxacin, trimethoprim, nitrofurantoin, fosfomycin, and fusidic acid . For the majority of antibiotics, Brevibacterium casei was the least susceptible species and Turicella otitidis the most susceptible taxon . The present study describes a database on the natural susceptibility of Brevibacterium casei, Dermabacter hominis, and Turicella otitidis to a wide range of antibiotics . This database can be applied for the validation of susceptibility testing results of these recently established coryneform bacteria.

Antimicrob Agents Chemother, 2001 Aug, 45(8), 2390 - 2
Activities of gemifloxacin and five other antimicrobial agents against Listeria monocytogenes and coryneform bacteria isolated from clinical samples; Martinez-Martinez L et al.; The in vitro activities of gemifloxacin, ciprofloxacin, ampicillin, doxycycline, gentamicin, and vancomycin were evaluated against 15 Listeria monocytogenes strains and 205 coryneform bacteria isolated from clinical samples . The percentages of strains inhibited by gemifloxacin at 0.5 microg/ml were 100% (L . monocytogenes), 93.3% (Brevibacterium spp.), 90% (Corynebacterium minutissimum), 42.5% (Corynebacterium amycolatum), 20% (Corynebacterium striatum), 12.5% (Corynebacterium jeikeium), and 10% (Corynebacterium urealyticum) . One hundred percent of the L . monocytogenes strains were inhibited by 0.25 microg of gemifloxacin per ml, whereas 0% of the strains were inhibited by 0.25 microg of ciprofloxacin per ml . Vancomycin at 2 microg/ml inhibited all strains . Doxycycline and gentamicin at 4 microg/ml inhibited 94 and 49% of the strains, respectively, while ampicillin at 0.5, 2, and 8 microg/ml inhibited 24, 61, and 66% of the strains, respectively . It is concluded that gemifloxacin shows good in vitro activity against L . monocytogenes and coryneform bacteria except C . jeikeium and C . urealyticum.

Biosci Biotechnol Biochem, 2001 May, 65(5), 1090 - 4
Digested bacterial cell powder (DBCP) as a source of reduced-form folates for pigs: use of a trimethoprim-resistant strain and the bioavailability of folates in DBCP; Toride Y et al.; The production of digested bacterial cell powder (DBCP) as a source of reduced-form folates for pigs was studied . Trimethoprim-resistant mutants of Brevibacterium lactofermentum ATCC 13869 accumulated a significantly higher amount of the reduced form of folate in the cells than the wild-type strain . DBCPs were prepared from the resistant mutant strain and the wild-type strain . The utilization of the reduced-form of folate in DBCP was evaluated by measuring the plasma folate level after orally administering DBCP to Gottingen minipigs . The folates in both DBCPs proved to have equally high bioavailability in the pigs.

Ann Agric Environ Med, 2001, 8(1), 81 - 90
Response of sawmill workers to work-related airborne allergens; Dutkiewicz J et al.; The aim of this work was to determine the reactivity of sawmill workers to biological allergens associated with wood dust . Allergological examinations by skin and precipitin tests were performed in 43 workers employed in a sawmill processing coniferous wood (pine), in 90 workers employed in two sawmills processing deciduous wood (oak), and in 32 healthy urban dwellers not exposed to organic dusts (referents) . The skin test was performed by the intradermal method with the saline extracts of wood dust and of the cultures of three microbial species (Rahnella sp., Brevibacterium linens and Penicillium citrinum) isolated from the air polluted with wood dust . Sawdust from pine was used for testing of the pine processing workers and referents while sawdust from oak was used for testing of the oak processing workers . Skin reactions were recorded after 20 minutes, 8 hours and 24 hours . The agar-gel test for the presence of precipitins in serum was performed with the extract of pine wood dust and extracts of 17 microbial isolates . The workers processing pine showed a very high frequency of positive skin reactions to the extract of wood dust at all time intervals, significantly greater compared to the workers processing oak and referents (p < 0.001) . The early skin reactions to the extracts of dust-borne bacteria and fungi were very common among sawmills workers and showed a significant relationship with the degree of exposure . The frequency of reactions to Gram-negative bacterium Rahnella sp . was significantly greater in the pine processing workers than in the oak processing workers and referents (p < 0.001) . By contrast, the oak processing workers reacted significantly more frequently to Penicillium citrinum, compared to the pine processing workers and referents (p < 0.01) . These results conform to the prior study of airborne microflora in which the dominancy of Gram-negative bacteria was stated in the pine processing sawmill while mould fungi were most common in the oak processing sawmills . The antibody response of sawmill workers to work-related antigens was much weaker compared to skin reactions . As many as 41 sawmill workers reported the occurrence of work-related symptoms . A significant relationship was found between the occurrence of symptoms and frequency of allergic reactions, but only with a limited number of antigens . The obtained results suggest that early allergic reactions to coniferous wood and to microorganisms associated with wood dust are common among sawmill workers, posing a potential risk of work-related disease in this occupational group.

J Hazard Mater, 2001 Jun 29, 84(2-3), 253 - 64
Microorganism selection and biosurfactant production in a continuously and periodically operated bioslurry reactor; Cassidy DP et al.; A continuous-flow reactor (CSTR) and a soil slurry-sequencing batch reactor (SS-SBR) were maintained in 8l vessels for 180 days to treat a soil contaminated with diesel fuel (DF) . Concentrations of Candida tropicalis, Brevibacterium casei, Flavobacterium aquatile, Pseudomonas aeruginosa, and Pseudomonas fluorescens were determined using fatty acid methyl ester (FAME) analysis . DF removal (biological and volatile) and biosurfactant concentrations were measured . The SS-SBR encouraged the growth of biosurfactant-producing species relative to the CSTR . Counts of biosurfactant-producing species (C . tropicalis, P . aeruginosa, P . fluorescens) relative to total microbial counts were 88% in the SS-SBR and 23% in the CSTR . Biosurfactants were produced in the SS-SBR to levels of nearly 70 times the critical micelle concentration (CMC) early in the cycle, but were completely degraded by the end of each cycle . No biosurfactant production was observed in the CSTR . DF biodegradation rates were over 40% greater and DF stripping was over five times lower in the SS-SBR than the CSTR . However, considerable foaming occurred in the SS-SBR . Reversing the mode of operation in the reactors on day 80 caused a complete reversal in microbial consortia and reactor performance by day 120 . These results show that bioslurry reactor operation can be manipulated to control overall reactor performance.

Can J Microbiol, 2001 May, 47(5), 457 - 9
Rapid detection of Brevibacillus formosus BN53-1 in chicken feces; Nakada Y et al.; Here we describe a rapid method for detecting the hydrogen sulfide-decomposing bacterium Brevibacillus formosus BN53-1 in chicken feces . The method, which can be adapted to the specific detection of a variety of useful eubacteria, is based on blot hybridization and polymerase chain reaction (PCR), and makes use of the genus or species hypervariable region of eubacterial 16S rDNA . The approximate limit of detection under the conditions we tested was 1.0 x 10(3) cells in 10 mg of chicken feces.

Appl Microbiol Biotechnol, 2001 May, 55(4), 466 - 70
Isolation of ftsI and murE genes involved in peptidoglycan synthesis from Corynebacterium glutamicum; Wijayarathna CD et al.; Corynebacterium glutamicum is known to excrete large amounts of L-glutamic acid upon treatment by penicillin . However, the mechanism of L-glutamate overproduction by penicillin treatment is still unknown . A 5.3-kb HindIII fragment was isolated by directly introducing the C . glutamicum (Brevibacterium lactofermentum) ATCC 13869 gene library into the temperature-sensitive Escherichia coli murE mutant and selecting temperature resistant clones . Two open reading frames (ORFs) were found in this fragment: (1) murE, encoding UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate ligase, and (2)ftsI, encoding septum-peptidoglycan synthetase, one of the targets of penicillin (penicillin-binding protein 3) . Both ORFs were involved in peptidoglycan synthesis . Proteins were synthesized from the C . glutamicum murE and ftsI genes, 55 kDa and 73 kDa respectively, in an in vitro protein synthesis system, using E . coli S30 extracts.

J Biol Chem, 2001 Aug 10, 276(32), 30435 - 41 Epub 2001 Jun 07.
Oxygen access to the active site of cholesterol oxidase through a narrow channel is gated by an Arg-Glu pair; Coulombe R et al.; Cholesterol oxidase is a monomeric flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one . Two forms of the enzyme are known, one containing the cofactor non-covalently bound to the protein and one in which the cofactor is covalently linked to a histidine residue . The x-ray structure of the enzyme from Brevibacterium sterolicum containing covalently bound FAD has been determined and refined to 1.7-A resolution . The active site consists of a cavity sealed off from the exterior of the protein . A model for the steroid substrate, cholesterol, can be positioned in the pocket revealing the structural factors that result in different substrate binding affinities between the two known forms of the enzyme . The structure suggests that Glu(475), located at the active site cavity, may act as the base for both the oxidation and the isomerization steps of the catalytic reaction . A water-filled channel extending toward the flavin moiety, inside the substrate-binding cavity, may act as the entry point for molecular oxygen for the oxidative half-reaction . An arginine and a glutamate residue at the active site, found in two conformations are proposed to control oxygen access to the cavity from the channel . These concerted side chain movements provide an explanation for the biphasic mode of reaction with dioxygen and the ping-pong kinetic mechanism exhibited by the enzyme.

J Biol Chem, 2001 May 25, 276(21), 18024 - 30 Epub 2001 Feb 28.
Cholesterol oxidase from Brevibacterium sterolicum . The relationship between covalent flavinylation and redox properties; Motteran L et al.; Brevibacterium sterolicum possesses two forms of cholesterol oxidase, one containing noncovalently bound FAD, the second containing a FAD covalently linked to His(69) of the protein backbone . The functional role of the histidyl-FAD bond in the latter cholesterol oxidase was addressed by studying the properties of the H69A mutant in which the FAD is bound tightly, but not covalently, and by comparison with native enzyme . The mutant retains catalytic activity, but with a turnover rate decreased 35-fold; the isomerization step of the intermediate 3-ketosteroid to the final product is also preserved . Stabilization of the flavin semiquinone and binding of sulfite are markedly decreased, this correlates with a lower midpoint redox potential (-204 mV compared with -101 mV for wild-type) . Reconstitution with 8-chloro-FAD led to a holoenzyme form of H69A cholesterol oxidase with a midpoint redox potential of -160 mV . In this enzyme form, flavin semiquinone is newly stabilized, and a 3.5-fold activity increase is observed, this mimicking the thermodynamic effects induced by the covalent flavin linkage . It is concluded that the flavin 8alpha-linkage to a (N1)histidine is a pivotal factor in the modulation of the redox properties of this cholesterol oxidase to increase its oxidative power.

Mikrobiologiia, 2000 Sep-Oct, 69(5), 686 - 93
{Establishment of the phylogenetic relationship between the microbial producers of cyclodextrin glucanotransferases using their complete amino acid sequences}; Bikbulatova SM et al.; A phylogenetic tree was constructed on the basis of the amino acid sequences of the known cyclodextrin glucanotransferases (CDGTs), including those deduced from the nucleotide sequences of Bacillus sp . strain 6.6.3 and Paenibacillus macerans IB-7 genes encoding alpha- and beta-CDGTs . The tree clearly demonstrates the existence of distinct phylogenetic groups of CDGT-producing microorganisms and the divergence of the alpha-, beta-, and gamma-CDGT produced by microorganisms from the genera Bacillus, Paenibacillus, Brevibacillus, and Thermoanaerobacter from a common ancestor, whereas the CDGT of Klebsiella pneumoniae is independent and results from the convergence of different ancestors . The degree of homology of the leader peptide sequences of CDGTs may serve as a criterion of intraspecies relatedness between CDGT-producing microorganisms.

Biochem Biophys Res Commun, 2001 Mar 23, 282(1), 21 - 7
Cloning and expression of glucose 3-dehydrogenase from Halomonas sp . alpha-15 in Escherichia coli; Kojima K et al.; The gene encoding glucose 3-dehydrogenase (G3DH) from Halomonas sp . alpha-15 was cloned and expressed in Escherichia coli . An open reading frame of 1686 nucleotides was shown to encode G3DH . The flavine adenine dinucleotide binding motif was found in the N-terminal region of G3DH . The deduced primary structure of G3DH showed about 30% identity to sorbitol dehydrogenase from Gluconobacter oxydans and 2-keto-d-gluconate dehydrogenases from Erwinia herbicola and Pantoea citrea . The folding prediction of G3DH suggested that the 3D structure of G3DH was similar with cholesterol oxidase from Brevibacterium sterolicum or glucose oxidase from Aspergillus niger .

J Dairy Sci, 2001 Feb, 84(2), 354 - 60
Assessment of the coloring strength of brevibacterium linens strains: spectrocolorimetry versus total carotenoid extraction/quantification; Dufosse L et al.; Brevibacterium linens is a major component of red-smear cheese microflora and imparts color to the cheese rind . The present study was done to evaluate carotenoid contents in 29 strains of this bacterium and to relate the color of the strains to the carotenoids present . A spectrocolorimeter was used to determine the color, and the carotenoid contents were determined by spectrophotorimetric measurement at 450 nm . Regression analysis was carried out on the color values a*, b*, and C* to determine which color value could be used to express the contents of carotenoids in B . linens biomass . The C* color value appeared to be the best estimate for correlation.

Microbios, 2001, 104(407), 7 - 15
Interrelation between synthesis and uptake of ectoine for the growth of the halotolerant Brevibacterium species JCM 6894 at high osmolarity; Nagata S et al.; The growth of a halotolerant Brevibacterium sp . JCM 6894 was examined in the presence of compatible solutes such as glycine betaine, ectoine (2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine) and ectoine derivatives . The effect of competition between their uptake and synthesis in the cells was subjected to osmotic shift towards the higher salinity . Among each solute examined the supplement of ectoine or hydroxyectoine exhibited a remarkable stimulation on the growth of strain JCM 6894, regardless of the range of osmotic shifts, where the largest was 0-->2 M NaCl, the intermediate was 1-->2 M NaCl, and no shift was 2-->2 M NaCl . The growth rates of this strain were dependent on the amount of ectoine taken up, which was conspicuous for the largest osmotic shift and during the first few hours of incubation after transfer . The cells subjected to 1-->2 M NaCl and 2-->2 M NaCl transfers took up less ectoine and this resulted in lower growth rates than those of cells with the largest osmotic shift (0-->2 M NaCl) . The role of other compatible solutes which accumulated is discussed in relation to growth stimulation of strain JCM 6894.

Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1687 - 92 Epub 2001 Feb 06.
A single gene for lycopene cyclase, phytoene synthase, and regulation of carotene biosynthesis in Phycomyces; Arrach N et al.; Previous complementation and mapping of mutations that change the usual yellow color of the Zygomycete Phycomyces blakesleeanus to white or red led to the definition of two structural genes for carotene biosynthesis . We have cloned one of these genes, carRA, by taking advantage of its close linkage to the other, carB, responsible for phytoene dehydrogenase . The sequences of the wild type and six mutants have been established, compared with sequences in other organisms, and correlated with the mutant phenotypes . The carRA and carB coding sequences are separated by 1,381 untranslated nucleotides and are divergently transcribed . Gene carRA contains separate domains for two enzymes, lycopene cyclase and phytoene synthase, and regulates the overall activity of the pathway and its response to physical and chemical stimuli from the environment . The lycopene cyclase domain of carRA derived from a duplication of a gene from a common ancestor of fungi and Brevibacterium linens; the phytoene synthase domain is similar to the phytoene and squalene synthases of many organisms; but the regulatory functions appear to be specific to Phycomyces.

Lett Appl Microbiol, 2001 Feb, 32(2), 93 - 8
Stereochemical applications of the expression of the L-2,3-butanediol dehydrogenase gene in Escherichia coli; Ui S et al.; The L-2,3-butanediol dehydrogenase (L-BDH) gene of Brevibacterium saccharolyticum was strongly expressed in Escherichia coli using the tac promoter . However, the stereospecificity of the resulting L-BDH was reduced . The region upstream of the meso-BDH gene of Klebsiella pneumoniae was also involved in the expression of the B . saccharolyticum gene . However, in this case, the resulting L-BDH exhibited more stable stereospecificity . A stereospecificity recognition region was located within the rear sequence (Hpa I site, carboxy terminal) of the BDH open reading frame . Using a transformed strain of E . coli, the conversion of L-acetoin (L-AC), in the commercially available racemic mixture of AC, to L-2,3-butanediol (L-BD) was attempted . As a result, 0.37% L-BD was formed from 1% AC added to the culture.

Curr Microbiol, 2000 Jul, 41(1), 50 - 5
Genetic fingerprinting of Brevibacterium linens by pulsed-field gel electrophoresis and ribotyping; Lima PT et al.; Members of Brevibacterium linens display physiological features that are relevant for cheese production . The genomes of five B . linens strains deposited on culture collections were compared by examining large restriction fragments on pulsed-field gel electrophoresis and detection of polymorphism at the level of 16S rRNA genes . Pulsed-field analysis with the endonucleases DraI and AsnI showed a characteristic restriction profile for each strain and allowed the calculation of genome sizes ranging between 3.2 and 3.9 Mbp . No linear genomic elements were detected . Polymorphisms at the level of 16S rRNA genes were revealed by hybridization with an oligonucleotide probe complementary to a universal domain of the 16S genes . An EcoRI fragment of 1.4 kb was identified as common to all strains under study . According to the number of positive bands detected by the probe, at least four rRNA operons must be present on the genome of the B . linens strains here studied.

Mem Inst Oswaldo Cruz, 2000, 95 Suppl 1, 201 - 6
Mosquitocidal bacterial toxins: diversity, mode of action and resistance phenomena; Charles JF et al.; Bacteria active against dipteran larvae (mosquitoes and black flies) include a wide variety of Bacillus thuringiensis and B . sphaericus strains, as well as isolates of Brevibacillus laterosporus and Clostridium bifermentans . All display different spectra and levels of activity correlated with the nature of the toxins, mainly produced during the sporulation process . This paper describes the structure and mode of action of the main mosquitocidal toxins, in relationship with their potential use in mosquito and/or black fly larvae control . Investigations with laboratory and field colonies of mosquitoes that have become highly resistant to the B . sphaericus Bin toxin have shown that several mechanisms of resistance are involved, some affecting the toxin/receptor binding step, others unknown.

Appl Environ Microbiol, 2000 Dec, 66(12), 5514 - 7
Diversity of L-methionine catabolism pathways in cheese-ripening bacteria; Bonnarme P et al.; Enzymatic activities that could be involved in methanethiol generation in five cheese-ripening bacteria were assayed, and the major sulfur compounds produced were identified . L-Methionine and alpha-keto-gamma-methyl-thio-butyric acid demethiolating activities were detected in whole cells and cell extracts (CFEs) of all the bacteria tested . No L-methionine deaminase activity could be detected in any of the ripening bacteria and L-methionine aminotransferase was detected in CFEs of Brevibacterium linens, Micrococcus luteus, and Corynebacterium glutamicum . The results suggest that several pathways for L-methionine catabolism probably coexist in these ripening bacteria.

Appl Microbiol Biotechnol, 2000 Oct, 54(4), 581 - 8
Zinc biosorption by a zinc-resistant bacterium, Brevibacterium sp . strain HZM-1; Taniguchi J et al.; A zinc-resistant bacterium, Brevibacterium sp . strain HZM-1 which shows a high Zn2+ -adsorbing capacity, was isolated from the soil of an abandoned zinc mine . Kinetic analyses showed that Zn2+ binding to HZM-1 cells follows Langmuir isotherm kinetics with a maximum metal capacity of 0.64 mmol/g dry cells and an apparent metal dissociation constant of 0.34 mM . The observed metal-binding capacity was one of the highest values among those reported for known microbial Zn2+ biosorbents . The cells could also adsorb heavy metal ions such as Cu2+ . HZM-1 cells could remove relatively low levels of the Zn2+ ion (0.1 mM), even in the presence of large excess amounts (total concentration, 10 mM) of alkali and alkali earth metal ions . Bound Zn2+ ions could be efficiently desorbed by treating the cells with 10 mM HCl or 10 mM EDTA, and the Zn2+ -adsorbing capacity of the cells was fully restored by treatment of the desorbed cells with 0.1 M NaOH . Thus, HZM-1 cells can serve as an excellent biosorbent for removal of Zn2+ from natural environments . The cells could grow in the presence of significant concentrations of ZnCl2 (at least up to 15 mM) and thus is potentially applicable to in situ bioremediation of Zn2+ -contaminated aqueous systems.

Lett Appl Microbiol, 2000 Nov, 31(5), 395 - 9
Selective medium based on tyrosine metabolism for the isolation and enumeration of Brevibacillus brevis (Bacillus brevis); Edwards SG et al.; AIMS: To develop a selective medium for the enumeration of Brevibacillus brevis Nagano spores from soil and plant material . METHODS AND RESULTS: Tyrosine agar was developed as a selective medium and compared with nutrient agar for the enumeration of B . brevis Nagano spores from sterile and non-sterile plant and soil extracts . Brevibacillus brevis Nagano colonies could be easily identified only on tyrosine agar due to their clear halo and distinct colony morphology . Identification was confirmed by thin layer chromatography of the antibiotic, gramicidin S, produced by this strain . CONCLUSIONS: Tyrosine agar was shown to be a suitable selective medium for the enumeration of B . brevis Nagano . SIGNIFICANCE AND IMPACT OF THE STUDY: The medium developed, tyrosine agar, can be used to monitor the population of the biological control agent, B . brevis Nagano, and will allow detailed studies within the crop environment.

Biochemistry, 2000 Nov 7, 39(44), 13383 - 9
Use of the parallax-quench method to determine the position of the active-site loop of cholesterol oxidase in lipid bilayers; Chen X et al.; To elucidate the cholesterol oxidase-membrane bilayer interaction, a cysteine was introduced into the active site lid at position-81 using the Brevibacterium enzyme . To eliminate the possibility of labeling native cysteine, the single cysteine in the wild-type enzyme was mutated to a serine without any change in activity . The loop-cysteine mutant was then labeled with acrylodan, an environment-sensitive fluorescence probe . The fluorescence increased and blue-shifted upon binding to lipid vesicles, consistent with a change into a more hydrophobic, i.e., lipid, environment . This acrylodan-labeled cholesterol oxidase was used to explore the pH, ionic strength, and headgroup dependence of binding . Between pH 6 and 10, there was no significant change in binding affinity . Incorporation of anionic lipids (phosphatidylserine) into the vesicles did not increase the binding affinity nor did altering the ionic strength . These experiments suggested that the interactions are primarily driven by hydrophobic effects not ionic effects . Using vesicles doped with either 5-doxyl phosphatidylcholine, 10-doxyl phosphatidylcholine, or phosphatidyl-tempocholine, quenching of acrylodan fluorescence was observed upon binding . Using the parallax method of London {Chattopadhyay, A., and London, E . (1987) Biochemistry 26, 39-45}, the acrylodan ring is calculated to be 8.1 +/- 2.5 A from the center of the lipid bilayer . Modeling the acrylodan-cysteine residue as an extended chain suggests that the backbone of the loop does not penetrate into the lipid bilayer but interacts with the headgroups, i.e., the choline . These results demonstrate that cholesterol oxidase interacts directly with the lipid bilayer and sits on the surface of the membrane.

J Clin Microbiol, 2000 Nov, 38(11), 4292 - 3
Peritonitis due to Brevibacterium otitidis in a patient undergoing continuous ambulatory peritoneal dialysis; Wauters G et al.; Brevibacterium otitidis is a coryneform rod and, as far as is known, is isolated only from infected ears . We report the first known case of peritonitis caused by B . otitidis in a patient undergoing continuous ambulatory peritoneal dialysis.

Appl Environ Microbiol, 2000 Nov, 66(11), 4620 - 4
Trehalose synthesis by sequential reactions of recombinant maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase from Brevibacterium helvolum; Kim YH et al.; A DNA fragment encoding two enzymes leading to trehalose biosynthesis, maltooligosyltrehalose synthase (BvMTS) and maltooligosyltrehalose trehalohydrolase (BvMTH), was cloned from the nonpathogenic bacterium Brevibacterium helvolum . The open reading frames for the two proteins are 2,331 and 1,770 bp long, respectively, and overlap by four nucleotides . Recombinant BvMTS, BvMTH, and fusion gene BvMTSH, constructed by insertion of an adenylate in the overlapping region, were expressed in Escherichia coli . Purified BvMTS protein catalyzed conversion of maltopentaose to maltotriosyltrehalose, which was further hydrolyzed by BvMTH protein to produce trehalose and maltotriose . The enzymes shortened maltooligosaccharides by two glucose units per cycle of sequential reactions and released trehalose . Maltotriose and maltose were not catalyzed further and thus remained in the reaction mixtures depending on whether the substrates had an odd or even number of glucose units . The bifunctional in-frame fusion enzyme, BvMTSH, catalyzed the sequential reactions more efficiently than an equimolar mixture of the two individual enzymes did, presumably due to a proximity effect on the catalytic sites of the enzymes . The recombinant enzymes produced trehalose from soluble starch, an abundant natural source for trehalose production . Addition of alpha-amylase to the enzyme reaction mixture dramatically increased trehalose production by partial hydrolysis of the starch to provide more reducing ends accessible to the BvMTS catalytic sites.

Int J Food Microbiol, 2000 Sep 25, 60(2-3), 231 - 9
Properties of Bacillus cereus and other bacilli contaminating biomaterial-based industrial processes; Pirttijarvi TS et al.; This paper is an overview on bacilli in industrial processes, with focus on food grade paper and paperboard production . Paperboards mainly contain sporeforming bacteria belonging to the genera Bacillus, Paenibacillus and Brevibacillus, usually found in quantities from < 50 to 250 cfu g(-1) homogenized paperboard . Of those frequently found, Bacillus cereus group, B . licheniformis, B . subtilis and Brevibacillus brevis are important for food hygiene because of their hydrolytic activities on food components and the ability of some strains to produce food poisoning toxins or to grow at refrigerated temperatures . We found that the phenotypic properties (lecithinase activity, nitrate reduction) used in standard methods (e.g., ISO, FDA, IDF) to recognize B . cereus, were unreliable for industrial isolates . Whole cell fatty acid composition of a group of the industrial isolates deviated so much from those in a widely used commercial database that the strains were not or only poorly recognized as B . cereus . Industrial isolates, including toxigenic ones, often missed one or more of these characters, even in cases where 100% 16S rDNA identity was found with B . cereus or with B . thuringiensis . 11-Methyldodecanoic acid and trans-9-hexadecenoic acid were found without exception in over 200 industrial B . cereus group isolates and in over 30 culture collection strains . The detection of these fatty acids is a secure method for the identification of B . cereus . Negative reaction for starch hydrolysis and for BCET-RPLA test and a specific ribotype were found in all B . cereus strains producing the emetic toxin.

J Dairy Sci, 2000 Aug, 83(8), 1674 - 83
Behavior of Brevibacterium linens and Debaryomyces hansenii as ripening flora in controlled production of soft smear cheese from reconstituted milk: protein degradation; Leclercq-Perlat MN et al.; Model smear soft cheeses, prepared with Debaryomyces hansenii and Brevibacterium linens as ripening starters, were ripened under aseptic conditions . Results of the cheese-making trials, in triplicate, were similar and showed similar patterns of protein degradation . In all of the trials, the acid-soluble nitrogen and nonprotein nitrogen (NPN) indexes and NH3 concentrations of the rind were low until d 10 . The acid-soluble nitrogen and NPN of the rind then increased to 100 and 18% of total nitrogen, respectively, at d 76 . The NH3 concentrations remained low until d 24 and increased until d 70, reaching about 1.8 g of NH3/kg of DM, and then remained constant . The acid-soluble nitrogen and NPN indexes and NH3 concentrations in the inner cheese mass were lower than in the rind . They showed the same evolution, reaching about 18% for acid-soluble nitrogen, 10% for NPN, and 1.5 g of NH3/kg of DM . It was shown that the inner cheese pH and populations of D . hansenii and B . linens have an effect on proteolysis . Viable cell counts of D . hansenii and B . linens were correlated with the environmental conditions and with proteolytic products . The determining role of carbon source and NH3 diffusions on the cheese ripening process were confirmed.

J Dairy Sci, 2000 Aug, 83(8), 1665 - 73
Behavior of Brevibacterium linens and Debaryomyces hansenii as ripening flora in controlled production of smear soft cheese from reconstituted milk: growth and substrate consumption dairy foods; Leclercq-Perlat MN et al.; Experimental cheeses inoculated with Debaryomyces hansenii and Brevibacterium linens were ripened for 76 d under aseptic conditions . Triplicate cheese-making trials were similar as a result of efficient control of the atmosphere . In all trials, D . hansenii grew rapidly during the first 2 d and then slowed, but growth remained exponential until d 10 (generation time around 70 h) . Total cell counts were higher than the number of viable cells, and after 10 d they remained around 3 x 10(9) yeast/g of DM . This difference resulted from the nonviability of a fraction of D . hansenii . After d 15, the pH of the rind was close to 7, and B . linens grew exponentially until d 25 (generation time around 70 h) . The growth rate subsequently decreased but remained exponential (generation time around 21 d) . Cell counts of D . hansenii and B . linens were correlated with the environmental technical conditions . Total D . hansenii counts were also correlated with total B . linens counts . Viable B . linens counts were related to rind lactate, and total counts depended on rind pH, internal lactate, and D . hansenii viable counts . The internal pH of the cheese depended on lactate concentrations, whereas surface pH was related to internal lactose, temperature, and relative humidity . These results suggest a determining role of the diffusion of the carbon sources in the ripening of smear soft cheese.

J Clin Microbiol, 2000 Sep, 38(9), 3513 - 4
Brevibacterium casei sepsis in an 18-year-old female with AIDS; Brazzola P et al.; Brevibacterium sp . was isolated from the blood of an acutely ill 18-year-old female with AIDS . The isolate was identified as Brevibacterium casei by use of carbohydrate assimilation tests . Treatment was successful with intravenously administered ciprofloxacin . To our knowledge, this is the first report of sepsis caused by B . casei in a human immunodeficiency virus-infected patient.

Syst Appl Microbiol, 2000 Jun, 23(2), 174 - 84
Propionicin SM1, a bacteriocin from Propionibacterium jensenii DF1: isolation and characterization of the protein and its gene; Miescher S et al.; We purified a bacteriocin from the cell-free supernatant of Propionibacterium jensenii DF1 isolated from Swiss raw milk, and named it propionicin SM1 . The heat-stable protein was strongly bactericidal against P . jensenii DSM20274 . On the basis of the N-terminal amino acid sequence of the purified protein, a degenerate oligonucleotide probe was designed to locate and clone the corresponding gene of P . jensenii DF1 . It hybridized exclusively with the DF1l-resident plasmid pLME106, but not with chromosomal DNA . Sequencing of the 6.9-kb plasmid revealed the targeted amino acid sequence within an open reading frame (ORF4) of 207 amino acids (molecular mass, 22,865 Da) . The corresponding gene was named ppnA . It encodes the prepeptide PpnA that is processed to the mature protein (19,942 Da) propionicin SM1 . No sequence homology is detectable with known proteins . However, the proposed leader peptide sequence containing 27 amino acids has typical signal peptide features and shows good homology to the leader peptide of Usp45, a protein excreted from Lactococcus lactis (VAN ASSELDONK et al., 1993) . Plasmid pLME106 contains at least 9 ORFs, some exhibiting significant homologies to plasmid-encoded functions from other bacteria . The highest identity values were found for ORF1 with the theta replicase (acc . no . U39878) of Brevibacterium linens (58.8%) and ORF6 with the recombinase/invertase (acc . no . AF060871) found in Rhodococcus rhodochrous (46.4%).

J Bacteriol, 2000 Aug, 182(15), 4241 - 8
Simultaneous identification of two cyclohexanone oxidation genes from an environmental Brevibacterium isolate using mRNA differential display; Brzostowicz PC et al.; The technique of mRNA differential display was used to identify simultaneously two metabolic genes involved in the degradation of cyclohexanone in a new halotolerant Brevibacterium environmental isolate . In a strategy based only on the knowledge that cyclohexanone oxidation was inducible in this strain, the mRNA population of cells exposed to cyclohexanone was compared to that of control cells using reverse transcription-PCR reactions primed with a collection of 81 arbitrary oligonucleotides . Three DNA fragments encoding segments of flavin monooxygenases were isolated with this technique, leading to the identification of the genes of two distinct cyclohexanone monooxygenases, the enzymes responsible for the oxidation of cyclohexanone . Each monooxygenase was expressed in Escherichia coli and characterized . This work validates the application of mRNA differential display for the discovery of new microbial metabolic genes.

Int J Food Microbiol, 2000 Jun 15, 57(3), 201 - 10
Characterization of Brevibacterium linens pigmentation using spectrocolorimetry; Guyomarc'h F et al.; Spectrocolorimetry was used for the evaluation of Brevibacterium linens pigmentation . A total of 23 strains of Brevibacterium linens, from various sources, were plated onto solid agar media and their color coordinates were measured and expressed by the L*a*b* colorimetric system . All the strains were located along a straight line with a hue of 66 degrees (orange color) . The effect of light exposure versus storage in the dark was also investigated using this approach . Three groups with distinct behaviors were demonstrated for the 23 B . linens strains.

Biosci Biotechnol Biochem, 2000 Apr, 64(4), 761 - 6
Development of an improved assay for purine nucleoside kinase activity in cell extracts and detection of inosine kinase activity in Brevibacterium acetylicum ATCC 953, related species, and Corynebacterium flaccumfaciens ATCC 6887; Kawasaki H et al.; An improved assay was developed to detect direct purine nucleoside phosphorylating activity in cell-free extracts . Direct inosine phosphorylating activity was detected in 2 of 70 species tested . Both activities, which depended on magnesium ion and ATP, phosphorylated a hydroxyl group at the 5' position of inosine . The new assay was shown to be useful for screening of direct purine nucleoside phosphorylating activity and have the potential to detect inosine kinase in the presence of a background of nucleoside phosphorylase and purine phosphoribosyltransferase activities . Previously, the latter two activities made it difficult to correctly detect direct phosphorylation of inosine by inosine kinase.

Biochim Biophys Acta, 2000 May 23, 1478(2), 211 - 20
Salvage pathway for NAD biosynthesis in Brevibacterium ammoniagenes: regulatory properties of triphosphate-dependent nicotinate phosphoribosyltransferase; Dulyaninova NG et al.; As the rate-limiting enzyme, catalyzing the first reaction in NAD salvage synthesis, nicotinate phosphoribosyltransferase (NAPRTase, EC 2.4.2.11) is of important interest for studies of intracellular pyridine nucleotide pool regulation . We have purified NAPRTase 520-fold from Brevibacterium ammoniagenes ATCC 6872 without using an over-expression system by applying acid treatment, salt fractionation, Ca-phosphate gel treatment, anion exchange column chromatography and size-exclusion gel filtration . Unlike this enzyme from other sources, B . ammoniagenes NAPRTase was found to be controlled by the feedback inhibition by the end product NAD with K(i)=0.7+/-0.1 mM . The reaction products, pyrophosphate and nicotinate mononucleotide, also decreased the enzyme activity, as did other intermediates of NAD synthesis, such as AMP, ADP and a NAD direct precursor, nicotinate adenine dinucleotide or deamido NAD . The enzyme was observed to require a nucleoside triphosphate for its activity and showed the maximum affinity for ATP . The specificity, however, turned out to be poor, and ATP could be substituted by other nucleoside triphosphates as well as by sodium triphosphate . The kinetic characteristics of the enzyme are reported . For the first time, our data have experimentally revealed such complicated stimulatory and inhibitory effects by the intermediates of NAD biosynthesis on one of its salvage enzymes, NAPRTase . On the basis of these data, the key role of NAPRTase is discussed in light of the regulation of NAD metabolism in B . ammoniagenes.

Mol Gen Genet, 2000 Apr, 263(3), 423 - 32
A carotenogenic gene cluster from Brevibacterium linens with novel lycopene cyclase genes involved in the synthesis of aromatic carotenoids; Krubasik P et al.; The carotenogenic (crt) gene cluster from Brevibacterium linens, a member of the commercially important group of coryneform bacteria, was cloned and identified . An expression library of B . linens genes was constructed and a fragment of the crt cluster was obtained by functional complementation of a colourless B . flavum mutant, screening transformed cells for production of a yellow pigment . Subsequent screening of a cosmid library resulted in the cloning of the whole crt cluster from B . linens . All genes necessary for the synthesis of the aromatic carotenoid isorenieratene were identified on the basis of sequence homologies . In addition a novel type of lycopene cyclase was identified by complementation of a lycopene-accumulating B . flavum mutant . Two genes, named crt Yc and crt Yd, which code for polypeptides of 125 and 107 amino acids, respectively, are necessary to convert lycopene to beta-carotene . The amino acid sequences of these polypeptides show no similarity to any of the known lycopene cyclases . This is the first example of a carotenoid biosynthetic conversion in which two different gene products are involved, probably forming a heterodimer.

J Chromatogr B Biomed Sci Appl, 2000 Jan 28, 738(1), 181 - 5
Detection of mycoloylglycerol by thin-layer chromatography as a tool for the rapid inclusion of corynebacteria of clinical origin in the genus Corynebacterium; Yague G et al.; A chemotaxonomic study of some corynebacteria isolated from clinical samples revealed characteristic thin-layer chromatographic patterns for meso-diaminopimelic acid containing species included in the genera Corynebacterium, Dermabacter and Brevibacterium . Notably, a specific compound was consistently detected in mycolic acid containing species of the genus Corynebacterium . This compound was composed by glycerol and mycolic acids and structural analyses carried out by fast atom bombardment mass spectrometry in C . minutissimum confirmed its identification as mycoloylglycerol . The chain length of mycoloyl groups in this molecule ranged from 28 to 34 carbon atoms, being mono-, di- or triunsaturated . Detection of mycoloylglycerol by thin-layer chromatography may be thus useful for the rapid inclusion of a great variety of corynebacteria of clinical origin in the genus Corynebacterium in laboratories employing chromatographic techniques as an adjunct for the identification of these microorganisms.

FEMS Microbiol Lett, 2000 Apr 15, 185(2), 199 - 204
A Brevibacterium lactofermentum 16S rRNA gene used as target site for homologous recombination; Amador E et al.; Genes for rRNA are highly conserved and present in multiple copies in most prokaryotic organisms increasing the number of theoretical sites for homologous recombination . They might be targets for integration events between unrelated microorganisms providing that an efficient genetic transfer is present . We have used a plasmid containing a portion of the 16S rRNA gene from the rrnD operon of Brevibacterium lactofermentum to transform the same strain resulting in non-essential inactivation of various rrn operons . Integration of the transforming DNA occurs in all cases . The system may be used to test possible gene transfer at least among closely related strains and is of great interest for integration of foreign DNA and for mapping.

J Clin Microbiol, 2000 Apr, 38(4), 1676 - 8
Identification of coryneform bacterial isolates by ribosomal DNA sequence analysis; Tang YW et al.; Identification of coryneform bacteria to the species level is important in certain circumstances for differentiating contamination and/or colonization from infection, which influences decisions regarding clinical intervention . However, methods currently used in clinical microbiology laboratories for the species identification of coryneform bacteria are often inadequate . We evaluated the MicroSeq 500 16S bacterial sequencing kit (Perkin-Elmer Biosystems, Foster City, Calif.), which is designed to sequence the first 527 bp of the 16S rRNA gene for bacterial identification, by using 52 coryneform gram-positive bacilli from clinical specimens isolated from January through June 1993 at the Mayo Clinic . Compared to conventional and supplemented phenotypic methods, MicroSeq provided concordant results for identification to the genus level for all isolates . At the species level, MicroSeq provided concordant results for 27 of 42 (64.3%) Corynebacterium isolates and 5 of 6 (83.3%) Corynebacterium-related isolates, respectively . Within the Corynebacterium genus, MicroSeq gave identical species-level identifications for the clinically significant Corynebacterium diphtheriae (4 of 4) and Corynebacterium jeikeium (8 of 8), but it identified only 50.0% (15 of 30) of other species (P < 0.01) . Four isolates from the genera Arthrobacter, Brevibacterium, and Microbacterium, which could not be identified to the species level by conventional methods, were assigned a species-level identification by MicroSeq . The total elapsed time for running a MicroSeq identification was 15.5 to 18.5 h . These data demonstrate that the MicroSeq 500 16S bacterial sequencing kit provides a potentially powerful method for the definitive identification of clinical coryneform bacterium isolates.

Vaccine, 2000 Jan 18, 18(13), 1236 - 43
Comparative study of the effects of peptidoglycan monomer and structurally related adamantyltripeptides on humoral immune response to ovalbumin in the mouse; Tomasic J et al.; Peptidoglycan monomer, GlcNAc-MurNAc-L-Ala-D-isoGln-mesoDAP(omega NH2)-D-Ala-D-Ala (PGM) originating from Brevibacterium divaricatum and synthetic adamantyltripeptides, diastereoisomers of D,L-(adamant-2-yl)-Gly-L-Ala-D-isoGln (AdTP1 and AdTP2) exhibit immunomodulating activity . An experimental model in the mouse has been established with suboptimal amounts of ovalbumin (OVA) as the immunogen, and parallel testing of adjuvant activity of these three immunomodulators was carried out in Balb/c, C57B16 or CBA mice . Tested compounds (100 or 200 micrograms/mouse) mixed with OVA in saline (50 micrograms/mouse) were administered s.c . Anti-OVA was assayed by ELISA in the sera of mice taken 7 days after the boosters (given on days 14 and 28) . The treatment with PGM and one of the diastereoisomers, AdTP2, resulted in significantly higher increase in anti-OVA IgG levels (stimulation index up to 46) with respect to controls and groups treated with AdTP1 . The effect of AdTP2 treatment was comparable to that of PGM in most experiments after the first booster, but after the second booster PGM exhibited markedly better effect . PGM and AdTP2 also induced markedly higher levels of IgG1 and IgG2 anti-OVA subclasses than detected in controls and AdTP1 treated mice, indicating that these two immunomodulators might upregulate both Th1-like and Th2-like immune responses.

Arch Microbiol, 2000 Jan, 173(1), 78 - 82
Indole-inducible proteins in bacteria suggest membrane and oxidant toxicity; Garbe TR et al.; Oxidant toxicity of indole was demonstrated by the induction of alkylhydroperoxide reductase subunit C (AhpC) in Escherichia coli K12 and by the constitutive overproduction of AhpC in a variant of E . coli JM109 with enhanced resistance to indole . Oxidant toxicity was also indicated in an indole-adapted variant of Brevibacterium flavum by the indole-inducible overproduction of a novel 36-kDa protein with N-terminal sequence similarity to proteins involved in superoxide and singlet oxygen resistance . It is proposed that indole dissolved in membrane lipids, which caused membrane derangement and enabled direct interaction of redox-cycling isoprenoid quinones and dioxygen, resulting in the generation of superoxide . A direct indication of membrane derangement in E . coli may be the indole-inducible overproduction of spheroplast protein y (Spy).

Appl Microbiol Biotechnol, 1999 Dec, 53(1), 98 - 107
Degradation of phenanthrene by different bacteria: evidence for novel transformation sequences involving the formation of 1-naphthol; Samanta SK et al.; Four polycyclic aromatic hydrocarbon (PAH)-degrading bacteria, namely Arthrobacter sulphureus RKJ4, Acidovorax delafieldii P4-1, Brevibacterium sp . HL4 and Pseudomonas sp . DLC-P11, capable of utilizing phenanthrene as the sole source of carbon and energy, were tested for its degradation using radiolabelled phenanthrene . {9-14C}Phenanthrene was incubated with microorganisms containing 100 mg/l unlabelled phenanthrene and the evolution of 14CO2 was monitored: within 18 h of incubation, 30.1, 35.6, 26.5 and 2.1% of the recovered radiolabelled carbon was degraded to 14CO2 by RKJ4, P4-1, HL4 and DLC-P11, respectively . When mixtures of other PAHs such as fluorene, fluoranthene and pyrene, in addition to phenanthrene, were added as additional carbon sources, there was a 36.1 and 20.6% increase in 14CO2 production from {9-14C}phenanthrene in the cases of RKJ4 and HL4, respectively, whereas P4-1 and DLC-P11 did not show any enhancement in 14CO2 production . Although, a combination of many bacteria enhances the degradation of organic compounds, no enhancement in the degradation of {9-14C}phenanthrene was observed in mixed culture involving all four microorganisms together . However, when different PAHs, as indicated above, were used in mixed culture, there was a 68.2% increase in 14CO2 production . In another experiment, the overall growth rate of P4-1 on phenanthrene could be enhanced by adding the non-ionic surfactant Triton X-100, whereas RKJ4, HL4 and DLC-P11 did not show any enhancement in growth . Pathways for phenanthrene degradation were also analysed by thin-layer chromatography, gas chromatography and gas chromatography-mass spectrometry . Common intermediates such as o-phthalic acid and protocatechuic acid were detected in the case of RKJ4 and o-phthalic acid was detected in the case of P4-1 . A new intermediate, 1-naphthol, was detected in the cases of HL4 and DLC-P11 . HL4 degrades phenanthrene via 1-hydroxy-2-naphthoic acid, 1-naphthol and salicylic acid, whereas DLC-P11 degrades phenanthrene via the formation of 1-hydroxy-2-naphthoic acid, 1-naphthol and o-phthalic acid . Both transformation sequences are novel and have not been previously reported in the literature . Mega plasmids were found to be present in RKJ4, HL4 and DLC-P11, but their involvement in phenanthrene degradation could not be established.

Biosci Biotechnol Biochem, 1999 Nov, 63(11), 1965 - 9
Structural conversion from non-native to native form of recombinant human epidermal growth factor by Brevibacillus choshinensis; Miyauchi A et al.; Brevibacillus choshinensis (Bacillus brevis) HPD31 is a very efficient producer of recombinant human epidermal growth factor (EGF) . The produced EGF is secreted into the medium with high efficiency . However part of the EGF that accumulates in the medium, exists as multimeric forms which are biologically inactive . We found the bacterium has the activity to structurally convert multimeric forms to the monomeric, native ones . Optimal temperature and pH for the conversion were 40 degrees C and pH 9, respectively . The reaction was promoted in the presence of reduced glutathione or cysteine . But the cells which had been sonicated or exposed to moderate heat treatment completely lost the activity . Thus, it was presumed that the activity might be due to the enzyme(s) that catalyze the protein disulfide exchanging reaction, and that they resides on the surface of viable cells.

Biosci Biotechnol Biochem, 1999 Sep, 63(9), 1582 - 8
Molecular cloning of isomaltotrio-dextranase gene from Brevibacterium fuscum var . dextranlyticum strain 0407 and its expression in Escherichia coli; Mizuno T et al.; The gene encoding an extracellular isomaltotrio-dextranase (IMTD), designed dexT, was cloned from the chromosomal DNA of Brevibacterium fuscum var . dextranlyticum strain 0407, and expressed in Escherichia coli . A single open reading frame consisting of 1923 base pairs that encoded a polypeptide composed of a signal peptide of 37 amino acids and a mature protein of 604 amino acids (M(r), 68,300) was found . The primary structure had no significant similarity with the structure of two other reported exo-type dextranases (glucodextranase and isomalto-dextranase), but had high similarity with that of an endo-dextranase isolated from Arthrobacter sp . Transformed E . coli cells carrying the gene encoding mature protein of IMTD overproduced IMTD under the control of the T7 phage promoter induced by IPTG . The purified recombinant enzyme showed the same optimum pH, lower specific activity, and similar hydrolytic pattern, as to those of native IMTD.

Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1527 - 30
Brevibacterium avium sp . nov., isolated from poultry; Pascual C et al.; Two strains of a Brevibacterium-like bacterium originating from bumble-foot lesions of domestic fowls were subjected to a polyphasic taxonomic study . The phenotypic characteristics of the bacterium were consistent with its assignment to the genus Brevibacterium although comparative 16S rRNA gene sequencing showed that the organism represents a distinct subline within the genus . Chromosomal DNA-DNA pairing studies confirmed that the unidentified bacterium was genomically distinct and worthy of separate species status . Based on the phenotypic and genotypic distinctiveness of the bacterium from poultry, a new species, Brevibacterium avium, is proposed . The type strain of Brevibacterium avium is NCIMB 703055T.

Appl Environ Microbiol, 1999 Nov, 65(11), 5182 - 5
Genotypic diversity among Brevibacillus laterosporus strains; Zahner V et al.; In comparison with other entomopathogenic Bacillus species, the genome of Brevibacillus laterosporus is poorly characterized . The aim of this study was to examine genetic variability in B . laterosporus by using a range of typing methodologies . Strains of B . laterosporus were examined for variation in 13 chromosomal genes encoding enzymes by multilocus enzyme electrophoresis . Optimal conditions of pulsed-field gel electrophoresis and randomly amplified polymorphic DNA were established that allowed analysis of the genome of B . laterosporus . None of these techniques allowed the identification of a convenient molecular marker for entomopathogenic strains, although one specific primer amplified only DNA from almost all mosquitocidal strains.

Antonie Van Leeuwenhoek, 1999 Jul-Nov, 76(1-4), 247 - 61
Sulfur metabolism in bacteria associated with cheese; Weimer B et al.; Metabolism of sulfur in bacteria associated with cheese has long been a topic of interest . Volatile sulfur compounds, specifically methanethiol, are correlated to desirable flavor in Cheddar cheese, but their definitive role remains elusive . Only recently have enzymes been found that produce this compound in bacteria associated with cheese making . Cystathionine beta- and gamma-lyase are found in lactic acid bacteria and are capable of producing methanethiol from methionine . Their primary function is in the metabolism of cysteine . Methionine gamma-lyase produces methanethiol from methionine at a higher efficiency than the cystathionine enzymes . This enzyme is found in brevibacteria, bacilli, and pseudomonads . Addition of brevibacteria containing this enzyme improves Cheddar cheese flavor . Despite recent progress in sulfur metabolism more information is needed before cheese flavor associated with sulfur can be predicted or controlled.

Izv Akad Nauk Ser Biol, 1999 Jul-Aug, (4), 403 - 13
{The biosynthesis of 2H-labelled inosine by Bacillus subtilis bacteria in a heavy-hydrogen medium}; Mosin OV et al.; We studied biosynthesis of the purine ribonucleoside inosine by the strain of Bacillus subtilis, which was adapted to deuterium through plating to individual colonies on 2% agar with 2H2O and subsequent selection . For growing the strain, a special heavy hydrogen medium with a high level of deuteration (89-90 at . % 2H) based on 2% hydrolyzate of the biomass of the methylotrophic Brevibacterium methylicum as a source of 2H-labeled growth substrates was obtained on 98 vol % 2H2O and 2 vol % {U-2H} methanol . We provide experimental data on the strain growth and accumulation of inosine in the culture medium . A study of the level of inosine deuteration using mass-spectroscopy of fast atoms has shown a polymorphism of deuterium incorporation in the molecule (isotope composition of inosine: 4 at . 2H, 20%; 5 at . 2H, 38%; 6 at . 2H, 28%; 7 at . 2H, 14%), deuterium being incorporated in the ribose and hypoxanthine fragments of the molecules.

Mol Gen Genet, 1999 Jul, 261(6), 1032 - 44
The eff-482 locus of Sinorhizobium meliloti CXM1-105 that influences symbiotic effectiveness consists of three genes encoding an endoglycanase, a transcriptional regulator and an adenylate cyclase; Sharypova LA et al.; The mutant T482 of Sinorhizobium meliloti CXM1-105, which carries a Tn5 insertion on megaplasmid 1, exhibits an enhanced symbiotic efficiency phenotype . Three genes, eglC, cya3 and syrB2, were identified in the eff-482 region tagged by the Tn5 insertion in T482 . The eglC gene encodes an endoglycanase which contributes to the depolymerization of the exo-polysaccharide succinoglycan . The N-terminal region of the predicted cya3 gene product was similar to eukaryotic-type adenylate cyclases from Brevibacterium liquefaciens and Streptomyces coelicolor . Four contiguous tetratricopeptide repeats which are known to mediate protein-protein interactions were identified in the C-terminal portion of Cya3 . Complementation analysis demonstrated that cya3 indeed encodes a functional adenylate cyclase . A central helix-turn-helix DNA-binding motif and a putative C-terminal coiled-coil structure implicated in protein oligomerization were found in SyrB2 . Extra copies of the syrB2 gene negatively affect transcription of both syrB2 itself and cya3 . The Tn5 insertion in T482 was localized between the divergently transcribed genes eglC and syrB2 . It eliminated eglC function and slightly stimulated transcription of both syrB2 and cya3, which lies downstream of syrB2 . Mutants carrying insertions of the lacZ-Gm interposon in the genes eglC, syrB2 and cya3 exhibit the same phenotype as mutant T482, indicating that these three genes influence symbiotic efficiency.

Biotechnol Appl Biochem, 1999 Aug, 30 ( Pt 1), 27 - 33
Cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum: effect of surfactants and organic solvents on activity; Pollegioni L et al.; We have studied systematically the effect of the non-ionic surfactants Thesit and Triton X-100, and of propan-2-ol (used as a substrate solubilizer) on the activity of the cholesterol oxidases from Streptomyces hygroscopicus (SCO) and Brevibacterium sterolicum (BCO) . Low concentrations of Thesit lead to an activity increase with both enzymes; at higher surfactant concentrations the opposite effect occurs . Triton X-100 inactivates both enzymes at all concentrations . It is deduced that these surfactants exert their effects by interaction with the enzymes and not by affecting micellar phenomena . The effect of propan-2-ol on SCO, in contrast with that on BCO, depends on the buffer concentration (potassium phosphate) . Other organic solvents induce results similar to those obtained with SCO and propan-2-ol . A significant difference between the two cholesterol oxidases emerges when stability is tested at 25 degrees C and in the presence of different concentrations of propan-2-ol: BCO activity is rapidly inactivated, whereas SCO still has 70% of the initial activity after 5 h in the presence of 30% propan-2-ol . From our results, SCO seems to be the catalyst of choice in comparison with BCO for the exploitation of cholesterol oxidases in biotechnology and applied biochemistry.

FEBS Lett, 1999 Jul 30, 456(1), 215 - 9
Propeptide of the metalloprotease of Brevibacillus brevis 7882 is a strong inhibitor of the mature enzyme; Serkina AV et al.; A metalloprotease gene of Brevibacillus brevis (npr) was expressed in Escherichia coli in a soluble form as native Npr precursor . A significant fraction of the precursor was spontaneously processed, producing the N-terminal propeptide and the mature enzyme . A strong inhibition of the mature Npr by its own propeptide in the crude lysate was observed even in the absence of the covalent linkage between them . Pure precursor, propeptide and the mature Npr were isolated and kinetic parameters of the mature enzyme inhibition by the propeptide were determined . The inhibition is of the tight-binding competitive type with Ki 0.17 nM . Inhibition of metalloproteases from Brevibacillus megaterium and thermolysine by the heterologous propeptide of the Npr from B . brevis was much weaker or none.

Eur J Biochem, 1999 Aug, 264(1), 140 - 51
Kinetic mechanisms of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum; Pollegioni L et al.; The kinetic properties of two cholesterol oxidases, one from Brevibacterium sterolicum (BCO) the other from Streptomyces hygroscopicus (SCO) were investigated . BCO works via a ping-pong mechanism, whereas the catalytic pathway of SCO is sequential . The turnover numbers at infinite cholesterol and oxygen concentrations are 202 s-1 and 105 s-1 for SCO and BCO, respectively . The rates of flavin reduction extrapolated to saturating substrate concentration, under anaerobic conditions, are 235 s-1 for BCO and 232 s-1 for SCO (in the presence of 1% Thesit and 10% 2-propanol) . With reduced SCO the rate of Delta5-6-->Delta4-5 isomerization of the intermediate 5-cholesten-3-one to final product is slow (0.3 s-1) . With oxidized SCO and BCO the rate of isomerization is much faster ( approximately 300 s-1), thus it is not rate-limiting for catalysis . The kinetic behaviour of both reduced COs towards oxygen is unusual in that they exhibit apparent saturation with increasing oxygen concentrations (extrapolated rates approximately 250 s-1 and 1.3 s-1, for BCO and SCO, respectively): too slow to account for catalysis . For BCO the kinetic data are compatible with a step preceding the reaction with oxygen, involving interconversion of reactive and nonreactive forms of the enzyme . We suggest that the presence of micelles in the reaction medium, due to the necessary presence of detergents to solubilize the substrate, influence the availability or reactivity of oxygen towards the enzyme . The rate of re-oxidation of SCO in the presence of product is also too slow to account for catalysis, probably due to the impossibility of producing quantitatively the reduced enzyme-product complexes.

Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 961 - 7
Comparative phylogeny of rrs and nifH genes in the Bacillaceae; Achouak W et al.; The rrs (16S rDNA) gene sequences of nitrogen-fixing endospore-forming bacilli isolated from the rhizosphere of wheat and maize were determined in order to infer their phylogenetic position in the Bacillaceae . These rhizosphere strains form a monophyletic cluster with Paenibacillus azotofixans, Paenibacillus polymyxa and Paenibacillus macerans . Two of them (RSA19 and TOD45) had previously been identified as Bacillus circulans (group 2) by phenotypic characterization (API 50CH) . Evidence for nitrogen fixation by P . azotofixans, P . polymyxa, P . macerans and putative B . circulans strains RSA19 and TOD45 was provided by acetylene-reduction activity, and confirmed by amplifying and sequencing a nifH fragment (370 nt) . The phylogenetic tree of nifH-derived amino acid sequences was compared to the phylogenetic tree of rrs sequences . All Paenibacillus nifH sequences formed a coherent cluster distinct from that of related nitrogen-fixing anaerobic clostridia and Gram-positive high-G+C-content frankiae . The nifH gene was neither detected in the B . circulans type strain (ATCC 4513T) nor in the type strains of Bacillus subtilis, Bacillus cereus, Bacillus alcalophilus, Bacillus simplex, Brevibacillus brevis and Paenibacillus validus . Accordingly, nitrogen fixation among aerobic endospore-forming Firmicutes seems to be restricted to a subset of species in the genus Paenibacillus.

J Antimicrob Chemother, 1999 Jun, 43 Suppl C, 27 - 32
In-vitro activity of levofloxacin, ofloxacin and D-ofloxacin against coryneform bacteria and Listeria monocytogenes; Martinez-Martinez L et al.; The objective of this study was to evaluate the in-vitro activity of levofloxacin, ofloxacin and D-ofloxacin compared with ciprofloxacin, norfloxacin and sparfloxacin against coryneform bacteria and Listeria monocytogenes isolated from clinical samples . The following organisms (and number of strains) were studied: Corynebacterium jeikeium (20), Corynebacterium urealyticum (20), Corynebacterium minutissimum (20), Corynebacterium striatum (20), Corynebacterium amycolatum (30), Brevibacterium spp . (15) and Listeria monocytogenes (15) . Antimicrobial activity was determined by microdilution using cation-adjusted Mueller-Hinton broth supplemented with 0.5% Tween 80 when testing C . jeikeium or C . urealyticum . Fluoroquinolones were used in the range 0.015-16 mg/L . Plates were incubated in air at 35 degrees C for 18-20 h (24 h when testing C . jeikeium or C . urealyticum) . The following MIC50 values were obtained for all 140 organisms tested: levofloxacin, 1 mg/L; ofloxacin, 2 mg/L; D-ofloxacin, > 16 mg/L; ciprofloxacin, 1 mg/L; norfloxacin, 16 mg/L; sparfloxacin, 1 mg/L . MIC90 values were > 16 mg/L for all test antibiotics with the exception of levofloxacin, which had an MIC90 value of 16 mg/L . At a concentration of 2 mg/L, levofloxacin inhibited all L . monocytogenes strains and 35-93% of the remaining species . MIC90 values of ofloxacin were one dilution step higher than those of levofloxacin against C . minutissimum, C . striatum, Brevibacterium spp . and L . monocytogenes . Levofloxacin showed similar (C . jeikeium, C . urealyticum, C . amycolatum and Brevibacterium spp.) or greater (C . minutissimum and C . striatum) activity than ciprofloxacin and sparfloxacin, and higher than D-ofloxacin or norfloxacin against all species studied . In conclusion, levofloxacin was the most active of the six fluoroquinolones evaluated against coryneform bacteria isolated from clinical samples and could therefore be a promising treatment option in this setting.

FEMS Microbiol Lett, 1999 Jun 15, 175(2), 193 - 6
Isolation of the murI gene from Brevibacterium lactofermentum ATCC 13869 encoding D-glutamate racemase; Malathi KC et al.; The murI gene encoding D-glutamate racemase plays an important role in the biosynthesis of D-glutamic acid, an essential component of cell wall peptidoglycan of almost all eubacteria . A DNA fragment that could rescue the auxotrophy of D-glutamic acid in the Escherichia coli murI mutant strain WM335 was isolated from Brevibacterium lactofermentum ATCC 13869 belonging to the coryneform bacteria . DNA sequencing reveals that it encodes a protein of 284 amino acid residues, which shows a high level of homology with D-glutamate racemases from several other bacteria.

Biosci Biotechnol Biochem, 1999 Apr, 63(4), 688 - 97
A new carboxylesterase from Brevibacterium linens IFO 12171 responsible for the conversion of 1,4-butanediol diacrylate to 4-hydroxybutyl acrylate: purification, characterization, gene cloning, and gene expression in Escherichia coli; Sakai Y et al.; A carboxylesterase that is responsible for conversion of 1,4-butanediol diacrylate (BDA) to 4-hydroxybutyl acrylate (4HBA) was found in Brevibacterium lines IFO 12171, and purified to homogeneity . The purified enzyme was active toward a variety of diesters of ethylene glycol, 1,4-butanediol, and 1,6-hexanediol . The K(m) and kcat of the enzyme for BDA were 3.04 mM and 203,000 s-1, respectively . The reaction with the purified enzyme gave 98 mM 4HBA from 100 mM BDA for 60 min . The enzyme gene was cloned from the chromosomal DNA of the bacterium . The open reading frame encoding the enzyme was 1176 bp long, corresponding to a protein of 393 amino acid residues (molecular mass = 42,569 Da) . The deduced amino acid sequence contained the tetra peptide motif sequence, STTK, and the serine residue was confirmed to be the catalytic center of BDA esterase by site-directed mutagenesis for several amino acid residues . The gene was expressed in Escherichia coli under the control of the lac promoter, and the gene product (a fusion protein with 6 amino acid residues from beta-galactosidase) showed the same catalytic properties as the enzyme from the parent strain.

Int J Food Microbiol, 1999 Mar 1, 47(1-2), 89 - 97
Analysis of the bacterial surface ripening flora of German and French smeared cheeses with respect to their anti-listerial potential; Carnio MC et al.; The anti-listerial potential of 19 different French smeared cheese bacterial consortia was analyzed semi-quantitatively . Comparison of the total viable cell count to the direct microscopic count yielded no indication that non-culturable bacteria contribute significantly to the undefined, complex ripening floras . From 2613 isolates, 48 showed clear inhibition of one or more Listeria monocytogenes strains on solid medium, while only three isolates excreted an anti-listerial, soluble substance when grown in liquid culture . From a total of 299 strains isolated from German dairy products, 30 strains inhibited at least one L . monocytogenes indicator strain on solid medium . Bacteria with antilisterial potential were members of the genera Arthrobacter, Brevibacterium, Corynebacterium, Enterococcus, Micrococcus and Microbacterium . Cluster analysis of inhibition pattern revealed that similar anti-listerial substances are produced by species belonging to different genera . Half of the strains inhibited six or more out of 12 Listeria indicator strains and are promising candidates for the development of a defined red smear cheese ripening flora . L . monocytogenes originating from red-smear cheese had a tendency to be more resistant towards anti-listerial activity than those isolated from other sources.

J Microbiol Methods, 1999 May, 36(1-2), 115 - 22
Actinomycetes in Karstic caves of northern Spain (Altamira and Tito Bustillo); Groth I et al.; A variety of isolation procedures were carried out to study the involvement of bacteria in the colonisation and biodeterioration of Spanish caves with paleolithic rock art (Altamira and Tito Bustillo) . The applied techniques mainly aimed to isolate heterotrophic bacteria such as streptomycetes, nocardioform and coryneform actinomycetes, and other gram-positive and gram-negative bacteria . The results demonstrated that actinomycetes were the most abundant gram-positive bacteria in the caves . Actinomycetes revealed a great taxonomic diversity with the predominant isolates belonging to the genus Streptomyces . Members of the genera Nocardia, Rhodococcus, Nocardioides, Amycolatopsis, Saccharothrix, Brevibacterium, Microbacterium, and coccoid actinomycetes (family Micrococcaceae) were also found.

Lett Appl Microbiol, 1999 May, 28(5), 394 - 400
Improved cytotoxicity assay for Bacillus cereus diarrhoeal enterotoxin; Fletcher P et al.; An improved McCoy cell cytotoxicity assay for Bacillus cereus diarrhoeal toxin, which includes a staining procedure in addition to visual examination, was developed and the method was compared with two commercially available kits (Oxoid BCET-RPLA and Tecra BDE-VIA) . A total of 71 strains of 15 different Bacillus, Brevibacillus and Paenibacillus species, including 16 strains of B . cereus from outbreaks of food-borne illness, were evaluated for toxin production . Eleven of the outbreak strains exhibited cytotoxicity, including all six B . cereus strains associated with diarrhoeal illness . Several other isolates of B . cereus, and its relatives B . anthracis, B . mycoides and B . thuringiensis, exhibited similar cytotoxicity . The other species showed no cytotoxicity, with the exception of certain B . subtilis strains . The cytotoxicity assay was more sensitive than the Oxoid kit and unlike the Tecra kit, did not give false positive results with supernatant fluids heat-treated to destroy the toxin.

Appl Environ Microbiol, 1999 Jun, 65(6), 2520 - 6
Microbial system for polysaccharide depolymerization: enzymatic route for xanthan depolymerization by Bacillus sp . strain GL1; Nankai H et al.; An enzymatic route for the depolymerization of a heteropolysaccharide (xanthan) in Bacillus sp . strain GL1, which was closely related to Brevibacillus thermoruber, was determined by analyzing the structures of xanthan depolymerization products . The bacterium produces extracellular xanthan lyase catalyzing the cleavage of the glycosidic bond between pyruvylated mannosyl and glucuronyl residues in xanthan side chains (W . Hashimoto et al., Appl . Environ . Microbiol . 64:3765-3768, 1998) . The modified xanthan after the lyase reaction was then depolymerized by extracellular beta-D-glucanase to a tetrasaccharide, without the terminal mannosyl residue of the side chain in a pentasaccharide, a repeating unit of xanthan . The tetrasaccharide was taken into cells and converted to a trisaccharide (unsaturated glucuronyl-acetylated mannosyl-glucose) by beta-D-glucosidase . The trisaccharide was then converted to the unsaturated glucuronic acid and a disaccharide (mannosyl-glucose) by unsaturated glucuronyl hydrolase . Finally, the disaccharide was hydrolyzed to mannose and glucose by alpha-D-mannosidase . This is the first complete report on xanthan depolymerization by bacteria . Novel beta-D-glucanase, one of the five enzymes involved in the depolymerization route, was purified from the culture fluid . This enzyme was a homodimer with a subunit molecular mass of 173 kDa and was most active at pH 6.0 and 45 degrees C . The enzyme specifically acted on xanthan after treatment with xanthan lyase and released the tetrasaccharide.

Curr Microbiol, 1999 Jun, 38(6), 320 - 3
A gene (tmpA) for an efflux protein of the transporter family III from Brevibacterium linens OC2, an antibacterial substance-producing strain; Boucabeille C et al.; A gene (tmpA) encoding a putative transmembrane protein has been cloned from B . linens OC2, an antibacterial substance-producing strain . The deduced TmpA protein sequence shares similarities to members of the transporter family III exploiting the transmembrane proton gradient to provide export of toxic compounds such as antiseptics or antibiotics . Northern blot analysis indicated that tmpA gene is expressed . Length of RNA messenger and overlapping of ORFs upstream tmpA gene suggested that it might belong to an operon . The tmpA gene is unusual among B . linens species since it was not detected among eight B . linens collection strains and 40 B . linens industrial strains.

J Dairy Sci, 1999 May, 82(5), 891 - 909
Aspects of enzymology and biochemical properties of Brevibacterium linens relevant to cheese ripening: a review; Rattray FP et al.; Brevibacterium linens is a major surface microorganism that is present in the smear of surface-ripened cheeses . The enzymology and biochemical characteristics of B . linens influence the ripening and final characteristics of smear surface-ripened cheeses . Proteolytic, peptidolytic, esterolytic, and lipolytic activities, which are of particular importance in the ripening process, are discussed in detail . This review also describes the production of volatile compounds, especially sulfur-containing ones, by B . linens, which are thought to be important in respect to the flavor of smear surface-ripened cheeses . The unique orange-colored carotenoids and the factors effecting their production by B . linens are also presented . The catabolism of aromatic amino acids, bacteriocin production, plasmids, and miscellaneous biochemical and physiological properties (peptidoglycan type, antibiotic resistance, insecticide degradation, and biotechnological applications) of B . linens are discussed . The problem associated with the current taxonomical classification of B . linens strains caused by strain variation is evaluated . Finally, the application of B . linens cell extracts or its proteolytic enzymes as cheese ripening accelerants for semi-hard or hard cheese varieties is considered.

Biochemistry (Mosc), 1999 Apr, 64(4), 384 - 9
Isolation and primary characterization of an amidase from Rhodococcus rhodochrous; Kotlova EK et al.; Amidase (EC 3.5.1.4) was purified to homogeneity from Rhodococcus rhodochrous M8 using isopropanol fractionation and exchange chromatography on Mono Q . The isolated amidase consists of four identical subunits with molecular weight 42+/-2 kD . The activity of the enzyme is maximal at 55-60 degrees C and within the pH range 5-8 . The amidase from R . rhodochrous M8 is highly sensitive to such sulfhydryl reagents as Hg2+ and Cu2+ . Chelators (EDTA and o-phenanthroline) and serine proteinase inhibitors (PMSF and DIFP) did not inhibit the activity of the enzyme . The enzyme exhibits hydrolytic and acyl transferase activity and does not possess urease activity . Aliphatic amides (acetamide and propionamide) were the best substrates for the amidase from R . rhodochrous M8, whereas bulky aromatic amides were poor substrates of this enzyme . The properties of the isolated enzyme are similar to those found in the corresponding amidase from Arthrobacter sp . J-1 and an amidase with wide substrate specificity from Brevibacterium sp . R312.

Appl Environ Microbiol, 1999 May, 65(5), 2232 - 4
Biodegradation of cyclohexylamine by Brevibacterium oxydans IH-35A; Iwaki H et al.; A bacterial strain capable of growing on cyclohexylamine (CHAM) was isolated by using enrichment and isolation techniques . The strain isolated, strain IH-35A, was classified as a member of the genus Brevibacterium . The results of growth and enzyme studies are consistent with degradation of CHAM via cyclohexanone (CHnone), 6-hexanolactone, 6-hydroxyhexanoate, and adipate . Cell extracts obtained from this strain grown on CHAM contained CHAM oxidase, and the model for CHAM oxidation by this enzyme was similar to the model for deamino oxidation of amine by amine oxidase.

Microbiology, 1999 Apr, 145 ( Pt 4), 915 - 24
Structure and organization of the rrnD operon of 'Brevibacterium lactofermentum': analysis of the 16S rRNA gene; Amador E et al.; Five rRNA operons (rrn) were found by hybridization in the genome of 'Brevibacterium lactofermentum' ATCC 13869 and Corynebacterium glutamicum ATCC 13032 . 'B . lactofermentum' DSM 20412 differed from the other corynebacteria tested in showing six hybridizing BamHI bands . Two of the rrn operons (rrnD and rrnE) were located in a single cosmid . Sequencing of the rrnD operon showed that it contains a complete 16S rRNA-23S RNA-5S rRNA gene cluster . Phylogenetic studies using the complete 16S rRNA sequence showed that 'B . lactofermentum' is closely related to several species of the genus Corynebacterium but only distantly related to the type species Brevibacterium linens and the authors suggest that it should be reclassified as Corynebacterium lactofermentum . The 5' end of mature 16S rRNA was identified by primer extension . Sequence elements similar to those of mycobacteria implicated in transcription antitermination (Boxes A, B, C) and in processing of the pre-rRNA to 16S rRNA were identified . An open reading frame encoding an rpoD-like sigma factor (named SigC) different from the previously reported SigA and SigB proteins was found upstream of rrnD in the opposite orientation . Both rpoD and sigC seem to be expressed from a bidirectional promoter region.

Microbiology, 1999 Mar, 145 ( Pt 3), 529 - 38
Organization of genes for tetrapyrrole biosynthesis in gram--positive bacteria; Johansson P et al.; Clusters of genes encoding enzymes for tetrapyrrole biosynthesis were cloned from Bacillus sphaericus, Bacillus stearothermophilus, Brevibacillus brevis and Paenibacillus macerans . The sequences of all hemX genes found, and of a 6.3 kbp hem gene cluster from P . macerans, were determined . The structure of the hem gene clusters was compared to that of other Gram-positive bacteria . The Bacillus and Brevibacillus species have a conserved organization of the genes hemAXCDBL, required for biosynthesis of uroporphyrinogen III (UroIII) from glutamyl-tRNA . In P . macerans, the hem genes for UroIII synthesis are also closely linked but their organization is different: there is no hemX gene and the gene cluster also contains genes, cysG8 and cysG(A)-hemD, encoding the enzymes required for synthesis of sirohaem from UroIII . Bacillus subtilis contains genes for three proteins, NasF, YInD and YInF, with sequence similarity to Escherichia coli CysG, which is a multi-functional protein catalysing sirohaem synthesis from UroIII . It is shown that YInF is required for sirohaem synthesis and probably catalyses the precorrin-2 to sirohaem conversion . YInD probably catalyses precorrin-2 synthesis from UroIII and NasF seems to be specific for nitrite reduction.

Biochemistry, 1999 Apr 6, 38(14), 4277 - 86
Crystal structure determination of cholesterol oxidase from Streptomyces and structural characterization of key active site mutants; Yue QK et al.; Cholesterol oxidase is a monomeric flavoenzyme which catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one . The enzyme interacts with lipid bilayers in order to bind its steroid substrate . The X-ray structure of the enzyme from Brevibacterium sterolicum revealed two loops, comprising residues 78-87 and residues 433-436, which act as a lid over the active site and facilitate binding of the substrate {Vrielink et al . (1991) J . Mol . Biol . 219, 533-554; Li et al . (1993) Biochemistry 32, 11507-11515} . It was postulated that these loops must open, forming a hydrophobic channel between the membrane and the active site of the protein and thus sequestering the cholesterol substrate from the aqueous environment . Here we describe the three-dimensional structure of the homologous enzyme from Streptomyces refined to 1.5 A resolution . Structural comparisons to the enzyme from B . sterolicum reveal significant conformational differences in these loop regions; in particular, a region of the loop comprising residues 78-87 adopts a small amphipathic helical turn with hydrophobic residues directed toward the active site cavity and hydrophilic residues directed toward the external surface of the molecule . It seems reasonable that this increased rigidity reduces the entropy loss that occurs upon binding substrate . Consequently, the Streptomyces enzyme is a more efficient catalyst . In addition, we have determined the structures of three active site mutants which have significantly reduced activity for either the oxidation (His447Asn and His447Gln) or the isomerization (Glu361Gln) . Our structural and kinetic data indicate that His447 and Glu361 act as general base catalysts in association with conserved water H2O541 and Asn485 . The His447, Glu361, H2O541, and Asn485 hydrogen bond network is conserved among other oxidoreductases . This catalytic tetrad appears to be a structural motif that occurs in flavoenzymes that catalyze the oxidation of unactivated alcohols.

Appl Environ Microbiol, 1999 Apr, 65(4), 1811 - 2
Semiautomated metabolic staining assay for Bacillus cereus emetic toxin; Finlay WJ et al.; This paper describes a specific, sensitive, semiautomated, and quantitative Hep-2 cell culture-based 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay for Bacillus cereus emetic toxin . Of nine Bacillus, Brevibacillus, and Paenibacillus species assessed for emetic toxin production, only B . cereus was cytotoxic.

Appl Microbiol Biotechnol, 1999 Feb, 51(2), 223 - 8
A murC gene from coryneform bacteria; Wachi M et al.; The upstream flanking region of the ftsQ and ftsZ genes of Brevibacterium flavum MJ233, which belongs to the coryneform bacteria, was amplified by the inverse polymerase chain reaction method and cloned in Escherichia coli . Complementation analysis of E . coli mutant with a defective cell-wall synthesis mechanism with the cloned fragment and its DNA sequencing indicated the presence of the murC gene, encoding UDP-N-acetylmuramate:L-alanine ligase involved in peptidoglycan synthesis, just upstream from the ftsQ gene . The B . flavum murC gene could encode a protein of 486 amino acid residues with a calculated molecular mass of 51 198 Da . A 50-kDa protein was synthesized by the B . flavum murC gene in an in vitro transcription/translation system using E . coli S30 lysate . These results indicate that the genes responsible for cell-wall synthesis and cell division are located as a cluster in B . flavum similar to the E . coli mra region.

Genet Anal, 1999 Mar, 15(1), 9 - 13
Cloning and nucleotide sequencing of the secA gene from coryneform bacteria; Kobayashi M et al.; Taking advantage of highly conserved domains present in the secA gene from Escherichia coli and Bacillus subtilis, we designed degenerate oligonucleotides (oligos) corresponding to these regions . These oligos were used as primers in PCR in order to amplify DNA sequences from Brevibacterium flavum MJ233 chromosomal DNA . The PCR product was used as a probe to recover genomic fragments from a lambda library of Br . flavum MJ233 . The complete nucleotide sequence (nt) of the cloned 5.3-kb EcoR1 fragment containing the secA homolog from Br . flavum MJ233 indicated that the deduced gene product of the Br . flavum secA homolog is composed of 845 amino acids (aa) with a deduced molecular weight (MW) of 95429 . Comparison of this aa sequence to the corresponding sequences from E . coli and B . subtilis revealed a high degree of conservation and suggested that the Br . flavum secA homolog has putative ATP binding regions.

Int J Syst Bacteriol, 1999 Jan, 49 Pt 1, 185 - 8
Reclassification of Brevibacterium incertum (Breed 1953) as Desemzia incerta gen . nov., comb . nov; Stackebrandt E et al.; Phylogenetic analysis of 16S rDNA indicates that Brevibacterium incertum is not a member of the genus Brevibacterium but related to species of the genus Carnobacterium . Hence, Brevibacterium incertum is not a member of the class Actinobacteria but belongs to the phylogenetically defined broad Bacillus-Lactobacillus cluster . Based upon properties that taxonomically clearly distinguishes Brevibacterium incertum from species of the phylogenetic sister genus Carnobacterium, Brevibacterium incertum is reclassified as Desemzia incerta gen . nov., comb . nov.

Int J Syst Bacteriol, 1999 Jan, 49 Pt 1, 175 - 7
Reclassification of Brevibacterium oxydans (Chatelain and Second 1966) as Microbacterium oxydans comb . nov; Schumann P et al.; Phylogenetic and chemotaxonomic analyses indicate that Brevibacterium oxydans is closely related to species of the genus Microbacterium, namely Microbacterium liquefaciens, Microbacterium luteolum and Microbacterium saperdae . DNA-DNA reassociation values of less than 60% between Brevibacterium oxydans and these three Microbacterium species support the distinctness of this misclassified Brevibacterium species, which is reclassified as Microbacterium oxydans comb . nov.

Br J Dermatol, 1998 Dec, 139 Suppl 53, 9 - 12
Skin bacteriology and the role of Staphylococcus aureus in infection; Noble WC; Many of the staphylococci and coryneforms that inhabit normal human skin do not cause skin disease . Amongst the remainder the mechanisms of pathogenicity vary widely . For Proteus, Pseudomonas and Brevibacterium species proteolysis is a major determinant . The precise role of Corynebacterium minutissimum in erythrasma and the propionibacteria in acne is not known . Staphylococcus aureus, however, produces a wide range of non-specific agents, such as haemolysins and leucocidins as well as highly specific toxins such as the epidermolytic toxins involved in bullous impetigo and scalded skin syndrome . Most of the current attention, however, is devoted to the role of the enterotoxins and toxic shock toxin as superantigens, with emphasis on their role in atopic dermatitis . Molecularly similar toxins in the streptococci play a similar role and may also have a role in the aetiology of psoriasis.

Biochem Mol Biol Int, 1998 Dec, 46(6), 1153 - 60
Complementation of the growth defect of an rnpA49 mutant of Escherichia coli by overexpression of arginine tRNA(CCG); Kim MS et al.; We previously found that overexpression of arginine tRNA(CCG) from Brevibacterium albidum complements the rnpA49 mutation, which is responsible for the thermosensitivity of Escherichia coli RNase P function . In this present work, we show that the E . coli homologue tRNA also complements the same mutation, but other tRNAs do not . These results suggest that the rnpA49 mutation causes a major cellular defect in an RNase P reaction to generate the mature arginine tRNA(CCG).

FEBS Lett, 1998 Dec 4, 440(3), 393 - 8
Homomultimeric protease in the hyperthermophilic bacterium Thermotoga maritima has structural and amino acid sequence homology to bacteriocins in mesophilic bacteria; Hicks PM et al.; A novel homomultimeric protease (> 669 kDa), based on 31 kDa subunits, was purified from cell extracts of the hyperthermophilic bacterium Thermotoga maritima . This protease exhibits activity toward chymotrypsin and trypsin substrates, optimally at 90 degrees C and pH 7.1, and has a half-life of 36 min at 95 degrees C . Transmission electron microscopy established that the protease consists of a large globular assembly which appears circular from the front view . The function of this protease in T . maritima remains unclear, although putative homologs include a 29 kDa antigen from Mycobacterium tuberculosis and a 31 kDa monomer of a high molecular weight bacteriocin produced by Brevibacterium linens {Valdes-Stauber, N . and Scherer, S . (1996) Appl . Environ . Microbiol . 62, 1283-1286} . The relationship of these mesophilic proteins to the T . maritima protease suggests that their antibacterial activity may involve elements of proteolysis, and raises the prospect for antimicrobial ecological strategies in hyperthermophilic niches.

Antimicrob Agents Chemother, 1998 Dec, 42(12), 3290 - 2
In vitro activities of ketolide HMR 3647, macrolides, and clindamycin against Coryneform bacteria; Martinez-Martinez L et al.; The in vitro activity of ketolide HMR 3647 against coryneform bacteria isolated from clinical samples was evaluated . Except against Corynebacterium jeikeium and C . urealyticum, HMR 3647 showed high activity against Corynebacterium spp., being more active than 14- and 16-membered macrolides, azithromycin, or clindamycin . HMR 3647 also had high in vitro activity against Brevibacterium spp . and Listeria monocytogenes.

Biochim Biophys Acta, 1998 Nov 8, 1442(2-3), 373 - 9
Molecular characterization of guanosine kinase gene from a facultative alkalophile, Exiguobacterium aurantiacum ATCC 35652; Usuda Y et al.; Guanosine kinase activity was identified in an alkalophile, Exiguobacterium aurantiacum ATCC 35652, and the gsk gene encoding it was isolated as a homologous fragment of the Brevibacterium acetylicum gsk . The cloned chromosomal fragment contained an open reading frame which encodes a polypeptide of 308 amino acids with a molecular mass of 33349 Da . The Ex . aurantiacum Gsk shows strong sequence similarity to B . acetylicum Gsk, but possesses five additional amino acids at the C-terminus . An Mr 39200 protein synthesized in Escherichia coli harboring plasmids carrying Ex . aurantiacum gsk was purified and characterized . A 0.9 kb transcript revealed by Northern blot analysis and the presence of a factor-independent terminator structure suggest that Ex . aurantiacum gsk is monocistronic, although B . acetylicum gsk has been shown to be bicistronic . The striking alterations in the 3'-untranslated region resulted in a different gene organization.

Microbiology, 1998 Oct, 144 ( Pt 10), 2827 - 36
pBLA8, from Brevibacterium linens, belongs to a gram-positive subfamily of ColE2-related plasmids; Leret V et al.; A 3.1 kb DNA fragment from pBLA8, a Brevibacterium linens cryptic plasmid, containing all the information required for autonomous replication was cloned and sequenced . Using deletion analysis, the fragment essential and sufficient for autonomous replication was delimited to 1.5 kb . This fragment is characterized by the presence of an ori site located upstream of an operon encoding two proteins, RepA and RepB, both essential for replication . Based on structural similarities and a strong conservation of ori, RepA and RepB, pBLA8 was assigned to a new subfamily of the ColE2 plasmid family . This subfamily is distinguished by the requirement for two Rep proteins and the location of an ori site upstream of the repAB operon . RepA is thought to encode primase activity, whereas RepB could be a DNA-binding protein . An Escherichia coli-B . linens shuttle vector, derived from pBLA8, was constructed . Its host spectrum was extended to Arthrobacter species.

Res Microbiol, 1998 Mar, 149(3), 211 - 9
Carnitine acts as a compatible solute in Brevibacterium linens; Jebbar M et al.; Carnitine is a trimethyl amino acid found at relatively high concentrations in materials of animal origin . Exogenously provided L-carnitine was found to stimulate growth of Brevibacterium linens ATCC 19391 in media with inhibitory osmotic strength . Its osmoprotective ability was as potent as that of glycine betaine . Electrophoretic and spectroscopic (NMR) analysis showed that this compound is only transiently accumulated, but in significant amounts, by B . linens under hyperosmotic stress and is converted into glycine betaine . The L-carnitine/glycine betaine pathway is inducible by L-carnitine in B . linens . The D-enantiomer did not improve growth of B . linens, even though this solute is accumulated by B . linens at the same level as glycine betaine . The two isomeric forms of carnitine repress the build-up of ectoine, the main endogenous osmolyte in B . linens.

Appl Environ Microbiol, 1998 Oct, 64(10), 3641 - 7
Intracellular changes in ions and organic solutes in halotolerant brevibacterium sp . Strain JCM 6894 after exposure to hyperosmotic shock
Nagata S, Adachi K, Sano H.
In the present study we aimed to observe the intracellular responses when there was a hyperosmotic shock with a large shift in ionic strength in nutrient-rich and nutrient-poor external environments in order to clarify the availability of substrates . To do this, we used the halotolerant organism Brevibacterium sp . strain JCM 6894, which is able to grow in the presence of a wide range of salt concentrations . Hyperosmotic shock was induced by transferring cells in the late exponential phase of growth in a complex medium containing 0.5 M NaCl into either old or fresh culture medium containing 2 M NaCl . Changes in the growth rate, in the pH of the medium, and in the internal cation or organic solute concentrations in the cytosol after an upshock were analyzed as a function of incubation time . The cells exhibited very different responses to upshocks in fresh culture medium and in old culture medium; in fresh culture medium, growth was stimulated and the medium became more acidic, whereas the old culture medium repressed growth and the medium became more alkaline . The intracellular free Na+ concentrations remained low (80 nmol mg of protein-1) after an upshock in fresh culture medium, although they quickly increased twofold in the old culture medium . In contrast, K+ ions immediately accumulated in the cells in fresh culture medium, whereas K+ ions were taken up quite slowly in old culture medium . Furthermore, the cells placed in fresh culture medium transiently accumulated alanine and glutamine in response to the upshock, but the cells placed in old culture medium did not . Growth of the Brevibacterium strain at higher levels of salinity was supported by ectoine synthesis but was not observed after the shift to high-osmolarity conditions in the old culture . In the fresh culture, however, ectoine was vigorously synthesized in cells for more than 5 h after the upshock; the concentration of ectoine in cells was more than 3,500 nmol mg of protein-1 at 10 h, which corresponded to a ninefold increase compared to the concentration before the shock . These findings are consistent with the results of an analysis of the extracellular medium composition before and after the upshock.

Mol Gen Genet, 1998 Jul, 259(1), 97 - 104
Identification, characterization, and chromosomal organization of the ftsZ gene from Brevibacterium lactofermentum; Honrubia MP et al.; The ftsZ gene was cloned from the chromosomal DNA of Brevibacterium lactofermentum by the polymerase chain reaction (PCR) using two oligonucleotides designed from two conserved regions found in most of the previously cloned and sequenced ftsZ genes from other microorganisms . ftsZ is a single-copy gene in corynebacteria and is located downstream from ftsQ and murC, indicating linkage between genes involved in peptidoglycan synthesis (mur genes) and genes involved in cell division (fts genes) . The organisation of the cluster is similar to that in Streptomyces and different from those of Escherichia coli or Bacillus subtilis because ftsA is not located upstream of ftsZ . The gene was expressed in E . coli using the T7 expression system; the calculated molecular weight of the expressed protein was 50 kDa . Expression of the B . lactofermentum ftsZ gene in E . coli inhibited cell division and led to filamentation . The ftsZ gene of this organism does not complement ftsZ mutations or deletions in E . coli, when cloned on low or high-copy-number vectors.

Appl Environ Microbiol, 1998 Sep, 64(9), 3327 - 31
Purification and characterization of L-methionine gamma-lyase from brevibacterium linens BL2
Dias B, Weimer B.
L-Methionine gamma-lyase (EC 4.4.1.11) was purified to homogeneity from Brevibacterium linens BL2, a coryneform bacterium which has been used successfully as an adjunct bacterium to improve the flavor of Cheddar cheese . The enzyme catalyzes the alpha,gamma elimination of methionine to produce methanethiol, alpha-ketobutyrate, and ammonia . It is a pyridoxal phosphate-dependent enzyme, with a native molecular mass of approximately 170 kDa, consisting of four identical subunits of 43 kDa each . The purified enzyme had optimum activity at pH 7.5 and was stable at pHs ranging from 6.0 to 8.0 for 24 h . The pure enzyme had its highest activity at 25 degreesC but was active between 5 and 50 degreesC . Activity was inhibited by carbonyl reagents, completely inactivated by DL-propargylglycine, and unaffected by metal-chelating agents . The pure enzyme had catalytic properties similar to those of L-methionine gamma-lyase from Pseudomonas putida . Its Km for the catalysis of methionine was 6.12 mM, and its maximum rate of catalysis was 7.0 &mgr;mol min-1 mg-1 . The enzyme was active under salt and pH conditions found in ripening Cheddar cheese but susceptible to degradation by intracellular proteases.

Appl Environ Microbiol, 1998 Sep, 64(9), 3320 - 6
Conversion of methionine to thiols by lactococci, lactobacilli, and brevibacteria
Dias B, Weimer B.
Methanethiol has been strongly associated with desirable Cheddar cheese flavor and can be formed from the degradation of methionine (Met) via a number of microbial enzymes . Methionine gamma-lyase is thought to play a major role in the catabolism of Met and generation of methanethiol in several species of bacteria . Other enzymes that have been reported to be capable of producing methanethiol from Met in lactic acid bacteria include cystathionine beta-lyase and cystathionine gamma-lyase . The objective of this study was to determine the production, stability, and activities of the enzymes involved in methanethiol generation in bacteria associated with cheese making . Lactococci and lactobacilli were observed to contain high levels of enzymes that acted primarily on cystathionine . Enzyme activity was dependent on the concentration of sulfur amino acids in the growth medium . Met aminotransferase activity was detected in all of the lactic acid bacteria tested and alpha-ketoglutarate was used as the amino group acceptor . In Lactococcus lactis subsp . cremoris S2, Met aminotransferase was repressed with increasing concentrations of Met in the growth medium . While no Met aminotransferase activity was detected in Brevibacterium linens BL2, it possessed high levels of L-methionine gamma-lyase that was induced by addition of Met to the growth medium . Met demethiolation activity at pH 5.2 with 4% NaCl was not detected in cell extracts but was detected in whole cells . These data suggest that Met degradation in Cheddar cheese will depend on the organism used in production, the amount of enzyme released during aging, and the amount of Met in the matrix.

Appl Environ Microbiol, 1998 Sep, 64(9), 3416 - 21
Mode of action of linenscin OC2 against Listeria innocua; Boucabeille C et al.; Linenscin OC2 is a small hydrophobic substance produced by the orange cheese coryneform bacterium Brevibacterium linens OC2 . Linenscin OC2 inhibits growth of gram-negative bacteria with an altered outer membrane permeability and gram-positive bacteria . It is also able to lyse eucaryotic cells . The mode of action of linenscin OC2 on the Listeria innocua cytoplasmic membrane and the effects of environmental parameters were investigated . Addition of low doses of linenscin OC2 resulted in an immediate perturbation of the permeability properties of the cytoplasmic membrane and of the bacterial energetic state . Linenscin OC2 induced a loss of cytoplasmic potassium, depolarization of the cytoplasmic membrane, complete hydrolysis of internal ATP, efflux of inorganic phosphate, and transient increase in oxygen consumption . Potassium loss occurred in the absence of a proton motive force and was severely reduced at low temperatures, presumably as a result of increased ordering of the lipid hydrocarbon chains of the cytoplasmic membrane . We propose that linenscin OC2 interacts with the cytoplasmic membrane and that the permeability changes observed at low doses reflect the formation of pore-like structures in this membrane.

J Biol Chem, 1998 Aug 28, 273(35), 22334 - 9
A novel phosphopantetheine:protein transferase activating yeast mitochondrial acyl carrier protein; Stuible HP et al.; In Saccharomyces cerevisiae, the low molecular weight acyl carrier protein (ACP) of mitochondrial type II fatty acid synthase (FAS) and the cytoplasmic type I FAS multienzyme contain 4'-phosphopantetheine as a prosthetic group . Sequence alignment studies with the recently isolated phosphopantetheine:protein transferase (PPTase), Ppt1p, from Brevibacterium ammoniagenes revealed the yeast open reading frame, YPL148C, as a potential PPTase gene (25% identical and 43% conserved amino acids) . In accordance with this similarity, pantetheinylation of mitochondrial ACP was lost upon disruption of YPL148C . In contrast, biosynthesis of cytoplasmic holo-FAS remained unaffected by this mutation . According to these characteristics, the newly identified gene was designated as PPT2 . Similar to ACP null mutants, cellular lipoic acid synthesis and, hence, respiration were abolished in PPT2 deletants . ACP pantetheinylation, lipoic acid synthesis, and respiratory competence were restored upon transformation of PPT2 mutants with cloned PPT2 DNA . In vitro, holo-ACP synthesis was achieved by incubating apo-ACP with coenzyme A in the presence of purified Ppt2p . The homologous yeast enzyme could be replaced, in this assay, by the ACP synthase (EC 2.7.8.7) of Escherichia coli but not by the type I FAS-specific PPTase of B . ammoniagenes, Ppt1p . These results conform with the inability of Ppt2p to activate the cytoplasmic type I FAS complex of yeast.

FEMS Microbiol Lett, 1998 Jul 15, 164(2), 411 - 8
Cloning and sequencing of a cyclodextrin glycosyltransferase gene from Brevibacillus brevis CD162 and its expression in Escherichia coli; Kim MH et al.; A cyclodextrin glycosyltransferase (CGTase) gene of Brevibacillus brevis CD162 was cloned into Escherichia coli using pUC19 as a vector . Determination of the nucleotide sequence showed the presence of an open reading frame of 2079 bp encoding a polypeptide of 693 amino acid residues, composed of a 20-amino acid signal sequence and a 673-amino acid mature enzyme . Neither a TATA- nor a TTGA-like sequence was observed within the cloned DNA fragment . However, the fragment was expressed in Escherichia coli by the lac promoter of pUC19 and 74% of the total activity was secreted into the fermentation medium . The amino acid sequence of the mature CGTase showed the highest homology of 86% to that of Bacillus sp . KC201 . The CGTase purified to homogeneity from the recombinant E . coli exhibited the same properties as those of native CGTase from Brevibacillus brevis CD162 in terms of molecular mass, reaction conditions, stability and the production of cyclodextrins.

J Food Prot, 1998 Jul, 61(7), 874 - 8
Degradation of histamine and tyramine by Brevibacterium linens during surface ripening of Munster cheese; Leuschner RG et al.; Red smear formation during fermentation of Munster cheese was started by using three different strains of Brevibacterium linens as surface inocula . The cheeses were produced with and without supplementation of histamine and tyramine . After smearing the cheese surface for the first time with B . linens viable counts of 10(7) CFU/g were detected . At the end of the logarithmic growth phase cell numbers increased to 10(10) CFU/g and remained constant during the whole ripening period . During a 4-week ripening period strains of B . linens reduced histamine and tyramine content by 55 to 70% . B . linens LTH 456 and LTH 3686 degraded histamine and tyramine in a phosphate buffer (pH 7) containing 0.54 M histamine and 0.58 M tyramine when incubated with agitation at 30 degrees C . B . linens LTH 3813 did not reveal any amine degradation activity in a buffer system . The pH on the cheese surface increased from 5 to 7, whereas it increased in the center only to 5.3 after a 3-week ripening period.

J Basic Microbiol, 1998, 38(2), 101 - 6
New shuttle vectors for Rhodococcus sp . R312 (formerly Brevibacterium sp . R312), a nitrile hydratase producing strain; Duran R; Two shuttle vectors named pRC52 (10.7 Kb) and pRK52 (12.2 Kb) carrying chloramphenicol (Cm) and chloramphenicol plus kanamycin (Km) resistance genes, respectively, were constructed by fusion of a cryptic plasmid pBL13869 (replicon pBL1, 5.8 Kb) from Brevibacterium lactofermentum ATCC13869 with pBR328 E . coli plasmid . Transformation of Rhodococcus sp . R312 (formerly Brevibacterium sp . R312) protoplasts was realised with an efficiency of 28 transformants per micrograms of DNA.

Biochemistry, 1998 Jun 2, 37(22), 7992 - 6
A divalent metal site in the small subunit of the manganese-dependent ribonucleotide reductase of Corynebacterium ammoniagenes; Griepenburg U et al.; Based on its metallo-cofactor, the manganese-dependent ribonucleotide reductase (Mn-RRase) responsible for delivery of DNA precursors in the Mn-requiring Gram-positive bacterium Corynebacterium (formerly Brevibacterium) ammoniagenes ATCC 6872 is no longer considered as a simple analogue of the aerobic Fe-RRase of Escherichia coli but as the prototype of the class IV enzymes (1) . Deliberate dissociation of the Mn-RRase holoenzyme and an improved sample preparation of the dimeric CA2 protein allowed further characterization of the inherent metallo-cofactor by Q-band electron paramagnetic resonance (EPR) spectroscopy . At 40 K, a distinct hyperfine sextet (I = 5/2,55Mn) pattern with a weak zero-field splitting was detected in the CA2 protein prepared from manganese-sufficient cells displaying high RRase activity as expected . This Q-band Mn(II) signal was absent in the apo-CA2 protein obtained from manganese-depleted cells devoid of this enzymatic activity . The presence of a mixed valence manganese cluster in the C . ammoniagenes RRase is excluded since no complex multiline EPR signals were detected in the CA2 protein even at very low (8 K) temperature . The observed Mn(II) spectrum indicates a protein-bound manganese which was modified in the presence of 5.7 mM p-methoxyphenol, but is insensitive toward 10 mM EDTA . Thus, the manganese appeared to be either strictly bound or buried within a hydrophobic pocket of the CA2 protein, inaccessible for EDTA.

Mol Gen Mikrobiol Virusol, 1998, (2), 35 - 8
{Novel site-specific endonucleases from Brevibacterium species}; Sokolov NN et al.; New site-specific endonucleases BecAI and BecAII have been detected in Brevibacterium species A . Endonuclease BecAII free from contaminating nonspecific endonucleases, exonucleases, and phosphatases was isolated by column chromatography on phosphocellulose, heparin sepharose, and DNA cellulose . It recognizes and cleaves the 5'-GG decreases CC-3' sequence and is a true isoschizomer of HaeIII restriction enzyme . The other restriction endonuclease, BecAI, cleaves Ad2 DNA at least by 2 sites but not the DNA of phage lambda, T7, SV40, phiX174, and plasmides pBR322 and pUC19 . The substrate specificity of BecAI indicates its appurtenance to the super rare restriction endonucleases.

Infect Immun, 1998 Jun, 66(6), 2728 - 35
Identification and characterization of a 29-kilodalton protein from Mycobacterium tuberculosis culture filtrate recognized by mouse memory effector cells; Rosenkrands I et al.; Culture filtrate proteins from Mycobacterium tuberculosis induce protective immunity in various animal models of tuberculosis . Two molecular mass regions (6 to 10 kDa and 24 to 36 kDa) of short-term culture filtrate are preferentially recognized by Th1 cells in animal models as well as by patients with minimal disease . In the present study, the 24- to 36-kDa region has been studied, and the T-cell reactivity has been mapped in detail . Monoclonal antibodies were generated, and one monoclonal antibody, HYB 71-2, with reactivity against a 29-kDa antigen located in the highly reactive region below the antigen 85 complex was selected . The 29-kDa antigen (CFP29) was purified from M . tuberculosis short-term culture filtrate by thiophilic adsorption chromatography, anion-exchange chromatography, and gel filtration . In its native form, CFP29 forms a polymer with a high molecular mass . CFP29 was mapped in two-dimensional electrophoresis gels as three distinct spots just below the antigen 85 complex component MPT59 . CFP29 is present in both culture filtrate and the membrane fraction from M . tuberculosis, suggesting that this antigen is released from the envelope to culture filtrate during growth . Determination of the N-terminal amino acid sequence allowed cloning and sequencing of the cfp29 gene . The nucleotide sequence showed 62% identity to the bacteriocin Linocin from Brevibacterium linens . Purified recombinant histidine-tagged CFP29 and native CFP29 had similar T-cell stimulatory properties, and they both elicited the release of high levels of gamma interferon from mouse memory effector cells isolated during the recall of protective immunity to tuberculosis . Interspecies analysis by immunoblotting and PCR demonstrated that CFP29 is widely distributed in mycobacterial species.

Protein Expr Purif, 1998 Apr, 12(3), 347 - 52
Increased expression of Brevibacterium sterolicum cholesterol oxidase in Escherichia coli by genetic modification; Sampson NS et al.; To improve expression of Brevibacterium sterolicum cholesterol oxidase in Escherichia coli, we utilized the T7lac promoter and modified the gene to encode the first 21 amino acids with high-expression E . coli codons . These changes resulted in a 60-fold improvement of expression level . N-terminal sequencing revealed that the E . coli produced cholesterol oxidase signal peptide is cleaved 6 amino acids closer to the N-terminus than in B . sterolicum . The recombinant E . coli produced protein is composed of 513 amino acids with a calculated Mr of 55,374 . The kinetic rate constants of the recombinant protein and the B . sterolicum produced cholesterol oxidase are identical .

Lett Appl Microbiol, 1998 Mar, 26(3), 183 - 8
Typing of Bacillus sporothermodurans and other Bacillus species isolated from milk by repetitive element sequence based PCR; Herman L et al.; When analysing raw milk for the presence of Bacillus sporothermodurans, 11 Bacillus strains were isolated which could be differentiated from known Bacillus, Brevibacillus and Paenibacillus spp . by different primer specificity in PCR experiments, indicating that they probably belong to Bacillus species as yet undescribed . Using repetitive element sequence based PCR (REP-PCR), these 11 strains could be clearly distinguished from B . sporothermodurans as well as from each other . Eighty-five B . sporothermodurans strains were characterized by a typical REP-pattern . Using REP-PCR combined with separation on non-denaturing polyacrylamide gel electrophoresis and silver staining, individual B . sporothermodurans strains could be discriminated, which was not possible by methods previously published.

Int J Food Microbiol, 1998 Jan 6, 39(1-2), 1 - 10
Histamine and tyramine degradation by food fermenting microorganisms; Leuschner RG et al.; Microorganisms suitable for food fermentation were examined with regard to their potential to degrade histamine and tyramine . Out of 64 lactic acid bacteria evaluated in this study, 27 degraded histamine and one tyramine, respectively, with low activity . Among 32 strains of Brevibacterium linens and coryneform bacteria, 21 exhibited histamine and tyramine oxidase activity . None of 20 strains of Staphylococcus carnosus tested degraded histamine or tyramine . One strain out of nine strains of Geotrichum candidum degraded tyramine slightly . Among 44 strains of Micrococcus sp . examined, 17 degraded either one or two biogenic amines . In this study Micrococcus varians (M . varians) LTH 1540 exhibited the highest tyramine oxidase activity of all strains tested and was therefore investigated in detail . The enzyme was found to be located in the cytoplasm and was not membrane bound . The reaction end product p-hydroxyphenyl acetic acid was detected by HPLC analysis . An activity staining for the amine oxidase in a native polyacrylamide gel based on the formation of H2O2 during amine oxidation was developed . Resting cells of the strain exhibited optimal tyramine oxidase activity at a pH of 7 at 37-40 degrees C . The enzyme in the cell free extract had a pH optimum between 7-8 . The enzyme activity was decreased by NaCl, glucose and hydralazine . Phenylethylamine and tryptamine were oxidized at lower concentrations than tyramine . The potential for amine degradation was not found to be associated with that of formation of biogenic amines, as 23 microorganisms with the ability to metabolise biogenic amines exhibited no decarboxylase activity toward histidine, tyrosine, phenylalanine, lysine or ornithine.

Eur J Biochem, 1998 Feb 15, 252(1), 90 - 9
Glucose oxidase from Penicillium amagasakiense . Primary structure and comparison with other glucose-methanol-choline (GMC) oxidoreductases; Kiess M et al.; The complete amino acid sequence of glucose oxidase from Penicillium amagasakiense was determined by Edman degradation and mass spectrometry of peptide fragments derived from three different specific proteolytic digests and a cyanogen bromide cleavage . The complete sequence of each monomer comprises 587 amino acid residues, contains three cysteine residues, and seven potential N-glycosylation sites, of which at least five were confirmed to be glycosylated . Glucose oxidase from P . amagasakiense shows a high degree of identity (66%) and 79% similarity to glucose oxidase from Aspergillus niger, and is a member of the glucose-methanol-choline (GMC) oxidoreductase family . The tertiary structures of glucose oxidase from A . niger and cholesterol oxidase from Brevibacterium sterolicum were superimposed to provide a template for the sequence comparison of members of the GMC family . The general topology of the GMC oxidoreductases is conserved, with the exception of the presence of an active site lid in cholesterol oxidase and the insertion of additional structural elements in the substrate-binding domain of alcohol oxidase . The overall structure can be divided into five distinct sequence regions: FAD-binding domain, extended FAD-binding domain, flavin attachment loop and intermediate region, FAD covering lid, and substrate-binding domain . The FAD-binding and the extended FAD-binding domains are composed of several separate sequence regions . The other three regions each comprise a single contiguous sequence . Four major consensus patterns have been identified, including the nucleotide-binding consensus sequence close to their N-termini . The functions of the two motifs recently selected by the Genetics Computer Group, Madison, Wisconsin, as additional signature patterns of the GMC oxidoreductases are discussed . The other consensus patterns belong to either the FAD-binding or the extended FAD-binding domain . In addition, the roles of conserved residues are discussed wherever possible.

Protein Expr Purif . 1998 Mar;12(2):iv.
Papers to Appear in Forthcoming Issues
Purification and characterization of the 2-ketoaldonate reductase from Brevibacterium ketosoreductum ATCC21914.
Bioprocess Engineering Division, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Taejon, Korea2-Ketoaldonate reductase, which is involved in ketogluconate catabolism, was purified to homogeneity from Brevibacterium ketosoreductum ATCC21914 . The enzyme was found to catalyze the reduction of 2,5-diketo-D-gluconate to 5-keto-D-gluconate, and to a lesser extent, 2-keto-D-gluconate to D-gluconate, and 2-keto-L-gluconate to L-idonate . The molecular mass of the reductase was 35 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 72 kDa by gel filtration, indicating that the native enzyme may exist as a dimer . The reductase was optimally active at pH 6.0 with NADPH as a preferred electron donor . The pI of 4.7 was measured for the enzyme . The apparent Km for 2,5-diketo-D-gluconate and NADPH were 5 microM and 10 microM, respectively . The amino-terminal amino acid sequence was NH2-Ala-Ser-Ile-Ser-Val-Ser-Val-Pro-Ser-Ala- Arg-Leu-Ala-Glu-Asp-Leu-Ser-Asp-Ile-Glu.

J Clin Microbiol, 1998 Feb, 36(2), 543 - 7
Evaluation of the RapID CB Plus system for identification of Corynebacterium species and other gram-positive rods; Hudspeth MK et al.; Due to the difficulty of identifying Corynebacterium spp . with standard methods, we compared them with the RapID CB Plus system (Remel, Lenexa, Kans . {formerly Innovative Diagnostic Systems, Norcross, Ga.}), which consists of 4 carbohydrate and 14 preformed enzyme tests, for the identification of 98 clinical isolates of Corynebacterium sp., other coryneforms, Listeria monocytogenes, and 17 ATCC strains . Forty (95%) of 42 strains of Corynebacterium spp . were accurately identified to the species level by the RapID CB Plus system, and two additional strains of C . striatum were identified with one additional conventional test for lipid requirement . Twenty-seven (75%) of the 36 coryneform strains tested were identified correctly to the species level . However, three of four strains of Brevibacterium sp . and all seven of the L . monocytogenes strains were identified to the genus level only . Actinomyces strains had variable results, and the one strain of Arcanobacterium haemolyticum tested was not identified . Overall, the RapID CB Plus system compared favorably with the conventional methods, was easy to inoculate and interpret, and is promising as a new method for identification of gram-positive bacilli.

Eur J Biochem, 1997 Dec 1, 250(2), 369 - 76
Characterization of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum; Gadda G et al.; The FAD-containing enzyme cholesterol oxidase catalyzes the oxidation and isomerization of 3beta-hydroxysteroids having a trans double bond at delta5-delta6 of the steroid ring backbone to the corresponding delta4-3-ketosteroid . Two representative enzymes of this family, namely cholesterol oxidase from Streptomyces hygroscopicus (SCO) and the recombinant enzyme from Brevibacterium sterolicum (BCO) expressed in Escherichia coli, have been characterized herein in their chemical, physical, and biochemical properties . In the native form, both enzymes are monomeric (55 kDa), acidic (pI 4.4-5.1) and contain oxidized FAD (peaks in the 370-390-nm and 440-470-nm regions) . Marked differences exist between the oxidized, reduced, and (red) anion semiquinone spectra of the two enzymes, suggesting substantial differences in the flavin microenvironment . Both enzymes form reversibly flavin N(5)-sulfite adducts via measurable k(on) and k(off) steps . BCO has a higher affinity for sulfite (Kd approximately 0.14 mM) compared to SCO (approximately 24 mM) . This correlates well with the midpoint redox potentials of the bound flavin, which in the case of BCO are about 100 mV more positive than for SCO . Both enzymes show a high pKa (approximately 11.0) for the N(3) position of FAD . With both enzymes, the rearrangement of 5-cholesten-3-one to 4-cholesten-3-one is not rate limiting indicating that the rate-limiting step of the overall reaction is not the isomerization . The absence of the double bond in the steroid molecule does not significantly affect turnover and affinity for the substrate, whereas both these parameters are affected by a decreasing length of the substrate C17 chain.

Biochemistry, 1997 Dec 9, 36(49), 15526 - 37
One- and two-dimensional ESEEM spectroscopy of flavoproteins; Martinez JI et al.; One- and two-dimensional (1D and 2D) electron spin echo envelope modulation (ESEEM) spectroscopy was applied to study the flavin cofactors in the neutral semiquinone states of flavodoxin and ferredoxin-NADP+ reductase (FNR) from the cyanobacterium Anabaena PCC 7119, and the anionic semiquinone state of cholesterol oxidase from Brevibacterium sterolicum . High-resolution crystal structures are available for all these proteins . Three- and 4-pulse ESEEM and hyperfine sublevel correlation spectroscopy (HYSCORE) techniques at X-band were used . HYSCORE spectra showed correlations between transitions caused by interaction of the isoalloxazine unpaired electronic spin present in the semiquinone state with several nitrogen and hydrogen nuclei . Measurements of isotopic labeled samples ({15N}FMN flavodoxin and {2H}flavodoxin) allowed the assignment of all the detected transitions to nuclei belonging to the FMN cofactor group . Interactions of nitrogens in positions 1 and 3 of the isoalloxazine ring were determined to have isotropic hyperfine coupling constants in the 1-2 and 0.5-1 MHz ranges for all the different flavoprotein semiquinones studied . Information about the quadrupolar term of these nuclei was also obtained . An intense correlation in the negative quadrant was detected . It has been associated to the strongly interacting N(10) nucleus . The complete hyperfine term parameters (including the sign) were obtained from detailed analysis of this signal, being the quadrupolar parameter, K, also estimated . Another correlation in the HYSCORE spectra, corresponding to hydrogen bound to the N(5) position in neutral flavin semiquinones, was detected . Its interaction parameters were also determined . This study demonstrates that ESEEM spectroscopy, and in particular the HYSCORE technique, are of particular utility for detecting and assigning nuclear transition frequencies in flavoprotein semiquinones . Moreover, the results reported here are complementary to ENDOR studies, and both techniques together provide an important tool for obtaining information about spin distribution in the flavin ring of flavoproteins in the semiquinone state.

Appl Environ Microbiol, 1997 Dec, 63(12), 4812 - 7
Growth reduction of Listeria spp . caused by undefined industrial red smear cheese cultures and bacteriocin-producing Brevibacterium lines as evaluated in situ on soft cheese; Eppert I et al.; The undefined microbial floras derived from the surface of ripe cheese which are used for the ripening of commercial red smear cheeses have a strong impact on the growth of Listeria spp . In some cases, these microbial consortia inhibit Listeria almost completely . From such undefined industrial cheese-ripening floras, linocin M18-producing (lin+) (N . Valdes-Stauber and S . Scherer, Appl . Environ . Microbiol . 60:3809-3814, 1994) and -nonproducing Brevibacterium linens strains were isolated and used as single-strain starter cultures on model red smear cheeses to evaluate their potential inhibitory effects on Listeria strains in situ . On cheeses ripened with lin+ strains, a growth reduction of L . ivanovii and L . monocytogenes of 1 to 2 log units was observed compared to cheeses ripened with lin strains . Linocin M18 activity was detected in cheeses ripened with lin+ strains but was not found in those ripened with lin strains . We suggest that production of linocin M18 contributes to the growth reduction of Listeria observed on model red smear cheeses but is unsufficient to explain the almost complete inhibition of Listeria caused by some undefined microbial floras derived from the surface of ripe cheeses.

J Bacteriol, 1997 Nov, 179(22), 6959 - 64
Molecular cloning and transcriptional analysis of a guanosine kinase gene of Brevibacterium acetylicum ATCC 953; Usuda Y et al.; The Brevibacterium acetylicum gsk gene, which encodes guanosine kinase (ATP:guanosine 5'-phosphotransferase), a kinase that is involved in guanosine salvage pathways, has been cloned by using the N-terminal amino acid sequence of the purified protein . The cloned chromosomal fragment containing the gsk gene was sequenced and shown to encode a polypeptide of 303 amino acids with a molecular mass of 32,536 Da, which is in good agreement with the measured molecular weight of the purified enzyme . Recombinant Escherichia coli strains harboring plasmids carrying the B . acetylicum gsk gene overexpressed both guanosine and inosine kinase activities . The primary structure of the gsk gene shows similarity to amino acid sequences of sugar kinases classified in the ribokinase family stronger than to those of the E . coli gsk gene encoding guanosine kinase and other nucleoside kinases . Northern blot analysis and primer extension analysis revealed a 1.4-kb transcript and promoter sequences, like the E . coli sigma70 and B . subtilis sigmaA consensus sequences, respectively . These results, together with the nucleotide sequence of the downstream region of gsk, suggested that the organization of B . acetylicum gsk is bicistronic . The second gene, orf2, shows significant similarity to the mutT mutator genes of several organisms, although its function has not yet been identified . The gsk gene was specifically transcribed in the early exponential growth phase, which seems to correspond to the specific guanosine kinase activity profile and suggests a role in controlling the nucleoside monophosphate level by efficiently recycling guanosine when cells are in the early exponential phase.

Gene, 1997 Oct 1, 198(1-2), 217 - 22
Cloning, sequencing and expression of the gene encoding elongation factor P in the amino-acid producer Brevibacterium lactofermentum (Corynebacterium glutamicum ATCC 13869); Ramos A et al.; The Brevibacterium lactofermentum EF-P gene, encoding the elongation factor protein P, was cloned and sequenced . According to DNA sequence analysis of this gene, the B . lactofermentum EF-P protein consists of 187 amino acids with a calculated molecular weight of 20,584 . Southern hybridization of an internal fragment of the EF-P gene from B . lactofermentum with chromosomal DNAs from different microorganisms reveals that it is a unique gene product in B . lactofermentum and Corynebacterium glutamicum . The EF-P gene was expressed in E . coli using the T7 expression system and the calculated molecular weight of the expressed protein was 23,000 . Disruption experiments using an internal fragment of the EF-P gene or a disrupted EF-P gene in suicide plasmids always failed, suggesting that the gene is needed for cell viability.

Biosci Biotechnol Biochem, 1997 Oct, 61(10), 1760 - 2
Sequence analysis of functional regions of homoserine dehydrogenase genes from L-lysine and L-threonine-producing mutants of Brevibacterium lactofermentum; Sugimoto M et al.; The homoserine dehydrogenase (HD) genes from Brevibacterium lactofermentum lysine- and threonine-producing mutants were cloned, using the polymerase chain reaction, and sequenced . We found the amino acid substitutions, Val104Ile in the lysine-producing mutants in which HD may cause leaky mutation and Ser393Phe in the threonine-producing mutant with feedback-insensitive HD.

Biochim Biophys Acta, 1997 Sep 5, 1341(2), 200 - 6
Characterization of guanosine kinase from Brevibacterium acetylicum ATCC 953; Usuda Y et al.; Guanosine kinase (GKase) activity was identified in a cell-free extract of Brevibacterium acetylicum ATCC 953 . We have purified the enzyme 4000-fold from the cell-free extract to apparent homogeneity . Molecular weight of 71,300 and 36,300 determined by gel filtration and sodium dodecyl sulfate gel electrophoresis, respectively, revealed that the enzyme is a dimer molecule . Maximum activity of the GKase was attained when the magnesium/ATP concentration ratio was close to 1 . The GKase activity was dependent on the presence of a divalent cation . The Km values for guanosine, inosine, and 2'-deoxyguanosine as phosphate acceptors were determined to be 0.022, 0.87, and 2.83 mM, respectively . In addition to ATP and dATP, GTP and dGTP were shown to be effective phosphate donors for the GKase . The optimal pH seemed to be around pH 8.3, although relatively high activity was observed in the alkaline pH range of 7.5-9.8 . The addition of 0.1 mM pyrimidine nucleotides, especially CMP and CTP, activated the GKase activity . On the other hand, the addition of 1 mM AMP, ADP, and GMP inhibited the GKase activity . It is thus likely that the GKase activity might be regulated in vivo by nucleotide concentrations and may control the nucleoside monophosphate level by efficiently recycling guanosine.

Eur J Biochem, 1997 Sep 1, 248(2), 481 - 7
Identification, isolation and biochemical characterization of a phosphopantetheine:protein transferase that activates the two type-I fatty acid synthases of Brevibacterium ammoniagenes; Stuible HP et al.; Upon heterologous expression of the Brevibacterium ammoniagenes type-I fatty acid synthase FAS-A in Escherichia coli, only the pantetheine-free apoenzyme is synthesized . Activation of FAS-A to its holoform was achieved by transformation with a second B . ammoniagenes gene, PPT1, encoding a type-I FAS-specific phosphopantetheine transferase . PPT1 was identified as a coding sequence located immediately downstream of the second FAS gene present on the B . ammoniagenes genome, fasB . Due to this linkage, PPT1 was part of the cloned fasB DNA region and, consequently, FAS-B but not FAS-A was synthesized as holoFAS in E . coli . PPT1 encodes a protein of 153 amino acids and has a calculated molecular mass of 16,884 Da . The PPT1 gene product contains 25% identical and 42% conserved amino acids compared with the type-II acyl-carrier-protein-activating enzyme of E . coli . Although there is essentially no intergenic region between fasB and PPT1, the PPTase gene is autonomously expressed in E . coli if flanked by 200 bp of its endogenous 5' DNA . The structural independence of Ppt1p was confirmed immunologically, as specific antibodies react with the purified PPTase but not with FAS-B . Overexpression and purification of the His-tagged Ppt1p allowed the in vitro activation of apoFAS-A . This holoenzyme synthesis requires, in addition to Ppt1p, CoA and Mg2+ and leads to a specific FAS activity comparable to that of natural B . ammoniagenes FAS-A . The reactivity of the in vitro-activated FAS-A was verified by the optical FAS assay and by analysis of its in vitro products . In agreement with the known overall colinearity of B . ammoniagenes FAS-B and the Saccharomyces cerevisiae FAS1 and FAS2 gene products, a PPT1-like sequence is also observed at the C terminus of FAS2 . However, in contrast to B . ammoniagenes PPT1, this sequence is an integral part of the yeast FAS2 gene . Thus, activation of type-I fatty acid synthases may be accomplished by distinct trans-acting PPTase enzymes and by intrinsic cis-acting PPTase domains.

Biochemistry, 1997 Sep 23, 36(38), 11504 - 13
Crystal structure and resonance Raman studies of protocatechuate 3,4-dioxygenase complexed with 3,4-dihydroxyphenylacetate; Elgren TE et al.; The crystal structure of the anaerobic complex of Pseudomonas putida protocatechuate 3,4-dioxygenase (3,4-PCD) bound with the alternative substrate, 3,4-dihydroxyphenylacetate (HPCA), is reported at 2.4 A resolution and refined to an R factor of 0.17 . Formation of the active site Fe(III).HPCA chelated complex causes the endogenous axial tyrosinate, Tyr447 (147beta), to dissociate from the iron and rotate into an alternative orientation analogous to that previously observed in the anaerobic 3,4-PCD.3,4-dihydroxybenzoate complex (3, 4-PCD.PCA) {Orville, A . M., Lipscomb, J . D., & Ohlendorf, D . H . (1997) Biochemistry 36, 10052-10066} . Two orientations of the aromatic ring of HPCA related by an approximate 180 degrees rotation within the active site are consistent with the electron density . Resonance Raman (rR) spectroscopic data from Brevibacteriumfuscum 3,4-PCD.HPCA complex in solution reveals low frequency rR vibrational bands between 500 and 650 cm-1 as well as a band at approximately 1320 cm-1 which are diagnostic of a HPCA . Fe(III) chelate complex . 18O labeling of HPCA at either the C4 or C3 hydroxyl group unambiguously establishes the vibrational coupling modes associated with the five-membered chelate ring system . Analysis of these data suggests that the Fe(III)-HPCAO4 bond is shorter than the Fe(III)-HPCAO3 bond . This consequently favors the model for the crystal structure in which the C3 phenolic function occupies the Fe3+ ligand site opposite the endogenous ligand Tyr408(Oeta) (108beta) . This is essentially the same binding orientation as proposed for PCA in the crystal structure of the anaerobic 3,4-PCD.PCA complex based solely on direct modeling of the 2Fo - Fc electron density and suggests that this is the conformation required for catalysis.

FEMS Microbiol Lett, 1997 Aug 15, 153(2), 295 - 301
Antibacterial and hemolytic activities of linenscin OC2, a hydrophobic substance produced by Brevibacterium linens OC2; Boucabeille C et al.; Linenscin OC2 is an antibacterial substance produced by the orange cheese coryneform bacterium Brevibacterium linens OC2 . It inhibits the growth of Gram-positive bacteria but it is inactive against Gram-negative bacteria . The intact outer membrane of Gram-negative bacteria was shown to be an effective permeability barrier against linenscin OC2 . At high dosage the effect of linenscin OC2 was bacteriolytic on Listeria innocua . Bacteriostasis was observed at low dosage and peptidoglycan biosynthesis was affected at an early step upstream of the UDP-N-acetylglucosamine . Hemolytic activity of this substance on sheep erythrocytes suggested a common mode of action on prokaryotic and eukaryotic cells . It also suggested that the cytoplasmic membrane might be the primary target of linenscin OC2.

FEMS Microbiol Lett, 1997 Aug 1, 153(1), 111 - 7
Transcriptional analysis of the sigA and sigB genes of Brevibacterium lactofermentum; Oguiza JA et al.; Transcription of the sigA and sigB genes of Brevibacterium lactofermentum, encoding the principal sigma factors of RNA polymerase, has been investigated by Northern blot and primer extension analysis . Northern hybridizations revealed that sigA is transcribed as a monocistronic mRNA of 1.7 kb and sigB gives two transcripts of 1.1 and 1.5 kb . Similar transcription patterns of sigA and sigB genes in nutrient-rich medium were observed; transcripts of both genes occurred simultaneously throughout the exponential growth phase and decayed clearly when the stationary phase was reached . Primer extension analysis of B . lactofermentum RNA showed that the sigA and sigB transcription initiation sites are located 17 bp and 24 bp upstream from the first nucleotide of the respective translation initiation codons . Alignment of the sigA and sigB promoter regions provided evidence for highly conserved sequences in both of them.

Arch Microbiol, 1997 Aug, 168(2), 164 - 8
The biotransformation of t-butylacetonitrile and its boron-containing analogue trimethylamine-cyanoborane by Brevibacterium R312; Millais AJ et al.; The ability of the nitrile hydratase/amidase system from Brevibacterium R312 to biotransform tert-butylacetonitrile was studied with a view to their utilisation in the production of novel amino acids from isostructural compounds . Brevibacterium R312 was able to transform nitriles with this structure; however, the wide spectrum amidase from this organism was unable to biotransform the corresponding amide to the carboxylic acid.

Biochem Biophys Res Commun, 1997 Jul 18, 236(2), 383 - 8
Cloning, sequencing, and characterization of the ftsZ gene from coryneform bacteria; Kobayashi M et al.; Taking advantage of highly conserved domains present in the ftsZ genes from Escherichia coli, Rhizobium meliloti, and Bacillus subtilis, we designed degenerate oligonucleotides (oligos) corresponding to these regions . These oligos were used as primers in PCR in order to amplify DNA sequences from Brevibacterium flavum MJ233 chromosomal DNA . The PCR product was used as a probe to recover genomic fragments from a lambda library of Br . flavum MJ233 . The complete nucleotide sequence (nt) of the cloned 4.2-kb EcoRI fragment containing the ftsZ homolog from Br . flavum MJ233 indicated that the deduced gene product of the Br . flavum ftsZ homolog is composed of 438 amino acids (aa) with a deduced molecular weight of 46.9 kDa . This size of molecular weight was also confirmed by the in vitro protein synthesis assay . Comparison of this aa sequence to the corresponding sequences from E . coli, Rh . meliloti, B . subtilis, and Streptomyces coelicolor revealed a high degree of conservation and suggested that the Br . flavum ftsZ homolog has a putative GTP binding motif and a GTP hydrolizing region . Expression of Br . flavum ftsZ gene in E . coli, JM109 inhibited its cell division, leading to filamentation . This suggested that the Br . flavum ftsZ product competed with the E . coli ftsZ product.

Biosci Biotechnol Biochem, 1997 Jul, 61(7), 1109 - 12
Relationship between the glutamate production and the activity of 2-oxoglutarate dehydrogenase in Brevibacterium lactofermentum; Kawahara Y et al.; Enzyme activities of 2-oxoglutarate dehydrogenase complex and glutamate dehydrogenase of wild type Brevibacterium lactofermentum, one of the typical glutamate-producing coryneform bacteria, were investigated by using cells cultured under glutamate-productive and glutamate-non-productive conditions . Significant reduction of the former enzyme activity was observed in the cells under the several glutamate-productive conditions, namely, in the cells cultured in media containing a) limited concentrations of biotin, b) sub-lethal amounts of penicillin, and c) sub-optimal amounts of a surface-active agent, as compared with those under the non-productive conditions . The activity of the latter enzyme was essentially unchanged in every condition . The relationship between glutamate production and the enzyme activities as well as permeability of glutamate through cell membrane was discussed from the results obtained.

Eur J Biochem, 1997 Jul 1, 247(1), 268 - 73
Heterologous expression and biochemical characterization of two functionally different type I fatty acid synthases from Brevibacterium ammoniagenes; Stuible HP et al.; The coryneform bacterium, Brevibacterium ammoniagenes, contains two structurally related but functionally differentiated type I fatty acid synthases, FAS-A and FAS-B . Isolation of homogeneous preparations of both enzymes was achieved by constructing specific fasA and fasB expression systems . In B . ammoniagenes, insertional mutagenesis of fasB allowed the specific production of enzymatically active FAS-A . The corresponding fasA mutant was not suited for FAS-B purification as the level of this enzyme was extremely low, in the fasA-disruptants . Instead, FAS-B could be efficiently expressed in the heterologous host, Escherichia coli . Using specific antisera against each of the two FAS variants, FAS-A was shown to be the predominant FAS protein in B . ammoniagenes . In contrast the two enzymes are expressed at comparable rates in E . coli even though the same upstream sequences were associated with fasA and fasB, as in B . ammoniagenes . Due to their differential capacities of being activated to the phosphopantetheine-containing holo-enzyme in the heterologous host, only FAS-B but not FAS-A exhibited overall FAS activity when isolated from E . coli . Irrespective of their origin, the purified FAS-A and FAS-B proteins were indistinguishable with respect to their flavin fluorescence, their subunit size and their sucrose density gradient sedimentation characteristics . Nevertheless, the in vitro products of both enzymes differ characteristically: while FAS-A synthesizes mainly the 18-carbon fatty acids oleate and stearate with only traces of palmitate, the major product of FAS-B is palmitic acid . No unsaturated fatty acids are produced by FAS-B . Thus, the two B ammoniogenes type I fatty acid synthases differ, in spite of their very similar overall protein structure, in both their ability to synthesize oleic acid and in their chain-length specificities.

J Appl Microbiol, 1997 Jul, 83(1), 53 - 8
Microbiological composition of raw milk from selected farms in the Camembert region of Normandy; Desmasures N et al.; Raw milk from 27 farms was sampled over 6 months for listerias, salmonellas, Yersinia enterocolitica and campylobacters . Total bacterial counts and somatic cell counts were measured . Lactococci, lactobacilli, dextran-producing leuconostocs, Brevibacterium linens, yeasts and moulds, Staphylococcus aureus and other Micrococcaceae, Pseudomonas, coliforms, Escherichia coli, enterococci, Clostridium perfringens and spores of anaerobic lactate-fermenting bacteria were also counted . Pseudomonas (2000 cfu ml-1), lactococci (760 cfu ml-1) and Micrococcaceae (720 cfu ml-1) were the most numerous groups . Lactic acid bacteria were detected in all samples . Coliforms were present in most samples, but 84% of samples had counts < 100 cfu ml-1 . Staphylococcus aureus was detected in 62% of milks, the average count was 410 cfu ml-1 . About 80% of supplies had < or = 10 E . coli cfu ml-1 and all samples had < or = 1 Cl . perfringens cfu ml-1 . Two of the tested milks were positive for salmonellas (2.9%), four were positive for Listeria monocytogenes (5.8%), 25 for Yersinia enterocolitica (36%) and one for campylobacters (1.4%).

Steroids, 1997 Jun, 62(6), 482 - 6
A direct stereoselective synthesis of 7 beta-hydroxytestosterone; Labaree D et al.; Although 7 beta-hydroxytestosterone is a known product of hepatic androgen metabolism, there are no published methods for its chemical synthesis except from the equally difficult to obtain 7 beta-hydroxy-4-androstene-3,17-dione . We found that several seemingly straightforward routes for its synthesis failed . Consequently, we tried to produce 7 beta-hydroxytestosterone by enzymatic oxidation of 5-androstene-3 beta, 7 beta, 17 beta-triol with cholesterol oxidase (Brevibacterium sp.), a procedure previously used to synthesize 7 beta-hydroxy-4-cholesten-3-one from 3 beta, 7 beta-dihydroxycholesterol (Alexander and Fisher 1995) . However, 5-androstene-3 beta, 7 beta, 17 beta-triol was, at best, a very poor substrate for the enzyme leading to the production of 7 beta-hydroxytestosterone in only trace amounts . Thus, we explored a strategy for the enzymatic synthesis in which a C8-ester at C-17 (5-androstene-3 beta, 7 beta, 17 beta-triol 17-caprylate) would serve to mimic the bulky and hydrophobic side chain of cholesterol and thus allow the C19-steroid to act as an effective substrate . When this ester was incubated with cholesterol oxidase, it was converted efficiently to 7 beta-hydroxytestosterone-17-caprylate . Attempts to remove the ester group by several mild hydrolytic procedures caused elimination of the 7 beta-hydroxyl group; we, therefore, obtained 7 beta-hydroxytestosterone by incubation of the intermediate ester with porcine lipase.

Protein Expr Purif, 1997 Jun, 10(1), 1 - 9
Cloning, overexpression, and mutagenesis of the gene for homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum; Wang YZ et al.; Homoprotocatechuate (hpca, 3,4-dihydroxyphenylacetate) is a central intermediate for the bacterial degradation of aromatic compounds . Homoprotocatechuate 2,3-dioxygenase (HPCD) catalyzes the key ring cleavage step in the metabolism of hpca by the Gram (+) bacterium Brevibacterium fuscum to yield alpha-hydroxy-delta-carboxymethyl cis-muconic semialdehyde . A genomic DNA library of B . fuscum was constructed in Escherichia coli using a cosmid vector and screened by spraying the cells with hpca . One clone was found to contain the gene for HPCD based on its ability to convert hpca into the yellow-colored product . This cosmid clone was further subcloned and the gene for HPCD was localized and sequenced . The open reading frame codes for a protein with 365 amino acids and M(r) = 41,699, in accord with the characteristics of the previously purified wild-type enzyme . The gene for HPCD was overexpressed in E . coli to approximately 30% of the total soluble protein, and purification of the recombinant enzyme to apparent homogeneity was achieved by a two-step procedure . Iron was the only abundant metal found in the purified recombinant enzyme, and the specific activity per iron was comparable to that observed for the wild-type enzyme . The deduced amino acid sequence of HPCD has a very high level of homology (78.6% identity in the 337-aa overlap) to the manganese-dependent homoprotocatechuate 2,3-dioxygenase (MndD) from Arthrobacter globiformis CM-2 . The basis for the difference in metal selection by HPCD and MndD was investigated by mutagenesis of a 50-base-pair region of the HPCD gene containing three frame shifts relative to the MndD gene . The purified triple mutant of HPCD did not exhibit a significant change in the metal content; therefore, other factors must contribute to the selection of the active site metal.

Appl Environ Microbiol, 1997 Jun, 63(6), 2468 - 71
Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine beta-casein; Rattray FP et al.; The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine beta-casein was studied . Hydrolysis was monitored over time by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and urea-PAGE . The major pH 4.6-soluble peptides were isolated by high-performance liquid chromatography and identified by N-terminal amino acid sequencing and mass spectrometry . The major sites of hydrolysis were Ser-18-Ser-19, Glu-20-Glu-21, Gln-56-Ser-57, Gln-72-Asn-73, Leu-77-Thr-78, Ala-101-Met-102, Phe-119-Thr-120, Leu-139-Leu-140, Ser-142-Trp-143, His-145-Gln-146, Gln-167-Ser-168, Gln-175-Lys-176, Tyr-180-Pro-181, and Phe-190-Leu-191 . The proteinase had a broad specificity for the amino acid residues present at the P1 and P'1 positions but showed a preference for hydrophobic residues at the P2, P3, P4, P'2, P'3, and P'4 positions.

Biochem Biophys Res Commun, 1997 May 8, 234(1), 157 - 61
A dtsR gene-disrupted mutant of Brevibacterium lactofermentum requires fatty acids for growth and efficiently produces L-glutamate in the presence of an excess of biotin; Kimura E et al.; A dtsR gene encoding a homolog of the beta subunit of some biotin-containing enzymes suppresses a detergent-sensitive mutation of Brevibacterium lactofermentum (E . Kimura et al., 1996, Biosci . Biotech . Biochem . 60, 1565-1570), which has been used for the fermentative production of L-glutamate . When the dtsR gene was disrupted, the organism exhibited strict fatty acid auxotrophy; oleate or oleate ester, but not palmitate ester or stearate ester, supported the growth of the delta dtsR mutant . Immunoblotting with an anti-DtsR antibody revealed that no intact DtsR was present in the cytosol of the delta dtsR mutant . In the presence of an excess of biotin, the wild type strain did not produce L-glutamate whereas the delta dtsR mutant efficiently produced it . The mechanism underlying the efficient production of L-glutamate by the delta dtsR mutant is discussed as to the possible role of dtsR in fatty acid metabolism.

Mol Gen Genet, 1997 Apr 28, 254(4), 464 - 8
The Brevibacterium albidum gene encoding the arginine tRNACCG complements the growth defect of an Escherichia coli strain carrying a thermosensitive mutation in the rnpA gene at the nonpermissive temperature; Kim MS et al.; The Escherichia coli rnpA gene encodes C5 protein, the protein component of RNase P . The rnpA49 mutation renders the C5 protein thermosensitive, which results in thermosensitivity of RNase P function . The chromosomal DNA region from Brevibacterium albidum that complements the rnpA49 mutation was analysed . The gene capable of complementing the growth defect of an rnpA49 mutant strain at nonpermissive temperature was identified as the gene for an arginine tRNA with anticodon CCG by a deletion analysis combined with complementation assays . Transcription of the arginine tRNA gene carried on a multicopy plasmid was correlated with the complementation of the rnpA49 mutation, indicating that the gene product is indeed responsible for complementation of the rnpA49 mutation.

Curr Microbiol, 1997 Apr, 34(4), 233 - 7
Reclassification of "Bacillus pulvifaciens" group II as Brevibacillus agri; Nakamura LK; Organisms formerly identified as strains of Paenibacillus pulvifaciens group II were reclassified as members of the species Brevibacillus agri . The reclassification was based on phylogenetic analysis of 16S rRNA gene sequences from selected Bacillaceae . The analysis strongly supported placement of group II strains in the genus Brevibacillus . High DNA relatedness of 87-100% and close phenotypic similarity demonstrated that group II strains were members of the species Brevibacillus agri.

Lett Appl Microbiol, 1997 Mar, 24(3), 211 - 3
Site-directed mutagenesis of the aspartokinase gene lysC and its characterization in Brevibacterium flavum; Lu JH et al.; Using overlap extension polymerase chain reaction (PCR), five transformants of Escherichia coli containing site-directed mutagenized lysC beta gene were generated and analysed . Exchange of C to A and C to T at nucleotide 1118 of the mutated lysC beta gene causes a substitution of serine301 in the wild-type enzyme for tyrosine301 and phenylalanine301 in the mutant enzymes, respectively . Enzyme assays showed that Brevibacterium flavum cells harbouring pSUMN18 with mutated lysC beta genes exhibited 16-20 fold lower specific activities of aspartokinase as compared to that of host containing wild-type lysC gene . The mutation introduced into lysC beta of B . flavum CCRC 18271 resulted in partial feedback-resistant aspartokinase activity.

Biochem Mol Biol Int, 1997 Feb, 41(2), 311 - 5
Molecular cloning and nucleotide sequencing of bioF (7-keto-8-amino pelargonic acid synthetase), bioC and bioD (dethiobiotin synthetase) genes of Erwinia herbicola; Wu CH et al.; The biotin operon of Erwinia herbicola Eho 10 was cloned and characterized by complementation of E . coli biotin mutants . The operon was found to contain five genes arranged in the order, bioABFCD . The nucleotide sequences of bioF (7-keto-8-aminopelargonic acid synthetase), bioC and bioD (dethiobiotin synthetase) were determined and analyzed . The nucleotide sequences and deduced amino acid sequences of bioFCD were compared with the corresponding sequences from Escherichia coli, Bacillus sphaericus, Serratia marcescens and Brevibacterium flavum.

Int J Food Microbiol, 1997 Feb, 34(2), 115 - 29
Identification of yeasts and coryneform bacteria from the surface microflora of brick cheeses; Valdes-Stauber N et al.; Coryneform bacteria and yeasts of 21 brick cheeses from six German dairies, produced by using undefined ripening cultures, were identified . Arthrobacter nicotianae, Brevibacterium linens, Corynebacterium ammoniagenes, Corynebacterium variabilis and Rhodococcus fascians were found in significant numbers . Out of 148 coryneform isolates 36 could not be identified at the species level . With the exception of a large rennet cheese, the coryneform microflora of rennet and acid cured cheeses were similar, but the cheeses had clearly different yeast populations . Debaryomyces hansenii and Galactomyces geotrichum prevailed in rennet cheeses while Kluyveromyces marxianus and Pichia membranaefaciens were the main species found in acid cured cheese . The dominance of Yarrowia lipolytica probably indicates an improper yeast population, resulting in poor cheese quality . Some of the species identified are potential candidates for designing a defined ripening culture for rennet red smear cheese.

FEBS Lett, 1997 Jan 3, 400(2), 247 - 51
Electron spin echo envelope modulation studies of the semiquinone anion radical of cholesterol oxidase from Brevibacterium sterolicum; Medina M et al.; The electron spin echo envelope modulation (ESEEM) technique of pulsed EPR spectroscopy was applied to the anionic semiquinone of the cholesterol oxidase flavin cofactor, formed when the enzyme was photoreduced in the presence of 5-deazariboflavin and EDTA . Fourier transforms of the three-pulse ESEEM spectra showed the presence of 14N nuclei magnetically coupled to the paramagnet . In 2H2O buffer the surroundings of the flavin ring were shown to be accessible to solvent exchange, with a deuterium population in close proximity to the paramagnetic centre . Upon binding of the pseudosubstrate, dehydroisoandrosterone, subtle changes were observed in the coupling to nitrogen nuclei, which are interpreted as changes in the electron density distribution of the flavin ring system . The results are discussed in terms of the three-dimensional structure reported for the protein and the flavin ring architecture.

Plasmid, 1997, 38(1), 61 - 9
Cryptic plasmid pKA22 isolated from the naphthalene degrading derivative of Rhodococcus rhodochrous NCIMB13064; Kulakov LA et al.; Cryptic plasmids were found in Rhodococcus rhodochrous NCIMB13064 derivatives which had lost the ability to utilize short-chain 1-chloroalkanes (chain length C3-C10) and had acquired the ability to degrade naphthalene . The reversions of these derivatives to the original phenotype were accompanied by the loss of the cryptic plasmids . The 4969-bp pKA22 plasmid was cloned in Escherichia coli and sequenced . This plasmid encodes a putative 33,200-Da protein which contains motifs typical of theta replicase proteins and shows a high degree of similarity to a putative theta replicase from Brevibacterium linens plasmid pRBL1 and to a putative protein encoded by ORF1 of the plasmid pAL5000 from Mycobacterium fortuitum . Two sets of long direct repeats were found in pKA22 which may be involved in the replication of the plasmid and recombination processes.

Microbiol Immunol, 1997, 41(6), 453 - 60
Genetic identification of members of the genus Corynebacterium at genus and species levels with 16S rDNA-targeted probes; Hou XG et al.; 16S rRNA gene-targeted probes were designed for the identification of corynebacteria at the genus and species levels . The genus-specific probe hybridized all clinically important members of the genus Corynebacterium and could distinguish them from other coryneform bacteria and phylogenetically related high G+C% gram-positive bacteria, including Actinomyces, Rhodococcus, Gordona, Nocardia, Streptomyces, Brevibacterium and Mycobacterium . The species-specific probes for C . jeikeium and C . diphtheriae could differentiated these two species from other members of this genus . The probes were used to select corynebacteria among gram-positive clinical isolates which had been tentatively identified as corynebacteria by biochemical tests . We screened 59 strains with the genus-specific probe; 51 strains hybridized to the genus-specific probe, 8 did not . Of the 51 strains that hybridized to the genus-specific probe, 1 hybridized to the C . diphtheriae species probe and 13 hybridized to the C . jeikeium species probe . The 8 strains that did not hybridize to the genus probe were further characterized by analyzing cell wall diaminopimelic acid and partial 16S rRNA sequencing . The results indicated that these strains were distributed in the genera Arthrobacter and Brevibacterium.

Biosci Biotechnol Biochem, 1997 Jan, 61(1), 105 - 8
Effects of an Escherichia coli ilvA mutant gene encoding feedback-resistant threonine deaminase on L-isoleucine production by Brevibacterium flavum; Hashiguchi K et al.; A mutated ilvA gene of Escherichia coli encoding a threonine deaminase that is resistant to feedback inhibition by L-isoleucine was obtained . It was functional in Brevibacterium flavum, and a wild strain of B . flavum into which it was introduced became able to convert exogeneous L-homoserine and L-threonine to L-isoleucine . When it was introduced into a L-threonine-producing B . flavum strain, the transformant accumulated 20 g/liter L-isoleucine from 100 g/liter glucose.

What Is Biotechnology?, What Is Bioassay?, What Is Bioengineering?, What Is Botulism?, What Is Bioreactor?, r, Microorganisms, i, Microbes, o, Microbiology, e, Microorganism, o, Bacteriology, r, Bacillus, r, Cell cultures, a, Nitrosomonas, a, Escherichia coli, a, Microbiological, s, Yeasts, o, Yeasts, s, Staphylococcus, r, Proteus, c, Bacteriological, i, Salmonella, c, Streptococci, n, Salmonella, e, Streptococcal, r, Pichia, c, Culture medium, e, Escherichia coli, e, Bacillus, i, Antimicrobials, a, Citrobacter, n, Fermentations

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005