Clin Microbiol Rev, 1997 Jan, 10(1), 125 - 59 Clinical microbiology of coryneform bacteria; Funke G et al.; Coryneform bacteria are aerobically growing, asporogenous, non-partially-acid-fast, gram-positive rods of irregular morphology . Within the last few years, there has been a massive increase in the number of publications related to all aspects of their clinical microbiology . Clinical microbiologists are often confronted with making identifications within this heterogeneous group as well as with considerations of the clinical significance of such isolates . This review provides comprehensive information on the identification of coryneform bacteria and outlines recent changes in taxonomy . The following genera are covered: Corynebacterium, Turicella, Arthrobacter, Brevibacterium, Dermabacter . Propionibacterium, Rothia, Exiguobacterium, Oerskovia, Cellulomonas, Sanguibacter, Microbacterium, Aureobacterium, "Corynebacterium aquaticum," Arcanobacterium, and Actinomyces . Case reports claiming disease associations of coryneform bacteria are critically reviewed . Minimal microbiological requirements for publications on disease associations of coryneform bacteria are proposed. Antimicrob Agents Chemother, 1996 Dec, 40(12), 2874 - 8 Antimicrobial susceptibility patterns of some recently established coryneform bacteria; Funke G et al.; The susceptibility patterns of 480 isolates representing six recently defined species of coryneform bacteria (Corynebacterium amycolatum {n = 101}, Corynebacterium auris {n = 48}, Corynebacterium glucuronolyticum {n = 86}, Brevibacterium casei {n = 50}, Dermabacter hominis {n = 49}, and Turicella otitidis {n = 146}) to 17 antimicrobial agents were determined by an agar dilution method . Most significantly, for C . amycolatum strains the MICs at which 90% of isolates are inhibited were > or = 32 micrograms/ml for nearly all agents . However, all 480 strains examined were susceptible to glycopeptide antibiotics. Appl Microbiol Biotechnol, 1996 Dec, 46(5-6), 554 - 8 Construction of L-lysine-overproducing strains of Brevibacterium lactofermentum by targeted disruption of the hom and thrB genes; Fernandez-Gonzalez C et al.; The mobilization of plasmids from gram-negative Escherichia coli to gram-positive Brevibacterium lactofermentum, mediated by P-type transfer functions, was used to construct disrupted mutants blocked specifically in the homoserine branch of the aspartate pathway . The mutant strain B . lactofermentum R31 showed an efficiency of conjugal transfer two to three orders of magnitude higher than that of the wild-type strain B . lactofermentum ATCC 13869 . The hom- and thrB-disrupted mutants of B . lactofermentum ATCC 13869 were lysine overproducers . B . lactofermentum R31 mutants do not overproduce lysine because R31 is an alanine-overproducing strain and channels the pyruvate needed for lysine biosynthesis to the production of alanine. Microbiology, 1996 Dec, 142 ( Pt 12), 3347 - 54 Molecular cloning of the Corynebacterium glutamicum ('Brevibacterium lactofermentum' AJ12036) odhA gene encoding a novel type of 2-oxoglutarate dehydrogenase; Usuda Y et al.; The Corynebacterium glutamicum ('Brevibacterium lactofermentum' AJ12036) odhA gene, encoding 2-oxoglutarate dehydrogenase (E1o subunit of the 2-oxoglutarate dehydrogenase complex), has been isolated and identified as an homologous counterpart of the Escherichia coll sucA and Bacillus subtilis odhA genes . The nucleotide sequence of a 4394 bp chromosomal fragment containing the C . glutamicum odhA gene was determined . The odhA gene comprised 3771 bp (1257 codons, including the initiation codon) and a molecular mass of 138656 Da was predicted for the OdhA polypeptide . Northern blot analysis revealed a 3.9 kb transcript . The size of the transcript, together with the presence of a rho-independent terminator-like structure, suggests that C . glutamicum odhA is monocistronic . Cells harbouring plasmids carrying C . glutamicum odhA showed a threefold increase in specific 2-oxoglutarate dehydrogenase complex activity and expression of a protein with an apparent molecular mass of 136 kDa, in good agreement with the predicted size of the OdhA polypeptide . The C-terminal region of the C . glutamicum OdhA protein shows strong sequence similarity to E1os from other organisms . C . glutamicum OdhA has an N-terminal extension not found in previously reported E1os . The amino acid sequence of this extension shows similarity to that of the C-terminal region of dihydrolipoamide S-succinyltransferase (E2o) subunits of 2-oxoglutarate dehydrogenase complexes and dihydrolipoamide S-acetyltransferase (E2p) subunits of pyruvate dehydrogenase complexes . It suggests that the C . glutamicum odhA gene might encode a novel bifunctional protein with E1o and E2o activities. Gene, 1996 Oct 17, 176(1-2), 265 - 7 Cloning and sequencing of the secY homolog from Streptomyces lividans 1326; Ostiguy S et al.; Two conserved regions of SecY proteins from six Gram+ bacteria were exploited in a PCR-based strategy for isolating a secY homolog from Streptomyces lividans (Sl) . The nucleotide sequence of part of a 3.8-kb fragment showed that the secY homolog is flanked, at the 5' end, by the gene encoding ribosomal protein L15 and, at the 3' end, by an adenylate kinase-encoding gene . The deduced gene product of secY would have 437 amino acids (aa) and an M(r) of 47,200 . Sl SecY shows 89.5, 56.1, 42 and 40% identity to its homologs from Streptomyces scabies, Brevibacterium flavum, Bacillus subtilis and Escherichia coli, respectively . Promoterprobe analyses indicated that the secY gene probably contains its own promoter. Biosci Biotechnol Biochem, 1996 Oct, 60(10), 1565 - 70 Molecular cloning of a novel gene, dtsR, which rescues the detergent sensitivity of a mutant derived from Brevibacterium lactofermentum; Kimura E et al.; Several strains of Corynebacterium and Brevibacterium are known for their ability to secrete large amounts of amino acids, especially L-glutamate . We focused on the mechanism of L-glutamate secretion triggered by a detergent, namely polyoxyethylenesorbitan monopalmitate (PESP) . A mutant strain, AJ11060, derived from Brevibacterium lactofermentum ATCC 13869 indicates the sensitivity to PESP . A multicopy suppresser gene that compliments the sensitivity of AJ11060 to the detergent was derived from a gene library of B . lactofermentum AJ12036 . A 2855-bp DNA fragment was cloned and sequenced . An open reading frame was found that coded for the rescuer gene of the sensitivity to PESP of AJ11060 and was designated dtsR . The expression of the dtsR gene in B . lactofermentum was confirmed by using anti-DtsR antibody . The deduced DtsR protein indicated significant homology with some biotin enzymes such as the beta chain of propionyl-CoA carboxylase from rat (48.3%) and human (48.7%), or a 12S chain of methylmalonyl-CoA carboxyltransferase from Propionibacterium freudenreichii (43.1%). Int J Syst Bacteriol, 1996 Oct, 46(4), 939 - 46 Proposal for two new genera, Brevibacillus gen . nov . and Aneurinibacillus gen . nov; Shida O et al.; 16S rRNA gene sequences of the type strains of 11 species belonging to the Bacillus brevis and Bacillus aneurinolyticus groups were determined . On the basis of the results of gene sequence analyses, these species were separated into two clusters . The B . brevis cluster included 10 species, namely, Bacillus brevis, Bacillus agri, Bacillus centrosporus, Bacillus choshinensis, Bacillus parabrevis, Bacillus reuszeri, Bacillus formosus, Bacillus borstelensis, Bacillus laterosporus, and Bacillus thermoruber . Bacillus aneurinolyticus and Bacillus migulanus belonged to the B . aneurinolyticus cluster . Moreover, the two clusters were phylogenetically distinct from other Bacillus, Amphibacillus, Sporolactobacillus, Paenibacillus, and Alicyclobacillus species . On the basis of our data, we propose reclassification of the B . brevis cluster as Brevibacillus gen . nov . and reclassification of the B . aneurinolyticus cluster as Aneurinibacillus gen . nov . By using 16S rRNA gene sequence alignments, two specific PCR amplification primers were designed for differentiating the two new genera from each other and from other aerobic, endospore-forming organisms. FEMS Microbiol Lett, 1996 Oct 1, 143(2-3), 103 - 14 Molecular control mechanisms of lysine and threonine biosynthesis in amino acid-producing corynebacteria: redirecting carbon flow; Malumbres M et al.; Threonine and lysine are two of the economically most important essential amino acids . They are produced industrially by species of the genera Corynebacterium and Brevibacterium . The branched biosynthetic pathway of these amino acids in corynebacteria is unusual in gene organization and in the control of key enzymatic steps with respect to other microorganisms . This article reviews the molecular control mechanisms of the biosynthetic pathways leading to threonine and lysine in corynebacteria, and their implications in the production of these amino acids . Carbon flux can be redirected at branch points by gene disruption of the competing pathways for lysine or threonine . Removal of bottlenecks has been achieved by amplification of genes which encode feedback resistant aspartokinase and homoserine dehydrogenase (obtained by in vitro directed mutagenesis). J Bacteriol, 1996 Oct, 178(19), 5768 - 75 Integration of narrow-host-range vectors from Escherichia coli into the genomes of amino acid-producing corynebacteria after intergeneric conjugation; Mateos LM et al.; Conjugative transfer of mobilizable derivatives of the Escherichia coli narrow-host-range plasmids pBR322, pBR325, pACYC177, and pACYC184 from E . coli to species of the gram-positive genera Corynebacterium and Brevibacterium resulted in the integration of the plasmids into the genomes of the recipient bacteria . Transconjugants appeared at low frequencies and reproducibly with a delay of 2 to 3 days compared with matings with replicative vectors . Southern analysis of corynebacterial transconjugants and nucleotide sequences from insertion sites revealed that integration occurs at different locations and that different parts of the vector are involved in the process . Integration is not dependent on indigenous insertion sequence elements but results from recombination between very short homologous DNA segments (8 to 12 bp) present in the vector and in the host DNA . In the majority of the cases (90%), integration led to cointegrate formation, and in some cases, deletions or rearrangements occurred during the recombination event . Insertions were found to be quite stable even in the absence of selective pressure. Eur J Biochem, 1996 Aug 15, 240(1), 239 - 44 Key role of alkanoic acids on the spectral properties, activity, and active-site stability of iron-containing nitrile hydratase from Brevibacterium R312; Kopf MA et al.; Interaction of n-butyric acid with dialyzed nitrile hydratase from Brevibacterium R312, which is characterized by a charge-transfer band at 680 nm and EPR signals typical of a low-spin Fe(III) with delta g = 0.22, leads to a form displaying different spectral properties (lambda = 710 nm, delta g = 0.31) . Butyric acid also acts as a competitive inhibitor of nitrile-hydratase-catalyzed hydration of acrylonitrile with a Ki value of 0.9 mM . Formation of the complex between the enzyme and butyric acid is highly dependent on the concentration of the latter and on pH . When stored with high levels of butyric acid, nitrile hydratase is completely inactive . The active uncomplexed enzyme is restored under the high dilution conditions used for the enzymatic assays, while the complexed form is favored at acidic pH and is not formed at pH above 8 . Furthermore, the inhibitory potency of butyric acid decreases upon increasing pH (IC50 increases from 0.8 mM at pH 6.2 to 12 mM at pH 8.2) . These data show that nitrile hydratase interacts with the acid form of butyric acid with a high affinity (Ki' approximately 4 microM at pH 7.2) . At pH < 3, the visible spectrum of the enzyme disappears, presumably because of demetallation, whereas that of the complex exhibits a charge-transfer band shifted to 800 nm, the presence of butyric acid preventing nitrile hydratase from demetallation . Other linear carboxylic acids such as valeric and hexanoic acids behave similarly; they act as inhibitors of nitrile hydratase and protect the enzyme during storage . A structure of the nitrile hydratase active site interacting with butyric acid is tentatively proposed in which the latter is hydrogen-bonded to the Fe(III)-OH moiety . This interaction between butyric acid and nitrile hydratase should be considered when deducing the nature of nitrile hydratase active site and mechanisms, from spectral and enzymatic data, since most results published previously have been obtained on nitrile hydratase containing large amounts of butyric acid and interpreted without taking into account the presence of this acid in the active site. Biochemistry, 1996 Aug 6, 35(31), 10078 - 88 X-ray spectroscopy of nitrile hydratase at pH 7 and 9; Scarrow RC et al.; The iron K-edge X-ray absorption spectrum of Rhodococcus sp . R312 (formerly Brevibacterium sp . R312) nitrile hydratase in frozen solutions at pH 7 and 9 has been analyzed to determine details of the iron coordination . EXAFS analysis implies two or three sulfur ligands per iron and overall six coordination; together with previous EPR and ENDOR results, this implies an N3S2O ligation sphere . The bond lengths from EXAFS analysis {rav(Fe-S) = 2.21 A at pH 7.3; rav(Fe-N/O) = 1.99 A} support cis coordination of two cysteine ligands and conclusively rule out nitric oxide coordination to the iron, a possibility proposed on the basis of an FTIR difference experiment {Noguchi, T., Honda, J., Nagamune, T., Sasabe, H., Inoue, Y., & Endo, I . (1995) FEBS Lett . 358, 9-12} . The higher-frequency EXAFS can be simulated well by inclusion of multiple scattering from two or three imidazole ligands; the fit to the data is improved if first-sphere multiple scattering pathways are also included . A slight shortening (by 0.02 +/- 0.01 A) of one or both Fe-S bonds when the pH is raised from 7.3 to 9.0 is consistent with shifts observed in the Raman spectrum {Brennan et al . (1996) Biochemistry 35, 10068-10077}. Biochemistry, 1996 Aug 6, 35(31), 10068 - 77 Resonance Raman spectroscopy of nitrile hydratase, a novel iron-sulfur enzyme; Brennan BA et al.; Resonance Raman spectra of Rhodococcus sp . R312 (formerly Brevibacterium sp . R312) nitrile hydratase, a novel non-heme iron enzyme, have a large number of peaks in the 300-500 cm-1 region; observation of shifts in these peaks after labeling with 34S shows that they arise from cysteine coordinated to the ferric ion in the protein . The rich Raman spectra result from coupling of the Fe-S stretch with cysteine side chain deformation modes; the observation of 15N isotope shifts in most of these peaks suggests participation of N-donor metal ligands and peptide backbone amide nitrogens in these modes as well . The aggregate 34S isotope shift is too large to result from a single cysteine ligand, consistent with the analysis of EXAFS data that shows two or three S-donor ligands {Scarrow et al . (1996) Biochemistry 35, 10078-10088} . Widespread 2H isotope shifts seen after exchange of the protein into 2H2O suggest the presence of hydrogen bonds to the coordinated cysteine sulfurs . Comparison of the resonance Raman spectra of nitrile hydratase prepared at pH 7.3 and 9.0 shows a shift of intensity into the higher-energy peaks in the spectra of the latter sample . This is interpreted as resulting from an increase in Fe-S bond strength at the higher pH and is supported by observation of a small decrease in Fe-S bond length in the EXAFS analysis {Scarrow et al . (1996) Biochemistry 35, 10078-10088} . Such a decrease in Fe-S bond length is also consistent with pH dependent changes in EPR spectra and could reflect the loss of one or more hydrogen bonds to sulfur ligands. J Bacteriol, 1996 Aug, 178(16), 4787 - 93 Identification and functional differentiation of two type I fatty acid synthases in Brevibacterium ammoniagenes; Stuible HP et al.; The fatty acid synthase (FAS) from Brevibacterium ammoniagenes is a homohexameric multienzyme complex that catalyzes the synthesis of both saturated and unsaturated fatty acids . By immunological screening of a B . ammoniagenes expression library, an fas DNA fragment was isolated and subsequently used to clone the entire gene together with its flanking sequences . Within 10,525 bp of sequenced DNA, the 9,189-bp FAS coding region was identified, corresponding to a protein of 3,063 amino acids with a molecular mass of 324,910 Da . This gene (fasA) encodes, at its 5' end, the same amino acid sequence as is observed with purified B . ammoniagenes FAS . A second reading frame encoding another B . ammoniagenes FAS variant (FasB) had been identified previously . Both sequences are colinear and exhibit 61 and 47% identity at the DNA and protein levels, respectively . By using specific antibodies raised against a unique peptide sequence of FasB, this enzyme was shown to represent only 5 to 10% of the cellular FAS protein . Insertional inactivation of the FasB coding sequence causes no defective phenotype, while fasA disruptants require oleic acid for growth . Correspondingly, oleate-dependent B . ammoniagenes cells obtained by ethyl methanesulfonate mutagenesis were complemented by transformation with fasA DNA but not with fasB DNA . The data indicate that B . ammoniagenes contains two related though differently expressed type I FASs . FasA represents the bulk of cellular FAS protein and catalyzes the synthesis of both saturated and unsaturated fatty acids, while the minor variant, FasB, cannot catalyze the synthesis of oleic acid. Proc Natl Sci Counc Repub China B, 1996 Jul, 20(3), 87 - 91 Cloning of the tryptophan operon of Brevibacterium divaricatum and its expression in E . coli; Su YC et al.; A genomic bank from Brevibacterium divaricatum has been prepared using lambda EMBL3 as a vector . The genomic bank's titers are 2.2 x 10(6) pfu/micrograms . Through screening by plaque hybridization, a 9.6 kb NcoI fragment which contains the entire trp operon has been isolated . Polymerase chain reaction amplification and restriction endonuclease analysis of PCR fragments indicated that there is homology between the coryneform bacteria; however, some genetic diversity among the species still exists . By complementation tests using subcloning of the 9.6 kb NcoI fragments and various E . coli tryptophan auxotrophs, this fragment was found to contain a gene cluster composed of trpE, trpD, trpC, trpB and trpA in this order . This revealed that the tryptophan biosynthesis genes in B . divaricatum may be an operon. Plasmid, 1996 Jul, 36(1), 62 - 6 A cryptic plasmid pBL1 from Brevibacterium lactofermentum causes growth inhibition and filamentation in Escherichia coli; Goyal D et al.; A shuttle vector composed of pBL1, a 4.46-kb cryptic plasmid from coryneform bacterium Brevibacterium lactofermentum, and Escherichia coli vector pHSG298 was found to inhibit growth and cause cell filamentation in E coli . After construction of several deletions of pBL1, a 1.23-kb AccI-HindIII fragment was found responsible . DNA sequence analysis showed that this fragment contained a 726-bp-long open reading frame (ORF3), encoding a protein with 242 amino acid residues . Corresponding to ORF3, a 28-kDa protein was detected in an in vitro protein synthesis system . ORF3 was dispensable for the stable maintenance of pBL1 in coryneform bacteria. Plasmid, 1996 Jul, 36(1), 36 - 41 A Brevibacterium linens pRBL1 replicon functional in Corynebacterium glutamicum; Ankri S et al.; Brevibacterium linens RBL strain cryptic plasmid pRBL1 (8.0 kb) is described . A region involved in pRBL1 autonomous replication in Corynebacterium glutamicum was identified by insertion and deletion mapping and partially sequenced . This region encodes for a hypothetical 310-amino acid (aa) protein closely related to the replicases of plasmids pXZ10142 (C . glutamicum) and pAL5000 (Mycobacterium fortuitum) . The 310-aa protein also shows significant homology to proteins of pColE5-099 (Shigella sonnei) and pJD1 (Neisseria gonorrhoea) . At least one of these proteins, the Rep protein of pColE5-099, is known to be involved in theta replication. Zentralbl Bakteriol, 1996 Jul, 284(2-3), 246 - 54 An identification scheme for rapidly and aerobically growing gram-positive rods; von Graevenitz A et al.; An identification scheme for aerobically growing Gram-positive rods (genera Actinomyces, Arcanobacterium, Aureobacterium, Bacillus, Brevibacterium, Cellulomonas, Corynebacterium, Dermabacter, Erysipelothrix, Gardnerella, Lactobacillus, Listeria, Microbacterium, Oerskovia, Propionibacterium, Rhodococcus, Rothia, Turicella, as well as unnamed CDC groups, Clostridium tertium, and Mycobacterium fortuitum/chelonae) is presented . It is derived from the Hollis-Weaver scheme and uses catalase, oxidative/fermentative carbohydrate metabolism and motility as primary reactions . Tests for lipophilism, nitrate reduction, urease, esculin hydrolysis, the CAMP reaction, acid formation from five carbohydrates, as well as for some facultative reactions should lead to a correct diagnosis based on information available at the end of 1995. Nucleic Acids Res, 1996 Jun 15, 24(12), 2268 - 70 Cloning and expression of the BalI restriction-modification system; Ueno H et al.; BalI, a type II restriction-modification (R-M) system from the bacterium, Brevibacterium albidum, recognizes the DNA sequence 5'-TGGCCA-3' . We cloned the genes encoding the BalI restriction endonuclease and methyltransferase and expressed them in Escherichia coli . The two genes were aligned tail-to-tail and their termination codons overlapped . BalI restriction endonuclease and methyltransferase comprise 260 and 280 amino acids, respectively, and have molecular weights of 29 043 and 31 999 Da . The amino acid sequence of BalI methyltransferase is similar to that of other m6A MTases, although it has been categorized as a m5C methyltransferase . A high expression system for the BalI restriction endonuclease was constructed in E . coli for the production of large quantities of enzyme. Gene, 1996 Apr 17, 170(1), 91 - 4 Cloning and characterization of an IS-like element present in the genome of Brevibacterium lactofermentum ATCC 13869; Correia A et al.; A repetitive DNA element of the Gram+ Brevibacterium lactofermentum (Bl), cloned by a modification of the subtractive hybridization method, contained a 1.4-kb IS-like element, IS13869, which included an open reading frame (ORF) inside a perfect 26-bp terminal inverted repeat (TIR) . An 8-bp direct repeat (DR) was found outside each TIR . The ORF encoded a deduced protein of 436 amino acids (49 380 Da) with extensive similarity to other known transposases of insertion elements of Mycobacterium smegmatis (IS1096) . Pseudomonas sp . (tpnA) and Corynebacterium glutamicum (IS31831) . Distinct patterns were observed in different strains of Bl by hybridization with a probe internal to IS13869: four copies of IS13869 occurred in the wild type (wt) and R31 strains, but only three of them were observed in a recA derivative of the wt . Analysis by pulsed-field gel electrophoresis suggested that at least one copy of IS13869 had changed its position inside the chromosome during the lineage of a Bl derivative. FEMS Microbiol Lett, 1996 Apr 1, 137(2-3), 265 - 8 Genomic organization of purK and purE in Brevibacterium ammoniagenes ATCC 6872: purE locus provides a clue for genomic evolution; Chung SO et al.; From the genomic library of Brevibacterium ammoniagenes ATCC6872, the purE locus encoding 5'-phosphoribosyl-5-aminoimidazole (AIR) carboxylase (EC 4.1.1.21) was cloned and its nucleotide sequence was determined . From the sequence analysis, two distinct open reading frames (ORFs) in the sequence of the purE locus were identified as purK and purE genes (purK-purE) . An in vivo translation experiment reconfirmed the purK and purE genes to be independent . The genomic organization in the purE locus of B . ammoniagenes is opposite to that of the bacteria Escherichia coli and Bacillus subtilis . However, it coincides with the fused genes (purKE) of higher organisms Saccharomyces cerevisiae, Schizosaccharomyces pombe and Vigna aconitifolia . This suggests that the purE locus might be an intermediate form for genomic evolution of bacteria to higher organisms. Appl Environ Microbiol, 1996 Apr, 62(4), 1283 - 6 Nucleotide sequence and taxonomical distribution of the bacteriocin gene lin cloned from Brevibacterium linens M18; Valdes-Stauber N et al.; Linocin M18 is an antilisterial bacteriocin produced by the red smear cheese bacterium Brevibacterium linens M18 . Oligonucleotide probes based on the N-terminal amino acid sequence were used to locate its single copy gene, lin, on the chromosomal DNA . The amino acid composition, N-terminal sequence, and molecular mass derived from the nucleotide sequence of an open reading frame of 798 nucleotides coding for 266 amino acids found on a 3-kb BamHI restriction fragment correspond closely to those obtained from the purified protein (N . Valdes-Stauber and S . Scherer, Appl . Environ . Microbiol . 60:3809-3814, 1994) . No sequence homology to any protein or nucleotide sequences deposited in databases was found . Comparison of the nucleotide sequence and the N-terminal amino acid sequence derived from the protein suggests that B . linens M18 produces an N-formyl-methionyl-CAC tRNA . A wide taxonomical distribution of the gene within coryneform bacteria has been demonstrated by PCR amplification . The structural gene from linocin M18 is present at least in three Brevibacterium species, five Arthrobacter species, and five Corynebacterium species. J Bacteriol, 1996 Apr, 178(7), 1996 - 2004 Genetic characterization of site-specific integration functions of phi AAU2 infecting "Arthrobacter aureus" C70; Le Marrec C et al.; All the essential genetic determinants for site-specific integration of corynephage phi AAU2 are contained within a 1,756-bp DNA fragment, carried on the integrative plasmid p5510, and are shown to be functional in Escherichia coli . One open reading frame, ORF4, encoding a protein of 266 amino acids was shown to represent the phi AAU2 integrase . The nucleotide sequence of the phi AAU2 attachment site, attP, and the attB, attL, and attR sequences in the host "Arthrobacter aureus" C70 were determined . Identical nucleotide sequences were shown to be responsible for the integration of p5510 in the chromosomes of Corynebacterium glutamicum, Brevibacterium divaricatum, and B . lactofermentum, and a sequence almost identical to attB was found to be present in these three strains . In contrast to other phage site-specific recombination systems, a plasmid encompassing only int-attP failed to integrate into the host chromosome . This led to the identification of an 800-bp noncoding region, immediately upstream of int, absolutely required for site-specific integration of p5510. FEMS Microbiol Lett, 1996 Mar 15, 137(1), 63 - 8 Construction of new cloning vectors for Brevibacterium lactofermentum; Cadenas RF et al.; Two plasmid cloning vectors (pULMJ55 and pULMJ95) were constructed for Brevibacterium lactofermentum using the origin of replication of the endogenous plasmid pBL1 . Plasmid pULMJ55 is a replacement vector with transcriptional terminators from the B . lactofermentum trp operon flanking the BglII cloning sites . Religation of the BglII digested vector without insert creates a 376 bp perfect palindrome that is not tolerated in B . lactofermentum, giving positive selection for recombinant plasmids with inserts . Plasmid pULMJ95 contains the promoter-less alpha-amylase gene from Streptomyces griseus downstream of the trp terminator and is particularly suitable for the detection of promoters which are activated late during the growth phase . alpha-Amylase is secreted and its activity can be detected using simple plate tests. J Biol Chem, 1996 Mar 8, 271(10), 5524 - 35 Homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum . A dioxygenase with catalase activity; Miller MA et al.; Homoprotocatechuate 2,3-dioxygenase (2,3-HPCD) cleaves the aromatic ring of its substrate with insertion of both atoms of oxygen from O2 to form alpha-hydroxy- delta-carboxymethyl cis-muconic semialdehyde . The enzyme has been purified from the Gram-positive bacterium Brevibacterium fuscum and characterized . The enzyme appears to have a range of quaternary structures with predominant components of alpha4 and alpha6 (alpha subunit Mr = 42500 +/- 1500) and binds approximately 1 Fe(II)/subunit . Although the substrate Km values are similar to those of other Fe(II) ring cleaving dioxygenases, the turnover number is lower by 90-97%, and the enzyme exhibits much higher stability to metal chelators and H2O2 . The stability to H2O2 is shown to derive from an endogenous catalase activity of 2,3-HPCD (stoichiometry: 2 H2O2 --> 2 H2O + O2) that is novel for dioxygenases . H2O2 is a mixed-type inhibitor of the dioxygenase activity, suggesting that dioxygenase and catalase activities are both catalyzed by the enzyme, but at distinguishable sites . In contrast, catecholic substrates, including homoprotocatechuate and p-nitrocatechol, are nonessential activators of the catalase activity . The plot of 1/vi of catalase activity versus 1/{H2O2} is parabolic in the absence of catecholic substrates and linear in their presence, indicating that these reactions proceed by different mechanisms . A mechanism for catalase activity is proposed in which 2 H2O2 molecules bind simultaneously to the iron to account for the observed parabolic kinetic plot . Electron transfer between the peroxides mediated by the iron would yield 2 H2O and O2 . Catecholic substrates are proposed to modify this reaction by excluding one H2O2 from the Fe(II), thereby causing the kinetic plots to appear linear . Electron donation by the catecholic substrates would facilitate O O bond cleavage of H2O2, but outer sphere electron transfer from a second H2O2 in another step would be necessary to complete the reaction . p-Nitrocatechol is shown to bind differently to 2,3-HPCD than to other Fe(II) ring cleavage dioxygenases . Possible explanations for this observation are considered in the context of the proposed catalase and normal dioxygenase mechanisms which may also have bearing on the unique catalase activity and low dioxygenase turnover number of the enzyme. FEMS Microbiol Lett, 1996 Mar 1, 136(3), 309 - 15 Analysis of nucleotide methylation in DNA from Corynebacterium glutamicum and related species; Jang KH et al.; Plasmid DNA (pCSL17) isolated from Corynebacterium glutamicum transformed recipient McrBC+ strains of Escherichia coli with lower efficiency than McrBC- strains, confirming a previous report by Tauch et al . (FEMS Microbiol . Lett . 123 (1994) 343-348) which inferred that C . glutamicum DNA contains methylcytidine . Analysis of nucleotides in C . glutamicum-derived chromosomal and plasmid DNA failed to detect significant levels of methylated adenosine, but methylated cytidine was readily detected . Restriction enzymes which are inhibited by the presence of methylcytidine in their recognition sequence failed to cut pCSL17 from C . glutamicum, whereas enzymes which require methylation at adenosine in GATC sequences failed to cut . Failure of HaeIII to cut two specific sites of C . glutamicum-derived pCSL17 identified the first cytidine in the sequence GGCCGC as one target of methylation in this species, which contains the methyltransferase recognition sequence . Although Brevibacterium lactofermentum-derived DNA showed a similar methylation pattern by HPLC analysis, HaeIII cleaved these GGCCGC sites, suggesting differences in the specificity of methylation between these two species . Results for all analyses of B . flavum DNA were identical to those for C . glutamicum. J Struct Biol, 1996 Mar-Apr, 116(2), 317 - 9 Crystallization and preliminary X-ray analysis of cholesterol oxidase from Brevibacterium sterolicum containing covalently bound FAD; Croteau N et al.; Single crystals of cholesterol oxidase from Brevibacterium sterolicum containing a covalently bound form of the FAD cofactor have been obtained . The crystals are grown by vapor diffusion using the hanging drop technique from 12% polyethylene glycol, Mr 8000, and 75 mM MnSO4 as the precipitant at pH 5.2 . In order to obtain large diffraction quality crystals, nucleation must occur at 22 degrees C with subsequent growth at 17 degrees C . The crystals belong to the monoclinic space group P21 with cell dimensions a = 78 . 5 A, b = 126.7 A, c = 82.4 A and beta = 108.9 degrees with two protein molecules per asymmetric unit . Diffraction of these crystals has been observed to at least 2.2 A resolution and they are suitable for an X-ray structure analysis. Appl Environ Microbiol, 1996 Feb, 62(2), 501 - 6 Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein; Rattray FP et al.; The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-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 time course of peptide formation indicated that His-8-Gln-9, Ser-161-Gly-162, and either Gln-172-Tyr-173 or Phe-23-Phe-24 were the first, second, and third bonds cleaved, respectively . Other cleavage sites included Asn-19-Leu-20, Phe-32-Gly-33, Tyr-104-Lys-105, Leu-142-Ala-143, Phe-150-Arg-151, Gln-152-Phe-153, Leu-169-Gly-170, and Thr-171-Gln-172 . The proteinase had a broad specificity for the amino acid residues 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. Pneumonol Alergol Pol, 1996, 64 Suppl 1, 38 - 44 {Microflora of the in furniture factors as a potential occupational hazard: concentration and composition of microflora and immunologic reactivity of workers to microbial aeroallergens}; Krysinska-Traczyk E et al.; Microbiologial studies of the air were performed in two furniture factories . The concentration of microorganisms in the air was low, being of the order 10(3) cfu/m3 . The most common organisms were corynebacteria (Arthrobacter, Corynebacterium, Brevibacterium, Microbacterium) and fungi (Aspergillus fumigatus, Rhodotorula rubra) . Some of the species found in this environment possess known allergenic properties . Allergological examinations of the workers with environmental aeroallergens have been performed in three departments of one factory . The highest frequency of positive skin reactions were observed among the workers of the varnishing department which may be due to synergistic effects of chemical pollutants . The incidence of precipitin reactions was low among all workers. Pneumonol Alergol Pol, 1996, 64 Suppl 1, 25 - 31 {Microflora of the air in sawmills as a potential occupational hazard: concentration and composition of microflora and immunologic reactivity of workers to microbial aeroallergens}; Dutkiewicz J et al.; Microbiologial studies of the air and allergological examinations of the workers were performed in two sawmills processing deciduous wood (mainly oak) and in one sawmill processing coniferous wood (mainly pine) . The concentration of microorganisms in the air was of the order 10(3)-10(4) cfu/m3 . The most common organisms were corynebacteria (Arthrobacter, Corynebacterium, Brevibacterium, Microbacterium), spore-forming bacilli (Bacillus), Gram-negative bacteria (rahnella) and filamentous fungi (Aspergillus, Penicillium) . The workers responded to the extract of pine dust with much higher frequency than to the extract of oak dust . The workers processing pine were often sensitized to Rahnella while those processing oak were commonly sensitized to Penicillium . Precipitin reactions were rare and occurred only with the antigen of Rahnella. Folia Microbiol (Praha), 1996, 41(1), 10 - 4 Construction and characterization of new corynebacterial plasmids carrying the alpha-amylase gene; Ugorcakova J et al.; Novel corynebacterial plasmids carrying alpha-amylase gene from Bacillus have been constructed . The level of alpha-amylase expression depends on the size of the vector . The highest expression levels were measured in brevibacteria harboring pA61 plasmid. J Bacteriol, 1996 Jan, 178(2), 550 - 3 Multiple sigma factor genes in Brevibacterium lactofermentum: characterization of sigA and sigB; Oguiza JA et al.; Four rpoD hybridizing signals have been identified in the chromosome of Brevibacterium lactofermentum . Two rpoD-like genes, sigA and sigB, have been cloned and sequenced, and they encode principal sigma factors of the RNA polymerase . The deduced amino acid sequences of SigA and SigB showed very high similarities to those of Mycobacterium smegmatis MysA and MysB proteins, respectively, and also to those of HrdB proteins from different Streptomyces species . SigA and SigB maintain the conserved motifs of sigma 70-like principal sigma factors . sigB is closely linked to the dtxR gene (encoding a repressor of iron-regulated promoters homologous to the diphtheria toxin repressor from Corynebacterium diphtheriae. Eur J Clin Microbiol Infect Dis, 1995 Dec, 14(12), 1082 - 5 Recurrent bacteremia due to Brevibacterium casei in an immunocompromised patient; Reinert RR et al.; A case of an immunocompromised patient who experienced two episodes of septicemia caused by a coryneform bacterium is reported . Biochemical characteristics and analysis of cellular fatty acids and of cell wall components showed two identical strains of Brevibacterium casei to be responsible for these infections . The lack of easy-to-perform methods for identification may have led, in the past, to an underestimation of the role of this bacterium, especially in immunocompromised patients. Eur J Clin Microbiol Infect Dis, 1995 Sep, 14(9), 801 - 4 Bacteremia caused by Brevibacterium species in an immunocompromised patient; Kaukoranta-Tolvanen SS et al.; A 46-year-old woman suffering from non-Hodgkin's lymphoma was admitted to the hospital because of high fever . Multiple blood cultures revealed an unusual finding, a Brevibacterium species, which was reisolated 16 days later from the tip of her long-term central venous catheter . This case indicates that Brevibacterium species isolated from normally sterile sites should be considered as a potential pathogen, especially in immunocompromised patients. FEMS Microbiol Lett, 1995 Sep 1, 131(2), 121 - 6 Isolation of promoters from Brevibacterium flavum strain MJ233C and comparison of their gene expression levels in B . flavum and Escherichia coli; Zupancic TJ et al.; A promoter probe shuttle vector suitable for the isolation of promoter elements from coryneform bacteria was constructed . This vector carried the neomycin phosphotransferase (NPTII) gene from transposon Tn5 as a reporter gene, and was capable of replication in both Escherichia coli and Brevibacterium flavum . The vector was used in the construction of a B . flavum library of 899 independently isolated promoter clones . Promoters with a wide range of activities in B . flavum, including some very strong promoter elements, were isolated . Comparative analysis suggests that significant differences between B . flavum and E . coli may exist in the determinants of promoter strength. Antonie Van Leeuwenhoek, 1995 Aug, 68(2), 173 - 9 Sizing of the Rhodococcus sp . R312 genome by pulsed-field gel electrophoresis . Localization of genes involved in nitrile degradation; Bigey F et al.; The two restriction enzymes AsnI and DraI were found to produce DNA fragment sizes that could be used for mapping the Rhodococcus sp . R312 (formerly Brevibacterium sp . R312) genome by pulsed-field gel electrophoresis . AsnI produced 24 fragments (4 to 727 kb) and DraI yielded 15 fragments (8.5 to 2400 kb) . The fragment lengths in each digest were summed, indicating that the size of the chromosome ranged from 6.31 to 6.56 Mb, with a mean of 6.44 Mb . In addition, the wide-spectrum amidase gene (amiE) and the operon containing the enantiomer-selective amidase gene (amdA) and the nitrile hydratase structural gene (nthA, nthB) were localized on the AsnI and DraI fragments. Acta Derm Venereol, 1995 Jul, 75(4), 280 - 2 The influence of the pH-value on the growth of Brevibacterium epidermidis in continuous culture; Lukacs A et al.; Brevibacterium epidermidis is a major component of the bacterial flora of certain skin surface biotopes, characterized by a comparatively high pH-value . The presence of Brevibacterium epidermidis seems to be linked to the production of malodour . Skin surface pH has been found to be a major factor of bacterial growth on the skin . In order to find out if this might also apply to Brevibacterium epidermidis, this microorganism was grown in vitro in continuous culture using a chemostat . Specific growth rate and density of colony forming units were well correlated . While the organism grew readily from pH 5.5 to 8.5, this was not the case with a pH of 5.0 . Thus pH-shifts induced by cosmetic procedures can only prevent unpleasant body odour due to abundant growth of bacteria if the pH-value is decreased to 5.0 or less. Res Microbiol, 1995 Jul-Aug, 146(6), 493 - 505 Prophage distribution in coryneform bacteria; Moreau S et al.; Four temperate bacteriophages of corynebacteria were isolated after UV induction . Phages phi 304L and phi 304S were both induced from Corynebacterium glutamicum ATCC 13058, ATCC 21488, ATCC 21649 and ATCC 21650 strains, and have no known sensitive host . Phages phi 15 and phi 16 were both induced from ATCC 14020 and ATCC 21792 . Phage phi 15 formed turbid plaques on Corynebacterium sp . ATCC 21857 and on C . glutamicum ATCC 13058, ATCC 21488, ATCC 21649 and ATCC 21650 . Phage phi 16 produced turbid plaques only on C . glutamicum ATCC 21792 cured of prophage phi 16 . All these phages belong to the Siphoviridae family . Their genomes consist of a double-stranded DNA with cohesive ends and share no homology with each other . Prophages phi 16, phi 304L and phi 304S were integrated into their respective host chromosomes, whereas prophage phi 15 seemed to persist free in the cell . Cross-hybridizations between phage DNAs and total cellular DNA obtained from 20 strains belonging to the genera Corynebacterium and Brevibacterium did not show the presence of these prophages in strains other than their respective hosts. Brain Res, 1995 Jun 12, 683(1), 36 - 42 Cholesterol oxidation reduces Ca(2+)+MG (2+)-ATPase activity, interdigitation, and increases fluidity of brain synaptic plasma membranes; Wood WG et al.; These experiments examined effects of cholesterol oxidation on Ca(2+)+Mg(2+)-ATPase activity, Na(+)+K(+)-ATPase activity, and membrane structure of brain synaptic plasma membranes (SPM) . Cholesterol oxidase {E.C.1.1.3.6 from Brevibacterium sp.} was used to oxidize cholesterol . Two cholesterol pools were identified in synaptosomal membranes based on their accessibility to cholesterol oxidase . A rapidly oxidized cholesterol pool was observed with a 1t1/2 of 1.19 +/- 0.09 min and a second pool with a 2t1/2 of 38.30 +/- 4.16 min . Activity of Ca(2+)+Mg(2+)-ATPase was inhibited by low levels of cholesterol oxidation . Ten percent cholesterol oxidation, for example, resulted in approximately 35% percent inhibition of Ca(2+)+Mg(2+)-ATPase activity . After 13% cholesterol oxidation, further inhibition of Ca(2+)+Mg(2+)-ATPase activity was not observed . Activity of Na(+)+K(+)-ATPase was not affected by different levels of cholesterol oxidation (5%-40%) . SPM interdigitation was significantly reduced and fluidity was significantly increased by cholesterol oxidation . The relationship observed between SPM interdigitation and Ca(2+)+Mg(2+)-ATPase activity was consistent with studies using model membranes {7} . Brain SPM function and structure were altered by relatively low levels of cholesterol oxidation and is a new approach to understanding cholesterol dynamics and neuronal function . The sensitivity of brain SPM to cholesterol oxidation may be important with respect to the proposed association between oxygen free radicals and certain neurodegenerative diseases. Gene, 1995 May 26, 158(1), 87 - 90 Cloning and sequence determination of the aspartase-encoding gene from Brevibacterium flavum MJ233; Asai Y et al.; A 2.5-kb EcoRI fragment containing the aspartase-encoding gene (aspA) of Brevibacterium flavum MJ233 was cloned into plasmid pUC18 using Southern hybridization with the Escherichia coli aspA gene as a probe . The complete nucleotide (nt) sequence of the cloned DNA indicated that the deduced gene product of the Br . flavum aspA is composed of 526 amino acids (aa) . Comparison of the aa sequence to the corresponding sequences from E . coli, Bacillus subtilis and Pseudomonas fluorescens revealed 63, 47 and 57% homology, respectively . The aspA product was determined to have a size of approx . 57 kDa by SDS-PAGE. Appl Environ Microbiol, 1995 May, 61(5), 1847 - 52 Activity and purification of linenscin OC2, an antibacterial substance produced by Brevibacterium linens OC2, an orange cheese coryneform bacterium; Maisnier-Patin S et al.; An orange cheese coryneform bacterium isolated from the surface of Gruyere of Comte and identified as Brevibacterium linens produces an antimicrobial substance designated linenscin OC2 . This compound inhibits gram-positive food-borne pathogens including Staphylococcus aureus and Listeria monocytogenes but is not active against gram-negative bacteria . Linenscin OC2 caused viability loss and lysis of the test organism, Listeria innocua . Electron microscopy showed that linenscin OC2 induces protoplast formation and cell lysis . The native substance is resistant to proteolytic enzymes, heat, and organic solvents and stable over a wide range of pH . The molecular weight of the native linenscin OC2 was estimated by gel chromatography to be over 285,000 . Linenscin OC2 was purified by ammonium sulfate precipitation, 2-propanol extraction, and reverse-phase chromatography . Direct detection of antimicrobial activity on a sodium dodecyl sulfate-polyacrylamide gel suggested an apparent molecular mass under 2,412 Da . Molecular mass was determined to be 1,196.7 Da by mass spectrometry . Amino acid composition analysis indicated that linenscin OC2 may contain 12 residues. Biofactors, 1995 May, 5(1), 1 - 4 Heat treatment of Corynebacterium ammoniagenes leads to aeration dependent accumulation of 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate; Ostrovsky D et al.; 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEC) identified as a new bacterial oxidative stress substance (Ostrovsky D . et al . (1993) Biochem . J., 295, 901-902) was shown to accumulate in Corynebacterium (Brevibacterium) ammoniagenes cells aerobically cultivated in peptone-yeast extract-glucose broth on heating for 1 hour at 45 degrees C . The enzyme(s) responsible for MEC biosynthesis is evidently oxidized for activation and is completely loosing its activity on anaerobic incubation at this temperature in an hour . Salt stress or drying did not provoke the MEC biosynthesis. Proc Natl Sci Counc Repub China B, 1995 Apr, 19(2), 113 - 22 Construction of lysine-producing strains by gene disruption and replacement in Brevibacterium divaricatum; Su YC et al.; Gene disruption and replacement techniques were applied to block the biosynthesis of threonine and methionine and thus to construct genetically stable lysine producers in a glutamate-producing bacteria, Brevibacterium divaricatum . The homoserine dehydrogenase gene (hom), homoserine kinase gene (thrB) and hom-thrB operon were amplified as 1.8, 1.25 and 2.8 kb fragments from B . divaricatum by polymerase chain reaction (PCR) and cloned in an E . coli-coryneform bacteria shuttle vector, pSUMN18 . These genes were disrupted by inserting a kanamycin resistant gene (kan) from pUC4-KISS into the structural genes . Integrative plasmids were constructed and transformed into B . divaricatum . Integrative transformants could be obtained only when the integrative plasmids were constructed from the plasmids which had escaped from the restriction barrier of the hosts . The resulting integrative transformants showed kanamycin resistance and contained no plasmids . About 1-10% of the transformants were auxotrophs . By checking the nutritional requirement, it was found that all of these transformants required threonine and/or homoserine as expected . Southern blot analysis confirmed the integrations, and both single and double crossover homologous recombination mechanisms were proposed to explain the integration and replacement . These auxotrophic integrative transformants which were derived from double crossover events accumulated 1-3% lysine in culture broth only when the added threonine was limited . Integrative transformants which were site-specifically inactivated in hom or hom-thrB genes produced more lysine than did those only inactivated at the thrB gene . These transformants were extremely stable, and the reversion frequency was below 10(-9) per generation . It is suggested that this technique will be useful in the construction of stable auxotrophic mutants. Steroids, 1995 Mar, 60(3), 290 - 4 A convenient synthesis of 7 alpha-hydroxycholest-4-en-3-one by the hydroxypropyl-beta-cyclodextrin-facilitated cholesterol oxidase oxidation of 3 beta,7 alpha-cholest-5-ene-3,7-diol; Alexander DL et al.; The initial biosynthetic conversions of cholesterol to the bile acids involve sequential 7 alpha-hydroxylation (catalyzed by cholesterol 7 alpha-hydroxylase) followed by C-3 oxidation and concomitant double bond migration (to a delta 4-configuration, catalyzed by 3 beta-delta 5-C27-steroid oxidoreductase) to provide 7 alpha-hydroxycholest-4-en-3-one . A straightforward, and economical, preparation (on a 0.1 g scale) of this pivotal biosynthetic intermediate has been devised . Reduction of 3 beta-(benzoyloxy)-cholest-5-en-7-one with LiB(sec-butyl)3H provided a 4:1 mixture, respectively, of the 7 alpha- and 7 beta-hydroxy diastereomers, which were separated chromatographically . Solvolytic removal of the C-3 benzoyl group gave 3 beta,7 alpha-cholest-5-ene-3,7-diol . A suspension of the 1:1 (v/v) complex (formed by mutual dissolution in MeOH, followed by evaporation of the solvent) of this diol with hydroxypropyl-beta-cyclodextrin, at a concentration of 1 mg mL-1 (in neutral phosphate buffer), was converted by Brevibacterium sp cholesterol oxidase (0.25 U mg-1 of substrate) and catalase (70 U mg-1 of substrate, to recover O2 from the H2O2 produced by the enzymatic oxidation) to a suspension of 7 alpha-hydroxycholest-4-en-3-one and the hydroxypropyl-beta-cyclodextrin . The yield for the enzymatic conversion was in excess of 90% . A much poorer and less reproducible yield (< 20%) was seen in the absence of the hydroxypropyl-beta-cyclodextrin . Routine extraction of this aqueous suspension, and chromatographic purification (85:15 CHCl3/acetone v/v on silica) of the residue, gave pure 7 alpha-hydroxycholest-4-en-3-one in 68% isolated yield . This route is a significant improvement, in terms of reaction scale and convenience, over the previous procedures for the preparation of this steroid. Biosci Biotechnol Biochem, 1995 Mar, 59(3), 512 - 3 Degradation of 2-ketoarginine by guanidinobutyrase in arginine aminotransferase pathway of Brevibacterium helvolum; Yorifuji T et al.; Guanidinobutyrase (EC 3.5.3.7) involved in the arginine oxygenase pathway of Brevibacterium helvolum IFO 12073 was found to catalyze also the hydrolysis of 2-ketoarginine (2-keto-5-guanidinovalerate) to 2-ketoornithine (2-keto-5-aminovalerate) and urea, the second step of the arginine aminotransferase pathway . No other enzyme that degraded 2-ketoarginine was found in cells grown on L-arginine . The enzyme hydrolyzed 2-ketoarginine with a relative rate of about 0.7% of that toward 4-guanidinobutyrate . The Km for 2-ketoarginine was 33 mM. Microbiology, 1995 Mar, 141 ( Pt 3), 729 - 36 23Na NMR spectroscopy of free Na+ in the halotolerant bacterium Brevibacterium sp . and Escherichia coli; Nagata S et al.; 23Na NMR spectroscopy was used to determine free Na+ concentrations in a halotolerant bacterium, Brevibacterium sp., and Escherichia coli . The internal Na+ concentration of both strains depended little on the growth phases and was unchanged after 5 d storage at 2 degrees C . In Brevibacterium sp . the level of intracellular sodium increased gradually at higher extracellular NaCl concentrations in both the presence and absence of yeast extract in the growth medium . E . coli cells accumulated a higher concentration of free Na+ than those of Brevibacterium sp . The change of Na+ concentration in both strains was inverse to that of growth rate . When appropriate amounts of osmoprotectants (proline, glycine betaine, or gamma-aminobutyrate) were added with the NaCl, internal free Na+ levels in Brevibacterium sp . were lowered, but those of E . coli were unchanged . While addition of KCl to medium containing NaCl increased the intracellular level of free Na+, the total sodium concentration in the cells remained unchanged, indicating that sodium that had been bound or attached was made free in the cytosol . In Brevibacterium sp . grown in the presence of 0.5 M NaCl, free and bound sodium concentrations in the cytosol were estimated to be 0.14 and 0.23 mumol (mg protein)-1, respectively . As a result, visibility by 23Na NMR was 38%. Gene, 1995 Feb 27, 154(1), 77 - 9 Brevibacterium linens pBL33 and Rhodococcus rhodochrous pRC1 cryptic plasmids replicate in Rhodococcus sp . R312 (formerly Brevibacterium sp . R312); Bigey F et al.; The replication of two cryptic plasmids from Brevibacterium linens ATCC 9174 (pBL33) and Rhodococcus rhodochrous ATCC 4276 (pRC1) was investigated in Rhodococcus sp . R312 (formerly Brevibacterium sp . R312) . The recombinant plasmids pSP33 (pBL33 derivative) and pSPC1 (pRC1 derivative) were found to be suitable for establishing new host-vector systems for Rhodococcus sp . R312 . They all carry the Tn903 neomycin-resistance-encoding gene (aphI). FEMS Microbiol Lett, 1995 Feb 1, 126(1), 1 - 6 Isolation of insertion elements from gram-positive Brevibacterium, Corynebacterium and Rhodococcus strains using the Bacillus subtilis sacB gene as a positive selection marker; Jager W et al.; The sacB gene of Bacillus subtilis was successfully applied in various Arthrobacter, Brevibacterium, Corynebacterium and Rhodococcus strains for the isolation of transposable elements . Three different insertion sequence (IS) elements entrapped in sacB were isolated . The IS elements IS-Bl and IS-Cg isolated from Brevibacterium lactofermentum and Corynebacterium glutamicum, respectively, were found to be similar in size (1.45 kb) and generated target duplications of 8 bp . Their inverted repeats showed homology . In contrast, the IS element IS-Rf isolated from Rhodococcus fascians was only 1.3 kb long and generated a 3-bp target duplication . IS-Cg and IS-Rf were not restricted to their original host strains, and we also found strains harbouring more than one element. Biochem Biophys Res Commun, 1995 Jan 17, 206(2), 688 - 93 The isomerization catalyzed by Brevibacterium sterolicum cholesterol oxidase proceeds stereospecifically with one base; Kass IJ et al.; We have demonstrated that the isomerization reaction catalyzed by Brevibacterium sterolicum (ATCC 81387) cholesterol oxidase (EC 1.1.3.6) proceeds via a stereospecific proton transfer from the 4 beta carbon to the 6 beta carbon to form 4-cholestene-3-one using deuterated and nondueterated substrates . This result implies that there is one active site base, positioned over the beta-face, responsible for isomerization . On the basis of X-ray crystallographic evidence {Li, J., Vrielink, A., Brick, P . & Blow, D . M . Biochemistry 32, 11507-11515 (1993)}, glutamate-361 is the most likely candidate for this general base. Folia Microbiol (Praha), 1995, 40(6), 595 - 606 Molecular cloning of the hom-thrC-thrB cluster from Bacillus sp . ULM1: expression of the thrC gene in Escherichia coli and corynebacteria, and evolutionary relationships of the threonine genes; Malumbres M et al.; A 6.5 kb DNA fragment containing the gene (thrC) encoding threonine synthase, the last enzyme of the threonine biosynthetic pathway, has been cloned from the DNA of Bacillus sp . ULM1 by complementation of Escherichia coli and Brevibacterium lactofermentum thrC auxotrophs . Complementation studies showed that the thrB gene (encoding homoserine kinase) is found downstream from the thrC gene, and analysis of nucleotide sequences indicated that the hom gene (encoding homoserine dehydrogenase) is located upstream of the thrC gene . The organization of this cluster of genes is similar to the Bacillus subtilis threonine operon (hom-thrC-thrB) . An 1.9 kb BclI fragment from the Bacillus sp . ULM1 DNA insert 351 amino acids was found corresponding to a protein of 37462 Da . The thrC gene showed a low G + C content (39.4%) and the encoded threonine synthase is very similar to the B . subtilis enzyme . Expression of the 1.9 kb BcI DNA fragment in E . coli minicells resulted in the formation of a 37 kDa protein . The upstream region of this gene shows promoter activity in E . coli but not in corynebacteria . A peptide sequence, including a lysine that is known to bind the pyridoxal phosphate cofactor, is conserved in all threonine synthase sequences and also in the threonine and serine dehydratase genes . Amino acid comparison of nine threonine synthases revealed evolutionary relationships between different groups of bacteria. Ukr Biokhim Zh, 1995 Jan-Feb, 67(1), 96 - 100 {Heterogeneity of peptidoglycans of bacterial origin}; Pozur VK et al.; Heterogeneity of peptidoglycans of Staphylococcus aureus of strains 3272 and Wood-46, Brevibacterium flavum, Micrococcus lysodeikticus has been studied by the method of electrophoresis in the system polyacrylamide gel--sodium dodecylsulfate . It is shown that peptidoglycans of different strains of S . aureus and other species of gram-positive bacteria treated with ultrasound are a complex of fractions with 3 basic high-molecular components with M 100, 92 and 84 kDa, while two main fractions with Mm 92 and 84 kDa are identified in peptidoglycans drugs treated with ultrasound. J Bacteriol, 1995 Jan, 177(2), 465 - 7 Molecular cloning, DNA sequence analysis, and characterization of the Corynebacterium diphtheriae dtxR homolog from Brevibacterium lactofermentum; Oguiza JA et al.; A homolog of the Corynebacterium diphtheriae dtxR gene was isolated from Brevibacterium lactofermentum . The product of the B . lactofermentum dtxR gene was immunoreactive with polyclonal anti-DtxR antibodies and functioned as an iron-activated repressor capable of regulating the expression of beta-galactosidase from a diphtheria tox promoter/operator transcriptional fusion in recombinant Escherichia coli . The extents of induction by increasing concentrations of the chelator 2,2'-dipyridyl were identical in cells expressing DtxR from either C . diphtheriae or B . lactofermentum. Biokhimiia, 1995 Jan, 60(1), 14 - 9 {Oxidative stress and organic cyclopyrophosphates in bacteria}; Ostrovskii DN; Oxidative stress in nocardioform bacteria--Corynebacterium (Brevibacterium) ammoniagenes, Micrococcus luteus and Mycobacterium smegmatis--is accompanied by a significant accumulation of 2-C-methyl-D-erythrol-2,4-cyclopyrophosphate (MEC), which is correlated with the ability of the producer to grow under stress . Metabolic stability of MEC in bacterial cells, its spontaneous isomerization into 1,2-cyclophospho-4-phosphate and the possibility of a genetic transfer of the MEC-synthesizing capacity from Corynebacterium to E . coli have been demonstrated . The involvement of MEC in the response to oxidative stress via the complex formation between MEC and bivalent cations has been postulated. Microbios, 1995, 81(327), 73 - 83 Lethal effect of carbonyl cyanide m-chlorophenylhydrazone on Escherichia coli and a halotolerant Brevibacterium species; Nagata S; An attempt was made to examine quantitatively the survival of Escherichia coli and the halotolerant Brevibacterium species, as a function of the exposure time to carbonyl cyanide m-chlorophenylhydrazone (CCCP), a proton conductor . Growth rates, viability, and protein concentrations of E . coli grown in the absence of glucose were unaffected by the addition of 100 microM CCCP . In the presence of glucose the viability was reduced after 24 h incubation with CCCP . Such a high efficiency of CCCP lethality for E . coli cells in the presence of glucose was attributed to not only the growth phase but also the acidic pH of the culture due to metabolites from glucose, mainly lactic acid . The culture of E . coli appeared to be in a syncopic state hovering between life and death when it was exposed to CCCP in the presence of more than 30 mM glucose . In contrast, growth rates of Brevibacterium were reduced in proportion to the exposure time to CCCP . The lethal effect of CCCP to Brevibacterium was slightly enhanced by the addition of glucose into the culture. J Bacteriol, 1994 Dec, 176(23), 7362 - 71 Transcriptional analysis and regulatory signals of the hom-thrB cluster of Brevibacterium lactofermentum; Mateos LM et al.; Two genes, hom (encoding homoserine dehydrogenase) and thrB (encoding homoserine kinase), of the threonine biosynthetic pathway are clustered in the chromosome of Brevibacterium lactofermentum in the order 5' hom-thrB 3', separated by only 10 bp . The Brevibacterium thrB gene is expressed in Escherichia coli, in Brevibacterium lactofermentum, and in Corynebacterium glutamicum and complements auxotrophs of all three organisms deficient in homoserine kinase, whereas the Brevibacterium hom gene did not complement two different E . coli auxotrophs lacking homoserine dehydrogenase . However, complementation was obtained when the homoserine dehydrogenase was expressed as a fusion protein in E . coli . Northern (RNA) analysis showed that the hom-thrB cluster is transcribed, giving two different transcripts of 2.5 and 1.1 kb . The 2.5-kb transcript corresponds to the entire cluster hom-thrB (i.e., they form a bicistronic operon), and the short transcript (1.1 kb) originates from the thrB gene . The promoter in front of hom and the hom-internal promoter in front of thrB were subcloned in promoter-probe vectors of E . coli and corynebacteria . The thrB promoter is efficiently recognized both in E . coli and corynebacteria, whereas the hom promoter is functional in corynebacteria but not in E . coli . The transcription start points of both promoters have been identified by primer extension and S1 mapping analysis . The thrB promoter was located in an 87-bp fragment that overlaps with the end of the hom gene . A functional transcriptional terminator located downstream from the cluster was subcloned in terminator-probe vectors. Appl Microbiol Biotechnol, 1994 Dec, 42(4), 575 - 80 Construction and characterization of recA mutant strains of Corynebacterium glutamicum and Brevibacterium lactofermentum; Fitzpatrick R et al.; An internal fragment of the Corynebacterium glutamicum recA gene was amplified by the polymerase chain reaction (PCR) using degenerate primers corresponding to two short sequences that are well conserved in procaryotic RecA proteins . The deduced amino acid sequence of the amplified fragment shared significant homology with RecA sequences from other bacteria including the "invariant" and functionally conserved amino acids Leu-126, Asp-144, Gly-157, Arg-169 and Asn-193 . Highest identity (91%) was shared with the gram-positive Mycobacterium tuberculosis RecA sequence . The amplified fragment was cloned into a conditional suicide vector, pBGS, and used to generate recA deficient strains of C . glutamicum and Brevibacterium lactofermentum by insertional inactivation . These strains exhibited classical RecA phenotypes including reduced recombinational activity and increased sensitivity to DNA-damaging agents such as UV irradiation, mitomycin C and methyl-methanesulphonate. Mol Gen Genet, 1994 Nov 15, 245(4), 397 - 405 Transposon mutagenesis of coryneform bacteria; Vertes AA et al.; The Corynebacterium glutamicum insertion sequence IS31831 was used to construct two artificial transposons: Tn31831 and miniTn31831 . The transposition vectors were based on a gram-negative replication origin and do not replicate in coryneform bacteria . Strain Brevibacterium flavum MJ233C was mutagenized by miniTn31831 at an efficiency of 4.3 x 10(4) mutants per microgram DNA . Transposon insertions occurred at different locations on the chromosome and produced a variety of mutants . Auxotrophs could be recovered at a frequency of approximately 0.2% . Transposition of IS31831 derivatives led not only to simple insertion, but also to cointegrate formation (5%) . No multiple insertions were observed . Chromosomal loci of B . flavum corresponding to auxotrophic and pigmentation mutants could be rescued in Escherichia coli, demonstrating that these transposable elements are useful genetic tools for studying the biology of coryneform bacteria. Appl Microbiol Biotechnol, 1994 Nov, 42(2-3), 300 - 3 Construction of a new host-vector system in Arthrobacter sp . and cloning of the lipase gene; Morikawa M et al.; Arthrobacter sp . strain MIS38 was transformed with a shuttle vector containing the kanamycin resistant gene kan (derived from Tn5) by an electroporation method . This shuttle vector is from Brevibacterium lactofermentum and Escherichia coli, pULRS8 . The following optimal condition of electroporation was determined . A square wave pulse of 1 kV/cm electric field strength for 0.5 ms duration yielded 3 x 10(5) transformants/micrograms plasmid DNA . The number of transformants increased with the amount of DNA over the range 0.01-5 micrograms . This host-vector system was then used successfully to clone and express a lipase gene from Arthrobacter sp . strain MIS38 into both Arthrobacter sp . MIS38 and E . coli JM109. Microbiology, 1994 Oct, 140 ( Pt 10), 2841 - 7 Pulsed-field gel electrophoresis analysis of the genome of amino acid producing corynebacteria: chromosome sizes and diversity of restriction patterns; Correia A et al.; A large number of species of corynebacteria are known to be amino acid producers, including members of the genera Corynebacterium and Brevibacterium . Pulsed-field gel electrophoresis (PFGE) of DNA fragments obtained by using endonucleases which recognize AT-rich hexanucleotide or octanucleotide sequences produces a discrete pattern of bands useful for fingerprinting and physical mapping of the chromosome . Using Pacl and Swal endonucleases the genome of Brevibacterium lactofermentum ATCC 13869 (genome size 3052 kb) was consistently cut into 26 and 20 bands, respectively, and the genome of Corynebacterium glutamicum ATCC 13032 (2987 kb) yielded 27 and 26 fragments, respectively . The pattern of restriction fragments was identical for related strains (B . lactofermentum ATCC 13869, B . lactofermentum BLO, B . lactofermentum R31) but different from the pattern of fragments of other soil isolates of the same species (B . lactofermentum DSM 20412) or from closely related organisms such as C . glutamicum; the different pattern of restriction fragments may be used to differentiate taxonomically related species . Brevibacterium linens showed a different behaviour, due to its high G+C content; its genome (3105 kb) was resolved into 8 or 15 fragments, respectively, by digestion with the hexanucleotide-recognizing endonucleases DraI and AseI . PFGE of DNA fragments obtained using these enzymes is a powerful technique for quick resolution of the corynebacteria genome into a small number of large fragments. Appl Environ Microbiol, 1994 Oct, 60(10), 3809 - 14 Isolation and characterization of Linocin M18, a bacteriocin produced by Brevibacterium linens; Valdes-Stauber N et al.; Brevibacterium linens M18, isolated from red smear cheese, produces a substance that inhibits the growth of Listeria spp . and several coryneform and other gram-positive bacteria . No gram-negative bacteria were inhibited . The substance is heat labile, sensitive to proteolytic enzymes, and stable between pH 3 and 12 . High levels of this bacteriocin, named Linocin M18, were obtained in the stationary growth phase . Linocin M18 was purified by ultrafiltration, ultracentrifugation, and gel filtration chromatography . In its native form, it is a proteinaceous aggregate with a high molecular weight . Fractions with Linocin M18 activity contained particles of 20 to 30 nm in diameter . The bacteriocin consists of a single protein subunit with a molecular mass of 31 kDa and an isoelectric point of 4.5 N-terminal sequence analysis yielded Met-Asn-Asn-Leu-Tyr-Arg-Glu-Leu-Ala-Pro-Ile-Pro-Gly-Pro-Ala-Ala-Ala-Glu- Ile . Significant homology with published sequences was lacking. FEMS Microbiol Lett, 1994 Sep 15, 122(1-2), 129 - 36 Cloning of the wide spectrum amidase gene from Brevibacterium sp . R312 by genetic complementation . Overexpression in Brevibacterium sp . and Escherichia coli; Azza S et al.; The amiE gene of Brevibacterium sp . R312 encoding wide spectrum amidase was isolated by complementation of a Brevibacterium sp . mutant using a plasmid gene bank of chromosomal DNA . The amiE structural gene and its promoter were localized on a 1.8-kb fragment by subsequent subcloning and complementation studies . Another promoter localized in the pSR 1 fragment of the cloning vector was shown to be able to control amiE gene expression . In Brevibacterium sp., the investigation of amidase activities related to one copy of the gene suggested that the regulation of the amiE gene expression was under negative control . High expression levels have been obtained in Brevibacterium sp . and, after substitution of the amiE promoter by the tac promoter, in Escherichia coli. J Am Acad Dermatol, 1994 Sep, 31(3 Pt 2), S31 - 3 Tinea pedis pathophysiology and treatment; Leyden JL; Fungal infections of the foot can be divided into three major varieties, all of which have differing pathophysiologic aspects with therapeutic implications . Interdigital infections involve an ecological interplay between dermatophytes and bacteria . Simple scaling types of infection are caused by dermatophyte invasion of the stratum corneum, whereas macerated, erosive infections are caused by selection and overgrowth of bacteria, particularly Brevibacterium epidermidis, Micrococcus sedantarius, and various gram-negative species . Bacterial production of methanethiol and other sulfur compounds leads to inhibition of dermatophytes and accounts for the lower recovery of dermatophytes from the most severe cases . Plantar surface infections consist of widespread, moccasin-type infection caused by Trichophyton rubrum and localized scaling infections with episodes of intense inflammation caused by Trichophyton mentagrophytes . The former is particularly associated with an atopic background . Therapy is difficult because of poor immune responses and difficulty in delivering a sufficient quantity of drugs to the lower layers of a thick stratum corneum . Intense inflammation in T . mentagrophytes infections is the result of an immune, contact allergic response to fungal antigens. Appl Environ Microbiol, 1994 Sep, 60(9), 3343 - 8 Purification and characterization of an amidase from an acrylamide-degrading Rhodococcus sp; Nawaz MS et al.; A constitutively expressed aliphatic amidase from a Rhodococcus sp . catalyzing acrylamide deamination was purified to electrophoretic homogeneity . The molecular weight of the native enzyme was estimated to be 360,000 . Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified preparation yielded a homogeneous protein band having an apparent molecular weight of about 44,500 . The amidase had pH and temperature optima of 8.5 and 40 degrees C, respectively, and its isoelectric point was pH 4.0 . The amidase had apparent K(m) values of 1.2, 2.6, 3.0, 2.7, and 5.0 mM for acrylamide, acetamide, butyramide, propionamide, and isobutyramide, respectively . Inductively coupled plasma-atomic emission spectometry analysis indicated that the enzyme contains 8 mol of iron per mol of the native enzyme . No labile sulfide was detected . The amidase activity was enhanced by, but not dependent on Fe(2+), Ba(2+), and Cr(2+) . However, the enzyme activity was partially inhibited by Mg(2+) and totally inhibited in the presence of Ni(2+), Hg(2+), Cu(2+), Co(2+), specific iron chelators, and thiol blocking reagents . The NH2-terminal sequence of the first 18 amino acids displayed 88% homology to the aliphatic amidase of Brevibacterium sp . strain R312. Appl Environ Microbiol, 1994 Jul, 60(7), 2209 - 19 Analysis and expression of the thrC gene of Brevibacterium lactofermentum and characterization of the encoded threonine synthase; Malumbres M et al.; The thrC gene of Brevibacterium lactofermentum was cloned by complementation of Escherichia coli thrC auxotrophs . The gene was located by deletion mapping and complementation analysis in a 2.9-kb Sau3AI-HindIII fragment of the genome . This fragment also complemented a B . lactofermentum UL1035 threonine auxotroph that was deficient in threonine synthase . A 1,892-bp DNA fragment of this region was sequenced; this fragment contained a 1,446-bp open reading frame that encoded a 481-amino-acid protein having a deduced M(r) of 52,807 . The gene was expressed in E . coli, by using the phage T7 system, as a 53-kDa protein . The promoter region subcloned in promoter-probe plasmids was functional in E . coli . A Northern analysis revealed that the gene was expressed as a monocistronic 1,400-nucleotide transcript . The transcription start point of the thrC gene was located by S1 mapping 6 bp upstream from the translation initiation codon, which indicated that this promoter was one of the leaderless transcription-initiating sequences . The threonine synthase overexpressed in B . lactofermentum UL1035 was purified almost to homogeneity . The active form corresponded to a monomeric 52.8-kDa protein, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The purified enzyme required pyridoxal phosphate as its only cofactor to convert homoserine phosphate into threonine. J Clin Microbiol, 1994 Jul, 32(7), 1729 - 32 Differentiation of Brevibacterium spp . encountered in clinical specimens; Funke G et al.; Forty-three strains belonging to the genus Brevibacterium which were encountered in clinical materials over 2 decades were compared with reference strains, including the type strains, of B . casei, B . epidermidis, B . mcbrellneri, B . iodinum, and B . linens . By means of carbohydrate assimilation tests (CATs) the 43 clinical isolates could be assigned to the species B . casei (n = 41) and B . epidermidis (n = 2) . DNA-DNA hybridizations were performed for 20 clinical isolates and confirmed the species identification of the isolates . Cellular fatty acid profiles of all strains were determined and found to have less discriminative power than CATs . This is the first report indicating that most clinical Brevibacterium isolates are B . casei and that CATs provide an easy-to-perform method for species determination within the genus, thus avoiding nucleic acid techniques. Int J Syst Bacteriol, 1994 Jul, 44(3), 583 - 5 Phylogenetic analysis of species of the meso-diaminopimelic acid-containing genera Brevibacterium and Dermabacter; Cai J et al.; 16S rRNA gene sequencing studies were performed on Dermabacter hominis and four meso-diaminopimelic acid-containing species of the genus Brevibacterium . Phylogenetic analysis revealed a close association between Dermabacter hominis and representatives of the lysine-containing genera Arthrobacter, Micrococcus, and Renibacterium . By contrast, the genus Brevibacterium formed a distinct line of descent within the high-guanine-plus-cytosine-containing actinomycetes, displaying no specific affinity with any other organism examined. Eur J Biochem, 1994 Jun 15, 222(3), 941 - 7 ESR and electron nuclear double resonance characterization of the cholesterol oxidase from Brevibacterium sterolicum in its semiquinone state; Medina M et al.; Reduction of the flavin of cholesterol oxidase from Brevibacterium sterolicum, at pH values above 7, by sodium dithionite or light irradiation in the presence of EDTA (either in the presence or absence of deazariboflavin) was found to occur through a stable intermediate state . This intermediate had an optical spectrum characteristic of a flavin anionic semiquinone . The rate and extent of reduction were pH-dependent . No semiquinone intermediate was detected during reduction by these agents at pH values of 6.5 or below or at any pH when dehydroisoandrosterone, a protein substrate analogue, was used as reductant . No intermediate radical was detected during the reoxidation process . Treatment of cholesterol oxidase semiquinone with dehydroisoandrosterone did not convert the semiquinone intermediate to the fully reduced state . The absorption coefficient of oxidised cholesterol oxidase at 470 nm is 10.3 M-1 cm-1 . The ESR signal of Brevibacterium sterolicum cholesterol oxidase semiquinone is centred at g = 2.004 . The linewidth of the signal was 1.48 mT when the protein was studied in H2O or D2O . These data are in agreement with those reported for anionic semiquinones . The linewidths were the same when measured either at X-band or at S-band frequencies, indicating that line broadening is due to hyperfine interactions . The linewidth decreased to 1.43 mT when the substrate, dehydroisoandrosterone, was added . Electron nuclear double resonance (ENDOR) spectroscopy of cholesterol oxidase semiquinone provided further information about the interactions of the flavin radical with protons . A group of signals, with couplings of 9-12 MHz, is attributed to protons on 8-CH3 (Aiso = 10.9 MHz) and on C6 (Aiso = 9 MHz) of the flavin ring . No change in these hyperfine coupling constants was detected when the protein was studied in D2O . However, the hyperfine coupling constant attributed to protons on 8-CH3 decreased by 0.98 MHz when the ENDOR spectrum of the cholesterol oxidase semiquinone was studied in the presence of dehydroisoandrosterone (Aiso = 9.92 MHz) . A second group of signals was observed with hyperfine couplings less than 2.5 MHz . Some of these weak couplings disappeared when the protein was transferred to D2O, or when the substrate, dehydroisoandrosterone, was present . These signals are attributed to displaced water protons, or to exchangeable protons from amino-acid residues on the protein near the flavin binding site, involved in substrate stabilisation. J Bacteriol, 1994 Jun, 176(11), 3154 - 61 Characterization of a region of plasmid pBL1 of Brevibacterium lactofermentum involved in replication via the rolling circle model; Fernandez-Gonzalez C et al.; The minimal region for autonomous replication of pBL1, a 4.5-kb cryptic plasmid of Brevibacterium lactofermentum ATCC 13869 that has been used to construct a variety of corynebacterium vectors, was shown to be contained on a 1.8-kb HindII-SphI DNA fragment . This region contains two open reading frames (ORFs) (ORF1 and ORF5) which are essential for pBL1 replication in B . lactofermentum . Accumulation of single-strand intermediates in some of the constructions indicates that plasmid pBL1 replicates via the rolling circle replication model; its plus strand and minus strand were identified by hybridization with two synthetic oligonucleotide probes complementary to each pBL1 strand . ORF1 seems to encode the Rep protein and showed partial homology with sequences for Rep proteins from Streptomyces plasmids which replicate via rolling circle replication such as pIJ101, pSB24, and pJV1. J Clin Microbiol, 1994 Jun, 32(6), 1511 - 8 Human infections caused by Brevibacterium casei, formerly CDC groups B-1 and B-3; Gruner E et al.; Forty-one clinical strains of CDC coryneform groups B-1 and B-3 were compared biochemically, by analysis of cell wall sugars, amino acids, and cellular fatty acids, and by DNA relatedness to the type strains of Brevibacterium casei, Brevibacterium epidermidis, and Brevibacterium linens . Twenty-two strains were shown to be B . casei, while five other strains formed a phenotypically inseparable genomospecies in the same genus . The remaining isolates were genetically heterogeneous, and most are probably members of the genus Brevibacterium . They were not further identified, but they were biochemically distinguishable from B . casei . Eleven of the clinical strains of B . casei were isolated from blood, and two each were isolated from cerebrospinal fluid and from pleural fluid . At least five isolates were from multiple blood or cerebrospinal fluid cultures . To our knowledge, these strains are the first described clinical isolates identified as B . casei, which was previously considered to be a nonpathogenic species. Appl Environ Microbiol, 1994 Jun, 60(6), 1993 - 2002 Influence of changing temperature on growth rate and competition between two psychrotolerant Antarctic bacteria: competition and survival in non-steady-state temperature environments; Rutter M et al.; Competition between two psychrotolerant bacteria was examined in glycerol-limited chemostat experiments subjected to non-steady-state conditions of temperature . One bacterium, a Brevibacterium sp . strain designated CR3/1/15, responded rapidly to temperature change, while a second, Hydrogenophaga pseudoflava, designated CR3/2/10, exhibited a lag in growth after a shift-down during a square-wave temperature cycle but not after a shift-up . The effects on competition and survival by these bacteria of both sine-wave and square-wave temperature changes between 2 and 16 degrees C over a 24-h cycle time were examined, as well as square-wave cycles over 12 and 96 h . The changing proportion of each bacterium in the chemostat was determined by plate counting at regular intervals . Under a sine-wave temperature cycle H . psedoflava outcompeted the Brevibacterium sp., but under square-wave temperature cycles the two bacteria coexisted because the lag by H . pseudoflava after the temperature shift-down favored the faster-responding Brevibacterium sp . The two bacteria thus exhibited different survival strategies, with H . pseudoflava adapted to effective competition under steady-state conditions and the Brevibacterium sp . adapted to rapid adaptation and survival in a changing environment . The degree of perturbation of the bacteria, expressed as a temperature challenge index (delta temp/delta time), was greater under a square-wave temperature cycle than under a sine-wave cycle of equivalent amplitude and frequency, and higher-temperature challenge favored the Brevibacterium sp . A computer model was developed to examine competition between the bacteria in transient environments . The frequency of the temperature cycle influenced competition, as with a longer cycle (96 h) the significance of the lag by H . pseudoflava decreased compared with that of a 24-h cycle, and H . pseudoflava predominated in a mixed culture with a 96-h cycle . The shift-down lag by H . pseudoflava, during which it adapted to low temperature, disadvantaged it in a changing temperature environment, but at a short cycle time (12 h) this disadvantage was countered by the incomplete loss of low-temperature adaptation between cycles and thus the carryover of some low-temperature adaptation . Also, it was demonstrated that, as well as consideration of the effect of temperature changes on inducing lags in growth, the loss of adaptation to low temperature between cycles had to be taken into account in the computer model if it was to reproduce the trends in the experimental data. Appl Environ Microbiol, 1994 Jun, 60(6), 1984 - 92 Influence of temperature on growth rate and competition between two psychrotolerant Antarctic bacteria: low temperature diminishes affinity for substrate uptake; Nedwell DB et al.; The growth kinetics of two psychrotolerant Antarctic bacteria, Hydrogenophaga pseudoflava CR3/2/10 (2/10) and Brevibacterium sp . strain CR3/1/15 (1/15), were examined over a range of temperatures in both batch culture and glycerol-limited chemostat cultures . The maximum specific growth rate (mu max) and Ks values for both bacteria were functions of temperature, although the cell yields were relatively constant with respect to temperature . The mu max values of both strains increased up to an optimum temperature, 24 degrees C for 2/10 and 20 degrees C for 1/15 . Strain 1/15 might therefore be considered to be more psychrophilic than strain 2/10 . For both bacteria, the specific affinity (mu max/Ks) for glycerol uptake was lower at 2 than at 16 degrees C, indicating a greater tendency to substrate limitation at low temperature . As the temperature increased from 2 to 16 degrees C, the specific affinity of 1/15 for glycerol increased more rapidly than it did for 2/10 . Thus 1/15, on the basis of this criterion, was less psychrophilic than was 2/10 . The steady-state growth kinetics of the two strains at 2 and 16 degrees C imply that 1/15 would be able to outgrow 2/10 only at relatively low substrate concentrations (< 0.32 g of glycerol.liter-1) and high temperatures (> 12 degrees C), which suggests that 1/15 has a less psychrotolerant survival strategy than does 2/10 . Our data were compared with other data in the literature for bacteria growing at low temperatures . They also showed an increase of substrate-specific affinity with increasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS) Bioorg Med Chem, 1994 Jun, 2(6), 447 - 55 Microbial hydrolysis of glutaronitrile derivatives with Brevibacterium sp . R 312; Kerridge A et al.; The enantiomerically pure (S)-cyano acids 3 and 4 can be obtained by biotransformation with Brevibacterium sp . R 312 of the corresponding prochiral dinitriles 5 and 6, respectively . The hydrolysis is probably a two step process involving a nitrile hydratase and an amidase . In connection with these investigations a facile method for the synthesis of racemic 4-cyano-3-hydroxybutanoic acid derivatives was developed. J Clin Microbiol, 1994 May, 32(5), 1223 - 8 Characteristics of CDC group 3 and group 5 coryneform bacteria isolated from clinical specimens and assignment to the genus Dermabacter; Funke G et al.; Over a 1-year period, 11 isolates (including 5 from blood cultures) of the recently described CDC group 3 and group 5 coryneform bacteria were derived from clinical specimens and compared with reference strains . Biochemical characteristics indicated a very close relationship between CDC group 3 and group 5 coryneform bacteria . The ability of CDC group 3 and the inability of CDC group 5 coryneform bacteria to ferment xylose were the only reactions that were different for the two taxa . Chemotaxonomic features of the two groups included the presence of meso-diaminopimelic acid, a lack of mycolic acids, and the presence of predominantly branched cellular fatty acids, a combination found among gram-positive rods only in Brevibacterium spp., Brachybacterium faecium, and Dermabacter hominis . 16S rRNA gene sequence analysis revealed that CDC group 3 and group 5 coryneform bacteria are members of the genus Dermabacter, which to date has been isolated exclusively from human skin. Biofactors, 1994 May, 4(3-4), 155 - 9 Bacterial oxidative stress substance spontaneously recyclizes to form 2-methylbutane-1,2,3,4-tetraol-1,2-cyclophospho-4-phosphate; Ostrovsky D et al.; The cells of Corynebacterium (Brevibacterium) ammonia-genes cultivated in a medium supplemented with diquat or benzylviologen accumulate 2-methylbutane-1,2,3,4-tetraol-2,4- cyclopyrophosphate as revealed by 31P-NMR spectroscopy . On heating at 120 degrees C for 30 min the cells still maintain a substantial portion of this compound and acquire new cyclic phosphates characterized by 31P-NMR chemical shifts of +17.3 and +20 p.p.m . The +17.3 p.p.m . component was isolated from the preparation of the purified cyclopyrophosphate kept for some time at pH above 7 and it was shown to be 2-methylbutane-1,2,3,4-tetraol-1,2,- cyclophospho-4-phosphate on the grounds of two-dimensional NMR spectroscopy. Biofactors, 1994 May, 4(3-4), 151 - 4 Bacteria and pesticides: a new aspect of interaction--involvement of a new biofactor; Ostrovsky D et al.; Positively charged hydrophobic pesticides of the dipyridyl family {diquat, paraquat, benzylviologen (BV++), etc.} were shown to provoke accumulation of 2-methylbutane-1,2,3,4-tetraol-2,4- cyclopyrophosphate in the cells Corynebacterium (Brevibacterium) ammoniagenes while neutral dipyridyls were not . Hydrophobicity was also an important factor in this phenomenon . Of the other pesticides tested, only linuron was effective . BV++ also induced biosynthesis of the compound in Rhodococcus rhodochrous, Rh.ruber, Rh.sp . (Nocardia corynebacteroides) . These microorganisms as well as most of the previously identified oxidative stress activated producers of this new cyclopyrophosphate were able to synthesize free radical generating compounds . The microorganisms concerned belong mainly to the order Actinomycetales. C R Acad Sci III, 1994 Apr, 317(4), 299 - 303 {Change of cholesterol oxidase of Brevibacterium sterolicum and substrate specificity}; Loubat-Hugel C et al.; Cholesterol oxidase modified by hydrogen peroxide is inactive with cholesterol solubilized in buffer with surfactants . Pregn-5-en-3 beta-ol when solubilized in the same conditions and substrates soluble in buffer, like 3 beta-hydroxy-androst-5-en-17-one or 3 beta-hydroxy-androst-5-en-17 beta-carboxylic acid are substrates of the modified enzyme . The observed loss of activity on cholesterol could be due to the inability of the oxidized cholesterol oxidase to extract cholesterol from mixed cholesterol/surfactant aggregates . Cholesterol oxidase on storage undergoes modifications close to those with hydrogen peroxide and care should be taken for the use of cholesterol oxidase as cholesterol probe. Biosci Biotechnol Biochem, 1994 Apr, 58(4), 768 - 70 Factors improving L-threonine production by a three L-threonine biosynthetic genes-amplified recombinant strain of Brevibacterium lactofermentum; Ishida M et al.; When a Brevibacterium lactofermentum L-threonine producer that accumulated L-lysine as well, was transformed with a recombinant plasmid carrying the indigenous hom, thrB, and thrC genes, delayed growth and plasmid instability were observed . Addition of organic nutrients, vitamins (thiamine.HCl and d-biotin), and NaCl under the optimal culture conditions solved these problems and further increased threonine production in a small jar fermentor. Biochemistry, 1994 Mar 1, 33(8), 2329 - 34 Cholesterol distribution in renal epithelial cells LLC-PK1 as determined by cholesterol oxidase: evidence that glutaraldehyde fixation masks plasma membrane cholesterol pools; el Yandouzi EH et al.; Treatment with cholesterol oxidases has shown that cholesterol is heterogeneously distributed in brush border membranes isolated from the apical domain of the renal and intestinal epithelial cells {Bloj, B., & Zilversmit, D . B . (1982) J . Biol . Chem . 257, 7608-7614; El Yandouzi, E . H., & Le Grimellec, C . (1992) Biochemistry 31, 547-551} . Cholesterol distribution between plasma membrane and intracellular membranes of the corresponding cells remains unexplored . The effects of Brevibacterium sp . cholesterol oxidase on the cholesterol content of LLC-PK1 cells, an epithelial cell line with multiple differentiated characteristics of the renal proximal tubule, were investigated . In confluent living cells grown as a monolayer on solid support, a small but significant fraction (13%) of the cholesterol was oxidized during the first hour of the oxidase treatment . Glutaraldehyde fixation prior to treatment resulted in a nearly complete (86.1 +/- 1.8) oxidation of the cellular cholesterol according to first-order kinetics . Filipin labeling and oxidation at 15 degrees C confirmed that cholesterol was essentially confined to the plasma membrane in LLC-PK1 cells . When adding the oxidase either on the apical or on the basolateral side of cells grown on permeant support and fixed with glutaraldehyde, a comparable monophasic oxidation of cholesterol was observed, despite the presence of efficient tight junctions . Adding the oxidase to both sides simultaneously did not increase the rate of oxidation . Finally, fixation of isolated renal brush border membranes with glutaraldehyde rendered undiscernible their cholesterol pools . We conclude that glutaraldehyde fixation, a commonly used process in the analysis of cholesterol distribution in cells, can mask the existence of cholesterol pools in plasma membranes. Gene, 1994 Feb 11, 139(1), 99 - 103 Cloning and sequencing of the secY homolog from Coryneform bacteria; Kobayashi M et al.; A conserved domain of the secY genes from Bacillus subtilis, Mycoplasma capricolum and Escherichia coli was used to design degenerate oligodeoxyribonucleotides . These synthetic DNA sequences were used to screen a lambda library of Brevibacterium flavum MJ233 . A 1.5-kb KpnI fragment of a recombinant lambda phage containing the secY homology from Br . flavum MJ233 was subsequently subcloned into plasmid pUC118 . The complete nucleotide (nt) sequence of the cloned fragment indicated that the deduced gene product of the Br . flavum secY homolog is composed of 440 amino acids (aa) with a deduced M(r) of 47,871 . 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 secY homolog is a membrane protein containing ten transmembrane segments . In addition, we could identify, downstream from secY, a putative coding sequence of the enzyme adenylate kinase . This gene organization is identical to that observed in the B . subtilis genome. J Mol Biol, 1994 Feb 11, 236(1), 374 - 6 Preliminary crystallographic study of protocatechuate 3,4-dioxygenase from Brevibacterium fuscum; Earhart CA et al.; The enzyme protocatechuate 3,4-dioxygenase from the Gram positive organism Brevibacterium fuscum crystallizes in the triclinic space group P1 with unit cell dimensions a = 96.1 A, b = 97.2 A, c = 118.1 A and alpha = 113.9 degrees, beta = 90.7 degrees, gamma = 117.8 degrees . The rod-like crystals diffract to 2.4 A resolution . Rotation function analysis suggests that there are six promoters arranged with local 32 symmetry in the asymmetric unit rather than the previously proposed pentameric complex. J Bacteriol, 1994 Feb, 176(3), 789 - 95 Degradation of fluorene by Brevibacterium sp . strain DPO 1361: a novel C-C bond cleavage mechanism via 1,10-dihydro-1,10-dihydroxyfluoren-9-one; Trenz SP et al.; Angular dioxygenation has been established as the crucial step in dibenzofuran degradation by Brevibacterium sp . strain DPO 1361 (V . Strubel, K . H . Engesser, P . Fischer, and H.-J . Knackmuss, J . Bacteriol . 173:1932-1937, 1991) . The same strain utilizes biphenyl and fluorene as sole sources of carbon and energy . The fluorene degradation sequence is proposed to be initiated by oxidation of the fluorene methylene group to 9-fluorenol . Cells grown on fluorene exhibit pronounced 9-fluorenol dehydrogenase activity . Angular dioxygenation of the 9-fluorenone thus formed yields 1,10-dihydro-1,10-dihydroxyfluoren-9-one (DDF) . A mechanistic model is presented for the subsequent C-C bond cleavage by an NAD(+)-dependent DDF dehydrogenase, acting on the angular dihydrodiol . This enzyme was purified and characterized as a tetramer of four identical 40-kDa subunits . The following Km values were determined: 13 microM for DDF and 65 microM for 2,3-dihydro-2,3-dihydroxybiphenyl . The enzyme also catalyzes the production of 3-(2'-carboxyphenyl)catechol, which was isolated, and structurally characterized, in the form of the corresponding lactone, 4-hydroxydibenzo-(b,d)-pyran-6-one . Stoichiometry analysis unequivocally demonstrates that angular dioxygenation constitutes the principal pathway in Brevibacterium sp . strain DPO 1361. Appl Microbiol Biotechnol, 1994 Feb, 40(6), 864 - 6 Transfer of the broad-host-range IncQ plasmid RSF1010 and other plasmid vectors to the gram-positive methylotroph Brevibacterium methylicum by electrotransformation; Nesvera J et al.; Gram-positive facultative methylotrophic coryneform bacterium Brevibacterium methylicum was efficiently transformed with various plasmids using electroporation of intact cells . In addition to the plasmid vectors pEC71 and pZ6-1 constructed on the basis of cryptic plasmids from coryneform bacteria, broad-host-range plasmids pLS5 (derivative of plasmid pMV158 from Streptococcus agalactiae) and RSF1010 belonging to the incompatibility group IncQ from Gram-negative bacteria were found to be present as autonomous structurally unchanged DNA molecules in B . methylicum transformants . With the exception of pZ6-1, all these plasmids were stably maintained in B . methylicum cells grown under non-selective conditions . When plasmid DNAs isolated from B . methylicum were used, the highest efficiency of transformation (10(5) transformants/micrograms DNA) was achieved. Gene, 1994 Jan 28, 138(1-2), 35 - 41 Directed mutagenesis of a regulatory palindromic sequence upstream from the Brevibacterium lactofermentum tryptophan operon; Guerrero C et al.; A cloned 9.6-kb fragment of Brevibacterium lactofermentum DNA, carrying the entire trp operon and upstream regulatory sequences, produces a polycistronic 7.0-kb transcript as detected by hybridization with an internal probe . The transcription start point (tsp) was identified by S1 mapping . The operator-promoter (OP) region subcloned in Escherichia coli and B . lactofermentum promoter-probe vectors exhibited about tenfold higher activity in B . lactofermentum . A 14-bp wild-type (wt) palindrome located at bp -15 to -28 was mutated to change the conserved adenine adjacent to the axis of symmetry . The wt and mutated OP regions were coupled to the amy reporter gene (encoding alpha-amylase {Amy}) or to the 5' region (trpE and trpG genes) of the trp operon, for expression studies . Constructions with the regulatory signals coupled to the wt trpE-trpG genes were introduced in a B . lactofermentum trpE mutant (obtained by gene disruption) . The mutation in the palindrome did not affect the promoter activity in B . lactofermentum or E . coli when grown in minimal medium . Tryptophan repressed the OP as assayed by the anthranilate synthase (AS) activity in B . lactofermentum in constructions with the wt OP region, but surprisingly, caused a large stimulation of either AS or the Amy reporter activity, in constructions with the mutated OP . The palindromic sequence is, therefore, involved in a dual repression-stimulation control of expression of the trp operon. Int J Syst Bacteriol, 1994 Jan, 44(1), 74 - 82 Phylogenetic interrelationships of round-spore-forming bacilli containing cell walls based on lysine and the non-spore-forming genera Caryophanon, Exiguobacterium, Kurthia, and Planococcus; Farrow JA et al.; The 16S rRNA gene sequences of "Bacillus aminovorans" and several species considered to be phylogenetically related to the group 2 bacilli of Ash et al . (C . Ash, J . A . E . Farrow, S . Wallbanks, and M . D . Collins, Lett . Appl . Microbiol . 13:202-206, 1991) were determined . A comparative analysis of the sequence data revealed that the round-spore-forming group 2 bacilli, together with some asporogenous taxa (the genera Caryophanon, Exiguobacterium, Kurthia, Planococcus), form a phylogenetically distinct cluster that is only remotely related to Bacillus subtilis, the type species of the genus Bacillus . Within this cluster, planococci, kurthiae, Caryophanon spp., and two lines defined by Bacillus sphaericus and Bacillus fusiformis and by Sporosarcina ureae, Bacillus pasteurii, Bacillus globisporus, and Bacillus psychrophilus were found to be distinct genera . Exiguobacterium aurantiacum and Brevibacterium acetylicum were found to form a distinct clade, which was peripherally related to this cluster . "B . aminovorans" exhibited no specific relationship with the group 2 bacilli or with any of the other reference species examined. Semin Dermatol, 1993 Dec, 12(4), 280 - 4 Tinea pedis; Leyden JJ et al.; Tinea pedis is a term used to encompass several clinically distinctive infections of the skin of the foot . Dermatophytic fungi are primarily responsible for these infections . Several nondermatophytes have been implicated in some patients, particularly for nail infections . The major clinical variants are (1) interdigital infections in which dermatophytes initiate the process by damaging the stratum corneum while the subsequent maceration and leukokeratosis results from overgrowth of bacteria such as Micrococcus sedantarius, Brevibacterium epidermidis, Corynebacterium minutissimum and gram-negative organisms; (2) plantar mocasin type of hyperkeratosis due to T rubrum and found primarily in those with an atopic background; (3) vesiculo-bullous infections in the arch and side of the foot due to an immune response of delayed hypersensitivity to T mentagrophytes. FEMS Microbiol Lett, 1993 Dec 1, 114(2), 145 - 51 The adenylate cyclase catalytic domain of Streptomyces coelicolor is carboxy-terminal; Danchin A et al.; A DNA fragment of Streptomyces coelicolor encoding the carboxy-terminal catalytic domain of adenylate cyclase was cloned, sequenced and expressed in an Escherichia coli cya-defective strain where it produced nanomole levels of cAMP . The amino acid sequence of the enzyme displays similarities with the Brevibacterium liquefaciens pyruvate regulated adenylate cyclase. Gene, 1993 Nov 30, 134(1), 15 - 24 Codon preference in corynebacteria; Malumbres M et al.; The codon usage (CU) of 34 genes from the closely related species, Brevibacterium lactofermentum and Corynebacterium glutamicum (BLCG), was analysed and compared with that of 23 genes from other Brevibacterium and Corynebacterium species . The G+C content of the BLCG genes ranged from 50 to 62% . A wider range was found in other corynebacterial genes (25-71%) . The G+C contents of non-coding regions in glutamic acid bacteria are lower than those of the coding regions and both values are lower than the G+C content of ribosomal RNA (rRNA) sequences, suggesting an unusual biased mutation pressure . The CU and synonymous codon usage (SCU) analysis showed several common characteristics among the sequenced corynebacterial genes, consistent with the close relatedness of B . lactofermentum and C . glutamicum . A subset of 25 preferred codons were deduced from the presumably highly expressed genes and they encode most of the amino acid (aa) residues of the BLCG group . An analysis of the effective number of codons (Nc) was carried out in order to check the GC3s (G+C content at the silent third position of sense codons) dependence of the CU in corynebacteria . Nc values showed differences between the BLCG group and other corynebacterial sequences . A comparison of the most used codons for each aa showed a stronger similarity to Streptomyces than to Escherichia coli . The CU/SCU tables of corynebacteria are useful for identification of protein-coding regions, including start codons when they are uncertain, and for designing oligodeoxyribonucleotide probes from an aa sequence. Biochemistry, 1993 Nov 2, 32(43), 11507 - 15 Crystal structure of cholesterol oxidase complexed with a steroid substrate: implications for flavin adenine dinucleotide dependent alcohol oxidases; Li J et al.; Cholesterol oxidase from Brevibacterium sterolicum is a flavin-dependent enzyme that catalyzes the oxidation and isomerization of 3 beta-hydroxy steroids with a double bond at delta 5-delta 6 of the steroid ring backbone . The crystal structure of the free enzyme in the absence of a steroid substrate has previously been determined . In this paper we report the crystal structure of the complex of cholesterol oxidase with the steroid substrate dehydroisoandrosterone, refined at 1.8-A resolution . The final crystallographic R-value is 15.7% for all reflections between 10.0- and 1.8-A resolution . The steroid is buried within the protein in an internal cavity which, in the free enzyme crystal structure, was occupied by a lattice of water molecules . The conformations of a number of side chains lining the active-site cavity have changed in order to accommodate the steroid substrate . A loop region of the structure between residues 70 and 90 differs significantly between the substrate-free and substrate-bound forms of the enzyme, presumably to facilitate binding of the steroid . The hydroxyl group of the steroid substrate is hydrogen-bonded to both the flavin ring system of the FAD cofactor and a bound water molecule . FAD-dependent cholesterol oxidase shares significant structural homology with another flavoenzyme, glucose oxidase, suggesting that it might also be a member of the glucose-methanol-choline (GMC) oxidoreductase family . Although there is only limited sequence homology, a superposition of these two structures reveals a conserved histidine residue within hydrogen-bonding distance of the active-site water molecule.(ABSTRACT TRUNCATED AT 250 WORDS) Plasmid, 1993 Nov, 30(3), 284 - 8 Expression of a synthetic DNA region containing a consensus promoter and two lac operators in Brevibacterium sp; Chan Kwo Chion CK et al.; The cat reporter gene was used to assess expression of two promoters, previously strongly expressed in Escherichia coli, in Brevibacterium sp . R312 strain . The tac promoter (de Boer et al., 1983, Proc . Natl . Acad . Sci . USA 80, 21-25) was poorly expressed in Brevibacterium sp . In contrast, the AatII-SalI fragment of plasmid pYEJ001 (Pharmacia LKB Biotechnology, Sweden) containing two lac operators, a consensus sequence promoter and the cat structural gene clearly revealed chloramphenicol acetyltransferase activity and the presence of a 25,600-kDa protein, corresponding to the monomeric CAT protein, in cell extracts. J Bacteriol, 1993 Nov, 175(22), 7356 - 62 A gene encoding arginyl-tRNA synthetase is located in the upstream region of the lysA gene in Brevibacterium lactofermentum: regulation of argS-lysA cluster expression by arginine; Oguiza JA et al.; The Brevibacterium lactofermentum argS gene, which encodes an arginyl-tRNA synthetase, was identified in the upstream region of the lysA gene . The cloned gene was sequenced; it encodes a 550-amino-acid protein with an M(r) of 59,797 . The deduced amino acid sequence showed 28% identical and 49% similar residues when compared with the sequence of the Escherichia coli arginyl-tRNA synthetase . The B . lactofermentum enzyme showed the highly conserved motifs of class I aminoacyl-tRNA synthetases . Expression of the argS gene in B . lactofermentum and E . coli resulted in an increase in aminoacyl-tRNA synthetase activity, correlated with the presence in sodium dodecyl sulfate-polyacrylamide gels of a clear protein band that corresponds to this enzyme . One single transcript of about 3,000 nucleotides and corresponding to the B . lactofermentum argS-lysA operon was identified . The transcription of these genes is repressed by lysine and induced by arginine, showing an interesting pattern of biosynthetic interlock between the pathways of both amino acids in corynebacteria. Enzyme Microb Technol, 1993 Nov, 15(11), 979 - 84 Bench-scale production of acrylamide using the resting cells of Brevibacterium sp . CH2 in a fed-batch reactor; Lee CY et al.; Effects of various organic acids and salts on the stabilization of nitrile hydratase were investigated . The stability of the nitrile hydratase of Brevibacterium CH2 during storage was greatly enhanced by the addition of n-butyric acid . Effects of temperature, pH, and concentrations of acrylonitrile and n-butyric acid on acrylamide production by the resting cells were also investigated . Acrylamide production per unit dry weight of the cells increased 1.33 times by the addition of 0.05% n-butyric acid . A 20% acrylamide solution was successfully produced in a bench-scale reactor (12 l) with only a trace amount of salts after 10 h of hydration reaction under optimum reaction conditions without using an isotonic substrate . The conversion yield was nearly 100%, and acrylic acid as a by-product was not produced . Final acrylamide production of 400 g g-1 cells and productivity of 20 g/(g cells l-1 x h-1) were obtained. J Med Microbiol, 1993 Oct, 39(4), 255 - 61 A new Brevibacterium sp . isolated from infected genital hair of patients with white piedra; McBride ME et al.; A new aerobic gram-positive non-sporeforming bacillus has been isolated from infected genital hair of patients with white piedra in association with Trichosporon beigelii . This species has been characterised morphologically, nutritionally, by DNA base composition, cell-wall analysis and cellular fatty-acid profile on the basis of 14 isolates . The G+C content of DNA is 63.05 mol% . Cell walls possess meso-diaminopimelic acid (Type IV) and the sugars glucose, galactose, xylose and ribose; mycolic acids are not present . The species has a distinct colonial and microscopic morphology, is strongly proteolytic and produces methanethiol . These findings and the cellular fatty-acid profile are compatible with the genus Brevibacterium . A new species is proposed based on the following characters: colonial and microscopic growth and morphology; conditions for rod-to-coccus cycle; ribose utilisation; and tellurite reduction . The type strain has been named Brevibacter