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The phage BFK20 replication origin was identified using bioinformatics tools and a fragment with the origin nucleotide sequence was cloned into the tetracycline resistance gene of Escherichia coli vector pBR328, to make the plasmid pBOS. After transformation into the host strain Brevibacterium flavum CCM 251, pBOS was able to replicate, showing that the cloned region may function as a replication origin. The presence of the BFK20 origin sequence in a pBOS plasmid isolated from B. flavum CCM 251 was confirmed by Southern hybridisation. Monitoring pBOS stability in corynebacterial hosts showed that pBOS was stable in Corynebacterium glutamicum RM3 for 20 generations and in B. flavum CCM 251 for 10 generations. The effect of the cloned BFK20 replication origin on host resistance to BFK20 infection was tested. Growth of a B. flavum CCM 251 strain harbouring pBOS stopped after phage infection, but without complete lysis. Five hours after infection, the viability of the modified strain was about five times higher than the viability of wild-type B. flavum CCM 251. Thus, the ability of the BFK20 replication origin to confer the origin-derived phage-encoded resistance phenotype to B. flavum CCM 251 was confirmed.

Linenscin OC2 is an antibacterial substance produced by the orange cheese coryneform bacterium Brevibacterium linens OC2. It inhibits the growth of Gram-positive bacteria but it is inactive against Gram-negative bacteria. The intact outer membrane of Gram-negative bacteria was shown to be an effective permeability barrier against linenscin OC2. At high dosage the effect of linenscin OC2 was bacteriolytic on Listeria innocua. Bacteriostasis was observed at low dosage and peptidoglycan biosynthesis was affected at an early step upstream of the UDP-N-acetylglucosamine. Hemolytic activity of this substance on sheep erythrocytes suggested a common mode of action on prokaryotic and eukaryotic cells. It also suggested that the cytoplasmic membrane might be the primary target of linenscin OC2.

A gene encoding a cholesterol oxidase from Brevibacterium sterolicum nov. sp. ATCC21387 was isolated by an expression cloning method and highly expressed by a recombinant strain Escherichia coli MM294/pnH10. The purified cholesterol oxidase was a typical flavoprotein with a molecular mass of 46.5 kDa, absorption peaks at 280, 360, and 450 nm. Optimum pH and temperature were found at pH 6.5 and 55 degrees C, respectively. The enzyme acted on 3beta-hydroxysteroids such as cholesterol, pregnenolone, and beta-sitosterol at high rates, but on dehydro-epi-androsterone to a lesser degree. The molecular and catalytic properties were different from those of cholesterol oxidase I, which was initially discovered in B. sterolicum nov. sp. ATCC21387. The new enzyme, designated cholesterol oxidase II, was distinguished by its high affinity toward cholesterol (K(m)=30 microM).

We have previously cloned a gene encoding a SigB, a principal-like sigma factor in Brevibacterium flavum, which was induced by several stress conditions. To clarify the in vivo function of this sigma factor, the sigB gene was disrupted by a homologous recombination, replacing the internal essential coding region in B. flavum chromosome by a kanamycin resistance marker gene. This mutation dramatically decreased vegetative growth rates of B. flavum. Studies of the effect of the sigB mutation on growth and viability of the cells under conditions of stress showed that the sigB mutant had increased susceptibility to acid, salt, alcohol, heat and cold stress. The plasmid-born wild-type sigB gene complemented the mutation. Based on the results, we propose that SigB has a role in vegetative growth and in response to various environmental stresses.

Corynebacteria are pleomorphic, asporogenous, Gram-positive bacteria. Included in this group are nonpathogenic soil corynebacteria, which are widely used for the industrial production of amino acids and detergents, and in biotransformation of steroids. Other members of this group are plant and animal pathogens. This review summarizes the current information available about the plasmids of corynebacteria. The emphasis is mainly on the small plasmids, which have been used for construction of vectors for expression of genes in these bacteria. Moreover, considerable information is now available on their nucleotide sequence, gene organization and modes of replication, which would make it possible to further manipulate these plasmids. Other plasmid properties, such as incompatibility and host range, are also discussed. Finally, use of these plasmids as cloning vectors for the expression of heterologous proteins using corynebacteria as hosts is also summarized to highlight the potential of these bacteria as hosts for recombinant DNA.

The electro-optical (EO) properties of a microbial cell suspension of Brevibacterium sp. strain 13 PA were examined during acrylamide and acrylic acid metabolism and during culture of cells in a mineral medium without added carbon sources. The dependence of the suspension turbidity changes due to cellular orientation on the frequency of an orienting electric field (orientational spectra, OSs) over the range of 10–10000 kHz were used. Alterations in the OSs, depending on cellular amidase activity, were recorded during growth with acrylamide as the role source of carbon. However, no amidase activity was observed in cells growing with acrylic acid as the sole carbon source. Growth on the carbon-free mineral medium resulted in a reduction in amidase activity. It is apparent that the OS changes reflect the corresponding changes in cell polarisability, which may be associated with acrylamide hydrolysis. The data generated from these studies can be taken as a basis for the EO determination of the enzyme activity of microbial biocatalysts.

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).

Directed integration of heterologous DNA into the insertion sequence IS13869 of Brevibacterium lactofermentum has been achieved by using integrative vectors that contain a 600 bp fragment from IS13869 and a kanamycin resistance cartridge with unique PacI, SwaI and PmeI sites. The incorporation of these restriction sites for rare-cutting enzymes, which recognize octanucleotide sequences, allowed rapid mapping of the location of plasmid insertion into the chromosome. Mapping by pulsed-field gel electrophoresis and hybridization with an IS13869 probe showed four types of transformants. In three of these types, integration of the vector occurred at each of three different sites in the chromosome. These vectors have the advantage of increasing the number of integration events due to the presence of multiple targets in the genome.

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.

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.

We have detected cyclic AMP in the culture medium of amino acid-producing coryneform bacteria. Extracellular concentrations of cyclic AMP vary depending on the nutritional medium, the growth phase and the carbon source. It is also shown that the activity of Brevibacterium flavum adenylate cyclase in intact cells is stimulated in the presence of glucose. Furthermore, addition of cyclic AMP to B. flavum cultures decreased amino acid production similarly to the effect produced by the addition of inorganic phosphate.

The aim of our investigation was to study the haloadaptation of a number of species of Gram-positive bacteria belonging to the genera Brevibacterium and Corynebacterium. We used two different HPLC-techniques and 13C-NMR spectroscopy for the identification of osmolytes (compatible solutes). The tetrahydropyrimidines (‘ectoines’) are the main compatible solutes in the genus Brevibacterium, whereas accumulation of glycine-betaine and accumulation of synthesis of glutamine is mainly responsible for osmoadaptation in the genus Corynebacterium. Pipecolic acid, formerly described as a potential osmolyte synthesized de novo in C. ammoniagenes, does not contribute markedly to the solute pool, unless supplemented to the medium.

New promoter-probe and terminator-probe shuttle vectors for Escherichia coli and corynebacteria were constructed. These vectors, working with the uidA gene of E. coli as reporter gene, are very useful for the cloning and subsequent analysis of transcriptional regulatory signals. The beta-glucuronidase encoding activity of uidA can be easily detected on agar plates containing chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-gluc). In the terminator-probe vector pUT2, uidA is expressed from a promoter of the Brevibacterium lactofermentum cryptic plasmid pBL1. Multiple cloning sites (MCS) located immediately upstream of uidA allow introduction and selection of terminators or regulatory signals. In the promoter-probe vector pUT3, transcription readthrough from vector promoters is prevented by a terminator of B. lactofermentum isolated using pUT2. We have successfully used pUT2 and pUT3 to isolate several terminators and promoter regions active in B. lactofermentum and shown that the E. coli strong terminator cartridge omega appears less efficient in corynebacteria.

A mutant of Brevibacterium ammoniagenes producing large quantities of UMP and uracil is described. The mutations render bacteria braditrophic for arginine, sensitive to adenine, resistant to rifampicin and pyrimidine analogues 5-fluorouracil, 5-fluorouridine, azauracil and thiouracil. The activities of enzymes involved in the UMP biosynthesis, i.e. orotate phosphoribosyl-transferase, orotate-5-monophosphate decarboxylase, dihydroorotate oxidase, are 4-, 3.5- and 4.5-fold higher in the mutant than in the parent strain when grown in minimal medium. The synthesis of these enzymes in mutant cells is not repressed in the presence of exogenous Ura. True revertants, which completely restore the wild-type phenotype, occur among the Arg+ clones. The nature of the mutation is discussed.

A rapid and effective method is described for electroporation of Brevibacterium sp. R312, a coryneform strain producing nitrile hydratase and amidase. The transformation efficiency of the method is 108 transformants per μg of plasmid under optimal conditions. Parameters optimised included field strength (11.8 kV cm−1), pulse length (2.4 ms), plasmid DNA concentration (0.25 μg ml−1 and cell density (1010 cells ml−1). Surprisingly, the transformation efficiency did not vary with the growth stage, in contrast to results in the literature. A shuttle vector was constructed containing several unique cloning sites down-stream of the SP6 RNA polymerase promoter.

In order to facilitate genetic engineering in amoni-acid producing bacteria we have isolated two restriction-deficient Brevibacterium lactofermentum strains. They have been selected for their ability to obtain a high yield of plaques from CL31 phage which was grown on Corynebacterium lilium. These mutant strains do not restrict either phage DNA by transfection or DNA from the shuttle vector pBLA extracted from Escherichia coli by protoplast transformation. These mutants have also lost modification activity. We also report the presence of a restriction modification system in C. lilium ATCC 15990.

Bouyant density gradient centifugation of ‘crude membranes’ of the coryneform bacterium Brevibacterium ammoniagenes yielded two distinct membrane fractions which differed significantly in equilibrium densities (1.15 and 1.18 g/cm3), NADH dehydrogenase activity (0.29 and 0.09 μmol·min−1·mg−1), protein composition and association of ribosomes. As determined by one dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) proteins unique to either the free membrane (FM) fraction or the denser ribosome-containing complexed membrane (CM) fraction were identified.

A 20-kDa Brevibacterium lactofermentum protein was detected when purifying the His-tagged FtsZBL. The protein was identified by matrix-assisted laser desorption/ionisation time of flight as the inorganic pyrophosphatase encoded by the ppa gene, which is present as a single copy in the genome of Corynebacterium glutamicum. The ppa gene was cloned from B. lactofermentum chromosomal DNA by polymerase chain reaction; it seemed to be an essential gene and it might represent an attractive target for drug discovery. The cloned ppa gene complemented a ppa- Escherichia coli mutant and a ppa-gfp gene fusion revealed that the gene product mainly accumulated at the cell poles in both E. coli and B. lactofermentum.

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

The purpose of the work reported here was to experimentally clarify the interconnection between changes in the electro-physical characteristics of microbial suspensions and processes of the metabolism of certain toxic compounds (acrylamide and p-nitrophenol (PNP)) in cells containing enzyme systems of the initial metabolism of these compounds. In this work, we used cells of two strains, Acinetobacter calcoaceticum A-122 and Brevibacterium sp. 13PA, which are capable of utilising PNP and acrylamide, respectively, as the sole source of carbon. Suspensions of these cells exhibited appreciable decreases in the magnitude of the electro-optical signal when the microbial metabolism of PNP and acrylamide was inhibited under conditions of limited O2 supply. This attests to a relationship between the electro-physical characteristics of microbial suspensions and cellular metabolic reactions and also to the negligibly small effect that the non-specific interaction of substrate with the cells has on the suspensions’ electro-optical properties.

The murI gene encoding d-glutamate racemase plays an important role in the biosynthesis of d-glutamic acid, an essential component of cell wall peptidoglycan of almost all eubacteria. A DNA fragment that could rescue the auxotrophy of d-glutamic acid in the Escherichia coli murI mutant strain WM335 was isolated from Brevibacterium lactofermentum ATCC 13869 belonging to the coryneform bacteria. DNA sequencing reveals that it encodes a protein of 284 amino acid residues, which shows a high level of homology with d-glutamate racemases from several other bacteria.

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

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.

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-BI 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.

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.

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.

Different strains of Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum were analysed for restriction fragment length polymorphism using the homoserine dehydrogenase gene (hom) as a probe. The hybridization patterns obtained PvuII- or Asp700-restriction of chromosomal DNA were specific and distinguishable for each of the three species and identical for the different strains of each species. Thus, the method employed allows rapid distinction of Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum. The former species could also be discriminated from the latter two by its resistance to 0.5 g/l of the methionine analog ethionine.

In order to facilitate genetic engineering in amino-acid producing bacteria we have isolated two restriction-deficient Brevibacterium lactofermentum strains. They have been selected for their ability to obtain a high yield of plaques from CL31 phage which was grown on Corynebacterium lilium. These mutant strains do not restrict either phage DNA by transfection or DNA from the shuttle vector pBLA extracted from Escherichia coli by protoplast transformation. These mutants have also lost modification activity. We also report the presence of a restriction modification system in C. lilium ATCC 15990.

Plasmid DNA was efficiently electro-transformed into intact cells of nine Corynebacteria strains belonging to Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium glutamicum and Corynebacterium melassecola. Relationships were explored between transformation efficiency and parameters such as electric field strength and pulse length, DNA concentration, physiological state and concentration of the cells. In optimal conditions, more than 10(7) transformants per microgram of DNA could be obtained. Electro-transformation with plasmid DNA isolated from different sources indicates that DNA modification may play a role in transformation efficiency.

A screening of plasmids in 25 nonpathogenic coryneform bacteria was carried out. 11 Strains showed at least one plasmid, ranging in size from 4.2 to 55 kb. These plasmids did not encode bacteriocin production or resistance to a number of antibiotics or to ions such as arsenite, mercury(II) and cobalt(II). A detailed study of plasmid pBL100 from Brevibacterium linens is presented. pBL100 has a size of 7.75 kb, and contains single sites for the endonucleases: HindIII; PstI, BglII, EcoRI and BamHI. B. linens is easily and efficiently transformed with vectors derived from pBL1 isolated from Brevibacterium lactofermentum.

Fourteen species of non-pathogenic corynebacteria have been examined for the presence of DNases. Seven of the species were found to be DNase positive when assayed in Toluidine Blue-DNA-containing agar whereas only one, Corynebacterium callunae, could be detectable as DNase positive when the test was performed in DNA-methyl green-containing agar. Electrophoretic patterns obtained from sodium dodecyl sulfate-polyacrylamide gels containing DNA showed the presence of one or two bands of nucleolytic activity in two species of Brevibacterium and of several bands in C. callunae. Degradation of DNases by cellular proteases seem to occur in this species.

Growth of Brevibacterium R312 on acetonitrile results in the appearance of acetate and ammonia in the medium, whereas incubation of harvested bacteria with acetonitrile results in appearance of acetamide, acetate and ammonia in the medium. Acetonitrilase and acetamidase activities were found to be located in the cytosolic fraction of the cell, suggesting that acetonitrile and acetamide readily enter and leave the bacterium. This was confirmed by the inability of these compounds to osmotically stabilise protoplasts, whereas acetate does so. The small size and neutral non-ionic structure of acetonitrile and acetamide suggest they probably permeate by simple diffusion.

The substrate specificity of cystathionine γ-synthase (EC 4.2.99.9) in various bacteria was examined. O -Succinyl- l -homoserine was used preferably as a substrate by facultative anaerobic Gram-negative bacteria belonging to such genera as Escherichia, Klebsiella, Enterobacter, Citrobacter, Erwinia, Serratia, Proteus and Salmonella . Among Gram-negative aerobic bacteria, bacteria belonging to the genera Pseudomonas, Xanthomonas and Alcaligenes also used O -succinyl- l -homoserine in preference to O -acetyl- l -homoserine. On the other hand, bacteria belonging to the genera Agrobacterium and Flavobacterium used O -acetyl- l -homoserine preferably. As to Gram-positive aerobic bacteria, bacteria of the genus Bacillus used O -acetyl- l -homoserine exclusively. Bacteria belonging to the genera Micrococcus, Corynebacterium, Brevibacterium and Arthrobacter used both O -acetyl- and O -succinyl- l -homoserine to similar extents.

The quantitative importance of the 3,4-dihydroxyphenylacetatemeta-cleavage pathway (the principal pathway of tyrosine catabolism) in the catabolism ofl-phenylalanine inBrevibacterium linens 47 was evaluated using tyrosine-negative mutants. Less than 5% of phenylalanine was catabolised through the tyrosine pathway. The results presented show that inB. linens the two structurally analogous amino acids are catabolised principally by separate pathways. Also, the initial pathway of phenylalanine catabolism in this bacterium has been established.

Cells from mid-exponential cultures of Brevibacterium lactofermentum were successfully transformed with pUL340 plasmid DNA using a standard (BioRad) electroporation protocol. However, growth in the presence of glycine plus tween 80 greatly increased the transformation frequencies and selecting transformants on an osmotically-protective medium resulted in further improvements. Under these optimal conditions, 4 × 107 transformants/μg DNA were isolated per 109 cells electroporated. Similar transformation frequencies were obtained for Corynebacterium glutamicum when tween 80 was added to the growth medium in addition to glycine and isonicotinic acid hydrazide, conditions which had been found previously to be essential for obtaining significant electrotransformation levels in this species.