Identification of plasmid partition function in coryneform bacteria.

We have identified and characterized a partition function that is required for stable maintenance of plasmids in the coryneform bacteria Brevibacterium flavum MJ233 and Corynebacterium glutamicum ATCC 31831. This function is localized to a HindIII-NspV fragment (673 bp) adjacent to the replication region of the plasmid, named pBY503, from Brevibacterium stationis IFO 12144. The function was independent of copy number control and was not associated directly with plasmid replication functions. This fragment was able to stabilize the unstable plasmids in cis but not in trans.     

Identification key for coryneform bacteria derived by numerical taxonomic studies

Six main groups were formed from a complete linkage dendrogram on 557 bacteria tested for 53 physiological features. The organisms were obtained from culture collections and included representatives of the following genera: Arthrobacter, Brevibacterium, Caseobacter, Cellulomonas, Corynebacterium, Curtobacterium, Micrococcus, Microbacterium, Mycobacterium, Nocardia, Oerskovia and Rhodococcus. The six groups were individually subjected to a numerical taxonomic analysis based on linkage maps, which resulted in a total of 33 subclusters. An identification key to determine the affiliation of the bacteria to the six main clusters and five group-specific schemes is presented. Reference strains are proposed for the 33 subclusters.     

Transposon mutagenesis of coryneform bacteria

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 10⁴ 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.     

High-frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria.

We report on the mobilization of shuttle plasmids from gram-negative Escherichia coli to gram-positive corynebacteria mediated by P-type transfer functions. Introduction of plasmids into corynebacteria was markedly enhanced after heat treatment of the recipient cells. High-frequency plasmid transfer was also observed when the restriction system of the recipient was mutated. On the basis of our data, we conclude that efficient DNA transfer from gram-negative to gram-positive bacteria, at least to coryneform bacteria, is conceivable in certain natural ecosystems.     

Genetic engineering of coryneform bacteria

The coryneforms are a diverse group of bacteria which includes animal and plant pathogens as well as non-pathogenic bacteria. Although they are of significant economic and health importance, their genetics is poorly understood. The development of genetic engineering techniques for coryneforms and initial gene cloning studies are discussed.     

Plasmids in coryneform bacteria of human origin

A study of plasmids in coryneform bacteria isolated from human sources is reported. Seventy of 269 strains possessed a total of 89 plasmids. These were shown to be of varying sizes and in some cases of varying structures by endonuclease restriction digest. In six of 20 strains antibiotic resistance was cured with loss of the plasmid. The diversity of plasmids is emphasized.     

Aromatic aminotransferases in coryneform bacteria.

Species of coryneform bacteria (Corynebacterium glutamicum, Brevibacterium flavum, and B. ammoniagenes) are capable of transaminating all three of the aromatic pathway intermediates; prephenate, phenylpyruvate, and 4-hydroxy-phenylpyruvate. Two molecular species of aromatic aminotransferase (denoted aminotransferase I and aminotransferase II) were partially purified from C. glutamicum and B. flavum, whereas a single aromatic aminotransferase was isolated from B. ammoniagenes. In both C. glutamicum and B. flavum, aromatic aminotransferase I and aromatic aminotransferase II have molecular weights of about 155,000 and 260,000 respectively. The two aromatic aminotransferases from C. glutamicum and B. flavum, although exhibiting a similar spectrum of overlapping specificities, differ substantially in substrate preference. Pyridoxal-5'-phosphate is tightly associated with these aminotransferases, since little loss of activity was detected when partially purified enzyme preparations were assayed in the absence of exogenous pyridoxal-5'-phosphate. The aminotransferases are quite sensitive to inhibition by phenylhydrazine. This has practical application when assay of prephenate dehydratase is desired in the presence of aromatic aminotransferase activity since potentially trivial interference can be negated by selective phenylhydrazine inhibition of aromatic aminotransferase activity. At 0.1 mM concentrations of phenylhydrazine, 90% inhibitions of aminotransferase activities were achieved in partially purified preparations of B. flavum and C. glutamicum.     

Morphology and physiology of coryneform bacteria

Four groups of coryneform bacteria, viz. soil arthrobacters, orange cheese coryneforms, orange sea-fish coryneforms and non-orange cheese coryneforms, were studied as regards morphological and physiological features. The soil arthrobacters can be divided into a simplex and a globiformis group on the basis of their ability of utilizing a number of carbon sources. The group of the orange cheese coryneforms was found to be rather homogeneous, in contrast to the groups of the orange sea-fish coryneforms and the non-orange cheese coryneforms, some strains of which deviated from the others of their group as to the majority of the characteristics tested. Mainly the physiological characteristics of each of the groups justify the division of the coryneforms into the four chief groups mentioned.     

Plasmids in coryneform bacteria of human origin

A study of plasmids in coryneform bacteria isolated from human sources is reported. Seventy of 269 strains possessed a total of 89 plasmids. These were shown to be of varying sizes and in some cases of varying structures by endonuclease restriction digest. In six of 20 strains antibiotic resistance was cured with loss of the plasmid. The diversity of plasmids is emphasized.     

Numerical taxonomy of certain coryneform bacteria

da Silva, G. A. Nigel (Iowa State University, Ames), and John G. Holt. Numerical taxonomy of certain coryneform bacteria. J. Bacteriol. 90:921-927. 1965-An electronic computer was used to sort 32 strains of coryneforms into groups with the tree-sort program. The similarity values obtained in this procedure were then used to construct a dendrogram depicting the phenetic resemblance among the taxa. The results indicated that all the phytopathogens studied were sufficiently distinct from the type species, Corynebacterium diphtheriae, to be excluded from the genus Corynebacterium. The grouping of some of the phytopathogens with Microbacterium lacticum is discussed. C. fascians appeared so distinct from the other strains studied that it should probably be excluded from the Corynebacteriaceae. The phenetic resemblance of Brevibacterium linens and Arthrobacter globiformis was emphasized, and the new combination, Arthrobacter linens, was proposed. In addition, because of distinct dissimilarity from the type species, it would seem desirable to exclude Arthrobacter tumescens from the genus Arthrobacter. The justification for classifying Kurthia zopfii in a family separate from the Corynebacteriaceae would appear to be open to serious question. It was concluded that the present taxonomic positions of Listeria monocytogenes, Cellulomonas biazotea, and Cellulomonas fimi are satisfactory.     

Development cycles of coryneform and Nocardia-like bacteria

The growth cycles and the types of cell separation were studied in a microchamber with the collection strains of Brevibacterium ammoniagenes ATTC 6871, B. helvolum ATCC 19239, B. linens CCM 47 and ATCC 9174, B. maris VKM B-464 and B. stationis ATCC 14403, as well as with the strains of the genus Rhodococcus isolated from soils, viz. R. maris sp. nov. IMB 283 and R. luteus sp. nov. IMB 385. According to the increasing complexity of cellular morphological transformation in the life cycle, the organisms may be arranged in a series: R. maris -- B. ammoniagenes -- B. stationis -- B. linens -- B. helvolum -- R. luteus. The first three organisms are characterized by the snapping type of separation of short rod-like daughter cells. The cells of B. linens separate by both the snapping and bending types. The coccoid cells of B. helvolum ATCC 19239 produce many buds which are transformed into rod-like cells in the course of growth. In the log phase of growth, both true and false branching of the cells is observed; the latter is the result of a peculiar growth of the ends in the separated cells of B. helvolum. The cells of R. luteus form a rudimentary, rapidly fragmenting mycelium whose rod-like elements divide then by binary fission; the daughter cells separate the bending and snapping types.     

Polyamines in representative taxa of coryneform and other bacteria

The composition of polyamines is studied for the first time in representatives of the genus Micrococcus and taxon "conglomeratus", strains Staphylococcus aureus CCM 209, Deinococcus erythromyxa CCM 706 as well as of Erwinia carotovora ATCC 15713 polyamines, which are not extracted by perchloric acid. Considerable amounts of spermine and rarely of spermidine are revealed in cells of Gram positive microorganisms, that differs them from Gram negative bacteria possessing high concentrations of putrescine, spermidine and their derivatives. A procedure is developed for detection of polyamines in cells of Gram positive microorganisms. It is recommended to use the hydrolysis of their cells by 6N HCl for 4 at 120 degrees C or for 8-10h at 100 degrees C with the subsequent electrophoretic separation. Putrescine, as well as comparable with it amount of agmatine and spermidine traces are found in Erwinia carotovora ATCC 15713 cell hydrolyzates, whereas putrescine and agmatine traces are found in perchloric extracts of intact cells. Spermine is not observed in the cells. The binding of polyamines with biopolymers of cells of Gram positive bacteria and their difference by the given character from the Gram negative procaryotes are under discussion.     

植物病原棒形细菌的RAPD分析

采用RAPD分析技术对萎蔫短小杆菌落葵致病变种（Curtobacterium flaccumfaciens pv. basellae pv. Nov.）和萎蔫短小杆菌糖甜菜致病变种（Curtobacterium flaccumfaciens pv. beticola pv. Nov.）及植物病原棒形细菌4个属的15个菌株进行分类研究：从50条随机引物中筛选出20条,共产生225条带,多态性条带占804%。遗传相似矩阵及UPGMA聚类分析,表明这两个新致病变种与萎蔫短小杆菌属亲缘关系近,最小相似系数为0.6511;与其他属细菌亲缘关系较远;结合前人研究结果对植物病原棒形细菌新近提出的分类地位的改变进行了探讨。     

Secretion of Streptomyces mobaraensis pro-transglutaminase by coryneform bacteria

We previously reported on the secretion of Streptomyces mobaraensis transglutaminase by Corynebacterium glutamicum ATCC13869 (formerly classified as Brevibacterium lactofermentum). In the present work, we investigated whether any other coryneform bacteria showed higher productivity than C. glutamicum ATCC13869. We found that most coryneform species secreted pro-transglutaminase efficiently. Moreover, we confirmed that Corynebacterium ammoniagenes ATCC6872 produced about 2.5 g/l pro-transglutaminase over a 71-h period in a jar fermentor. Our findings suggest that some other coryneform bacteria, especially C. ammoniagenes ATCC6872, are potential hosts for industrial scale protein production.     

Phenylalanine and tyrosine catabolism in some cheese coryneform bacteria

Abstract 23 cheese coryneform bacteria (14 orange, 3 white, and 6 yellow-pigmented) were examined for five enzymes of two branch-point steps in the catabolic pathways of l -phenylalanine and l -tyrosine. Orange cheese coryneforms ( 'Brevibacterium linens' ) catabolized th amino acids by transamination and the benzene ring was cleaved by 3, 4-dihydroxyphenylacetate-2, 3-dioxygenase. Both enzymes appear to be inducible. Yellow and white strains possessed non-inducible low activity of aminotransferase and lacked completely benzene ring cleavage enzymes.     

Industrial production of amino acids by coryneform bacteria

In the 1950s Corynebacterium glutamicum was found to be a very efficient producer of L-glutamic acid. Since this time biotechnological processes with bacteria of the species Corynebacterium developed to be among the most important in terms of tonnage and economical value. L-Glutamic acid and L-lysine are bulk products nowadays. L-Valine, L-isoleucine, L-threonine, L-aspartic acid and L-alanine are among other amino acids produced by Corynebacteria. Applications range from feed to food and pharmaceutical products. The growing market for amino acids produced with Corynebacteria led to significant improvements in bioprocess and downstream technology as well as in molecular biology. During the last decade big efforts were made to increase the productivity and to decrease the production costs. This review gives an overview of the world market for amino acids produced by Corynebacteria. Significant improvements in bioprocess technology, i.e. repeated fed batch or continuous production are summarised. Bioprocess technology itself was improved furthermore by application of more sophisticated feeding and automatisation strategies. Even though several amino acids developed towards commodities in the last decade, side aspects of the production process like sterility or detection of contaminants still have increasing relevance. Finally one focus of this review is on recent developments in downstream technology.     

Identification and characterization of an active plasmid partition mechanism for the novel Lactococcus lactis plasmid pCI2000

The replication region of the lactococcal plasmid pCI2000 was subcloned and analyzed. The nucleotide sequence of one 5.6-kb EcoRI fragment which was capable of supporting replication when cloned on a replication probe vector revealed the presence of seven putative open reading frames (ORFs). One ORF exhibited significant homology to several replication proteins from plasmids considered to replicate via a theta mode. Deletion analysis showed that this ORF, designated repA, is indeed required for replication. The results also suggest that the origin of replication is located outside repA. Upstream and divergently transcribed from repA, an ORF that showed significant (48 to 64%) homology to a number of proteins that are required for faithful segregation of chromosomal or plasmid DNA of gram-negative bacteria was identified. Gene interruption and transcomplementation experiments showed that this ORF, designated parA, is required for stable inheritance of pCI2000 and is active in trans. This is the first example of such a partitioning mechanism for plasmids in gram-positive bacteria.