Comparative genomics identified two conserved DNA modules in a corynebacterial plasmid family present in clinical isolates of the opportunistic human pathogen Corynebacterium jeikeium

Investigation of 62 clinical isolates of the opportunistic human pathogen Corynebacterium jeikeium revealed that 17 possessed plasmids ranging in size from 7.6 to 14.9 kb. The plasmids formed four groups on DNA restriction analysis. The complete nucleotide sequence of a representative from each group (pK43, pK64, pCJ84, and pB85766) was subsequently determined. Additionally, two plasmids (pCo455 and pCo420) were shown to be derivatives of pK43 and pK64 carrying insertion sequences of the IS3 family. Comparative genomics identified a conserved plasmid backbone consisting of two distinct DNA modules. Conserved motifs in the parAB-repA module indicated that the sequenced plasmids from C. jeikeium are new members of the pNG2 family. Recombinant derivatives of pK43 were shown to replicate in the soil bacterium Corynebacterium glutamicum and in the human pathogen Corynebacterium diphtheriae. The second plasmid module most likely encodes a novel type of DNA invertase. The respective gene is flanked by highly conserved 112-bp inverted repeats. All plasmids are 'loaded' with a characteristic set of genes encoding products of unknown function. Plasmids indistinguishable from pK43 by DNA restriction analysis were identified in different C. jeikeium strains, which revealed 16S-23S rDNA spacer length polymorphisms and specific antibiotic susceptibility profiles, implying a wide dissemination of the plasmid in clinical isolates of C. jeikeium.     

The 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum encodes the aminoglycoside adenyltransferase gene cassette aadA9 and the regulated tetracycline efflux system Tet 33 flanked by active copies of the widespread insertion sequence IS6100

We determined the complete nucleotide sequence of the 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum LP-6 which encodes streptomycin, spectinomycin, and tetracycline resistance. The antibiotic resistance determinant of pTET3 comprises an intI1-like gene, which was truncated by the insertion sequence IS6100, and the novel aminoglycoside adenyltransferase gene cassette aadA9. The deduced AADA9 protein showed 61% identity and 71% similarity to AADA6 of integron In51 from Pseudomonas aeruginosa. In addition, pTET3 carries the novel repressor-regulated tetracycline resistance determinant Tet 33 which revealed amino acid sequence homology to group 1 tetracycline efflux systems. The highest level of similarity was observed to the tetracycline efflux protein TetA(Z) from the C. glutamicum plasmid pAG1 with 65% identical and 77% similar amino acids. Each antibiotic resistance region of pTET3 is flanked by identical copies of the widespread insertion sequence IS6100 initially identified in Mycobacterium fortuitum. Transposition assays with a cloned copy of IS6100 revealed that this element is transpositionally active in C. glutamicum. These data suggest a central role of IS6100 in the evolutionary history of pTET3 by mediating the cointegrative assembly of resistance gene-carrying DNA segments.     

1,25(OH)2 vitamin D3-stimulated osteoclast formation in spleen-osteoblast cocultures is mediated in part by enhanced IL-1 alpha and receptor activator of NF-kappa B ligand production in osteoblasts

We examined the ability of 1,25 (OH)(2) vitamin D(3) (Vit D) to stimulate osteoclast-like cell (OCL) formation in cocultures of spleen cells and primary calvarial osteoblasts from wild-type (WT) and IL-1R type 1-deficient (knockout; KO) mice. Vit D dose dependently increased OCL in cocultures containing WT osteoblasts. In contrast, there was a 90% reduction in OCL numbers in cocultures containing KO osteoblasts. In cocultures with either WT or KO osteoblasts, treatment with Vit D increased receptor activator of NF-kappaB ligand mRNA by 17-, 19-, or 3.5-fold, respectively. Vit D decreased osteoprotegerin mRNA to undetectable in all groups. Intracellular IL-1alpha protein increased after Vit D treatment in cocultures containing WT, but not KO osteoblasts. We also examined direct effects of Vit D, IL-1alpha, and their combination on gene expression in primary osteoblasts. In WT cells, Vit D and IL-1 stimulated receptor activator of NF-kappaB ligand mRNA expression by 3- and 4-fold, respectively, and their combination produced a 7-fold increase. Inhibition of osteoprotegerin mRNA in WT cells was partial with either agent alone and greatest with their combination. In KO cells, only Vit D stimulated a response. IL-1 alone increased IL-1alpha protein expression in WT osteoblasts. However, in combination with Vit D, there was a synergistic response (100-fold increase). In KO cultures, there were no effects of IL-1, Vit D, or their combination on IL-1alpha protein. These results demonstrate interactions between IL-1 and Vit D in primary osteoblasts that appear important in both regulation of IL-1alpha production and the ability of Vit D to support osteoclastogenesis.     

Two-dimensional electrophoresis patterns of total proteins,esterases and acid phosphatases from pollen,seeds and leaves of three cultivars of Helianthus annuus L

Total proteins, esterases and acid phosphatases from pollen, seeds and leaves of three sunflower cultivars were separated by 2-D electrophoresis. The characteristic peptides for each cultivar were identified. The seeds and pollen of the cultivar Wielkopolski contained 45 and 37 characteristic peptides, respectively, while the seeds and pollen of Coril contained 73 and 35 characteristic peptides. The cultivar Frankasol had the lowest total number of stained peptides in seeds and pollen, and the number of the characteristic peptides was only 61 and 25, respectively. Analyses of esterases and acid phosphatases from young leaves and pollen led to identification of isoenzymes characteristic of the three cultivars. Only for Frankasol no specific acid phosphatase was observed, both in leaves and in pollen.     

Differential influence of bacitracin on plant proteolytic enzyme activities

The effect of bacitracin on the activity of proteases extracted from pollen and sprouts of various plant species and compared to five commercially available proteases was studied. Bacitracin stimulates some pollen proteolytic enzyme activities, contrary to its inhibitory influence on proteases from the other sources. Proteases from maize pollen, inhibited by pepstatin and phenylmethylsulfonyl fluoride, immediately accelerate their activities after addition of bacitracin to the reaction mixture. The stimulating influence of peptide antibiotic on pollen proteases of some plants is unexpected and molecular mechanism of this phenomenon requires a further elucidation. The augmentation of allergenic response caused by pollen enzymes and drugs containing bacitracin is discussed.     

The 51,409-bp R-plasmid pTP10 from the multiresistant clinical isolate Corynebacterium striatum M82B is composed of DNA segments initially identified in soil bacteria and in plant, animal, and human pathogens

The 51,409-bp DNA sequence of the multiresistance plasmid pTP10 from the gram-positive opportunistic human pathogen Corynebacterium striatum M82B has been determined. Fully automated genome interpretation led to the identification of 47 ORFs. Analysis of the genetic organization of pTP10 suggests that the plasmid is composed of eight DNA segments, the boundaries of which are represented by transposons and insertion sequences. The DNA segments of pTP10 are highly similar to (1) a plasmid-encoded erythromycin resistance region from the human pathogen Corynebacterium diphtheriae; (2) a chromosomal DNA region from Mycobacterium tuberculosis; (3) a plasmid-encoded chloramphenicol resistance region from the soil bacterium Corynebacterium glutamicum; (4) transposable elements from phytopathogenic gram-negative Pseudomonas, Xanthomonas and Erwinia species; and (5) a plasmid-encoded aminoglycoside resistance region from the gram-negative fish pathogen Pasteurella piscicida. The complete DNA sequence of pTP10 provides genetic information regarding the mechanisms of resistance to 16 antimicrobial agents that belong to six structural classes. In addition, the mosaic structure of pTP10 represents the evolutionary consolidation into a single plasmid molecule of antimicrobial resistances from microorganisms found in different habitats by means of mobile elements, resulting in the generation of a multiresistant bacterium that can infect humans. Received: 5 August 1999 / Accepted: 4 November 1999     

TetZ, a new tetracycline resistance determinant discovered in gram-positive bacteria, shows high homology to gram-negative regulated efflux systems

The complete nucleotide sequence of the tetracycline resistance plasmid pAG1 from the gram-positive soil bacterium Corynebacterium glutamicum 22243 (formerly Corynebacterium melassecola 22243) was determined. The R-plasmid has a size of 19,751 bp and contains at least 18 complete open reading frames. The resistance determinant of pAG1 revealed homology to gram-negative tetracycline efflux and repressor systems of Tet classes A through J. The highest levels of amino acid sequence similarity were observed to the transmembrane tetracycline efflux protein TetA(A) and to the tetracycline repressor TetR(A) of transposon Tn1721 with 64 and 56% similarity, respectively. This is the first time a repressor-regulated tet gene has been found in gram-positive bacteria. A new class of tetracycline resistance and repressor proteins, termed TetA(Z) and TetR(Z), is proposed.     

Rational design of a Corynebacterium glutamicum pantothenate production strain and its characterization by metabolic flux analysis and genome-wide transcriptional profiling

A "second-generation" production strain was derived from a Corynebacterium glutamicum pantothenate producer by rational design to assess its potential to synthesize and accumulate the vitamin pantothenate by batch cultivation. The new pantothenate production strain carries a deletion of the ilvA gene to abolish isoleucine synthesis, the promoter down-mutation P-ilvEM3 to attenuate ilvE gene expression and thereby increase ketoisovalerate availability, and two compatible plasmids to overexpress the ilvBNCD genes and duplicated copies of the panBC operon. Production assays in shake flasks revealed that the P-ilvEM3 mutation and the duplication of the panBC operon had cumulative effects on pantothenate production. During pH-regulated batch cultivation, accumulation of 8 mM pantothenate was achieved, which is the highest value reported for C. glutamicum. Metabolic flux analysis during the fermentation demonstrated that the P-ilvEM3 mutation successfully reoriented the carbon flux towards pantothenate biosynthesis. Despite this repartition of the carbon flux, ketoisovalerate not converted to pantothenate was excreted by the cell and dissipated as by-products (ketoisocaproate, DL-2,3,-dihydroxy-isovalerate, ketopantoate, pantoate), which are indicative of saturation of the pantothenate biosynthetic pathway. Genome-wide expression analysis of the production strain during batch cultivation was performed by whole-genome DNA microarray hybridization and agglomerative hierarchical clustering, which detected the enhanced expression of genes involved in leucine biosynthesis, in serine and glycine formation, in regeneration of methylenetetrahydrofolate, in de novo synthesis of nicotinic acid mononucleotide, and in a complete pathway of acyl coenzyme A conversion. Our strategy not only successfully improved pantothenate production by genetically modified C. glutamicum strains but also revealed new constraints in attaining high productivity.     

Population structure of Atlantic salmon (Salmo salar L.): a range-wide perspective from microsatellite DNA variation

Atlantic salmon (n = 1682) from 27 anadromous river populations and two nonanadromous strains ranging from south-central Maine, USA to northern Spain were genotyped at 12 microsatellite DNA loci. This suite of moderate to highly polymorphic loci revealed 266 alleles (5-37/locus) range-wide. Statistically significant allelic and genotypic heterogeneity was observed across loci between all but one pairwise comparison. Significant isolation by distance was found within and between North American and European populations, indicating reduced gene flow at all geographical scales examined. North American Atlantic salmon populations had fewer alleles, fewer unique alleles (though at a higher frequency) and a shallower phylogenetic structure than European Atlantic salmon populations. We believe these characteristics result from the differing glacial histories of the two continents, as the North American range of Atlantic salmon was glaciated more recently and more uniformly than the European range. Genotypic assignment tests based on maximum-likelihood provided 100% correct classification to continent of origin and averaged nearly 83% correct classification to province of origin across continents. This multilocus method, which may be enhanced with additional polymorphic loci, provides fishery managers the highest degree of correct assignment to management unit of any technique currently available.