Identification and growth of Leuconostoc lactis SHO-54, producing high amounts of NAD-specific 6-phosphogluconate dehydrogenase

The G+C content of the DNA of strain SHO-54 which produces a large amount of NAD-specific 6-phosphogluconate dehydrogenase was 41.0 mol%. The extent of the DNA–DNA homology between this strain and Leuconostoc lactis NRIC 1540 ranged from 72.8% to 95.5%. A minimal medium in which strain SHO-54 grew well was determined, and a high activity of the enzyme (410 nkat/ml) was obtained.     

Lactobacillus alvi sp. nov., isolated from the intestinal tract of chicken

Strain R54(T) was isolated from the gizzard of hens. The isolate was Gram-positive, facultative anaerobic, gas-forming, catalase-negative, nonmotile, nonspore-forming and short-rod-shaped. The optimal temperature for growth was 40 °C and the DNA G+C content was 42.7 mol%. The 16S rRNA gene sequences similarity showed that strain R54(T) was most closely related to Lactobacillus ingluviei LMG 20380(T) (97.5%), followed by Lactobacillus coleohominis CIP 106820(T) (96.1%), Lactobacillus secaliphilus DSM 17896(T) (95.6%) and Lactobacillus gastricus LMG 22113(T) (95.4%). The DNA-DNA relatedness between strain R54(T) and L. ingluviei LMG 20380(T) , was 43.3%. The predominant cellular fatty acids of strain R54(T) were C(18:1 ω9c) (64.9%) and C(16:0) (20.0%), and the major polar lipid group was phospholipids. On the basis of polyphasic taxonomy approach, strain R54(T) represents a novel species of the genus Lactobacillus, for which the name Lactobacillus alvi sp. nov. is proposed (type strain R54(T) = KCCM 90099(T) = JCM 17644(T)).     

A Study on the Mechanism of DNA Excretion from P. aeruginosa KYU-1—Effect of Mitomycin C on Extracellular DNA Production—

The mechanism of excretion of DNA was investigated using strain KYU-1. This strain produced a considerable amount of DNA extracellularly (7.8mg/ml), and the amount of extracellular DNA per ml of the culture was scores of times more compared to that of intracellular DNA per ml of the culture. DNA synthesis was repressed by the addition of inhibitors (nalidixic acid, 5-fluoro uracil or mitomycin C), their inhibition rates were 35 to 54%. With the addition of 100 μg/ml of mitomycin C at the middle of the logarithmic growth phase, only 0.89 mg of extracellular DNA per ml of the culture was obtained, the inhibition rate was 95%. However, the amount of organic phosphate synthesized in the culture was almost the same no matter whether with or without an inhibitor.

Furthermore, the excretion of DNA from the cell surface of strain KYU-1 was observed with on electron microscope and the DNA molecules excreted were found to be double-stranded.     

Segregation into and Replication of Plasmid Deoxyribonucleic Acid in Chromosomeless Segregants of Escherichia coli

The progeny cells of Escherichia coli strain P678-54, which normally do not contain deoxyribonucleic acid (DNA), were found to carry DNA when the parental cell carried colicin factor E1 (Col E1). The DNA found in the normally DNA-less segregants was shown to be Col E1 DNA, which is present primarily as a covalently closed circular molecule that can undergo more than one complete cycle of replication.     

<Emphasis Type="Italic">Saccharothrix</Emphasis> sp. PAL54, a new chloramphenicol-producing strain isolated from a Saharan soil

An actinomycete strain designated PAL54, producing an antibacterial substance, was isolated from a Saharan soil in Ghardaïa, Algeria. Morphological and chemical studies indicated that this strain belonged to the genus Saccharothrix. Analysis of the 16S rDNA sequence showed a similarity level ranging between 96.9 and 99.2% within Saccharothrix species, with S. longispora DSM 43749^T, the most closely related. DNA–DNA hybridization confirmed that strain PAL54 belonged to Saccharothrix longispora. It showed very strong activity against pathogenic Gram-positive and Gram-negative bacteria responsible for nosocomial infections and resistant to multiple antibiotics. Strain PAL54 secreted the antibiotic optimally during mid-stationary and decline phases of growth. One antibacterial compound was isolated from the culture broth and purified by HPLC. The active compound was elucidated by uv-visible and NMR spectroscopy and by mass spectrometry. The results showed that this compound was a d(?)-threo chloramphenicol. This is the first report of chloramphenicol production by a Saccharothrix species.     

Lactobacillus harbinensis sp. nov., consisted of strains isolated from traditional fermented vegetables 'Suan cai' in Harbin, Northeastern China and Lactobacillus perolens DSM 12745

Taxonomical analysis of two genetically distinguished Lactobacillus strains isolated from traditional Chinese fermented vegetables ‘Suan cai’ was performed. They formed l-lactate from glucose, were facultatively heterofermentative, and had a DNA G+C content of 53–54 mol%. They fermented d- and l-arabinose. They produced lactate, ethanol and acetate from gluconate at a molar ratio of 1.1:0.4:0.7. Phylogenetic analysis of 16S rDNA revealed that the two strains were closely related to L. perolens. DNA–DNA hybridization analysis revealed that the two strains were different from L. perolens type strain DSM 12744 and formed a separate cluster with L. perolens DSM 12745. G+C molar content of DNA of the former is 51%, whereas those of the latter strains were in the range of 53–54%. Based on the results, we propose that the new species be named L. harbinensis sp. nov. and that L. perolens DSM 12745 be reclassified as L. harbinensis DSM 12745. The type strain of L. harbinensis DSM 16991^T (=AHU 1762^T=SBT 10908^T).     

Improvement of penicillin yields in solid-state and submerged fermentation of Penicillium chrysogenum by amplification of the penicillin biosynthetic gene cluster

Penicillium chrysogenum low and high penicillin producing strains were transformed with a cosmid containing the whole penicillin biosynthetic gene cluster. The cosmid library was constructed in a newly developed cosmid vector, IztapaCos, which allows cloning and direct introduction of large DNA fragments in fungal recipients using phleomycin resistance as selection marker. The effect of increased gene dosage on penicillin production was evaluated both in submerged (SmF) and solid-state fermentation (SSF). Transformants from the low-producing strain Wis 54-1255, showed a 67.3 and 28.3% increased penicillin titer in SSF and SmF, respectively. In transformants from the high-producing strain P2-32 the increase was 92.9 and 158.4% respectively. Strain P2-32 already contains originally about 14 copies of the penicillin biosynthetic cluster, which shows that the strategy of increasing the gene dosage is still valid for high copy-number strains. The different behavior of the two strains in each type of culture is discussed, along with the practical implications for industrial penicillin production.     

The role of the Rdh54 protein in regulation of DNA repair in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

This work provides evidence that the product of the RDH54 gene participates in the coordination of some repair pathways of DNA lesions. The unique point mutation rdh54–29 described in our previous works confers the phenotype markedly differing from that of the strain with a full deletion of gene RDH54. The epistatic type of interaction between mutations rdh 54–29 and apn 1Δ allowed the product of gene RDH54 to be attributed to the base excision repair pathway. However, a pleiotropic effect of mutation rdh54–29 manifested as sensitivity to a wide spectrum of DNA-damagi ng agents suggests that Rdh54 is involved in the regulation of several DNA repair pathways. To verify this hypothesis, the direct influence of mutation rdh54–29 on recombination and mutagenesis was evaluated. The results obtained led to the assumption that, in addition to the involvement in base excision repair, Rdh54p may play a certain role in the coordination of DNA lesion repair by various systems, including recombinational and mutagenic repair pathways or nucleotide excision repair. This function supposedly is mediated through modification of chromatin structure at the location of DNA lesion, in particular, by alleviation of DNA-hi stone bonds, thus rendering DNA more susceptible to the action of various repair proteins.     

Adenoviruses with nonidentical terminal sequences are viable.

Adenovirus genomes consist of linear DNA molecules containing inverted terminal repeat sequences (ITRs) of 100 to 200 base pairs. The importance of identical termini for viability of adenoviruses was investigated. The viral strains used in this study were wild-type adenovirus type 5 (Ad5) and a variant Ad2 strain with termini which were distinct from those of all other human adenoviruses sequenced to date. A hybrid virus (sub54), obtained by recombination between Ad2 and Ad5, derived the left 42 to 52% of its genome from Ad2 and the right 58 to 48% from Ad5. Southern blotting analysis with labeled oligodeoxynucleotides indicated that both Ad2 and Ad5 ITRs were present in sub54 viral DNA preparations, and successive plaque purifications of sub54 demonstrated that viruses with nonidentical terminal sequences were viable but were rapidly converted to viruses with identical ends. Cloning of the sub54 genome as a bacterial plasmid supported the observations made by analysis of sub54 virion DNA. A plasmid, pFG154, was isolated which contained the entire adenovirus genome with an Ad2 ITR at the left terminus covalently linked to an Ad5 ITR at the right terminus. Upon transfection of mammalian cells with pFG154, viral progeny were obtained which had all possible combinations of termini, thus confirming that molecules with nonidentical termini are viable. Pure populations of viruses with nonidentical termini could not be isolated, suggesting efficient repair of one end with the opposite terminus used as a template. A model for this process is proposed involving strand displacement replication and emphasizing the importance of panhandle formation (annealing of terminal sequences) as a replicative intermediate.     

Endophytic Bacillus sp. Isolated from the Interior of Balloon Flower Root

A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C_15:0 (37%), C_17:0 (5.1%), and iso-C_15:0 (27.7%). DNA G + C content was 54 mol%. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.     

Cloning and sequence analysis of cnaA gene encoding the catalytic subunit of calcineurin from Aspergillus oryzae

Calcineurin has been implicated in ion-homeostasis, stress adaptation in yeast and for hyphal growth in filamentous fungi. Genomic DNA and cDNA encoding the catalytic subunit of calcineurin (cnaA) were isolated from Aspergillus oryzae. The cnaA open reading frame extended to 1727 bp and encoded a putative protein of 514 amino acids. Comparative analysis of the nucleotide sequence of cnaA genomic DNA and cDNA confirmed the presence of three introns and a highly conserved calmodulin binding domain. The deduced amino acid sequence was homologous to calcineurin A from Aspergillus nidulans (92%), Neurospora crassa (84%), human (67%), Saccharomyces cerevisiae (58%) and Schizosaccharomyces pombe (54%). Further, A. oryzae cnaA cDNA complemented S. cerevisiae calcineurin disruptant strain (Deltacmp1 Deltacmp2), which was not viable in the presence of high concentrations of NaCl (1.2 M) and at alkaline pH 8.5.     

Endophytic bacillus sp. isolated from the interior of balloon flower root

A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C15:0 (37%), C17:0 (5.1%), and iso-C15:0 (27.7%). DNA G+C content was 54 mol%. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.     

Characterization and sequencing of the lac Y54-41 "uncoupled" mutant of the lactose permease

The Escherichia coli strain carrying the lac Y54-41 gene encodes a mutant lactose permease which carries out normal downhill transport of galactosides but is defective in uphill accumulation. In this study, the mutant lac Y54-41 gene was cloned onto the multicopy vector pUR270. As expected, the cloned gene was shown to express normal downhill transport activity but was markedly defective in the uphill transport of methyl-beta-D-thiogalactopyranoside. Direct measurements of H+ transport revealed that the mutant permease can transport H+ with methyl-beta-D-thiogalactopyranoside but at a significantly reduced capacity compared to the wild-type strain. However, under conditions where the mutant and wild-type strains both transport lactose at similar rates, no detectable H+ transport was observed in the mutant strain. The entire cloned lac Y54-41 gene was subjected to DNA sequencing, and a single base substitution was found which replaces glycine 262 in the protein with a cysteine residue. Inhibition experiments showed that the mutant permease is dramatically more sensitive to three different sulfhydryl reagents: N-ethylmaleimide, p-hydroxymericuribenzoate, and p-hydroxymercuriphenylsulfonic acid. However, the lactose analogue, thiodigalactoside, was only marginally effective at protecting against inhibition in the mutant strain. The results are consistent with the idea that the sulfhydryl reagents are inhibiting the mutant permease activity by reacting with cysteine 262.     

A Na+/H+ antiporter gene of the moderately halophilic bacterium Halobacillus dabanensis D-8T: cloning and molecular characterization

A gene encoding a Na(+)/H(+) antiporter was cloned from a chromosomal DNA of Halobacillus dabanensis strain D-8(T) by functional complementation. Its presence enabled the antiporter-deficient Escherichia coli strain KNabc to survive in the presence of 0.2 M NaCl or 5 mM LiCl. The gene was sequenced and designated as nhaH. The deduced amino-acid sequence of NhaH consists of 403 residues with a calculated molecular mass of 43,481 Da, which was 54% identical and 76% similar to the NhaG Na(+)/H(+) antiporter of Bacillus subtilis. The hydropathy profile was characteristic of a membrane protein with 12 putative transmembrane domains. Everted membrane vesicles prepared from E. coli cells carrying nhaH exhibited Na(+)/H(+) as well as Li(+)/H(+) antiporter activity, which was pH-dependent with highest activities at pH 8.5-9.0 and at pH 8.5, respectively. Moreover, nhaH confers upon E. coli KNabc cells the ability to grow under alkaline conditions.     

Dual control of quorum sensing by two TraM-type antiactivators in Agrobacterium tumefaciens octopine strain A6

Agrobacterium tumefaciens wild-type strains have a unique quorum-sensing (QS)-dependent Ti plasmid conjugative transfer phenotype in which QS signaling is activated by corresponding conjugative opine inducers. Strain K588, with a nopaline-type chromosomal background harboring an octopine-type Ti plasmid, however, is a spontaneous mutant displaying a constitutive phenotype in QS. In this study, we show that a single amino acid mutation (L54P) in the QS antiactivator TraM encoded by the traM gene of Ti plasmid is responsible for the constitutive phenotype of strain K588. Introduction of the L54P point mutation to the TraM of wild-type strain A6 by allelic replacement, however, failed to generate the expected constitutive phenotype in this octopine-type strain. Intriguingly, the QS-constitutive phenotype appeared when the pTiA6 carrying the mutated traM was placed in the chromosomal background of the nopaline-type strain C58C1RS, suggesting an unknown inhibitory factor(s) encoded by the chromosomal background of strain A6 but not by C58C1RS. Low-stringency Southern blotting analysis showed that strain A6, but not strain C58 and its derivatives, contains a second traM homologue. The homologue, designated traM2, has 64% and 65% identities with traM at the DNA and peptide levels, respectively. Similar to TraM, TraM2 is a potent antiactivator that functions by blocking TraR, the QS activator, from specific binding to the tra gene promoters. Deletion of traM2 in strain A6 harboring the mutated traM confers a constitutive QS phenotype. The results demonstrate that the QS system in strain A6 is subjected to the dual control of TraM and TraM2.     

Isolation and characterization of a carbon monoxide utilizing strain of the acetogen Peptostreptococcus productus

From sludge obtained from the sewage digester plant in Marburg-Cappel a strictly anaerobic bacterium was enriched and isolated with carbon monoxide as the sole energy source. Based on morphological and physiological characteristics the isolate was identified as a strain of Peptostreptococcus productus, which was called strain Marburg. The organism was able to grow on CO (50% at 200 kPa) as the sole energy source at a doubling time of 3 h and converted this substrate to acetate and CO₂. The type strain of P. productus was not able to grow at the expense of CO. Electron microscopic investigations of strain Marburg cells revealed a cell wall which was different from that of other Gram-positive prokaryotes. DNA:DNA hybridization studies of the DNA isolated from strain Marburg and the type strain as well as some morphological and physiological properties of both strains confirmed the low degree or relatedness between the two strains.     

Cloning and characterization of the gene encoding RNA polymerase sigma factor sigma(54) of deep-sea piezophilic Shewanella violacea

We have recently reported that a sigma(54)-like factor recognizes a DNA element, designated as region A, upstream of a pressure-regulated operon in piezophilic Shewanella violacea strain DSS12 (Nakasone et al., FEMS Microbiology Lett. 176 (1999) 351-356). In this study, we isolated and characterized the rpoN gene of this piezophilic bacterium. The rpoN gene was found to encode a putative protein consisting of 492 amino acid residues with a predicted molecular mass of 55359 Da. Significant homology was evident comparing the rpoN sequence of S. violacea with that of Escherichia coli (62.8% identity), Vibrio anguillarum (61.7% identity) and Pseudomonas putida (57.0% identity). The DNA-binding domain at the C-terminus of sigma(54) is well conserved in the case of the S. violacea rpoN gene product and the helix-turn-helix motif and the RpoN box are also present. In addition, the conserved glutamine-rich domain is present at the N-terminus. sigma(54) in S. violacea was expressed at a relatively constant level under various growth conditions as determined by both primer extension and Western blotting analyses. By means of a recombinant plasmid, a hexahistidine-tagged derivative of the sigma(54) from strain DSS12 was overexpressed in Escherichia coli and purified to near homogeneity. An electrophoretic mobility shift assay demonstrated that the purified sigma(54) protein specifically recognizes region A in the above-mentioned pressure-regulated operon.     

Detection of measles virus-induced apoptosis of human monocytic cell line (THP-1) by DNA fragmentation ELISA

Abstract Measles virus (wild strain, Toyoshima strain)-induced cell death is characterized by cell shrinkage, chromatin condensation, and nuclear fragmentation in a human monocytic cell line (THP-1). DNA fragmentation of measles virus-infected THP-1 cells was demonstrated by DNA agarose gel electrophoresis as well as by DNA fragmentation ELISA. When measles virus-infected THP-1 cells were cultured on monolayers of fibroblasts or human umbilical vein endothelial cells (HUVEC), the percentage of measles virus antigen-positive THP-1 cells and DNA fragmentation were significantly decreased. Addition of anti-intercellular adhesion molecule (ICAM)-1 (CD54) monoclonal antibody to culture of measles virus-infected THP-1 cells reduced significantly DNA fragmentation induced by measles virus. These findings suggest that inhibition of virus spread by fibroblasts and HUVEC reduces apoptosis, and ICAM-1 (CD54) may participate in the DNA fragmentation pathway.