Analysis of a kanamycin resistance gene (kmr) from Streptomyces kanamyceticus and a mutant with increased aminoglycoside resistance

Kanamycin resistance gene (kmr) from the overproducing mutant strain of Streptomyces kanamyceticus ISP1375 (strain 1) and from its gentamicin-resistant mutant were cloned into the high copy number vector pIJ702 and transformed into S. lividans 66. This gene provides resistance to kanamycin, gentamicin, sisomicin and tobramicin. The resistance of the transformed recipient strains was higher than the resistance level of the donor S. kanamyceticus 1. Sequencing of the kmr gene (EMBL Nucleotide Sequence Database accession no. Y15838) revealed 53.9% identity in 274 aa with the kgmB gene product (16S rRNA methylase) of S. tenebrarius. Hybridisation analysis using a 0.85 kb fragment carrying the kmr gene revealed that other gentamicin-resistant mutants of S. kanamyceticus 1 and unstable kanamycin-nonproducing mutant had a high level of kmr amplification. We found no homology between the kmr gene and the total DNA of the neomycin producer S. fradiae IFO3718; the sisomicin producer M. zionensis IFO14116 and the gentamicin producer M. purpurea ATCC15835.     

Fatty acids and production of tylosin-like compounds in Streptomyces fradiae

The composition of fatty acids and the spectrum of macrolide antibiotics produced in 7 mutant strains of Streptomyces fradiae, a tylosin producer, were investigated. The strains under investigation differed in the production level and representation of individual tylosin-like compounds. The composition of fatty acids in the mycelium did not depend on the total production. However, the strains producing relomycin in addition to tylosin produced a significantly higher fraction of fatty acids with a higher melting point, and, on the contrary, the strains producing only tylosin or tylosin and desmycosin synthesized a significantly lower proportion of these acids. The results obtained indicate that in addition to the activity and substrate specificity of secondary metabolism enzymes, the composition of the tylosin-like compounds produced can be influenced by the cell membrane and its function.     

Detection of a novel aph(2") allele (aph[2"]-Ie) conferring high-level gentamicin resistance and a spectinomycin resistance gene ant(9)-Ia (aad 9) in clinical isolates of enterococci

Aminoglycoside-modifying enzymes (AMEs) are major factors that confer aminoglycoside resistance to enterococci. In an epidemiologic study on distribution of 12 AME genes in 534 recent clinical strains isolated from a Japanese hospital, two uncommon AME genes, ant(9)-Ia and a novel aph(2") allele, aph(2")-Ie, were detected. ant(9)-Ia had been reported only in Staphylococcus aureus and encodes spectinomycin adenylyltransferase ANT(9)-I, which confers resistance to spectinomycin. The ant(9)-Ia gene was detected in three strains, a single strain each of Enterococcus faecalis, E. faecium, and E. avium. Nucleotide sequences of ant(9)-Ia from these three enterococcal species were identical to that reported for S. aureus and considered to be located on Tn 554. The new aph(2") allele, designated aph(2")-Ie, was identified in three E. faecium strains. The aph(2")-Ie allele was genetically close to aph(2")-Id reported in E. casseliflavus (93.7% amino acid sequence identity; 96.3% similarity), while distant from aph(2")-Ia, aph(2")-Ib, or aph(2")-Ic (26.3-29.5% amino acid sequence identity). Sequence divergence between APH(2")-Id and APH(2")-Ie was mostly located in amino-terminal half. In contrast, sequences corresponding to the three motifs required for aminoglycoside phosphotransferase were conserved except for a single amino acid. Three E. faecium strains having aph(2")-Ie showed high-level resistance to gentamicin and streptomycin, but not to kanamycin, dibekacin, and tobramycin, unlike enzyme specificity described for aph(2")-Id in E. casseliflavus. Such a difference in resistance phenotype was suggested to be related to amino acid sequence divergence between APH(2")-Id and APH(2")-Ie.     

Unusual features in familial asphyxiating thoracic dysplasia (Jeune''s disease)

Asphyxiating thoracic dysplasia or Jeune's disease is a hereditary condition characterized by typical roentgenologic changes of the ribs, pelvis and extremities. Affected infants have very narrow chests and suffer from respiratory insufficiency which often leads to early death.
A large family of Arabic and Greek origin is described. Two siblings were dwarfed, had narrow chests and skeletal changes similar to those seen in Jeune's disease. In both infants widening and shortening of the left lower ribs and a trident appearance of the pelvis was demonstrated. One child who was severely mentally retarded died in infancy from bronchopneumonia and the other suffers from respiratory difficulties. Other members of the family had roentgenologic changes of the pelvis, spine and skull and mental retardation, features which have not previously been described in this disease.     

Involvement of the bacterial phosphotransferase system in diverse mechanisms of transcriptional regulation

A large number of genes in bacteria appear to be expressed in processes regulated by very different mechanisms dependent on the activities of the proteins of the phosphoenolpyruvate/sugar phosphotransferase system. These mechanisms include protein phosphorylation, antitermination, enhancement, antagonistic repression/activation, sensory detection involving two component systems, and other processes not yet understood.     

Unusual features of protein interaction in human immunodeficiency virus (HIV) virions

Summary The treatment of HIV-1 virions with ionic and nonionic detergents (NP 40, ocytlglucoside, Na deoxycholate) resulted in an effect unusual for enveloped viruses: instead of solubilization of glycoproteins, the core protein p24 was solubilized while envelope glycoproteins with other structural proteins were found in subviral particles. These date are consistent with a model of HIV structural organization in which glycoproteins are included in the matrix formed by the protein p17 and suggest that p24 is neither involved in the matrix nor closely bound to any viral proteins.     

Biochemical mechanism of keratin degradation by the actinomycete Streptomyces fradiae and the fungus Microsporum gypseum: A comparison

Two keratinolytic organisms, the procaryote Streptomyces fradiae and the fungus Microsporum gypseum, were cultured on sterile sheep's wool in a mineral solution. The loss in substrate was recorded and the degradation products in the cultivation fluid were analyzed. In M. gypseum the key reaction was the cleaving of the substrate disulfide bridges by means of sulfite excreted into the medium. Keratin denatured by ?sulfitolysis’? was further attacked by extracellular proteases. A typical finding was the accumulation of peptides containing S-sulfocysteine, the product of sulfitolysis of cystine. The overall excess of sulfur was removed by oxidation to sulfite and to sulfate, which was the main and final product. In S. fradiae the degradation was faster. The results did not prove that sulfite formed and the concentration of sulfate in the medium remained negligible. Neither could cysteine desulfhydration and hydrogen sulfide excretion be demonstrated. The medium was found to contain relatively high concentrations of sulfhydryl compounds, evidently cysteine-containing peptides. Therefore, in this microorganism, keratin was most likely denatured by the direct reduction of cystine bridges. The main product of the elimination of excess sulfur was inorganic thiosulfate, which accumulated in the medium.     

Sequence and expression of the genes for HPr (ptsH) and enzyme I (ptsI) of the phosphoenolpyruvate-dependent phosphotransferase transport system from Streptococcus mutans.

We report the sequencing of a 2,242-bp region of the Streptococcus mutants NG5 genome containing the genes for ptsH and ptsI, which encode HPr and enzyme I (EI), respectively, of the phosphoenolpyruvate-dependent phosphotransferase transport system. The sequence was obtained from two cloned overlapping genomic fragments; one expresses HPr and a truncated EI, while the other expresses a full-length EI in Escherichia coli, as determined by Western immunoblotting. The ptsI gene appeared to be expressed from a region located in the ptsH gene. The S. mutans NG5 pts operon does not appear to be linked to other phosphotransferase transport system proteins as has been found in other bacteria. A positive fermentation pattern on MacConkey-glucose plates by an E. coli ptsI mutant harboring the S. mutans NG5 ptsI gene on a plasmid indicated that the S. mutans NG5 EI can complement a defect in the E. coli gene. This was confirmed by protein phosphorylation experiments with 32P-labeled phosphoenolpyruvate indicating phosphotransfer from the S. mutans NG5 EI to the E. coli HPr. Two forms of the cloned EI, both truncated to varying degrees in the C-terminal region, were inefficiently phosphorylated and unable to complement fully the ptsI defect in the E. coli mutant. The deduced amino acid sequence of HPr shows a high degree of homology, particularly around the active site, to the same protein from other gram-positive bacteria, notably, S. salivarius, and to a lesser extent with those of gram-negative bacteria. The deduced amino acid sequence of S. mutans NG5 EI also shares several regions of homology with other sequenced EIs, notably, with the region around the active site, a region that contains the only conserved cystidyl residue among the various proteins and which may be involved in substrate binding.     

Unusual (epithelioid) leiomyoblastoma of the stomach

The authors present clinical and morphological characteristics of gastric leiomyoblastoma (25 cases) nominated by Soviet investigators as a myoid or angioleiomyomatous variant of a glomus tumor. The tumor was composed of leiomyomatous sites, clusters of epithelioid cells with apparently empty cytoplasm and frequently odd nuclei. Morphological criteria are suggested referring leiomyoblastoma to mature, infiltrative and malignant. Electron-microscopic examination in 8 cases provided evidence for myogenic origin of the neoplasm. Smooth muscle differentiation is shown to resolve with growing of cell proliferation and relevant atypia.