The Enterococcus Cassette Chromosome, a Genomic Variation Enabler in Enterococci.

Enterococcus faecium has a highly variable genome prone to recombination and horizontal gene transfer. Here, we have identified a novel genetic island with an insertion locus and mobilization genes similar to those of staphylococcus cassette chromosome elements SCCmec This novel element termed the enterococcus cassette chromosome (ECC) element was located in the 3' region of rlmH and encoded large serine recombinases ccrAB similar to SCCmec Horizontal transfer of an ECC element termed ECC::cat containing a knock-in cat chloramphenicol resistance determinant occurred in the presence of a conjugative reppLG1 plasmid. We determined the ECC::cat insertion site in the 3' region of rlmH in the E. faecium recipient by long-read sequencing. ECC::cat also mobilized by homologous recombination through sequence identity between flanking insertion sequence (IS) elements in ECC::cat and the conjugative plasmid. The ccrABEnt genes were found in 69 of 516 E. faecium genomes in GenBank. Full-length ECC elements were retrieved from 32 of these genomes. ECCs were flanked by attR and attL sites of approximately 50 bp. The attECC sequences were found by PCR and sequencing of circularized ECCs in three strains. The genes in ECCs contained an amalgam of common and rare E. faecium genes. Taken together, our data imply that ECC elements act as hot spots for genetic exchange and contribute to the large variation of accessory genes found in E. faeciumIMPORTANCEEnterococcus faecium is a bacterium found in a great variety of environments, ranging from the clinic as a nosocomial pathogen to natural habitats such as mammalian intestines, water, and soil. They are known to exchange genetic material through horizontal gene transfer and recombination, leading to great variability of accessory genes and aiding environmental adaptation. Identifying mobile genetic elements causing sequence variation is important to understand how genetic content variation occurs. Here, a novel genetic island, the enterococcus cassette chromosome, is shown to contain a wealth of genes, which may aid E. faecium in adapting to new environments. The transmission mechanism involves the only two conserved genes within ECC, ccrABEnt, large serine recombinases that insert ECC into the host genome similarly to SCC elements found in staphylococci.

[Surgical treatment of vaginal agenesia]. / Kirurgisk behandling ved vaginal agenesi.

BACKGROUND: Vaginal agenesia appears among 1 in 5,000-10,000 newborn girls, i.e. 3-6 patients per year in Norway. MATERIAL AND METHODS: From 1975 to 1998, 14 women (mean age 20 years) were operated for vaginal agenesia by McIndoe's method at Haukeland University Hospital. Twelve patients were examined 13 years later by questionnaire, gynaecological examination, ultrasound examination of the urinary tract, and blood and urinary test concerning kidney function and diabetes mellitus. RESULTS: Five patients had been re-operated within nine months because of contracture. In all patients (except one), the constructed vagina had good elasticity, width and depth. The donor area at the medial thigh was unaesthetic in four patients. No abnormalities were found in the urinary tract by ultrasound. One patients had a familial syndrome with Müllerian aplasia, progressive renal disease and mild diabetes mellitus. All patients found the operation important, despite memories of a painful postoperative period. INTERPRETATION: This study shows functional and psychological satisfactory results of construction of vagina in women with vaginal agenesia.

Correlations between transcription of a yeast tRNA gene and transcription factor-DNA interactions.

A partly purified fraction from Saccharomyces cerevisiae has a RNA polymerase III transcription factor activity and contains protein which binds specifically to two internal promoter regions of RNA polymerase III-transcribed genes. The influence of ionic strength and of dimethyl sulfoxide on specific binding to a S. cerevisiae tRNAleu3 gene has been analyzed by DNase I protection (footprinting) and by nitrocellulose filter binding. The effects of these agents on binding correlate with their effects on transcription and on the stability of transcription complexes. Dimethyl sulfoxide stabilizes binding and transcription complexes against dissociation by NaCl, with 10% dimethyl sulfoxide compensating for the addition of 50-60 mM NaCl. Binding of protein in the 5' proximal part of the S. cerevisiae tRNAleu3 gene is more NaCl-sensitive than binding in the 3' proximal part.

Studies on the structure and expression of Escherichia coli pyrC, pyrD, and pyrF using the cloned genes.

The Escherichia coli pyrC, pyrD and pyrF genes were cloned on multicopy plasmids derived from pBR322 and analysed by means of restriction endonucleases. It was found that the pyrC gene is destroyed by cutting with the restriction endonuclease BamHI, that the entire pyrD gene can be isolated on a 1300-base pairs DNA fragment generated by EcoRI cleavage and that cutting with EcoRI removes the promotor and probably also the translational start site from the pyrF gene. More details on the restriction maps are presented. Further, it was found that the presence of a pyr gene in multiple copies on a plasmid does not significantly interfere with the activity of the chromosomal pyr genes. Using the 'minicell' technique, the polypeptides encoded by the three cloned pyr genes were identified. The relative molecular masses for the pyrC-encoded and pyrD-encoded polypeptides are 38 000-40 000 and 36 000-38 000, respectively. Thus in their native form, dihydroorotase and dihydroorotate oxidase appear to be dimeric proteins. The 'minicell' experiments positively identified a protein chain of Mr 23 000-24 000 as being a subunit of OMP decarboxylase encoded by pyrF. Moreover, the coding frame for this polypeptide seems to be expressed as the first gene in the operon with the coding frame for another protein chain of Mr 13 000-14 000. Since, however, the native OMP decarboxylase during sedimentation and gel filtration behaves as a protein of Mr 45 000 +/- 4000, this latter polypeptide (Mr 13 000-14 000) is hardly a component of the enzyme. Pyr-lac+ operon fusions were constructed by the Mu d1 procedure. By integrating an F'lac episome into the lac part of the fusions and determining the direction of chromosomal transfer from the resultant Hfr strains, the direction of pyrC transcription was found to be counter-clockwise, while pyrD and pyrF were found to be transcribed in a clockwise direction.

An unusual transposon encoding heat shock inducible and developmentally regulated transcripts in Dictyostelium.

We have isolated and characterized a transposable element from Dictyostelium discoideum denoted Tdd-1. There are approximately 50 complete copies of the element per haploid genome and approximately 100-150 partial elements. Southern blots of DNA from different Dictyostelium discoideum strains show that Tdd-1 is a mobile element. Tdd-1 is 4.9 kb long, with 313 bp inverted repeats. These repeats lie near the termini, but unlike other transposable elements one end of Tdd-1 extends 36 bp past the repeat and the other extends 1 bp. Tdd-1 encodes a series of developmentally regulated transcripts, all with the same polarity, that increase dramatically in abundance after approximately 10 hr of development. We have also identified a heat shock inducible transcript of the opposite polarity.