In this work, we report cDNA cloning of two type I interferons (IFNs) from the head kidney of Atlantic salmon, called SasaIFN-alpha1 (829 bp) and SasaIFN-alpha2 (1290 bp). Both translate into 175 amino acid precursor molecules showing 95% amino acid sequence identity. The precursors have a putative 23 amino acid signal peptide, which suggests that the mature Atlantic salmon IFNs contain 152 amino acids (18.2 kDa). Salmon IFN appears to have five alpha-helices, similar to mammalian and avian type I IFNs, and showed 45% sequence identity with zebrafish IFN, up to 29% identity with mammalian IFN-alpha sequences, and 17%-18% sequence identity with mammalian IFN-beta and chicken type I IFNs. Human embryonic kidney 293 cells transfected with the SasaIFN-alpha1 cDNA gene produced high titers of acid-stable antiviral activity, which protected salmonid cells against infectious pancreatic necrosis virus (IPNV) and also induced Mx protein in the cells. Poly(I)-poly(C) induced two IFN transcripts in head kidney of Atlantic salmon. Genomic IFN sequences contained four introns and five exons, which is different from the intronless type I IFN genes of birds and mammals.

Mx proteins form a family of interferon (IFN)-induced

The bet regulon allows Escherichia coli to synthesize the osmoprotectant glycine betaine from choline. It comprises a regulatory gene, betI, and three structural genes: betT (choline porter), betA (choline dehydrogenase), and betB (betaine aldehyde dehydrogenase). The bet genes are regulated by oxygen, choline, and osmotic stress. Primer extension analysis identified two partially overlapping promoters which were responsible for the divergent expression of the betT and betIBA transcripts. The transcripts were initiated 61 bp apart. Regulation of the promoters was investigated by using cat (chloramphenicol acetyltransferase) and lacZ (␤-galactosidase) operon fusions. Mutation of betI on plasmid F2 revealed that BetI is a repressor which regulates both promoters simultaneously in response to the inducer choline. Both promoters remained inducible by osmotic stress in a betI mutant background. On the basis of experiments with hns and hns rpoS mutants, we conclude that osmoregulation of the bet promoters was hns independent. The bet promoters were repressed by ArcA under anaerobic growth conditions. An 89-bp promoter fragment, as well as all larger fragments tested, which included both transcriptional start points, displayed osmotic induction and BetI-dependent choline regulation when linked with a cat reporter gene on plasmid pKK232-8. Flanking DNA, presumably on the betT side of the promoter region, appeared to be needed for ArcA-dependent regulation of both promoters.Hyperosmotically stressed cells of Escherichia coli build up the cytoplasmic osmolarity by accumulation of potassium glutamate and various osmoprotectants (12). The highest osmotolerance is achieved by the accumulation of glycine betaine (hereafter called betaine) (49). Betaine can either be taken up by the ProU and ProP systems (7,8,37) or be synthesized by the Bet system, i.e., the choline-to-betaine pathway (31). Synthesis of betaine requires an external supply of choline or the intermediate metabolite betaine aldehyde. At low external concentrations, choline is mainly taken up by the high-affinity choline porter BetT (K m ϭ 8 M), whereas at higher concentrations choline is also taken up by ProU (K m ϭ 1.5 mM) (30, 50). Oxygen-dependent choline dehydrogenase (BetA) catalyzes both steps in the oxidation of choline to betaine by the way of betaine aldehyde, whereas NAD-dependent betaine aldehyde dehydrogenase (BetB) is specific for the last step (1, 31).Biochemical data have previously revealed that expression of the Bet system is reduced under anaerobic conditions. For aerobic cells, osmotic stress gives a partial induction, but for full expression, the cells also need an external supply of choline (31). Experiments with lacZ fusions showed that the regulation occurs at the level of transcription (16).The DNA sequence of the bet region has revealed that in addition to the structural genes, the bet system encodes a regulatory protein called BetI. Albeit BetI shares some sequence homology with the TetR family of bacterial regulatory proteins (29), Bet...

VanB-type vancomycin resistance is encoded by the vanB gene cluster, which disseminates by horizontal gene transfer and clonal spread of vancomycinresistant enterococci (VRE). Genetic linkage of the vanB gene cluster to transposon Tn5382 and the insertion sequences IS16 and IS256-like has previously been shown. In this study linkage of defined vanB gene cluster subtypes to these elements was examined. All the vanB2 subtype strains studied (n l 14) revealed co-hybridization of vanB and Tn5382, whereas the strains of vanB1 (n l 8) and vanB3 (n l 1) subtypes were Tn5382 negative. Conjugative cotransfer of the vanB2 gene cluster and Tn5382 was demonstrated for two strains. DNA sequencing of the vanX B -ORFC region in vanB2 strains confirmed that the vanB2 gene cluster is an integral part of Tn5382. No general pattern of linkage was observed with regard to IS16 and IS256-like. Two novel insertion sequences were identified in specific vanB2 subtype strains. (i) A 1611 bp element (ISEnfa110) was detected in the left flank of Tn5382. Its insertion site, lack of terminal inverted and direct repeats, and two conserved motifs in its putative transposase all conform to the conventions of the IS110 family. (ii) A 787 bp element (ISEnfa200) was detected in the vanS B -vanY B intergenic region. Its ORF encoded a putative protein with 60-70 % identity to transposases of the IS200 family. No further copies of ISEnfa110 were found by colony hybridization of 181 enterococcal isolates, whereas ISEnfa200 was found in four additional vanB2 strains from the USA. The five strains had identical ISEnfa200 element insertion sites, and Tn5382 was located downstream from a pbp5 gene conferring high-level ampicillin resistance. These isolates showed related PFGE patterns, suggesting possible clonal spread of a VRE strain harbouring a Tn5382-vanB2-ISEnfa200 element linked to a pbp5 gene conferring ampicillin resistance.

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