Poul Andersson

A family of genes encoding a cell-killing function may be conserved in all Gram-negative bacteria

The relF gene in Escherichia coli is related to the hok gene on plasmid R1. Both genes encode small proteins which, when overexpressed In E. coli lead to collapse of the membrane potential and cell death. A third gene, designated gef, which encodes a homologous cell‐toxic protein, has been isolated from E. coli DNA. Both gef and relF are transcribed in E. coli and subject to post‐transcriptional regulation which, in the case of gef, is coupled to translation of a leader sequence. The finding of homologous sequences in such distantly related bacteria as Agrobacterium and Rhizobium species suggests an important physiological role.

The gef gene from Escherichia coli is regulated at the level of translation

We describe post‐transcriptional regulation of the chromosomal gene, gef, from Escherichia coli. The gef gene is a member of a gene family consisting of the chromosomal gef and relF genes from Escherichia coli and the hok, flmA, smB, and pndA genes, which are situated on conjugative plasmids. All the genes encode small, toxic proteins of approximately 50 amino acids which are functionally and structurally homologous. Furthermore, the gene family shares post‐transcriptional regulation of expression, albeit by different mechanisms. We demonstrate here that translation of gef is coupled to an upstream open reading frame which, in turn, is regulated by a transacting factor, probably an antisense RNA.

Analysis of an Escherichia coli mutant strain resistant to the cell-killing function encoded by the gef gene family

The chromosomal genes gef and relF from Escherichia coli and the plasmid‐encoded genes hok, flmA, srnB, and pndA constitute the gef qene family, which encodes a cell‐killing function. In order to investigate the mechanism of cell killing we have isolated an E. coli mutant strain that is resistant to the overexpression of the toxic proteins encoded by the gef gene family. This phenotype requires at least two mutations, one of which has been mapped to 55.2 minutes. This mutation was sequenced and shown to represent a single base substitution in an open reading frame (ORF178) encoding a putative membrane protein having a molecular mass of 20.1 kDa. ORF178 and an upstream frame, ORF190, probably constitute an operon.

Topographic analysis of the toxic Gef protein from Escherichia coli

The chromosomal gef gene of Escherichia coli is a member of the gef gene family which encodes strongly toxic proteins of about 50 amino acids. We demonstrate here that the Gef protein is detectable by anti‐peptide antibodies. Furthermore, we show that Gef is anchored in the cytoplasmic membrane by the N‐terminal part of the protein, and that the C‐terminal part is localized in the periplasm in a dimeric form with at least one disulphide bond. By mutagenesis of gef it is shown that the periplasmic portion of Gef encodes the toxic domain and that the dimerization of Gef is not essential for the toxic effect.

Biological Containment of Bacteria and Plasmids to be Released in the Environment

The design of a biological containment system to be employed in a broad spectrum of bacteria useful in connection with release to the external evironment is reported. The key element is a gene, hok, encoding a small polypeptide of 52 amino acids which, when expressed, is lethal to several different bacterial species. Through construction of various combinations of regulatable promoters and this toxin gene we have achieved containment, since cells accidentally escaping to the outside environment, or any other new combination of a bacterium and the toxin carrying plasmid, will be killed. A specific application of the containment system with respect to deliberate release is based on a fusion between an invertible promoter (flip-flop sequence) and the toxin gene. This results in a stochastic induction of the killing function which eventually will lead to the non-conditional elimination of the organism as a consequence of the competition with related bacteria in the environment.

Delta Infection and Hepatitis B Virus Replication in Danish Patients with Fulminant Hepatitis B

The presence of hepatitis B virus and delta agent markers was investigated in 41 patients referred during the years 1970-1985 with fulminant hepatitis classified as type B or non-A non-B and compared to findings in patients with uncomplicated hepatitis B and chronic hepatitis B infection. 13 patients had no markers of hepatitis B and delta infection and were classified as non-A non-B hepatitis. The remaining 28 patients were all HBsAg and IgM anti-HBc positive and 14 (50%) had evidence of delta infection. In contrast, only 13/71 patients (18%) with acute benign hepatitis B had evidence of delta coinfection (p less than 0.005). This corresponds to an odds ratio of 4.5 for development of fulminant hepatitis among patients with hepatitis B and delta coinfection. In 100 chronic HBsAg carriers 29% were positive for delta markers. 12 of the delta infected patients with fulminant hepatitis were positive for total antibody to the delta antigen, and 2 were delta antigen positive. Three were HBeAg positive/anti-HBe negative. None had hepatitis B virus DNA. Among the 14 patients without delta infection, hepatitis B virus DNA was found in 2/4 HBeAg positive/anti-HBe negative patients and in 1/8 patients negative for both markers. The present data indicate that a high proportion of Danish patients with fulminant hepatitis B have hepatitis B and delta agent coinfection. Further, the findings suggest that hepatitis B and delta coinfection may be associated with an increased risk of development of fulminant hepatitis as compared to that of hepatitis B alone.