We present evidence that the expression of rho is regulated by rho-dependent attenuation of transcription. Gene fusion analysis with nested series of deletions of rho indicated that the transcription of rho is attenuated in a rho-dependent manner in the leader region and that neither a read-through transcription from the upstream gene, trxA, nor a modulation of transcription initiation of the rho promoter is involved in the self-control of rho. Si mapping and Northern hybridization analyses localized at least six transcription attenuation or termination sites in the region ranging from the 3' end of the trxA structural gene to the middle of the rho structural gene. Among them, the most upstream site overlapping the rho promoter sequence was assigned to the terminator for the trxA gene, and the second and third sites, mapping about 80 and 50 nucleotides upstream from the start codon of rho, were suggested to function as the major attenuation sites for regulation of the rho expression. Further, the start points of the trxA and rho RNAs were determined in an in vitro transcription system to be located 111 nucleotides (U) and 255 nucleotides (G) Kushner, Gene 32:399408, 1984).The termination of transcription plays profound roles in the regulation of gene expression in procaryotes. Termination of RNA synthesis generally occurs at the ends of operons so as to ensure independent expression of neighboring genes. In addition, it can also occur within operons to allow differential expression of genes within an operon. In Escherichia coli, several protein factors which participate in transcription termination or antitermination have been identified: Rho, NusA, NusB, and NusE (14,21,22,25,26,30,47,56).Rho protein causes termination of transcription catalyzed by E. coli RNA polymerase at certain specific sites on DNA template (47). These sites are called Rho-dependent terminators, as opposed to Rho-independent terminators which are recognized by RNA polymerase without the aid of Rho. Several Rho-dependent terminators have been found at different positions in various operons. For example, trp-t' (57) and the tyrT terminator (33) are located at the ends of their operons, while tLl, tRl, and tR2 are within the leftward and rightward operons of bacteriophage X, respectively (47). It has also been shown that mutational polarity involves Rho-dependent terminators residitng within a structural gene.Such terminators are normally nonfunctional, but are activated when preceding translation is arrested at a nonsense codon introduced by mutation (1, 13). A number of rho mutants of E. coli have been isolated that are defective in Rho-dependent transcription termination (12,28,29,31,46). Some of them are conditional-lethal, suggesting that Rho is essential for cell growth. (27, 45). Conversely, decreased Rho levels have been observed with a new class of rho mutants which is hyperactive in termination (53). These observations suggest that the expression of rho is autogenously regulated. In view of the role of Rho as a transcription term...

Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) causes hemorrhagic colitis and hemolytic uremic syndrome. TMA-15 is a humanized monoclonal antibody against Stx2, a major pathogenic factor. In a mouse infection model that used B2F1, a virulent STEC strain, the efficacy of TMA-15 was assessed when it was administered after bacterial and toxin exposure. In this model, a time-course analysis of the serum Stx2 level showed that the toxin was detectable from 24 h after infection. In an evaluation of the time-dependent efficacy, treatment with TMA-15 up to 24 h after infection ameliorated the lethal challenge, although treatment at 48 h showed no efficacy. To determine the effective dose, escalating doses were administered at 24 h after infection. The number of mice that survived after doses of 0, 0.25, 0.5, 1.0, and 2.0 mg/kg were 0/20, 11/20, 17/20, 20/20, and 20/20, respectively. These findings suggest that TMA-15 shows potential for prevention of severe complications associated with STEC infection.

Because analysis of this factor is important to understanding the pathology induced by Shiga toxin-producing Escherichia coli, we purified this factor from human plasma and identified it. Purification was carried out by serially subjecting human plasma to Con A-Sepharose, DEAE-Sepharose, hydroxyapatite, and gel-filtration high performance liquid chromatography (HPLC), using Stx2-neutralizing activity as the indicator. The gel-filtration HPLC fraction yielded a single band on SDSpolyacrylamide gel electrophoresis. Twenty N-terminal amino acid residues of this fraction were analyzed and found to correspond perfectly to human serum amyloid P component (HuSAP). Because commercially available HuSAP also showed Stx2 binding and neutralizing activity, we identified this factor as HuSAP.

No abstract

No abstract