It has been known for several decades that iron inhibits the production of diphtheria toxin by Corynebacterium diphtheriae by preventing expression at maximal levels. We examined the inhibition kinetics of toxin production after the addition of either iron or rifampin to iron-limited cultures of C7(/tlx+). Ironmediated inhibition of toxin production was found to be linear within the range of 16 nM to 16 ,.M. The inhibition kinetics following the addition of iron or rifampin was almost identical. [3H]RNA extracted from iron-limited toxigenic C. diphtheriae was found to hybridize to a greater extent to corynephage ,8 DNA than either [3H]RNA extracted from toxigenic C. diphtheriae before the onset of toxin production or [3H]RNA extracted from nonlysogenic, nontoxigenic C. diphtheriae.Uchida et al. ( 28) by the isolation of phage mutants that code for the production of nontoxic proteins that are serologically related to diphtheria toxin have demonstrated that the structural gene for toxin, tox, was carried on the corynebacteriophage ,8 genome. The diphtheria
A synthetic gene encoding human interleukin-2 (IL-2) was designed such that the codon usage bias resembled that found in highly expressed Escherichia coli genes. The percentage of preferred codons was increased from 43% in the native cDNA sequence to 85% in the synthetic sequence. The cDNA and synthetic IL-2 genes were placed under the control of the trc promoter and expressed in E. coli JM101. While Northern blot analysis of IL-2 mRNA from each genetic construct demonstrated equivalent message half-lives, immunoblot and bioactivity analyses showed the synthetic gene to direct the synthesis of up to 16 times more IL-2 than the native cDNA sequence.
The toxigenic corynebacteriophage wo/ was isolated from the hypertoxigenic Park-Williams no. 8 (PW8) strain of Corynebacterium diphtheriae and compared with the toxigenic corynebacteriophage Ptox. The physical size and host range of both phages were found to be identical. An endonuclease restriction map of tox+ was constructed, and the locations of the cohesive ends (cos), phage attachment site (attP), and the diphtheria tox operon were identified. The genome of w0 + was found to differ from that of 1tOx in three regions. In addition, w'+ was shown to be integrated into two nontandem corynebacterial phage attachment sites (attBI, attB2) in the PW8 chromosome. The differences in the restriction endonuclease digestion maps of wfox and 1toxX and the contribution of double lysogeny are discussed in relation to the hypertoxigenicity of the PW8 strain. Although the structural gene for diphtheria toxin, tox, is carried by a family of temperate corynebacteriophages, the final yield of toxin obtained from Corynebacterium diphtheriae grown under optimal conditions is dependent upon several bacterial host-determined factors (10, 22). The most toxigenic C. diphtheriae strain ever described is Park-Williams no. 8 (PW8) (24). Pappenheimer et al. (23) have shown that the hypertoxigenicity of the PW8 strain is due, in part, to a defective cytochrome system and to the ability to continue to'grow for three doublings after iron becomes the rate-limiting substrate. However, little is known of the relationship between PW8 and its toxigenic corynebacteriophage wtox (13) or the relationship between oMX and the well-studied corynebacteriophage POX. We have isolated a spontaneous, clear-plaqueforming mutant of wOX, wctox, and report its restriction endonuclease map and the location of its cohesive ends (cos), attachment site (attP), and the diphtheria tox operon. We compare corynebacteriophages Wctox and 3c'ox with respect to their restriction map, morphology, and corynebacterial host range. In addition, we show that wtOX is integrated into two nontandem bacterial attachment sites (attBl, attB2) on the C. diphtheriae strain PW8 chromosome and is able to form nontandem double lysogens by integrating into two attB sites on the C. diphtheriae (Belfanti) 1030(-)'x chromosome. This
Transcription of the tox gene in lysogenic Corynebacterium diphtheriae strains C7(,B tox+), C7(y tox) and the hypertoxigenic PW8 (w tox+) was analyzed and compared with transcription of the C. diphtheriae tox gene in the recombinant strain Escherichia coli(pDT201). In anl cases Si nuclease mapping localized the 5' terminus of the tox mRNA to a site 8 or 9 base pairs (bp) downstream of a region similar to the-10 consensus sequence of E. coli promoters. In C. diphtheriae the tox transcript was observed only in strains that were grown under iron-limiting conditions; in the presence of excess iron, transcription beyond bp 38 of the tox coding region was not observed. In contrast, in E. coli(pDT201) tox was expressed at equivalent levels in both iron-depleted and iron-supplemented media. The DNA insertion in the tox gene of the nontoxigenic corynephage y was found to occur at bp 54 of the tox coding region. The insertion event resulted in the duplication of a 7-bp target sequence, and the ends of the insert were found to constitute an imperfect inverted repeat of approximately 26 bp. Transcription from the tox promoter in C7(y tox) was found to initiate at the same nucleotides as in C7(0 tox+),
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