YieJ (CbrC) Mediates CreBC-Dependent Colicin E2 Tolerance in Escherichia coli |
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Authors: | S. James L. Cariss Chrystala Constantinidou Mala D. Patel Yuiko Takebayashi Jon L. Hobman Charles W. Penn Matthew B. Avison |
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Affiliation: | Department of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TD, United Kingdom,1. School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom2. |
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Abstract: | Colicin E2-tolerant (known as Cet2) Escherichia coli K-12 mutants overproduce an inner membrane protein, CreD, which is believed to cause the Cet2 phenotype. Here, we show that overproduction of CreD in a Cet2 strain results from hyperactivation of the CreBC two-component regulator, but CreD overproduction is not responsible for the Cet2 phenotype. Through microarray analysis and gene knockout and overexpression studies, we show that overexpression of another CreBC-regulated gene, yieJ (also known as cbrC), causes the Cet2 phenotype.Colicins are protein antibiotics that have various modes of action. They are usually encoded on plasmids and, in many cases, alongside genes encoding colicin immunity factors, which protect colicin-producing cells from the colicin they produce. Of the enzymatic (E) colicins, some carry nuclease activity, including colicin E2, colicin E9, and colicin E3. These three proteins bind to susceptible cells via the surface protein BtuB (the vitamin B12 importer) and, through a series of events that are poorly understood, cross the cell envelope to enter the cytoplasm, where they degrade nucleic acids: colicins E2 and E9 target DNA; colicin E3 targets rRNA (11).Cells can readily become tolerant of E colicins. Mutants usually have lost either the colicin receptor or some protein involved in colicin import. Loss-of-function mutations in btuB confer tolerance of high levels of colicins E2, E9, and E3. Almost 40 years ago, Escherichia coli mutants having a colicin E2-tolerant (Cet2) phenotype were identified. The Cet2 phenotype confers tolerance of colicins E2 and E9 only, while cells remain susceptible to colicin E3, and BtuB is intact (8, 9). Cet2 mutants were shown to overproduce an inner membrane protein (26), and the cet2 mutation was found to be dominant in trans and mapped at 99.9 min on the E. coli chromosome (8, 9). Using the Cet2 mutant RB208 as a source of genomic DNA, a clone able to transform E. coli cells to a Cet2 phenotype was identified. Since this clone carried a gene predicted to encode an inner membrane protein with properties identical to those overproduced in Cet2 mutants, the gene was named cet (15).The cet gene is the last gene in the four-gene cre locus, so cet is also known as creD. The other genes in this locus are creA (hypothetical open reading frame [ORF]); creB, encoding a response regulator; and creC, encoding a sensor kinase. CreB and CreC form a classical two-component regulatory system, and we recently showed that CreBC are activated upon fermentation of glucose in minimal medium or during aerobic growth on minimal medium containing fermentation products, such as pyruvate, lactate, or acetate, as the sole carbon and energy source (10). CreBC controls the expression of a number of genes (the Cre regulon), some of which encode metabolic functions but several of which are hypothetical. One of the most tightly controlled Cre regulon genes is creD (5).We have previously shown that the Cet2 strain RB208 has a point mutation in creC but that creD itself is wild type (5). Since the RB208 genomic clone capable of transforming cells to a Cet2 phenotype carries the whole cre locus, not just creD (15), our hypothesis is that the Cet2 phenotype of the transformant was due to a trans-dominant mutation in the cloned creC mutant allele activating one or more Cre regulon genes and that the Cet2 phenotype may or may not be caused by overexpression of creD. The aims of the experiments described in this paper were to test our hypothesis that the Cet2 phenotype is caused by activating mutations in CreBC and to definitively identify the Cre regulon gene that encodes the colicin E2 tolerance (Cet) protein. |
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