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. 1998 Jul;66(7):3088–3094. doi: 10.1128/iai.66.7.3088-3094.1998

FIG. 4.

FIG. 4

Construction of vacA::xylE transcriptional fusions in Tox+ H. pylori 60190. Sequences derived from Tox+ strain 60190 are represented by open boxes. Sequences derived from Tox− strain 86-313 are indicated by black boxes. Vector sequences are shown as thin, single lines. The vacA transcriptional start point (+1) is represented by a bent arrow. The directional arrow in the xylE/km cassette denotes the orientation of xylE. The kanamycin resistance gene (km) is transcribed under the control of its native promoter and in the same direction as xylE. (A) Construction of a vacA transcriptional reporter strain. The xylE/km cassette was cloned into the BglII site of pCTB6, which contains a 3.2-kb vacA fragment from Tox+ strain 60190 (yielding pCTB6::xylE/km). This construct was introduced into the chromosome of strain 60190 by natural transformation and allelic exchange, and the resultant strain (60190 vacA::xylE) was designated 60190 VX-1. (B) Introduction of Tox− vacA promoter region sequences into Tox+ strain 60190. To place the vacA::xylE fusion in 60190 VX-1 under the control of a heterologous vacA promoter from Tox− strain 86-313, the cat gene was cloned into a fragment from strain 86-313 containing the entire cysS-vacA intergenic region (to yield pBW4cat). Natural transformation and allelic exchange were used to introduce this sequence into the 60190 VX-1 chromosome. The extent of vacA sequence exchange in the chimera was experimentally determined to be up to +87 relative to the vacA TSP. The resultant strain, which now contains the vacA promoter region from strain 86-313, was designated 60190 VXC-1. (C) Construction of a chloramphenicol-resistant control strain. The construction of the chimeric reporter strain outlined in panel B required the use of a marker for chloramphenicol resistance. To determine the effect of the cat gene alone on vacA transcription, an isogenic control strain was constructed by transforming Tox+ vacA reporter strain 60190 VX-1 with plasmid pCTB2cat. The resultant Cmr Kmr strain bears the cat gene at the same location and in the same orientation as in the chimeric reporter strain, 60190 VXC-1, described above. This isogenic control strain was designated 60190 VX-1 cat control.