Operon organization of the <i>cagH</i>, <i>cagI</i> and <i>cagL</i> genes.

<p>(A) Gene arrangement of the <i>cag</i> pathogenicity island in <i>H. pylori</i> strain P12. Genes are represented as arrows, and their designations are indicated below. The operon containing <i>cagI</i> may comprise five (<i>cagF</i>, <i>cagG</i>, <i>cagH</i>, <i>cagI</i> and <i>cagL</i>) or even more genes, but <i>cagH</i>, <i>cagI</i> and <i>cagL</i> are particularly tightly associated since they have overlapping reading frames or Shine-Dalgarno (SD) sequences. Translational starts are indicated by arrows. (B) A <i>cagI</i> deletion mutant containing a terminatorless chloramphenicol resistance cassette <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035341#pone.0035341-Fischer2" target="_blank">[16]</a> was generated by transformation with plasmid pSO171. A polyclonal rabbit antiserum raised against CagI was used for immunoblot analysis of whole cell lysates of the P12 wild-type strain or isogenic <i>cagA</i> or <i>cagI</i> mutants. The same lysates were examined by Western blot for the presence of CagL using the polyclonal rabbit antiserum AK271. CagI and CagL protein bands, respectively, are marked by arrowheads. Note that both antisera recognize cross-reactive bands, but anti-CagI does not react with CagL or vice versa (data not shown). (C) Plasmid constructs used for generation and complementation of <i>cagI</i> mutants. A counterselection strategy was used for generating marker-free deletion mutants. Mutants generated with plasmids pWS320 and pWS326 retain the 3′ part of <i>cagI</i>, whereas the mutant generated with plasmid pWS327 has a complete deletion without any <i>cagI</i> traces. For complementation of <i>cagI</i> mutants <i>in trans</i>, the <i>cagI</i> gene was cloned under the control of the <i>cagA</i> promoter and integrated into the <i>recA</i> gene (plasmid pWS322).