Similarly, Marche`s et al reported that cif is not universally present in pathogenic EPEC and EHEC, and that some strains encode a truncated variant that is inactive. Variation in the repertoire of Type III secreted effectors is well known and it is NS 1643 possible that CHBP is non-essential for virulence, that functional redundancy may exist, or that presence or absence of Cif is related to subtle differences in virulence. Reactivity was restored when cloned chbP was introduced into the mutant on an inducible plasmid. In contrast to BopE, which was readily detected in the supernatant of LB-grown B. pseudomallei as before, we were unable to detect CHBP despite evidence that the protein was present in the whole-cell fraction. Despite the absence of CHBP in the secreted fraction when BopE was detected, appearance of cytosolic CHBP in NI 42 infected cells was dependent on a functional Bsa system, as it was absent in a bsaQ mutant previously reported to be deficient in Type III secretion. Though it is tempting to speculate that the failure of CHBP to appear in lysates of U937 cells infected with the bsaQ mutant is evidence that CHBP is secreted via the Bsa apparatus, it should be noted that Bsa is required for the bacteria to escape endosomes. It remains a possibility that CHBP is secreted only once B. pseudomallei enters the cytosol in a Bsa-dependent way. To separate these possibilities we repeatedly attempted to detect the Bsa-dependent appearance of CHBP in cells infected with B. pseudomallei wild-type and mutant strains in the presence of cytochalasin D to prevent bacterial uptake. By Western blotting we were unable to detect injection of CHBP into cells where B. pseudomallei was prevented from uptake, though this may reflect low levels of injection or the sensitivity of the detection method, The cytosolic staining obtained with a CHBP-specific antibody is in contrast to observations with E. coli Cif, where ectopic expression leads to accumulation of the protein in the nucleus. CHBP is predicted to act on nuclear targets, but we cannot preclude the possibility that it enters the nucleus at lower levels, or that it may be enriched in the nucleus at time intervals beyond those studied here. E. coli Cif induces the accumulation of p21 and p27 that inhibit CDK1-CyclinB and CDK2-CyclinA/E, leading to cell cycle arrest at the G2/M and G1/S transitions. Cui et al demonstrated that this and other activities of Cif require glutamine deamidation of ubiquitin or the ubiquitin-like protein NEDD8 that regulates Cullin-RING ubiquitin ligases. We repeatedly attempted to detect CHBP-dependent inhibition of the cell cycle during B. pseudomallei infection as previously demonstrated by E. coli Cif by flow cytometric analysis of propidium iodide-stained cells, but were hindered by our inability to completely remove B. pseudomallei from the culture system owing to its intrinsic high level of resistance to antibiotics and induction of cell death 24�C48 h post-inoculation.