The ripA transcript levels were evaluated by RT-PCR in replicates of four independent cultures and normalized to tul4 [22]. Primers internal to ripA

and tul4 were designed with matched melting temperatures and amplification product sizes. Total RNA was collected from F. tularensis LVS cultures at mid exponential stage growing in Chamberlains defined media at pH 5.5 and pH 7.5. cDNA was generated from the RNA samples using random primers in a reverse transcriptase reaction. Samples lacking reverse transcriptase were used to monitor DNA contamination. Quantization of ripA transcripts was Ganetespib mw achieved by SHP099 supplier densitometry of gene-specific products isolated by agarose electrophoresis. Mean normalized expression of ripA ± standard deviation at pH 5.5 was 1.527 ± 0.1656 and 2.448 ± 0.2934 at pH 7.5 (Fig. 6c) representing a 1.6 fold expression differential (P = 0.0033). The concentration Momelotinib clinical trial of RipA protein present at pH 5.5 and pH 7.5 was measured by FlAsH™ labeling of RipA-TC present in whole cell lysates of the chromosomal fusion strain (Table 1). Six μg of total protein was incubated with TC specific FlAsH™ reagents, separated by SDS-PAGE and subjected to in-gel fluorescence. Mean intensity of RipA-TC ± standard deviation of four independent samples at pH

5.5 was 1.083 × 107 ± 6.340 × 105 arbitrary units as compared to 1.551 × 107 ± 8.734 × 105 arbitrary units at pH 7.5 (Fig. 6d), representing a 1.43 fold change in expression (P = 0.00031) as compared to the 1.8 fold difference expressed by the ripA’-lacZ1 translational fusion. Results from

the four different measures of ripA expression revealed pH – affected increases ranging from 1.3 to 1.8 fold. While the increased ripA expression at pH 7.5 as compared to 5.5 is mathematically statistically significant, it remains to be seen if Phospholipase D1 is biologically relevant. F. tularensis LVS ripA expression during intracellular growth The pH effect on ripA expression parallels the location-specific requirement for functional RipA within the host cell. That is, RipA is dispensable for the early stages of invasion and phagosome escape where the pH is likely to be relatively acidic, but is required for replication in the more neutral pH of the cytoplasm, a condition where ripA expression is elevated. To see if this correlation exists throughout the course of infection we measured β-galactosidase produced by the F. tularensis LVS chromosomal transcriptional ripA-lacZ2 fusion strain at different stages of intracellular growth. Since the iglA gene is induced during intracellular growth [28], we therefore constructed and used an iglA-lacZ transcriptional reporter for control and comparison purposes. The iglA-lacZ fusion was cloned into pBSK aphA1 (Table 1) and integrated into the F. tularensis LVS chromosome as described earlier for ripA. The insertion of pBSK iglA’-lacZ into the chromosome likely has polar effects on iglB, iglC, and iglD.