Induction and identification of HilA in S. typhimurium
SPI1 gene expression is induced in high osmolarity and low oxygen conditions [10]. Although standing cultures have been used to achieve these inducing conditions, such cultures require 14 hours for growth and are not convenient for following SPI1 regulatory events as they occur. We developed an alternative high osmolarity, low oxygen culture condition and examined the induction of hilA expression. We diluted a stationary phase EE658 (hilA080:: Tn5lacZY) culture 1:100 into poorly aerated LB (see Methods). As seen in Figure 1A, such a bacterial culture achieves mid-log, late-log, and early-stationary phases during an 8 hour period. Measurement of β-galactosidase, in Miller Units, expressed from the chromosomal hilA080:: Tn5lacZY fusion, shows that there is a significant level of β-galactosidase already present in the stationary phase inoculum (Fig. 1B). Interestingly, soon after inoculation, β-galactosidase levels decrease, presumably due to poor hilA expression during the first 2 hours post-inoculation and dilution of the pre-existing β-galactosidase by cell growth. Approximately 3 hours post-inoculation, conditions apparently become favorable for hilA expression as evidenced by an increase in Miller Units. Previous studies would suggest that reduced oxygen levels, caused by the increasing density and insufficient aeration of these bacterial cultures, trigger the induction of hilA[12–14].
In order to detect the HilA protein in S. typhimurium, we solubilized and purified a His-tagged version of HilA (HilA-His) in 0.5% SDS for injection into a rabbit. The resultant antiserum was used to identify and characterize the HilA protein in a hilA+ strain. Western blots show that our antiserum recognizes two S. typhimurium proteins (Fig. 2). The 65.5 kDa reactive protein is present in both the hilA+ and the hilA- strain, whereas the 63 kDa reactive protein is clearly hilA-specific. Interestingly, similar to the β-galactosidase levels expressed from the hilA080:: Tn5lacZY fusion, the hilA-specific protein is present early after dilution of the stationary phase inoculum, disappears by 2 hours post-inoculation, and then increases 3 hours post-inoculation. These results suggest that production of the HilA protein correlates with hilA gene expression in these growth conditions.
Effect of a shift to aerobic growth conditions on SPI1 gene expression and HilA protein levels in S. typhimurium
Previous studies have shown that SPI1 gene expression is repressed by steady-state aerobic growth conditions [10, 15]. We decided to examine SPI1 gene expression and HilA protein production in S. typhimurium after a shift from inducing low oxygen to repressing aerobic growth conditions. First, we grew strains CL87 and EE656 in our high osmolarity, low aeration growth conditions. CL87 contains the iagB87::lacZY fusion, which is located immediately downstream of hilA and is used to monitor chromosomal hilA expression [11]. EE656 contains the prgH020:: Tn5lacZY fusion [10]. HilA binds upstream of and directly activates the prgH promoter [4]. So, we used the prgH020:: Tn5lacZY fusion to monitor HilA-activated SPI1 gene expression. After growing these strains for 3.5 hours to induce hilA expression and HilA protein production, we diluted the bacteria 1:10 into fresh media and shifted the cultures to a high aeration vs. a low aeration growth condition, for 0.5, 1.5 and 2.5 hours. Figure 3A shows that the diluted bacteria continue to grow in both conditions, with the high aeration condition promoting more rapid bacterial growth. Figure 3B shows that β-galactosidase levels decrease in CL87 and EE656 shifted to the high aeration condition, presumably due to aerobic repression of hilA and prgH transcription, and dilution of pre-existing β-galactosidase by cell growth. In contrast, bacteria shifted to the low aeration condition appear to maintain high, induced levels of hilA and prgH expression.
Western blots show that high levels of HilA are present in the pre-shifted cells as well as in the bacteria shifted to the low aeration condition (Fig. 3C). However, HilA levels decrease in bacteria shifted to the high aeration condition, which correlates with the reduced transcription of hilA and prgH. These results suggest that repression of hilA transcription by oxygen leads to a decrease in HilA protein production, which in turn reduces the expression of HilA-activated genes, such as prgH.
SDS-PAGE mobility of native HilA vs. engineered forms of HilA
The hilA ORF contains two possible AUG start codons that are separated by 22 codons. We examined native HilA in SDS-PAGE to see if it corresponds to the predicted AUG1-TAA translation product (553 amino acids, 63 kDa) or to the predicted AUG2-TAA translation product (531 amino acids, 60.4 kDa). We compared the migration of HilA produced by wild-type S. typhimurium with engineered derivatives of HilA. pCR53 was designed to express the AUG1-TAA product (63 kDa). pCH112 was designed to express a HilA derivative tagged with an extra 22 amino acids (LEQKLISEEDLNSAVDHHHHHH), which we call HilA-Myc-His (65.5 kDa). As shown in Figure 4, native HilA co-migrates with the engineered 63 kDa form of HilA. Our electrophoresis conditions can clearly distinguish the migration of HilA proteins that differ by 2.5 kDa, indicating that native HilA does not correspond to the AUG2-TAA 60.4 kDa product. Instead, our results indicate that native HilA is produced by translation of the hilA ORF from the first AUG codon. This conclusion is consistent with results showing that pCR53 expresses transcriptionally active HilA (data not shown). Unfortunately, we have not been able to construct a plasmid that expresses the AUG2-TAA translation product and so cannot examine this form in SDS-PAGE or in functional assays.
Sub-cellular fractionation of native HilA vs. HilA-Myc-His
Previous studies have shown that HilA-Myc-His, expressed from an arabinose-inducible promoter, binds to DNA containing the HilA box and activates gene expression [3]. Interestingly, HilA-Myc-His was found to pellet with bacterial membrane fractions [3]. The regulatory properties of many bacterial transcription factors are determined by their ability to associate with membranes [16–19]. We examined whether native HilA associates with the membrane by separating S. typhimurium cells into soluble and membrane fractions. We found native HilA in the soluble fraction (Fig. 5A). To investigate why HilA-Myc-His pelleted with the membrane in previous experiments, we induced the expression of HilA-Myc-His in E. coli by addition of arabinose as was done previously. We examined the subcellular fractions of cells harvested at one hour and 2 hours post-induction. Interestingly, we found that HilA-Myc-His is soluble at the one hour time period, but appears in both the soluble and membrane pellet fractions at the 2 hour time period (Fig. 5B). Previously, cell extracts in which HilA-Myc-His was only found in the membrane fraction had been prepared from cells induced with arabinose for 4–5 hours. These results indicate that native HilA and the engineered HilA-Myc-His protein are inherently soluble. The fractionation of HilA-Myc-His with membranes may be an artifact of overproduction.