Environmental and intracellular regulation of Francisella tularensis ripA
© Fuller et al; licensee BioMed Central Ltd. 2009
Received: 13 April 2009
Accepted: 12 October 2009
Published: 12 October 2009
Francisella tularensis is a highly virulent, facultative intracellular pathogen and the etiologic agent of the zoonotic disease Tularemia. RipA is a cytoplasmic membrane protein that is conserved among Francisella species and is required for intracellular growth. F. tularensis ripA deletion mutants escape the phagosome of infected cells, but unlike wild type organisms fail to replicate in the host cell cytoplasm.
Further analysis of ripA with respect to environmental effects on the growth of mutant strains and expression levels revealed that RipA is required for optimal growth at pH 7.5 but not pH 6.5. Using a combination of RT-PCR, ripA-lacZ transcriptional and translational fusions, and a RipA-tetracysteine tag fusion protein we found that both ripA transcription and RipA protein levels were elevated in organisms grown at pH 7.5 as compared to organisms grown at pH 5.5. A number of genes, including iglA, that are required for intracellular growth are regulated by the transcriptional regulators MglA and SspA, and are induced upon infection of host cells. We quantified ripA and iglA expression at different stages of intracellular growth and found that the expression of each increased between 1 and 6 hours post infection. Given the similar intracellular expression patterns of ripA and iglA and that MglA and SspA are positive regulators of iglA we tested the impact of mglA and sspA deletions on ripA and iglA expression. In the deletion mutant strains iglA expression was reduced dramatically as expected, however ripA expression was increased over 2-fold.
Expression of ripA is required for growth at neutral pH, is pH sensitive, and is responsive to the intracellular environment. The intracellular expression pattern of ripA coincided with iglA, which is positively regulated by MglA and SspA. However, in contrast to their positive impact on iglA expression, MglA and SspA negatively impacted ripA expression in vitro.
Francisella tularensis is a highly virulent Gram negative bacterial pathogen and the etiologic agent of the zoonotic disease tularemia. The bacteria are spread via multiple transmission routes including arthropod bites , physical contact with infected animal tissues , contaminated water [3, 4], and inhalation of aerosolized organisms . Inhalation of as few as 10 colony forming units (CFU) are sufficient to initiate lung colonization [6, 7] and the subsequent development of pulmonary tularemia, which is the most lethal form of the disease exhibiting mortality rates as high as 60% .
F. tularensis is a facultative intracellular pathogen that invades, survives and replicates within numerous cell types including, but not limited to, macrophages [9, 10], dendritic cells , and alveolar epithelial cells . Intracellular growth is intricately associated with F. tularensis virulence and pathogenesis, and the intracellular lifestyle of F. tularensis is an active area of investigation. Following uptake or invasion of a host cell wild type F. tularensis cells escape the phagosome and replicate within the cytoplasm [13–15] of infected cells. The phagosome escape mechanism employed by F. tularensis remains essentially unknown, but this property is clearly necessary for F. tularensis intracellular growth since mutants that fail to reach the cytoplasm are essentially unable to replicate within host cells [16, 17].
Following phagosome escape F. tularensis must adapt to the cytoplasmic environment. Purine auxotrophs , acid phosphatase , clpB protease , and ripA mutants  reach the cytoplasm but are defective for intracellular growth. RipA is a cytoplasmic membrane protein of unknown function that is conserved among Francisella species .
Notably, the majority of attenuating mutations described to date impart intracellular growth defects on the mutant strains. We recently identified a locus, ripA, that encoded a cytoplasmic membrane protein that was conserved among Francisella species. Mutant strains lacking ripA entered host cells and escaped the phagosome, but were defective for intracellular growth . The deletion mutants had no apparent affect on F. tularensis growth with respect to doubling time or final density when propagated in Chamberlains chemically defined media or complex nutrient rich BHI. Thus, expression of ripA appeared to be required for adaptation and growth in the cytoplasmic environment of a host cell.
The expression of a number of Francisella virulence factors required for phagosomal escape and intracellular replication are induced in the intracellular environment by a process involving the positive transcriptional regulators MglA and SspA [16, 22–24]. Data on whether MglA regulates ripA expression is contradictory. Microarray analysis of MglA regulated loci indicated that ripA expression was unaffected by MglA, , whereas results from a proteomics study suggested that RipA was repressed by MglA .
Given the ripA deletion mutant phenotype with respect to intracellular growth, that MglA and SspA regulate numerous genes required for intracellular growth and that there is a discrepancy between the microarray and proteomic results with respect to MglA affects on ripA expression, we applied multiple approaches to investigate environmental requirements for, and influences on, F. tularensis ripA expression.
Characterization of the ripA locus and transcriptional unit
Individual primer sets were designed to amplify coding regions from each of the three genes, and another set was designed to amplify any RNA transcripts that bridged adjacent genes (Fig. 1a). Twenty ng of synthesized first strand cDNA was subjected to 25 or 30 cycles of amplification to synthesize intragenic and potential gene bridging (intergenic) products, respectively. There was no detectible product following amplification with primers bridging FTL_1912 and FTL_1913 (Fig. 1b), suggesting that a predicted Rho independent terminator located between the two genes was functional. A faint amplification product was present in reactions using FTL_1913 - ripA bridging primers (Fig. 1b). However, the band intensity was significantly lower than that of the ripA amplicon and was detectable only after the additional cycles of amplification. This result suggests that the FTL_1913 transcript terminates, albeit less efficiently than that of FTL_1912, and that ripA expression was initiated independently from the FTL_1912 promoter.
Bacterial strains and plasmids.
Strains or Plasmid
Source or Reference
Francisella tularensis LVS
F. tularensis live vaccine strain
CDC, Atlanta, GA
Tn5 ripA transposon mutant
ripA:: pBSK Φ(ripA'-lacZ)2
iglA:: pBSK Φ(iglA'-lacZ)
Inframe deletion of mglA
ΔmglA ripA:: pBSK Φ(ripA'-lacZ)2
ΔmglA iglA:: pBSK Φ(iglA'-lacZ)
Inframe deletion of sspA
ΔsspA ripA:: pBSK Φ(ripA'-lacZ)2
ΔsspA iglA:: pBSK Φ(iglA'-lacZ)
Inframe gene deletions
ΔmglA ΔsspA ripA:: pBSK Φ(ripA'-lacZ)2
ΔmglA ΔsspA iglA:: pBSK Φ(iglA'-lacZ)
pBSK bla lacZ
pBluescript cloning vector
pBSK lacZ aphA1 bla
Transcriptional lacZ fusion
pBSK lacZ cat bla
Translational lacZ fusion
pBSK Φ(ripA'-lacZ)2 aphA1 bla
Francisella suicide vector
pBSK Φ(ripA'-lacZ)1 cat
Francisella suicide vector
pBSK Φ(iglA'-lacZ)2 aphA1
Francisella suicide vector
Francisella sacB suicide vector
mglA allelic exchange vector
sspA allelic exchange vector
Φ(ripA'-TC) suicide vector
Francisella shuttle vector
mglA+ with native promoter
sspA+ with native promoter
Francisella shuttle vector
pKK MCS Φ(ripA'-lacZ)1
pKK MCS Φ(ripA'-lacZ)1a
pKK MCS Φ(ripA'-lacZ)1b
pKK MCS Φ(ripA'-lacZ)1c
pKK MCS Φ(ripA'-lacZ)1d
ripA core promoter
pKK MCS Φ(ripA'-lacZ)2
pKK MCS Φ(ripA'-TC)
ripA-CT tetracysteine tag fusion
Quantifying ripA expression with transcriptional and translational lacZ fusions
We examined the effects of specific mutations in the predicted ripA promoter, ribosome binding site, and translation frame on the expression of β-galactosidase. Mutations in the predicted -10 sequence, RBS, and the introduction of a frameshift mutation (Fig. 2a) in the translational fusion construct each resulted in decreased β-galactosidase activity as compared to the wild type reporter (Fig. 2c).
The β-galactosidase activity expressed by the chromosomal reporters was less than 25% of that produced by the plasmid reporters (Fig. 2b). The ripA'-lacZ 1 translational fusion produced significantly less activity than the ripA'-lacZ 2 transcriptional fusion in both the chromosomal and plasmid version of the reporter (Fig. 2b). These differences might reflect post transcriptional regulation of expression or simply a difference in the efficiency of translation initiation between the two constructs.
Quantification of RipA protein
Whole cell lysates prepared from mid exponential phase bacteria growing in Chamberlains defined media were suspended in FlAsH™ loading buffer containing biarsenical fluorescein and subjected to SDS-PAGE. The RipA-TC fusion protein was detected and quantified by relative mean fluorescence with wild type F. tularensis LVS lacking any TC fusion protein serving as a control to identify background and non-specific fluorescence. To determine the detection limits of the TC tag fusion protein assay, whole cell lysates (6000 ng to 60 ng total protein) of LVS expressing chromosomal (Fig. 4a) or plasmid ripA'-TC fusion alleles were incubated with FlAsH™ reagent, separated via SDS-PAGE and subjected to in - gel fluorescence measurement. There were 3 nonspecific biarsenical fluorescein binding proteins between 22 kDa and 30 kDa in size in wild type F. tularensis LVS lysates, which were easily distinguishable from RipA-TC which migrated at approximately 18 kDa (Fig. 4c). RipA-TC expressed from plasmid was detectable in the 60 ng whole cell lysate samples whereas chromosomally expressed was detected in 600 ng samples (Fig. 4c). The concentration of RipA-TC (plasmid) was approximately 6.5 fold greater than RipA-TC (chromosome). Thus, the use of the RipA-TC fusion in conjunction with biarsenical labeling provided a sensitive and reproducible method to detect and quantify RipA in Francisella.
Expression of ripA is affected by pH
RT-PCR and FlAsH™ labeling of RipA-TC were used as complementary assays for comparison to the lacZ fusion results. The ripA transcript levels were evaluated by RT-PCR in replicates of four independent cultures and normalized to tul4 . 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 achieved by 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 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 is biologically relevant.
F. tularensis LVS ripA expression during intracellular growth
We predicted that the conditions under which the cultures were prepared might affect the ripA and iglA expression levels prior and subsequent to internalization by host cells. Therefore, the activities of ripA'-lacZ 2 and iglA'-lacZ transcriptional fusions were measured from cultures grown in BHI and CDM to assess the impact of complex nutrient rich and chemically defined minimal media, respectively, on their expression. The mean activity of each reporter was ca. 1.6 fold higher in CDM relative to BHI (P < 0.01) (Fig 7ab). Given the effect of growth media on ripA and iglA we measured and compared the expression of these genes in cells infected with the reporter strains propagated in each of these media.
To initiate the intracellular expression analyses host cell entry was synchronized by centrifugation of reporter strains onto chilled J774A.1 monolayers as described . β-galactosidase activity was measured in the inoculums, and at 1, 6, and 24 hours post inoculation using a modified β-galactosidase assay similar in concept to the Miller assay but based on the rate and amount of CPRG conversion per CFU.
The mean β-galactosidase activity (± standard deviation) of F. tularensis LVS ripA'-lacZ2 at 0 (inoculum), 1, 6, and 24 hours post infection when the inoculum was prepared from BHI cultures was 199.7 (± 13.32), 155.9 (± 12.96), 193.5 (± 23.99), and 80.6 (± 17.83), respectively (Fig. 7a). The activity-galactosidase level remained similar to that of the inoculum at 1 hour post infection, increased slightly at 6 hours then dropped at 24 hours to a level that was significantly less than for all other time points (P < 0.05). When prepared in CDM the β-galactosidase levels started at a much higher value than that of the BHI-grown samples, and steadily decreased until the lowest measurement at 24 hours post inoculation (Fig. 7b).
Expression of iglA prepared in BHI was 135.0 (± 9.59), 97.8 (± 9.59), 199.4(± 26.24), and 112.0 (± 24.21) for the inoculum, 1, 6, and 24 hours post inoculation, respectively (Fig. 7a). The most significant change was a two fold increase at 6 hours post inoculation relative to 1 and 24 hours post inoculation (P < 0.01). By 24 hours post inoculation the relative activity returned to levels similar to that of the inoculum and at 1 hour post inoculation. The 6 hour post inoculation spike of iglA expression did not occur when the bacteria were prepared in CDM (Fig. 7b). As with the ripA fusion strain, β-galactosidase levels were significantly higher in the inoculums and throughout the course of infection. Both fusion strains invaded and replicated in the J774A.1 cells (Fig. 7c) demonstrating that the reporter integrations did not impact intracellular replication. Also, even though growth media significantly impacted ripA and iglA expression levels throughout the experiment, it had no discernable effect on host cell invasion or replication.
The effects of mglA and sspA deletions on ripA expression
MglA and SspA are transcriptional regulators that associate with DNA and RNA-polymerase and modulate the expression of a number of stress response and virulence associated genes, including iglA, in F. tularensis [22–25]. In a recent study comparing protein expression profiles of wild type and mglA mutant strains both IglA and RipA protein levels were affected in the mglA mutant . We investigated further the relationship between these regulators and RipA expression using the ripA'-lacZ2 and iglA'-lacZ transcriptional fusions in ΔmglA and ΔsspA mutant strains (Table 1).
As expected the mglA and sspA deletions had the opposite effect on iglA expression. The mean expression (± standard deviation) of F. tularensis LVS iglA'-lacZ was substantially decreased in both the ΔmglA (80 ± 2.2) and ΔsspA (67 ± 0.9) strains versus wild type (2757 ± 98) (Fig. 8b). The differences of iglA expression in the mutant backgrounds were all significantly different from wild type (P < 0.01), and near wild type levels of expression were restored by complementation with mglA and sspA in trans (Fig. 8b). Together, these results confirm that mglA and sspA expression positively influence iglA expression, and conversely demonstrate that these two regulators negatively influence ripA expression.
As a facultative intracellular pathogen, F. tularensis is able to survive and replicate within several different types of eukaryotic cells as well as in a number of extracellular environments [9, 11, 12, 29–32]. Other facultative intracellular pathogens such as Salmonella typhimurium , Legionella pneumophila , and Listeria moncytogenes [35, 36] are similarly capable of adapting to multiple environments. These organisms exhibit differential gene expression in response to entering or exiting host cells, and even as they transition between intra-vacuolar and cytoplasmic niches. Mapping the gene expression profiles that accompany different stages of infection have helped to identify environmental cues that impact gene expression and virulence.
Studies on intracellular gene expression by Francisella species have revealed a number of genes including iglC , iglA  and mglA , that are induced upon entry and growth in macrophages. IglC protein concentrations increased between 6 hours and 24 hours post host cell invasion . Similarly IglA protein concentrations increased between 8 hours and 12 hours post invasion as measured by Western blot . In the current study we found that iglA expression was increased during intracellular growth, but only for a limited period of time. This increase in expression did not occur immediately after host cell invasion, but rather coincided with the time frame associated with the early stage of replication following phagosome escape.
We found that the laboratory growth media used to propagate the bacteria affected both ripA and iglA expression levels. Reporter activity of ripA'-lacZ and iglA-lacZ transcriptional fusions were each significantly higher in inoculums prepared in CDM vs. those prepared in BHI. As a consequence, the results of intracellular expression assays were dependent on the type of media in which the organisms were grown prior to infection. Since the initial expression levels of ripA and iglA were significantly higher in CDM grown organisms, the relative in vivo expression levels of these genes actually decreased throughout the course of infection. Modest increases in iglA and ripA expression during intracellular growth were observed only when organisms were propagated in BHI prior to infection. These observations are in line with that of Hazlett et. al. who found that Francisella virulence genes are variably expressed in different types of media, some of which more closely replicate intracellular expression profiles than others .
When infected with BHI-grown organisms, F. tularensis ripA and iglA gene expression changes coincided with the transitions from vacuolar, to early cytoplasmic, and then late cytoplasmic stages of infection. The expression of ripA was repressed during the early stage of infection when the bacteria are reportedly associated with a phagosome [13–15]. Expression of both ripA and iglA increased during the early phase of cytoplasmic growth then decreased during the latter stages of infection. The ripA expression levels associated with these sites and stages of intracellular growth corresponded to our observed effects of pH on ripA expression in CDM and the reported pH of the relevant intracellular environment. A number of studies have shown that the early Francisella - containing phagosome is acidified prior to bacterial escape [40, 41]. Interestingly, we found that acidic pH repressed ripA. Additionaly, ripA expression was dispensable for growth at acidic pH in vitro, and was likewise dispensable for survival and escape from the phagosome. The pH of the cytosol of a healthy macrophage is reportedly ca. 7.4. Neutral to mildly basic pH resulted in increased ripA expression in vitro. The ripA deletion mutant was defective for growth both at neutral pH in vitro, and within the cytoplasm of host cells. Finally, the pH of the cytosol during late stages of Francisella infection has not been measured, however during apoptosis the pH reportedly drops to 5.8 . Since Francisella has been demonstrated to induce apoptosis in macrophages  this might explain, at least in part, the drop in ripA expression during the late stage of infection. We are currently investigating the scope and mechanisms of pH associated gene regulation in Francisella and its role in host cell adaptation and virulence.
Given that growth media and the stage of infection had similar affects on iglA and ripA expression we thought it reasonable to determine if the two genes were subject to the same or overlapping regulatory mechanism(s). Earlier microarray and proteomic [22–25] analyses revealed that the expression of iglA and IglA, respectively, as well as a number of other genes and proteins, are regulated by two related transcriptional regulators, MglA and SspA [23, 44].
Transcriptional profiling studies of mglA and sspA mutant strains by microarray  gave no indication that either of these regulators affected ripA expression. However, in complementary proteomic studies, RipA (FTN_0157) was present in 2 - fold higher amounts in a F. novicida mglA mutant strain as compared to wild type . This result suggested that MglA has a direct or indirect repressive effect on RipA expression. Our analysis using ripA'-lacZ fusion reporter strains revealed that ripA expression was increased in both ΔmglA and ΔsspA mutants, and therefore correlated with the proteomics analysis of MglA mediated gene regulation. Thus, MglA and SspA positively affect iglA, but have a negative effect on ripA expression in vitro. If the intracellular regulation of iglA does indeed occur through the activities of MglA and SspA it is likely that in the early stages of F. tularensis intracellular replication, the increase in ripA expression is mediated by a mechanism that is independent of, or ancillary to, the MglA/SspA regulon.
Studies focusing on intracellular gene expression are an important aspect of discerning Francisella pathogenesis mechanisms. We found that ripA, which encodes a cytoplasmic membrane protein that is required for replication within the host cell cytoplasm, is transcribed independently of neighbouring genes. Further, ripA is differentially expressed in response to pH and during the course intracellular infection. The intracellular expression pattern of ripA mirrored that of iglA and other Francisella virulence - associated genes that are regulated by MglA and SspA. However, in the transcriptional regulator deletion mutants, there were opposing effects on iglA and ripA expression in vitro. Since ripA is essentially repressed by MglA and SspA, the increase in ripA expression that corresponds with increased MglA/SspA activity in vivo suggests that this gene is responsive to an as-of-yet unknown complementary regulatory pathway in Francisella.
Bacterial strains and cell culture
F. tularensis Live Vaccine Strain (LVS) (Table 1) was propagated on chocolate agar (25 g BHI l-1, 10 μg hemoglobin ml-1, 15 g agarose l-1) supplemented with 1% IsoVitaleX (Becton-Dickson), BHI broth (37 g BHI l-1, 1% IsoVitalex), or Chamberlains defined media . All bacterial strains cultured on chocolate agar were grown at 37°C. Broth cultures were incubated in a shaking water bath at 37°C. J774A.1 (ATCC TIB-67) reticulum cell sarcoma mouse macrophage-like cells were cultured in DMEM plus 4 mM L-glutamine, 4500 mg glucose l-1, 1 mM sodium pyruvate, 1500 mg sodium bicarbonate l-1, and 10% FBS at 37°C and 5% CO2 atmosphere.
Reverse transcriptase PCR
Total RNA was isolated from mid exponential phase cultures using a mirVana RNA isolation kit (Ambion) and procedures. DNA was removed by incubation with RQ1 DNase (Promega) for 1 hour at 37°C. First strand cDNA was generated using SuperScript III Reverse transcriptase (Invitrogen) and random primers. cDNA was quantified using a ND-1000 spectrophotometer (Nanodrop). PCR analysis of ripA and tul4 expression was accomplished using 20 ng cDNA per 50 μl PCR reaction. As a control for DNA contamination, a Reverse transcriptase reaction was conducted without the Reverse transcriptase enzyme. Ten percent of each reaction was analyzed by agarose gel electrophoresis, ethidium bromide staining, and densitometry using BioRad Quantity One software. Trace intensity (Int mm) of ripA was normalized to the mean tul4 expression . Mean normalized expression and standard deviation were calculated based on RT-PCR of four samples of RNA derived from independent cultures. Significance was determined using an unpaired two tailed t test with unequal variance.
Agarose formaldehyde electrophoresis and Northern analysis
Total RNA was harvested from mid exponential phase F. tularensis LVS grown in Chamberlains defined media using RNAeasy columns (Qiagen), concentrated by ethanol/sodium acetate precipitation and quantified with a ND-1000 spectrophotometer (Nanodrop). RNA was separated using agarose-formaldehyde (2% agarose, 2.2 M Formaldehyde) electrophoresis followed by capillary transfer to nitrocellulose as described . Additional lanes of the membrane containing duplicate samples were stained with methylene blue to assess rRNA bands for degradation and equality of loading. Digoxigenin labeled RNA probes were generated using a Northern Starter Kit (Roche). Probe generation, hybridization, washing, and detection were performed using the manufacturer's (Roche) protocols.
Reporter fusion construction and mutagenesis
Specific F. tularensis LVS DNA fragments were produced by PCR amplification of genomic DNA using Pfu turbo DNA polymerase (Stratagene). Three DNA fragments were PCR amplified, cloned, and the DNA sequenced for conformity to the published F. tularensis LVS DNA sequence. (1) 1300 bp amplicon (primers TTTGGTGTGTTTATCGGTCTTGAAGGCGGTATTGATG and CACGATATCCATTTTATTCCTTTCTAATCCATTTATCC) for the generation of the in-frame ripA'-lacZ1 translational fusion of the ripA start codon to lacZ . (2) 1000 bp amplicon (primers atagcggccgccaggtaaagtgactaaagtacaagataatggtgc and gcgttaattaacctttctaatccatttatccaaaagaatttacac) for the generation of the ripA'-lacZ2 transcriptional fusion. (3) 740 bp amplicon (primers agttGCGGCCGCtattccaaccagtgcatttttcactttagtg and TTCCttaattaaCTTATTGTCCTTTTTTTCACAACACCTTATAAGC) for the generation of the iglA'-lacZ transcriptional fusion. The lacZ reporter vectors pALH109 and pALH122 were used as the source of the translational and gene transcriptional lacZ fusion constructs . The translational gene fusion (pALH109) was ligated with a pBSK vector containing the cat gene driven by the F. tularensis groEL promoter to construct pBSK lacZ cat. The transcriptional gene fusion (pALH122) was ligated with a pBSK vector containing the aphA1 allele driven by the F. tularensis groEL promoter to construct pBSK lacZ aphA1. A KpnI/EcoRV fragment containing the ripA promoter was ligated to a SmaI/KpnI fragment of pBSK lacZ cat to form pBSK ripA'-lacZ1. NotI/PacI fragments of the cloned promoters were ligated to a NotI/PacI fragment of pBSK lacZ aphA1 to form pBSK ripA'-lacZ2 and pBSK iglA'-lacZ. KpnI/NotI fragments from pBSK reporters were ligated to KpnI/NotI fragments of pKK MCS to construct pKK ripA'-lacZ1 and pKK ripA'-lacZ2. All plasmids used in these studies are listed in Table 1.
Francisella chromosomal and multicopy reporter strains were generated by transformation of pBSK suicide vectors or pKK shuttle vectors containing the fusion constructs into the F. tularensis LVS strains as described . Wild type and reporter alleles of each gene are present in the reporter strains. Site directed mutagenesis of pKK ripA'-lacZ1 was performed using the Stratagene QuickChange XL kit and the manufacturers protocols. All ripA promoter mutations were confirmed by DNA sequence analysis.
Measuring β-galactosidase activity expressed by intracellular organisms
Significance was calculated using an unpaired two tailed t test assuming unequal variance. P values of less than 0.05 were considered significant.
A ripA'-TC fusion was made by Splice Overlap Extension (SOE) PCR  using primers designed to insert the tetracysteine (TC) tag sequence with a glycine linker between the last ripA codon and the stop codon (Fig. 4b). Deletion constructs made by SOE PCR retained the start and stop codons of mglA (fusion of 1st four and last two codons) and sspA (fusion of 1st four and last 4 codons) in frame with 0.8 kb of flanking sequence. The constructs were cloned into pMP590 (Table 1) and sequenced to confirm the integrity of the flanking DNA sequence. Allelic exchange was achieved by transformation, selection for plasmid co-integrates, counter selection on sucrose containing media and confirmed via PCR analysis for replacement of the wild type with the deletion mutant allele as described . Each mutation was confirmed by DNA sequence analysis.
Extracellular β-galactosidase assay
Statistical analysis was performed to determine the mean Miller units and standard deviation from three independent cultures and significance calculated using an unpaired two tailed t test with unequal variance.
SDS-PAGE and FlAsH™ labelling
Proteins were separated by SDS-PAGE. Total protein loaded in each sample was equivalent as determined by a BCA assay (Pierce). FlAsH™ labeling was accomplished using the manufactor's protocols (Invitrogen). In gel fluorescence of the arsenical fluoriscein and total protein stain was conducted on a Typhoon 9200 laser scanner (488 nm laser/520 nm BP 40 filter and 633 nm laser/670 nm BP 30 filter). Densitometry was conducted using ImageQuant XL software and sample comparisons made using the same gel and scan. Mean intensity and standard deviation of four samples from independent cultures was calculated and significance determined using an unpaired two tailed t test with unequal variance.
We thank Allen Honeyman for sending us the lacZ containing plasmids pALH109 and pALH122. This work was supported by a Southeast Regional Center of Excellence in Biodefense and Emerging Infections grant (NIH/NIAID U54-AI057157) and by the National Institutes of Health (AI069339).
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