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Department of Microbiology, University of Georgia, Athens, GA 30602, USA
Correspondence
Robert J. Maier
rmaier{at}uga.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Salmonella species are some of the most common pathogens associated with sporadic diarrhoea in adults living in developed countries (de Bruyn, 2000). According to the World Health Organization, the cost associated with Salmonella infections alone is estimated at three billion dollars annually in the United States.
It was recently shown that hydrogen gas (H2) is an important energy source for the survival of pathogens in vivo (Mehta et al., 2005; Olson & Maier, 2002). H2 is produced in the host by colonic bacterial fermentations and diffuses throughout the animal's body (Maier, 2003). Bacterial NiFe uptake-type hydrogenases catalyse the H2-oxidizing reaction H2
2e–+2H+, yielding protons that can contribute to energy production via generation of a proton gradient across the cytoplasmic membrane. In addition, electrons produced in the H2-splitting reaction can be passed to other membrane-bound electron carriers, eventually contributing to ATP production (Maier et al., 2004).
Physiological studies indicated that Salmonella enterica serovar Typhimurium has at least two Ni-containing hydrogenases that are responsible for hydrogen uptake (Jamieson et al., 1986; Sawers et al., 1986). The genomic sequence (TIGR; http://www.tigr.org) indicates that three NiFe membrane-bound hydrogenases exist in the S. enterica strain SGSC1412 (McClelland et al., 2001). The results from mouse infection studies suggest that these hydrogenases are individually important for virulence (Maier et al., 2004), and the removal of all three hydrogenases (creating a triple mutant) resulted in a strain that was avirulent and unable to colonize the mouse liver or spleen. However, the presence of any one hydrogenase on a low-copy-number plasmid restored some virulence characteristics to the triple mutant and each double-mutant strain with a deletion in two hydrogenases was less virulent than the parent strain (Maier et al., 2004).
The reason that S. enterica serovar Typhimurium has three very similar NiFe hydrogenases is unknown, but it may be expected that they are utilized under different environmental conditions. If so, the mechanism of gene expression of these operons is likely to be different. Park et al. (1999) studied a reporter gene fusion to one of the S. enterica serovar Typhimurium NiFe hydrogenase promoters that is homologous to hya of a similar bacterium, Escherichia coli. They determined that the hydrogenase operon was acid-inducible, repressed by nitrate at acidic pH, but not at neutral or alkaline pH, and induced by anaerobiosis. Cyclic AMP receptor protein (CRP) and tyrosine were required for expression (Park et al., 1999). Several studies that measured hydrogenase protein levels in S. enterica serovar Typhimurium under different growth conditions suggested that hydrogenase protein levels are affected by anaerobiosis and catabolite repression (Jamieson et al., 1986; Sawers et al., 1986).
The nomenclature of S. enterica serovar Typhimurium hydrogenase genes is somewhat confusing. Therefore, the Salmonella hydrogenases were renamed in this paper, according to nomenclature used for similar uptake-type hydrogenases in E. coli. The E. coli hydrogenase genes are located in the hya operon (containing the hydrogenase 1 genes) and hyb operon (containing the hydrogenase 2 genes). Amino acid sequence alignments using the BLAST program from the NCBI website have shown that the S. enterica serovar Typhimurium hydrogenase Hyb large subunit has about 94 % sequence similarity to the Hyb large subunit of E. coli. The other two S. enterica serovar Typhimurium uptake-type hydrogenases (both annotated as Hyd) are similar to the single E. coli Hya (www.tigr.org). The S. enterica serovar Typhimurium hyd STM1786–STM1787 was called hya in this study because the hydrogenase large subunit has a high percentage sequence similarity to E. coli Hya large subunit (91 %). The S. enterica serovar Typhimurium hyd genes STM1538–1539 were not renamed, since the hydrogenase large subunit has a much lower percentage sequence similarity to the E. coli Hya large subunit (66 %). In a recently published study on decreased virulence in strains lacking the hydrogenases, the hyb genes STM3147–STM3150 were called Group I hydrogenase, hyd genes STM1538–STM1539 were Group II hydrogenase, and hya genes STM1786–STM1787 were called Group III hydrogenase (Maier et al., 2004). Here, the nomenclature was modified for ease of comparison with previous work done in E. coli and Salmonella.
Studies of reporter genes fused to E. coli hya and hyb promoters have shown that their expression is regulated by anaerobiosis, nitrate, catabolite repression and pH. Several proteins are involved in this regulation, including fumarate nitrate respiration (FNR) protein and aerobic respiration control (Arc) protein. The hyb operon in E. coli is affected by catabolite repression, mediated by CRP (Richard et al., 1999). Both hyb and hya operons are regulated by the NarP and NarL proteins, which repress the operons in the presence of nitrate (Richard et al., 1999). Since E. coli and S. enterica are closely related, these studies led us to test whether the hydrogenases in S. enterica serovar Typhiumurium were regulated in a similar fashion.
Here we examine the hydrogenase gene expression level in S. enterica serovar Typhimurium using reporter gene fusions. We show that the hydrogenases are each expressed under different growth conditions, and are differentially regulated by FNR, ArcA and NarL. The genes hyb and hya had the highest expression under anaerobic conditions and were indirectly regulated by FNR, and hyb may be glucose repressed. Our study shows that hya was nitrate repressed, and the repression was mediated by NarL, but NarP was not involved. ArcA activated hyb expression. S. enterica serovar Typhimurium hya was not under the control of ArcA. Unlike what has been shown for either hya or hyb in E. coli, the hyd operon was expressed highest aerobically and was repressed by ArcA.
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METHODS |
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. Strains were maintained in Luria–Bertani (LB) broth or on agar plates with appropriate antibiotics: 50 µg ampicillin ml–1, 34 µg chloramphenicol ml–1, 25 µg kanamycin ml–1. CR-HYD medium was used where indicated (Sawers et al., 1986). The medium was supplemented with glucose (0.4 %), mannose (0.4 %), glycerol (0.4 %), sodium formate (20 mM), sodium fumarate (20 mM) or sodium nitrate (20 mM), where indicated. Cells were grown at 37 °C either anaerobically or aerobically. Anaerobic conditions were established by sparging sealed 165 ml bottles with N2 for 15 min, then anaerobic mix (10 % H2, 5 % CO2 and 85 % N2) for 20 min. ONPG was purchased from Pierce. Primers were purchased from Integrated DNA Technologies.
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) to generate 300–500 bp fragments. Primers were engineered to create a HindIII site on one side of the PCR product and a NotI site on the other end. PCR fragments and a low-copy-number vector pNN387, which contains a promoterless lacZ gene and the lacY gene, were sequentially digested with HindIII and NotI. Each PCR fragment was ligated into the digested pNN387 and transformed into E. coli TOP10 cells, yielding plasmids pZBL1, pZBL2 and pZBL3, respectively (Table 1
). To facilitate transformation of plasmids into strain JSG210, plasmids were first introduced into the restriction-deficient strain E218 before being transformed into JSG210. Fusions were confirmed by PCR using a lacZ-specific primer, Lac, and HybNot1, HydNot1, or HyaNotI to generate a 2.7 kb fragment. The resulting PCR products were sequenced, using the promoter-specific primer (Integrated Biotech Labs, University of Georgia).
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Mutant strain construction.
Deletion mutants of the arcA, fnr, narL, narP and iscR genes were constructed using the lambda Red system, as described by Datsenko & Wanner (2000). In this system, the target gene is replaced by an antibiotic-resistance cassette, which is then removed by site-specific recombination at the FRT [flippase (FLP) recognition target] sites that flank the resistance gene. Gene deletions were confirmed by PCR using primers homologous to regions flanking the deleted gene (Table 2) and by sequencing across the deletion (DNA Sequencing Core, University of Michigan). The mutant strains are listed in Table 1.
The double mutant was constructed using the fnr deletion mutant and an arcA mutant in which the kanamycin cassette replaced the arcA gene. The phage P22Htint (J. Gunn, The Ohio State University, Columbus) was used to transduce the antibiotic marker from the 
arcA : : kan strain into the fnr mutant, making an arcA/fnr double mutant. The kanamycin cassette was removed as described above. The double mutant was confirmed by PCR.
Hydrogenase deletion mutants were constructed using the lambda Red system as described previously (Maier et al., 2004). Here, deletion of the Group I genes (STM3147–3150) resulted in the hyb mutant, deletion of the Group II genes (STM1538–1539) resulted in the hyd mutant, and deletion of the Group III genes (STM1786–1787) resulted in the hya mutant (Table 1).
Enzyme assay.
Gene expression was determined using β-galactosidase assays according to Miller (1992). S. enterica serovar Typhimurium fusion strains were grown overnight anaerobically or aerobically in sealed bottles containing LB broth or CR-HYD medium. Cultures were assayed for β-galactosidase activity at 37 °C as described previously (Miller, 1992).
Growth studies.
Wild-type S. enterica serovar Typhimurium JSG210, or hyd, hyb or hya deletion mutant strains were grown in bottles containing CR-HYD medium supplemented with glucose, or glycerol and sodium fumarate, as indicated. Cells were grown aerobically or anaerobically overnight at 37 °C. Growth yield was determined by measuring OD600.
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RESULTS |
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) directly upstream of a promoterless lacZ gene. The resulting plasmids are listed in Table 1. Strain JSG210 was transformed with the plasmids.
To examine the possible roles of these hydrogenases, promoter activity was determined after cells were grown under different environmental conditions in CR-HYD medium. The hyb promoter expression was about 13-fold higher when cells were grown under anaerobic respiration conditions (glycerol plus fumarate), compared to fermentation (anaerobic glucose) conditions (Table 3), suggesting that the Hyb proteins are used during anaerobic respiration. The hyb mutant had a 16 % lower growth yield compared to wild-type when grown anaerobically with glycerol and fumarate; however, this reduced growth yield was not significant. The hyd promoter had more than seven times more activity under aerobic conditions, compared to anaerobic glucose levels. The hya promoter had the highest activity when grown under fermentative conditions (anaerobic glucose or mannose), and the hya mutant had about 25 % lower growth yield than wild-type when grown fermentatively with glucose, indicating that this hydrogenase is used during fermentation. The presence of nitrate resulted in a twofold reduction in β-galactosidase activity for the hya promoter (when grown with glucose), suggesting that hya is repressed by nitrate.
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). Such an effect was not observed for the hyd and hya genes, indicating that they are not glucose repressed.
Anoxic regulation of hydrogenase promoters
A mutant analysis approach was used to further examine the regulation of S. enterica serovar Typhimurium hydrogenase operons in response to oxygen availability. Upon sequence analysis, several putative ArcA-binding sites were found in all three hydrogenase promoters. Although no putative FNR-binding sites were found in the S. enterica serovar Typhimurium hydrogenase promoters, FNR was previously shown to regulate E. coli hydrogenase genes (Richard et al., 1999). Therefore, non-polar deletions were made in the arcA and fnr genes using the lambda Red system (Datsenko & Wanner, 2000). Mutant strains that contained the hydrogenase promoter fusion plasmids were grown overnight in LB medium and β-galactosidase activity was measured. LB broth was used in this experiment because specific growth conditions were not required, and since overall hydrogen uptake activity is higher when cells are grown in rich media (Maier et al., 2004). β-Galactosidase activity was also assayed using wild-type and mutant strains grown anaerobically in CR-HYD medium supplemented with glucose.
β-Galactosidase activity from anaerobically grown cells decreased at least threefold for the hyb promoter in the arcA mutant background as compared to the wild-type (Table 4), suggesting that hyb is upregulated by ArcA. Interestingly, an opposite effect was seen for hyd. β-Galactosidase activity tripled for the hyd fusion in the arcA mutant background. This result indicates that ArcA represses the hyd genes. ArcA did not appear to regulate hya gene expression when cells were grown in LB, as β-galactosidase activity remained unchanged in the mutant as compared to the wild-type.
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). Likewise, β-galactosidase activity decreased fourfold for the hya fusion in the fnr mutant background, indicating that both hyb and hya are regulated by FNR.
Sequence analysis did not reveal any FNR-binding sites in either promoter [using the consensus sequence TTGATNNNNATCAA (Zhang & Ebright, 1990)]. It has been shown that FNR can upregulate arcA expression in E. coli (Compan & Touati, 1994). In order to examine the possibility that the FNR effect is mediated by ArcA in Salmonella, an arcA/fnr double mutant was constructed using the lambda Red method. If FNR activates ArcA, it would be expected that β-galactosidase activity in the double mutant would be the same as in the FNR or ArcA mutant. In contrast, hyb expression decreased about 14-fold in the double mutant, 11-fold lower than in either the arcA or fnr single mutant. This result suggests that both FNR and ArcA are separately involved in hyb expression. Expression of hyd was similar between the arcA/fnr double mutant and the arcA mutant, supporting the conclusion that fnr does not regulate hyd. Likewise, hya expression was similar in the arcA/fnr double mutant compared to the fnr mutant when cells were grown in LB, indicating that arcA does not regulate hya.
A recent study reported that E. coli hya and hyb genes were repressed by IscR (iron–sulphur cluster regulator) under aerobic conditions, but not anaerobically (Giel et al., 2006). A non-polar iscR deletion mutant was made in order to determine whether S. typhimurium hyb, hyd and hya genes are regulated similarly. Aerobic hyb expression in the iscR mutant was about twofold greater than in wild-type cells, and near the anaerobically achieved levels. These results indicate that hyb is repressed by IscR aerobically; however, unlike in E. coli, S. typhimurium hya genes do not seem to be regulated by IscR.
Similar trends were seen for hyd and hya when arcA, fnr, arcA/fnr, iscR mutant and wild-type cells were grown in CR-HYD medium compared to LB, although the actual values varied. This variation is likely due to the fact that LB is a richer medium than CR-HYD. There were a few differences when cells were grown in CR-HYD, namely a significant increase in hyd expression in the fnr mutant and a significant decrease in hya expression in the arcA mutant (data not shown). This may indicate a role for FNR in the regulation of hyd expression and a role for ArcA in the regulation of hya expression under some conditions. Overall hyb expression was low for both wild-type and mutant strains when cells were grown in CR-HYD plus glucose, and only slight differences were observed between the mutant and wild-type cells. It seems that LB is a more appropriate medium than CR-HYD to assay hyb expression levels. Clearly, growth conditions play a role in the regulation of these genes, and the ArcA and FNR regulation of these operons may depend on other factors present under certain growth conditions. Gel-shift and DNA-binding studies will be useful to address whether this regulation is direct, or dependent on other factors.
Regulation of hya by nitrate
The hya promoter in the wild-type background was shown to be downregulated about twofold in the presence of nitrate (Table 3). NarX/NarL and NarQ/NarP are common two-component systems that control the nitrate response in S. enterica serovar Typhimurium (Rabin & Stewart, 1993). narL and narP deletion mutants were constructed using the lambda Red system. Wild-type and mutant strains were grown on CR-HYD with glucose and nitrate, and β-galactosidase assays were then performed. Nitrate repression was abolished in the narL strain (Table 5), indicating that NarL mediates the nitrate regulation of hya. NarP did not appear to play a role in the regulation of this gene, since β-galactosidase activity in the narP strain was not significantly different from wild-type levels (Table 5).
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. The entire binding site in the hya promoter was replaced by a random sequence of nucleotides (AGCAAAAGTC) using a two-step PCR approach. Cells containing lacZ driven by the mutant promoter were grown overnight in CR-HYD with glucose and nitrate and β-galactosidase activity was subsequently determined. We found that the strain containing the mutant NarL-binding site was not repressed by nitrate (Table 5). Expression was likely reduced in this strain because ten base pairs were replaced in the promoter, which could affect the binding of other regulatory proteins. DNA-binding and gel-shift assays will be useful to confirm whether NarL binds to the potential binding site in the hya promoter, or whether the nitrate regulation is indirect. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The expression of the hyd genes was unique in comparison to either hya or hyb in the similar bacterium, E. coli, and it appears that hyd is absent from E. coli.
The results agree with previous work in which researchers analysed the active hydrogenase enzyme levels of cells grown under different conditions (Jamieson et al., 1986; Sawers et al., 1986). Those studies were based on immunoreaction to antibodies raised against E. coli hydrogenases, and it was concluded that there were at least two H2-oxidizing hydrogenase isoenzymes. Isoenzyme 2 was necessary for anaerobic respiration-dependent hydrogen uptake, since this activity was greatly reduced in a strain lacking that isoenzyme (Sawers et al., 1986). Isoenzyme 2, so named because of its sequence similarity to E. coli hydrogenase 2, is called Hyb in this work. Therefore, it is logical that hyb would be transcribed maximally when cells are grown with glycerol and fumarate. In light of these results, it seems that S. enterica serovar Typhimurium Hyb, like hydrogenase 2 in E. coli (Menon et al., 1994), is used for hydrogen oxidation coupled to fumarate respiration. Nevertheless, based on mutant analysis (Maier et al., 2004), this hydrogenase was capable of coupling H2 oxidation to O2 as well.
An early report using immunoelectrophoretic methods to study the active hydrogenase activity of wild-type and mutant strains indicated that isoenzyme 2 was catabolite repressed by glucose (Jamieson et al., 1986). This repression was mediated by CRP, since, in a crp mutant, levels of isoenzyme 2 could not be increased by the addition of cAMP (Jamieson et al., 1986). Our study took a different approach to examine catabolite repression of the uptake-type hydrogenases. We determined the gene-expression activity of each hydrogenase grown with different sugars and found that the hyb genes may be repressed in the presence of glucose (Table 3). Our results agree qualitatively with the early S. enterica serovar Typhimurium report, showing that hyb is glucose repressed and hya is not. In addition, our work shows that hyd is not glucose repressed, which was not examined previously.
Sawers et al. (1986) reported that S. enterica serovar Typhimurium isoenzyme 1 (analogous to both Hya and Hyd) was necessary for hydrogen uptake under fermentative growth in the absence of nitrate. Our results support this conclusion, since hya had the highest activity under fermentative conditions, and it was repressed by nitrate (Table 3). These results indicate that the function of Hya may be to oxidize H2 while the cell is undergoing fermentation. Such hydrogen oxidation would be expected to generate additional energy for metabolism under this condition, and may be necessary for optimal growth, since growth yield was decreased in the hya mutant. Previous studies did not distinguish between Hya and Hyd, because it was not known at that time that S. enterica serovar Typhimurium had three uptake-type hydrogenases.
Although early work examined hydrogenase production under anaerobic conditions, we report here that hyd has the highest expression when cells are grown aerobically (Table 3). It has been shown that there is significant respiratory hydrogenase activity coupled to oxygen in S. enterica serovar Typhimurium cells grown under low oxygen conditions (O2 levels were below 0.2 % partial pressure) (Maier et al., 2004). Therefore, we suggest that Hyd is used to completely oxidize hydrogen in a low oxygen environment.
Several transcriptional regulators of each operon were identified in this study. Expression of hyb was shown to be downregulated in both the arcA and fnr mutants (Table 4). These results agree with an early study by Jamieson et al. (1986), which reports that the presence of isoenzyme 2 was fnr dependent, based on immunoreaction to E. coli anti-hydrogenase 2 antibodies. In addition, Jamieson et al. (1986) showed that protein levels of isoenzyme 1 (analogous to both Hya and Hyd) were dependent on fnr. Our work clarifies this result, since we found that hya expression was downregulated in an fnr mutant, yet hyd expression was unaffected by FNR (Table 4). Interestingly, ArcA was shown to repress hyd expression under anaerobic conditions (Table 4). This is further evidence that Hyd is used under aerobic conditions, since ArcA usually represses genes used for aerobic growth and induces anaerobic genes (Lynch & Lin, 1996). As in E. coli, IscR repressed hyb expression aerobically; however, hya expression was not regulated by IscR under the conditions tested.
Using sequence analysis, we were able to find several potential binding sites for ArcA in the region directly upstream from the first gene in the hyb operon, indicating that ArcA may directly regulate hyb transcription. DNA-binding studies with purified ArcA will be needed to confirm this result. We were unable to find a binding site for FNR in either the hyb or hya promoter, indicating that FNR regulation is indirect. It was shown that FNR indirectly regulates the hydrogenase genes in E. coli, and was speculated that this may occur through altering nickel metabolism (Richard et al., 1999). To test this hypothesis in S. enterica serovar Typhimurium, we added up to 1 mM nickel to the growth medium, but such nickel supplementation did not significantly change hyb, hyd or hya promoter activity in fnr mutants (data not shown). Therefore, it appears that the FNR effect is not due to nickel availability in S. enterica serovar Typhimurium. In order to test the hypothesis that the FNR effect is mediated by ArcA, an fnr/arcA double mutant was constructed. Our results demonstrated that both FNR and ArcA are needed for full expression of hyb (Table 4).
The hya operon was shown to be regulated by nitrate (Table 3). Nitrate repression of S. enterica serovar Typhimurium hydrogenase genes is not well understood. Previous knowledge is limited to the study by Sawers et al. (1986), which showed that hydrogen uptake was abolished when cells were grown with nitrate. However, the mechanism of nitrate repression of E. coli hydrogenases has been studied. Richard et al. (1999) demonstrated that both E. coli hya and hyb operons are repressed by nitrate, mediated by both NarL and NarP. In contrast, we found that nitrate repression of hya was only mediated by NarL, and not by NarP (Table 5). We found a potential NarL heptamer recognition sequence in the promoter region of the hya operon. The heptamer was not in the 7-2-7 arrangement, as described by Darwin et al. (1997), which provides further evidence that NarL was involved in the observed repression, but NarP was not. When this site was mutated, gene expression was no longer repressed by nitrate (Table 5).
In summary, the three uptake-type hydrogenases in S. enterica serovar Typhimurium are transcribed under different physiological conditions and they are differently regulated. Although some of these results are similar to those described for the homologous enzymes in E. coli, other aspects are unique. For example, the regulation by ArcA and the nitrate repression is different in S. enterica serovar Typhimurium. In addition, the regulation and expression of hyd is unique.
The uptake-type hydrogenases are essential for virulence in S. enterica serovar Typhimurium (Maier et al., 2004). Since the cell encounters a variety of environments during infection, it may be beneficial to be able to express a hydrogenase capable of oxidizing H2 at different stages of colonization. Perhaps Hyd is used to conserve energy by oxidizing hydrogen in the initial stages of infection, when the cell encounters a low oxygen environment. As the bacterium progresses through the digestive system into a highly competitive and putatively anaerobic environment, hyb expression may be favoured to exploit H2 as an energy source. Maier and co-workers reported H2 levels to be 43 µM in the stomach and 118–239 µM in the small intestine of mice, levels much higher than needed to saturate the hydrogenase enzymes (Maier et al., 2004; Olson & Maier, 2002). When the bacterium reaches the bowel, it probably both ferments available substrates and encounters large amounts of H2 produced by the colonic flora. Hya may be very important in this environment. Future investigation examining the transcriptional level of these hydrogenase operons in the animal model and experiments that examine the H2 affinity of each S. enterica serovar Typhimurium enzyme will be useful to test these ideas.
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ACKNOWLEDGEMENTS |
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Edited by: R. G. Sawers
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Park, K. R., Giard, J. C., Eom, J. H., Bearson, S. & Foster, J. W. (1999). Cyclic AMP receptor protein and TyrR are required for acid pH and anaerobic induction of hyaB and aniC in Salmonella typhimurium. J Bacteriol 181, 689–694.
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Received 12 April 2007;
revised 29 June 2007;
accepted 8 July 2007.
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