Differential regulation of amidase- and formamidase-mediated ammonia production by the Helicobacter pylori Fur repressor*

The production of high levels of ammonia allows the human gastric pathogen Helicobacter pylori to survive the acidic conditions in the human stomach. H. pylori produces ammonia through urease-mediated degradation of urea, but it is also able to convert a range of amide substrates into ammonia via its AmiE amidase and AmiF formamidase enzymes. Here data are provided that demonstrate that the iron-responsive regulatory protein Fur directly and indirectly regulates the activity of the two H. pylori amidases. In contrast to other amidase-positive bacteria, amidase and formamidase enzyme activities were not induced by medium supplementation with their respective substrates, acrylamide and formamide. AmiE protein expression and amidase enzyme activity were iron-repressed in H. pylori 26695 but constitutive in the isogenic fur mutant. This regulation was mediated at the transcriptional level via the binding of Fur to the amiE promoter region. In contrast, formamidase enzyme activity was not iron-repressed but was significantly higher in the fur mutant. This effect was not mediated at the transcriptional level, and Fur did not bind to the amiF promoter region. These roles of Fur in regulation of the H. pylori amidases suggest that the H. pylori Fur regulator may have acquired extra functions to compensate for the absence of other regulatory systems.

INTRODUCTION 1 transferred to nylon membranes (Roche, Basel, Switzerland) using standard protocols (25,27). 1 Following transfer, RNA was covalently bound to the membrane by cross-linking with 0.120 2 J/cm 2 UV light of 254 nm wavelength. RNA was visualized by methylene blue staining (25), 3 and RNA samples were normalized based on 16S and 23S rRNA band intensities. Internal 4 fragments of the amiE and amiF genes were PCR amplified with primers listed in Table 1. 5 The resulting PCR fragments contained a T7 promoter sequence on the non-coding strand, 6 and were used for the production of antisense RNA probes labeled with DIG by in vitro 7 transcription using T7 RNA polymerase (Roche). Northern hybridization and stringency 8 washes were performed at 68°C, and bound probe was visualized with the DIG-Detection Kit 9 (Roche) and the chemiluminescent substrate CPD-Star (Amersham Pharmacia) (25). 10

Recombinant DNA techniques 12
Restriction enzymes and modifying enzymes were purchased from New England Biolabs 13 (Beverly, MA) and Promega (Madison, WI), and standard protocols were used for manipulation 14 of DNA and transformation of E. coli (27) and H. pylori (26). Plasmid DNA was prepared using 15 Qiaprep spin columns (Qiagen, Valencia, CA). PCR was carried out using Taq polymerase 16 (Promega). 17 18

Gel retardation assay 19
Recombinant H. pylori Fur protein was purified from E. coli with the pASK-IBA Streptag 20 system (IBA, Göttingen, Germany) as described previously (29). DIG-labelled amiE and 21 amiF promoter fragments were amplified with primer combinations 22 AmiE-PrF/AmiE-PrR-DIG and AmiF-PrF/AmiF-PrR-DIG, respectively ( In the amidase-positive bacteria Pseudomonas aeruginosa and Mycobacterium 4 smegmatis, amidase activity is controlled by substrate availability via the AmiR-AmiC and 5 AmiA proteins, respectively (30,31). These proteins mediate induction of amidase expression 6 upon supplementation of growth media with the amide substrate (30,31). While orthologs of 7 the corresponding amidase regulatory proteins are absent in H. pylori, inspection of the H. 8 pylori amiE and amiF promoters indicated the presence of sequences resembling Furboxes, 9 suggesting iron-responsive regulation of these genes (22,32). 10 In order to determine whether amidase and formamidase activity was substrate-inducible 11 or iron-regulated, we determined the effect of substrate supplementation and varying iron-12 availability on amidase and formamidase activity of H. pylori strain 26695. The highest 13 concentrations of amidase substrates that still allowed growth of H. pylori 26695 were 5 mM 14 acrylamide and 100 mM formamide (data not shown). Unlike other bacterial amidases, 15 supplementation with these concentrations of amide substrates did not result in induction of 16 amidase or formamidase enzyme activity (Fig. 1). However, changing iron-availability had a 17 pronounced effect on amidase activity, which was high in iron-restricted conditions, but was 18 almost absent in iron-replete conditions (Fig. 1A). In contrast, formamidase activity was not 19 changed in iron-restricted conditions when compared to iron-replete conditions (Fig. 1B). 20 Thus we conclude that amidase and formamidase activity in H. pylori 26695 is not substrate-21 inducible, but that amidase activity is regulated by iron-availability while formamidase 22 activity seems constitutive. 23 24 Amidase expression and activity, and formamidase activity is regulated by Fur van Vliet et al.

Fur-regulation of Helicobacter pylori amidases
The AmiE (HP0294) protein was previously identified as a protein of approx. 45 kDa 1 with a pI of 6.4 (33). A protein of similar molecular mass and pI was identified when 2 comparing 2D-protein profiles for identification of Fur-and iron-regulated proteins of H. 3 pylori 26695 (Fig. 2). Wild-type cells expressed this protein when grown in iron-restricted 4 conditions, but not in iron-replete conditions. This iron-repression was absent in the fur 5 mutant strain (Fig. 2), suggesting that iron regulation was mediated by Fur. Subsequent 6 identification of the protein by mass spectometry confirmed that this iron-and Fur-repressed 7 protein was indeed AmiE (13,14). Since the AmiF protein has not been identified on 2D-gels 8 yet (33), we were unable to compare AmiF protein expression levels. 9 To assess whether the effect of Fur and iron on AmiE at the protein expression level was 10 also present at the enzyme activity level, we determined amidase activity in lysates of H. 11 pylori 26695 and its isogenic fur mutant, grown in iron-restricted and iron-replete conditions 12 ( Fig. 3). As control we also determined formamidase activity in both strains and medium 13 conditions. Amidase activity displayed identical regulation as observed at the protein 14 expression level: in wild-type cells amidase activity was high at iron-restricted conditions and 15 absent in iron-replete conditions (P < 0.01), whereas in the fur mutant activity was always 16 high, independent of iron-availability (P = 0.56; Fig. 3A). Surprisingly, formamidase activity 17 was also affected by the fur mutation: formamidase activity did not differ between cells 18 grown in iron-restricted and iron-replete conditions, but differed significantly between the 19 wild-type and fur mutant cells (P < 0.02; Fig. 3B). In wild-type cells formamidase activity 20 was low but present, whereas formamidase activity was increased almost threefold in the fur 21 mutant (Fig. 3B). These results were reproduced with a second, independently constructed H. 22 pylori 26695 fur mutant (data not shown), indicating that the increase in formamidase activity 23 is not caused by a secondary mutation. 24

Fur mediates regulation of amiE, but not amiF at the transcriptional level 1
Regulation via iron and Fur is usually mediated at the transcriptional level (32). The 2 observed iron-and Fur-responsive regulation of AmiE expression was indeed reflected at the 3 mRNA level, as demonstrated by Northern hybridization (Fig. 4). There was no amiE mRNA 4 detected in the wild-type strain under iron-replete conditions, but transcription of a 1 kb 5 mRNA was clearly apparent in iron-restricted conditions. In contrast, in the fur mutant amiE 6 mRNA was present irrespective of the iron-availability of the medium (Fig. 4). The effect of 7 the fur mutation on formamidase activity is however not mediated at the transcriptional level, 8 since the small changes in the levels of amiF mRNA observed on Northern hybridizations 9 ( Fig. 4) did not correlate with the changes in enzyme activity observed (Fig. 4). 10 Specific binding of Fur to the amiE promoter, but not to the amiF promoter 12 The Fur protein normally functions by metal-dependent binding to a binding sequence 13 (Furbox) located in the promoter region of the regulated gene (32). Analysis of the sequence 14 directly upstream of the amiE and amiF genes had already indicated the presence of putative 15 Furboxes (Fig. 5A). To confirm that amiE and amiF transcription were indeed differentially 16 regulated by Fur, we performed gel retardation assays using recombinant H. pylori Fur (29)  17 and DIG-labelled amiE and amiF promoter regions. Addition of recombinant H. pylori Fur 18 with the metal cofactor Mn 2+ to the amiE promoter region shifted mobility of the amiE 19 promoter, consistent with binding of Fur to this promoter (Fig. 5B). Gel retardation was 20 dependent on the presence of the Mn 2+ metal cofactor (not shown). To check sequence 21 specificity, we also used an internal fragment of the amiE gene, whose mobility was not 22 affected by Fur (not shown). Finally, as predicted from the Northern hybridization 23 experiments, but despite the presence of Furbox-like sequence, mobility of the amiF promoter 24 was not affected by Fur (Fig. 5B). animals, and in this respect they represent unique pathogens (1). Colonization is dependent on 4 acid-resistance, and while this process is multifactorial, the production of high levels of ammonia 5 is essential to allow initial infection as well as subsequent colonization. Acid resistance of H. 6 pylori has long been considered to be solely based on unregulated production of large amounts of 7 urease, but recent studies have shown that acid resistance of H. pylori is based on multifactorial, 8 interactive and probably well regulated processes (3,25,(34)(35)(36). In these processes metal-9 responsive regulatory proteins play an important role, with the NikR protein regulating urease 10 expression (25,34) and the Fur protein regulating iron homeostasis, acid resistance (20-24) and 11 amidase-and formamidase-mediated ammonia production (this study) (Fig. 6). 12 Under physiological conditions, the optimal pH for H. pylori growth lies between 4 and 6. 13 health (40). The acrylamide can be produced after Strecker degradation of asparagine or 5 methionine in the presence of dicarbonyl compounds via the Maillard reaction (41,42). Of 6 special interest in the gastric environment may be the route via methionine since this reaction 7 has a requirement for ammonia, as produced by H. pylori (41,42). Furthermore, although it is 8 possible that the amidases function in protection against toxic amides, our preliminary data 9 indicate that production of the AmiE amidase does not increase protection against toxic 10 concentrations of acrylamide in a disc assay (data not shown). 11 amide substrates. Both the urease-and amidase enzymatic reactions lead to the production of 1 ammonia, but while the urease reaction results in alkalinization of the environment (39), the 2 amidase reaction is pH-neutral (13,14). Thus amidase-generated ammonia is probably not 3 sufficient for acid-resistance of H. pylori (44), but may still be used to form urea through the 4 previously suggested urea cycle of H. pylori (12,45), and thus amidase activity may be 5 important when urea availability is low. Alternatively, since ammonia also plays an important 6 role in nitrogen metabolism, the pH-neutral production of ammonia by both amidases may 7 allow production of sufficient intracellular concentrations of ammonia without alkalinization 8 of the cellular environment. 9 Finally, a coupling between iron-availability and substrate-availability is supported by 10 studies on the function and secretion of the H. pylori vacuolating cytotoxin VacA (46,47). 11 Firstly, the VacA protein has been suggested to function as a urea permease, promoting urea 12 diffusion from epithelial cells (46). Secondly, VacA is present in outer-membrane vesicles 13 which are thought to deliver pro-inflammatory proteins to the epithelial cells, but only in iron-14 van Vliet et al.
Fur-regulation of Helicobacter pylori amidases second, independent fur mutant in H. pylori strain 26695 which contains a promoterless 1 chloramphenicol cassette in fur (23). This independent fur mutant also displayed derepressed 2 amidase activity and increased formamidase activity (data not shown), thus excluding the 3 possibility that the effect on formamidase activity result from a secondary mutation or polar 4 effects of the antibiotic cassette inserted in the fur gene. The Fur protein showed specific 5 binding to the amiE promoter, but not to the amiF promoter, despite both promoters having 6 sequences resembling Furboxes (Fig. 5A). This again demonstrates the limitations of Furbox 7 predictions that are based solely on sequence similarity (48). 8 In conclusion, we have identified a novel type of gene regulation for bacterial amidases, 9 which is mediated by Fur at the transcriptional and enzyme activity level (for AmiE) and at 10 the enzyme activity level (for AmiF). The diverse roles of the Fur regulatory protein in 11 metabolic and pathogenic processes of H. pylori indicate that this bacterium is able to use 12 several intricately linked mechanisms to survive and thrive in the gastric mucosa, and is able 13 to sense and cope with the variable conditions and multiple stresses occuring there despite its 14 relatively limited range of regulatory proteins. 15 Table 1 Oligonucleotide primers used in this study Primer sequences were derived from the H. pylori 26695 genome sequence (19) b) Primers contained a 5´-extension with T7 promoter sequence (in lowercase letters), for the creation of an antisense RNA probe (25).

c)
Primer was labelled at the 5' end with DIG, for use in gel retardation assays.

Figure 1
Amidase and formamidase activity in H. pylori is not substrate-inducible, but amidase activity is iron-repressed.  iron-replete (+Fe) conditions, were compared on 2D-protein gels. The relevant part of the protein gel is magnified for each gel, and the iron-and Fur-repressed AmiE protein is circled.
The estimated molecular mass and pI are indicated.

Figure 5
The Fur protein binds specifically to the amiE promoter, but not to the amiF promoter.