Use of digoxigenin-labelled ampicillin in the identification of penicillin-binding proteins in Helicobacter pylori

Andrew G. Harrisa, Stuart L. Hazellb,* and Andrew G. Nettingc

a School of Microbiology and Immunology, University of New South Wales, Sydney 2052, NSW; b Department of Biological Sciences, Faculty of Informatics, Science and Technology, University of Western Sydney, Macarthur, PO Box 555, Campbelltown 2560, NSW; c School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney 2052, NSW, Australia


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Amoxycillin is used in current therapeutic regimens to treat the infection caused by the human gastric pathogen, Helicobacter pylori. The penicillin-binding proteins (PBPs) are the primary targets for the ß-lactam antibiotics, such as amoxycillin, and are involved in the terminal stages of peptidoglycan synthesis. They also play active roles in the determination and maintenance of cellular morphology. It was believed that an organism with a complex morphology, such as H. pylori, would have more than the three PBPs previously suggested. Using digoxigenin-labelled ampicillin (DIG-ampicillin), we report the identification of eight PBPs in H. pylori with masses of 72, 62, 54, 50, 44, 33.5, 30.5 and 28 kDa. A smaller (21 kDa) ninth band was also detected, which may represent another PBP. However, the relatively small size of this apparent PBP raises questions as to whether this is a true PBP. In an attempt to identify the PBPs to which amoxycillin preferentially binds, amoxycillin was used in competition assays with DIG-ampicillin. It appeared that amoxycillin inhibited the binding of DIG-ampicillin to only the 72 kDa PBP. The experimental data were also compared with the seven putative PBPs identified in the two published H. pylori genomes, most of which correlate with the experimental data. To investigate further the properties of these PBPs, the seven putative PBP genes identified in the H. pylori genomes were examined. The derived amino acid sequences of the putative PBPs were examined for the three characteristic motifs found in all conventional PBPs, SXXK, SXN and KTG. We were able to determine that all of the putative PBPs had at least one of these motifs, but none possessed all three motifs with the characteristics of conventional PBPs. These findings suggest that the PBPs of H. pylori are unique.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
The penicillin-binding proteins (PBPs), in the conventional sense, are a set of enzymes found in the cytoplasmic membrane of bacteria that are involved in the terminal stages of peptidoglycan synthesis. The PBPs are integral components in the determination and maintenance of cellular morphology and are, as the nomenclature suggests, the target proteins for penicillin and other ß-lactam antibiotics.1,2 Covalent linkage of the ß-lactam antibiotics to specific PBPs in sensitive organisms leads to rapid cell lysis and death.1

Amoxycillin, a ß-lactam antibiotic, is used in current combination therapeutic regimens to treat infection caused by Helicobacter pylori. H. pylori is a Gram-negative, microaerophilic, helix-shaped pathogen that infects the human gastric mucosa. H. pylori infection is associated with active chronic gastritis,3 peptic ulcer disease4 and is a predisposing factor for gastric adenocarcinoma and B-cell mucosaassociated lymphoid tissue (MALT) lymphoma.5,6 Successful treatment of the infection has been shown to significantly reduce gastritis,7 facilitates the cure of duodenal ulcer disease and could lower the risk of gastric carcinomas forming as a result of the infection.58

In vitro, H. pylori is sensitive to many antibiotics. However, the efficacy of these in vivo has been disappointing.9,10 This has led to the use of combination therapies based on acid suppressive drugs in combination with two antibiotics, a macrolide (clarithromycin) and/or a nitroimidazole (metronidazole) and/or amoxycillin to treat the infection.11,12 These combination therapies are far from ideal and are further complicated by H. pylori developing resistance to metronidazole and clarithromycin,1315 two of the key antibiotics used in a number of combination therapies. There have been limited reports of H. pylori developing resistance to the ß-lactam antibiotics and resistance to amoxycillin does not appear to represent an important clinical problem.16,17

The intrinsic ß-lactamase activity that is observed in many Gram-negative bacteria appears absent and there is no apparent chromosomally encoded ß-lactamase in H. pylori.18,19 There are, however, two coding regions identified within the H. pylori genome that have 55.4 and 55.5% similarity to the ‘penicillin tolerance protein’ (lytB) (HP0400/JHP981) of Haemophilus influenzae and the ‘methicillin resistance protein’ (llm) (HP1581/JHP1488) of Staphylococcus aureus, respectively.18,19 The low similarity, and the fact that H. pylori remains inherently sensitive to the ß-lactam class of antibiotics, indicates that the physiological function of the proteins encoded by these putative genes in H. pylori may not relate to resistance.

PBPs within a cell have various affinities for different ß-lactam antibiotics, often resulting in selective inhibition of particular PBPs.20 This selective inhibition can result in gross morphological changes, as opposed to rapid cell lysis. This has been shown by the selective inhibition of PBPs 2 and 3 of Escherichia coli inducing spherical and filamentous forms of the bacterium, respectively.21,22 In H. pylori, it has been shown that amoxycillin and cefixime both induce the premature formation of cells with coccoid morphology in vitro.23,24 It has also been demonstrated that amoxycillin kills H. pylori relatively slowly.23,25 The formation of coccoid cells and the slow killing kinetics indicate that amoxycillin may not be the ß-lactam antibiotic of choice for the treatment of H. pylori infection. A possible alternative may be a ß-lactam antibiotic that is more active against slow-growing bacteria, such as a derivative of imipenem.2628

Traditionally, PBPs have been detected by use of radiolabelled ß-lactams and autoradiography. The use of benzyl [14C]penicillin exposed to membrane preparations has detected over nine PBPs in E. coli29 and has been used to identify three PBPs in H. pylori, which were designated A, B and C by Ikeda et al.24 The shape of H. pylori and the active role that the PBPs play in the determination of cellular morphology led to the hypothesis that this organism, with a more complex morphology, may possess more PBPs than those with non-complex morphologies. The simple rod-shaped organism, E. coli, has at least nine PBPs.29 The hypothesis regarding further undetected PBPs of H. pylori was supported by data obtained from the H. pylori genome, with several possible regions encoding more than three putative PBPs.18,19

To detect further PBPs in H. pylori, digoxigenin-(DIG) labelled ampicillin was synthesized and exposed to membrane preparations of H. pylori. This led to the identification of eight PBPs in H. pylori, five of which have not been identified previously by direct examination. In addition, we examined the effect of amoxycillin on the PBPs when in competition with the DIG-labelled ampicillin, and analysed the putative PBPs identified within the two published H. pylori genomes.18,19


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Bacterial cultures

H. pylori strain NCTC 11637 was grown in brain–heart infusion broth supplemented with 5% (v/v) horse serum (Oxoid, Basingstoke, UK), trimethoprim 2.5 g/L, polymyxin B 161.5 mg/L (Sigma, St Louis, MO, USA), vancomycin 5 g/L (Eli Lilly, Indianapolis, IN, USA) and amphotericin B 2 mg/L (E. R. Squibb and Sons, Princeton, NJ, USA) at 37°C with constant shaking for 36 h, in 2.5 L anaerobic jars with a microaerophilic environment generated by CampyGen (Oxoid). The purity of the cultures was determined by positive catalase and urease reactions, Gram's stain and phase contrast microscopy. Cultures of E. coli K12 were grown in Luria-Bertani Broth at 37°C with constant shaking for 12–16 h.

Preparation of bacterial membranes

Cells were harvested and cooled on ice. The cells were centrifuged at 6000g for 10 min at 4°C and washed with 100 mM sodium phosphate buffer, pH 7.2. The remaining pellet was resuspended and the cells were disrupted by two passages through a French pressure cell. Large debris and unfractured cells were removed by centrifugation at 8000g for 20 min at 4°C. The supernatant was removed and centrifuged at 105 000g for 40 min at 4°C. The resultant pellet consisted of both inner and outer membranes and peptidoglycan (referred to as membranes for brevity). The membranes were washed a further two times with 100 mM sodium phosphate buffer, pH 7.2, and finally resuspended such that the final protein concentration was 5–10 g/L.

Digoxigenin (DIG) labelling of ampicillin and purification

Ampicillin (Sigma) was labelled with DIG (Boehringer– Mannheim, Indianapolis, IN, USA) as described by Weigel et al.30 Purification of the DIG-ampicillin conjugate was by reversed-phase high-performance liquid chromatography (RP-HPLC). The separation was carried out on a Hewlett Packard series II 1090 liquid chromatograph with a Zorbax C18 reversed-phase column (Hewlett Packard, Palo Alto, CA, USA), 250 mm in length, inner diameter of 9.4 mm with pore size of 80 Å. Ampicillin was detected at a wavelength of 206 nm. The mobile phase, set at a constant rate of 2 mL/min, consisted of 100 mM sodium phosphate buffer, pH 6.1, and acetonitrile, both degassed and filter sterilized before use. For the initial 10 min, acetonitrile was kept at a constant 10% of the total mobile phase. A gradient was then set up beginning with 10% acetonitrile and increasing to 40% over a period of 25 min. The final composition of the mobile phase consisted of 40% acetonitrile and this concentration was maintained for a further 10 min. After fractionation, acetonitrile was removed from the samples by passing gaseous N2 over the surface until evaporated. The newly synthesized DIG-ampicillin conjugate was stored as a freeze-dried powder until required.

Detection of PBPs

Based on the method of Spratt,29 50 µL aliquots of the membrane preparation were pre-incubated at 30°C for 10 min. This was followed by the addition of 10 µL of the DIG-ampicillin conjugate (50 mg/L) and incubation at 30°C for a further 10 min. The labelling reaction was terminated by the addition of 5 µL of non-labelled ampicillin (120 g/L), followed by 10 µL of 20% (w/v) Sarkosyl (sodium lauroyl sarcosinate) selectively dissolving the inner membranes. Page & Taylor31 have previously verified this method of membrane solubilization in Campylobacter spp. The reaction mixture was centrifuged at 108 000g for 40 min at 10°C. The supernatant was added to SDS–PAGE sample buffer and boiled for 10 min. Samples (20 µL) were fractionated by SDS–PAGE (4% stacking, 12% separating) based on the method of Laemmli.32 The fractionated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane by electroblotting with a Milliblot graphite electroblotter (Millipore, Bedford, MA, USA), using a protocol established by Towbin et al.33 To determine the efficacy of transfer, the gels were stained with 300 mM CuCl2 as described by Lee et al.34 The PVDF membranes were soaked in maleic acid buffer (100 mM maleic acid, 150 mM NaCl, pH 7.5) for 30 min at room temperature with gentle agitation (all steps, unless stated otherwise, were under these conditions). This was followed by soaking in blocking buffer [maleic acid buffer plus 1% (w/v) proteolytic fragments of casein] to prevent nonspecific binding by the anti-DIG antibodies. The PVDF membranes were labelled by exposure to anti-DIG Fab fragments, conjugated to alkaline phosphatase (Boehringer– Mannheim) at 75 U/L in blocking buffer for 45 min. The PVDF membranes were washed four times with maleic acid buffer plus 0.3% (v/v) polyoxyethylene sorbitan monolaurate (Tween 20) for 10 min (each wash). They were allowed to equilibrate for 5 min in detection buffer (100 mM Tris–HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.5), to which the chemiluminescent substrate 1% (v/v) di-sodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5-chloro) tricyclo[3.3.1.13,7]decan}-4-yl)phenylphosphate (CSPD; Tropix, Bedford, MA, USA) was added and incubated for 10 min at 37°C without agitation. The PVDF membranes were then exposed to a pre-flashed X-ray film overnight and developed.

Amoxycillin competition assays

Before the labelling of PBPs with DIG-ampicillin, the membrane fractions were pre-incubated at 30°C for 10 min in the presence of amoxycillin at a concentration equal to or greater than ten times the MIC (MIC = 0.025 mg/L). The DIG detection process was identical to that described above.

Densitometry

Densitometry was carried out on a Scanmaster 3+ (Howtek, Hudson, NH, USA) on the transmissive scanning setting. The image was analysed using a Bioimage whole band analyser (Millipore).

Protein concentration determination

The protein concentration was determined by the bicinchoninic acid assay (Pierce, Rockford, IL, USA). We found that when phosphate buffer was used as a diluent, the membranes seemed to mask the actual protein concentration by a factor of six. To overcome this problem, 20% (w/v) Sarkosyl was used as the diluent. This selectively dissolves the cytoplasmic membrane, releasing all of the inner membrane proteins. The membrane preparation was stored at –20°C until required.

Computer-aided analysis

The derived amino acid sequences from the putative PBP genes in the H. pylori genome, identified by Tomb et al.18 and Alm et al.,19 were examined using the GCG-defined programs ‘Findpatterns', ‘Bestfit' and ‘Pepstats' through the Australian National Genomic Information Service (Sydney, Australia). ‘Findpatterns' searches for particular sequences, motifs or patterns within a peptide or DNA sequence. ‘Bestfit' aligns two peptide or nucleotide sequences and determines the percentage similarity and identity. ‘Pepstats' determines the mass of a protein based on its amino acid sequence.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Identification of PBPs

The PBPs of E. coli are well studied and have previously been detected using DIG-labelled ampicillin.30 Our DIG-ampicillin conjugate produced PBP profiles similar to those achieved by Weigel et al.30 including the intense signals when bound to PBPs 5 and 6 of E. coli (data not shown).

Using DIG-labelled ampicillin, nine bands were detected in the membrane extracts from H. pylori. The nine bands detected had apparent molecular masses of 72, 62, 54, 50, 44, 33.5, 30.5, 28 and 21 kDa (FigureGo). Strong signals were produced from the DIG-ampicillin bound to the 62, 44, 33.5, 30.5 and 28 kDa PBPs.



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Figure. PBPs of H. pylori. Lane A shows PBPs detected when membranes were treated with DIG-ampicillin. Lane B shows PBPs detected when membranes were pretreated with amoxycillin.

 
Amoxycillin competition assays

It was anticipated that amoxycillin would selectively bind to one or more of the H. pylori PBPs, thus inhibiting the binding of DIG-ampicillin. Under the conditions stipulated for the competition assays, amoxycillin bound to the 72 kDa protein inhibiting the DIG-ampicillin from binding to this protein. It is also apparent that the 54 kDa protein is less intense in the competition assay; however, the amoxycillin does not completely inhibit the binding of the DIG-ampicillin to this PBP (FigureGo). This apparent lowering of intensities is also evident with the amoxycillin-pretreated 21 kDa band. This was repeated three times and confirmed by densitometry. As a control, unlabelled ampicillin was used in competition with the DIG-labelled ampicillin. This completely inhibited the binding of the DIG-labelled ampicillin. No bands were detected, thus eliminating the possibility of the DIG-ampicillin probe labelling non-PBPs (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
ß-Lactam antibiotics act on the PBPs, inhibiting their enzymic function. The PBPs are involved in the terminal stages of peptidoglycan synthesis and play significant roles in the determination and maintenance of cellular morphology.21,22 In an attempt to characterize the PBPs of H. pylori, Ikeda et al.24 used benzyl[14C]penicillin and were able to detect three PBPs that they designated A, B and C. In the current study, benzyl[14C]penicillin was used initially to identify the PBPs of H. pylori. However, we found the sensitivity of this compound to be too low for the identification of additional bands we believed to be present. It has been shown by Weigel et al.30 that DIG-labelled ampicillin is extremely sensitive and was therefore used in preference to radiolabelled penicillin.

Proteins with molecular masses of 72, 62, 54, 50, 44, 33.5, 30.5, 28 and 21 kDa were detected with DIG-labelled ampicillin. Based on the PBP profile obtained by Ikeda et al.24 for both E. coli and H. pylori, we believe that the three previously identified PBPs, A, B and C, correspond respectively to the 62, 54 and 50 kDa PBPs detected here by DIG-ampicillin. When DIG-labelled ampicillin was used to detect the PBPs of H. pylori, we observed intense bands of mass 62, 44, 33.5, 30.5 and 28 kDa. This could be explained by DIG-ampicillin having a higher affinity for these PBPs or could relate to the concentration of each PBP within the cell.

The molecular masses of the H. pylori PBPs are relatively low in comparison with the PBPs of other organisms. The low-intensity 21 kDa protein detected may be a real PBP, as Borrelia burgdorferi has PBPs with masses of 22, 20 and 13 kDa.35 However, PBPs are generally larger than 21 kDa and this band may be the result of proteolytic degradation of a larger PBP. The fact that the PBPs of H. pylori are relatively small may have an effect on the division of the PBPs into their respective classes. With other bacteria, in particular E. coli, the PBPs have been divided into two classes, the high molecular mass PBPs and the low molecular mass PBPs. By definition, the high molecular mass PBPs are considered to be essential for survival and are those in E. coli that are greater than 60 kDa. The PBPs identified in H. pylori can also be divided into high (79, 62, 54, 50 and 44 kDa) and low (33.5, 30.5, 28 and 21 kDa) molecular mass proteins. The definitions by which the PBPs are classified may have to be refined because of the relatively small size of the PBPs in H. pylori. Until the enzymic activities of each PBP are fully characterized, the utility of these allocations remains limited.

When H. pylori is exposed to amoxycillin (or cefixime) the helical shape of the bacterium is lost and coccoid forms are generated.23,24 This suggests that these antibiotics are selectively binding to a PBP or to PBPs that play active roles in the maintenance of the spiral/rod morphology. Ikeda et al.24 reported that, in competition assays, cefixime bound selectively to PBP B (the 54 kDa PBP detected by DIG-ampicillin), and concluded that this PBP was involved in morphological maintenance. When amoxycillin was used in competition with the DIG-ampicillin, the intensity of this band (54 kDa) was reduced. Although the amoxycillin did not completely inhibit the binding of the DIGampicillin it indicates that some form of competition exists. This may help to explain the coccoid morphologies of H. pylori that are generated in the presence of cefixime and amoxycillin in that these agents may both target the PBPs that are involved in maintaining the helical/rod-shaped morphology that is characteristic of H. pylori.

An interesting finding of this investigation was that under the conditions used, amoxycillin was able to compete successfully with DIG-ampicillin for the 72 kDa PBP (HP0579/JHP544—PBP 1A; Table IGo). Although we were able to determine that amoxycillin bound to the 72 kDa PBP with a higher affinity than the DIG-ampicillin, it has been shown by Dore et al.17 that amoxycillin resistance development in H. pylori is dependent upon a previously undetected PBP, PBP D (~32 kDa correlating with either HP0400 or HP1372; Table IGo), having a lowered affinity for amoxycillin. This indicates that amoxycillin must also interact with this PBP, along with other PBPs involved in maintaining the rod/helical structure of H. pylori. It was also noted that the intensity of the 21 kDa PBP decreased when the membranes were pretreated with amoxycillin. However, there is a degree of uncertainty as to whether this band is a true PBP.


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Table I. Comparison of putative PBPs derived from the amino acid sequences obtained from the TIGR and AstraZeneca H. pylori genome databases
 
The conditions used in this study may not have been ideal for amoxycillin binding to all of the PBPs in H. pylori. Chambers et al.36 have shown previously in S. aureus that to obtain 90% acylation of the PBPs by the ß-lactam ring of penicillin, exposure periods from 13 min to in excess of 3 h are required. Although DIG-ampicillin appeared to be binding effectively in this study, the conditions may not have been ideal for amoxycillin binding. Norgard et al.35 found that in recombinant PBPs of B. burgdorferi, the presence of Zn2+ improved the binding of DIG-ampicillin. They suggested that some PBPs may rely on the presence of divalent cations for the binding of ß-lactam antibiotics.

To gain further insight into the attributes of the H. pylori PBPs, the amino acid sequences of the seven putative PBP genes were examined. When the amino acid sequences of corresponding genes from the TIGR and AstraZeneca genome databases were compared, similarities ranging from 97.789 to 100% were obtained. There is some discrepancy in the name and function of these putative PBPs. The results of these alignments are summarized in Table IGo. There were three PBPs detected experimentally (54, 50 and 21 kDa) that were not similar to the predicted mass of the putative PBPs identified in either of the genomes. There is still a large number of coding sequences within the genome of H. pylori that have no known functions or database matches,18,19 which may encode these proteins, especially when considering that the percentage similarities between the PBPs identified by database comparisons are rather low.

Three characteristic motifs common to all conventional PBPs (including some classes of ß-lactamases) have been identified.37,38 The first motif, SXXK, has the active serine residue responsible for the nucleophilic attack on the ß- lactam ring.37,39 This motif is always found at the N terminus of an {alpha}-helix.37 This motif is not found within the metallo or zinc-dependent ß-lactamases. The second motif, SXN, is also found at the N terminus of an {alpha}-helix downstream of the first motif, from which it is usually separated by 57–95 amino acids although a separation of up to 243 amino acids has also been reported.37 The third motif found in all proteins that interact with ß-lactams is KTG. This motif forms part of a ß-sheet and is separated by 100–160 amino acids downstream from the second motif. The latter two motifs are believed to be involved in the formation and stabilization of an active site cleft.37 The amino acid sequences were also examined for other less common motifs. These motifs include YXN and HTG as the second and third elements, respectively, found in the Streptomyces R61 DD-peptidase,40 or RTG or RSG as the third element as found in some of the class A ß-lactamases.41 Using computer-aided analysis, the amino acid sequences of the putative PBPs of H. pylori were examined for the presence of these motifs. The results of the motif searches for each of the putative PBPs are summarized in Table IIGo. All of the proposed proteins had at least one of the motifs described, but not one of them had all three motifs that matched the properties of ‘conventional’ PBPs. HP0743/JHP680 did not have the important SXXK motif, which is considered essential for the acylation of the ß-lactam ring and hence covalent linkage. However, the SXN motif was identified at the N terminus of a predicted {alpha}-helix. The absence of the SXXK motif is unusual, raising questions as to whether this gene encodes for a putative PBP, but it is possible that the ß-lactam ring of the antibiotic is interacting with a motif similar to the SXXK motif. In the absence of the SXN or KTG motif, an attempt to identify alternative motifs was made. In every case where the above-mentioned motifs were absent, no alternative motifs were identified.


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Table II. Identification of the SXXK, SXN and KTG motifs found within each of the putative genes encoding PBPs in H. pylori
 
It has been shown in other bacteria with spiral or helical morphology that their PBPs appear to have unique properties. Norgard et al.35 demonstrated in B. burgdorferi that OspA, B and D, initially considered outer membrane lipoproteins, were PBPs on the basis of covalent binding of benzylpenicillin. Weigel et al. found that Treponema pallidum possesses a lipoprotein that has carboxypeptidase activity, one of the enzymic activities of PBPs.39 Further, OspA, B and D are similar to the putative PBPs identified in H. pylori in that none of the latter have all of the characteristic motifs that are associated with ‘conventional’ PBPs. OspC in B. burgdorferi was not considered to be a PBP, because of the unusual relative positioning of the motifs present: SXXK at the extreme C terminus and the SXN at the extreme N terminus of the protein.35 The unusual positioning of these motifs is also apparent in amino acid sequences of HP1372/JHP1287 and HP1556/JHP1464.

In this investigation we have revealed that the PBPs of H. pylori may be unique in many respects. In total, nine bands were detected by DIG-ampicillin, although it is possible that only eight are PBPs. Seven putative genes encoding PBPs have been identified in the H. pylori genomes based on sequence similarity with other PBP genes. Not all of these appear to correlate with the experimental data. Within the two complete genomes of H. pylori there are many coding sequences that have no database matches.18,19 Further unidentified genes encoding PBPs could be present in the genome. PBPs are an area of research that has been neglected in H. pylori and further work addressing the issues of ß-lactam binding and the nature of the PBPs is required. There appear to be many properties that are common to the PBPs of spiral/helical organisms that differ from the properties of the ‘conventional’ PBPs. The common and unique properties of these PBPs warrant further investigation, from not only a clinical but also a physiological perspective, including the understanding of the generation and maintenance of spiral morphology.


    Note added in proof
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
A gene which previously had no known function in H. pylori has recently been shown to be a penicillin-binding protein by Krishnamurthy et al.,44 who identified four PBPs in H. pylori 84-183. The larger PBPs were considered to be A, B and C as described by Ikeda et al.24 The fourth protein had a mass of 32 kDa and the protein sequence matched the predicted amino acid sequence of HP0160/JHP148, originally annotated as ‘conserved with no known function’. The unusual positioning of the SXXK, SXN and KTG motifs within this protein was noted and is concurrent with our study. However, this gene was not identified in our study owing to the lack of similarity of this protein with other PBPs.


    Acknowledgments
 
This work was supported in part by a grant from the Clive and Vera Ramaciotti Foundation and from the National Health and Medical Research Council of Australia. Part of this work was presented as an abstract at the XIth international workshop on gastroduodenal pathology and Helicobacter pylori, Budapest, 1998.


    Notes
 
* Corresponding author. Tel: +61-2-4620-3242; Fax: +61-2-4620-3793; E-mail: s.hazell{at}uws.edu.au Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
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Received 18 February 1999; returned 19 August 1999; revised 16 September 1999; accepted 3 January 2000