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Journal of Bacteriology, February 2001, p. 1259-1268, Vol. 183, No. 4
Department of Anti-Infective
Research1 and Department of
Bioinformatics,2 SmithKline Beecham
Pharmaceuticals, Collegeville, Pennsylvania 19426, and
Department of Biology, Pennsylvania State University,
University Park, Pennsylvania 168023
Received 27 September 2000/Accepted 17 November 2000
A comparative genomic approach was used to identify
Helicobacter pylori 26695 open reading frames (ORFs) which
are conserved in H. pylori J99 but highly diverged in other
eubacteria. A survey of selected pathways of central intermediary
metabolism was also carried out, and genes with a potentially selective
role in H. pylori were identified. Forty-five ORFs
identified in these two analyses were screened using a rapid
vector-free allelic replacement mutagenesis technique, and 33 were
shown to be essential in vitro. Notably, 13 ORFs gave essentiality
results which are unexpected in view of their known or proposed
functions, and phylogenetic analysis was used to investigate the
annotation of 7 such ORFs which are highly diverged. We propose that
the products of a number of these H. pylori-specific
essential genes may be suitable targets for novel anti-H.
pylori therapies.
The gastric pathogen
Helicobacter pylori causes one of the most common infections
of humans. In developed nations up to half of all individuals are
infected with H. pylori, and this number rises to more than
90% in many developing nations (22). Although infected
individuals are often asymptomatic, H. pylori is a causal agent of chronic gastritis in humans and is strongly associated with
the development of both duodenal and gastric ulcers and the rare cancer
gastric mucosa-associated lymphoid tissue lymphoma. It has also been
established that there is a connection between H. pylori
infection and gastric cancer, which is the second most common
neoplastic cause of death worldwide. Various studies have even
suggested that H. pylori infection has a role in nongastric diseases, including coronary heart disease and hypertension, although supporting evidence in these cases is often rather inconclusive. Eradication therapy is recommended for H. pylori-infected
ulcer patients (39). Antibiotic monotherapies achieve only
a low clearance rate, so the usual treatments are combinations of acid
suppressors such as proton pump inhibitors with two antibiotics, often
from metronidazole, tetracycline, amoxicillin, or clarithromycin. While they can achieve eradication rates of 85 to 90%, the efficacy of these
treatments is being eroded by the rise in frequency of antibiotic-resistant isolates. Although resistance to amoxicillin and
tetracycline is rare, substantial and increasing rates of resistance to
metronidazole and clarithromycin are a significant cause of treatment
failure (1). A further concern is that the use of
broad-spectrum drug combinations to treat this chronic infection may
promote resistance in other pathogenic bacteria, thereby compromising
their utility in the treatment of more serious infections. As
H. pylori produces an organ-specific infection which is not
normally complicated by coinfection with other pathogens, it would seem
ideally suited to treatment with a narrow-spectrum therapeutic agent
which would not promote resistance in endogenous flora. Hence, there is
considerable interest in developing novel therapies which are both more
effective and specific for H. pylori.
The search for new ways to eradicate H. pylori has been
greatly facilitated by the publication of the complete genome sequences of two H. pylori strains, 26695 (50) and J99
(2). We have used this information to drive two distinct
approaches to identifying essential genes which are potential targets
for H. pylori-specific antibiotics. Although the genome is
only 1.65 Mb in size, it encodes many catabolic and anabolic
capabilities which are equivalent to those of bacteria with much larger
genomes, suggesting that H. pylori must have few functional
redundancies and limited metabolic diversity. Hence, it is predicted
that a relatively high percentage of genes involved in central
metabolic pathways will be essential for its survival even if their
direct equivalents in other organisms are not. Although the H. pylori 26695 and J99 genomes contain just 1,552 and 1,495 open
reading frames (ORFs), respectively, the translational products of more
than a third of these are only distantly related to their nearest
eubacterial homologs or are of completely unknown function. Elucidation
of the cellular roles of these unique genes may lead to the
identification of completely novel, essential functions which are
H. pylori specific. We therefore examined pathways of
central intermediary metabolism for genes which are uniquely essential
to H. pylori, and we also used a bioinformatic genome
prioritization analysis to identify highly diverged H. pylori ORFs. We describe the allelic replacement mutagenesis of
these genes and discuss the biological implications of our findings.
Bacterial strains and culture conditions.
H.
pylori 26695 (ATCC 700392) was obtained from the American Type
Culture Collection. H. pylori cultures were grown under microacrobic conditions (5% [vol/vol] O2, 5%
[vol/vol] CO2, 90% [vol/vol] N2) at 37°C
on Columbia agar (Oxoid, Basingstoke, United Kingdom) supplemented with
5% (vol/vol) defibrinated horse blood (Becton Dickinson, Sparks, Md.)
and containing amphotericin B, vancomycin, and polymyxin B, each at 10 µg/ml. For selection of transformants, 20 µg of chloramphenicol per
ml was also added. Supplements were used during the allelic replacement
mutagenesis of particular ORFs as follows: 0.2 to 1 mM uracil with and
without 0.3 mM orotidine monophosphate (OMP), 0.3 mM UMP, and/or 0.3 mM orotate (HP1257 [pyrE]); 0.01 to 1 mM
p-aminobenzoic acid and 0.1 to 1 mM tyrosine, 0.02 to 1 mM
tryptophan, and 1 to 4 mM phenylalanine (HP1038 [aroQ]);
and 1 mM each adenine, guanine, xanthine, and hypoxanthine (HP0735
[gpt]). We found that increasing the concentrations of the
medium supplements used during mutagenesis of HP0735 and HP1257 above
these levels was toxic to wild-type cells.
Identification of highly diverged H. pylori
ORFs.
A previously described relational bacterial genomic database
was used to identify high-priority H. pylori gene targets in silico (11). The core of this database is an array of
ORF-by-ORF sequence similarity scores (smallest sum probabilities)
across multiple genomes as calculated by the Basic Local Alignment
Search Tool (BLASTP) version 2.0 (3). Database queries in
SQL language allow data mining of this database for various
intergenomic protein evolutionary relationships. Predicted protein
sequences of H. pylori 26695 were compared to those of
H. pylori J99 (2), Campylobacter jejuni NCTC11168 (41), Escherichia coli
K-12 (8), Bacillus subtilis (36),
Haemophilus influenzae Rd (24),
Enterococcus faecalis V583 (preliminary sequence data were
obtained from The Institute for Genomic Research [TIGR] website at
http://www.tigr.org), Yersinia pestis CO-92 biovar
Orientalis (Y. pestis Sequencing Group, Sanger Centre,
ftp://ftp.sanger.ac.uk/pub/pathogens/yp/), Klebsiella
pneumoniae 573 (Genome Sequencing Center, Washington University,
St. Louis, Mo., personal communication), Saccharomyces cerevisiae S288-C (28), Archeoglobus
fulgidis VC-16 (35), Methanobacterium
thermoautotrophicum Membrane protein predictions.
Average hydropathy profiles
Phylogenetic analyses.
Publicly available databases
including partial genomic sequences were searched for homologs of the
highly divergent H. pylori ORFs HP1159 (fic),
HP0785 (lolA), HP1553 (recB), HP0020
(nspC), HP1257 (pyrE), HP0735 (gpt),
and HP1164 (trxB) using BLASTP, TBLASTN, and PSI-BLAST
(3). Amino acid sequences were aligned using the program
CLUSTALW (49) and then manually edited. Nonconserved indels (highly variable regions which constitute insertions or deletions with reference to the conserved blocks of the alignment) were
removed from multiple-sequence alignments prior to phylogenetic analysis. Maximum-parsimony (MP) analysis was performed using PAUP*
(47), while neighbor-joining (NJ) trees were based on pairwise distances between amino acid sequences calculated by the
programs PROTDIST and NEIGHBOR of PHYLIP version 3.57c
(23) (http://evolution.genetics.washington.edu/phylip.html). For both methods, confidence limits of branch points were estimated by 1,000 bootstrap replications. In PROTDIST the Dayhoff program option, which
estimates the expected amino acid replacements per position using a
model based on the Dayhoff 120 matrix, was invoked (18).
Trees from all analyses were visualized and, where appropriate, converted into figures using TREEVIEW version 1.6.1 (40).
Annotation of target ORFs.
We utilized ORF annotations
assigned by TIGR (further details on TIGR gene annotations can be
obtained at http://www.tigr.org), with the following
exceptions. The oor (HP0588-0591) and por
(HP1108-1111) genes and HP0086 (mqo) are annotated as
described by subsequent authors (31, 32). HP1138 is
annotated as spo0J based on the homology of the
translational product to B. subtilis Spo0J and its proximity
to a soj homolog, HP1139. HP1563 is annotated as ahpC, which is the more usual nomenclature for bacterial
alkyl hydroperoxide reductase enzymes. HP1553 and HP0785 are annotated as recB and lolA, respectively, based on
phylogenetic analyses performed during this work (see Results and Discussion).
Generation of H. pylori allelic replacement
mutants.
An invariant resistance cassette containing a
Campylobacter coli chloramphenicol resistance gene
(cat) (53) was PCR amplified using primers
designated C1 (5'-GATATAGATTGAAAAGTGGAT-3') and C2
(5'-TTATCAGTGCGACAAACTGGG-3'). H. pylori 26695 genomic DNA was isolated by washing cells scraped from five 48-h
confluent plates, resuspending them in 3.5 ml of Qiagen (Valencia,
Calif.) buffer B1, and then using Qiagen Genomic DNA kits as per the
manufacturer's instructions. Two pairs of gene-specific primers
designated P1-P2 and P3-P4 were used to PCR amplify the up- and
downstream regions, respectively, of each target gene using
Pwo DNA polymerase (Roche, Mannheim, Germany), in order to
produce fragments on the order of 500 bp flanking the region to be
deleted. P2 and P3 contained 5' leaders complementary to C1 and C2,
respectively, followed by 20 bp of gene-specific sequence. Each PCR
product was purified using Qiaquick PCR purification cartridges. A
template mixture containing 1 µg of each of the three purified
products was then PCR amplified using primers P1 and P4 in a single
reaction, to generate a linear construct in which the up- and
downstream fragments are fused to the central cat gene. In
order to maximize product recovery, the PCR conditions used, following
a standard hot start, were 2 cycles of 94°C for 1 min, 45°C for 1 min, and 72°C for 5 min, followed by 40 cycles of 94°C for 45 s,
40°C for 1 min, and 72°C for 3 min, and finally 70°C for 10 min.
The resulting allelic replacement construct was confirmed by sequencing
and introduced into H. pylori 26695 by transformation
(52), by scraping cells from 24-h confluent plates into
0.2 ml of brucella broth (Becton Dickinson) and spotting 100 µl of
this high-density inoculum onto Columbia agar plates made as described
above. After incubation with 2 µg of DNA for 12 h, the
transformed cell mix was spread over the plates and incubated for a
further 24 h. The cells were then scraped onto selective Columbia
agar plates containing 20 µg of chloramphenicol per ml and incubated
for 3 to 7 days.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.4.1259-1268.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Systematic Identification of Selective Essential
Genes in Helicobacter pylori by Genome Prioritization
and Allelic Replacement Mutagenesis
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
H (45), Methanococcus janaschii (12), Pyrococcus abyssi 29292 (R. Helig, unpublished data, http://www.genoscope.cns.fr.), and
Pyrococcus horikoshii OT3 (34). H. pylori 26695 ORFs were selected for further target evaluation if
they showed both highly significant homology to the top hit in H. pylori J99 (P[N] 
1.0e
30)
and low homology (P[N] 1.0e15)
to any ORF from most other bacterial genomes in the comparison profile.
H
(51) were generated using GES
hydropathy values (21) weighted using a trapezoid window. Using a process similar to the initial steps of the TopPred II algorithm (16), helical transmembrane segments (TMS) were
predicted for each peptide sequence by selecting 19 amino acids
centered on the highest H value (MaxH),
masking these from further consideration, and repeating the process
until no peaks with a H of >0.5 remained. Subcellular locations were assigned based on the peak MaxH value, number of segments with a H of >1.0, and
distribution and peak H values of the putative
TMS. A MaxH cutoff of 1.15 was chosen to maximize the discrimination
between two SwissProtein release 34 test datasets containing
transmembrane and cytoplasmic proteins, respectively (10).
Proteins with a MaxH of <1.15 were classified as cytoplasmic, while
those with a MaxH of >1.15 and at least three possible TMS were
classified as membrane proteins. Anchored proteins were defined as
having exactly two TMS, one starting before amino acid (aa) 35 and one
having a H of >1.15 with the other having a
H not lower than 0.5. Proteins containing a
single N-terminal TMS with a H of >1.15 were
classified as secreted, although no test for a signal sequence cleavage
site was performed. Proteins not fitting into any other category were
classified as unknown. This method does not detect gram-negative outer
membrane proteins, which do not have multiple transmembrane helices,
but outer membrane proteins identifiable by homology were eliminated on
the grounds of gene prevalence or protein function at other stages of
our analysis.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Identification of ORFs unique to H. pylori.
In
order to identify H. pylori-specific ORFs for essentiality
testing, we performed a global bioinformatics analysis of the H. pylori 26695 genome using a series of prioritization steps based
on various biological criteria. H. pylori strains are
genetically diverse, and a previous comparison of the H. pylori 26695 and J99 genomes showed that approximately 7% of the
genes are strain specific (2), so a requirement for the
chosen ORFs was that they be present in both available genomes at a
BLASTP confidence level of 1.0e30 or less (3).
For the same reason, genes associated with the variable cag
pathogenicity island (14) were excluded. The translational
products of ORFs common to both strains were screened against 13 other
microbial genomes to identify unique and highly diverged ORFs. An ORF
was defined as having a homolog in another genome if the top hit had a
BLASTP score equal to or less than 1.0e15. Homology to
archeabacterial or yeast ORFs was recorded for information but was not
used as an exclusion criterion. H. pylori 26695 ORFs were
retained if they were either unique or had homologs only in the related
organism C. jejuni or in only one or two additional
eubacterial genomes. These first two steps reduced the number of target
ORFs from 1,552 to 606. TMS predictions using hydropathy profiles
showed that H. pylori 26695 encodes 1,221 cytoplasmic
proteins, while the remaining 21% of ORF translational products are
either noncytoplasmic or of unknown subcellular localization (E. coli contains 27% noncytoplasmic proteins). Since membrane proteins tend to be poorly soluble outside the membrane and therefore less amenable to in vitro biochemical study, ORFs encoding probable integral membrane proteins were excluded from the gene set (with the
exception of HP0740 [murF]), further reducing the number
of target ORFs to 505. ORFs whose only detectable homologs were genes of unknown or hypothetical function were also excluded, since the
absence of clues to function would make biochemical exploitation difficult, leaving 172 ORFs. Finally, ORFs known to encode proteins involved in nonessential processes (such as flagellar biosynthesis, hemolysis, urease production, capsular biosynthesis, and
restriction-modification), or whose highest homologs in the public
databases were genes encoding such proteins, were excluded. From the
remaining 73 highly diverged potential target ORFs, 29 were chosen at
random for essentiality testing.
Identification of key genes in central intermediary metabolism. Biochemical and in silico pathway reconstruction studies have indicated that there are gaps in some H. pylori central intermediary metabolic pathways. We selected 13 ORFs encoding key enzymes in these pathways for further study. These included HP1099 (eda) and HP1100 (edd), which are thought to encode the Entner-Doudoroff (ED) pathway enzymes 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) and 6-phosphogluconate dehydratase (EC 4.2.1.12), respectively; HP1385 (fbp), which is thought to encode the Embden-Meyerhof pathway enzyme fructose-1,6-bisphosphatase (EC 3.1.3.11); HP0086 (mqo), encoding malate quinone oxidoreductase (EC 1.1.99.16); and HP0509 (glcD), which is thought to encode glycolate oxidase subunit (EC 1.1.3.15). We also selected genes which encode the Krebs cycle enzymes pyruvate:flavodoxin oxidoreductase (EC 1.2.7.1) (HP1108-1111 [porCDAB]) and 2-oxoglutarate oxidoreductase (EC 1.2.7.3) (HP0588-0591 [oorDABC]). The porB (HP1111) and oorA (HP0590) genes have previously been shown to be essential (31). Some of these ORFs (oorDABC [HP0588-0591], porCDAB [HP1108-1111], and mqo [HP0086]) are highly diverged and also ranked highly in our global genome bioinformatic analysis, but HP0509 (glcD), HP1099 (eda), HP1100 (edd), and HP1385 (fbp) are moderately or highly conserved in other bacterial species.
Design of allelic replacement mutagenesis cassettes.
As a
result of these analyses, 29 highly diverged ORFs and 13 ORFs encoding
key products in central intermediary metabolism were chosen for further
study. Three more genes whose essentiality or nonessentiality in
H. pylori has already been reported in the literature
(HP1027 [fur], HP1021 [response regulator], and HP1364 [histidine kinase]) were also included as controls. Allelic
replacement constructs targeting each of these 45 ORFs were made by PCR
amplification. In order to minimize potential polar effects,
oligonucleotide primers were chosen so that flanking genes and
intergenic regions including potential promoters would remain intact in
the deletion mutant. The translation start sites of each ORF assigned
by TIGR were checked for optimal spacing from potential ribosome
binding sites, and where possible potential promoter sequences
conforming to the H. pylori 10 region consensus TAtaaT
were identified (25). Flanking genes were identified and
in most cases were consistent with the gene order given by TIGR.
However for certain ORFs with several possible start sites and those
adjacent to short unannotated ORFs, the most conservative option was
used when designing the primers.
Allelic replacement mutagenesis of potential target
genes.
In order to determine gene essentiality, the allelic
replacement constructs were directly transformed into H. pylori 26695. Typically, essential genes were so annotated
following three independent, controlled transformations in which no
mutants were recovered. However, in the case of 14 genes which we have
designated essential (HP0020, HP0509, HP0588, HP0589, HP0590, HP0808,
HP0975, HP1108, HP1109, HP1418, HP1563, HP1553, HP0740, and
HP1159), a small number of chloramphenicol-resistant colonies was
recovered following transformation with the allelic replacement
cassette. In each case PCR and Southern blot analysis of 20 to 30 putative mutants demonstrated that they contained an apparently intact
wild-type copy of the target gene in addition to the correct mutation,
suggesting that only mutants which contain a functional second gene
copy are viable. During allelic replacement mutagenesis studies for a
further three ORFs (HP1257 [pyrE], HP1038
[aroQ], and HP0735 [gpt]), the transformed
cells were plated onto media with and without nutritional supplements
appropriate to the proposed gene function. A few
chloramphenicol-resistant aroQ and gpt putative mutant colonies were obtained on supplemented media, but further subculture demonstrated that they did not require the presence of
supplements for growth, and they too were found to contain a copy of
the wild-type target gene. This type of gene duplication has been
observed during similar allelic replacement mutagenesis studies with
Streptococcus pneumoniae (15), and it was
postulated that this might be explained by the stabilization under
selective pressure of spontaneous tandem unstable chromosomal
duplications, similar to those which have been reported to occur
between repetitive sequences at moderately high frequency in E. coli and Salmonella enterica serovar Typhimurium
(29). The essentiality results for all 45 ORFs tested are
shown in Table 1.
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Control allelic replacement studies. Four of the five genes whose essentiality in H. pylori has previously been reported behaved as expected in our allelic mutagenesis system. We confirmed the essentiality of porB (HP1111) and oorA (HP0589) (31) and additionally demonstrated that each of the six remaining por and oor subunit genes is individually required for cell viability in vitro. We also recovered and confirmed mutants of the nonessential genes HP1027 (fur) (7) and HP1364 (histidine kinase) (6). However, we found that the orphan response regulator gene HP1021 is not essential for viability, in contrast to a previous study in which putative HP1021 mutant colonies were recovered but could not be further passaged (6). It is possible that the discrepancy is due to some difference in our respective protocols, and indeed we note that those authors used clinical isolate strains as mutation hosts and a more oxygen-rich atmosphere (5% CO2, 95% air) for cell culture. However we were unable to test the hypothesis that HP1021 is essential only under conditions of higher oxygen tension, as in our hands neither the HP1021 mutants nor wild-type H. pylori 26695 grew under these conditions.
Overview of target gene essentiality data. Of the remaining 40 ORFs, 31 were essential and 9 were nonessential in vitro. In five cases (HP1140 [birA], HP0657 [ymxG], HP0830 [gatA], HP0658 [gatB], and HP0975 [gatC]) this analysis represents to our knowledge the first description of essentiality of the gene or its homologs in any bacterial species. In most other cases the essentiality data are consistent with the proposed function of the ORF. The results for these 27 ORFs are listed in Table 1 together with their putative function but are not further discussed. However, the behavior of 13 ORFs in our allelic mutagenesis system (shown in boldface in Table 1) was unexpected based on existing knowledge of the gene products or their orthologs. Six of these ORFs either have been biochemically characterized in H. pylori (HP0086 [mqo] and HP1038 [aroQ]), or are well conserved among eubacteria (HP1099 [eda], HP1100 [edd], HP1385 [fbp], and HP1563 [ahpC]). The remaining seven (HP1257 [pyrE], HP0735 [gpt], HP1553 [recB], HP1159 [fic], HP1164 [trxB], HP0020 [nspC], and HP0785 [lolA]) are highly diverged, so their primary structural relationships were investigated using phylogenetic analysis.
Essentiality of H. pylori genes of known function. HP0086 has been shown in complementation studies to encode malate:quinone oxidoreductase (Mqo) (32). Although malate dehydrogenase activity has been reported in H. pylori extracts (43), no mdh gene has been detected in the H. pylori genomes, so our finding that HP0086 is essential is consistent with the argument that H. pylori is solely dependent on Mqo for malate oxidation.
The product of HP1038 (aroQ) has been purified and confirmed to be a type II dehydroquinase from the shikimate pathway (9). Disruption of shikimate pathway genes is well documented in other bacteria and has been shown to cause auxotrophy for aromatic amino acids and p-aminobenzoic acid, but we were unable to obtain HP1038 mutants on rich media even when these supplements were supplied exogenously. It therefore appears either that H. pylori is impaired in its ability to take up these supplements or that elimination of the shikimate pathway is lethal. Chorismate is a precursor for synthesis of the quinone components of the electron transport chain, and our results could be explained if H. pylori cannot obtain these molecules from the horse blood-supplemented Columbia plates used here.Essentiality of conserved genes. The ED pathway is responsible for catabolism of glucose, the only carbohydrate utilized by H. pylori (37). The pathway is constitutively expressed in H. pylori, and genes for key enzymes in the alternative glycolytic and pentose phosphate pathways (phosphofructokinase, pyruvate kinase, and 6-phosphogluconate dehydrogenase) cannot be detected in the genome, suggesting that it has a particular importance in this organism. The ED enzymes 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase have been detected in H. pylori extracts (38) and are thought to be encoded by HP1099 (edd) and HP1100 (eda), respectively. Neither gene is essential in E. coli, although the accumulation of 2-keto-3-deoxy-6-phosphogluconate in eda mutants has a bacteriostatic effect on growth (26). The fact that we were unable to recover mutants of HP1099 or HP1100 is consistent with the hypothesis that catabolism of glucose in H. pylori occurs exclusively via the ED pathway.
The conserved ORF HP1385 is thought to encode fructose-1,6-bisphosphatase (Fbp). The relative activities in H. pylori extracts of this enzyme and the phosphofructokinase enzyme, which catalyzes the reverse reaction, suggest that gluconeogenesis is favored over glycolysis (30), and indeed no phosphofructokinase gene has been detected in the genome (37). The fbp gene is not essential in E. coli or B. subtilis (27), but in Pseudomonas aeruginosa, where Fbp is additionally required for catabolism of glyceraldehyde-3-phosphate, fbp null mutants could not be recovered (5). We found that HP1385 is essential, suggesting that Fbp may have a similarly enhanced cell role in H. pylori. The conserved ORF HP1563 is thought to encode the AhpC subunit of alkylhydroperoxide reductase (AhpR), which catalyzes the reduction of organic hydroperoxides and hydrogen peroxide. ahpC is not essential in C. jejuni (4), but as C. jejuni ahpC mutants are hypersusceptible to oxidative stress, it is possible that HP1563 mutants were generated but were not able to proliferate in the 5% O2 microaerobic atmosphere used. It therefore appears either that the mechanisms for controlling oxidative stress in H. pylori are exquisitely sensitive to oxygen concentration or that they play an unusually critical role in cell viability.Essentiality of highly diverged ORFs.
Phylogenetic analysis
(Fig. 1A) supports the annotation of
HP1257 as pyrE, encoding orotate phosphoribosyl transferase
(PRTase), although it is very distantly related to other
pyrE genes. This enzyme catalyzes the production of OMP,
which is a precursor for UMP, a central intermediate in pyrimidine
nucleotide metabolism. Bacterial pyrE mutants are usually
uracil auxotrophs (54), but we were unable to obtain
HP1257 mutants on rich media in the presence of exogenous uracil, even
with supplemental OMP, UMP, and orotate. H. pylori has no
homologs of the pyrimidine salvage enzymes uracil PRTase (Upp) or
uridine kinase (Udk), which convert uracil to UMP (37).
Although our results could be explained by poor uptake of medium
supplements, they are consistent with recent reports that the conserved
gene HP1084 (pyrB), encoding aspartate carbamoyltransferase, is also essential (13) and that inhibitors of
dihydroorotate dehydrogenase (PyrD) are lethal to H. pylori
(17). Taken together these data appear to demonstrate that
de novo pyrimidine biosynthesis is required for survival of H. pylori.
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(C, or D enzymes, which
provide precursors for spermidine synthase SpeE, and as we have shown
that HP0832 (speE) is not essential, it is possible that
NspC is solely responsible for spermidine biosynthesis
(19).
All of the ORFs described above are essential in H. pylori
even though their orthologs in other organisms are nonessential. HP0785
is the only example of a nonessential ORF which might be expected to be
essential. The product of HP0785 is homologous to the periplasmic
lipoprotein chaperone LolA, although phylogenetic analysis (Fig. 1C)
shows that the H. pylori protein is highly divergent from
its proteobacterial equivalents. LolA localizes outer membrane-specific
lipoproteins following their translocation to the periplasmic side of
the inner membrane, in conjunction with the LolB receptor protein. The
lolA gene is essential in E. coli
(48). Our result that the lipoprotein signal peptidase gene HP0074 is essential demonstrates that lipoproteins are important for H. pylori survival, so our finding that HP0785 is not
essential suggests that H. pylori may encode more than one
such chaperone.
Perspectives. The data presented here demonstrate that stepwise prioritization of genome ORFs using simple biological criteria, particularly in the case of small genomes such as that of H. pylori, can be an effective way of quickly reducing the number of genes of interest to an experimentally manageable number. The essentiality testing of a relatively large number of gene targets was accomplished by the application in H. pylori of a rapid and robust vector-free allelic replacement mutagenesis method. When combined with a set of initial selection criteria which are directly relevant to antibacterial drug discovery, this process is an efficient way to enrich for potential target genes and identify those which are critical for normal cell function.
The essentiality results reported for most of the ORFs are consistent with their proposed functions. However, we have identified 12 essential genes which might be expected to be nonessential by analogy with orthologous genes in other bacteria, including two genes whose products have previously been directly characterized in H. pylori (aroQ and mqo) and one nonessential ORF (lolA) whose closest homolog is essential in E. coli. It is possible that the most highly diverged of these ORFs have been misannotated, but in general our phylogenetic analyses support the ORF annotation given by TIGR. The exceptions are HP0785 and HP1553, which we have designated lolA and recB, respectively, but this refinement of the functional annotation has not clarified the reason for their essentiality. These results reflect the fact that H. pylori has a relatively small genome with little room for functional redundancy and must therefore contain a higher proportion of essential genes with core functions. The high degree of sequence divergence of many of the essential gene products described here suggests that it may be possible to design specific chemical inhibitors which do not affect the growth of other organisms. This is recognized as a desirable attribute for any new anti-H. pylori therapeutic agent, as it would reduce the severity of gastrointestinal sequelae caused by destruction of the normal flora, and also minimize the spread of drug resistance to other pathogens. Importantly, a further degree of selectivity may be conferred by the fact that the nearest neighbors of some of these genes are not required for viability in other bacteria. The potential of this type of approach has been graphically illustrated by the recent identification of pyrazole-based anti-H. pylori compounds which specifically inhibit the pyrimidine biosynthesis enzyme PyrD in H. pylori but are inactive against orthologous enzymes in other bacteria and humans (17). We therefore suggest that the products of these highly diverged essential genes merit further investigation as potential targets for novel, highly specific anti-H. pylori agents.| |
ACKNOWLEDGMENTS |
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We are grateful to John Throup for useful discussions, Peter Morrison for assistance with phylogenetic analysis, Thomas Mathie for synthesis of oligonucleotide primers, and Stephanie Van Horn for sequence confirmation. We thank the Genome Sequencing Center, Washington University, St. Louis, Mo., for communication of K. pneumoniae DNA sequence data prior to publication.
Sequencing of H. pylori 26695 was accomplished with support from TIGR. Sequencing of E. faecalis V583 was accomplished with support from NIAID.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Anti-Infective Research, SmithKline Beecham Pharmaceuticals, 1250 South Collegeville Rd., Collegeville, PA 19426-0989. Phone: (610) 917-6366. Fax: (610) 917-7901. E-mail: Alison_F_Chalker{at}sbphrd.com.
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Journal of Bacteriology, February 2001, p. 1259-1268, Vol. 183, No. 4
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.4.1259-1268.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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