COMMUNICATION
The Largest Subunits of RNA Polymerase from Gastric Helicobacters Are Tethered*

Natalya ZakharovaDagger , Paul S. Hoffman§, Douglas E. Berg, and Konstantin SeverinovDagger parallel **

From the Dagger  Waksman Institute, Piscataway, New Jersey 08854, the § Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada, the  Departments of Molecular Microbiology and of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110, and the parallel  Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The rpoB and rpoC genes of eubacteria and archaea, coding respectively for the beta - and beta '-like subunits of DNA-dependent RNA polymerase, are organized in an operon with rpoB always preceding rpoC. The genome sequence of the gastric pathogen Helicobacter pylori (strain 26695) revealed homologs of two genes in one continuous open reading frame that potentially could encode one 2890-amino acid-long beta -beta ' fusion protein. Here, we show that this open reading frame does in fact encode a fused beta -beta ' polypeptide. In addition, we establish by DNA sequencing that rpoB and rpoC are also fused in each of four other unrelated strains of H. pylori, as well as in Helicobacter felis, another member of the same genus. In contrast, the rpoB and rpoC genes are separate in two members of the related genus Campylobacter (Campylobacter jejuni and Campylobacter fetus) and encode separate RNA polymerase subunits. The Campylobacter genes are also unusual in overlapping one another rather than being separated by a spacer as in other Gram-negative bacteria. We propose that the unique organization of rpoB and rpoC in H. pylori may contribute to its ability to colonize the human gastric mucosa.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

DNA-dependent RNA polymerase (RNAP)1 is the central enzyme of gene expression and a major target for regulation. RNAPs are large, multisubunit protein complexes. The best studied RNAP, from Escherichia coli (>400 kDa), contains four core polypeptides: beta ' (155 kDa), beta  (150 kDa), a dimer of alpha  (37 kDa), and one of several possible sigma  (specificity) subunits. RNAPs from other bacteria have similar subunit composition and exhibit striking and co-linear sequence similarities with the E. coli enzyme (1). The two largest RNAP core subunits comprise 60% of the RNAP mass and appear to be responsible for most of the functions of the enzyme.

The synthesis of RNAP subunits is coordinately regulated (2), but the exact mechanisms at play are unknown. In most eubacteria and archaea, genes encoding the beta - and beta '-like subunits are organized in an operon with the gene for the beta -like subunit (rpoB) always preceding that for the beta '-like subunit (rpoC) (3, 4). The two genes are separated by a short, untranslated linker (3, 5, 6),2 whereas in archaea they overlap by several codons (4).

The genome sequence of the gastric pathogen Helicobacter pylori (strain 26695) revealed one continuous open reading frame containing the homologs of rpoB and rpoC, potentially encoding one fused 2890-amino acid-long beta -beta ' polypeptide (8). Our previous analysis using E. coli RNAP showed that such a beta -beta ' fusion is compatible with RNAP function: (i) the product of artificially fused rpoB and rpoC genes of E. coli could assemble into a functional RNAP in vivo and in vitro and (ii) an E. coli strain containing the fused rpoBC gene as its only source for RNAP was viable and contained RNAP of the expected (beta -beta ')alpha 2 subunit composition (9). This tethering of E. coli beta  and beta ' increased the efficiency of RNAP assembly in vitro and suppressed an rpoCts assembly mutation in vivo.3 It thus seemed that natural tethering could be advantageous for an organism like H. pylori that needs to colonize the stomach, an intrinsically acid-rich and putatively hostile environment (10). However, RNAP had never been purified from H. pylori, and therefore post-translational proteolysis of fused beta -beta ' protein followed by assembly into "normal" RNAP with a beta beta 'alpha 2 subunit composition could not be ruled out a priori.

H. pylori belongs to the epsilon  group of proteobacteria (10). With the exception of H. pylori, no rpoBC gene sequences from this group of bacteria were known. Thus, it seemed possible that rpoBC fusion could be (i) characteristic of epsilon  proteobacteria in general; (ii) a specific feature of the Helicobacter genus; (iii) an accidental feature of the H. pylori species; or (iv) an accidental feature of the particular H. pylori strain that was sequenced. To assess these possibilities, we sequenced the rpoB-rpoC junction in four different strains of H. pylori, in an isolate of Helicobacter felis (11), and in two species of the related genus Campylobacter. In addition, we purifed and characterized the product of rpoBC gene from H. pylori 26695.

Our results establish that in H. pylori 26695 the rpoB-rpoC gene does in fact encode a fused beta -beta ' polypeptide. In addition, we find that translational fusion of rpoBC genes is characteristic of two gastric Helicobacters but not of Campylobacter jejuni and Campylobacter fetus, members of a related genus that colonize nongastric sites. We suggest that the beta -beta ' tethering in Helicobacter might (i) be an accident of evolution because of a frameshift mutation in an ancestor that, like current Campylobacters, contained overlapping but separate rpoB and rpoC genes or (ii) help gastric organisms cope with their acid- and urea-rich niche.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Bacterial Growth and DNA Preparation-- H. pylori were grown under microaerobic conditions (5% O2, 10% CO2, 85% N2) on Brucella agar medium supplemented with 5% horse blood, 1% Isovitalex, amphotericin B (8 mg/liter), trimethoprim (5 mg/liter), vancomycin (6 mg/liter), essentially as in Ref. 12. For biochemical purification of RNAP H. pylori 26695 liquid cultures were grown in Brucella broth with 10% fetal calf serum in 500-ml screw capped flasks; the medium was equilibrated with 7% O2, 5% CO2 in the microaerobic incubator for 1 h prior to inoculation, and then the bacteria were added, and the flasks were sealed and placed on a rotary shaker at 150 rpm. The bacteria were harvested in late log phase (OD660 = ~0.8), and the cell pellets were stored at -70 °C. C. jejuni strain H840 and C. fetus strain have been previously described (13) and were grown at 37 °C on Brucella agar plates in a microaerobic incubator maintained at 7% O2, 5% CO2.

Helicobacter genomic DNA was extracted from confluent cultures using the Qiamp tissue kit (Qiagen). Genomic DNA was purifed from 50-100 mg of Campylobacter cell paste by resuspending cells in 200 µl of buffer containing 25 mM Tris-HCl, 1 mM EDTA, pH 7.9, and 0.1 mg/ml RNase A. Cell suspension was extracted three times with equal volume of phenol, followed by chloroform extraction and ethanol precipitation. DNA was dissolved in 500 µl of water and used in PCR.

Molecular Biology-- The following primers were used for PCR: the upstream primer (GGGGGTCAAAGGTTTGGGGAAATGGAAGTGTGGGC) corresponds to H. pylori 26695 rpoBC positions 3893-3926 (beta  conserved segment I, see Ref. 1 for nomenclature); the downstream primer, TTTGGAGTGCGTGATCGCCACGCCGCATTTTTCGCA is complimentary to rpoBC positions 4393-4428 (beta ' conserved segment A). Standard PCR reactions contained in 100 µl 200 ng of genomic DNA, 4 pmol of each primer, 1 mM dNTPs, and 5 units of Taq DNA polymerase in the standard PCR II buffer (Perkin-Elmer) supplied with 2. 5 mM MgCl2. 30 amplification cycles (1 min at 94 °C, 1 min at 48 °C, and 1.5 min at 72 °C) were performed. PCR fragments (~500 bp) were cloned in pT7blue blunt vector (Novagene, Inc., Madison, WI) and sequenced using T7 promoter and U-19 sequencing primers (Novagene, Inc.) primers on both strands at the Rockefeller University Protein-DNA Technology Center.

Protein Purification and Sequencing-- 4 g of H. pylori (strain 26695) cells were resuspended in 15 ml of lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, pH 7.9, 1 mM beta -mercaptoethanol) and lysed by passage through an Emulsiflex C-5 homogenizer (Avestin). The lysate was cleared by low speed centrifugation, and PEI was added to a final concentration of 0.8%. The PEI pellet was collected by low speed centrifugation, washed by 20 ml of lysis buffer, and extracted with 20 ml of lysis buffer containing 1 M NaCl. Proteins in 1 M NaCl extract were precipitated with ammonium sulfate (0.7 g/ml extract), and the pellet was recovered by centrifugation, dissolved in 20 ml of lysis buffer, and loaded on a 1-ml heparin HiTrap cartridge (Amersham Pharmacia Biotech) equilibrated in the same buffer and attached to a Waters 650 chromatographer. The column was washed with the buffer + 0.3 M NaCl and eluted with buffer + 0.6 M NaCl. Fractions containing 300-kDa band (monitored by SDS-PAGE) were pooled concentrated on a C-100 concentrator (Amicon) to ~1 mg/ml, diluted 2-fold with glycerol, and stored at -20 °C.

For protein sequencing, heparin affinity chromatography fraction containing ~5 µg of 300-kDa band was blotted on polyvinylidene difluoride membrane after SDS-PAGE and submitted to the Rockefeller University Protein-DNA Techonology Center.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

rpoBC Genes Are Fused in Helicobacter but Not in Campylobacter-- Two primers that target the rpoB-rpoC junction and that are complimentary to highly conserved sequences in the 3' end of the rpoB portion and the 5' end of the rpoC portion of the H. pylori 26695 rpoBC gene were used for PCR amplification with genomic DNA from the following organisms: H. pylori strains Hp1 (14), J-166 (15), SS1 (16), and NCTC11638 (17); an isolate of Helicobacter felis; and isolates of two different Campylobacter species: C. jejuni, strain H840, and C. fetus (13). In all cases, a single major PCR fragment ~500 bp in length was amplified. The fragment was cloned, and its sequence was determined. Alignment of sequences at and around the rpoB-rpoC junction site is shown on Fig. 1. Each H. pylori strain differed from the 26695 sequence in ~10 of 474 positions (italicized in Fig. 1). This relatively high (~2%) level of DNA polymorphism between different H. pylori strains is consistent with published data on the extent of polymorphism within H. pylori (18, 19). Most of these differences involved third codon positions, and none resulted in changes in the deduced amino acid sequence of the protein. Thus, the rpoB-rpoC fusion is maintained in all four strains of H. pylori.


View larger version (89K):
[in this window]
[in a new window]
 
Fig. 1.   rpoB and rpoC genes are fused in five independent H. pylori strains and in H. felis but are separate in C. jejuni and C. fetus. DNA sequence of the rpoB-rpoC junction from H. pylori strains 26695 (8), corresponding to codons 1308-1465, is aligned to corresponding sequences from H. pylori strains Hp1, J-166, SS1, and NCTC11638 (11638); H. felis (H.f.); C. jejuni H840 (C. j.); and C. fetus (C.f.). The deduced protein sequences are also shown. Sites in H. pylori DNA that differ from the published (8) 26695 sequence are indicated in capital italic letters. Amino acid positions that differ from the deduced H. pylori sequence are also indicated in capital letters. In campylobacters, the deduced initiating codon is shown in bold, and the likely ribosome-binding site is underlined.

Equivalent DNA sequencing of 3' and 5' ends of the rpoB and rpoC homologs from H. felis (11), another gastric Helicobacter, showed that it also encoded a beta -beta ' fusion protein, although the deduced amino acid sequence of this portion of the H. felis protein differed from that of H. pylori in 20 of 158 positions (shaded in Fig. 1). Hence we conclude that such organization is not unique to H. pylori but may be a common feature of gastric Helicobacters.

C. jejuni and C. fetus are members of a genus closely related to Helicobacter, but they colonize the small intestine not gastric tissue (20). Equivalent DNA sequence analysis showed that rpoB and rpoC are separate genes in these two species (Fig. 1) in contrast to H. pylori and H. felis. In most Gram-negative bacteria, rpoB and rpoC are separated by a short, untranslated linker of 50-100 bp with potential for extensive intrastrand pairing (3, 5, 6).2 The Campylobacter genes are unusual in this context, because they overlap by two codons (no linker). We infer that the ATG codon shown in bold type in Fig. 1 probably encodes the first methionine of Campylobacter beta ' because: (i) it is near an appropriately spaced A/G-rich sequence that could serve as a ribosome-binding site (underlined in Fig. 1) and (ii) the next methionine residue that might possibly serve as initiator is at position 80, in the middle of the universally conserved segment A (1).

Helicobacter rpoBC Encodes a Fused beta -beta ' RNAP Subunit-- The results of DNA sequencing experiments presented above do not test directly whether Helicobacter actually produces a fused RNAP subunit. To critically test this issue, we purified RNAP from H. pylori strain 26695.

RNAP beta  and beta ' are among the largest proteins in bacterial cells and can be detected in whole cell extracts by SDS-PAGE. Fig. 2 shows SDS-PAGE of proteins from whole cell lysates of E. coli, C. jejuni strain H840, and H. pylori 26695. The E. coli and C. jejuni lysates (lanes 1 and 2) contained a characteristic double band that comigrated with the beta  and beta ' subunits of purified E. coli RNAP (lane 4). In contrast, H. pylori lysates (lane 3) contained no such beta  and beta ' bands. Rather, a single band with an apparent mobility of ~300 kDa was observed. No such 300-kDa bands were detected in E. coli or C. jejuni lysates.


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 2.   H. pylori lysates do not contain proteins with mobility of split RNAP beta  and beta '. ~50 µg of E. coli XL1-blue, C. jejuni H840, and H. pylori 26695 were resuspended in Laemmli loading buffer containing SDS, and the proteins from whole cell lysates were resolved by SDS-PAGE on 8% Tris-glycine gel and visualized by silver staining.

To establish whether this 300-kDa band in H. pylori lysates is indeed fused beta -beta ', we began H. pylori RNAP purification using a standard procedure that involved PEI precipitation and extraction of RNAP from the PEI pellet with 1 M NaCl, followed by heparin affinity chromatography (see "Experimental Procedures"). The 300-kDa band was quantitatively precipitated by PEI at low (200 mM) NaCl concentration (Fig. 3, lane 3) and was extracted from the PEI pellet with buffer containing 1 M NaCl (Fig. 3, lane 4). Heparin affinity purification allowed further purification of the 300-kDa band (Fig. 3, lane 5). When heparin affinity chromatography fractions containing 300-kDa band were mixed with pure E. coli RNAP and loaded on a Superose-6 gel filtration column attached to fast protein liquid chromatography, the 300-kDa protein coeluted with the E. coli beta , beta ', and alpha  polypeptides (data not shown). Thus, the chromatographic behavior of the 300-kDa protein is consistent with its being part of RNAP of the beta -beta 'alpha 2 subunit composition.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 3.   Partial purification of the 300-kDa protein from H. pylori extracts. Proteins in H. pylori 26695 were fractionated as described under "Experimental Procedures," and the indicated fractions were resolved by SDS-PAGE and visualized by silver staining. Control lanes contain purified E. coli RNAP of beta beta 'alpha 2 composition (lane 6, E.c. RNAPWT) and fused E. coli RNAP of beta -beta 'alpha 2 subunit composition (lane 1, E.c. RNAPfused, Ref. 9).

When fractions containing the 300-kDa band were incubated in a standard E. coli transcription buffer in the presence of poly(dA-dT) and NTPs, no RNAP activity was observed. Control fractions containing E. coli RNAP of similar purity were highly active in this assay (data not shown). More importantly, 1 M extracts of C. jejuni PEI pellets also exhibited significant poly(A-U) synthesizing activity (data not shown). We were also unable to detect any transcription in crude H. pylori lysates with either poly(dA-dT) or strong E. coli promoters in the presence of exogenously added E. coli sigma 70 subunit. Control extracts from E. coli containing the same amount of total protein were highly active in this assay (data not shown)

Because no transcriptional activity was found associated with the 300-kDa band, we identified it by protein sequencing. The protein appeared to be N-terminally blocked, and therefore internal sequencing was performed after Lys-C protease digestion and high pressure liquid chromatography purification. The sequence obtained, IQQQYDQGLLTDQER, matches exactly to positions 2040-2054 of the predicted fused product of H. pylori rpoBC gene product. We conclude that the 300-kDa band is the fused beta -beta ' RNAP subunit.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The principal conclusion of this work is that a single fused RNAP rpoB-rpoC gene is a regular feature of at least two species of gastric helicobacters, H. pylori and H. felis, and that the gene product, a 300-kDa beta -beta ' fusion protein is the predominant or only form of this gene product in vivo. Because no proteins in H. pylori lysates that would comigrate with separate RNAP beta and beta ' subunits were detected, the fused subunit is probably not extensively proteolyzed; it is likely to be the only source of RNAP beta  and beta ' in the cell, as is also the case for an E. coli strain with a beta -beta ' fusion RNAP that we had engineered to study holoenzyme topology and assembly (9).

No transcriptional activity was found in fractions of H. pylori extract containing the beta -beta ' fused protein under standard conditions that had been optimized for E. coli RNAP transcription in vitro, although similarly prepared RNAP-containing fractions from extracts of E. coli and also of C. jejuni were active under our assay conditions. It is known that RNAPs from different eubacterial species can have markedly different requirements for efficient transcription in vitro (21-23), and hence, the inactivity of H. pylori RNAP under our present conditions may reflect an unusual buffer or salt requirement and conditions in the gastric environment in which it grows. Because H. pylori is a fastidious microbe and rather difficult to grow in large quantities, we have not yet purified the large amounts of H. pylori RNAP that should facilitate establishing a system for transcription by this RNAP in vitro.

Although H. pylori is extremely diverse as a species, our DNA sequencing results establish that the rpoBC fusion is not just a peculiar feature of the one H. pylori strain that was chosen for the genome project (26695), but rather it is a common feature of H. pylori in general and indeed of at least one other gastric helicobacter. In contrast, rpoB and rpoC are separate genes in the closely related genus, Campylobacter. The Campylobacter genus is also unusual among Gram-negative bacteria, however, because its rpoB and rpoC genes overlap by two codons. A frameshift mutation at or shortly before the overlap area might have created the continuous open reading frame found in present day helicobacters. Alternatively, a frameshift mutation in a Helicobacter-like ancestral rpoBC gene might have created separate but overlapping genes of present day campylobacters. A number of nongastric helicobacters and also members of closely related genera that colonize gastric and nongastric sites have been discovered recently (10, 24), and DNA sequence analyses similar to those carried out here should help us learn how these unusual arrangements of RNAP subunit genes have evolved.

The functional significance of the rpoBC fusion is not known. In organisms with transcriptional-translational coupling, the rpoB and rpoC genes are always found in the same operon, suggesting that RNAP assembly in the cell may occur contranslationally. Fusion of the two genes may further increase RNAP assembly efficiency. In E. coli, the beta -beta ' fusion appears to stabilize RNAP in vitro and in vivo.3 Thus the fusion could be advantageous for gastric helicobacters, which must grow in the putatively hostile, acid-rich stomach environment. On the other hand, many acidophilic archaea have separate rpoB- and rpoC-like subunits and in fact contain natural splits in their beta - and beta '-like subunits (7). Experiments aimed at directly testing the importance of the rpoBC fusion in H. pylori are in progress.

    ACKNOWLEDGEMENTS

We are grateful to Drs. Katherine M. Eaton and Ausra Raudonikiene for generously providing H. felis and H. pylori DNAs.

    FOOTNOTES

* This work was supported by a Burroughs Wellcome Fund Career Award in the Biomedical Sciences (to K. S.), grants from Astra Pharma, Canada (to P. H.), Medical Research Council of Canada Grant R-14292 (to P. H.), National Institutes of Health Grants DK48029, AI138166, and HG00820 (to D. B.), and American Cancer Society Grant VM-121 (to D. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence shall be addressed: Waksman Inst., State University of New Jersey, Rutgers, Piscataway, NJ 08854. Tel.: 732-445-6095; Fax: 732-445-5735; E-mail: severik{at}waksman.rutgers.edu.

1 The abbreviations used are: RNAP, RNA polymerase; PEI, polyethyleneimine; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; bp, base pairs.

2 O. J. Nolte (1995) GenBankTM accession number Z54353.

3 T. Naryshkina and K. Severinov, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Buhler, J.-M., Riva, M., Mann, C., Thuriaux, P., Memet, S., Micouin, J. Y., Treich, I., Mariotte, S., and Sentenac, A. (1987) in RNA Polymerase and the Regulation of Transcription (Reznekoff, W. S., Burgess, R. R., Dahlberg, J. E., Gross, C. A., Record, M. T., Jr., and Wickens, M. P., eds), pp. 25-36, Elsevier Science Publishing Co., Inc., New York
  2. Dykxhoorn, D. M., St. Pierre, R., and Linn, T. (1996) Mol. Microbiol. 19, 483-493[Medline] [Order article via Infotrieve]
  3. Fleischmann, R. D., Adams, M. D., White, O., Clayton, R. A., Kirkness, E. F., Kerlavage, A. R., Bult, C. J., Tomb, J. F., Dougherty, B. A., Merrick, J. M., et al.. (1995) Science 269, 496-512[Medline] [Order article via Infotrieve]
  4. Bult, C. J., White, O., Olsen, G. J., Zhou, L., Fleischmann, R. D., Sutton, G. G., Blake, J. A., FitzGerald, L. M., Clayton, R. A., Gocayne, J. D., Kerlavage, A. R., Dougherty, B. A., Tomb, J. F., Adams, M. D., Reich, C. I., Overbeek, R., Kirkness, E. F., Weinstock, K. G., Merrick, J. M., Glodek, A., Scott, J. L., Geoghagen, N. S. M., Weidman, J. F., Fuhrmann, J. L., Venter, J. C., et al.. (1996) Science 273, 1058-1073[Abstract]
  5. Ovchinnikov, Y. A., Monastyrskaya, G. S., Gubanov, V. V., Guryev, S. O., Chertov, O. Yu., Modyanov, N. N., Grinkevich, V. A., Makarova, I. A., Marchenko, T. V., Polovnikova, I. N., Lipkin, V. M., and Sverdlov, E. D. (1981) Eur. J. Biochem. 116, 621-629[Abstract]
  6. Borodin, A. M., Danilkovich, A. V., Chernov, I., Azhikina, T. L., Rostapshov, V. M., and Monastyrskaya, G. S. (1988) Bioorg. Khim. 14, 1179-1182[Medline] [Order article via Infotrieve]
  7. Pühler, G., Leffers, H., Gropp, F., Palm, P., Klenk, H.-P., Lottspeich, F., Garrett, R. A., and Zillig, W. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4569-4573[Abstract]
  8. Tomb, J. F., White, O., Kerlavage, A. R., Clayton, R. A., Sutton, G. G., Fleischmann, R. D., Ketchum, K. A., Klenk, H. P., Gill, S., Dougherty, B. A., Nelson, K., Quackenbush, J., Zhou, L., Kirkness, E. F., Peterson, S., Loftus, B., Richardson, D., Dodson, R., Khalak, H. G., Glodek, A., McKenney, K., FitzGerald, L. M., Lee, N., Adams, M. D., Venter, J. C., et al.. (1997) Nature 388, 539-547[CrossRef][Medline] [Order article via Infotrieve]
  9. Severinov, K., Mooney, R., Darst, S. A., and Landick, R. (1997) J. Biol. Chem. 272, 24137-24140[Abstract/Free Full Text]
  10. Fox, J. G. (1997) Semin. Gastrointest. Dis. 8, 124-141[Medline] [Order article via Infotrieve]
  11. Eaton, K. A., Dewhirst, F. E., Radin, M. J., Fox, J. G., Paster, B. J., Krakowka, S., and Morgan, D. R. (1993) Int. J. Syst. Bacteriol. 43, 99-106[Abstract]
  12. Akopyants, N. S., Clifton, S. W., Kersulyte, D., Crabtree, J. E., Youree, B. E., Reece, C. A., Bukanov, N. O., Drazek, E. S., Roe, B. A., and Berg, D. E. (1998) Mol. Microbiol. 28, 37-54[CrossRef][Medline] [Order article via Infotrieve]
  13. Goodman, T. G., and Hoffman, P. S. (1983) J. Clin. Microbiol. 18, 825-829[Medline] [Order article via Infotrieve]
  14. Guruge, J. L., Falk, P. G., Lorenz, R. G., Dans, M., Wirth, H. P., Blaser, M. J., Berg, D. E., and Gordon, J. I. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3925-3930[Abstract/Free Full Text]
  15. Xu, Q., Peek, R. M., Jr., Miller, G. G., and Blaser, M. J. (1997) J. Bacteriol. 179, 6807-6815[Abstract]
  16. Lee, A., O'Rourke, J., De Ungria, M. C., Robertson, B., Daskalopoulos, G., and Dixon, M. F. (1997) Gastroenterology 112, 1386-1397[Medline] [Order article via Infotrieve]
  17. Bukanov, N. O., and Berg, D. E. (1994) Mol. Microbiol. 11, 509-523[Medline] [Order article via Infotrieve]
  18. Akopyanz, N., Bukanov, N. O., Westblom, T. U., and Berg, D. E. (1992) Nucleic Acids Res. 20, 6221-6225[Abstract]
  19. Garner, J. A., and Cover, T. L. (1995) J. Infect. Dis. 172, 290-293[Medline] [Order article via Infotrieve]
  20. Paster, B. J., Lee, A., Fox, J. G., Dewhirst, F. E., Tordoff, L. A., Fraser, G. J., O'Rourke, J. L., and Taylor, N. S. (1991) Int J. Syst. Bacteriol. 41, 31-38[Abstract]
  21. Stetter, K. O., and Zillig, W. (1974) Eur. J. Biochem. 48, 527-540[Medline] [Order article via Infotrieve]
  22. Murray, C. L., and Rabinowitz, J. C. (1981) J. Biol. Chem. 256, 5153-5161[Abstract]
  23. Whipple, F. W., and Sonenshein, A. L. (1992) J. Mol. Biol. 223, 399-414[Medline] [Order article via Infotrieve]
  24. Suarez, D. L., Wesley, I. V., and Larson, D. (1997) J. Vet. Microbiol. 57, 325-336 [CrossRef]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.