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Journal of Bacteriology, December 1999, p. 7285-7290, Vol. 181, No. 23
Department of Bacteriology, University of
Wisconsin-Madison, Madison, Wisconsin 53706
Received 6 July 1999/Accepted 16 September 1999
ApbE is a lipoprotein in Salmonella typhimurium, and
mutants unable to make this protein have a reduced ability to make
thiamine (vitamin B1) and require it as a supplement for
optimal growth in minimal glucose medium. Polyclonal antibodies
specific to ApbE were used to determine that wild-type ApbE is located
exclusively in the inner membrane. The periplasmic, monotopic topology
of ApbE was determined by using computer-based hydrophobicity plots, LacZ and PhoA gene fusions, and proteinase protection experiments. This
extracellular location of ApbE is required for its function, since a
cytoplasmic form (ApbEcyto) did not allow an
apbE mutant to grow in the absence of thiamine. A
periplasmic form of ApbE (ApbEperi) lacking the lipoprotein
modification allowed an apbE mutant to grow in the absence
of thiamine, indicating that soluble ApbE could function in thiamine
synthesis and that lipoation and membrane association were not
required. Alteration of the amino acid implicated in membrane sorting
for other lipoproteins did not result in a relocalization of ApbE to
the outer membrane, suggesting that additional sorting determinants
exist for ApbE.
Thiamine is synthesized in
Salmonella typhimurium by a low-flux biosynthetic pathway
that has recently been shown to be sensitive to a number of subtle
metabolic perturbations (7, 8, 10, 11, 13). This sensitivity
is manifested as a thiamine auxotrophy and has been exploited to
identify new aspects of metabolic integration. Through this approach
integration between thiamine synthesis, carbon catabolism, coenzyme A
biosynthesis, and the redox state of the cell has been uncovered
(7, 8, 10, 11, 13). It was possible to rationalize many of
the metabolic interactions, since precursors to vitamin biosynthetic
pathways are often metabolites diverted from major anabolic-catabolic
pathways (5, 11, 16, 26).
A number of loci that affect thiamine synthesis have been identified
based on a thiamine requirement of the respective mutants. Unlike those
alluded to above, several of these loci were uncharacterized open
reading frames, and thus an explanation for the resulting thiamine
requirement was not readily apparent. Of significance to this study was
the identification of two such loci, rseC (3) and
apbE (4), whose products have been shown to be
membrane proteins. The latter locus encodes a 36-kDa lipoprotein. These loci do not appear to encode biosynthetic enzymes involved directly in
thiamine synthesis, although membrane association of such enzymes might
not be unexpected. Compartmentalization of enzymes to the cytoplasm,
cytoplasmic membrane (the inner or outer face), periplasm, or outer
membrane has been shown to allow closer interactions with substrates,
and such compartmentalization might be warranted when substrate levels
are relatively low, as would be expected in a low-flux pathway such as
those involved in vitamin synthesis (20).
Membrane proteins are involved in a wide range of cellular functions,
including import and export of compounds, sensing environmental cues,
and electron transfer for energy generation and redox catalysis. Lipoproteins represent a specific class of membrane proteins that are
anchored to the lipid bilayer through their lipid modification. ApbE is
the first lipoprotein whose absence results in a nutritional requirement. Although the number of known bacterial lipoproteins has
increased to more than 130, the membrane location, topology, and
function of most lipoproteins remain unknown (24, 28, 31).
Experiments presented here initiate the structural characterization of
the ApbE lipoprotein and reveal it to be completely exposed to the
periplasmic space but anchored to the inner membrane. Additionally, we
show that the function of ApbE in thiamine synthesis is dependent on
its extracellular location. Further, the membrane sorting signals
reported for other lipoproteins are not sufficient to alter the
location of ApbE, suggesting additional determinants contribute to
membrane localization of lipoproteins.
Strains, media, and growth conditions.
All strains used in
this study are derivatives of S. typhimurium LT2 (except
Escherichia coli CC118) and are listed with their genotypes
in Table 1. Tn10d(Tc) refers
to the transposition-defective mini-Tn10 described by Way et
al. (30). Growth curves were performed by using cells that
were grown overnight in rich medium and then resuspended in saline.
Cell suspensions (10 µl) were used to inoculate 190 µl of medium in
individual wells of a 96-well microtiter dish. Growth was monitored as
the increase in absorbance at 650 nm by using a SpectraMAX Plus
Microplate Spectrophotometer (Molecular Devices, Sunnyvale, Calif.).
The culture plate was maintained at 37°C with periodic shaking for
aeration. NCE medium supplemented with MgSO4 (1 mM)
(9) and glucose (11 mM) was used as minimal medium. Difco
nutrient broth (8 g/liter) with NaCl (5 g/liter) added was used as rich
medium. Difco BiTek agar was added to a final concentration of 1.5%
for solid medium. Unless otherwise stated, the final concentrations of
adenine and thiamine were 0.4 mM and 100 nM, respectively. The final
concentrations of antibiotics in rich/minimal medium were as follows:
tetracycline (20/10 µg/ml), kanamycin (50/125 µg/ml), ampicillin
(30/15 µg/ml), and chloramphenicol (20/4 µg/ml).
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Periplasmic Location Is Essential for the Role of
the ApbE Lipoprotein in Thiamine Synthesis in Salmonella
typhimurium
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
TABLE 1.
Strains and plasmids
Plasmid construction. (i) Cytoplasmic ApbE (ApbEcyto). The coding sequence of the apbE gene excluding the signal sequence (amino acids 1 to 20) was amplified from the S. typhimurium LT2 chromosome by standard PCR techniques. An NdeI site was designed into the upstream (5'-end) primer and the downstream (3'-end) primer contained the stop codon for apbE. After blunt-end ligation into pSU19 (pApbE9) the 1.1-kb NdeI-HindIII fragment was cloned into pET20b (Novagen, Madison, Wis.), yielding pApbE10. The full-length apbE gene was cloned into pET20b by a similar method with a distinct upstream primer and resulted in pApbE12.
(ii) Periplasmic ApbE (ApbEperi). The coding sequence of the apbE gene that corresponded to amino acid positions 20 to 350 was amplified from the S. typhimurium chromosome by using standard PCR techniques. The upstream (5') primer contained a PstI site which replaced the cysteine at position 20 with an alanine; the downstream (3') primer was as described above. The amplified product was ligated into the SmaI site of pSU19, yielding pApbE(C20A). The ompF sequence for the OmpF-ApbE fusion was generated by annealing complementary sequences of the OmpF signal peptide that contained NdeI and PstI sites. The DNA encoding the OmpF signal peptide was ligated into the SmaI site of pSU19, yielding pOmpF. A 0.9-kb PstI fragment from pApbE(C20A) was ligated into pOmpF that had been digested with PstI. Sequence analysis was used to confirm a clone with the desired ompF::apbE(C20A) fusion. This fusion was then cloned into pT7-7 as a NdeI-HindIII fragment, resulting in pApbE14.
(iii) ApbE(D21S). The aspartate residue at position 21 of ApbE (+2 in the processed protein) was changed to a serine by the megaprimer method of Barik (1). Chromosomal DNA from S. typhimurium LT2 was used as the DNA template in this two-step amplification method. All plasmid constructs were confirmed by sequence analysis.
Construction of ApbE-His6 and Western analysis. A C-terminal His6 fusion to ApbE lacking the leader sequence was constructed by standard PCR amplification with primers specific to the ends of apbE. The appropriate fragment was cloned into the NdeI-HindIII sites of pET20b and protein expression was confirmed utilizing methods described by the manufacturer (Novagen). Purification of ApbE-His6 was achieved by passing extracts expressing the fusion protein over an Ni2+-affinity column (Novagen). ApbE-His6 was obtained in >90% homogeneity by purification under denaturing conditions. Rabbit polyclonal antibodies against ApbE-His6 were generated at the animal care unit of the University of Wisconsin Medical School. Antisera was prepared and titered according to the method of Harlow and Lane (14). Standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used and Western analysis were performed as described previously with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (Promega, Madison, Wis.) used to detect anti-ApbE bound to the membrane support (14).
Isolation of apbE::phoA
fusions.
Plasmid pApbE1 was introduced into E. coli
CC118. The method of Manoil and Beckwith (17) was used to
isolate alkaline phosphatase (phoA) gene fusions to
apbE, in addition to unsuccessful attempts to isolate
-galactosidase (lacZ) gene fusions to the same gene. PhoA
insertions in the apbE gene were identified on agar plates supplemented with 40 µg of the chromagenic indicator XP
(5-bromo-4-chloro-3-indolyl phosphate; Fluka, Milwaukee, Wis.) per ml.
The location of the insertions was estimated by PCR amplification with
a primer specific to the sequence upstream of the 5' end of the
apbE gene (5'-TATTCCGGCGTACAAATACG-3') and a
primer that annealed within the TnphoA sequence
(5'-AATATCGCCCTGAGCAGCCCG-3'). These primers were also used
to sequence the junction of the fusions and thus precisely determine
the location of the fusion. The alkaline phosphatase activities of the
E. coli CC118 cells expressing ApbE::PhoA fusions
were determined by the procedure described by Brickman, with a control
strain containing no fusion set to zero (6).
Protease accessibility of ApbE in intact spheroplasts. The accessibility of ApbE from S. typhimurium LT2 whole cells, spheroplasts (generated as described by Osborn and Munson [22]), and lysed spheroplasts to proteinase K was determined by a modification of the method of Randall and Hardy (25). After a 20-min treatment with proteinase K (100 µg/ml) at 4°C, protein was precipitated with 5% trichloroacetic acid. Each sample (equivalent to 0.05 A650) was analyzed by SDS-PAGE and Western hybridization.
Protein analysis and cell fractionation. Separation of cell protein into cytoplasmic and total membrane fractions and subsequently into inner and outer membrane fractions was carried out as described by Osborn and Munson (22). Briefly, cells were grown to mid-log phase (ca. 55 Klett units [red filter]), spheroplasted, and lysed by osmotic shock. Low-speed centrifugation was used to remove unbroken cells, and then the supernatant was centrifuged at 40,000 rpm for 2 h in a Beckman 70 Ti rotor. The pellet was saved as the total membrane fraction, and the supernatant contained the cytoplasmic-periplasmic fraction. The total membrane fraction was loaded onto a 50 to 35% stepwise sucrose gradient with a 55% sucrose cushion and centrifuged at 36,000 rpm for 16 h in a Beckman SW40 Ti rotor. Fractions were collected dropwise, and the location of the inner and outer membranes within the gradient was visualized by Coomassie staining and confirmed by detecting NADH oxidase activity (29) and 2-keto-deoxyoctonate (data not shown) (15), respectively.
Isolation of periplasmic and cytoplasmic fractions was performed by a modification of the osmotic shock procedures developed by Neu and Heppel (21) and Thorstenson et al. (29). A 5-ml culture was grown in minimal medium to mid-log phase, and 1.0 ml of the cells was resuspended in 100 µl of 0.5 M sucrose-0.2 M Tris-0.5 mM EDTA and kept on ice for 15 min. Then, 400 µl of TE (10 mM Tris [pH 8.0], 1 mM EDTA) was mixed into the suspension, which was kept on ice for 30 min. The cells were pelleted, and the supernatant was saved as the periplasmic fraction, while the pellet was further processed. The cell pellet was washed and resuspended in 0.1 M sucrose-40 mM Tris-0.1 mM EDTA and sonicated for 10 s at a 50% duty cycle by using a model 550 sonic dismembrator (Fisher, Itasca, Ill.), and a low-speed centrifugation was used to remove the unbroken cells. The supernatant was centrifuged at 100,000 rpm for 30 min in a Beckman TLA 100.2 rotor. The supernatant contained the cytoplasmic fraction, and the pellet was washed once with TE buffer to remove any contaminating periplasmic and peripheral proteins and then pelleted again. The final pellet was saved as the total membrane fraction. The relative purity of the periplasmic and membrane fractions was determined by using-lactamase or NADH oxidase, respectively, as marker enzymes.
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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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ApbE is located in the inner membrane. Our characterization of ApbE was driven by two assumptions: (i) by understanding its location and structural features, we could better address the role of this protein in thiamine synthesis, and (ii) we could further the general understanding of bacterial lipoproteins by topological and mutational characterization of another lipoprotein. Analysis of ApbE sequence in the context of characterized lipoproteins suggested that the mature protein would be lipoated and localized to the inner membrane. Immunoblot analyses were performed on wild-type and mutant strains to validate and extend this prediction. Comparison of whole-cell extracts of strain DM271(apbE) and wild-type strain LT2 blotted with antibodies specific to ApbE identified an appropriately sized band that was detected in the LT2 extract but not in extracts from the apbE mutant strain (data not shown). From these comparisons, we concluded this band corresponded to ApbE.
Western blot analyses with LT2 with ApbE-specific antisera showed that ApbE clearly separated with the membrane fraction, and none was detected in the soluble fraction (Fig. 1A). When the membrane was separated into inner and outer membrane fractions, ApbE was found exclusively in the inner membrane. Biochemical characterization of the membrane separation is documented in Fig. 1B and is representative of the separations in subsequent experiments.
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ApbE is exposed on the periplasmic face of the inner membrane. Although the lipoated N terminus of ApbE would be expected to be located in the inner membrane (31), the presence of a cytoplasmic domain was tested. Based on analyses with a number of computer programs (i.e., TMPRED, TMAP, and Kyte-Doolittle), ApbE does not have an apparent membrane-spanning region (data not shown), suggesting that the entire ApbE protein was located in the periplasm with the fatty acids tethering it to the cell membrane.
Two lines of genetic evidence confirmed that ApbE did not contain a cytoplasmic domain. Attempts to isolate translational fusions between ApbE and LacZ, producing functional-galactosidase that would
indicate a cytoplasmic domain, were unsuccessful. Conversely, five
active PhoA fusions to ApbE were isolated, and the fusion junctions
determined by DNA sequencing (Fig. 2A).
Since the activity of alkaline phosphatase requires the protein to be
extracellular, these results were consistent with the topology
predicted by computer analysis.
|
) proteinase K (100 µg/ml). The proteins in each
sample (equivalent to 0.05 A650) were separated
by SDS-PAGE and immunoblotted with a 1:5,000 dilution of ApbE
antisera.
The periplasmic location of ApbE is required for its function. Having identified the subcellular location of ApbE, we took advantage of the nutritional defect of an apbE mutant to explore the correlation of function with location. The extent of the nutritional defect caused by an apbE mutation is dependent on both the genetic background and the carbon source in the growth medium (4). Specifically, when there is low carbon flux through the purine biosynthetic pathway (e.g., purF), ApbE is required for thiamine synthesis regardless of the carbon source, hence its apb designation (23). In the nutritional studies described here, we took advantage of the fact that apbE mutations also result in a thiamine auxotrophy in an otherwise wild-type genetic background when glucose is the sole carbon and energy source.
The first experiment tested whether the role of ApbE in thiamine synthesis required export to the membrane or whether the protein might have a cytoplasmic function prior to its export. Two strains, carrying either pApbE10 (expressing ApbE without the signal peptide sequence and lipoation site) or pApbE12 (encoding the full-length ApbE protein) as the only source of ApbE, were assessed for their growth in the absence of thiamine and the cellular location of the respective plasmid-encoded ApbE. Data from Western blot analysis (Fig. 3B) confirmed that when the signal peptide sequence was removed (pApbE10) ApbE was located completely in the cytoplasm, whereas wild-type ApbE (pApbE12) was located solely in the membrane fraction. Significantly, the growth data in Fig. 3A showed that the leaderless construct was unable to restore thiamine-independent growth to an apbE mutant, while the wild-type protein completely complemented the growth defect (Fig. 3A, left panel). While we cannot eliminate the possibility that the cytoplasmic ApbE was partially functional, it did not result in significantly more thiamine-independent growth than strain DM271 carrying the vector alone (data not shown).
|
), or
pApbE10, encoding ApbE without the lipoprotein signal peptide (
), is
shown. Cells were grown in minimal medium with glucose as the sole
carbon and energy source and supplemented with 100 nM thiamine where
indicated. (B) Immunoblot showing the cellular location of ApbE
produced by pApbE10 and pApbE12. Cellular fractions were isolated as
described in Materials and Methods. Abbreviations: WC, whole-cell
fraction; SOL, soluble cytoplasmic and periplasmic proteins; TM, total
membrane proteins.
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The +2 amino acid is not sufficient to determine membrane target. An extension of the above experiment was to determine whether ApbE functioned when transported to the outer membrane. Such a result would impact the kind of models we could consider for the function of ApbE in thiamine synthesis. As noted previously, ApbE contains an aspartate residue at the +2 position in the processed protein (position 21 in the full-length protein). The lipoprotein studies of Yamaguchi et al. (32), Gennity and Inouye (12), and Matsuyama et al. (18, 19) predicted that substitution of a serine residue for this aspartate would target ApbE to the outer membrane. Construction of such a mutant was done to (i) test the significance of this consensus amino acid in another lipoprotein and (ii) if possible address the function of ApbE located in the outer membrane. Oligonucleotide-directed site-specific mutagenesis was used to generate a serine-to-aspartate substitution at position 21 in the full-length protein (+2 in the mature protein), and the resulting mutant ApbE protein was designated ApbE(D21S). The gene encoding the ApbE(D21S) mutant protein was cloned into pSU19 (2).
Growth analyses determined that ApbE(D21S) fully restored thiamine-independent growth to strain DM271(apbE). This result demonstrated that the D21S substitution did not significantly impair the function of the protein compared to wild-type ApbE (µ = 0.45 versus 0.53 h
1, respectively, in the absence of
thiamine). Unexpectedly, Western blot analyses showed that ApbE(D21S)
was located only in the inner membrane, a location indistinguishable
from the location of the wild-type protein (data not shown). The fact
that no ApbE(D21S) was detected in the outer membrane demonstrated
that, in contrast to some other lipoproteins, in ApbE the +2 position
is not the determinant for outer membrane targeting. Efforts by Gennity
and Inouye (12) indicate that the amino acids adjacent to
position +2 (positions +3 and +4) may influence its role as the sorting determinant. Specifically, a glutamate at position +3 was observed to
cause minor but significant inner membrane retention of
Lipo--lactamase(E3), a hybrid lipoprotein containing a serine at
position +2. Since ApbE has a glutamate at position +3, the effect of
the serine at position +2 could be minimized, but the complete lack of
movement to the outer membrane strongly suggests that there are
additional structural determinants involved in the inner membrane
localization of ApbE.
Conclusions. At present our thinking is that ApbE participates in thiamine synthesis indirectly, possibly as part of a complex that mediates a redox-sensitive step in thiamine synthesis. This thinking has been influenced by several recent results. First, RnfF, a membrane protein from Rhodobacter capsulatus, contains sequences orthologous to both ApbE and RseC, an additional inner membrane protein required for optimal thiamine synthesis in S. typhimurium (3). The function of RnfF in R. capsulatus physiology remains unclear but has been suggested to anchor a membrane-associated protein complex that passes electrons to nitrogenase for nitrogen fixation (27). Our finding that RnfF can complement both apbE and rseC mutants (data not shown) makes it tempting to suggest that ApbE and/or RseC may associate and perform a function similar to that of RnfF. Second, recent work in the lab has identified additional loci affecting thiamine synthesis that are involved in maintaining the redox environment of the cell. Lastly, growth under anaerobic conditions eliminates the need for ApbE in thiamine synthesis.
ApbE belongs to a growing class of proteins that are required for thiamine synthesis under conditions that strain this biosynthetic pathway (e.g., low flux through the purine biosynthetic pathway). Thus, these proteins may have a role in thiamine synthesis that can be satisfied by partially redundant cellular functions when substrates are plentiful. We suggest that many of the unknown open reading frames in various genome sequences may encode similar redundancies and that their metabolic role may be uncovered only when the relevant pathways are required to function efficiently. Current work in the lab seeks to address the specific thiamine biosynthetic enzyme(s) affected in apbE mutants and use this knowledge to identify the biochemical role of ApbE.| |
ACKNOWLEDGMENTS |
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This work was supported by Hatch grant WIS3734 from the U.S. Department of Agriculture and by National Institutes of Health grant GM47296 to D.M.D.
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FOOTNOTES |
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* Corresponding author. Mailing address: University of Wisconsin-Madison, Department of Bacteriology, 1550 Linden Dr., Madison, WI 53706. Phone: (608) 265-4630. Fax: (608) 262-9865. E-mail: downs{at}bact.wisc.edu.
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REFERENCES |
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|
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
|
|---|
| 1. | Barik, S. 1995. Site-directed mutagenesis by double polymerase chain reaction. Mol. Biotechnol. 3:1-7[Medline]. |
| 2. | Bartolomé, B., Y. Jubete, E. Martinez, and F. de la Cruz. 1991. Construction and properties of a family of pACYC184-derived cloning vectors compatible with pBR322 and its derivatives. Gene 102:75-78[Medline]. |
| 3. |
Beck, B. J.,
L. E. Connolly,
A. de las Penas, and D. M. Downs.
1997.
Evidence that rseC, a gene in the rpoE cluster, has a role in thiamine synthesis in Salmonella typhimurium.
J. Bacteriol.
179:6504-6508 |
| 4. |
Beck, B. J., and D. M. Downs.
1998.
The apbE gene encodes a lipoprotein involved in thiamine synthesis in Salmonella typhimurium.
J. Bacteriol.
180:885-891 |
| 5. | Begley, T. P., D. M. Downs, S. Ealick, F. McLafferty, D. van Loon, S. Taylor, H. Chiu, C. Kinsland, J. Reddick, J. Xi, and N. Campobasso. 1999. Thiamin synthesis in prokaryotes. Arch. Microbiol. 171:293-300[Medline]. |
| 6. |
Brickman, E., and J. Beckwith.
1975.
Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and  80 transducing phages.
J. Mol. Biol.
96:307-316[Medline].
|
| 7. | Christian, T., and D. M. Downs. 1999. Defects in pyruvate kinase cause a conditional increase of thiamine synthesis in Salmonella typhimurium. Can. J. Microbiol. 45:1-7[Medline]. |
| 8. | Claas, K., S. Weber, and D. M. Downs. Mutants defective in the energy conserving NADH dehydrogenase are unable to utilize the oxidative pentose phosphate pathway. Submitted for publication. |
| 9. | Davis, R. W., D. Botstein, and J. R. Roth. 1980. Advanced bacterial genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y |
| 10. |
Enos-Berlage, J. L., and D. M. Downs.
1997.
Mutations in sdh (succinate dehydrogenase genes) alter the thiamine requirement of Salmonella typhimurium.
J. Bacteriol.
179:3989-3996 |
| 11. |
Frodyma, M., and D. M. Downs.
1998.
The panE gene, encoding ketopantoate reductase, maps at 10 minutes and is allelic to apbA in Salmonella typhimurium.
J. Bacteriol.
180:4757-4759 |
| 12. |
Gennity, J. M., and M. Inouye.
1991.
The protein sequence responsible for the lipoprotein membrane localization in Escherichia coli.
J. Biol. Chem.
266:16458-16464 |
| 13. | Gralnick, J., and D. M. Downs. Unpublished data. |
| 14. | Harlow, E., and D. Lane. 1988. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y |
| 15. | Keleti, G., and W. H. Lederer. 1974. Handbook of micromethods for the biological sciences. Van Nostrand Reinhold Co., New York, N.Y |
| 16. |
Lam, H. M., and M. E. Winkler.
1990.
Metabolic relationships between pyridoxine (vitamin B6) and serine biosynthesis in Escherichia coli K-12.
J. Bacteriol.
172:6518-6528 |
| 17. |
Manoil, C., and J. Beckwith.
1986.
A genetic approach to analyzing membrane protein topology.
Science
233:1403-1408 |
| 18. | Matsuyama, S., T. Tajima, and H. Tokuda. 1995. A novel periplasmic carrier protein involved in the sorting and transport of Escherichia coli lipoproteins destined for the outer membrane. EMBO J. 14:3365-3372[Medline]. |
| 19. | Matsuyama, S., N. Yokota, and H. Tokuda. 1997. A novel outer membrane lipoprotein, LolB (HemM) involved in the LolA (p20)-dependent localization of lipoproteins to the outer membrane of Escherichia coli. EMBO J. 16:6947-6955[Medline]. |
| 20. | Mayer, F. 1993. "Compartments" in the bacterial cell and their enzymes. ASM News 59:346-350. |
| 21. |
Neu, H. C., and L. A. Heppel.
1965.
The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts.
J. Biol. Chem.
240:3685-3692 |
| 22. | Osborn, M. J., and R. Munson. 1974. Separation of the inner (cytoplasmic) and outer membranes of gram-negative bacteria. Methods Enzymol. 31:642-652[Medline]. |
| 23. | Petersen, L. A., J. E. Enos-Berlage, and D. M. Downs. 1996. Genetic analysis of metabolic crosstalk and its impact on thiamine synthesis in Salmonella typhimurium. Genetics 143:37-44[Abstract]. |
| 24. |
Pugsley, A. P.
1993.
The complete general secretory pathway in gram-negative bacteria.
Microbiol. Rev.
57:50-108 |
| 25. | Randall, L. L., and S. J. S. Hardy. 1986. Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in E. coli. Cell 46:921-928[Medline]. |
| 26. | Rondon, M. R., J. R. Trzebiatowski, and J. C. Escalante-Semerena. 1997. Biochemistry and molecular genetics of cobalamin biosynthesis, vol. 56. Academic Press, Inc., New York, N.Y |
| 27. | Schmehl, M., A. Jahn, A. Meyer zu Vilsendorf, S. Hennecke, B. Masepohl, M. Schuppler, M. Marxer, J. Oelze, and W. Klipp. 1993. Identification of a new class of nitrogen fixation genes in Rhodobacter capsulatus: a putative membrane complex involved in electron transport to nitrogenase. Mol. Gen. Genet. 241:602-615[Medline]. |
| 28. |
Sutcliffe, I. C., and R. R. B. Russell.
1995.
Lipoproteins of gram-positive bacteria.
J. Bacteriol.
177:1123-1128 |
| 29. |
Thorstenson, Y. R.,
Y. Zhang,
P. S. Olson, and D. Mascarenhas.
1997.
Leaderless polypeptides efficiently extracted from whole cells of osmotic shock.
J. Bacteriol.
179:5333-5339 |
| 30. | Way, J. C., M. A. Davis, D. Morisato, D. E. Roberts, and N. Kleckner. 1984. New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene 32:369-379[Medline]. |
| 31. | Wu, H. C. 1996. Biosynthesis of lipoproteins, 2nd ed., vol. 1. ASM Press, Washington, D.C. |
| 32. | Yamaguchi, K., F. Yu, and M. Inouye. 1988. A single amino acid determinant of the membrane localization of lipoproteins in E. coli. Cell 53:423-432[Medline]. |
| 33. | Zgurskaya, H. I., and H. Nikaido. 1999. ArcA is a highly asymmetric protein capable of spanning the periplasm. J. Mol. Biol. 285:409-420[Medline]. |
Journal of Bacteriology, December 1999, p. 7285-7290, Vol. 181, No. 23
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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(2003). Lack of the ApbC or ApbE Protein Results in a Defect in Fe-S Cluster Metabolism in Salmonella enterica Serovar Typhimurium. J. Bacteriol.
185: 98-106
[Abstract] [Full Text] -
Gralnick, J., Downs, D.
(2001). Protection from superoxide damage associated with an increased level of the YggX protein in Salmonellla enterica. Proc. Natl. Acad. Sci. USA
10.1073/pnas.151243198v1
[Abstract] [Full Text] -
Gralnick, J., Webb, E., Beck, B., Downs, D.
(2000). Lesions in gshA (Encoding gamma -L-Glutamyl-L-Cysteine Synthetase) Prevent Aerobic Synthesis of Thiamine in Salmonella enterica Serovar Typhimurium LT2. J. Bacteriol.
182: 5180-5187
[Abstract] [Full Text] -
Gralnick, J., Downs, D.
(2001). Protection from superoxide damage associated with an increased level of the YggX protein in Salmonella enterica. Proc. Natl. Acad. Sci. USA
98: 8030-8035
[Abstract] [Full Text]
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