JOURNAL OF BACTERIOLOGY, July 1982, p. 495-499
`0021-9193/82/070495-05$02.00/0
`
`Vol. 151, No. 1
`
`Identification of the Salmonella typhimurium cysB Gene
`Product by Two-Dimensional Protein Electrophoresis
`EDWARD W. BAPTISTt, SCOTT G. HALLQUIST, AND NICHOLAS M. KREDICH*
`Howard Hughes Medical Institute at Duke University and the Departments of Medicine and Biochemistry,
`Duke University Medical Center, Durham, North Carolina 27710
`
`Received 28 December 1981/Accepted 10 March 1982
`
`Examination of two-dimensional electropherograms of proteins from wild-type
`Salmonella typhimurium and 16 different cysB strains permitted the identification
`of a single 34,500-dalton polypeptide chain with a pl of 7.6 that was the product of
`cysB. Exclusion chromatography indicated that the native cysB protein is a
`multimer of at least two and probably four or more such subunits.
`
`Control of L-cysteine biosynthesis in enteric
`bacteria is achieved both by feedback inhibition
`of serine transacetylase by L-cysteine (10, 11)
`and by a system of gene regulation in which a
`combination of sulfur starvation, O-acetyl-L-
`serine, and the cysB regulatory gene is required
`for derepression (8, 9). Recent investigations in
`Salmonella typhimurium and Escherichia coli
`indicate that cysB consists of a single cistron (2,
`15), which codes for a protein (12, 14) that
`functions as an element of positive control (7).
`In this communication we provide additional
`direct chemical evidence for the protein nature
`and monocistronic origin of the cysB gene prod-
`uct in S. typhimurium by reporting the identifi-
`cation of wild-type and mutant cysB polypeptide
`chains on two-dimensional electropherograms of
`whole-cell extracts.
`Our rationale was to analyze protein extracts
`of various S. typhimurium strains by the two-
`dimensional electrophoresis method of O'Farrell
`(13) and then to identify the wild-type cysB
`polypeptide spot by its absence or altered mobil-
`ity in cysB mutants. Bacteria were grown in a
`sulfate-free minimal salts medium (16) contain-
`ing 5 g of glucose per liter, appropriate supple-
`ments for auxotrophs, and a sulfur source that
`was either 0.5 mM glutathione for derepression
`of the cysteine regulon or 0.5 mM L-cystine for
`repression (9).
`To radiolabel cell proteins, overnight cultures
`were diluted 20-fold into 10 ml of fresh medium
`containing 2 ,uCi of L-14C-amino acids (New
`England Nuclear Corp.) per ml and then incu-
`bated at 37°C with vigorous shaking until density
`reached 0.8 x I0 to 1.0 x 109 cells per ml. After
`centrifugation and one wash with 10 mM Tris-
`hydrochloride (pH 7.6) containing 1 mM MgC92,
`cells were disrupted by sonication in a small
`volume of this same buffer. This crude extract
`
`t Present address: Southern Biotech, Inc., Tampa, FL
`33613.
`
`AcysB403
`cysB+
`
`cysB45
`
`cysB18
`
`cysB484
`
`cysB27
`
`cysB25
`
`cysB12
`
`cysB16
`
`cysB88
`
`cysB1352
`
`TABLE 1. Gene product in cysB mutant strainsa
`Strain or
`Two-dimensional gel
`Sourceb
`cysB allele
`mutation
`protein spot
`cysB+
`Wild type
`Present with normal
`pIC
`Absent
`Present with normal
`pI
`AcysB403
`Absent
`AcysB17S3 Absent
`AcysB1767 Absent
`cysBS17
`Absent
`cysB661
`Absent
`cysB267
`Absent
`cysB70
`Present with basic
`p1 shift
`Present with basic
`pl shift
`Present with basic
`pl shift
`Present with basic
`pl shift
`Present with basic
`pl shift
`Present with acidic
`pl shift
`Present with normal
`pI
`Present with normal
`pI
`Present with normal
`pI
`Present with normal
`pI
`a Cells were grown on minimal medium with re-
`duced glutathione as the sole sulfur source, and ex-
`tracts were analyzed for a cysB spot by two-dimen-
`sional protein electrophoresis. All non-cysB mutants
`tested had a normal cysB spot and included cysA3,
`cysA20, cysA197, cysCD519, cysE2, cysE6, cysE8,
`cysEll, cysE396, cysG439, cysHIJ383, cysK1751 (4),
`cysK1772, and cysMi771 (3).
`b H/S, From either P. Hartman or K. E. Sanderson;
`DU, constructed in this laboratory.
`c In wild type the cysB+ protein spot is "present
`with normal pl" by definition.
`
`cysB403
`DW391
`
`DW392
`DW353
`DW367
`cysBS17
`cysB661
`DW81
`cysB70
`
`cysB45
`
`DW44
`
`DW46
`
`DW75
`
`cysB25
`
`cysB12
`
`DW43
`
`DW79
`
`DW25
`
`H/S
`DU
`
`DU
`(2)
`(2)
`H/S
`H/S
`DU
`H/S
`
`H/S
`
`(9)
`
`(9)
`
`DU
`
`H/S
`
`H/S
`
`(9)
`
`DU
`
`(9)
`
`495
`
`Mylan v. Genentech
`IPR2016-00710
`Merck Ex. 1100, Pg. 1
`
`

`

`496
`
`NOTES
`
`J. BACTERIOL.
`
`SSU:r ~ IASip
`
`cys
`
`I
`
`I
`
`,
`
`I IIII I
`
`5
`
`7
`
`FIG. 1. Two-dimensional electropherogram of proteins from a crude extract of wild-type S. typhimurium
`grown on L-`4C-amino acids and on glutathione as sole sulfur source. The autoradiograph was developed after 7
`days of exposure. The position of the spot corresponding to O-acetylserine sulflhydrylase A (OASS-A) was
`established from gels of purified enzyme. The cysB protein spot is estimated to have a molecular weight of 34,500
`and a pl of 7.6.
`
`was then analyzed on two-dimensional electro-
`phoretic gels with pH 5 to 8 ampholines (LKB
`Instruments, Inc.) in the initial isoelectric focus-
`ing dimension. After fixation and staining with
`
`Coomassie brilliant blue G-250, the gels were
`exposed to Kodak No-Screen X-ray film for 3 to
`14 days before development.
`Gels were run on extracts prepared from wild
`
`Merck Ex. 1100, Pg. 2
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`

`

`VOL. 151, 1982
`
`NOTES
`
`497
`
`A
`
`fr
`
`*.C
`
`.,.
`
`.-.
`
`FIG. 2. Comparison of the region around the cysB spot in wild type and in three different cysB mutants. The
`autoradiographs show the cysB spot to be present in wild type (A); absent in cysB403 (B); present with a more
`basic pl in cysB70 (C); and present with a more acidic pl in cysB25 (D). The cysB70 gel was exposed a shorter
`time than the others, and ordinarily gives an altered cysB spot that is much more intense than that of wild type or
`other mutants.
`
`type, from strains carrying 16 different cysB
`mutant alleles, and from strains carrying muta-
`tions in all known cys structural genes (Table 1).
`Extensive examination of the autoradiograms
`from these gels revealed only a single protein
`spot that could be identified as a product of the
`cysB gene by the following criteria: (i) present in
`wild type and all non-cysB mutant strains tested,
`whether cells were grown under conditions of
`repression or derepression for the cysteine regu-
`Ion; (ii) absent in all of the three cysB deletion
`strains tested as well as in some strains carrying
`different cysB point mutations; (iii) appearance
`in several cysB point mutation strains of a new
`protein spot with the same molecular weight as
`the missing spot, but with a slightly altered pl.
`The position of this protein spot, shown for wild
`type in Fig. 1, corresponded to an apparent pl of
`7.6 and a subunit molecular weight of approxi-
`mately 34,500. The cysB gene product of E. coli
`is reported to have a pI of 7 and a subunit
`molecular weight of 39,000 (12).
`Comparison of the area around the cysB pro-
`tein spot in gels of 16 cysB mutants (Table 1)
`revealed that in all three deletion strains and in
`three point mutants the wild-type cysB spot was
`absent, and no new spot of similar molecular
`weight could be found (Fig. 2B). However, in
`
`five cysB point mutants the wild-type spot was
`also absent, but a new spot with the same
`molecular weight appeared with a basic pI shift
`of 0.15 pH unit (Fig. 2C). Only one mutant,
`cysB25, had a pl shift of 0.15 pH unit toward the
`acidic end of the gel (Fig. 2D). Four cysB point
`mutants could not be distinguished from wild
`type by two-dimensional gel electrophoresis.
`When the isogenic strains DW391 (cysB+) and
`DW392 (cysB403) were compared by this tech-
`nique, they were identical except for the ab-
`sence in DW392 of the cysB protein spot and
`those proteins that were represssed by growth
`on L-cystine in the wild-type strain.
`To estimate the molecular weight of the native
`cysB protein, a crude extract of the cysB+ strain
`DW391 was fractionated with ammonium sul-
`fate, and the material precipitating at 229 mg/ml
`was chromatographed on Ultrogel AcA34 (LKB
`Instruments, Inc.) together with a small amount
`of purified O-acetylserine sulfhydrylase A.
`Semiquantitative analysis of the effluent frac-
`tions by means of two-dimensional gel electro-
`phoresis revealed that the cysB protein was
`eluted from the column at a K, approximately
`one-half that of native O-acetylserine sulfhydry-
`lase A, which is a dimer with a native molecular
`weight of 68,000 (1). This suggests that the
`
`Merck Ex. 1100, Pg. 3
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`

`

`498
`
`NOTES
`
`J. BACTERIOL.
`
`cysB
`
`AcysBI753
`AcyiB767
`FIG. 3. Genetic map of the cysB region showing the relative positions of mutations used in this study. The
`Roman numerals indicate segments defined by various deletions (2). The other numbers indicate mutant cysB
`alleles, which are grouped according to whether they give a cysB polypeptide chain with a normal pl or an
`abnormal pl, or no spot at all. The deletions cysBI753 and cysBI767 give no cysB spot.
`
`native cysB protein is a multimer of at least two,
`and most likely four or more, 34,500-dalton
`subunits, which agrees with data obtained from
`genetic experiments in S. typhimurium (5) and
`with studies on the E. coli cysB protein (12).
`Comparison of the data summarized in Table 1
`with a fine-structure map of the S. typhimurium
`cysB gene (2) shows that both a complete dele-
`tion and a partial deletion extending only a very
`short distance into the trp-proximal end of cysB
`result in loss of the cysB protein spot (Fig. 3).
`The same result was found in strains carrying
`either the internal deletion cysB403 or any of the
`three trp-distal point mutations. Although the
`data are compatible with a monocistronic struc-
`ture for this region, they might also be explained
`by polar effects on a multicistronic cysB. It is
`significant then that point mutations ranging in
`location from the trp-proximal cysB27 to very
`near the opposite end of cysB (e.g., cysB4S and
`cysB484) give cysB spots with altered pI values.
`Therefore, that portion of cysB extending from
`the trp-proximal end to cysB484, which includes
`about 80% of mapped point mutations (2), must
`be a single cistron, and it seems likely that all of
`cysB codes for a single polypeptide chain.
`Jagura-Burdzy and Hulanicka (6) have recent-
`ly reported that in E. coli cysB is autoregulated
`by a process that is independent of whether cells
`are grown under conditions of repression or
`derepression for the cysteine regulon. We too
`noted no appreciable differences in the intensity
`of the wild-type cysB spot between repressed
`and derepressed cells. We did find, however,
`that the cysB70 spot was consistently several-
`fold more intense than any other cysB spot. If
`
`there is a property of the cysB protein that is
`responsible for autoregulation it may be altered
`in such a way in cysB70 to allow overproduction
`of the mutant cysB gene product.
`
`This work was supported by grant AM12828 from the
`National Institute of Arthritis, Diabetes and Digestive and
`Kidney Diseases.
`
`LITERATURE CITED
`1. Becker, M. A., N. M. Kredich, and G. M. Tomkins. 1969.
`The purification and characterization of O-acetylserine
`sulfhydrylase A from Salmonella typhimurium. J. Biol.
`Chem. 244:2418-2427.
`2. Cheney, R. W., Jr., and N. M. Kredich. 1975. Fine-
`structure genetic map of the cysB locus in Salmonella
`typhimurium. J. Bacteriol. 124:1273-1281.
`3. Hulanicka, M. D., S. G. Haliquist, N. M. Kredkch, and T.
`Mojica-A. 1979. Regulation of O-acetylserine sulfhydry-
`lase B by L-cysteine in Salmonella typhimurium. J. Bac-
`teriol. 140:141-146.
`4. Hulanicka, M. D., N. M. Kredich, and D. M. Trelm.
`1974. The structural gene for O-acetylserine sulfhydrylase
`A in Salmonella typhimurium: identity with the trzA
`locus. J. Biol. Chem. 249:867-872.
`5. Jagura, G., D. Hulanicka, and N. M. Kredich. 1978.
`Analysis of merodiploids of the cysB region in Salmonella
`typhimurium. Mol. Gen. Genet. 165:31-38.
`6. Jagura-Burdzy, G., and D. Hulanicka. 1981. Use of gene
`fusions to study expression of cysB, the regulatory gene of
`the cysteine regulon. J. Bacteriol. 147:744-751.
`7. Jones-Mortimer, M. C. 1968. Positive control of sulphate
`reduction in Escherichia coli: the nature of the pleiotropic
`cysteineless mutants of E. coli K12. Biochem. J. 110:597-
`602.
`8. Jones-Mortlmer, M. C., J. F. Wheldrake, and C. A.
`Pasternak. 1968. The control of sulphate reduction in
`Escherichia coli by O-acetyl-L-serine. Biochem. J. 107:
`51-53.
`9. Kredich, N. M. 1971. Regulation of L-cysteine biosynthe-
`sis in Salmonella typhimurium: effects of growth on
`varying sulfur sources and O-acetyl-L-serine on gene
`expression. J. Biol. Chem. 246:3474-3484.
`
`Merck Ex. 1100, Pg. 4
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`VOL. 151, 1982
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`NOTES
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`499
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`10. Kredlch, N. M., M. A. Becker, and G. M. Tomkins. 1969.
`Purification and characterization of cysteine synthetase, a
`bifunctional protein complex, from Salmonella typhimu-
`rium. J. Biol. Chem. 244:2428-2439.
`11. Kredich, N. M., and G. M. Tomkins. 1966. The enzymatic
`synthesis of L-cysteine in Escherichia coli and Salmonella
`typhimurium. J. Biol. Chem. 241:4955-4965.
`12. Masarenhas, D. M., and M. D. Yudkln. 1980. Identifica-
`tion of a positive regulatory protein in Escherichia coli:
`the product of the cysB gene. Mol. Gen. Genet. 177:535-
`539.
`
`13. O'Farreil, P. H. 1975. High resolution two-dimensional
`electrophoresis ofproteins. J. Biol. Chem. 250:4007-4021.
`14. Tully, M., and M. D. Yudkin. 1975. The nature of the
`product of the cysB gene of Escherichia coli. Mol. Gen.
`Genet. 136:181-183.
`15. Tully, M., and M. D. Yudkin. 1977. Fine-structure map-
`ping and complementation analysis of the Escherichia coli
`cysB gene. J. Bacteriol. 131:49-56.
`16. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinase
`of Escherichia coli: partial purification and some proper-
`ties. J. Biol. Chem. 218:97-106.
`
`Merck Ex. 1100, Pg. 5
`
`