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ACKNOWLEDGEMENTS
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I would like to express my gratitude to the members of my committee whose advise and
encouragement have made this dissenation possible. Special thanks go to Dr. Jeter for
enduring five years of friendly irreverence. Thanks to Dr. Faust for introducing reality into
my experiments and for teaching me the true value of controls. You have all set standards
which have been difficult to achieve, and your expectations have been most flattering.
Collectively, you have led by example and you have my utmost respect as scientists and
individuals.
I would like to thank my wife Paige, who has contributed the most to this dissertation.
She has spent many nights alone and has patiently endured rambling conversations
regarding such obscure things as picomole ratios. Her understanding and support have
been the most important factors in the completion of this work.
Finally I would like to thank Karin Otto who has been much more than a lab panner.
You provided a sympathetic ear when the experiments would not work and served as a
friendly source of competetion. You and Elmer are treasured friends in spite of your
extremely poor taste in football teams.
11
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS
ii
ABSTRACT
v
LIST OF TABLES
vi
LIST OF FIGURES
vii
CHAPTER
I. LITERATURE REVIEW
1
Discovery of Lipoic Acid
1
Chemical Properties of Lipoic Acid
1
Lipoic Acid Dependent Biological Functions
2
Lipoic Acid Biosynthesis
4
Transport and Cellular Content of Lipoic Acid
5
Genetics of Lipoic Acid Biosynthesis
6
Research Objectives
6
References
16
II. REGULATION OF LIPOIC ACID BIOSYNTHESIS
m.
19
Introduction
19
Methods
20
Results
24
Discussion
28
References
49
CHARACTERIZATION OF lip GENE STRUCTURE
51
Introduction
51
Methods
52
iii
Results
61
Discussion
67
References
87
IV
ABSTRACT
Strains of the enteric bacterium Salmonella typhimurium which have an
auxotrophic requirement for lipoic acidwere isolated by mutagenesis with the
transposable element Mu dJ. The chromosomal location of these insertion mutations
was determined to be at 14 minutes by bacteriophage P22-mediated cotransduction.
The lip gene is transcribed in the clockwise direction with respect to the S.
typhimurium genetic map. Strains with lipvAac operon fusions were used to
characterize the transcriptional activity of the lip promoter. Transcription of the lip
gene is not regulated by catabolite repression or lipoic acid concentration. The data
indicate that the lip gene product is expressed constitutively at low level. An S.
typhimurium genomic library was constructed in the X replacement vector EMBL4 and
screened for clones which contain the lip biosynthetic gene. The lip gene was located
on a 1.3 kbp EcoR I fragment. The nucleotide sequence of this fragment was
determined in order to define the structural components of the lip gene. A contiguous
open reading frame was identified which encodes a polypeptide with a deduced
molecular mass of 22,247 daltons. Sequences with similarity to the -10 and -35
consensus sequences and a Shine-Dalgamo ribosomal binding site were also
identified.
LIST OF TABLES
2.1
Bacterial strains used in this study
30
2.2
Growth phenotypes of lip insertion mutants
32
2.3
6-Galactosidase specific activities of lipwlac operon fusions in
culture media designed to test the hypothesis that the lip gene is
regulated by catabolite repression.
33
2.4
6-Galactosidase specific activities of lipwlac operon fusions in
various culture media
34
3.1
List of bacterial strains used in this study
73
3.2
Deduced amino acid composition of the 22,247 dalton protein
encoded by the open reading frame ORF-2
74
VI
LIST OF FIGURES
1.1
1.2
Graphical representation of the interplanar bond angles formed
by disulfides.
8
Reaction sequence of the oxidative decarboxylation of pyruvate
by the pyruvate dehydrogenase complex illustrating the cofactor
role of lipoic acid.
10
Reaction sequence of the glycine cleavage reaction illustrating
the cofactor role of lipoic acid.
12
The proposed biosynthetic pathway for the de novo synthesis
of lipoic acid.
14
2.1
Growth curves of RTl 142.
35
2.2
Growth curves of RTl 148.
37
2.3
Cotransductional mapping of the lip insertion mutations with
regional genetic markers in the 14 minute region of the
S. typhimurium genome.
1.3
1.4
39
2.4
Effect of lipoic acid depletion on lip transcription.
41
2.5
Effect of excess concentrations of lipoic acid on lip transcription
43
2.6
Division of 6-galactosidase activities of strain RTl 142 and
RTl 148 into statistical groups by Student-Newman-Keuls
analysis of ordered means
45
2.7
Statistical analysis of the relationship between growth rate
and lip transcriptional activity.
47
3.1
Restriction digest of 0Eclip-4 DNA.
75
3.2
Restriction map of the 16 kbp genomic insert contained
in 0EcUp-4.
77
3.3
Restriction digest of recombinant pIBI30 plasmids.
79
3.4
Schematic representation of pVJl, which contains
the 1.3 kbp EcoK I insert.
81
vu
3.5
3.6
Sequencing strategy for obtaining the nucleotide sequence
of the lip gene by using as templates plasmids containing
nested deletions.
83
Composite nucleotide sequence of the 1.3 kbp EcoR I
genomic insert and the amino acid sequence of the protein
deduced form the open reading frame ORF-2.
85
viu
CHAPTER I
LITERATURE REVIEW
Discovery of Lipoic Acid
In 1950, Kline and Barker (1950) reported the existence of an unidentified factor
which the bacterium Butyribacterium rettgeri required for growth on minimal medium with
lactic acid as a sole source of carbon. This growth factor, designated B.R. Factor, was
found to be most abundant in acid hydrolysates of yeast extract. Reed et al (1951b)
showed that some biological preparations contained a substance which could replace
sodium acetate in stimulating the growth of lactic acid bacteria. This acetate-replacing
factor was found to be 440 times more active than an equivalent weight of acetate. In
1951, Lester Reed and his coworkers (Reed et ai, 1951a) processed the equivalent of 10
tons of beef Hver to produce 30 mg of a crystalline organic acid, designated a-lipoic acid,
which was highly active in acetate-replacement and pyruvate oxidation factor assays. The
following year, Kline (1952) demonstrated that B. R. Factor could be replaced by lipoic
acid.
Chemical Properties of Lipoic Acid
Lipoic acid (l,2-dithiolane-3-valeric acid) is an 8-carbon fatty acid chain that
terminates in a 6, 8-dithiolane group (Bullock et al., 1952). The disulfide moiety
O
II
CH^
/ CH^
\2/
\ 2 / CH^
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2
of open-chain disulfides that are free of strain form interplanar bond angles of 90^
(Schmidt etal., 1954) (Figure 1.2). In this configuration there is minimal repulsion
between lone P2 electron pairs, which allows them to freely associate with D level
electrons and gives the disulfide bond a double-bonded character {Pn-^n bonding)
(Schmidt et al., 1954). By contrast, the interplanar bond angles formed by sulfiu"
atoms in a dithiolane ring are reduced to 27^ (Figure 1.1). In this configuration the
repulsion between P2 electron pairs is at a maximum, which eliminates the doublebonded nature of the disulfide bond (Schmidt et al., 1954). The overall effect of the
ring strain is to destabilize the disulfide bond and increase chemical reactivity. During
nucleophilic attack on the disulfide bond, the dithiolane ring participates 10^ times
faster than disulfides with a linear arrangement (Schmidt et al., 1954). Upon the
opening of the dithiolane ring by nucleophilic attack, it passes through a transition
state in which the interplanar bond angles are 92°. This transition state has a greater
similarity to the ground state than the transition state of linear disulfides, and the
activation entropy is greatly reduced (Schmidt et al., 1954).
Lipoic Acid-Dependent Biological Functions
Lipoic acid participates in a variety of biochemical reactions, most notably in the
oxidative decarboxylation of pyruvate to acetate by the pyruvate dehydrogenase
enzyme complex (Reed et al., 1951a). Lipoic acid is enzymatically activated through
the formation of an amide linkage to the epsilon amino group of a specific lysine
residue on the dihydrolipoyl transacetylase subunit of the apoenzyme. This covalent
attachment creates a swinging arm 14 angstroms long with the dithiolane functional
group at the distal end. The mobility of the lipoic acid moiety allows the dithiolane ring
to contact the catalytic sites of the three enzyme subunits and participate in a cycle of
in the a-ketoglutarate dehydrogenase enzyme complex and the branched chain a-keto acid
dehydrogenase complexes.
Lipoic acid plays a critical acyl transfer role in the enzymatic cleavage of glycine to
ammonia, carbon dioxide, and A^,Nl0.jiiethylenetetrahydrofolate (Fujiwara etal, 1986a;
Fujiwara etal, 1986b; Kiochi and Kikuchi, 1976; Plamann etal, 1983)(Figure 1.3).
Lipoic acid is attached to the E2 subunit (also referred to as H-protein) of a four-protein
complex at a ratio of one lipoic acid molecule per E2 polypeptide (Kiochi and Kikuchi,
1976). This E2 subunit works in conjunction with the pyridoxal-containing subunit (El or
P-protein) to decarboxylate glycine, during which the lipoamide moiety is reductively
acylated with the decarboxylation product. The lipoamide-bound intermediate is
decomposed to ammonia and /V^,/VlO-methylenetetrahydrofolate through the catalytic
activity of the E3 polypeptide (T-protein). The reduced lipoamide is subsequently
reoxidized by a lipoamide dehydrogenase (E4 subunit or L-protein) and NAD"*" (Kiochi and
Kikuchi, 1976).
Lipoic acid may also participate in the binding protein-dependent transport of ribose,
galactose and maltose. Richarme (1985) demonstrated that the transport activities of these
carbohydrates were inhibited by lipoic acid deprivation in a lipoic acid auxotroph.
Arsenite, which inhibits thiol functions (Knowles and Benson, 1983), was shown to
inhibit transport activities in a lipoic acid auxotroph when supplemented with lipoic acid but
not when deprived of lipoic acid. Additionally, the compound 5-methoxyindole-2carboxylic acid produced complete inhibition of these sugar transport systems. The
compound 5-methoxyindole-2-carboxylic acid inhibits lipoamide dehydrogenase function
of the a-keto acid dehydrogenases (Hanson et al, 1969). These data suggest that lipoic
acid may participate in sugar transport as a bound enzyme cofactor in conjunction with
lipoamide dehydrogenase activity (Richarme, 1985; Richarme and Heine, 1986).
Lipoic Acid Biosynthesis
The enzyme activities responsible for the synthesis of lipoic acid are unknown,
although several key biosynthetic intermediates have been demonstrated by in vivo isotope
labeling experiments in bacteria (Eisenberg, 1988; Johnson and Collins, 1973) and rat liver
(Dupre et al., 1980). These studies were conducted by feeding either stable or radioactive
isotopic precursors to the cultures, followed by extraction and analysis of the lipoic acid
product (Parry, 1983; White, 1980b). The results indicate that octanoic acid serves as the
immediate precursor for the 8-carbon chain. Sulfur is directly inserted at the C-6 and C-8
positions with inversion of configuration at the prochiral center of the C-6 position. It has
not been determined whether the two sulfur atoms are inserted simultaneously or
sequentially. Cysteine appears to serve as the source of sulfur (Dupre et al., 1980). The
proposed biosynthetic reaction is indicated in Figure 1.4 (Eisenberg, 1988).
The enzymatic mechanism by which the thiolation of octanoic acid occurs is an
enigma. The most plausible mechanism would be one analogous to the conversion of
serine into cysteine with sulfide. This type of mechanism requires molecular oxygen to
form a hydroxylated intermediate (White, 1980b; Kusunose et al., 1964; Miura and Fulco,
1974). However, 8-hydroxyoctanoic acid is not a precursor of lipoic acid and Escherichia
coli can synthesize lipoic acid under anaerobic growth conditions (White, 1980b). A
reaction mechanism analogous to the insertion of oxygen into hydrocarbons by
monoxygenases, but using sulfur rather than oxygen, would also achieve the desired
result. Knight et al. (1963) showed that sulfur could be directly inserted into C-H bonds of
hydrocarbons (White, 1980b). However, most monoxygenase-catalyzed hydroxylations
proceed with retention of configuration, but the thiolation of octanoic acid must proceed
with inversion of configuration. To date there are no known examples of this type of
mechanism (White, 1980b).
Transport and Cellular Content of Lipoic Acid
Exogenous lipoic acid may be taken up and incorporated into intracellular pools by a
constitutive, energy-dependent active transport system which is similar to those for the
amino acids glycine and proline. The membrane-bound transport proteins are capable of
concentrating lipoic acid up to 100-fold intracellularly (Leach, 1970; Sanders and Leach,
1962; Sanders and Leach, 1964). The rate of incorporation is directly proportional to
incubation temperature with the maximum uptake occurring between 20 and 37°C.
Osmotic shock will also reduce the rate of transport. Octanoic acid competes with lipoic
acid for transport but l,2-dithiolane-3-caproic acid and l,2-dithiolane-3-butyric acid have
no effect (Leach, 1970). These compounds are the 9- and 7-carbon chain analogs of lipoic
acid.
It is relatively difficult to measure the lipoic acid content of microorganisms since its
intracellular concentration ranges from 0.5 to 25 ng / g dry weight (White, 1980b; Herbert
and Guest, 1975). Chemical quantitation involves acid hydrolysis of the sample to release
the enzyme-bound lipoic acid, followed by extraction with benzene and evaporation of the
solvent. The residue is resuspended in NaHCOa buffer (White, 1980a). Other methods of
quantitation include gas chromatography and bioassays. Gas chromatographic techniques
are sensitive to 50 ng, but the lipoic acid must first be derivatized prior to analysis (White,
1980a). Bioassay techniques employ bacterial strains which are dependent on lipoic acid
supplementation for growth (Herbert and Guest, 1970). Lipoic acid concentration is
estimated by measuring turbidity of these cultures supplemented with an unknown sample
and interpolating against a standard curve. The sensitivity of this technique approaches the
nanomolar range. Recently, a competitive binding immunoassay for lipoic acid was
developed which uses the dihydrolipoyl transacetylase polypeptide of the pyruvate
dehydrogenase complex as an enzyme-lipoic acid conjugant and anti-lipoic acid antibodies
as the binder. This technique has a sensitivity of 5 |iM (MacLean and Bachas, 1991).
Genetics of Lipoic Acid Biosynthesis
Previous studies with enteric bacteria have identified a single genetic locus (lip) that is
presumed to encode a lipoic acid biosynthetic enzyme (Herbert and Guest, 1968). The lip
locus is situated at 14 minutes on the Salmonella typhimurium genetic map and is
cotransducible with the leuS and nadD genes (Hughes et al., 1983; Sanderson and Roth,
1988). This enzyme, lipoic acid synthetase, would be responsible for the addition of both
sulfide groups to octanoic acid. Enzymatic assays which measure lipoic acid synthetase
activity have not yet been developed. Therefore, all data regarding the function of the lip
gene must be inferred from the growth phenotype. Strains with mutations at the lip locus
simultaneously lose the ability to synthesize acetyl-CoA (via the activity of the pyruvate
dehydrogenase complex) and succinyl-CoA (via the a-ketoglutarate dehydrogenase
complex). As a result, they are unable to grow on glucose as a sole carbon source. The
ability to metabolize glucose can be restored by supplementing the growth medium with
either lipoic acid, lysine and methionine, or acetate and succinate. Lipoic acid is not
required for anaerobic growth on glucose since the tricarboxylic acid cycle is not a critical
component of anaerobic metabolism.
Research Objectives
The primary goal of this study was to identify and characterize the genetic elements
responsible for biosynthesis of lipoic acid. The first phase of this study involved the
isolation of lipoic acid auxotrophs in the enteric bacterium S. typhimurium. The growth
phenotypes of these mutants were determined in order to define functions for the disrupted
genes. The chromosomal location of each mutation was determined by either
cotransduction with adjacent genetic markers or by conjugation in order to establish
whether or not the genes encoding lipoic acid biosynthetic functions were present at more
than one locus.
The second phase involved construction of in vivo operon fusions to the lip gene.
These fusions placed the expression of a reporter gene under the control of the lip
regulatory elements. The transcriptional activity of the lip gene was monitored under a
variety of growth conditions in an attempt to detect changes in lip expression. Several
models of gene regulation were evaluated in this manner. Among these were catabolite
repression, end-product repression, and substrate induction.
In the third and final phase, an S. typhimurium genomic library was constructed in
bacteriophage lambda and screened for recombinant phage which contained the lip gene.
A genomic fragment containing the lip gene was identified and then subcloned into a
plasmid vector in order to construct a restriction map. The lip gene was also cloned into an
expression vector in an attempt to overproduce the lip protein. Finally, the nucleotide
sequence of the lip gene was obtained and the coding and regulatory sequences were
identified.
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16
References
Bullock M. W., Brockman Jr. J. A., Patterson E. L., Pierce J. V., Stokstad E. L. (1952).
Synthesis of Compounds in the Thioctic Acid Series. Journal of the American
Chemical Society 74: 3455.
Dupre S., Spoto G., Matarese R. M., Oriando M., Cavallini D. (1980). Biosynthesis of
Lipoic Acid in the Rat: Incorporation of 35S- and I'^C-Labeled Precursers. Archives of
Biochemistry and Biophysics 202:361-365.
Eisenberg M. (1988). Biosynthesis of Biotin and Lipoic Acid, p. 544-550. In F. C.
Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E.
Umbarger (Eds.), Escherichia coli and Salmonella typhimurium: Cellular and
Molecular Biology. American Society for Microbiology, Washington, D. C.
Fujiwara K., Okamura-Ikeda K., Motokawa Y. (1986a). Chicken Liver H-Protein, a
Component of the Glycine Cleavage System. Joumal of Biological Chemistry
261:8836-8841.
Fujiwara K., Okamura-Ikeda K., Ohmura Y., Motokawa Y. (1986b). Mechanism of the
Glycine Cleavage Reaction: Retention of C-2 Hydrogens of Glycine on the
Intermediate Attached to H-Protein and Evidence for the Inability of Serine
Hydroxymethyltransferase to Catalyze the Glycine Decarboxylation. Archives of
Biochemistry and Biophysics 251:121-127.
HansonR.L.,RayP.D., Walter P., Lardy H. A. (1969). Mode of Action of
Hypoglycemic Agents: Inhibition of Gluconeogenesis by Quinaldic Acid and 5Methoxyindole-2-carboxylHc Acid. Joumal of Biological Chemistry 244:4351-4359.
Herbert A. A., Guest J. R. (1968). Biochemical and Genetic Studies with Lysine +
Methionine Mutants of Escherichia coli: Lipoic Acid and a-Ketoglutarate
Dehydrogenase-less Mutants. Joumal of General Microbiology 53:363-381.
Herbert A. A., Guest J. R. (1970). Turbidometric and Polarographic Assays for Lipoic
Acid Using Mutants of Escherichia coli. Methods in Enzymology 18a:269-272.
Herbert A. A., Guest J. R. (1975). Lipoic Acid Content of Escherichia coli and Other
Microorganisms. Archives of Microbiology 106:256-266.
Hughes K. T., Ladika D., Roth J. R., Olivera B. (1983). An Indispensable Gene for NAD
Biosynthesis in Salmonella typhimurium. Journal of Bacteriology 155:213-221.
Johnson M. G., Collins E. B. (1973). Synthesis of Lipoic Acid by Streptococcus faecalis
lOCl and End-Products Produced Anaerobically from Low Concentrations of
Glucose. Joumal of General Microbiology 78:47-55.
Kline L., Barker H. A. (1959). A New Growth Factor Required By Butyribacterium
rettgeri. Joumal of Bacteriology 60:349-363.
17
Kline L., Pine L., Gunsalus I. C , Barker H. A. (1952). Probable Identity of the Growth
Promoting Factor for Butyribacterium rettgeri with Other Biologically Active
Substances. Joumal of Bacteriology 64:467-472.
Knight A. R., Strausz O. P., Gunning H. E. (1963). Reactions of Sulfur Atoms. Ill The
Insertion in Carbon-Hydrogen bonds of Paraffinic Hydrocarbons. Joumal of the
American Chemical Society 85:2349-2355.
Knowles W. L., Benson A. A. (1983). The Biochemistry of Arsenic. Trends in
Biochemical Science 8:178-180.
Kochi H., Kikuchi G. (1976). Mechanism of Reversible Glycine Cleavage Reaction in
Arthrobacter globiformis: Function of Lipoic Acid in the Cleavage and Synthesis of
Glycine. Archives of Biochemistry and Biophysics 173:71-81.
Kusunose M., Kusunose E., Coon M. J. (1964). Enzymatic co-Oxidation of Fatty Acids.
I. Products of Octanoate, Decanoate, and Laurate Oxidation. Joumal of Biological
Chemistry 239:1374-1380.
Leach F.R. (1970). Lipoic Acid Transport. Methods in Enzymology 18:276-281.
MacLean A. I., Bachas L. G. (1991). Homogenous Enzyme Assay for Lipoic Acid Based
on the Pyruvate Dehydrogenase Complex: A Model for an Assay Using a Conjugate
with One Ligand per Subunit. Analytical Biochemistry 195:303-307.
Miura Y., Fulco A. J. (1974). (co-2) Hydroxylation of Fatty Acids by a Soluble System
from Bacillus megaterium. Joumal of Biological Chemistry 249:1880-1888.
Parry R. J. (1983). Biosynthesis of Some Sulfur Containing Natural Products.
Investigations of the Mechanism of Carbon-Sulfur Bond Formation. Tetrahedron
39:1215-1238.
Plamann M. D., Rapp W. D., Stauffer G. V. (1983). Escherichia coli K12 Mutants
Defective in the Glycine Cleavage Enzyme System. Molecular and General Genetics
192:15-20.
Reed L. J. (1957). The Chemistry and Function of Lipoic Acids, p. 319-347. In F. F.
Nord (Ed.), Advances in Enzymology, vol. 18. Interscience, New York.
Reed L. J., Yeaman S. J. (1987). Pymvate Dehydrogenase, p. 77-95. In P. D. Boyer
(Ed.), The Enzymes, vol. 18. Academic Press, New York.
Reed L. J., DeBusk B. G., Gunsalus I. C , Homberger Jr. C. S. (1951a). Crystalline aLipoic Acid: A Catalytic Agent Associated with Pymvate Dehydrogenase. Science,
114:93-94.
18
Reed L. J., DeBusk B. G., Johnston P. M., Getzendaner M. E. (1951b.) AcetateReplacing Factors for Lactic Acid Bacteria. Joumal of Biological Chemistry 192: 851858.
Richarme G. (1985). Possible Involvement of Lipoic Acid in Binding Protein-Dependent
Transport Systems in Escherichia coli. Joumal of Bacteriology 162:286-293.
Richarme G., Heine H-G. (1986). Galactose- and Maltose-Stimulated Lipoamide
Dehydrogenase Activities Related to the Binding Protein-Dependent Transport of
Galactose and Maltose in Toluenized Cells of Escherichia coli. European Joumal of
Biochemistry 156:399-405.
Sanders D. C , Leach F. R. (1962). Lipoic Acid Uptake in Streptococcus faecalis lOCl.
Biochemica et Biophysica Acta 62:604-605.
Sanders D. C , Leach F. R. (1964). Studies on Lipoic Acid Uptake by Bacteria.
Biochemica et Biophysica Acta 82:41-57.
Sanderson K. E., Roth J. R. (1988). Linkage Map of Salmonella typhimurium. Edition
VII. Microbiological Reviews 52:485-532.
Schmidt U., Grafen P., Altland K., Goedde H. W. (1954). Biochemistry and Chemistry
of Lipoic Acids, p. 423-469. In F. F. Nord (Ed.), Advances in Enzymology, vol. 32.
Interscience, New York.
White R. H. (1980a). A Gas Chromatographic Method for the Analysis of Lipoic Acid in
Biological Samples. Analytical Biochemistry 110:89-92.
White R. H. (1980b). Stable Isotope Studies on the Biosynthesis of Lipoic Acid in
Escherichia coli. Biochemistry 19:15-19.
CHAPTER II
REGULATION OF LIPOIC ACID SYNTHESIS
Introduction
Strains of Salmonella typhimurium which have an auxotrophic requirement for lipoic
acid were isolated by mutagenesis with the transposable element Mu dJ. This 4.5 kbp
transposon randomly inserts into the host genome and usually dismpts target gene activity.
The chromosomal locations of these insertion mutations were determined by bacteriophage
P22-mediated cotransduction. All insertion lesions were placed at 14 map units, thus
suggesting that lipoic acid biosynthesis is encoded by a single gene or gene cluster. The
transcriptional orientation of the lip gene was determined by constmcting Hfr derivatives of
lip::Mu dJ strains and evaluating their efficiency of conjugal transfer. The lip gene is
transcribed in the clockwise direction relative to the S. typhimurium genetic map. Strains
with lipr.lac operon fusions were used to characterize the transcriptional activity of the lip
promoter. These fusion strains express the lacZ gene from Escherichia coli under control
of the lip promoter and regulatory elements. Since 6-galactosidase, the enzyme encoded by
the lacZ gene, is not found in S.typhimurium, it can be easily assayed. The resulting
enzyme activity is a direct indicator of lip transcription. Data from these experiments show
that transcription of the lip gene is not regulated by catabolite repression or lipoic acid
concentration. The data indicate that the lip gene product is expressed constitutively at a
low level.
19
20
Methods
Bacterial and Bacteriophage Strains
The bacterial strains used in this study and their sources are listed in Table 2.1. All
strains are derivatives of 5. typhimurium LT2. Bacteriophage P22 HT 105/1 int-201 was
used for routine transductional crosses. Mu di [Mu ^ 1 7 3 4 (Kan, lac)] is a conditionally
transposition-defective derivative of Mu d\ (Casadaban and Cohen, 1979) which forms
operon fusions (Castilho etal, 1984).
Culture Media
NCE (no carbon E) minimal medium base (Davis et al, 1980), with the addition of
D-glucose (14 mM), was used routinely as the defined medium. Altemate carbon sources
included acetate (25 mM), succinate (25 mM), pymvate (25 mM), glycerol (14 mM),
maltose (14 mM), lactose (14 mM), and galactose (14 mM). Alanine, cysteine, glycine,
serine, and uracil were added as needed to defined medium at a final concentration of 0.10.5 mM (Davis et al, 1980). Luria-Bertani (LB) broth (Miller, 1972) and NCE medium
base with the addition of casamino acids (Difco, 0.8%) and 5 p.M lipoic acid served as the
complex media. Agar (Difco) was added at a final concentration of 1.5% to solidify
complex medium. Noble Agar (Difco, 1.5%) was added to solidify minimal medium.
Antibiotics were added in the following final concentrations to defined and complex culture
media respectively: sodium ampicillin 15 mg/L and 30 mg/L, tetracycline hydrochloride
10 mg/ L and 20 mg/ L, kanamycin sulfate 125 mg/ L and 50 mg/ L, and streptomycin
1000 mg/ L and 200 mg/ L. Inorganic chemicals were supplied by J.T. Baker Chemicals
and Fisher Scientific. Organic chemicals and biochemicals were supplied by Aldrich,
Boehringer Mannheim, and Sigma.
21
Transductional Methods
P22 lysates were prepared on donor strains of 5. typhimurium as previously
described (Davis et al, 1980). Donor bacteriophage and recipient cells were mixed at a
multiplicity of infection of 0.01 and spread onto selective medium. Phage-free
transductants were isolated by streaking non-selectively on modified green indicator
medium (Jeter, 1990). Phage-free clones were checked for P22 sensitivity by crossstreaking with the clear plaque-mutant phage P22 H5.
Mutant Isolation
A P22 lysate was grown on strain TT10288 ihisD9953::Mu dS hisA9944::Mu dl)
and used to create random Mu dJ chromosomal insertions in an LT2 recipient (Hughes and
Roth, 1988). Kanamycin-resistant mutants were selected on LB agar supplemented with
lipoic acid and kanamycin and were subsequentiy replica-plated onto NCE casamino acids
agar plates with and without lipoic acid. Colonies which required exogenous lipoic acid for
growth were chosen for additional characterization. Mutant strains containing lipv.lac
operon fusions were detected by testing for 6-galactosidase activity on LB agar containing
5-bromo-4-chloro-3-indolyl-6-D-galactopyranoside (X-gal) (Silhavy andBeckwith, 1985).
Lac"*" fusion strains formed blue colonies.
Cotransductional Mapping
Lip" strains were transduced to tetracycline resistance with P22 lysates grown on
donor strains TT7247 (zbe-1023::TnlO) and TT7252 {zbe-I028::TnlO). These strains
contain TnlO transposons that are cotransducible with the lip locus (Hughes et al, 1983).
Tetracycline-resistant colonies were replica-plated to LB agar with tetracycline and
kanamycin and scored for growth (i.e., retention of the lip::Mu dJ insertion).
22
Cotransduction frequencies were used to estimate the physical distances between the
genetic markers (Wu, 1966).
Formation of Duplications
Merodiploid strains containing duplications of the lip region were constmcted using
TnlO and TnlOd-Tet insertions near the lip gene as regions for homologous recombination
(Maloy and Roth, 1983). Strain RTl 142 (/ip-^7::Mu dJ) was transduced to tetracycline
resistance with a P22 lysate grown on pooled cells containing random chromosomal
insertions of TnlOd-Ttt (Way et al, 1984). Transductants were scored for loss of
kanamycin resistance and a Lip"*" phenotype (replacement of lip::Mu dJ with lip"^) by replica
plating. These strains contain TnlOd-Tet insertions near the ///?"•" allele. Several of these
strains were transduced to kanamycin resistance with a lysate grown on RTl208 {lip46::Mu di zbe-1023::TnlO). Kanamycin-resistant transductants were scored for retention
of the Lip"*" phenotype. Strains that contain both lip-46::Mu dJ and ///?"•" alleles were
presumed to be chromosomal duplications created by homologous recombination between
the Tn70 and the TnlOd-Tct insertions. These putative duplication strains were streaked
for purification on NCE glucose minimal medium containing tetracycline and kanamycin.
Overnight cultures of each strain were grown in LB broth, diluted by a factor of 1 x 10 8
and spread onto LB agar (no selective pressure). One hundred isolated colonies from each
culture were tested for segregation of the lip"*" and lip' alleles.
Conjugational Methods
Strain TR2647 {pyrC7 rpsLl / F' tsl 14 /ac"*") was inoculated into NCE lactose broth
with a uracil supplement and incubated ovemight at 30^0. Strains RT1241 (//p-50::Mu di
pncB185::TnlO) and RT1242 (lip-50::Mu di pro-1931::TnI0) were grown in NCE
23
glucose supplemented with proline and lipoic acid. The F 'lac plasmid was transferred by
spreading equal volumes of donor and recipient cultures onto NCE lactose agar
supplemented with proline and lipoic acid and incubating at 3(PC. Purified Lac"*" colonies
from each mating were streaked onto the same medium and incubated at 420C to select
colonies containing a stable Hfr integrate (Chumley et al, 1979). Hfr derivatives of
RT1241 and RT1242 served as donors in conjugal matings with RTl 182 (rpsLl). Equal
volumes of donor and recipient cells were spread onto NCE glucose medium containing
proline, lipoic acid, tetracycline, and streptomycin. The Tet^ Str^ colonies from each mating
were tested by relica plating for simultaneous inheritance of either an auxotrophic
requirement for proline (ProAB" phenotype) or resistance to 6-aminonicotinic acid (PncB"
phenotype).
6-Galactosidase Assays
6-Galactosidase enzyme activities were measured by the procedure described in Miller
(1972). Specific activities are expressed as nanomoles of orr/zo-nitrophenol (ONP) formed
/ minute / unit of absorbance at 650 nm. Wild-type S. typhimurium LT2 (Lac") served as
the negative control for all enzyme assays. 6-Galactosidase enzyme activities from RTl 146
(AproAB47 / F ' 128 pro"*" lac*") induced with isopropyl-6-D-thiogalactopyranoside
(IPTG) served as the positive control. All reported values are the means of five replicate
assays. 6-Galactosidase activities were compared by analysis of variance to test the
hypothesis that they were all equal (a=0.05). Homogeneity of variance was determined by
Box's small sample F approximation. Student-Newman-Keuls (SNK) post-hoc multiple
comparison test was also performed (a=0.05) where indicated.
24
Determmation of Generation Times
Strains RTl 142 and RTl 148 were inoculated into 2 ml of NCE minimal medium
supplemented with the appropriate test carbon source and incubated ovemight at 2n^C with
aeration. One hundred \i\ of each ovemight culture was inoculated into 100 ml of the same
medium pre-warmed to 37^0. The cultures were incubated at 37^0 with aeration (250
rpm). Aliquots were removed at 1-hour intervals and the turbidity was measured as the
absorbance at 600 nm. Growth curves were prepared for each medium and the generation
times were calculated from the time required for the absorbance to double during
exponential growth.
Lipoic Acid Deprivation
An ovemight culture of strain RTl 142 was grown in NCE succinate medium
supplemented with 5 |iM lipoic acid. The culture was washed twice in NCE buffer and
resuspended in NCE succinate medium without lipoic acid to an absorbance at 600 nm of
0.20. The cultures were incubated at 37^0 with aeration for an additional 8 hours.
6-Galactosidase activities were measured in samples taken from these cultures at 2-hour
intervals.
Results
Isolation and Characterization of Lip" Mutants
Seven strains which contained chromosomal Mu di insertions that conferred a Lip"
phenotype were isolated independently. These mutants were initially identified by their
lack of growth on NCE casamino acids medium without lipoic acid supplementation. The
ability to grow on defined solid medium with altemate carbon sources was also tested
(Table 2.2). Exogenous lipoic acid was required under all culture conditions tested, except
25
for anaerobic growth on glucose alone and aerobic growth on glucose supplemented with
either lysine and methionine or acetate and succinate, which is in agreement with previously
published results (Herbert and Guest, 1968). Growth rates of some cultures were
determined by measuring the increase in absorbance at 600 nm in defmed liquid medium
(Figures 2.1 and 2.2).
Genetic Mapping
The lip locus is situated at 14 map units on the S. typhimurium chromosome
(Sanderson and Roth, 1988). Transductional crosses with other genetic markers in the
region indicated that all seven of the Mu di insertions that conferred a Lip" phenotype were
located at the lip locus. All lip alleles tested in this study were 95-100% cotransducible
with zbe-1023::TnlO and 8-14% cotransducible with zbe-1028::TnlO, which is similar to
the results of a previous study (Hughes et al, 1983) (Figure 2.3).
Chromosomal Direction of Transcription
Orientation of the lip promoter was established by creating Hfr insertions of the
temperature-sensitive plasmid F 'tsl 14 /ac"*" in strains RTl241 {lip-50::Mu di
pncBI85::TnlO) and RT1242 (lip-50::Mu di pro-193I::TnI0d-Tct) and determining the
direction of chromosomal transfer in a conjugal mating. The lac genes carried by the Mu
di transposon served as a region of homology for recombination between the plasmid and
the bacterial chromosome. The orientation of the plasmid-encoded lac genes and its relation
to the direction of transfer have been described (Maloy and Roth, 1983). The lip-50::Mu
di insertion in the recipient strains confers a Lac" phenotype. RTl241 and RTl242 also
contain single insertions of TnlO or Tn/O^-Tet on either side of the lip gene. The direction
of transfer was determined by selecting Tet^ transconjugants from each mating and scoring
26
for simultaneous inheritance of the derivative phenotype. Two hundred seventeen
transconjugant colonies inherited the TnlOd-Tet located in the proAB genes (at 7 map
units), whereas one colony inherited the TnlO located in thepncB gene (at 20 map units).
These data indicate that the lip gene is transcribed in the clockwise direction with respect to
the S. typhimurium genetic map (Figure 2.3).
Transcriptional Activity
Transcription of the aceEF and sucAB genes, which encode the El and E2 subunits of
the pymvate dehydrogenase and a-ketoglutarate dehydrogenase complexes, is repressed by
glucose (Spencer and Guest, 1987). This repression is mediated by cyclic AMP
(adenosine 3',5'-cyclic monophosphate) and the cyclic AMP receptor protein. Strains
containing lip::lac fusions were grown in NCE casamino acids medium supplemented with
glucose, glycerol, or cyclic AMP, and 6-galactosidase levels were measured to determine
whether lip transcription was regulated by catabolite repression. Enzyme activity in NCE
casamino acids medium without any of these supplements was used as the baseline value.
Transcription of lip was not affected by the addition of glucose, glycerol, or cyclic AMP to
the medium (Table 2.3). Strains containing additional mutations in the crp and cya genes
(both null mutations and crp*, which makes expression of catabolite-repressed genes
independent of cyclic AMP) were also constmcted and tested. 6-Galactosidase activities in
these strains indicated that these mutations had no effect on lip transcription. These data
support the conclusion that the lip gene does not respond to catabolite repression and is not
regulated by cyclic AMP and the cyclic AMP receptor protein.
Transcription of the aceEF and sucAB genes is also sensitive to high levels of acetate
and succinate (Spencer and Guest, 1987). 6-Galactosidase activities in lipv.lac fusion
27
strains were measured in minimal media containing acetate and succinate as sole carbon
sources. These compounds had no effect on lip transcription (Table 2.4).
Lipoic acid functions as a cofactor in the conversion of glycine to serine and a onecarbon unit (glycine cleavage reaction) in Escherichia coli (Plamann et al, 1983). The
effects of glycine and serine supplementation on lip transcription were tested. Also, it has
been postulated that cysteine serves as the sulfur donor in the synthesis of lipoic acid from
octanoic acid. Thus, cysteine supplementation was also evaluated. None of these amino
acids had a specific effect on lip transcription (Table 2.4). However, a nonspecific increase
in 6-galactosidase activity was observed in some cases (see Discussion).
S. typhimurium does not require lipoic acid for anaerobic growth. However,
transcriptional activity of the lip gene under anaerobic culture conditions was similar to the
aerobic activity in the same medium (Table 2.4).
The effect of lipoic acid deprivation on lip transcription was determined by measuring
6-galactosidase activity in strain RTl 142 after a shift to lipoic acid-free medium. Lipoic
acid starvation did not induce lip transcriptional activity. On the contrary, a decrease in
transcriptional activity was observed, which probably reflects a general lowering of
metabolism in non-dividing cells. Addition of lipoic acid to the starved cultures resulted in
renewed cell division and a corresponding increase in transcriptional activity (Figure 2.4).
The effect of excess Upoic acid on lip transcription was evaluated by measuring 6galactosidase activity in a merodiploid strain (RTl 148) containing both a lipv.lac fusion and
a lip^ allele within a chromosomal duplication of the lip region. The lip^ allele eliminated
the requirement for an exogenous lipoic acid supplement by providing a functional gene
product. Supplementation with lipoic acid had no effect on lip transcription. No change
was observed over a 500-fold increase in exogenous lipoic acid concentration (Figure 2.5).
However, the presence of the wild-type ///?"*" allele in the merodiploid strain was correlated
28
with a lower level of 6-galactosidase activity than in the lipv.lac haploid strain (RTl 142) on
certain growth media (see Discussion).
Analysis of variance dcn^ post-hoc evaluation by Student-Newman-Keuls multiple
comparison test indicate that all 6-galactosidase activities could be divided statistically into
two different groups. The members of each group are indicated in Table 2.4.
Discussion
All known lip mutations, including the new Mu di insertions identified in this study,
occur at a single genetic locus (14 map units) on the S. typhimurium chromosome. This
region is presumed to encode a single lipoic acid biosynthetic enzyme that catalyzes the
incorporation of both sulfur atoms into octanoic acid. The location of the gene encoding
the lipoic acid-activating enzyme, which attaches lipoic acid to the E2 subunits of the
pymvate dehydrogenase and a-ketoglutarate dehydrogenase complexes, remains
unknown.
Several models for the transcriptional regulation of the lip gene were tested by
measuring 6-galactosidase levels in lipv.lac fusion strains under a variety of culture
conditions. The experimental data indicated that transcription initiation from the lip
promoter does not require cychc AMP or cyclic AMP receptor protein and is not sensitive
to glucose concentration. Thus, the lip gene is not regulated by catabolite repression.
Growth on acetate, succinate, or pymvate (rather than glucose) as sole carbon and energy
sources also had no differential effect. Anaerobic growth conditions did not cause a
decrease in lip expression. 6-Galactosidase enzyme activities in a merodiploid (duplication)
strain indicated that excess exogenous lipoic acid does not repress transcription. Thus,
end-product repression is not indicated as a mechanism for lip regulation. Recently, Ali
and Guest (1990) found that overexpression of the E2 subunit of the pymvate
29
dehydrogenase complex leads to accumulation of the unlipoylated form of the protein. This
observation, which suggests that the synthesis of lipoic acid is not induced in response to
an increased cellular demand for it, also supports the results of this study.
Statistical analysis of 6-galactosidase activities under all growth conditions tested in
this study suggests that the lip promoter is expressed constitutively at a low rate but that
there are two distinct levels of expression (Figure 2.6). These levels appear to be related in
some way to nutrient availability, but our data do not suggest the exact nature of this
relationship. Defined culture media containing a single carbon source and lipoic acid
produced lower activities. Supplementation of these same media with amino acids which
can serve as a source of carbon skeletons increased transcription from the lip promoter
approximately two-fold (Table 2.4). Growth rates were slower in defined media with a
single source of carbon (generation times of from 41 to 95 minutes) than in supplemented
media (26 to 35 minutes). Yet this enhancement of lip transcription does not appear to be a
simple function of growth rate alone. Statistical analysis indicated that the growth rates
were not significantly correlated to transciptional activity in the same medium (Figure 2.7).
The growth rates of the haploid strain (RTl 142) and the merodiploid strain (RTl 148) were
essentially identical in each medium tested. Nevertheless, the transcriptional activity in
RTl 142 increased in supplemented media while the transcriptional activity in RTl 148 did
not The chromosomal duplication provides both a functional gene product in trans to the
lipv.lac fusion and a second copy of the lip promoter. Either or both of these elements
might be modulating the level of lip transcription, but if so, the molecular mechanism for
this interaction remains to be determined.
30
31
<p
>>_>^ >> >> >» >^ >^ >» >-, _>»>»>> >» >>
"O 'O ' o "^3 'O 'O "Q 'O TiJ ' o 'O 'O "O "O
13 3 3 3 3 3 3 3 3 3 3 3 3 3 3
'O T J T) 'O
- 3 3 3 3
u
s
o
J3 J3 J3 J3 J3 . ' . N 4—1 4—• 4—> 4—>
O O O O O O -- ,_ ,^ ,^
o ^ o
pq
pd Pi: 0^ cti 0^ ci^ .2 .g .^ .^
4 . ^ 4 - < 4 — > 4 - > 4 - > 4 . ^ 4 — • 4 - > 4 - > 4 — • 4 - > 4 . ^
4_>
4-^
C/3
3
+
+
oo
8
"a
U^
3
*
^o
+
c
IT)
'^
3 3
3
"^-i '~-i <-^
H
H
C 3 C
s
H H
C5 O O
+ *
I
ON
HHH
I
"a
C9
--< r n ^
—J
O
C
- ^
. .
• •
5?.
c
H
^ tr> !r»
•• * •
r
-^
o
3 3 3
3:: 3 3 3 3 3
>--( u-^ VO 0 \ vo
*>• ^ 5:i '^
o t\ rv r v o o
*^ 'T T "^ "^ "^
oo , - O O O O O O O O O O O j : ^ j : ^ c q r q ^ ^ ^
ON r- cs (N o <^
C/3
5
32
Table 2.2. Growth phenotypes of lip insertion mutants. Growth was scored after 24 and
48 hours incubation by the presence or absence of isolated colonies.
NCE growth media
RT1142a
RT1148
LT2
supplemented with
lip::lac
lipv.lac lip+
wild type control
the following
5 jiM
No
5 }iM
No
No
carbon sources lipoic acid Lipoic acid Lipoic acid Lipoic acid Lipoic acid
Glucose
Acetate
Succinate
Pymvate
Glycerol
Citrate
Galactose
Maltose
Glucose -1- acetate
Glucose + succinate
Glucose +Acetate
+ succinate
Glucose + lysine
+ methionine
Glucose [anaerobic]
+
+
+
+
+
+
-I+
+
+
+
-f-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-I-
+
+
+
+
+
+
+
+
+
+
+
+
+
-1-
+
+
+
+
+
+
-1-
+
+
+
+
+
33
VOO ^
H
O
• • • •
oooo
u
.s ^
\D\0
.S»5
O so
%
0\ oo
»—•
oo
ON
en
ON
•s-
a
<S v^ <N ^ .
Tt 00 iri en
VO NO VO VO
en H
+
r^ '-H
ON
-^
§
,
,—I
• • • •
^-H T-H CO 1-H
H
J3
•S-
-HI -t-l -l-l -t-l
p pcnp
r-^ r-^ cs od
s
en <s "^ en
^ ^ ^ ^
M +1 +1 +1
^H oo
ON
oo
Tf O rj-* 00
ir> vo «r> -^
CJ
c
"5
&N
-<
<
U
o
O a,
s
^
fj
o a> o
u
3
O
CjO V}
u
O o V
ow S w
c2 3 ^
g +++
uuuu
CO
34
Table 2.4. 6-Galactosidase specific activities of lipv.lac operon fusions in various culture
media. 6-Galactosidase specific enzyme activities are presented as the mean values of 5
replicate trials. Standard error and statistical classification are included.
NCE minimai growth
medium with the following
carbon sources a
B-Galactosidase specific activity in strain:
RTl 142
RT1148
^^^..^^
^^^..^^^ ^.^+
Aerobic culture conditions
casamino acids (CAA)
CAA +glucose
CAA + glucose + cAMP
CAA + glycerol
glucose
acetate
succinate
pyruvate
glycerol
glucose + acetate + succinate [no lipoic acid]
glucose + lysine + methionine [no lipoic acid]
glucose + serine
glucose + glycine
glucose + cysteine
amino acid pool A^
amino acid pool B^
Anaerobic culture conditions
glucose [no lipoic acid]
glucose
Casamino acids [no lipoic acid]
Casamino acids
54.1 ± 3.9b
60.8 ± 4.4^
54.9 + 1.9b
48.8 ± 1.5^
30.5 ± 2.9^
28.4 ± 4.3^
22.7 ± 3.5^
23.9 ± 2.6^
28.5 ± 1.4^
23.9 ± 3.1^
22.9 ± 3.1^
44.2 ± 7.4^
54.7 ± 2.7^
49.0 ± 2.7^
29.5 ± 3.9^
28.6 ± 3.9^
37 g ± 1.7c
27.0 ±1.1^
423 + 3.9c
38.0 ± 1.1^
32.0 ± 6.9^
21.5 ± 4.6^
28.4 ± 2.5^
24.9 ± 0.2^
28.4 ± 5.0^
23.4 ± 0.4C
23.7 ± 3.4C
34.0 ± 3.7C
31.9 ± 3.8^^
29.1 ± 4.6^
36.9 ± 3.9^
32.2 ± 3.9^
37.8 ±
38.3 ±
54.9 ±
55.7 ±
25.5 ± 1.5'24.7 ± 4.6^
32.4 ± 3.2C
37.1 ± 4.0^
3.6C
8.0^
1.8^
0.7*'
a All media contained lipoic acid at a final concentration of 5 \xMIL unless indicated otherwise.
Superscripted ** and superscripted ^ B-galactosidase activities differed at P=0.05 by Student-Newman-Keuls
analysis of ordered means. RTl 142 has 6-galactosidase values which fall into both the high (**) and low
(C) activity groups, whereas all values from the duplication strain RTl 148 only fall into the low activity
group.
^ Composition of amino acid pool A: methionine, proline, alanine, cysteine, valine, serine, phenylalanine
and arginine.
® Composition of amino acid pool B: histidine, asparagine, glutamic acid, tryptophan, isoleucine, aspartate,
leucine, lysine, and threonine. Final amino acid concentrations in the culture medium were as described in
Davis e/fl/. (1980).
35
36
+
u
8
3
+
00
+
u
C/3
luco
u
M
luco
u
tn
u
CA
luco
u
tA
(U
uccin
U9
c
yst
a
c
u>
60
60
^
u
i
aea
a.
a
o
3
u
o
a
9i
(A
U
9
O
s
uio 009 )^ ssueqjosqv
37
38
u
00
+
U
+
COS
ste
.S
>.
u
o
3
u
M
o
u
3
00
00
<A
O
3
00
a
UCC ina
u
(A
o
u
3
a>
c
<u
Vi
+
-k
%
C/5
U
u
Oi
(0
I..
3
O
0)
E
«>
(0
Q.
Si
MX
I
I
O
..^
I
i I I '
O^
00
I
f~
d
c>
o
•
I
^
o
•
I '
''I
»
"^
o
o
uiu 009 l« ^J'suaa IB^IldQ
I • I • I •
CO
c>
CS
d
^
cs
O
d
39
40
(>5
3
4>
<B5
Q
•o
s
a
O
•
i
^5
a
t
A
•
I
t t
s
1-H
•s
t t
41
42
XJIAIPV 3SBplS0J3BIB9-g
d
d
d
d
uiu 009 90UBqjosqvv
o
d
43
44
."2
<
o
iCjiAipV
asepisopBiBO-a
45
46
70
60
-
50
-
40
-
30
-
<
a
•o
Vi
O
•^*
o
I
cs
20
High
Activity
Low
Activity
47
48
1
1
*
.
1
T
'
o
-
*
«
1
*
•
Generation Tinle (minutes)
•
*
*
I
•
—•
y = - 0.047534X + 65.784 I^ = 0.097
.
20
•
20
1
30
1
1
40
1
1
50
70
60
fi-Galactosidase Activity
B.
ANOVA Table
Source
Sum of
Squares
Degrees of
Freedom
Mean
Squares
Model
Error
17.293
4726.936
1
10
17.293
472.694
Total
0.081
11
Coefficient of Determination (R^)
Adjusted Coefficient ( R 2 )
Coefficient of Correlation (R)
Standard Error of the Estimate
Durbin-Watson Statistic
F Ratio
Probability> F
0.037
0.852
0.004
0.096
0.060
21.742
2.426
49
References
Ali S , Guest J. R. (1990). Isolation and Characterization of Lipoylated and Unlipoylated
Domains of the E2p Subunit of the Pyruvate Dehydrogenase Complex of Escherichia
coll. Biochemical Joumal 271:139-145.
Casadaban M. J., Cohen S. N. (1979). Lactose Genes Fused to Exogenous Promoters in
One Step Usmg Mu-lac Bacteriophage: In Vivo Probe for Transcriptional Control
Sequences. Proceedings of the National Academy of Sciences USA 76:4530-4533.
Castilho B. A., Olfson P . , and Casadaban M. J. (1984). Plasmid Insertion Mutagenesis
and lac Gene Fusion with Mini-Mu Bacteriophage Transposons. Joumal of
Bacteriology 158:488-495.
Chumley F. G., Menzel R., Roth J. R. (1979). Hfr Formation Directed by TnlO.
Genetics 91:639-655.
Davis R. W., Botstein D., Roth J. R. (1980). Advanced Bacterial Genetics. Cold Spring
Harbor Laboratory. Cold Spring Harbor N.Y.
Hughes K. T., Ladika D., Roth J. R., Olivera B. (1983). An Indispensable Gene for NAD
Biosynthesis in Salmonella typhimurium. Joumal of Bacteriology 155:213-221.
Hughes K. T., Roth J. R. (1985). Directed Formation of Deletions and Duplications Using
Mu d{Ap, lac). Genetics 109:263-282.
Hughes K. T., Roth J. R. (1988). Transitory Cis Complementation: A Method for
Providing Transposition Functions to Defective Transposons. Genetics 119:9-12.
Jeter R. M. (1990). Cobalamin-Dependent 1,2-Propanediol UtiUzation by Salmonella
typhimurium. Joumal of General Microbiology 136:887-896.
Maloy S. R., Roth J. R. (1983). Regulation of ProUne Utilization in Salmonella
typhimurium: Characterization ofput::Mu d{Ap, lac) Operon Fusions. Joumal of
Bacteriology 154:561-568.
Miller J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor Laboratory.
Cold Spring Harbor N.Y.
Sanderson K. E., Roth J. R. (1988). Linkage Map of Salmonella typhimurium. Edition
VIL Microbiological Reviews 52:485-532.
Schmid M. B., Roth J. R. (1983). Genetic Methods for Analysis and Manipulation of
Inversion Mutations in Bacteria. Genetics 105:517-537.
Silhavy T. J., Beckwith J. R. (1985). Uses of lac Fusions for the Study of Genetic
Problems. Microbiological Reviews 49:398-418.
50
Spencer M. E., Guest J. R. (1987). Regulation of Citric Acid Cycle Genes in Facultative
Bacteria. Microbiological Sciences 4(6): 164-168.
Way J. C , Davis M. A., Morisato D., Roberts D. E., Kleckner N. (1984). New TnlO
Derivatives for Transposon Mutagenesis and for Constmction of lacZ Operon Fusions
by Transposition. Gene 32:369-379.
Wu T. T. (1966). A Model for Three-Point Analysis of Random General Transduction.
Genetics 54:405-410.
CHAPTER III
CHARACTERIZATION OF lip GENE STRUCTURE
Introduction
A Salmonella typhimurium genomic library was constmcted in the X replacement
vector EMBL4 (Frischauf et al, 1983) and screened for recombinant bacteriophages
containing the lip biosynthetic gene. Since tiie protein encoded by the lip gene has not
yet been purified, nucleic acid probes for the lip gene are not available. Furthermore,
enzyme assays which measure the activity of the lip gene product have not been
developed. Therefore, a screen had to be devised which could exploit existing
phenotypic data to identify recombinant bacteriophages that express the lip gene
product. This type of screen requires that cells infected with recombinant
bacteriophages form colonies rather than plaques. Since X,EMBL4 is genetically
incapable of lysogenic growth (Frischauf et al, 1983), lysogenized bacteriophage >.l 12
was used as a helper to express the cloned genes (Maurer et al, 1980). The helper
bacteriophage A. 112 contains the Ximmll immunity region as well as the X CI
repressor. Thus, a strain lysogenized with XI12 possesses superinfection immunity
to X. The X.112-encoded CI repressor protein commits a superinfecting A.EMBL4
bacteriophage to lysogenic growth. Bacterial genes cloned within the EMBL4
bacteriophage genome are expressed from their own regulatory sequences. This has
the advantage that potentially toxic proteins from the cloned genes are not overexpressed.
The E. coli strain W1485 was selected as the host for the screen. This strain
contains an auxotrophic mutation in the lip gene which renders it incapable of growth
51
52
on NCE glucose minimal medium unless it is supplemented with exogenous lipoic acid
(Herbert and Guest, 1968). Since W1485 is derived from E. coli K12, it carries the
necessary receptors that make it susceptible to infection by bacteriophage X. The
library was screened for Lip+ clones by lysogenizing strain W1485 with
bacteriophage X,112 and superinfecting with the A.EMBL4 library. The transductant
colonies were detected by spreading the recipient cells onto NCE glucose minimal
medium. Colony formation indicated that complementation of the host lip mutation
with a DNA fragment from the library had occurred.
This screen resulted in the isolation of over 3000 independent lip clones, one of
which was selected for stmctural characterization. Subcloning experiments localized
the lip gene to a 1.3 kbp EcoR I fragment, which was then sequenced to identify the lip
promoter and define the lip stmctural gene.
Methods
Bacteria, Plasmids, and Bacteriophage Strains
The genotypes of the E. coli and S. typhimurium bacterial strains used in this
study are listed in Table 3.1. Bacteriophage P22 HT 105/1 int-201 was used for routine
transductional crosses in S. typhimurium. This derivative of bacteriophage P22
performs generalized transduction at a higher frequency than the wild-type P22
(Anderson and Roth, 1978; Schmeiger, 1971, 1972). Bacteriophage PI was used for
generalized transduction in E. coli (Silhavy et al, 1984). Bacteriophage EMBL4 was
used as the primary cloning vector for construction of the 5. typhimurium genomic
library. Plasmids pKK223-3 and pBR322 were obtained from Pharmacia and plasmids
pIBI30 and pIBI31 were obtained from Intemational Biotechnologies, Inc.
Bacteriophage X and A,112 were routinely grown in E. coli strains NM539 and LE392
53
in accordance with established protocols (Ausubel etal, 1989; Maniatis etal,
mi;
Davis et al, 1980). E. coli strain DH5a was used as a host for plasmid preparation
unless otherwise indicated.
Culture Media and Reagents
NCE (no carbon E) minimal medium base (Davis et al, 1980) supplemented
with D-glucose (14 mM) was routinely used as the defined medium. Luria-Bertani
(LB) broth (Miller, 1972) served as the complex broth. NZYC medium (Ausubel et
al, 1989), Lambda plates, and TB top agar (Davis et al, 1980) were used for
cultivation and maintenance of bacteriophage XmdXlU.
Lipoic acid was added to
culture media at a final concentration of 5 |LiM as needed. Agar (Difco) was added to
at a final concentration of 1.5% to solidify complex medium. Noble agar (Difco) or
agarose (Gibco BRL) was added at a final concentration of 1.5% to solidify minimal
medium. Antibiotics were added in the following final concentrations to defmed and
complex culture media respectively: sodium ampicillin 25 mg/L and 50 mg/L,
tetracycline hydrochloride 10 mg/L and 20 mg/L, and kanamycin sulfate 125 mg/L and
50 mg/L. Inorganic chemicals were supplied by J.T. Baker Chemicals and Fisher
Scientific. Organic chemicals and biochemicals were supplied by Aldrich, Boehringer
Mannheim, and Sigma. Restriction endonucleases and T4 DNA ligase were supplied
by Gibco BRL and Promega. Calf intestinal alkaline phosphatase (CIAP) was
supplied by Boehringer Mannheim Biochemicals. The in vitro bacteriophage packaging
kit (Packagene®) and the nested deletion (Erase-a-base®) kits were obtained from
Promega. Sequencing enzymes, including Sequenase® modified T7 DNA polymerase
and associated biochemicals were obtained from United States Biochemical
54
Corporation. HydroLink™ Long Ranger modified acrylamide was obtained from AT
Biochem.
Restriction Endonuclease Digestion
Restriction endonucleases were obtained from New England Biolabs, Gibco BRL,
Promega, and United States Biochemical Corporation. Digestion was accomplished
with 1-2 units of enzyme per microgram of substrate DNA for 1-2 hours at the
temperatures and in reaction buffers recommended by the manufacturers. Restriction
fragments were resolved by agarose gel electrophoresis in 0.4%-2.0% TAE (0.4 M
Tris-HCl, 10 mM EDTA, and 50 mM sodium acetate) agarose and visualized by
ethidium bromide staining.
Preparation of Genomic DNA
Genomic DNA was prepared from the 5. typhimurium strain RTl346 (zbd6166::Tnl0d-Kan).
This strain contains a mini-kan transposable element which is
>99.5% cotransducible to the lip gene and retains a Lip"*" phenotype. A 100-ml
saturated culture was centrifuged at 8000 x g for 20 minutes to collect the cells. The
bacterial pellet was resuspended in 9.5 ml of TE buffer (50mM Tris-HCl, lOmM
EDTA). Five hundred microliters of 10% sodium dodecyl sulfate (SDS) was added to
the cell suspension along with 50 [i\ of Proteinase K (20 mg/ml). The mixture was
incubated at 37^0 for 1 hour. Following incubation, 1.8 ml of 5 M NaCl and 1.5 ml of
CTAB solution (10% hexadecyltrimethyl ammonium bromide in 0.7 M NaCl) was
added and the mixture was incubated for 20 minutes at 650C. The mixture was
extracted twice with chloroform/ isoamyl alcohol (24:1) solution, and the nucleic acids
were precipitated by the addition of 0.6 volumes of isopropyl alcohol and centrifugation
55
at 15,000 rpm for 1 hour. The DNA was washed twice with 70% ethanol and
resuspended in TE buffer. The DNA solution was purified by ultracentrifugation in a
CsCl equilibirum gradient (4.3 g CsCl/4 ml) ovemight at 70,000 rpm. The genomic
DNA band was removed and extracted with water-saturated isobutanol to remove the
residual ethidium bromide. Contaminating cesium ions were removed by ovemight
dialysis against 2 liters of TE buffer (Ausubel et al, 1989).
Preparation of Bacteriophage DNA
Ovemight cultures of E. coli NM539 were grown in NZYC medium (Ausubel et
al, 1989) at 37^0 with aeration. One hundred |LI1 of the ovemight culture was mixed
with equal volumes of bacteriophage lysate and lOmM MgC^lOmM CaCl2 solution
and incubated for 20 minutes at 37^0. This mixture was added to 250 ml of prewarmed NZYC medium and incubated for 9-12 hours until complete lysis of the culture
had occured. The cell debris was removed from the lysate by centrifugation at 10,000
x g for 10 minutes. Aliquots (35 ml) of the supematant were passed through a sterile
0.45 |iM membrane filter prior to the addition of 7 |xl DNase (2 mg/ml) and 17.5 \i\ of
DNase-free RNase (2 mg/ml). The lysates were incubated ovemight at 25^0 followed
by centrifugation at 22,500 x g for 2 hours. The phage pellet was resuspended in 500
\i\ of TE buffer. The resulting phage suspension was extracted once with Tris-buffered
phenol (pH 8.0) followed by two successive chloroform extractions. The DNA was
precipitated from the sample volume by the addition of 0.1 volume of 3 M sodium
acetate pH 7.5 and 2 volumes of 100% ethanol (Ausubel et al, 1989; Maniatis et al,
1982; Davis era/., 1980).
56
Preparation of Plasmid DNA
Plasmid DNA was prepared from 50-ml cultures by anion exchange
chromatography on modified silica gel columns obtained from Qiagen. Small scale
preparations of plasmid DNA were obtained by the alkaline lysis procedure described
in Ausubel er a/. (1989).
Preparation of Competent Cells
Cultures of E. coli DH5a were made competent as described by Hanahan (1983).
Cultures of 5. typhimurium JR501 and RTl 142 were made competent by the
procedures of Tsai et al. (1989) and MacLachlan and Sanderson (1985).
Constmction of Genomic Library
Partial Digestion of Genomic DNA
Six hundred \ig of genomic DNA from strain RTl346 were treated with 8.4 units of
the restriction endonuclease Sau3A I and incubated at 37^0 for 30 minutes. The
reaction was terminated by extraction with an equal volume of phenol/chloroform
solution (1:1). The aqueous phase was subjected to two additional extractions with
chloroform and ethanol precipitation of the DNA. The DNA pellet was resuspended
into 100 ^1 of TE buffer (50 mM Tris-Cl pH 7.0,10 mM EDTA).
Fractionation bv Sucrose Densitv Gradient
A sucrose density gradient was prepared by underiaying 4.5 ml of 7 sucrose
solutions of increasing density (10%, 15%, 20%, 25%, 30%, 35%, and 40% sucrose in
10 mM Tris pH 7.5, 10 mM NaCl and 1 mM EDTA) in an SW-41 ultracentrifuge tube.
57
600 ^ig of DNA suspended in IX TE buffer were applied to the gradient and centrifuged
at 25,000 rpm for 24 hours. Following ultracentrifugation, 1 ml fractions were
collected. A 40-^1 aliquot from each fraction was electrophoresed in 0.4 % TAE
agarose to determine the size of the DNA fragments. Fractions containing DNA
molecules ranging from 10-20 kbp in size were pooled and the DNA was ethanolprecipitated. The DNA pellet was resuspended in IX TE buffer at a concentration of
250 ng/^il.
In Vitro Packaging into the X Replacement Vector EMBL4
Five |ig of EMBL4 DNA was digested with 10 units of the restriction
endonuclease BamH I for 24 hours at 4^0. The reaction mixture was extracted with
phenol-chloroform followed by ethanol precipitation. The DNA was resuspended in 20
|il of TE buffer and subsequently digested with 10 units of Sal I for 24 hours at 4^0. 20
|J.g of partially- digested genomic DNA was treated with 0.01 units of calf intestinal
alkaline phosphatase (CIAP) for 2 hours at 37^0. The efficiency of phosphatase
activity was evaluated by treating 500 ng of this DNA with T4 DNA ligase for 4 hours
at 25^0 and measuring the shift in electrophoretic mobility on 0.4% TAE agarose. No
change was detected, indicating that efficient removal of the 5' phosphate groups had
occurred. The integrity of the dephosphorylated cohesive ends was evaluated by
ligating the sample DNA to pBR322 DNA cut with BamU I. The ligation mixture was
electrophoresed and an increase in molecular weight was observed, indicating tiiat die
cohesive ends were intact and functional. CIAP-treated sample DNA was mixed with
EMBL4 arms at a picomole ratio of 5:1 (vector:insert) and incubated with T4 DNA
ligase for 12 hours at 25^0.
58
The ligation mixture was encapsidated using the in vitro packaging system
(Packagene®) supplied by Promega in accordance with the manufacturer's instmctions
and the procedures described by Enquist and Stemberg (1979). The resulting lysates
were titered on E. coli host strains LE392 and NM539.
Librarv Screening
E. coli strain W1485 was lysogenized with the helper phage XI12 by suspending
100 [\\ of an ovemight culture in a soft agar overlay and spotting 50 |il of diluted phage
on the surface (Silhavy et al, 1984). The plates were incubated for 24 hours at 37^0.
Several large colonies were purified by restreaking on LB agar. Lysogeny was
verified by cross-streaking pure cultures with lysates of 0X112 and 0XcI857 Sam7.
Colonies that were immune to lysis were inoculated into 5 ml of LB broth and
incubated ovemight at 37^0 with aeration. A 1-ml aliquot of the cell suspension was
centrifuged for 2 minutes in a microcentrifuge (12,000 x g) and the pellet was washed
three times in phage buffer (20 mM Tris-HCl pH 7.4, 100 mM NaCl, 10 mM MgS04).
The pellet was resuspended in 1 ml of phage buffer. A 100-|il volume of the cell
suspension was mixed with an equal volume of MgCl2/CaCl2 solution and infected
with the bacteriophage library at a multiplicity of infection of 5. The mixture was
incubated for 20 minutes at 37^0 to maximize adsorption and then spread onto NCE
glucose minimal medium. The plates were incubated for 24 hours at 37^0. The
resulting colonies were replica-printed onto NCE glucose minimal medium to verify the
Lip"*" phenotype.
59
Complementation hv Purified Phap^
Complementation of the lip auxotrophic mutation by purified bacteriophage was
tested by a modification of the procedure described by Daub et al (1988). A lOO-^il
volume of an ovemight culture of E. coli W1485 (XI12) was incubated with 25 |il of
bacteriophage lysate for 20 minutes at 37^0. The suspension was washed three
times by centrifugation for 2 minutes in a microcentrifuge (12,000 x g) and the pellet
was resuspended in 100 |xl of phage buffer. Twenty-five microliters of the cell
suspension were spotted onto a NCE glucose minimal medium plate and incubated for
24 hours at 37^0. Complementation was indicated by the presence of large colonies or
dense growth within the spotted area after incubation. Every phage lysate and
washed-cell suspension (25 |J.l of each) was plated separately as a negative control to
ensure that growth was a function of complementation by phage. None of the lysates
or cells formed colonies by themselves.
Construction of Nested Deletions
Nested deletions were obtained by using the Erase-a-base® nested deletion kit
containing Exonuclease III and SI Nuclease supplied by Promega. A preparation
containing 20 |ig of plasmid DNA (derivatives of pIBI30 or pIBI31) was cut with 20
units of Apa I and and 20 units of Xba I to generate the required 3' and 5' cohesive
ends. The reactions were performed under conditions recommended by the
manufacturer and Henikoff (1984). The extent of each deletion was evaluated by
digesting plasmids from each reaction time-point with EcoR V and separating the
linear fragments by TAE agarose electrophoresis.
60
Induction of the lip Gene Product
Ovemight cultures of E. coli DH5a bearing either plasmids pVJ6 or pVJ7 were
grown in LB containing 75 |ig/ml of ampicillin at 37^0 with aeration. These cells were
diluted 1:20 in fresh medium and incubated at 37^0 until the culture reached A600 =
0.50. The cultures were induced by the addition of isopropyl-6-Dthiogalactopyranoside (IPTG) to a final concentration of 100 mM. Samples were taken
at 30-minute intervals and analyzed for the induction of protein by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Identification of Protein
A 1-ml aliquot of each induced culture was collected and centrifuged for 2 minutes
at 12,000 X g in a microcentrifuge. The cell pellets were resuspended in 100 )LI1 of 50
mM MOPS (3-[A^-morpholinoJ propanesulfonic acid) buffer. All of the samples were
adjusted to the same absorbance by the addition of MOPS buffer. A 30-|il aliquot of
each adjusted suspension was resuspended in 90 |J,1 of 4X sample buffer (250 mM
Tris-HCl pH 6.8, 8% SDS, 40% glycerol, 20% 2-mercaptoethanol, and 0.05%
bromphenol blue) and incubated at lOO^C for 10 minutes. The prepared samples were
stored at -20^0 until needed.
Determination of Nucleotide Sequence
Sequence analysis was performed by the dideoxy chain termination method of
Sanger and Coulson (1977). Double-stranded plasmid DNA was used as a template.
Both strands were sequenced using either T3 (pIBI30 derivatives) or T7 (pIBI31
derivatives) primers, 35s-deoxyadenosine triphosphate, and modified T7 DNA
polymerase (Sequenase) (Tabor and Richardson, 1987, 1989). Extension products
61
were separated on a 5% urea-polyacrylamide denaturing gel using HydroLink™
chemically-modified acrylamide monomer.
Results
Cloning the Salmonella typhimurium lip Gene
DNA from a partial Sau3A I digest of 5. typhimurium genomic DNA was ligated
into tiie BamH I site of the lambda replacement vector EMBL4. The ligated fragments
were packaged in vitro into bacteriophage lambda capsids and transduced into the E.
coli host strain NM539. This strain is lysogenized with the unrelated bacteriophage
P2. The P2 lysogen interferes with plaque formation of lambda phage that expresses
functional red and gam gene products. These proteins are encoded on the 12-kbp
stuffer fragment which is enzymatically removed and replaced with the genomic insert.
Therefore, only recombinant Red" and Gam" bacteriophages are able to form plaques
(Zissler et al, 1971; Hendrix et al, 1983). A bacteriophage library was constmcted
by pooling the resulting plaques. This library was subsequentiy screened for
recombinant phages that expressed lip gene activity by transducing E. coli strain
W1485 (XI12) and selecting for colonies on NCE glucose minimal medium. W1485
carries the lip-2 mutation and is a lipoic acid auxotroph (Herbert and Guest, 1968).
The XI12 helper phage supplies the genes in trans that are necessary for lysogenic
growth of the recombinant phage EMBL4. Therefore, lysogenized strains that are
superinfected with the phage library will form colonies rather than plaques, and the
bacterial genes contained in the phage vector will be expressed.
Approximately 3000
Lip+ colonies were isolated independently from three successive screens of the X
library. These colonies were replica-plated onto NCE glucose minimal medium for
verification of the Lip+ phenotype and onto NCE glucose medium containing
62
kanamycin to detect the mini-kan genetic marker linked to the lip gene. Three hundred
seventy-four Lip+ Kan^ colonies were identified. Eight Lip+ Kans and eight Lip+ Kan^
colonies were selected for additional characterization.
Characterization of Lip+ Clones
Colonies of the sixteen lip+ clones that had been selected for additional
characterization were purified by successive streaking on NCE glucose minimal
medium (supplemented with kanamycin where appropriate). The lysogenized
bacteriophages (EMBL4 and XI12) from each strain were induced to lytic growth by
brief exposure to ultraviolet light. A lysate was prepared from each induced culture
and the titer was determined by plating on the E. coli host strain LE392 on X plates
containing 5-bromo-4-chloro-3-indolyl-6-D-galactopyranoside (X-gal) in the top agar.
Under these culture conditions, the induced XI12 bacteriophages will form turbid blue
plaques and the recombinant EMBL4 bacteriophages will form clear plaques (Maurer
et al, 1980). Both populations of bacteriophages were observed in each lysate. A
clear plaque from each lysate was picked as a plug and purified by three successive
rounds of plaque assays using NM539 as the host strain and plating onto X plates
containing X-gal in the top agar. Previous experiments confirmed that the Red+ Gam+
XI12 was unable to form plaques on NM539. A 5-ml transducing lysate was prepared
from each purified bacteriophage and used to transduce strain W1485 (XI12) to Lip+.
All sixteen of the Lip+ clones complemented the lip-2 auxotrophic mutation. Four
representative bacteriophages were selected from each population (4 Lip+ Kan^ and 4
Lip+ KanS) for additional characterization.
DNA was isolated from each of the eight complementing bacteriophages and
digested with EcoR I in order to verify the presence and size of insert DNA and
63
ensure that the complementing bacteriophage was not a product of in vivo
recombination between Xll2 and EMBL4. Both the EMBL4 vector and the XI12
helper phage have distinct electrophoretic banding pattems after EcoR I digestion
(Maurer et al, 1980; Frischauf et al, 1983). All eight of the clones produced EcoR I
fragments indicative of EMBL4 containing insert DNA. One Lip+ Kan^ clone
(designated Eclip-4) was selected for restriction analysis (Figures 3.1 and 3.2) and
for constmction of Lip+ plasmid subclones.
Constmction of Subclones
DNA fragments resulting from an EcoR I digestion of Eclip-4 were ligated into the
EcoR I site of the plasmid pIBI31 multiple cloning region (Dente et al, 1984). This
145- bp region lies within the plasmid-encoded lacZ stmctural gene. Ligation of an
insert fragment into the multiple cloning region creates a lacZ insertion mutation which
can be detected by the addition of X-gal to the culture medium. Transformant cells
that contain parental Lac"*" plasmids will form blue colonies whereas recombinant Lac"
plasmids will form white colonies. The tigation mixture was transformed into the E.
coli strain DH5a and ampicillin-resistant colonies were selected. Plasmid DNA was
isolated from each Lac" Amp^ colony and screened for insert DNA by digestion with
EcoR I and subsequent electrophoresis. Insert DNA was verified by the presence of 2
separate bands ( 2.9 kbp linear plasmid and genomic insert). Recombinant plasmids
were selected and purified for complementation studies.
64
Plasmid Complementation in E. coli
Plasmid subclones containing the lip gene were identified by their ability to
complement the lip-2 auxotrophic mutation in the E. coli strain W1485. This was
accomplished by preparing lysates of the generalized transducing bacteriophage PI on
strains of E. coli DH5a containing the recombinant plasmids. These plasmids were
then transduced into W1485. Lip+ transductants were selected by their ability to
grow on NCE glucose minimal medium containing ampicillin. Four plasmids, each
containing a 1.3 kbp insert, corrected the lipoic acid auxotrophy in E. coli.
Plasmid Complementation in S. typhimurium
All of the recombinant plasmids isolated from the subcloning experiment were
transformed into the 5. typhimurium strain JR501 (BuUas and Ryu, 1983). This strain
was used to modify the plasmid and insert DNA in preparation for its introduction into
the restriction-capable S. typhimurium strain RTl 142 (lip-47::Mu di). Transformant
colonies of JR501 were initially detected by ampicillin resistance and confirmed by
isolation and restriction digestion of plasmid DNA. A P22 transducing lysate was
grown on each strain containing the recombinant plasmids and used to transduce
strain RTl 142 to ampicillin resistance and Lip""". All four plasmids that complemented
the lip-2 mutation in E. coli W1485 also complemented the lip::Mu di insertion
mutation in RTl 142. Each of these plasmids contained a 1.3 kbp insert (Figure 3.3).
One plasmid, pVJl, was selected for further characterization (Figure 3.4).
Attempted Expression of the lip Gene Product
The 1.3-kbp EcoR I fragment was subcloned into the pKK223-3 expression vector
in order to place transcription of the lip gene under the control of the inducible tac
65
promotor which is regulated by the lad repressor. This plasmid contains the pUC18
multiple cloning site and the strong rrn ribosomal terminator downstream from the tac
promoter and which prevents over-expression of plasmid-encoded proteins. The 1.3kbp insert contained in pVJl was liberated from the vector by digestion with EcoR I.
The fragments were separated by agarose gel electrophoresis and a gel slice
containing the 1.3-kbp fragment was excised. The DNA was extracted from the
agarose matrix by centrifugation and subsequentiy purified by phenol extraction and
ethanol precipitation. This fragment was ligated into the EcoR I site of the prokaryotic
expression vector pKK223-3. The ligation mix was used to transform competent cells
of E. coli DH5a. Transformant colonies were identified by their resistance to
ampicillin. DNA from individual colonies was prepared and evaluated for the presence
of insert by digestion with EcoR I. Plasmids which contained insert DNA were
digested with EcoR V and Hind III to establish the orientation of the insert within the
vector. Two plasmids with opposite insert orientations (pVJ3 and pVJ4) were
selected for expression studies. Transcription of the insert DNA was induced by the
addition of the IPTG to the culture media. Proteins were extracted from lysed cells
and separated by SDS-PAGE. No induced proteins could be detected in either
orientation of the insert DNA.
Several underlying assumptions were made in regards to the ability to express
the lip protein in this vector system. First, it was assumed that the lip gene still
retained a functional ribosomal binding site and start codon. Since the boundaries of
the gene had not yet been ascertained, we could not rely upon the formation of an inframe tac::lip fusion. Second, since the sequence of the DNA was not available, it
was assumed that there were no residual transcription termination sequences
between the tac promoter and the lip initiation sequences. Third, since the in vivo
66
fusion data indicated that lip is expressed at very low levels (Chapter 2), it was
assumed that sufficient over-expression of the lip gene product in the multicopy
plasmid vector would occur to allow detection of the induced proteins by Coomasie
blue staining. Since it was not known which strand was the coding strand, the insert
fragment was inserted into the pKK223-3 vector in both orientations. The inability to
detect induced proteins in this experiment was probably due to the violation of one or
more of the aforementioned assumptions.
Nucleotide Sequence of the lip Gene
Two sets of unidirectional nested deletions were constmcted in plasmids pVJl
(pIBI30) and pVJ2 (pIBI31) for their use as double-stranded DNA templates for chain
termination sequencing. These deletions extended from the Xba I site in the multiple
cloning region of both plasmids into the insert DNA. Five plasmids derived from pVJl
were produced which contained deletions extending from 200 to 1150 base pairs into
the insert DNA. These plasmids were used to generate the sequence of one strand of
the insert from the T3 primer. Four additional plasmids derived from pVJ2 were
constmcted with insert deletions ranging from 300 to 800 base pairs. These plasmids
were used to generate the sequence of the complementary strand from the T7 primer.
The plasmid templates and sequencing strategy are illustrated in Figure 3.5.
The sequence of the noncoding strand was 1244 nucleotides in length and had a
GC content of 45% (Figure 3.6). Two sizeable open reading frames were identified
with Wisconson Package sequence analysis software. These open reading frames
were designated ORF-1 and ORF-2. ORF-1 begins at nucleotide 161 and ORF-2
begins at nucleotide 571. Both ORFs terminate at nucleotide 1147. They are
predicted to encode proteins with molecular masses of 37,741 and 22,247 daltons. By
67
comparison, the molecular mass of the E. coli lip gene product was estimated to be
35,000 daltons by protein gel electrophoresis in a previous study (Spratt et al, 1980).
The regions preceding the ATG initiation codons of each ORE were analyzed for
promoter sequences. ORF-1 did not appear to contain any sequences that were
similar to the well-described -10 and -35 consensus sequences (Hawley and McClure,
1983; Rosenberg and Court, 1979) or ribosomal binding sites (Gold et al, 1981; Childs
era/., 1985; Stormo^/fl/., 1982). A putative promoter was identified for ORF-2. The
deduced amino acid sequence of the polypeptide encoded by ORF-2 is presented in
Figure 3.6. Analysis of the amino acid composition is presented in Table 3.2.
Discussion
In this study, we describe the cloning, identification, and the nucleotide
sequencing of the Salmonella typhimurium lip gene. Previous studies have
determined that the lip gene is located at 14 minutes on the 5. typhimurium genetic
map (a minute is approximately 1% of the Salmonella chromosome and is equivalent to
about 45 kbp of DNA). Fine structure mapping by P22-mediated cotransduction
placed the lip gene 6.9 kbp from the nadD gene (14% cotransducible) and 16.0 kbp
from the leuS gene (50% cotransducible). We have isolated clones which express
lipoic acid biosynthetic activity on a 1.3 kbp EcoR I fragment of S. typhimurium
genomic DNA. The protein encoded by this fragment complements lipoic acid
auxotrophic mutations in both S. typhimurium (RTl 142) and E. coli (W1485). All
plasmids (derivatives of pIBI30, pIBI31, and pKK223-3) which contained this EcoR I
fragment in either orientation complemented the lip mutations equally well. These
data indicate that the lip gene product is expressed from its own functional promoter.
Furthermore, the ability to express a protein from an endogenous promoter indicates
68
that the stmctural gene must contain a ribosomal binding site. Analysis of the
nucleotide sequence revealed several open reading frames (ORFs). Two of these
appeared to be good candidates for the lip coding sequences based upon the predicted
mass of their encoded proteins. Comparison was made to the molecular mass of the
E. coli lip gene product, which was estimated to be 35,000 daltons in a previous study
(Spratt et al, 1980). The first open reading frame, ORF-1, initiates at nucleotide 161
and is presumed to encode a polypeptide with a mass of 37,741 daltons. The predicted
mass of the presumed protein encoded by ORF-2 is 22,247 daltons. Botii open reading
frames were evaluated for the presence of regulatory sequences.
A promoter is a sequence of DNA which is capable of binding the holoenzyme of
RNA polymerase (core enzyme and sigma subunit). The critical sequences which
facilitate this DNA-protein interaction are located in two regions, designated -10 and
-35, which precede the start site of transcription initiation. The "-10 region" is situated
5-9 nucleotides upstream from the start site. The "-35 region" usually precedes the
-10 sequence by 16-18 nucleotides (Reznikoff er a/., 1985; McClure, 1985). Greater
variations in spacing between the -35 and -10 regions and between the -10 region and
the ATG codon may decrease the efficiency of transcription initiation. The -10 region
is characterized by the consensus sequence TATA AT (McClure, 1985)(large capital
letters indicate those bases which are highly conserved, small capital letters indicate
the sequences which have the most variation). The -35 region is characterized by the
consensus sequence TTGACA (McClure, 1985).
These consensus sequences
represent the "perfect" promoter which is capable of initiating transcription with
maximum efficiency. Deviations from either consensus sequence contribute to a
decrease in polymerase binding efficiency. It should be noted that of all the promoters
69
that have been described to date, not one has a perfect match to both consensus
sequences (McClure, 1985).
The sequences that preceded the initiation codons of ORF-1 and ORF-2 were
examined for these consensus sequences. Two potential matches to the -35
consensus sequence were identified in the region upstream from ORF-1 (TTGATC at
nucleotide 58 and T T Q A A G at nucleotide 70, bases that match the consensus
sequence are underiined). No sequences which possessed similarity to the -10
consensus could be identified. This region was also searched for sequences that are
known to interact with altemate sigma factors (Cowing et al, 1985). No similar
sequences could be identified. ORF-2 was examined for sequences that were similar
to the canonical -10 and -35 sequences. Two sequences with reasonable similarity to
the -35 consensus were identified at nucleotides 460 (TTGAGC^ and 477 (TTTA£C).
A search for sequences with similarity to the -10 consensus as well as those that
recognize altemate sigma factors revealed a single region at nucleotide 507 with the
sequence T A T A T T .
The sequences centered at nucleotides 477 and 507 are the most likely candidates
for the -35 and -10 regions of a promoter which directs the transcription of ORF-2. A
search of all known E. coli and 5. typhimurium promoters that are maintained in the
GENEBANK data base was performed in an attempt to identify additional promoters
with similar sequences. The bacterial gene rplJ contains the -35 sequence TTTACG,
and the alaS gene contains the sequence TTTACC. A bacterial gene which contains a
-10 sequence of TATATT could not be found. However, this sequence has been
identified in several bacteriophage genes, most notably the ant gene of bacteriophage
P22 (Hawley and McClure, 1983). It is important to note that this search was
70
restricted to promoters which were confirmed by either primer extension experiments
or DNA protection assays.
The functional assays indicated that the lip gene contained a ribosomal binding
sequence in addition to a functional promoter. The sequence at the ribosomal binding
site of the mRNA transcript must hybridize with a complementary sequence of the 16S
rRNA molecule contained in the small ribosomal subunit. The rRNA region that
interacts with the mRNA transcript has the sequence 5'-GAUCACCUCCUUA-3' in E.
coli. The region preceding the ATG codon of ORF-2 was searched for sequences that
complement this rRNA sequence. Several likely candidates for ribosomal binding
sites were identified at nucleotides 549 (GGAGG^. 551 (AGGAi and 556
(AGGTGAcCl All of these sites are within an acceptable distance from the ATG
initiation codon and have been identified in a number of promoters (Gold et al, 1981).
The optimum space between a Shine-Dalgamo sequence and the ATG initiation codon
has been defined as 5-14 nucleotides with 7 nucleotides representing the ideal
position (Watson et al, 1987; Gold et al, 1981). The sequences which begin at
nucleotides 549 and 551 exceed this range, but they still fall within acceptable limits.
The best candidate for the ribosomal binding site of ORF-2 is the sequence that
initiates at nucleotide 556, which precedes the ATG codon by 5 nucleotides. No actual
sequences that resemble a Shine-Dalgamo sequence were found in the region
preceding the initiation codon of ORF-1.
The region that precedes the ATG initiation codon of ORF-2 contains sequences
which satisfy the criteria for a functional promoter. A putative -35 and -10 region has
been identified in addition to a Shine-Dalgarno sequence which facilitates ribosomal
binding. The inability to identify similar stmctres in the region preceding ORF-1
suggests that ORF-2 is the sequence that comprises the lip gene. The protein that is
71
specified by these sequences has a predicted molecular mass of 22,247 daltons. This
size falls well short of that predicted by Spratt et al (1980). However, it should be
noted that the protein which they detected was not conclusively identified as the
product of the lip gene. Since the conclusions of this study are based entirely upon
sequence data, one must also heed the caveat that unresolved ambiguities in the
sequence data may exist and that these errors may incorrectly indicate a termination
of the predicted mRNA transcript that is in fact premature.
Analysis of 6-galactosidase activity in lip::lac operon fusions suggests that the lip
gene is expressed constitutively and in the absence of any apparent regulatory
mechanism (Chapter II). The data from these experiments indicate that the stmcture
of the lip promoter may deviate from the established -10 and -35 consensus
sequences. Raibaud and Schwartz. (1984) demonstrated that promoters which have
strong similarity to the -10 consensus sequence, but have no similarity to the -35
consensus, usually require activation by a DNA-binding protein. This protein binds at
the -35 region and perturbs the tertiary stmcture of the DNA double helix in such a
way as to facilitate RNA polymerase binding. The putative promoter for ORF-2
contains sequences that are similar to both the -10 and -35 consensus sequences.
Thus, activation by a DNA-binding protein does not appear to be required. The
spacing between the -10 and -35 elements of the ORF-2 promoter do not agree with
the optimal values of the consensus promoter (16-18 bp between -35 and -10). This
spatial deviation may inhibit the ability of RNA polymerase to productively bind to tiie
promoter region, which would decrease the frequency of transcription initiation. This
observation is supported by the transcriptional activity measured in the lip::lac fusion
strains. Thus, the data from this study support the hypothesis that the rate of
transcription initiation from the lip promoter and subsequent expression of the lip gene
72
product is modulated by promoter stmcture and does not appear to require a DNAprotein interaction.
>.
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74
Table 3.2. Deduced amino acid composition of the 22,247 dalton protein
encoded by the open reading frame ORF-2.
Amino Acid Composition
Amino Acid Number
15
alanine
histine
4
8
threonine
5
glutamine
phenylalanine 6
asparagine
8
leucine
30
10
tyrosine
9
glycine
8
serine
% composition
Amino Acid
Number
% composition
8%
2%
4%
3%
3%
4%
15%
5%
5%
4%
cysteine
methionine
arginine
isoleucine
tryptophan
glutamate
proline
aspartate
lysine
valine
8
2
16
16
3
7
10
4
10
16
4%
1%
8%
8%
2%
4%
5%
2%
5%
8%
75
76
77
78
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82
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86
1- GAA TTC CCT ACG CCA TTT ACT TAC AAA GTA ATG GGG CAG GCG TTA CCT GAG CTG
-54
55- GTT GAT CAG GTG GTT GAA GTG GTA CAG CGC CAT GGC CCT GGT GAT TAC TCT CCG
-108
109- ACG GTA AAA CCG AGC AGC AAA GGC AAC TAC CAC TCG GTC TCT ATC ACC ATT AAT
-162
163- GCC ACC CAT ATT GAA CAG GTT GAA ACG CTG TAC GAA GAA CTG GGC AAT ATT GAT
-216
217- ATC GTG CGA ATG GTA TTG TAA TAC ATC CAG ACG GTT ACC CGG TCT TTA TCC GGT
-270
271- AAC TCC CCG CCT CCC CCG CTG TGA TAT ACT TTC CTC TCT TTT TTC ATT CTC TAC
-324
325- GGA GAT GCC GTT TTG TAT CAG GAT AAA ATC CTT GTC CGC CAG CTC GGC CTT CAG
-378
379- CCC TAT GAA GCT ATC TCT CAG GCC ATG CAT AAC TTT ACC GAT ATG CGC GAT
-35 region
433- AAC AGC CAT GAT GAA ATC TGG TTG GTT GAG CAT TAC CCG GTG TTT ACC AGG
-10 region
487- GGC CGG TAA AGC GGA ACA TAT ATT GAT GCC TTG GCG ATA TTC CCG TTG TGC
ribosome binding site
541- GCG ATC GTG GAG GAC AGG TGA CCT ATC ATG GTC CGG CCA ACA GGT AAT GTA
MET VAL ARG PRO THR GLY ASN VAL
GAA
-432
GAG
-486
AGA
-540
CGT
ARG
-594
595- TTT TTA CTT AAC CTG AAA CGA CGT AAA CTG GGC GTG CGT GAT TTA GTC ACC CTA
PHE LEU LEU ASN LEU LYS ARG ARG LYS LEU GLY VAL ARG ASP LEU VAL THR LEU
-648
649- CTT GAA CAG ACC GTG GTG AAT ACG CTG GCT GAA ATT GGT ATT GAG GCG CAT CCG
LEU GLU GLN THR VAL VAL ASN THR LEU ALA GLU ILE GLY ILE GLU ALA HIS PRO
-7 02
703- CGC CGA TGC GCG GGC GTC TAC GTG GTG AAA AGA AAA CTG CTC GTG GTT ACG ATC
ARG ARG CYS ALA GLY VAL TYR VAL VAL LYS ARG LYS LEU LEU VAL VAL THR ILE
-7 56
757- GCT CGC GCT GCT ATC CAG TTA CGC GTA ACG GTA CAT GGA TCT TCT CTT CCT GCG
ALA ARG ALA ALA ILE GLN LEU ARG VAL THR VAL HIS GLY SER SER LEU PRO ALA
-810
811- TAT CAA CCG TGG TGG TAT AGC CGG GGA CAT GGA ATG CAA AAT AAC GCA GTG AAG
TYR GLN PRO TRP TRP TYR SER ARG GLY HIS GLY MET GLN ASN ASN ALA VAL LYS
-864
865- GAA GAT GCA TAC CAA CGG ATA ATA TTG GCC GCT TGC TGG CGA ATA ATT TTA GCT
GLU ASP ALA TYR GLN ARG ILE ILE LEU ALA ALA CYS TRP ARG ILS ILS LEU ALA
-918
919- CTG CTA ATA ATC ACG TAT GAA TAT ATT TGC TGC TTT AAA TGC GTA AAA TAT AAT
LEU LEU ILE ILE THR TYR GLU TYR ILE CYS CYS PHE LYS CYC VAL LYS TYR ASN
-972
973- GGC CCA AGA AAA TTT TAT GGG TCA TTA ATC ATA TAT GAT TTG AAT GCT TTA CTT
GLY PRO ARG LYS PHE TYR GLY SER LEU ILE ILE TYR ASP LEU ASN ALA LEU LEU
-1026
1027- CTC CCC CCG CTT TCT ATA TTT TCG TTA TAT CCC TGC TGT ACC CGA GCG CGA TGC
LEU PRO PRO LEU SER ILE PHE SER LEU TYR PRO CYS CYS THE ARG ALA ARG CYS
-1080
1081- CTC TTC AGT CCT CAT GAT GAG GAA TTT TCC GTG AAC AAA ATC CCG GCC TTG CTG
LEU PHE SER PRO HIS ASP GLU GLU PHE SER VAL ASN LYS ILE PRO ALA LEU LEU
-1134
1135- TTA CTT ATC TTA TTG AAA TGA CTA AAA ATA CAT TCA TAA GAG TAA TTT TAA AAC
LEU LEU ILE LEU LEU LYS OPA
-1188
1189- CAA CCC ATA AAT GCA AAA TAT ACT ATT TCA TCT ATT ATT CAT AAT ACG AAT GAA
-1242
1243- TTC G
-1246
87
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