END PRODUCT REPRESSION OF COBALAMIN SYNTHESIS LESLIE …
Transcript of END PRODUCT REPRESSION OF COBALAMIN SYNTHESIS LESLIE …
END PRODUCT REPRESSION OF COBALAMIN SYNTHESIS
IN SALMONELLA TYPHIMURIUM
by
LESLIE B. GARRISON, B.S.
A THESIS
IN
MICROBIOLOGY
Submitted to the Graduate Faculty of Texas Tech University in
Partial Fulfillment of the Requirements for
the Degree of
MASTER OF SCIENCE
Approved
Accepted
December, 1989
73
/3^ IS3 ACKNOWLEDGMENTS
I am deeply indebted to Dr. Randall M. Jeter for his direction of the work for this
thesis and my graduate work, and to the other members of my committee, Drs. IDoris
Lefkowitz and Llewellyn Densmore, for their assistance in the final preparation of this
thesis. I would also like to express my appreciation to my parents, grandparents and wife
for their encouragement and support during my graduate wOTk.
u
CONTENTS
ACKNOWLEDGMENTS ii
ABSTRACT iv
LIST OF TABLES v
I. INTRODUCTION 1
n. MATERL\L AND METHODS 9
in. RESULTS 22
IV. DISCUSSION 34
REFERENCES 38
m
ABSTRACT
The purpose of this research is to elucidate the mechanism that regulates the end-
product repression of the genes for cobalamin biosynthesis (cob) in Salmonella
typhimurium. Twenty-nine insertion mutants which exhibited altered repression
phenotypes were isolated. The effect of these insertion mutations on the expression of the
cob genes was quantified by using P-galactosidase assays to monitor the transcription of a
coblr.lac operon fusion. The mutants were divided into two phenotypically distinct groups
which were designated positive and negative. In the 11 negative mutants, the cobl operon
was not expressed under any conditions. All of the insertion mutations in this group were
mapped in or near the cobl operon itself. In the 18 positive mutants, the cobl operon was
less sensitive to repression by cobalamin than in the parent strain. The positive mutants
were subdivided into four groups by cotransduction and conjugational mapping. One
group of mutations is located in the cobalamin transport genes (btuB and btuCED). A
second group lies near the distal end of the ethanolamine utilization (eut) operon at 50 map
units and prevents the mutants from utilizing ethanolamine as a sole carbon source. A third
group lies between 47 and 62 map units on the S. typhimurium chromosome. The fourth
group of positive mutations remains unmapped.
The insertion mutations that are located in the eut region appear to lie downstream
from the genes that encode the enzymes and a known regulatory protein for ethanolamine
utilization. We propose that these mutations affect the synthesis of a second regulatory
protein that binds cobalamin. This protein plays a dual regulatory role by repressing
transcription of the cobl operon and activating transcription of the eut operon.
IV
UST OF TABLES
TABLE 1 Salmonella typhimurium stTddns used mitds study 10
TABLE 2 Mini-tet insertion mutants generated in this study 24
TABLE 3 Results ofcotransductionalcrossses between the positive insertion mutations and markers in or near known cobalamin transport genes 26
TABLE 4 Results ofcotransductions between ewr mini-tet insertion mutants and maikers in and near eut operon 28
TABLE 5 Cojugational mapping of mini-tet insertion mutations in strain RT887 31
TABLE 6 p-Galactosidase enzyme activity in permeabilized cells 32
CHAPTER I
INTRODUCTION
Cobalamin (B12) is the largest and most complex of the vitamins (13). It is not
synthesized de novo by plants or animals; it is produced only by certain microorganisms
(18). Therefore, animals must depend on these microorganisms for their supply of
cobalamin.
Ruminant animals obtain the cobalamin they require from the microorganisms
which grow in the rumen. Other animals, including humans, must glean their supply of
cobalamin entirely fix)m dietary sources. In humans, prolonged dietary deficiency of
cobalamin, or a defect in cobalamin absorption, may result in pernicious anemia. This is an
often fatal disease which has no animal counterpart It can be treated by intravenous
injections of the vitamin form of cobalamin (14).
The Structure of Cobalamin
The cobalamin molecule is made up of two major components, the corrin ring and
a ribonucleotide that contains the heterocyclic base 5,6-dimethylbenzimidazole (DMBI) see
Fig. 1. The corrin ring is similar to the tetrapyrrole rings found in heme and chlorophyll
(7). However, the linkages between the four pyrroles in the porphyrin ring are all methene
bridges, while the A and D pyrroles of the corrin ring are joined by a simple covalent bond
between the a carbons. An atom of cobalt is held in the center of the corrin ring by a
covalent bond to the nitrogen of the A pyrrole, and by coordinate bonds to the other three
pyrrole nitrogens (20).
The nucleotide portion of the cobalamin molecule is joined to the corrin ring by a
D-l-amino-2-propanol side chain on the D pyrrole, and by a coordinate bond between
1
NH'
NH2OCCH2CH2
hfH^CCH2
" 3 ^ H3C NH2OCCH
.CH2CONH2
CH2CH2CONH2
CH2CH2CONH2
CH2OH
Figure 1: Adenosylcobalamin.
DMBI and the cobalt atom. The nucleotide lies below the plane of the ring (13,15). This
nucleotide is unusual for two reasons: the base is linked to the C-1 of the ribose by a 7-a-
glycoside bond (in lieu of the 9-p-glycoside bond found in other nucleotides), and DMBI
itself is found only in cobalamin (7,20).
The presence of the unique base is apparently necessary for the function of
cobalamin as a coenzyme. 0)balamin analogues, in which DMBI has been replaced by
other purine bases, are less effective in vertebrates, and analogues that have pyrimidine
bases are inert as coenzymes in humans (17,20).
The sixth substituent position of the cobalt atom, which lies above the plane of the
ring, may be occupied by a number of different groups, including NO2, OH, SO3H, CNS,
or CN (20). In the commercially-produced form of cobalamin, the sixth position is most
commonly occupied by a cyano (CN) group; thus, cyanocobalamin has become
synonymous with the term vitamin B12 (10). However, cyanocobalamin is formed only
during the industrial production of cobalamin and apparendy is not produced in vivo
(10,20). In the cofactor forms, which do occur in the cell, the sixth position is filled by
either a methyl group (methylcobalamin) or by 5'-deoxyadenosine (adenosylcobalamin)
(18). Cobalamin-dependent enzymes and their cofactors are discussed in greater detail
below.
Industrial Production
Cobalamin is one of only two vitamins that are produced industrially entirely by
microbial fermentations, riboflavin being the other (7). The majority of the cobalamin is
produced in the form of cyanocobalamin, with smaller amounts of hydroxocobalamin and
adenosylcobalamin being produced as well. The bacterial species most often used to
produce cobalamin industrially is Pseudomonas denitificans, which requires the addition of
DMBI to the culture medium (7).
The originally-isolated Pseudomonas strain was able to produce 0.6 mg of
cobalamin per liter. After 12 years of strain development, the yield was increased to 60 mg
per liter (7). Strain development usually involves generating mutants with ultraviolet
radiation or chemical mutagens, indirect selection, and the screening of mutants for
increased production. Yields may also be increased by varying the culture conditions (7).
Neither of these two methods for enhancing product yield requires a detailed knowledge of
the genes involved or their regulation.
The discovery that Salmonella typhimurium synthesizes cobalamin under
anaerobic conditions (16) allows established genetic and enzymatic techniques to be applied
in an organism that has a well-understood genetic make-up. The analysis of cobalamin
biosynthesis can therefore be facilitated by studying the process in this organism.
Cobalamin-Dependent Enzvmes in Salmonella
It has been estimated that the production of cobalamin may require as many as 30
unique enzymes (11); this is a considerable investment of the cell's resources. Yet none of
the cobalamin-dependent enzymes known to exist in Salmonella seem to be vital to the ceU
under most culture conditions. Four cobalamin-dependent enzymes have thus far been
described in Salmonella. They are N^-methyltetrahydrofolate-homocysteine
methyltransferase (EC 2.1.1.13), ethanolamine ammonia-lyase (EC 4.3.1.7), propanediol
dehydratase (EC 4.2.1.28), and an unnamed enzyme that reduces epoxyqueuosine to
queuosine (12).
The homocysteine methyltransf erase, which uses methylcobalamin, (11) transfers
a methyl group from N^-methyltetrahydrofolate to homocysteine to form methionine. This
enzyme is the product of the metH gene (29). The final step of methionine synthesis may
also be carried out by a cobalamin-independent methyltransferase, which is the product of
the metE gene (16). However, the metH enzyme is much more efficient than the metE
enzyme, which may constitute as much as 5 % of the total soluble protein in cells which
are using it to supply their need for methionine (32). The production rates of these two
enzymes are regulated by the availability of cobalamin. In the presence of cobalamin, the
expression of metH is increased while the activity of the metE gene is suppressed (25).
The suppression of metE by cobalamin requires the products of the metH and metF (5-
methylenetetrahydrofolate reductase) genes, which suggests that 5-methyltetrahydrofolate
must bind to the metH holoenzyme in order for metE to be repressed (22).
Ethanolamine ammonia-lyase is encoded within the eut operon, which lies at 50
map units on the Salmonella chromosome between the cysA and purC genes (24). The
coenzyme for ethanolamine ammonia-lyase is adenosylcobalamin (1,4). This enzyme
cleaves ethanolamine to yield acetaldehyde and ammonia (2,27). The acetaldehyde is then
converted into acetyl coenzyme A by acetaldehyde dehydrogenase, which is also encoded
within the eut operon (24).
In addition to the two enzymes, the eut operon also encodes a regulatory protein
that enhances the transcription of its own operon. This positive regulatory protein appears
to be transcribed finom the main promoter for the operon and also fh>m a weak secondary
promoter just in front of the regulatory gene (24). The main promoter of the eut operon is
induced by the simultaneous presence of cobalamin and ethanolamine in the culture medium
(3,24).
Propanediol dehydratase is another enzyme which uses adenosylcobalamin as a
coenzyme (1). This enzyme transforms 1,2-propanediol into propionaldehyde (19). This
is the first step in the utilization of propanediol as a carbon and energy source by
Salmonella (24).
The last of the four cobalamin-dependent enzymes reduces the 2,3-epoxy-4,5-
dihydroxycyclopentane ring of epoxyqueuosine to the 4,5-dihydroxycyclo-2-pentene ring
found in queuosine. C^euosine is a nucleoside which replaces guanosine in position 34 of
the anticodon of certain tRNA molecules in eubacteria and many eucaryotes. When
cobalamin is not available, all of the tRNAs will contain guanosine. If cobalamin is
available, then some of these tRNAs will contain queuosine at position 34 (12).
Transport of Cobalamin
Cells may obtain cobalamin for use as a cofactor either by transporting external
cobalamin into the cell or by internal synthesis. In Escherichia coli, the transport of
cobalamin into the cell requires the products of genes btuB^ btuC^ btuD, btuE and tonB
(5). The btuB gene product is an outer-membrane protein which binds cobalamin. The
tonB gene product is thought to be an energy-transducing protein that is responsible for the
active transport of cobalamin across the cell wall (23). The transport of cobalamin fix)m the
periplasmic space across the cytoplasmic membrane and into the cell is perfonned by the
btuC, btuD, and btuE proteins. Mutants that have lost one or more of these proteins are
only slightiy impaired in their ability to transport cobalamin as long as they have an intact
btuB-tonB system (5).
The Genes Involved in Cobalamin Synthesis
The biosynthesis of cobalamin begins in the porphyrin pathway, which also
produces heme and chlorophyll. The last intemoediate that the two pathways have in
common is uroporphyrinogen HI (7,33). The specifics are not fiilly understood, but the
general outline of the cobalamin pathway has been elucidated in nonenteric bacteria and
several of the intermediates are known (7,33).
While litUe is known about the enzymatic pathway that produces cobalamin, the
biosynthetic genes and their regulation in Salmonella have been described. The genes
involved in the production of cobalamin (cob) are located at 42 map units (between his and
supD). The cob genes appear to be organized into three discrete operons that arc
designated cob I, cob n, and cob HI. The order of the operons (in the counterclockwise
direction along the S. typhimurium genetic map) is cobl-cobUl-cobll. The contribution of
each of these operons to the production of cobalamin has been determined by nutritional
studies, cobl mutants cannot synthesize the corrin ring, and therefore require an
exogenous supply of the biosynthetic interatiediate cobinamide in order to produce
cobalamin. cobU mutants are unable to produce DMBI, and so an exogenous supply of
DMBI is required in order to produce cobalamin. cobUl mutants cannot join the corrin ring
and the DMBI portions of the molecule together, so that these mutants cannot produce
cobalamin even if cobinamide and DMBI are both provided (16).
Regulation of the cofe Genes
The expression of the cob genes is affected by a number of factors. These genes
are maximally expressed under conditions of anaerobic respiration with fumarate as the
electron acceptor. The presence of oxygen represses the transcription of the cob operons in
general and cobl in particular. The expression of cobl may be reduced 40-fold from its
maximal level in the absence of oxygen. The cob genes are also regulated by adenosine
3',5'-cyclic monophosphate (cAMP) (11). An increase in cAMP levels within the cell
causes the cAMP-catabolite regulatory protein complex to bind to and enhance the
expression of the cob genes. Conversely, a drop in the cAMP level allows the complex to
be released from the cob genes, which reduces their rate of transcription (28). This type of
regulation by cAMP is typical of genes involved in the utilization of carbon sources. The
cob genes are also subject to end-product repression by cobinamide (the ring portion of the
cobalamin molecule), DMBI, and cobalamin itself. Individually, DMBI and cobinamide
have only a slight effect on cobl, but together they decrease the operon's level of
transcription by half Cobalamin has the strongest repressive effea of all, decreasing the
expression of the cob I genes by up to 80 % (11).
8
I propose that the end-product lepression of the cob operon is effected by a
legulatofy protein ^ch binds cobalamin, changes conformation, then blocks transription
by binding to an operator sit on the operon. In this study we will use genetic and
enzymatic techniques to map the location of this regulatory gene and to quantify its effect
on thecob genes in S. typhimurium.
CHAPTER n
MATERIAL AND METHODS
Bacteria. Culture Media, and Growth Conditions
The bacterial strains used in this study were derivatives of Salmonella
typhimurium LT2; the genotypes are listed in Table 1. The bacteriophage used to perform
transductions was P22 HT 105/1 mr-201(10). The complex media used routinely in this
study were nutrient agar and nutrient broth (Difco Laboratories), and the minimal medium
was the E medium of Vogel and Bonner (31). NCE (No carbon E) minimal medium (8)
was used in experiments involving the utilization of specific carbon sources. The final
concentrations of various carbon sources added to NCE medium were ethanolamine,
14mM; fumarate, 40mM; glucose, llmM; glycerol, 22mM; lactose, 7mM; and 1,2-
propanediol, lOmM. Other supplements were spread onto the minimal medium plates as
required to support the growth of auxotrophic mutants (0.1 ml of these supplements were
added to each plate). The concentrations of the stock solutions were as follows:
cyanocobalamin, 4mg/l; cobinamide, 4mg/l; 5,6-dimethylbenzimidazole (DMBI), 50 mg/1;
and L-methionine, 9g/l. The concentrations of the antibiotics used in the complex media
were 30 mg/1 of ampicillin, 50mg/l of kanamycin, and 30 mg/1 of tetracycline. The
antibiotic concentrations used in the minimal media were 125 mg/1 of kanamycin, 1(X)0
mg/1 of streptomycin, and lOmg/1 of tetracycline.
Unless otherwise stated, all bacterial cultures were incubated at 37°C. Broth
cultures were grown aerobically at 200 rpm in a rotary incubator shaker (Series 25, New
Brunswick Scientific C!o.). Plate cultures were incubated in standard incubators (Model
330, National Appliance Co.). Anaerobic incubations were performed in an anaerobic
chamber (Model 1025, Forma Scientific Co.) under an atmosphere of 93% N2, 5% CO2,
9
10
TABLE I, Salmonella typhimurium stndns used in this study.
Strains
RT787 RT789 RT801 RT869 RT870
RT933 RT934 RT935 TR5654 TR5655
TR5656 TR5657 TR5658 TR5660 TR5662
TR5663 TR5666 TR5667 TR5668 TR5669
TR5671 TR5686 TR5688 TR6583 TT627
Tr628 TT10271 TT10423 TT10426 TT10427
TT10723 TT10852 Tni580 TT13775 TT13893
Genotype
metE205 ara-9 cob-21:: Mu dA aroDSSrpsU argfl87 purC7 cysA533
leu^7 cob-24::MudJ Aeut-237 cob-24::Mu dJ thrA9rpsLl leu^SSrpsLl
proA36 rpsLl purES rpsLl pyrCJrpsLl pyrF146 rpsLl his01242 hiS'2236 rpsLl
purFMSrpsLl serA13 rpsLl cysG439rpsLl cysE396 rpsLl ilV'508rpsLl
pyrB64 rpsLl aroDMOrpsU purAlSS rpsLl metE205 ara-9 purC7 rpsLI (Ftsl 14 lac^zrf'20::TnlO)
pyrC7 rpsLl (Ftsl 14 lac+zzf-21::TnlO ) eut-I8::Mu dJ proAB -^7(F128pro+ toc+ zzf'1831::TnlO Ll6M7TtX^ proAB47iFl2S pro+ lac+ zzf'1834::TnlO Ai6 AiTKan pNK972
metE205 ara-9 metH2355:'Mu dA metE205 ara-9 cdb-24:Ma dJ t^m-237 btu-2vM\x dJ em-20S:\lTdO
11
and 2% H2. Centrifugations were performed in a table-top clinical centrifuge (International
Equipment Co.). The 30°C incubations for the p-galactosidase assay were performed
using a Dri-Bath (Type 16500, Thermolyne Corp.), and the absorbance readings were
made with a Spectronic 20 spectrophotometer (Milton-Roy Co.).
The organic chemicals and antibiotics used in this study were obtained from Sigma
Chemical Co., with the exception of the ethanolamine and 1,2-propanediol, which were
purchased fiom Aldrich Chemical O).
Lysate Production and Transformations
Lysates were produced by adding a 0.3 ml sample of an overnight culture of S.
typhimurium (various mutant strains) to a tube containing 5 ml of nutrient broth and 0.03
ml of a stock P22 lysate (1 x 10 -1 x 10 ^ plaque-forming units/ml) which had been
grown on LT2. The cells were grown overnight, and then the culture was centrifuged at
2,500 ipm for 20 min. The supernatant containing the phage was poured into a sterile
tube, and 0.5 ml of chloroform was added. The supernatant and chloroform were mixed
using a vortex mixer, and the chloroform was allowed to settie out of the aqueous layer
ovemight at room temperature.
Transductions were perforated by adding 0.03 ml of a lysate (of phage grown on
the desired donor bacterial strain) to an ovemight culture of recipient cells. The culture was
incubated for 30 min to allow for phenotypic expression of the transduced marker.
Samples (0.1 ml) of the culture were then spread onto plates which would only allow the
cells that had inherited the desired trait (an antibiotic maricer or the repair of some
auxotropic mutation) to grow.
12
Gene Pool Construction
A TnlO Al6 A17Tet^ (mini-tet) gene pool was produced by using Salmonella
strains TT10423 and TT10427. The strain TT10423 contains an F plasmid which has a
mini-tet element (a TnlO with the gene for transposase removed) inserted into it (8,10).
TT10427 contains the plasmid pNK972 which carries only the transposase gene.
A P22 lysate grown on TT10423 was used to transduce an ovemight culture of
TT10427, and 0.1 ml aliquots of the cells were spread onto nutrient agar + tetracycline
plates. The plates were incubated overnight, and the antibiotic-resistant colonies (-100
colonies^late) were washed from the plates with 0.1 M phosphate buffer with 10 mM
ethyleneglycol-bis-(p-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) to bind free
calcium ions in the solution and thereby prevent P22 phage fix)m attaching to the uninfected
cells (8). The cells were collected in a 15 ml tube, centrifiiged at 2,500 rpm for 20 min,
and washed twice with phosphate buffer with EGTA. The cells were resuspended in
phosphate buffer without EGTA, and a lysate was grown on the pooled cells. This lysate
was the mini-tet gene pool which was used in later experiments. A similar procedure was
also used to prepare a TnlO A16A17 Kan^ (mini-kan) gene pool with TT10426 as the
donor strain.
Isolation of Mutants Using Mini-Tet Insertions
S. typhimurium strain TT10852 has a Mu dJ element inserted into the cobl
operon. Mu dJ contains a partial lac operon which has no promoter of its own, so that it
must depend on the promoter of the operon into which it is inserted for its transcription.
LT2, which is the parent strain of all the S. typhimurium strains that were used or
generated in this study, does not produce P-galactosidase. The only lac operon that is
contained by TT10852 is the one within the Mu dJ element. Thus, the expjression of the
cobl operon can be monitored by assaying for P-galactosidase enzyme activity.
13
Strain Tri0852 was grown ovemight in nutrient broth. The broth culture was
transduced with a mini-tet gene pool, and 0.1 ml aliquots were spread onto nutrient agar
plates with tetracycline to select for cells which had inherited the mini-tet elements by
homologous recombination into the chromosome. The plates were incubated overnight,
then replica-plated onto lactose MacConkey agar plates with tetracycline and with or
without a supplement of cyanocobalamin. If the colonies produced P-galactosidase, they
appeared red on the MacConkey agar plates; if they did not produce p-galactosidase, they
appeared white.
Two classes of regulatory mutants were detected by this selection. In the first
class, termed positive mutants, the cob genes were rendered insensitive to end-product
repression, and the cells were Lac"*" in both the presence and absence of cobalamin. In the
second class, termed negative mutants, the cob genes were not transcribed whether
cobalamin was present or absent, and these cells were Lac" under both sets of conditions.
All mutants of both classes were isolated, and their phenotypes were confirmed by
restreaking them onto the same type of indicator medium that had been used in the original
screening. The mutants were also streaked onto E glucose minimal medium plates and
incubated ovemight to determine if the production of cobalamin in these cells was still
repressed by oxygen. This test was performed in a metE (TR6583) background.
Determining the Proximity of the Putative Regulatory Mutations to the cobl Operon
The ability of the mini-tet insertions in each mutant to cotransduce with the
cobli'Mu dJ of TT10852 was determined. P22 lysates were grown on each the putative
regulatory mutants. Ovemight cultures of TT10852 were transduced with these lysates,
and 0.1 ml samples were spread onto nutrient agar plates with tetracycline. The plates were
incubated ovemight and the colonies were scored for kanamycin sensitivity, which
14
indicates that inheritance of the mini-tet results in simultaneous loss of the Mu dJ, and that
the two elements are cotransducible.
Mutants that carried cotransducible mini-tet insertions were tested to determine if
the insertions were located in the cob genes. All of the mutants, along with LT2 and
TT10852 controls, were streaked from nutrient agar stock plates onto four different sets of
E glucose minimal medium plates. The control set of plates was spread with cobalamin,
while the three test sets contained either no supplanent, cobinamide, or DMBI. All four
sets of plates were incubated anaerobically for 48 hr. Mutants in the cobl operon are
unable to produce cobinamide, and they grow only on the plates where cobinamide has
been supplied. Mutants in cobU are unable to produce the DMBI portion of the molecule,
and they require a supplement of DMBI in the medium for growth. Mutants which do not
grow on all three types of test medium are considered to have cobUl insertions (16).
Assay for Transport Mutant Phenotypes
The ability of the mutants to transport cobalamin across the cell envelope was
tested by the mutants' ability to grow with limited cobalamin (5). The positive mini-tet
insertion mutations were tested in a metE205 (TR6583) genetic background. Strain LT2
(mef^ btu^) and TT13775 (mef^ btu-2::Mu dJ) controls were streaked onto E glucose and
NCE ethanolamine minimal medium plates which had been spread with 0.1 ml of a 0.04
mg/1 solution of cyanocobalamin. This is 1/lOOth of the standard amount of cobalamin per
plate. These plates were incubated aerobically for 5 days.
Test for Cotransduction of the Positive Mutations with Markers In or Near Known Transport Genes
Three genetic loci have been identified that are involved in the uptake of cobalamin
in Salmonella: btuB, btuC, and tonB. S. typhimurium strain TT13775 has a kanamycin-
15
resistance marker at the btuC locus (5). This strain was transduced with positive mutant
lysates and inoculated onto nutrient agar plates with tetracycline. After ovemight
incubation, 100 colonies were patched to the same medium. The patch plates were
incubated ovemight and then replica-plated onto nutrient agar plates with tetracycline and
with or without kanamycin. These plates were incubated ovemight. The patches were then
scored for the loss of kanamycin resistance.
The ability of the remaining unmapped positive mutations to cotransduce with the
other transport genes was determined by using S. typhimurium strains RT801 and RT797,
which have auxotrophic markers near btuB and tonB, respectively. These strains were
transduced with the positive mutant lysates, inoculated onto nutrient agar plates with
tetracycline, and incubated ovemight Patch plates were prepared and replica-plated onto E
glucose minimal medium with tetracycline. The replica plates were incubated ovemight and
the patches were scored for repair of the auxotrophic mutations.
Test for Cotransduction of the Positive Mutations with a Marker in metH
S. typhimurium strain TT10723 contains an ampicillin-resistance marker in the metH
gene, which is responsible for the production of the methylcobalamin-dependent
homocysteine methyltransferase. Ovemight broth cultures of TT10723 were transduced
with the lysates of the remaining unmapped positive mutants. A 0.1 ml sample of each
transduced culture was spread onto nutrient agar plates with tetracycline, and these plates
were incubated ovemight. Patch plates were prepared and then replica-plated onto nutrient
agar plates with tetracycline, with or without ampicillin. These plates were incubated
ovemight and the patches were scored for loss of ampicillin resistance.
16
Assay for Conversion of the Vitamin Form of Cobalamin into the Cofactor Forms
Cyanocobalamin, the vitamin form of cobalamin, is modified to make the different
cofactors that are used by the cobalamin-dependent enzymes. Some of the mutations which
conferred a transport-deficient phenotype did not cotransduce with maikers near the three
loci known to be involved in cobalamin transport. These mutations were placed into a
TR6583 (metE) background by transduction. The mutants were then incubated in liquid E
glucose minimal medium with methionine to minimize the carryover of nutrients. The cells
were streaked onto three different sets of minimal medium plates with either glucose,
propanediol, or ethanolamine as a sole carbon source. All three types of media had been
spread with 0.1 ml of a 400 mg/1 solution of cyanocobalamin (1(X)X the usual
concentration) to allow transport mutants to grow on the plates. The plates were incubated
for 5 days and screened for the presence or absence of growth.
Test for Proximitv of the Positive Mutations to the Ethanolamine Genes
While performing the above experiment, two mutants were characterized which
grew on both the glucose and propanediol plates, but did not grow on ethanolamine as a
sole carbon source. Transductions were performed to determine if these mutations were in
the region of the ethanolamine utilization (eut) operon. S. typhimurium strain TT10271,
which has a Mu dJ inserted in the ethanolamine genes, was transduced with P22 lysates
grown on the two ethanolamine-negative mutants. The transductants were plated on
nutrient agar + tetracycline and these plates were incubated ovemight. Patch plates were
prepared and replica-plated onto nutrient agar plates with tetracycline with or without
kanamycin. These plates were incubated ovemight and the patches were then scored for
loss of kanamycin resistance.
17
Two other transductions were performed with the mutant strains RT869 and
RT870, which have auxotrophic mutations in the pwrC and cysA genes, respectively (Table
1). These two genes lie on either side of the ethanolamine genes (24). The procedure for
both cotransduction experiments was the same. Ovemight broth cultures of recipient cells
were transduced with the mini-tet insertion mutant lysates and then inoculated onto nutrient
agar plates with tetracycline. These plates were incubated ovemight Patch plates were
prepared and replica-plated onto E glucose minimal medium plates with tetracycline.
Repair of the auxotrophic mutation indicated that the mini-tet insertion and the auxotrophic
mutation are cotransducible.
To determine if the mini-tet insertions were in a control region that is known to
exist at the distal end of the ethanolamine operon (24), the phenotype of a previously-
isolated mutant in the control region was compared to the phenotype of the positive mutants
with insertions that were found to map near the ethanolamine genes. S. typhimurium strain
TT13893, with a TnlO insertion in this control region, was transduced to kanamycin
resistance with a P22 lysate grown on TT 10852 (coW::Mu dJ). The double insertion
mutant was streaked onto MacConkey plates with and without cyanocobalamin, and was
then checked for the positive phenotype (Lac"*" on both plates).
The ability of the two eut insertion mutants to repair a deletion of the eut operon in
strain TTl 1580 was also used as a method of determining if the mutants were inserted in
the eut control region. Two sets of ethanolamine minimal medium plates were prepared.
One set was spread with an ovemight culture of TTl 1580, while the control set was spead
with an ovemight culture of RT933 (leu-447). One drop of each P22 lysate grown on cells
containing the two eut insertion mutations was placed onto each plate. One drop of a lysate
grown on LT2 was also placed on each plate as a control. The plates were incubated for 2
days and then scored for transductants that inherited the ability to use ethanolamine as a
sole carbon source.
18
Arranging the Unmapped Mutations into Groups by Cotransduction
All of the mutants which were not successfully mapped by cotransduction were
arranged into groups by replacing the mini-tet insertions in a few of the mutants with a
linked mini-kan marker, and then transducing the cells containing the new mini-kan marker
with each of the remaining mini-tet insertions. If the mini-kan marker is at the same
chromosomal location as the mini-tet insertion with which it is being transduced,
inheritance of the mini-tet will cause the mini-kan to be lost from the chromosome at a
detectable frequency. If all of the colonies resulting fix)m a transduction are both
kanamycin- and tetracycline-resistant, then the two mutations are not P22-cotransducible
and they must be inserted into different parts of the chromosome. If, however, some of the
transductants are kanamycin-sensitive, then the mutations are P22-cotransducible and they
are located near to each other on the chromosome.
Certain mutants, chosen at random, were transduced with a mini-kan gene pool
and inoculated onto nutrient agar plates with kanamycin. These plates were incubated
ovemight Patch plates were prepared and replica-plated onto nutrient agar plates with
kanamycin and with or without tetracycline. Patches that were kanamycin-resistant but
tetracycline-sensitive were purified, grown ovemight in broth culture, and used as
recipients for transductions in which the donor lysates were grown on the other mini-tet
insertion mutants. If the inheritance of mini-tet caused the simultaneous loss of the mini-
kan marker, then that mini-tet mutant was placed in the same group as the mini-tet mutant
from which the min-kan had been derived originally. In this way, unmapped mutations
were grouped at separate chromosomal locations.
19
Mapping the Location of the Mutations bv Conjugation
Hfr donor strains were constructed using representative mutants taken from each
of the genetic groups defined in the above experiments. The Hfr strains were constmcted
using F' plasmids which had Tn70s inserted into them. The TnlOs in the plasmid created
an area of homology between the plasmid and the mini-tet insertions so that the plasmid
integrated into the chromosome at the point of the mini-tet insertion (6). Two plasmid
donor strains (TT627 and TT628) were used for the constructions. These two strains
contain Flac plasmids with Tn70 inserted in opposite orientations (6). Thus, two Hfr
donors could be constmaed for each mutant, one with a clockwise and the other with a
counterclockwise gradient of chromosomal transfer. The two Flac plasmid donors were
grown ovemight in NCE glucose minimal broth at 3(y*C. The donor and recipient cells
were then cross-streaked onto NCE lactose minimal plates with tetracycline. The colonies
that grew on the plates were composed of tetracycline-resistant recipient cells that had
inherited the Flac plasmid. A healthy colony was taken from each plate and grown
ovemight at 30°C in NCE lactose minimal broth. A 0.1 ml sample of this culture was
added to 2.9 ml of the same medium and incubated ovemight at 42°C. Because the
plasmid's replication is temperature-sensitive, only cells in which the plasmid has
integrated into the host chromosome will grow at 42°C (6). These cells were then streaked
onto NCE lactose minimal plates with tetracycline. The plates were incubated for 2 days at
42°C. Healthy colonies were taken from the plates, inoculated into NCE lactose minimal
broth, and were incubated ovemight at 42°C. These cells were the Hfr donors. Seventeen
different streptomycin-resistant auxotrophic mutants were used as recipients in the
conjugational matings (6). Aliquots (0.1 ml) of each Hfr stain were mixed with 0.1 ml of
each auxotropic recipient in 1 ml of nutrient broth. The matings were allowed to proceed
for 3 hrs. Samples (0.1 ml) were then taken from each tube and spread onto NCE glucose
20
minimal medium plates with streptomycin. The plates were incubated for 2 days, and the
numbers of recombinants on each plate were counted.
Determining the Effect of the Mutations on the Transcription of the cob Genes
p-galactosidase assays were performed on all the mutants that were not located in
the cobl stmctural genes. Four replicates of the assay were done for each mutant The
mini-tet insertions were placed into an LT2 background by transduction. The cells carrying
the mini-tets were transduced to kanamycin-resistance with a P22 lysate that had been
grown on strain TT10852 (coblvMn dJ) lysate. In these transductants, the Mu dJ element
was inserted into the cobl operon and the expression of the lac genes was direcdy related to
the transcription of the cobl operon. Strains LT2 (wild-type) and TTl 1580 (Aeut-237)
were also transduced to kanamycin resistance and used as controls. A wild-type
Escherichia coli K-12 strain (RT504) that was grown with and without an inducer for the
lac genes (isopropyl-P-D-thiogalactopyranoside; IPTG) was also used as a positive control
for p-galactosidase activity. For these assays, cells were grown in NCE glycerol-fumarate
minimal medium under anaerobic conditions so that the cob genes would be maximally
expressed (11).
One ml volumes of glycerol-fumarate medium were placed into test tubes and
incubated ovemight under anaerobic conditions to remove dissolved oxygen. The tubes
were inoculated with 0.1 ml of ovemight cultures of the 18 mini-tet strains, which had been
pre-grown in nutrient broth under aerobic conditions. The tubes were incubated
anaerobically for 6 hrs., then centrifuged at 2,500 rpm for 20 min. The cells were
resuspended and diluted 1:10 in sterile physiological saline (0.85%), and placed on ice for
20 min to prevent further growth. They were then diluted 1:10 with complete Z buffer
(21). Chloroform and 0.1% sodium dodecyl sulfate (SDS) were added to the tubes, and
21
the tubes were mixed with a vortex mixer for 10 sec to penneabilize the cells. The tubes
were equilibrated at 30°C for 2 min, and then o-nitrophenyl-P-D-galaaopyranoside
(ONPG) was added to each tube. The tubes were mixed again and the reaction was
allowed to proceed at 30°C. When a faint yellow color had developed, the reaction was
stopped by adding 1 M Na2C03 to the tubes. At this point, the tubes were placed in a
30°C shaking incubator for 5 min. Absorbance readings of the reaction mixture at 420 nm
and 550 nm were taken with a spectrophotometer. A reading of the diluted cells was also
taken at 650 nm. The reaction time and the three readings were used to calculate the
enzyme activity in nanomoles of ONPG produced/min/units of absorbance at 650 nm (11).
CHAPTER m
RESULTS
Major Classes of Mutants
Twenty-nine insertion mutants with altered phenotypes for the end-product
repression of cobalamin biosynthesis were isolated in S. typhimurium during this study.
All of the mutants were generated using the mini-tet insertion element This insertion
element was used for two reasons: the antibiotic resistance marker (Tet^ provides a means
for isolating the mutants by direct selection, and insertion mutations produce dismptions in
the coding sequence of a target gene, which usually results in complete loss of the gene's
activity.
There are no direct assays presendy available for any of the enzymes involved in
the biosynthesis of cobalamin. Therefore, the transcription of the cob genes in the mutant
cells was monitored by assaying for the activity of p-galactosidase in a cobl::lac gene
fusion strain. On MacConkey indicator medium, lactose-fermenting (Lac"*") colonies
appear deep red, and lactose-nonfermenting (Lac") colonies appear white. The fusion
strain is Lac" in the presence of cyanocobalamin and Lac"*" in its absence. Thus, the
production of p-galactosidase by the fusion is normally repressed when cyanocobalamin is
added to the culture medium. Following mutagenesis, the mini-tet insertion strains were
screened for changes in the mutant cells' ability to ferment lactose on MacConkey plates.
Based on the color of the colonies, it was possible to arrange the mini-tet insertion mutants
into two different classes. The first group of 18 mutants was Lac"*" on MacConkey agar
regardless of whether cyanocobalamin was present or absent in the medium. They
appeared to be constitutive for cob gene transcription. The second class of 11 mutants was
Lac" regardless of whether cyanocobalamin was present or absent in the medium. These
22
23
were designated negative mutants. All of the mutants produced in this study are listed in
Table 2.
Characterization of the Negative Mutants
The coblv.lac gene fusion was originally generated by the insertion of the defective
bacteriophage Mu dJ into the cobl operon. This genetic element carries a kanamycin
resistance maricer (Kan') in addition to the lac genes. All of the mini-tet insertions were
tested to determine whether or not they were cotransducible with Mu dJ by using
bacteriophage P22. Each mini-tet was transduced into the Mu dJ-containing fusion strain
(TTl0852), and the tetracycline-resistant transductant colonies were tested for simultaneous
loss of kanamycin resistance (indicating that the two markers are cotransducible). All of
the negative mutants were found to be cotransducible with the Mu dJ in the cobl operon.
These results indicated that the mini-tet insertions might also be somewhere in the cob
genes. A nutritional study was performed to determine if the mini-tets were also interfering
with the transcription of the cobU and cobTH operons. Each of the mini-tet mutants was
tested for growth under conditions that required cobalamin synthesis from the biosynthetic
intermediates cobinamide and DMBI. All of the negative mutants could make their own
DMBI and join it with cobinamide to form cobalamin; however, they were not able to
produce their own cobinamide. Thus, this result indicated that all the negative mutants
interfere only with the transcription of the cobl operon; transcription of the cobVi and coWII
operons is not disrupted.
Mapping the Positive Mutants
None of the 18 positive mutants were cotransducible with the Mu dJ insertion in the
cobl operon. These mutants were tested for the ability to produce cobalamin aerobically, in
order to ascertain if the repression of cob by oxygen had also been affected by these
24
TABLE 2. Mini-tet insertion mutants generated in this study.
Mutant Strains » Genotype or Description
RT871 RT872 RT873 RT874 RT875
RT876 RT877 RT878 RT879 RT880
RT881 RT882 RT883 RT884 RT885
RT886 RT887 RT888 RT889 RT890
RT891 RT892 RT893 RT894 RT895
RT896 RT897 RT898 RT899
btu-151 btu-152 btu-153 btu-154 btu-155
btu-156 loss of end-product repression for cob loss of end-product repression for cob loss of end-product repression for cob loss of end-product repression for cob
loss of end-product repression for cob loss of end-product repression for cob loss of end-product repression for cob loss of end-product repression for cob eut-lOOl
eut-1002 loss of end-product repression for cob loss of end-product repression for cob cob-2001 cob-2002
cob-2003 cob-2004 cob-2005 cob-2006 cob-2007
cob-2008 cob-2009 cob-2010 cob-2011
^ The mutations in strains RT877 through RT884 belong to a single group but have not been mapped by conjugation.
^ The mutations in strains RT887 and RT888 have been mapped by conjugation and are located between 47 and 62 map units on the 5. typhimurium chromosome.
25
mutations. This test was performed in a metE (TR6583) genetic background. None of the
positive mutants produced enough cobalamin to grow aerobically on glucose minimal
medium plates. This result indicated that Uie cob genes arc still sensitive to oxygen
repression in this group of mutants.
The ability of tiiese mutants to grow on limited (100-fold dilution) cobalamin was
also tested to determine if the appearance of the repression-insensitive phenotype in these
cells was being caused by a loss of cobalamin transport into the cell. The only positive
mutant which did not grow aerobically with limited cobalamin was RT871. However, this
is not conclusive evidence that the other mutants are not located in cobalamin transport
genes, since a mutation in the btuC locus might still allow the cells to grow with limited
cobalamin (5). Further experiments were performed to detemine if any of the other positive
mutants were cotransducible with markers near the three known loci for cobalamin
transport The results of these cotransduction experiments indicate that RT871 and RT874
are btuB mutants (they have lost the outer membrane-binding protein for cobalamin). Four
additional strains (RT872, RT873, RT875, and RT876) appear to be btuC mutants (they
have lost one or more of the cytoplasmic membrane proteins for cobalamin transport).
None of the strains appear to be tonB mutants (loss of an energy-transfer protein involved
in cobalamin transport). The results of these experiments are listed in Table 3.
The metE gene, which encodes the cobalamin-independent homocysteine
transferase, is repressed by cyanocobalamin (25). Mutations in or near the metH gene,
which encodes the cobalamin-requiring homocysteine methyltransferase, block this
repression (26). Thus, the ability of the positive insertion mutations to be cotransduced
with metH was also tested. None of the mutations was cotransducible with metH.
26 TABLE 3. Results of cotransductional crosses between the positive insertion mutations
and markers in or near known cobalamin transport genes.^
Mutant Strains
RT871 RT872 RT873 RT874 RT875
RT876 RT877 RT878 RT880 RT881
RT882 RT883 RT884 RT885 RT886
RT887 RT888
TT13775
(btu-2:Mu dJ)^
0 99 90 0
100
93 0 0 0 0
0 0 0 0 0
0 0
RT801 (argH87) c
48 ND ND 96 ND
ND 0 0 0 0
0 0 0 0 0
0 0
RT797 (trpA3)^
ND ND ND ND ND
ND 0 0 0 0
0 0 0 0 0
0 0
ND, not determined.
a The data are expressed as frequency of cotransduction [(number of colonies that inherit the unselected maricer/100 colonies that inherit the selected maricer) x 100].
b Inserted at the frmC locus.
c Cotransduccs with ^mB.
d Cotransduces with tonB.
27
Ability of Mutants tn Cnnvert Cobalamin into the r/>fantor^
The form of cobalamin (cyanocobalamin, metiiylcobalamin, or adenosycobalamin)
that mediates the end-product repression of the cob genes is unknown. Thus, a mutation
that affects the cells' ability to convert cyanocobalamin (used to supplement the plates) into
one of the cofactors could result in the positive phenotype. For the mutants to grow on
propanediol or ethanolamine as a sole cartx)n source, the cells would have to be able to
produce adenosylcobalamin; to grow on glucose in the absence of methionine, they must
produce methylcobalamin. The ability of the mutants to grow on minimal medium plates
with either glucose, propanediol, or ethanolamine as a sole carbon source was tested. The
plates were supplemented with a 100-fold concentrated solution of cobalamin to overcome
any transport deficiencies. All of the mutants were able to grow on the propanediol and the
glucose plates. However, two of the mutants were unable to grow on the ethanolamine
plates. This result indicated that these two mutants produce both methylcobalamin and
adenosylcobalamin, but that their ability to utilize ethanolamine as a carbon source is
impaired in some other way.
Mapping the Ethanolamine Mutants
The positive mutants that were unable to utilize ethanolamine as a sole cartx)n
source were characterized further. The eut operon encodes the enzymes for ethanolamine
utilization and requires the simultaneous presence of cobalamin and ethanolamine to induce
transcription (3,24). The operon also contains the gene (eutR) for a regulatory protein that
is required in order for the operon to be expressed.
Cotransdution experiment were performed to determine if the two ethanolamine-
deficient mutations were located within the eut operon. The data for this experiment are
shown in Table 4. These cotransduction experiments indicate that these mutations lie at the
28 TABLE 4. Results of cotransductions between Euf mini-tet insertion
mutants and markers in and near the eut operon a.
Mutants
TT10271
(eM/-18::MudJ)
RT869
(purCJ)^
RT870
(cysA533) ^
RT885 24 0 23
RT886 34 20
a The data are expressed as finequency of cotransduction (as defined in Table 3).
^ The purC gene is upstream from the proximal end and the cysA gene is downstream from the distal end of the eut operon.
29
distal end of the eut operon. The eut R regulatory gene has also been mapped at the distal
end of the operon (24). Therefore, the two mini-tet insertions may occur near or within the
eutR gene itself.
To determine if these two mutants were within the eutR gene, they were
transduced into S. typhimurium strain TTl 1580. This strain contains a deletion in the eut
operon which runs from die promoter through the eutR gene. If the mutations occuired
within the deleted area, then it would not be possible for the transduction to produce a Eut"*"
phenotype. However, both of the eut insertion mutations were able to repair the eut
operon. Thus, both of the mutations appear to lie outside of the deletion and downstream
from the eutR gene.
Mapping the Remaining Mutants
The 10 positive mutants which did not appear to be blocked in cobalamin
transport or ethanolamine catabolism were arranged into two additional groups. Mini-kan
insertions were isolated that were linked to several randomly-chosen mini-tet insertions.
Linkage to other unmapped mini-tet insertions was then tested by cotransduction. One
group contains two mutants (RT887 and RT888), while the other group contains the
remaining eight mutants (RT877-RT884). The mutations in the first group were mapped
by conjugation, they lie between 47 and 62 map units on the 5. typhimurium chromosome
(Table 5). The mutations in the second and larger group were not mapped.
p-Galactosidase Assavs
p-galactosidase assays were performed on all of the positive mutants in order to
quantify the effect that these mini-tet insertions have on the end-product repression of the
cob genes. Control assays were also performed on the following three control strains
30
TABLE 5. Cbnjugational mapping of mini-tet insertion mutations in strain RT887.
Auxotrophic recipient
TR5654 (thrA9)
TR5655 (leu-485)
TR5656 (proA36)
TR5657 (purE8)
TR5658 (pyrCT)
TR5660 (pyrF146)
TR5686 (aroDMO)
TR5662 (his01242 his-2236)
TR5663 (purFI45)
TR5666 (serA13)
TR5667 (cysG439)
TR5668 (cysE396)
TR5669 (ilv-508)
TR5671 (pyrB64)
TR5688 (PMM755)
Map units
0
2
6
11
23
33
36
42
47
62
72
79
83
98
96
Numbers of prototrophi ic colonies in matings with Hfr donor derived from
TT627
597
164
255
270
340
552
430
612
980
41
57
146
219
152
16
TT628
ND
ND
ND
ND
ND
ND
ND
76
31
752
623
ND
ND
ND
ND
ND, not determined.
a The Hfr donors were derived from strains containing F plasmids that were originally inherited from TT627 and TT628. The plasmids insert into the chromosome in opposite orientations.
31
RT504, RT934, and RT935. The RT934 strain contains a cobl.Mu dJ insertion but has an
otherwise wild-type (LT2) genetic background. This strain was used to establish the
normal level of cobl transcription in 5. typhimurium. In addition to the cobl:Mu dJ
insertion, the RT935 strain also contains a deletion which removes the entire eut operon.
This strain was used to determine what effect the eut operon has on the expression of the
cobl operon. RT504 is a wild-type E. coli K-12 strain which was grown in the presence
and absence of isopropyl-p-D-thiogalactoside (IPTG) as an inducer for the lac genes.
These control assays were used to establish the upper and lower limits for the entire set of
assays. The assays on all of the S. typhimurium strains were performed both in the
presence and absence of cyanocobalamin (Table 6).
32
TABLE 6. p-Galactosidase enzyme activity in permeabilized cells.a b
Strains ^
Parent strain
RT934
Transport mutants
RT871 RT872 RT873 RT874 RT875 RT876
Unmapped mutants
RT877 RT878 RT880 RT881 RT882 RT883 RT884
Eut" mutants
RT885 RT886 RT935
Cells grown with cobalamin
1396 ±87.55
1400 -1- 49.65 1835+92.16 1659+ 0.00 1708 + 99.02 1689 ±90.07 1658 ± 20.00
2030+ 79.00 2017+ 27.05 2103+ 57.88 2314+ 84.92 1993+ 90.29 1087 + 105.90 1619 ± 52.28
2121+ 22.91 1774+139.30 1707+ 56.60
Mutants mapped by conjugation
RT887 RT888
1202 + 59.25 914 ±32.65
Cells grown without cobalamin
235 ± 27.00
1450 + 72.22 1400 + 66.37 1861 +21.72 905 + 22.73
1084+ 0.00 1485 ±68.13
878 + 33.49 1422 + 87.53 1350 + 38.58 2105 + 26.86 1645 + 34.42 928 + 45.10
1070 ± 32.00
2062 + 21.48 1532 ± 4.30 1008 ± 56.60
854 + 26.51 781 ± 5.32
%
change in activity
83.17
-3.45 23.71 10.85 47.01 35.82 10.43
56.75 29.50 35.81 9.03
17.46 14.63 33.91
4.95 13.64 40.95
28.95 14.55
^ The specific activity is given in units of nmol of ONPG/min/units of absorbance at 650 nm. The numbers in this table represent the average of four replications + the standard deviation.
"v- .
33
TABLE 6, continued.
^ Two £. coli controls that were grown with and without IPTG as the inducer were also assayed. Four replications of each control were perfonned. The induced cells produced 9899 ± 331.20 units of activity, while die uninduced control produced 162.0 ± 19.04 units.
^ The cob'24v^yx dJ insertion was transduced from strain TT10852 into each of the strains listed in this table in order to assay for p-galactosidase activity.
CHAPTER IV
DISCUSSION
Mini-tet insertions which alter end-product repression of the cob genes were
isolated in this study. The effect of these mutations on the expression of die cob genes was
determined by performing growth studies, and by assaying for the p-galactosidase activity
of coblr.lac fusions in die mutant cells. The 29 different mutants were divided into two
distinct groups, based on the Lac phenotype that they exhibited on lactose MacConkey
indicator plates in the presence and absence of cyanocobalamin. These two groups were
designated positive and negative mutants.
The 11 negative mutants were shown by both cotransduction and nutritional
experiments to lie within or near the cobl operon. The mini-tet insertions that confer the
negative phenotype could be regulatory mutations that disrupt an operator site that is
involved in the end-product repression of the cobl operon. Alternatively, they might
interfere with the transcription of the operon by being inserted in the promoter or between
the promoter and the cobv.lac fusion. The exact location of these mini-tet insertions can be
determined more precisely by deletion mapping. This has not been done at this time for
lack of a detailed genetic map of the cobl genes.
The importance of this group of mutants lies in the fact that all of the mutations
that inactivate transcription of the cobl operon are inserted in or near the c^ron itself.
These mutations appear to produce the negative phenotype in the cells because they
physically block the transcription of the operon. If this is true, it suggests that the cob
genes are not regulated by an activation mechanism.
The 18 positive mutants can be subdivided into four different groups based on
their map locations, and the types of genes in which they are found. Six of these mutants
34
35
belong to the first group and are blocked in cobalamin transport. Two of die insertions
occur in die btuB gene,while the otfier four are in the btuCED genes. These mutations give
die cells a repression-insensitive phenotype because diey decrease die ability of die cells to
transport cobalamin across die cell wall. The remaining groups of positive mutants are
probably involved more direcdy in die regulation of cob. The insertions in die second
group of mutants have been mapped by cotransduction, and diey lie near die distal end of
die eut operon at 50 map units on die chromosome (Table 4). The insertions in die diird
group of mutants have been mapped more crudely by conjugation. They lie between 47
and 62 map units on the chromosome, but they are not cotransducible with eut genetic
markers. The insertions in the fourth and largest group of mutants have not yet been
mapped. Because of the larger number of mutants belonging to this group, the operon in
which these insertions lie may be larger than the operons in which the other insertions are
located.
The mutations in the eut operon render the ceU incapable of utilizing ethanolamine
as a sole carbon source. They are located near the region of the operon that codes for a
regulatory protein (EutR) which must be produced in order for the operon to be expressed.
This part of the eut operon has been designated region in (24). The induction of the eut
operon is dependent on the simultaneous presence of cobalamin and ethanolamine; cither
compound by itself does not stimulate transcription. I propose a regulatory model in which
two proteins are involved, one that binds to ethanolamine, and a second which binds to
cobalamin. After these proteins bind to their effectors, they change conformation and bind
to separate regulatory sites at the eut operon. This would allow the operon to be expressed.
In this model, the protein which binds to cobalamin would also bind to a regulatory site at
the cob genes. When this protein binds to the cob operator site, it blocks the transcription
of the cob operon. This would only occur when cobalamin was present in excess of the
cell's needs.
35
In order for this model to work, the cobalamin-binding protein would have to be
expressed by a weak promoter which is regulated independendy of die eut operon's main
promoter, so that a low level of this protein would always be present in the cell. A weak
promoter of this type is known to exist in the eut operon just upstream from the gene for
the EutR regulatory protein (24). However, the coblr.lac fusions are still partially
repressed in a strain (RT935) that also carries a deletion of the eut operon including the
eutR gene, and in strains (derivatives of RT885 and RT886) that carry the two eut insertion
mutations (Table 6). This in^lies diat the regulatory protein which is affecting the
transcription of both the eut and cob operons is not EutR but is produced by a region of the
eut operon that is downstream from the eutR gene. Further evidence for this conclusion is
the fact that P22 lysates that were grown on both strains containing the eut insertion
mutations could repair the eut deletion.
The effect of both the deletion and the insertion mutations on the end-product
repression of die cob genes seems to be diat they reduce die regulatory protein's rate of
syndiesis. They do not disrupt the regulatory gene itself, since die cob genes are still
partially repressed by cobalamin in all of diese strains. In die model stated above, diis
would mean that bodi of die regulatory proteins would have to have dieir own weak
promoters, and that the EutR protein is die ethanolamine-binding regulatory protein.
There are two remaining groups of mutants which have not been characterized at
this time. The role that these genes which have been inactivated by insertion mutations
normally play in die regulation of cob is not known, but diey may be involved in modifying
cobalamin into the chemical form diat is used as an effector for die regulatory protein. For
example, the regulatory protein may bind only adenosylcobalamin, but not
methylcobalamin or cyanocobalamin.
Research into die mechanism involved in die end-product repression of cob has just
begun. There are a number of questions left unanswered, and a number of additional
37
experiments that should be perfOTmed. The location of the other two groups of mutants
that affect the repression of cob should be mapped more precisely. A more detailed map of
the eut operon should be produced to determine whether the mutations affecting the
regulation of cob are in region HI. The regulatory protein itself should be characterized to
confirm that it does bind cobalamin, and to determine its binding characteristics.
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3. Bassford, P. J., Jr., and R. J. Kadner. Genetic analysis of components involved in vitamin Bn uptake in Escherichia coli. J. Bacteriol. 132: 795-805.
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6. Chumley, F. G., R. Menzel, and J. R. Rodi. 1979. Hfr formation directed by Tn70. Genetics 91: 639-655.
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