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Journal of Bacteriology, December 2001, p. 7058-7066, Vol. 183, No. 24
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.24.7058-7066.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Use of Transposon Tn5367 Mutagenesis and a
Nitroimidazopyran-Based Selection System To Demonstrate a Requirement
for fbiA and fbiB in Coenzyme
F420 Biosynthesis by Mycobacterium
bovis BCG
Kwang-Pil
Choi,
Thomas B.
Bair,
Young-Min
Bae,
and
Lacy
Daniels*
Department of Microbiology, University of
Iowa, Iowa City, Iowa 52242
Received 12 April 2001/Accepted 21 September 2001
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ABSTRACT |
Three transposon Tn5367 mutagenesis vectors (phAE94,
pPR28, and pPR29) were used to create a collection of insertion mutants of Mycobacterium bovis strain BCG. A strategy to select
for transposon-generated mutants that cannot make coenzyme
F420 was developed using the nitroimidazopyran-based
antituberculosis drug PA-824. One-third of 134 PA-824-resistant mutants
were defective in F420 accumulation. Two mutants that could
not make F420-5,6 but which made the biosynthesis intermediate FO were examined more closely. These mutants contained transposons inserted in two adjacent homologues of Mycobacterium tuberculosis genes, which we have named fbiA and
fbiB for F420 biosynthesis. Homologues of
fbiA were found in all seven microorganisms that have
been fully sequenced and annotated and that are known to make
F420. fbiB homologues were found in all but
one such organism. Complementation of the fbiA mutant
with fbiAB and complementation of the
fbiB mutant with fbiB both restored the
F420-5,6 phenotype. Complementation of the
fbiA mutant with fbiA or
fbiB alone did not restore the F420-5,6
phenotype, but the fbiA mutant complemented with
fbiA produced F420-2,3,4 at levels similar
to F420-5,6 made by the wild-type strain, but produced much
less F420-5. These data demonstrate that both genes are
essential for normal F420-5,6 production and suggest that
the fbiA mutation has a partial polar effect on
fbiB. Reverse transcription-PCR data demonstrated that fbiA and fbiB constitute an operon.
However, very low levels of fbiB mRNA are produced by
the fbiA mutant, suggesting that a low-level alternative
start site is located upstream of fbiB. The specific reactions catalyzed by FbiA and FbiB are unknown, but both function between FO and F420-5,6, since FO is made by both mutants.
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INTRODUCTION |
F420 is a
7,8-didemethyl-8-hydroxy 5-deazaflavin electron transfer coenzyme that
is present in few microorganisms. It was first described in detail in
methanogens (11, 12). Methanobacterium F420 contains two glutamates (12),
while Mycobacterium contains primarily five- and
six-glutamate forms (F420-5 and
F420-6). Figure 1A
shows the structure of F420-5 (4).
FO is a biosynthetic intermediate that contains the deazaflavin ring
and ribityl, but lacks phosphate, lactyl, and glutamate moieties. In
archaea, F420 is essential for
methylenetetrahydromethanopterin reductase, some hydrogenases, formate
and methylenetetrahydromethanopterin dehydrogenases, some alcohol
dehydrogenases, and quinone oxidoreductase activities (18, 21,
23, 26, 27, 47). F420 is used by
Streptomyces for tetracycline and lincomycin biosynthesis
(8, 31, 39) and perhaps in mitomycin C biosynthesis
(29). In Mycobacterium and Nocardia,
F420 is used by
F420-dependent glucose-6-phosphate dehydrogenase
(35, 36) and is required for activation of the experimental antituberculosis drug PA-824 by Mycobacterium
tuberculosis and Mycobacterium bovis strain BCG
(45). The green alga Scenedesmus, the
cyanobacterium Synechocystis, and the archaeon
Halobacterium use F420 in their
photolyase (14, 15, 32).
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FIG. 1.
(A) Structure of coenzyme F420-5 from
Mycobacterium spp.(4). (B) Overview of the
hypothesized pathway for F420 biosynthesis, which is based
on our modifications of the reactions proposed by Bacher and Thauer and
their colleagues (13, 22, 38), and including the role of
2-phospholactate proposed by Graupner and White (17).
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The pathway by which F420 is made has been
explored with labeling approaches in methanogens (13, 22,
38). Also, recent enzymatic work with methanogens has shown that
2-phospholactate serves as a precursor of the lactyl and phosphate
groups of F420 (17).
A simplified overview of the current hypothetical pathway is shown in
Fig. 1B. The genes used for F420 biosynthesis
have never been described. Because of our interest in
F420 biosynthesis and in the metabolism of
M. tuberculosis, we have undertaken a study to determine the
genes involved in F420 biosynthesis in
Mycobacterium spp. Identification of these genes will enable
us to create knockout mutants of M. tuberculosis to study in
animal models in order to determine if F420 is
important for virulence. We believe that a role for
F420 in virulence is possible because preliminary
studies have indicated that M. bovis mutants not producing
F420 are more sensitive than the wild-type strain
to some oxidative stress agents (K.-P. Choi, unpublished observations).
We anticipated that mycobacteria would be more amenable than most other
organisms for a study of F420 biosynthesis for
three reasons. First, since Streptomyces mutants lacking
F420 biosynthetic capabilities can grow without
F420 supplementation (8, 31, 39), we
expected that F420 would not be required for in
vitro growth of the closely related mycobacteria, whereas in several archaea F420 is essential for growth. Thus, with
mycobacteria, no supplementation with F420 should
be required in the screening medium (F420 is not
commercially available and is expensive to produce, e.g., compared to
the purchase of NADP.). Second, these organisms are considerably easier
to grow than methanogens or Archaeoglobus. Third, several
good systems exist for creating Tn5367 insertion mutants in
Mycobacterium species (5, 30, 34).
We describe here our development of a strategy to select transposon
insertion mutants of M. bovis which cannot make
F420. We then demonstrate the utility of this
method to identify the M. bovis homologues of the Rv3261 and
Rv3262 genes from M. tuberculosis as essential for a step or
steps in the pathway between FO and F420-5,6,
after the deazaflavin ring is formed.
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MATERIALS AND METHODS |
Bacteria, plasmids, and culture conditions.
The bacteria and
plasmids used in this study are given in Table
1. M. bovis was typically
grown in Middlebrook 7H9 liquid medium or on 7H10 agar plates
supplemented with 0.05% Tween 80 (liquid medium only), 0.2% glycerol,
and Bacto Middlebrook ADC enrichment. Escherichia coli JM109
and pGEM were used as recombinant host and vector, respectively.
E. coli was grown at 37°C on Luria-Bertani medium;
ampicillin (100 µg/ml),
isopropyl-
-D-thiogalactopyranoside (IPTG; 0.5 mM), and 3-indolyl--D-galactopyranoside
(X-Gal; 80 µg/ml) were incorporated to select for and identify
recombinants.
HPLC.
F420 and FO were separated by
high-pressure liquid chromatography (HPLC) using a Beckman System Gold
Nouveau 126 HPLC with a Shimadzu RF-10Axl fluorescence detector
(excitation at 400 nm, emission at 470 nm). HPLC was performed using a
method similar to that of Gorris (16), with modifications.
An 
-Bondpack C-18 (Supelco) column (3.9 mm by 300 mm) was eluted at
1 ml/min. Buffer A was 27.5 mM sodium acetate (pH 4.7) containing 2%
acetonitrile; buffer B was 100% acetonitrile. The portion of buffer B
in the elution buffer was varied as follows: 0 to 2 min, 0%; 2 to 6 min, 0 to 2% (linear gradient); 6 to 15 min, 2 to 10%; 15 to 22 min, 28%; 22 to 27 min, 28 to 0%. Extracts of cultures to be examined were
prepared by heating approximately equal volumes of a wet cell pellet
and buffer A at >90°C for 15 min, followed by centrifugation to
remove the pellet.
Molecular biology techniques.
Chromosomal DNA was purified
according to Husson et al. (19). Access reverse
transcription (RT)-PCR System and Wizard minicolumns for plasmid
purification were from Promega (Madison, Wis.). RNA was purified
according to Mangan et al. (28) and processed with an
RNeasy Kit (Qiagen). Standard E. coli cloning protocols were used (40). Primers are described in Table 1. Sequencing
was done at the University of Iowa DNA Facility using an Applied
Biosystems 373A sequencer. PA-824 (a nitroimidazopyran used to select
F420-minus mutants) was a gift of the
PathoGenesis Corporation.
Insertion mutants of
M. bovis were created with the phage
phAE94 using techniques described by Bardarov et al. (
5),
but
with the incorporation of PA-824 selection. Phage prepared from
Mycobacterium smegmatis grown at 30°C was incubated with
the target
M. bovis. The infected culture was then grown at
37°C on 7H9-ADC
agar medium containing 20 µg of kanamycin/ml, which
did not allow
growth of the phage due to a temperature-sensitive
mutation. Colonies
from these plates were combined and spread on plates
of 7H9-ADC
agar with kanamycin (20 µg/ml) and PA-824 (10 µg/ml).
Colonies
that grew on these plates were examined by PCR for the
presence
of uninterrupted
fgd (
fgd codes for
F
420-dependent glucose-6-phosphate
dehydrogenase
[
35,
36]). Insertion mutants with an intact
fgd that survived this selection were further propagated and
analyzed
by HPLC for the presence of 5-deazaflavins.
Transposon insertions in F
420-minus mutants
created with phAE94 were identified using a semirandom two-step PCR
procedure
modified from the original method (
7). The
primers that corresponded
to the known transposon sequence, HOPS1 and
HOPS2, were taken
from Bardarov et al. (
5). The random
primer Semi-rand 2-1 was
independently developed, but Semi-rand 4 was
the same as primer
4 of Chun et al. (
7). Semirandom PCR
was carried out as described
previously (
7), but with the
#3 buffer from the Boehringer
Mannheim Expand Long Template PCR kit,
and Platinum
Taq polymerase
(Gibco).
Insertion mutants were created in
M. bovis with plasmid
pPR28 or pPR29 using techniques similar to those described by Pelicic
et al. (
34), but with incorporation of PA-824 selection.
pPR28
(1 µg) produced in
E. coli JM109 was electroporated
into 0.1 ml
of electrocompetent
M. bovis cells.
Electroporated cells were
supplemented with 5 ml of 7H9-ADC liquid
medium and incubated
at 32°C for 1 day. Transformants were selected
by growth on 7H9-ADC
agar medium containing gentamicin (5 µg/ml) and
kanamycin (20
µg/ml) for 4 to 6 weeks at 32°C. One colony was used
to inoculate
100 ml of 7H9-ADC-kanamycin medium. After 3 weeks of
incubation
at 32°C with shaking at 170 rpm, 100 µl of this culture
was spread
on 7H9-ADC plates containing kanamycin (20 µg/ml), PA-824
(5 µg/ml),
and sucrose (2%, wt/vol) and then incubated at 39°C for
3 weeks.
Colonies were picked and grown in 7H9-ADC liquid
medium.
pPR29 (1 µg) produced in
E. coli JM109 was electroporated
into 0.4 ml of electrocompetent
M. bovis cells.
Electroporated cells
were supplemented with 4 ml of 7H9-ADC and
incubated at 32°C for
1 day. Transformants were selected by growth in
50 ml of 7H9-ADC
liquid medium containing gentamicin (5 µg/ml) and
kanamycin (20
µg/ml) for 4 to 6 weeks at 32°C. Cultures were
centrifuged, the
pellet (
0.3 ml of packed cells) was diluted with
0.1 ml of 10%
glycerol in water, and 0.2-ml fractions were
spread-plated onto
7H10-ADC agar plates containing kanamycin (20 µg/ml), PA-824 (5
µg/ml), and sucrose (2%, wt/vol). These plates
were grown at 39°C
for 4 to 6 weeks, and the resultant colonies were
used to inoculate
7H10-ADC agar medium or 7H9-ADC liquid medium
supplemented with
sucrose, kanamycin, and PA-824. Strains mutated by
pPR28 or pPR29
that survived this selection were further propagated and
analyzed
by HPLC for 5-deazaflavin. Dry weights were estimated by
measuring
the postboiling pellet volume (milliliters) and dividing by 8
ml/g (a conversion factor determined experimentally with
M. bovis).
Transposon insertion sites created with pPR28 and pPR29 were found by
an inverse PCR technique similar to that described by
David Bowtell
(
http://www.pmci.unimelb.edu.au/manual/molbiol).
With inverse
PCR, chromosomal DNA from mutants was digested with
EagI.
Two
EagI sites in the transposon of pPR28 and pPR29 allowed
a short internal section of the transposon to be excised, and
at some
distance upstream and downstream of the insertion other
EagI
sites resulted in chromosomal DNA cleavage. This resulted
in two
independent segments with
EagI-cut ends, with DNA adjacent
to the insertion site and either the left or right portion of
the
transposon. These segments were ligated to create circular
DNA. To
identify the pPR28 sites, inverse PCR was conducted with
primers YMB1
and YMB2. To identify the pPR29 sites, inverse PCR
was conducted using
TNP3 and TNP4 (for molecules containing the
left transposon component)
or HOPS1 and TNP2 (for molecules containing
the right transposon
component). Fragments of approximately 500
to 1,500 bp were usually
obtained and were then gel purified and
subcloned into pGEM, and one
round of single-strand sequencing
was
conducted.
M. bovis homologues of both Rv3261 and Rv3262 were amplified
by
Pfu DNA polymerase PCR using primers 3260-2 and 3260-3.
A
terminal A overhang was introduced onto both ends of this product
by
Taq polymerase, and the final product was cloned into pGEM
to make pFbiAB. Using this clone as a template, PCR products were
constructed with an
EcoRV site on the 5' end and a
HindIII site
on the 3' end. The insert containing both
fbiA and
fbiB that was
used to create p61-62 was
constructed using primers 3260C-1 and
3260C-2. The insert containing
fbiA used to create p3261 was constructed
using primers
3260C-1 and 3261C-2. The insert containing
fbiB used to
create p3262 was constructed using primers 3262C-1 and
3260C-2. These
inserts were subcloned in frame into pSMT3 after
digesting this plasmid
with both restriction enzymes. pSMT3 was
used as an expression vector
to complement insertion mutants (
33),
using
hygromycin-supplemented liquid or agar medium (50 µg/ml
for
M. bovis).
Electroduction/electroporation to confirm that pSMT3 containing an
insert was present in complemented cells was performed
by a
modification of the method described by O Gaora (
33). One
M. bovis colony was transferred to 100 µl of 10% glycerol
in water
along with
0.1 ml of 1-mm glass beads in a 1.5-ml screw-cap
plastic
tube, and the tube was shaken for 30 s in a Mini
Beadbeater (Biospec
Products, Bartlesville, Okla.) at 5,000 rpm to
disrupt clumps
but not break all cells. After the beads settled, 50 µl of the
supernatant was used for electroporation of
E. coli, which were
then plated on LB-hygromycin (250 µg/ml) to
select transformants.
Plasmids prepared from the
E. coli
were then examined by agarose
gel electrophoresis. The remaining
supernatant from the Beadbeater
was spread on 7H10-ADC medium
supplemented with sucrose and kanamycin,
and after growth, cells were
examined to confirm F
420 content.
RT-PCR was used to determine if genes were coexpressed in an operon.
The reverse transcriptase reaction was performed with
Access RTase at
48°C (45 min) according to instructions provided
by the manufacturer.
PCR was performed with primers pairs F1 and
R2, F3 and R4, and F5 and
R6 (Table
1), and the products were
analyzed by agarose gel
electrophoresis. RT-PCR to determine if
the
fbiB transcript
was made by
fbiA::Tn
5367 was conducted
with
primer pairs 3260-9 and 3262-2, and 3261-1 and 3262-2.
Computer analysis of sequences.
Comparison of derived amino
acid sequences to the NCBI protein database was performed by the NCBI
BlastP (1) and 
-Blast (2) programs
(http://www.ncbi.nlm.nih.gov). Comparison of M. tuberculosis
sequences to M. bovis sequences not in the NCBI database was
made by the TBLASTN program at the Sanger Centre
(http://www.sanger.ac.uk). Genes in the Mycobacterium leprae
genome were examined at the Institut Pasteur Leproma site
(http://genolist.pasteur.fr). Sequences were aligned using ClustalW
(46) or PIMA (43) multiple alignment programs
with default settings available at the Baylor College of Medicine
Search Launcher (http://dot.imgen.bcm.tmc.edu). Alignments were
prepared for examination by shading with the Boxshade program at the
Swiss Institute for Experimental Cancer Research
(http://www.isrec.isb-sib.ch). Relationships of protein sequences
were examined by the NCBI COGs program.
A computer program was written in C++ to examine any genome for sites
with high, medium, and low probability for Tn
5367 insertion
[4(A/T), 3(A/T), and 2(A/T), respectively] (
3). This
program
was used to examine the
M. tuberculosis nucleotide
sequence (
9)
at the Sanger Centre web site to identify the
frequency and locations
of all such sequences in the
M. tuberculosis genome and to calculate
maximum distances and average
distances between high-, medium-,
and low-probability sites. To
characterize Tn
5367 insertion sites,
the duplicated 8-bp
sequences of all 50 available sequences were
analyzed by the WebLogo
program (
41) (available at
http://www.bio.cam.ac.uk/cgi-bin/seqlogo/logo.cgi).
Chi square analysis
was used to examine the significance of the
AT
percentages.
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RESULTS AND DISCUSSION |
Transposon mutagenesis and selection for PA-824-resistant
F420-minus mutants of M. bovis.
Using
recently reported transposon mutagenesis methods (5, 34),
we were confident that we could create insertion mutants of
Mycobacterium spp. that would not produce
F420. However, it was not obvious how to identify
mutants which did not make F420 except to screen
thousands of colonies by HPLC. As a rapid alternative to HPLC, we tried
direct spectrofluorometric analysis of cell extracts, but the low
levels of F420 present compared to the amounts produced by methanogens and the presence of other fluorescent material
made this unsuitable for mutant screening (E. Schoenberger, unpublished).
We then encountered an approach to enrich the mutant population for
F
420-minus mutants using an experimental
antituberculosis
drug (a nitroimidazopyran, PA-824) (
45).
F
420-dependent glucose-6-phosphate
dehydrogenase
(FGD) (
35,
36) is required to activate PA-824,
which can
then kill members of the
M. tuberculosis complex; other
mycobacteria (e.g.,
M. smegmatis) are not inhibited
(
45). We
developed the hypothesis (Fig.
2) that PA-824 is activated by
a
reductive reaction that is dependent on reduced
F
420
(F
420H
2).
Thus, some
M. tuberculosis or
M. bovis mutants that are
resistant
to this drug would be defective in FGD activity or unable to
produce
F
420. This allowed us to substantially
reduce the number of mutants
to be examined by HPLC.
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FIG. 2.
Hypothesis for the role of F420 in PA-824
activation. Protein X is a hypothetical enzyme that transfers electrons
from F420H2 to PA-824.
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The transposon vectors phAE94, pPR28, and pPR29 all successfully
transformed
M. bovis, giving rise to colonies on plates
supplemented
with kanamycin and PA-824. While the phAE94 method was
initially
easier and less susceptible to generating sibling mutants,
the
plasmid method proved superior due to its ability to routinely
generate large numbers of mutants. We examined cell extracts from
134 cultures derived from these colonies by HPLC and compared
F
420 and FO levels to those in the wild-type
strain. FO levels
were considerably lower than
F
420 levels, and in some cases it
was difficult
to determine conclusively if FO was absent. About
one-third
(
n = 48) of the PA-824-resistant mutants were
defective
in F
420 accumulation.
Identification of genes interrupted in F420-minus
mutants.
With either inverse PCR or semirandom two-step PCR, we
located transposon insertion sites in PA-824-resistant mutants. Among 48 PA-824-resistant F420-minus mutants, we
identified 31 distinct genes with a transposon insertion. Several genes
were found more than once due to independent insertions or siblings.
Insertions in 19 genes were found in mutants which had an
F420-minus and FO-minus phenotype, and
interruptions in 12 genes were found in mutants with an
F420-minus and FO-plus phenotype.
Characteristics of Tn5367 insertion sites.
As
shown in Fig. 3, examination of our
transposon insertion sites and sites reported by others (5, 30,
34) showed that in the 8-bp duplicated region, positions 4 and 5 are nearly 100% A or T and positions 3 and 6 are very high in A+T;
this is a significant deviation from the 34% A+T genome content
( < 0.001). Position 7 is G+C rich (16% A+T; < 0.005). The inset sequence logo graph portrays the approximately equal
amounts of A and T at positions 3 to 6 and the predominance of C at
position 7. This agrees with the general observations (that the
duplicated region was AT-rich) by others who had each examined a
smaller number of insertions (5, 30, 34).
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FIG. 3.
Sequence characteristics of the Tn5367
transposon insertion sites. In the large graph, percent A+T at each
position to the right (positive values) and to the left (negative
values) of the insertion site is given for nine bases on both sides of
the 31 gene insertion sites observed in our experiments and the 8 duplicated bases (positive values 1 to 8) for an additional 19 insertion sites reported by others (5, 30, 34). The
horizontal dotted line indicates the A+T content of the M.
tuberculosis genome (34.4%). The inset graph provides a
sequence logo plot to graphically emphasize the incidence of individual
base types, if they predominate at positions 1 to 9.
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The sequence on the other side of the insertion site (the unduplicated
region corresponding to positions
1 through
9), for
which we have
data for only our mutants, was not conserved; it
was not AT-rich (Fig.
3), nor did any bases predominate (data
not shown). We cannot
confidently conclude if position
6 is GC
rich (
< 0.025).
We wondered if this transposon would insert into most mycobacterial
genes, which have a G+C content of 65.6%. We examined
the
M. tuberculosis H37Rv genome with a computer program to determine
how
many genes had sites that Tn
5367 would insert into with high
probability (four adjacent A's or T's) or with a moderate probability
(three adjacent A's or T's) (
3). Of the 3,924 genes in
the
genome, 103 (2.6%) do not contain at least one high-probability
site, but all have a moderate-probability site. All of our 31
transposon-inserted genes had a high-probability site. We conclude
that, although this transposon is not truly random for insertion
in
mycobacteria, insertion is very likely in >97% of
M. tuberculosis and
M. bovis genes, and ultimately all
genes will be interrupted
if a large number of mutants are
examined.
M. bovis mutants with an insertion in Rv3261 or
Rv3262 homolog.
Of 18 genes interrupted by pPR29 which gave an
F420-minus phenotype, one each had an independent
insertion in the adjacent M. bovis homolog of M. tuberculosis H37Rv sequences Rv3261 (790 bp downstream from the
start codon) and Rv3262 (801 bp downstream from the start codon), as
shown in Fig. 4. We have named these genes fbiA and fbiB, respectively, for
F420 biosynthesis. These mutants made no
F420 but did make FO. An example of the HPLC
profile of the fbiA mutant is given in Fig.
5, in comparison with the wild-type
strain. The fbiB mutant had about the same HPLC profile as
the fbiA mutant at the scale shown in Fig. 5 (data not
shown). Since FO is produced by both mutants, the products of these
genes were expected to be involved in steps between FO and
F420-5,6 (Fig. 1B).
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FIG. 4.
Gene arrangement of the fbiAB
cluster in M. bovis BCG, corresponding transposon
insertion sites, and alignment of these genes with homologs from other
microorganisms. Triangles indicate transposon insertion sites. CAB,
Streptomyces coelicolor; MTH, Methanobacterium
thermoautotrophicum; AF, Archaeoglobus fulgidus;
Vng, Halobacterium sp; SLL, Synechocystis
sp.; AAC, Nostoc sp. Numbers refer to gene designations
in the corresponding genome sequences. Rv3259 and Rv3263 are used to
indicate the M. bovis homologs of these M.
tuberculosis H37Rv genes, since the M. bovis
sequence is not yet annotated, and thus M. bovis genes
have not been assigned unique designations. The lines below the
M. bovis genes indicate the sizes and locations of
RT-PCR products expected; a solid line indicates that a product was
seen, and a dotted line indicates that no product was observed. The
primer pairs used were (from left to right) F1-R2, F3-R4, and F5-R6.
Boxes with solid lines indicate genes identified with default NCBI
Blast settings. Boxes with dashed lines indicate genes identified with
-Blast iterations. Boxes connected by bars indicate the genes are
adjacent, and those not connected are not adjacent. Figure is
approximately to scale, with boxes indicating the sizes of all putative
homologous genes.
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FIG. 5.
HPLC elution profiles of extracts made from M.
bovis BCG wild-type, the fbiA mutant (Rv3261
homolog interrupted), the fbiB mutant (Rv3262 homolog
interrupted) that was complemented with p3262, and the
fbiA mutant that was complemented with p3261.
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We cloned and sequenced a 2,718-bp region from
M. bovis
containing
fbiA and
fbiB, creating pFbiAB. Our
sequences for both
genes precisely matched the Rv3261 and Rv3262
sequences and the
Sanger database
M. bovis homologs of these
genes. As shown in
Table
2, Blast
analysis of the deduced sequence of FbiA against
the NCBI database
revealed that this gene has homologs in all
fully sequenced
microorganisms which make F
420, including
M. tuberculosis (
9),
M. leprae
(
10),
Archaeoglobus fulgidis (
25),
Halobacterium sp. (
32),
Methanococcus
jannaschii (
6),
Methanobacterium thermoautotrophicum (
42), and
Synechocystis sp. (
24). However,
the
Synechocystis homolog was not identified by default BlastP
settings, but was identified by
-Blast (
2).
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TABLE 2.
Blast analysis results when M. bovis BCG FbiA
and FbiB were compared to homologs from other F420
producersa
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When MTH1018 was analyzed by BlastP against
Synechocystis
alone, a weak hit was obtained with SLL0154 (score = 44; E = 10
5). The
-Blast analysis also revealed a
homolog from a
Nostoc sp. (also known as
Anabaena), which may contain F
420
(since it
is a cyanobacterium), although the genome of this organism
has
not been sequenced. We are cautiously optimistic that these weak
hits in cyanobacteria have identified true functional homologs
of FbiA,
but will wait for further supportive data (e.g., complementation
of our
fbiA mutant with
Synechococcus fbiA and the
M. bovis fbiB on the same plasmid) before reaching a final
decision.
Strong FbiA hits were also obtained with
Streptomyces
coelicolor (
37), another F
420
producer for which the genome sequence
is largely completed but not yet
annotated. Figure
4 provides
an overview of the arrangement of these
homologs (the
M. leprae homolog, ML0759, which was the same
size as Rv3261, is listed
in Table
2 but was omitted from Fig.
4 to
conserve space). Homologs
for FbiB were found in all sequenced
F
420-producing organisms
except
Synechocystis (Table
2 and Fig.
4). The
Mycobacterium, Streptomyces, and
Methanobacterium fbiA and
fbiB homologs were
adjacent, while in the other organisms
these two genes were distant
from each other.
M. tuberculosis and
M. bovis have exactly the
same gene
arrangement in the region shown in Fig.
4.
M. leprae has a
very similar arrangement, except that another open reading
frame (ORF)
(ML0756) was identified between
fbiB and the Rv3263
homolog.
Transcription in the fbiAB cluster.
As shown in
Fig. 4, the whiB gene upstream of fbiA is
transcribed in the opposite direction. Downstream of fbiB
there is a 296- or 371-bp gap (depending on the start codon chosen)
before the start of Rv3263. Downstream of Rv3263, rmlA2 (not
shown in Fig. 4) is transcribed in the opposite direction. This
arrangement left open the possibility that fbiA,
fbiB, and Rv3263 were transcribed together or that some or
all were transcribed independently. Thus, we used RT-PCR to examine
transcription from these three genes.
As shown in Fig.
4, mRNA species existed which coded for significant
sections of both
fbiA and
fbiB. The sizes of the
amplified
sections within these two genes were as predicted. However,
no
product was obtained when primers were used to see if mRNA arising
from both
fbiB and Rv3263 was present (Fig.
4). This
supports
the concept that
fbiA and
fbiB are
expressed as an operon which
does not include Rv3263. We also used
RT-PCR to examine mRNA production
by the
fbiA mutant, since
it was possible that significant
fbiB transcripts could
result from a promoter upstream of
fbiB. Using
mRNA at the
same concentration as above, we saw no
fbiB transcript,
but
when the mRNA level was increased 10-fold, a weak but significant
band
of the correct size was observed. This suggests that although
most
fbiB transcription occurs from a promoter upstream of
fbiA,
a small but possibly significant amount of
monocistronic
fbiB transcription may occur from an
alternative
site.
Complementation of fbiA and fbiB
mutants.
Using complementation, we confirmed that interruption of
these genes was responsible for the F420-minus
phenotype. PCR of the cloned M. bovis fbi genes yielded
subclones containing each gene separately (p3261 and p3262) and one
clone containing both genes (p61-62). The strong mycobacterial
promoter in pSMT3 located upstream of the cloned gene(s) ensured
adequate expression. Electroduction confirmed that pSMT3 in
complemented cells contained an insert of the correct size.
Figure
5 shows examples of the HPLC profiles of extracts from the
wild-type strain, the
fbiA mutant, the
fbiB
mutant complemented
with p3262, and the
fbiA mutant
complemented with p3261. Transformation
of the
fbiA mutant
with p61-62 (HPLC data not shown) and of the
fbiB mutant
with p3262 (HPLC data shown in Fig.
5) resulted in
full restoration of
F
420-5,6 production, as shown quantitatively
in
Table
3 for both complemented mutants.
The
fbiA mutant complemented
with p3262 did not make
F
420-5,6 (Table
3); FO was the only significant
5-deazaflavin peak observed.
View this table:
[in this window]
[in a new window]
|
TABLE 3.
F420 production by wild-type M. bovis BCG, by fbiA and fbiB mutants, and by
mutants complemented with these genesa
|
|
However, as shown in Table
3, complementation of the
fbiA
mutant with p3261 led to a slight but significant production of
F
420-5,6 (comprised only of
F
420-5). As shown in Fig.
5, this
complementation
also led to the accumulation of compounds that
are almost certainly
F
420-2,3,4 (F
420-2,
F
420-3, and F
420-4),
since
coinjection of standard F
420-2 and
F
420-5,6 showed precisely
coincident elution with
putative F
420-2 and F
420-5,
and the fluorescent
properties of these peaks are consistent with their
being F
420 species. Prior experience with
F
420-4 also agrees with this conclusion
(
4). It is possible that smaller peaks at 17 to 20 min
include
F
420-0 and F
420-1.
F
420-2,3,4 peaks of this magnitude were not
observed in the uncomplemented mutants or in mutants complemented
with
p3262 or p61-62, but the wild-type strain and some of the
mutants
often showed small peaks in this region that were about
one-tenth (or
less) of the size seen in the
fbiA mutant complemented
with
p3261. The appearance of several F
420 species
with fewer
than five or six glutamates also suggests that glutamates
are
not added exclusively as an already assembled five- or
six-glutamate
piece, but are instead added individually, at least after
F
420-2
is
formed.
The discovery of intermediates in the p3261-complemented
fbiA mutant led us to more carefully examine the HPLC
profiles of
the
fbiA and
fbiB mutants. As shown
in Fig.
6, aside from FO,
the
uncomplemented
fbiB mutant accumulated material showing up
as three fluorescent HPLC peaks (indicated by arrows) that eluted
between 16 and 22 min. The peaks at 18.5 and 22.5 min were not
always
seen in the
fbiB mutant. Note that the scale in Fig.
6 is
considerably magnified compared to Fig.
5, allowing these peaks
to be
seen. The three
fbiB mutant peaks were not seen with the
fbiA mutant. Peaks eluting in this region are expected to
contain
no glutamate, and thus these peaks could arise from
F
420-0 or
closely related structures. This would
be consistent with a role
for FbiA in making
F
420-0 (F0-phosphate-lactyl), and with the
inability of the
fbiB mutant to add glutamates to
F
420-0.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 6.
HPLC elution profiles of extracts made from the
fbiA mutant and the fbiB mutant. Compare
the scale to the HPLC data in Fig. 5; the scale here is enhanced by
13.3-fold.
|
|
The complementation data clearly show that FbiB is essential, since
replacing the inactive gene with an active one fully restores
the
original phenotype. The complication in interpreting these
data is the
behavior of the
fbiA mutant which has been complemented
with
p3261. If the genes are monocistronic, we would expect to
see full
restoration of F
420-5,6 production when the
fbiA mutant
was complemented with p3261, but instead,
although a little F
420-5
was made, mostly the
smaller species were made. If the two genes
were entirely transcribed
as an operon from a promoter or promoters
upstream of
fbiA,
then we should see no F
420-2,3,4,5 made when
the
fbiA mutant is complemented with p3261, since no FbiB could
be produced. A reasonable explanation of these data, in light
of the
transcriptional data, is that although the
fbiA mutant
makes
no FbiA, it can make a small amount of FbiB due to a leaky
polar
effect, i.e., a small amount of
fbiB transcript is made
from
an alternative start site just upstream of
fbiB.
Consideration of the roles of FbiA and FbiB.
Since the mutated
strains make FO, FbiA and FbiB must play roles in the later portion of
the pathway shown in Fig. 1B, between FO and
F420-n. This portion of the pathway
involves the construction, by mostly unknown mechanisms, of a side
group that is composed of a phosphate, a lactyl, and five or six
glutamyl groups. Although it would be logical to initially add a
phosphate to FO by an FO kinase (in analogy to flavin mononucleotide
biosynthesis), our repeated efforts to demonstrate such a reaction
using ATP or GTP have been unsuccessful (20). Recent work
with methanogens has demonstrated that 2-phospholactate is a precursor
of the phosphate and lactyl groups of F420
(17), suggesting that FO kinase may not be present in
mycobacteria. It also makes sense that an enzyme transfers individual
glutamyl groups to F420-0 (since we see
intermediates with two, three, four, five, and six glutamyl groups),
but we have not sought such an enzyme yet.
We have examined protein sequence databases with several search methods
(COGS, BlastP, and
-Blast) to deduce possible functions
for FbiA and
FbiB and have conducted multiple alignments of FbiA
and FbiB with
homologs from other F
420 producers. None of these
methods provided firm support for assignment of specific functions
to
FbiA and FbiB, which is not surprising because these enzymes
have never
been studied. However, taken together, our data suggest
three
hypotheses. (i) FbiA is involved in making
F
420-0, and FbiB
participates in the addition of
glutamates to F
420-0. (ii) FbiA
and FbiB are
subunits of one enzyme which adds glutamates to
F
420-0;
neither protein is active in glutamyl
transfer by itself. (iii)
FbiB is involved in making
F
420-0, and FbiA adds glutamates to
F
420-0. Our data cannot confidently distinguish
between the three
hypotheses. Nonetheless, we are most attracted to
hypothesis i,
that FbiA participates in the conversion of FO to
F
420-0, and
that FbiB plays a role in the
addition of glutamates. This hypothesis
is consistent with intermediate
accumulation in the
fbiB mutant,
a lack of intermediate
accumulation in the
fbiA mutant, and a
leaky polar effect
seen with the
fbiA-complemented
fbiA mutant.
Development of assays in
Mycobacterium species and with
overexpressed
FbiA and FbiB for the 2-phospholactate and glutamyl
transfer steps
will be very useful for examination of these
hypotheses.
| |
ACKNOWLEDGMENTS |
We thank C. Kendall Stover, Paul Warrener, David Sherman, and
Ying Yuan of the PathoGenesis Corporation for the gift of PA-824, an
expensive material that is not commercially available. We thank the
Brigitte Gicquel-Vladimir Pelicic group at the Institut Pasteur, Paris,
the William Jacobs Jr. HHMI group at the Albert Einstein College of
Medicine for providing phAE94, pPR28, and pPR29, and Larry Schlesinger
at Iowa for providing pSMT3. We are very appreciative of the excellent
technical work contributed by Seong-Ae Kang. We thank Elena
Schoenberger for screening for F420-minus mutants of
M. smegmatis. We thank Robert White and Marion Graupner
for making us aware of their work on the role of 2-phospholactate in
F420 biosynthesis prior to publication. We appreciate
advice from Frances Ufkes on statistical analysis of our insertion sites.
This work was supported by National Institutes of Health grant GM56177
and U.S. Department of Agriculture grant 4132008 to L.D.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, University of Iowa, Iowa City, IA 52242. Phone: (319)
335-7780. Fax: (319) 335-9006. E-mail: lacy-daniels{at}uiowa.edu.
Present address: Department of Microbiology, Changwon University,
Changwon, Kyungnam 641-773, South Korea.
| |
REFERENCES |
| 1.
|
Altschul, S. F.,
W. Gish,
W. Miller,
E. W. Myers, and D. J. Lipman.
1990.
Basic local alignment search tool.
J. Mol. Biol.
215:403-410[CrossRef][Medline].
|
| 2.
|
Altschul, S. F.,
T. L. Madden,
A. A. Schaffer,
J. Zhang,
Z. Zhang,
W. Miller, and D. J. Lipman.
1997.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res.
25:3389-3402[Abstract/Free Full Text].
|
| 3.
|
Bair, T. B.
2001.
Methods to identify genes involved in F420 biosynthesis by Mycobacterium. Ph.D. thesis.
University of Iowa, Iowa City.
|
| 4.
|
Bair, T. B.,
D. W. Isabelle, and L. Daniels.
2001.
Structures of coenzyme F420 in Mycobacterium species.
Arch. Microbiol.
176:37-43[CrossRef][Medline].
|
| 5.
|
Bardarov, S.,
J. Kriakov,
C. Carriere,
S. Yu,
C. Vaamonde,
R. A. McAdam,
B. R. Bloom,
G. F. Hatfull, and W. R. Jacobs, Jr.
1997.
Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis.
Proc. Natl. Acad. Sci. USA
94:10961-10966[Abstract/Free Full Text].
|
| 6.
|
Bult, C. J.,
O. White,
G. J. Olsen,
L. Zhou,
R. D. Fleischmann,
G. G. Sutton,
J. A. Blake,
L. M. FitzGerald,
R. A. Clayton,
J. D. Gocayne,
A. R. Kerlavage,
B. A. Dougherty,
J. F. Tomb,
M. D. Adams,
C. I. Reich,
R. Overbeek,
E. F. Kirkness,
K. G. Weinstock,
J. M. Merrick,
A. Glodek,
J. L. Scott,
N. S. M. Geoghagen,
J. F. Weidman,
J. L. Fuhrmann,
E. A. Presley,
D. Nguyen,
T. R. Utterback,
J. M. Kelley,
J. D. Peterson,
P. W. Sadow,
M. C. Hanna,
M. D. Cotton,
M. A. Hurst,
K. M. Roberts,
B. P. Kaine,
M. Borodovsky,
H.-P. Klenk,
C. M. Fraser,
H. O. Smith,
C. R. Woese, and J. C. Venter.
1996.
Complete genome sequence of the methanogenic archaeon Methanococcus jannaschii.
Science
273:1058-1073[Abstract].
|
| 7.
|
Chun, K. T.,
H. J. Edenberg,
M. R. Kelley, and M. G. Goebl.
1997.
Rapid amplification of uncharacterized transposon-tagged DNA sequences from genomic DNA.
Yeast
13:233-240[CrossRef][Medline].
|
| 8.
|
Coats, J. H.,
G. P. Li,
M. S. Kuo, and D. A. Yurek.
1989.
Discovery, production, and biological assay of an unusual flavenoid cofactor involved in lincomycin biosynthesis.
J. Antibiot. (Tokyo)
42:472-474[Medline].
|
| 9.
|
Cole, S. T.,
R. Brosch,
J. Parkhill,
T. Garnier,
C. Churcher,
D. Harris,
S. V. Gordon,
K. Eiglmeier,
S. Gas,
C. E. Barry, 3rd,
F. Tekaia,
K. Badcock,
D. Basham,
D. Brown,
T. Chillingworth,
R. Connor,
R. Davies,
K. Devlin,
T. Feltwell,
S. Gentles,
N. Hamlin,
S. Holroyd,
T. Hornsby,
K. Jagels,
A. Krogh,
J. McLean,
S. Moule,
L. Murphy,
K. Oliver,
J. Osborne,
M. A. Quail,
M. A. Rajandream,
J. Rogers,
S. Rutter,
K. Seeger,
J. Skelton,
R. Squares,
S. Squares,
J. E. Sulston,
K. Taylor,
S. Whitehead, and B. G. Barrell.
1998.
Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.
Nature
393:537-544[CrossRef][Medline].
|
| 10.
|
Cole, S. T.,
K. Eiglmeier,
J. Parkhill,
K. D. James,
N. R. Thomson,
P. R. Wheeler,
N. Honore,
T. Garnier,
C. Churcher,
D. Harris,
K. Mungall,
D. Basham,
D. Brown,
T. Chillingworth,
R. Connor,
R. M. Davies,
K. Devlin,
S. Duthoy,
T. Feltwell,
A. Fraser,
N. Hamlin,
S. Holroyd,
T. Hornsby,
K. Jagels,
C. Lacroix,
J. Maclean,
S. Moule,
L. Murphy,
K. Oliver,
M. A. Quail,
M. A. Rajandream,
K. M. Rutherford,
S. Rutter,
K. Seeger,
S. Simon,
M. Simmonds,
J. Skelton,
R. Squares,
S. Squares,
K. Stevens,
K. Taylor,
S. Whitehead,
J. R. Woodward, and B. G. Barrell.
2001.
Massive gene decay in the leprosy bacillus.
Nature
409:1007-1011[CrossRef][Medline].
|
| 11.
|
Eirich, L. D.,
G. D. Vogels, and R. S. Wolfe.
1979.
Distribution of coenzyme F420 and properties of its hydrolytic fragments.
J. Bacteriol.
140:20-27[Abstract/Free Full Text].
|
| 12.
|
Eirich, L. D.,
G. D. Vogels, and R. S. Wolfe.
1978.
Proposed structure for coenzyme F420 from Methanobacterium.
Biochemistry
17:4583-4593[CrossRef][Medline].
|
| 13.
|
Eisenreich, W.,
B. Schwarzkopf, and A. Bacher.
1991.
Biosynthesis of nucleotides, flavins, and deazaflavins in Methanobacterium thermoautotrophicum.
J. Biol. Chem.
266:9622-9631[Abstract/Free Full Text].
|
| 14.
|
Eker, A. P.,
J. K. C. Hessels, and J. van de Velde.
1988.
Photoreactivating enzyme from the green alga Scenedesmus acutus. Evidence for the presence of two different flavin chromophores.
Biochemistry.
27:1758-1765[CrossRef].
|
| 15.
|
Eker, A. P.,
P. Kooiman,
J. K. Hessels, and A. Yasui.
1990.
DNA photoreactivating enzyme from the cyanobacterium Anacystis nidulans.
J. Biol. Chem.
265:8009-8015[Abstract/Free Full Text].
|
| 16.
|
Gorris, L. G. M., and C. van der Drift.
1988.
Separation and quantitation of cofactors from methanogenic bacteria by high-performance liquid chromatography: optimum and routine analysis.
J. Microbiol. Methods
8:175-190[CrossRef].
|
| 17.
|
Graupner, M., and R. H. White.
2001.
Biosynthesis of the phosphodiester bond in coenzyme F420 in the Methanoarchaea.
Biochemistry, in press.
|
| 18.
|
Hartzell, P. L.,
G. Zvilius,
J. C. Escalante-Semerena, and M. I. Donnelly.
1985.
Coenzyme F420 dependence of the methylenetetrahydromethanopterin dehydrogenase of Methanobacterium thermoautotrophicum.
Biochem. Biophys. Res. Commun.
133:884-890[CrossRef][Medline].
|
| 19.
|
Husson, R. N.,
B. E. James, and R. A. Young.
1990.
Gene replacement and expression of foreign DNA in mycobacteria.
J. Bacteriol.
172:519-524[Abstract/Free Full Text].
|
| 20.
|
Isabelle, D. W.
2001.
Coenzyme F420 biosynthesis in Mycobacterium smegmatis. M.S. thesis.
University of Iowa, Iowa City.
|
| 21.
|
Jacobson, F. S.,
L. Daniels,
J. A. Fox,
C. T. Walsh, and W. H. Orme-Johnson.
1982.
Purification and properties of an 8-hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum.
J. Biol. Chem.
257:3385-3388[Abstract/Free Full Text].
|
| 22.
|
Jaenchen, R.,
P. Schonheit, and R. K. Thauer.
1984.
Studies on the biosynthesis of coenzyme F420 in methanogenic archaea.
Arch. Microbiol.
137:362-365[CrossRef][Medline].
|
| 23.
|
Jones, J. B., and T. C. Stadtman.
1980.
Reconstitution of a formate-NADP+ oxidoreductase from formate dehydrogenase and a 5-deazaflavin-linked NADP+ reductase isolated from Methanococcus vannielii.
J. Biol. Chem.
255:1049-1053[Abstract/Free Full Text].
|
| 24.
|
Kaneko, T.,
S. Sato,
H. Kotani,
A. Tanaka,
E. Asamizu,
Y. Nakamura,
N. Miyajima,
M. Hirosawa,
M. Sugiura,
S. Sasamoto,
T. Kimura,
T. Hosouchi,
A. Matsuno,
A. Muraki,
N. Nakazaki,
K. Naruo,
S. Okumura,
S. Shimpo,
C. Takeuchi,
T. Wada,
A. Watanabe,
M. Yamada,
M. Yasuda, and S. Tabata.
1996.
Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions.
DNA Res.
3:109-136[Abstract].
|
| 25.
|
Klenk, H. P.,
R. A. Clayton,
J. F. Tomb,
O. White,
K. E. Nelson,
K. A. Ketchum,
R. J. Dodson,
M. Gwinn,
E. K. Hickey,
J. D. Peterson,
D. L. Richardson,
A. R. Kerlavage,
D. E. Graham,
N. C. Kyrpides,
R. D. Fleischmann,
J. Quackenbush,
N. H. Lee,
G. G. Sutton,
S. Gill,
E. F. Kirkness,
B. A. Dougherty,
K. McKenney,
M. D. Adams,
B. Loftus, and J. C. Venter.
1997.
The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus.
Nature
390:364-370[CrossRef][Medline].
|
| 26.
|
Kunow, J.,
D. Linder,
K. O. Stetter, and R. K. Thauer.
1994.
F420H2: quinone oxidoreductase from Archaeoglobus fulgidus: characterization of a membrane-bound multisubunit complex containing FAD and iron-sulfur clusters.
Eur. J. Biochem.
223:503-511[Medline].
|
| 27.
|
Ma, K., and R. K. Thauer.
1990.
Purification and properties of N5,N10-methylenetetrahydromethanopterin reductase from Methanobacterium thermoautotrophicum (strain Marburg).
Eur. J. Biochem.
191:187-193[Medline].
|
| 28.
|
Mangan, J. A.,
K. M. Sole,
D. A. Mitchison, and P. D. Butcher.
1997.
An effective method of RNA extraction from bacteria refractory to disruption, including mycobacteria.
Nucleic Acids Res.
25:675-676[Abstract/Free Full Text].
|
| 29.
|
Mao, Y.,
M. Varoglu, and D. H. Sherman.
1999.
Molecular characterization and analysis of the biosynthetic gene cluster for the antitumor antibiotic mitomycin C from Streptomyces lavendulae NRRL 2564.
Chem. Biol.
6:251-263[CrossRef][Medline].
|
| 30.
|
McAdam, R. A.,
T. R. Weisbrod,
J. Martin,
J. D. Scuderi,
A. M. Brown,
J. D. Cirillo,
B. R. Bloom, and W. R. Jacobs, Jr.
1995.
In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis.
Infect. Immun.
63:1004-1012[Abstract].
|
| 31.
|
McCormick, J. R. D., and G. O. Morton.
1982.
Identity of cosynthetic factor 1 of Streptomyces aureofaciens and fragment FO from coenzyme F420 of Methanobacterium sp.
J. Am. Chem. Soc.
104:4014-4015[CrossRef].
|
| 32.
|
Ng, W. V.,
S. P. Kennedy,
G. G. Mahairas,
B. Berquist,
M. Pan,
H. D. Shukla,
S. R. Lasky,
N. S. Baliga,
V. Thorsson,
J. Sbrogna,
S. Swartzell,
D. Weir,
J. Hall,
T. A. Dahl,
R. Welti,
Y. A. Goo,
B. Leithauser,
K. Keller,
R. Cruz,
M. J. Danson,
D. W. Hough,
D. G. Maddocks,
P. E. Jablonski,
M. P. Krebs,
C. M. Angevine,
H. Dale,
T. A. Isenbarger,
R. F. Peck,
M. Pohlschroder,
J. L. Spudich,
K. H. Jung,
M. Alam,
T. Freitas,
S. Hou,
C. J. Daniels,
P. P. Dennis,
A. D. Omer,
H. Ebhardt,
T. M. Lowe,
P. Liang,
M. Riley,
L. Hood, and S. DasSarma.
2000.
Genome sequence of Halobacterium species NRC-1.
Proc. Natl. Acad. Sci. USA
97:12176-12181[Abstract/Free Full Text].
|
| 33.
|
O Gaora, P.
1998.
Expression of genes in mycobacteria, p. 472.
In
T. Parish, and N. G. Stoker (ed.), Mycobacteria protocols, vol. 101. Humana Press, Totowa, N.J.
|
| 34.
|
Pelicic, V.,
M. Jackson,
J. M. Reyrat,
W. R. Jacobs, Jr.,
B. Gicquel, and C. Guilhot.
1997.
Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis.
Proc. Natl. Acad. Sci. USA
94:10955-10960[Abstract/Free Full Text].
|
| 35.
|
Purwantini, E., and L. Daniels.
1996.
Purification of a novel coenzyme F420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis.
J. Bacteriol.
178:2861-2866[Abstract/Free Full Text].
|
| 36.
|
Purwantini, E.,
T. Gillis, and L. Daniels.
1997.
Presence of F420-dependent glucose-6-phosphate dehydrogenase in Mycobacterium and Nocardia species, but absence in Streptomyces and Corynebacterium species and methanogenic Archaea.
FEMS Microbiol. Lett.
146:129-134[CrossRef][Medline].
|
| 37.
|
Redenbach, M.,
H. M. Kieser,
D. Denapaite,
A. Eichner,
J. Cullum,
H. Kinashi, and D. A. Hopwood.
1996.
A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3(2) chromosome.
Mol. Microbiol.
21:77-96[CrossRef][Medline].
|
| 38.
|
Reuke, B.,
S. Korn,
W. Eisenreich, and A. Bacher.
1992.
Biosynthetic precursors of deazaflavins.
J. Bacteriol.
174:4042-4049[Abstract/Free Full Text].
|
| 39.
|
Rhodes, P. M.,
N. Winskill,
E. J. Friend, and M. Warren.
1981.
Biochemical and genetic comparison of Streptomyces rimosus mutants impaired in oxytetracycline biosynthesis.
J. Gen. Microbiol.
124:329-338.
|
| 40.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual.
Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
|
| 41.
|
Schneider, T. D., and R. M. Stephens.
1990.
Sequence logos: a new way to display consensus sequences.
Nucleic Acids Res.
18:6097-6100[Abstract/Free Full Text].
|
| 42.
|
Smith, D. R.,
L. A. Doucette-Stamm,
C. Deloughery,
H. Lee,
J. Dubois,
T. Aldredge,
R. Bashirzadeh,
D. Blakely,
R. Cook,
K. Gilbert,
D. Harrison,
L. Hoang,
P. Keagle,
W. Lumm,
B. Pothier,
D. Qiu,
R. Spadafora,
R. Vicaire,
Y. Wang,
J. Wierzbowski,
R. Gibson,
N. Jiwani,
A. Caruso,
D. Bush,
H. Safer,
D. Patwell,
S. Prabhaker,
S. McDougall,
G. Shimer,
A. Goyal,
S. Pietrokovski,
G. M. Church,
C. J. Daniels,
J.-I. Mao,
P. Rice,
J. Nolling, and J. N. Reeve.
1997.
Complete genome sequence of Methanobacterium thermoautotrophicum  H: functional analysis and comparative genomics.
J. Bacteriol.
179:7135-7155[Abstract/Free Full Text].
|
| 43.
|
Smith, R. F., and T. F. Smith.
1992.
Pattern-induced multisequence alignment (PIMA) algorithm employing secondary structure-dependent gap penalties for use in comparative protein modeling.
Protein Eng.
5:35-41[Abstract/Free Full Text].
|
| 44.
|
Snapper, S. B.,
R. E. Melton,
S. Mustafa,
T. Kieser, and W. R. Jacobs, Jr.
1990.
Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis.
Mol. Microbiol.
4:1911-1919[Medline].
|
| 45.
|
Stover, C. K.,
P. Warrener,
D. R. VanDevanter,
D. R. Sherman,
T. M. Arain,
M. H. Langhorne,
S. W. Anderson,
J. A. Towell,
Y. Yuan,
D. N. McMurray,
B. N. Kreiswirth,
C. E. Barry, and W. R. Baker.
2000.
A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis.
Nature
405:962-966[CrossRef][Medline].
|
| 46.
|
Thompson, J. D.,
D. G. Higgins, and T. J. Gibson.
1994.
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res.
22:4673-4680[Abstract/Free Full Text].
|
| 47.
|
Widdel, F., and R. S. Wolfe.
1989.
Expression of secondary alcohol dehydrogenase in methanogenic bacteria and purification of the F420-specific enzyme from Methanogenium thermophilum strain TCI.
Arch. Microbiol.
152:322-328[CrossRef].
|
Journal of Bacteriology, December 2001, p. 7058-7066, Vol. 183, No. 24
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.24.7058-7066.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
