Journal of Bacteriology, October 1998, p. 5473-5477, Vol. 180, No. 20
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Characterization of the mIHF Gene of
Mycobacterium smegmatis
Marisa L.
Pedulla
and
Graham F.
Hatfull*
Department of Biological Sciences, University
of Pittsburgh, Pittsburgh, Pennsylvania 15260
Received 6 May 1998/Accepted 5 August 1998
 |
ABSTRACT |
Integration of mycobacteriophage L5 requires the mycobacterial
integration host factor (mIHF) in vitro. mIHF is a 105-residue heat-stable polypeptide that is not obviously related to HU or any
other small DNA-binding proteins. mIHF is most abundant just prior to
entry into stationary phase and is essential for the viability of
Mycobacterium smegmatis.
| |
TEXT |
Site-specific integration of
mycobacteriophage L5 requires the phage-encoded integrase protein and
the host-encoded mycobacterial integration host factor (mIHF) protein
(13, 14, 17). mIHF is an unusual host factor in that it does
not bind specifically to L5 attP DNA (17), but is
required for formation of recombinogenic intasomes that contain
attP DNA, L5 integrase, and mIHF (17, 21, 22).
While its name reflects the requirement of the mIHF protein for L5
integration, mIHF is not closely related at the sequence level to
Escherichia coli IHF, the HU family of proteins, or any
other small DNA-binding proteins (10, 17). IHF is not essential for the viability of E. coli, although it is
implicated in a variety of cellular processes including gene expression
(6, 7), DNA metabolism (2, 8, 9, 12, 16), and
pathogenesis (15, 23); it also reaches its highest
intracellular level just prior to stationary phase (1, 3, 5)
and may be involved in the regulation of genes required for the
establishment of stationary phase (11). In this study, we
asked whether mIHF is required for the viability of Mycobacterium
smegmatis and whether its intracellular levels fluctuate with
growth of the bacteria.
Organization of the mIHF locus in M. smegmatis and Mycobacterium tuberculosis.
The fast-growing M. smegmatis and the slow-growing
Mycobacterium tuberculosis both contain a single gene
encoding mIHF (17). However, the nucleotide sequences beyond
the 5' and 3' ends of the mIHF genes (with the exception of
a small region to the 5' side) are not closely related (17).
Thus, while mIHF may play important roles in the mycobacteria, it is
unclear whether the genes occupy similar chromosomal locations.
Additional information on the sequence of the mIHF locus of
M. smegmatis shows that mIHF is located
approximately 1 kb downstream of the pyrF gene with no
identifiable genes within the intergenic space (Fig.
1A). In M. tuberculosis there
are two genes in the interval between pyrF and
mIHF; one encodes a small protein of unknown function, and the other encodes a protein that, while also of unknown function, bears
sequence similarity to a large family of paralogous proteins in
M. tuberculosis. This family has been designated the PPE
family of proteins (4). Members of this family in M. smegmatis have yet to be described.
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FIG. 1.
mIHF locus of M. smegmatis and
M. tuberculosis. (A) Arrows represent genes present at the
mIHF locus of M. smegmatis and M. tuberculosis H37Rv, and the shaded regions represent regions of
close sequence similarity between the two species. The M. tuberculosis H37Rv organization is taken from data on cosmid
MTCY21B4 (accession no. Z80108), and M. smegmatis
information was obtained from accession no. U75344 and U91572. (B)
Sequence similarity of the mIHF genes. A region of
approximately 250 bp around the beginning of the M. smegmatis mIHF gene is aligned with that from M. tuberculosis H37Rv; bases in common are indicated with an
asterisk, and gaps introduced for alignment are shown as dashes. The
position of the GTG translation initiation codon is indicated by a
horizontal line, and the sequences corresponding to the ribosome
binding sites are boxed. The position of the M. smegmatis
transcription initiation site is shown by an arrow, and the putative
 10 and 35 regions are indicated.
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|
The region of high sequence similarity of the M. smegmatis
and M. tuberculosis mIHF genes extends approximately 125 bp
upstream of the mIHF coding regions (Fig. 1B). Part of this
region corresponds to the putative ribosome binding sites, and
sequences further upstream may be important for promoter activity and
regulatory functions. We have identified a putative transcription
initiation site by S1 nuclease mapping at position 77 (relative to
the start of the mIHF coding sequence), which is within the
conserved region (Fig. 1B). Bases at the putative 10 and 35
positions upstream of the transcription initiation site are also well
conserved (Fig. 1B).
Intracellular levels of mIHF vary according to bacterial growth
phase.
The abundance of mIHF in M. smegmatis
and M. bovis bacillus Calmette-Guérin (BCG) as a
function of the state of bacterial growth was determined as shown in
Fig. 2. Following dilution of saturated
cultures, samples were removed at various times, and the mIHF levels
were determined by Western blotting with anti-mIHF serum (Fig. 2).
These data show that the abundance of mIHF is not constant throughout
the growth of the bacterial cultures and is most prevalent during late
logarithmic growth. Similar patterns were seen for M. smegmatis and BCG even though the growth rates for the cultures
are very different (Fig. 2). We note that E. coli IHF is
also most abundant just prior to stationary phase although the
magnitude of the effect (5- to 10-fold) (1, 3) is somewhat
greater than that observed for mIHF (Fig. 2).
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FIG. 2.
Growth phase dependency of mIHF. (A) Detection of
M. smegmatis mIHF by immunoblotting. Following dilution of a
saturated culture of M. smegmatis into fresh media, samples
were removed at the times indicated (in hours), and cells were
harvested by centrifugation. Samples were sonicated, normalized for
total protein content, and electrophoresed on a sodium dodecyl
sulfate-15% polyacrylamide gel. After transfer to polyvinylidine
difluoride, the filter was probed with anti-mIHF serum and proteins
were visualized by chemiluminescence. The marker lane (M) contains
purified mIHF protein. (B) The optical density at 600 nm
(OD600) of the bacterial culture used for panel A was
determined at various times. The mIHF levels shown in panel A were
quantitated by using NIH Image and are in arbitrary units. (C)
Detection of BCG mIHF by immunoblotting. Samples of M. bovis
BCG were removed at the indicated times (in days) after dilution of a
saturated culture; cells were harvested, normalized for total protein
content, and electrophoresed on a sodium dodecyl
sulfate-polyacrylamide gel. Following transfer to polyvinylidine
difluoride, the filter was probed with anti-mIHF serum and detected by
chemiluminescence. (D) The optical density at 600 nm
(OD600) of the bacterial culture used for panel C was
determined at various times. The mIHF levels shown in panel C were
quantitated by using NIH Image and are in arbitrary units.
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|
mIHF is essential for M. smegmatis
viability.
To determine whether mIHF is essential for
mycobacterial viability, allelic replacement experiments were
performed. The strategy used was similar to that described previously
(18-20) in which the Bacillus subtilis sacB gene
is used as a counterselectable marker; M. smegmatis is
normally resistant to sucrose but becomes sucrose sensitive when the
sacB gene is introduced. Introduction of a nonreplicating
plasmid carrying an interrupted mIHF gene (containing the
aph gene conferring kanamycin resistance) into M. smegmatis should give rise to kanamycin-resistant,
sucrose-sensitive colonies via a single crossover event at the
mIHF locus (Fig. 3A). Selection of sucrose-resistant
derivatives of this strain can either regenerate the initial strain
or
by recombination on the side of the aph gene opposite to
that which gave rise to the integrantgenerate a strain having only an
interrupted copy of the gene (Fig. 3A). However, the replacement of
mIHF by an interrupted gene will result in viable cells only
if mIHF is not required for growth of the bacteria; if the gene is
essential, then the replacement will produce viable cells only if a
second copy of mIHF is present elsewhere on the chromosome.
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FIG. 3.
Allelic replacement of mIHF. (A)
Strategy for allelic replacement of M. smegmatis mIHF.
Transformation of M. smegmatis mc2155 with
plasmid pMP32 generates a strain (MP1) containing an integrated copy of
the plasmid at the mIHF locus. Strain MP1 is resistant to
kanamycin but sensitive to sucrose due to the presence of the
sacB gene. Selection of sucrose-resistant colonies can give
rise to two alternative products depending on where recombination
occurs. If the recombination event is on the same side of
aph as the initial integration event, then the original
strain (mc2155) is regenerated; if it is on the other side,
then the mIHF gene is replaced by an interrupted copy. (B)
Schematic representation of strains used or generated in allelic
replacement experiments. At the top, the mIHF locus of
M. smegmatis mc2155 is represented with the
positions of restriction sites for BamHI (B),
PstI (P), and SalI (S) indicated; the unlinked
attB site is also shown. Also shown are relevant parts of
the chromosomes of strains MP1, MP6, MP2, MP3, MP4, and MP5. Strain MP1
is a derivative of mc2155 created by insertion of plasmid
pMP32 by homologous recombination at the mIHF locus; MP6 is
a derivative of MP1 that is similar to MP1 as determined by Southern
hybridization and PCR analyses but is sucrose resistant and probably
contains an inactivating mutation within the sacB gene (×).
Strain MP2 was derived from MP1 by transformation with an
integration-proficient plasmid containing the mIHF gene that
integrates site specifically at the attB locus. Strains MP3,
MP4, and MP5 are sucrose-resistant derivatives of MP2 that have an
inactivating mutation in sacB (MP3) or have undergone
recombination at the mIHF locus to leave only the wild-type
mIHF gene (MP5) or a replacement by an interrupted
mIHF gene (MP4). DNA fragments generated by restriction
enzyme digests that hybridize with an mIHF-specific DNA
probe are shown as thick horizontal lines with their sizes in
kilobases. The positions of primers used in PCR characterization
experiments are shown as arrowheads, and the sizes of PCR products are
shown in base pairs. Restriction sites originating from plasmid vector
sequences are shown with an asterisk. (C) PCR amplification of the
mIHF locus in M. smegmatis strains. DNAs from
various M. smegmatis strains were used for PCR amplication
with the primers shown in panel B, and the products were separated by
agarose gel electrophoresis. DNAs used were from pMP18 (containing the
wild-type mIHF gene) (lane 2), pMP32 (containing the
aph-interrupted mIHF gene) (lane 3), pMP28 (which
contains wild-type mIHF but lacks one of the primer binding
sites) (lane 4), mc2155 (lane 5), MP1 (lane 6), MP2 (lane
7), MP3 (lane 8), and MP4 (lane 9). Lane 1 contains no DNA. The
positions of the 513-bp fragment amplified from the wild-type
mIHF gene and the 1,476-bp fragment from the
aph-interrupted gene are indicated. Note that when both the
wild-type and interrupted mIHF loci are present in the same
strain (i.e., MP1, MP2, and MP3) the smaller product is preferentially
amplified. Plasmid pMP32 was constructed by insertion of a
sacB fragment into pMP27, a pUC119 derivative that contains
the mIHF gene interrupted by the aph gene at the
EcoNI site.
|
|
A plasmid (pMP32) which cannot replicate in mycobacteria and contains
the M. smegmatis mIHF gene with the aph
kanamycin-resistance gene inserted within the coding region was
constructed (Fig. 3A). This plasmid was introduced into M. smegmatis mc2155 by electroporation (24),
and kanamycin-resistant transformants were recovered. One of these
(MP1) (Fig. 3B) was characterized further and was shown to be sucrose
sensitive and to contain a single copy of the plasmid integrated at the
mIHF locus (Fig. 3C and data not shown). When MPI was
cultured and plated onto solid media containing sucrose (but without
kanamycin), sucrose-resistant colonies were generated at a frequency of
approximately 10
3 (Table
1). When these were tested for the
aph phenotype, 56% were found to be kanamycin resistant as
well. However, when examined by PCR amplification of the
mIHF locus (Fig. 3C), all of the more than 100 individual
sucrose-resistant kanamycin-resistant colonies tested retained the
wild-type mIHF locus (data not shown). Southern hybridization of a subset of these colonies indicated that there were
no additional recombination events in this region and that these
colonies most likely arose from either point mutations within sacB or suppressor mutations elsewhere in the chromosome
(e.g., MP6) (Fig. 3B). While the frequency of these events is somewhat higher than expected (Table 1), the recombinant strains containing the
sacB gene grow noticeably slower than the parent strain,
even in the absence of sucrose, providing a selective advantage for sacB mutants.
The inability to isolate sucrose-resistant recombinants that have lost
the wild-type mIHF gene suggests that either mIHF
is an essential gene or the recombination events that give rise to the
mIHF replacement are very infrequent events. To address this issue, we constructed a strain (MP2) that contains an additional copy
of the mIHF gene integrated at the phage L5 attB
attachment site (Fig. 3B). This was accomplished by introduction of an
integration-proficient plasmid containing the wild-type mIHF
gene (pMP28) into strain MP1 and selection of transformants resistant
to kanamycin and hygromycin (Fig. 3B). MP2 was cultured and plated on
solid media to select sucrose-resistant colonies as described above;
58% of these were shown to be kanamycin resistant (Table 1). The
mIHF loci of 12 of these sucrose-resistant
kanamycin-resistant colonies were tested by PCR, and 50% of these were
shown to have lost the normal wild-type mIHF locus (e.g.,
MP4) (Fig. 3B and 3C). The remaining 50% presumably have mutations
within the sacB gene (e.g., MP3) (Fig. 3B). These
experiments show that the recombination events that give rise to a
replacement of mIHF can indeed occur but produce viable
cells only if an additional wild-type copy of mIHF is
present. We conclude that mIHF is an essential gene in
M. smegmatis.
These experiments show that mIHF plays an important role in the
mycobacteria. It is clearly essential for the growth of M. smegmatis, and it seems likely that it is also essential in
slow-growing mycobacteria. Assuming that M. smegmatis also
contains HU-like and HupB DNA-binding proteins similar to those
identified in M. tuberculosis (4), these do not
appear to compensate for the loss of mIHF. It thus seems likely that
mIHF performs specialized functions in the mycobacteria. Since it is
most abundant prior to entry into the stationary phase, one of these
functions may be to regulate the expression of
stationary-phase-specific genes in a manner similar to that of E. coli IHF.
| |
ACKNOWLEDGMENTS |
This work was supported by NIH GM49647.
We thank Erica Shepard for assistance with DNA sequencing, G. Sarkis
for integrating plasmid pGS67, L. Pascopella and W. R. Jacobs,
Jr., for the pYUB415::mc2155 cosmid library, and M. Pavelka and W. R. Jacobs, Jr., for the sacB-containing plasmid
pYUB657. We also thank Carol Peña for helpful comments on the
manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260. Phone: (412) 624-6975. Fax: (412) 624-4870. E-mail:
gfh{at}vms.cis.pitt.edu.
Present address: Department of Biochemistry and Biophysics,
Washington State University, Pullman, WA 99164.
| |
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Journal of Bacteriology, October 1998, p. 5473-5477, Vol. 180, No. 20
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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