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J Bacteriol, February 1998, p. 746-748, Vol. 180, No. 3
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A New Transformation-Deficient Mutant of
Haemophilus influenzae Rd with Normal DNA Uptake
M. L.
Gwinn,*
R.
Ramanathan,
H. O.
Smith, and
J.-F.
Tomb
Department of Molecular Biology and Genetics,
School of Medicine, Johns Hopkins University, Baltimore, Maryland
21205
Received 8 May 1997/Accepted 17 November 1997
 |
ABSTRACT |
Haemophilus influenzae Rd is a gram-negative natural
transformer. A mutant strain, RJ248, that has normal DNA uptake and
translocation but whose transformation frequency is 300 times lower
than that of wild-type H. influenzae and whose phage
recombination is 8 times lower was isolated. The affected gene,
comM, is induced during competence development in wild-type
H. influenzae but not in RJ248.
| |
TEXT |
Haemophilus influenzae Rd
is a gram-negative bacterium capable of natural DNA transformation. It
achieves low-level competence in late log phase and can be induced to
high levels of competence by a nutritional downshift into MIV medium
(12) or by transient anaerobic growth (10).
Transformation involves several steps. The first is the uptake of naked
DNA into a membrane-protected compartment (13). Uptake of
DNA is facilitated by an uptake signal sequence (20). Following uptake, the DNA translocates slowly into the cytoplasm, where
the 5' strand is completely degraded and the 3' strand is partially
degraded (2). If homology exists between the incoming DNA
and the chromosome of the cell, the entering 3' strand will invade and
replace the chromosomal DNA by homologous recombination (2).
The comABCDEF operon is essential for transformation and is
induced during competence development but not during exponential growth
(21, 22). A 26-bp palindromic competence-regulatory element
(CRE) (also known as the dyad symmetry element) is required for
induction of the operon (9, 14, 22). This element has been
found upstream of three other known competence genes (14, 23) (pilABCD [8, 23], rec2
[7], and dprA [14]), all of which have been shown to be induced during competence development (14, 23). In all cases, the element is positioned about one turn of the helix upstream of either a known or a putative promoter (14, 22). It is likely that this sequence is the binding
site for a competence-specific, positively acting transcriptional
regulator. A known regulator of competence which could fill this role
is the product of the gene sxy (also known as
tfoX). This gene is required for competence development and
for the induction of both dprA and comF (14,
24, 26). In addition, certain point mutations of this gene which
allow cells to be competent all of the time exist (18). It
is not known whether the effect of sxy on expression of
competence genes is direct or indirect (14).
Strains with mutations in rec2, dprA, and
comF have normal DNA uptake but are transformation deficient
(7, 14, 23, 26). In rec2 mutants, the DNA remains
trapped in the membrane compartment and never translocates into the
cytoplasm (4). The H. influenzae homolog of the
Escherichia coli recA gene, rec1, is required for
homologous recombination of donor DNA into the chromosome
(16).
Here we report on RJ248, a new transformation mutant of H. influenzae Rd that has a transformation frequency 300-fold lower than that of wild-type H. influenzae and a deficiency in the
competence-induced increase in phage recombination. However, it has
normal DNA uptake and translocation. The affected gene has been
identified and named comM. It has a CRE site upstream of its
putative promoter. It is induced during competence development in
wild-type cells but not in RJ248.
Growth, transformation, and 
-galactosidase activity.
H.
influenzae was grown as described by Barcak et al. (3).
E. coli was grown as described by Sambrook et al.
(19). The MIV nutritional-downshift procedure was used to
measure transformation frequencies (3, 12). DNA uptake was
measured as described by Gwinn (11). -Galactosidase
activity was measured with a permeabilized-cell assay (17).
Mutagenesis and isolation of RJ248.
MIV-competent wild-type
cells were transformed with a plasmid carrying Tn916
(6, 15). Transposition events were selected for by
tetracycline resistance, which is encoded by Tn916.
Tetracycline-resistant colonies were screened by the cyclic AMP
transformation plate assay (25). One strain, RJ24, was
consistently transformation deficient. Genomic DNA from RJ24 was
transformed into wild-type cells to isolate a pure strain with only one
Tn916 insertion, resulting in RJ248. Southern and MIV
transformation analysis confirmed that RJ248 carries one
Tn916 insertion responsible for the transformation phenotype
and that the insertion was not in a known transformation gene.
UV sensitivity.
RJ248, KW20 (wild type), and DB117
(rec1 mutant; negative control) were grown and exposed to a
GE 615T8 germicidal lamp for 0, 3, or 6 s at a distance of 37 cm.
Plates were incubated at 37°C for 24 h, and survivors were
counted.
Phage recombination.
Exponential-phase cells and competent
cells were separately coinfected with ts1 and
ts3, temperature-sensitive point mutants of the H. influenzae phage Hp1c1. Recombinant progeny were identified by
their ability to grow at the nonpermissive temperature (40°C) (5).
Translocation.
A plasmid containing an H. influenzae genomic PstI fragment was cut with
PstI and end labeled with [32P]dCTP by using
T4 polymerase. Cells which had been made competent by the MIV method
were incubated with labeled plasmid. At 5, 10, 15, 30, and 60 min,
samples of the cells were removed and washed with MIV medium by
centrifugation. Total DNA was isolated. DNA samples were
electrophoresed on a 0.8% agarose gel and autoradiographed (4).
RNA isolation and Northern analysis.
All buffers used in RNA
isolation were made with diethyl pyrocarbonate-treated water. KW20
(wild type) and RJ248 were made competent by the MIV method. At 0, 50, and 100 min in MIV medium, 35 ml of cells was removed. Cells were
lysed, and RNA was isolated by hot-phenol extraction. Northern analysis
was performed with the Gene Images CDP-Start labeling and detection
kits (Amersham, Indianapolis, Ind.). Probes were gene-internal
fragments of HI1118 and HI1117 DNA.
Phenotype of RJ248.
RJ248 has a transformation frequency that
is 300-fold lower than that of the wild type. It has a wild-type DNA
uptake level and grows at the wild-type rate. Its resistance to UV
light is the same as that seen in the wild type (data not shown).
To test whether the mutation in RJ248 affected regulation, we
transformed RJ248 with genomic DNA from MGH100 (a strain containing lacZ kan in an operon fusion with comA), which
resulted in a strain with the RJ248 mutation and a -galactosidase
transcription indicator in comA. We found that induction of
comA, as measured by -galactosidase activity, is
unaffected in the RJ248 background (data not shown).
Competent cells exhibit a higher rate of phage recombination than cells
in exponential phase (
5). We found that with wild-type
cells
there is about a 40-fold increase in the recombination rate
in
competent cells over that in cells in exponential phase, while
with
RJ248 the increase is only 5-fold.
To test whether DNA translocation is normal in RJ248, we compared the
strain to several other mutant strains. Samples of competent
cells were
given radioactively labeled, digested plasmid DNA (two
fragments). In
wild-type cells, labeled donor DNA was taken up
and became associated
with the chromosome over time (Fig.
1).
As expected,
rec2 mutants did not translocate DNA into the
cytoplasm,
as indicated by the absence of accumulation of label in the
chromosome.
Cells with a mutation in
rec1 take DNA into the
cytoplasm; however,
since the DNA cannot undergo homologous
recombination, it is degraded
in the cytoplasm and the labeled
nucleotides are incorporated
into the chromosome through subsequent
reuse (
4). We found
that the accumulations of label in the
chromosome in the wild
type and RJ248 were indistinguishable,
indicating that translocation
in RJ248 is normal (Fig.
1). It is
theoretically possible that
in RJ248 the DNA becomes trapped in the
membrane compartment,
where it undergoes degradation and subsequent
entry via another
mechanism. However, we feel this to be unlikely,
since there is
substantial evidence indicating that large-scale
degradation of
donor DNA does not occur until it reaches the cytoplasm.
In addition,
although slow DNA degradation has been observed in the
membrane
compartment, this has not been shown to allow the DNA to then
enter the cytoplasm (
2,
4).
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FIG. 1.
Results of a translocation experiment. Translocation is
indicated by the appearance in the chromosome of labeled plasmid DNA.
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Identification of the affected gene.
Sequence analysis of
clones carrying the region of insertion revealed that Tn916
was inserted between the HI1117 and HI1118 genes (9), which
are in the same orientation (Fig. 2). The predicted products of both open reading frames have significant homology to hypothetical proteins in E. coli (see the
H. influenzae database at www.tigr.org). In addition, HI1117
has homology to a magnesium chelatase gene of Rhodobacter
capsulatus, bchI, involved in bacteriochlorophyll
biosynthesis (1) and to related genes from other
photosynthetic organisms. The putative promoter region of HI1117
contains a CRE sequence about one turn of the helix upstream of a
putative promoter (Fig. 2). The insertion point of Tn916 is
in the 
10 hexamer region of the possible promoter. This insertion is
beyond the stop codon of HI1118. We conclude that HI1117 is affected by
the disruption of its promoter and have named it comM.
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FIG. 2.
Sequence around the point of insertion of Tn916
with the putative comM promoter indicated. hyp.,
hypothetical. The asterisk denotes a stop codon. RBS, ribosomal binding
site.
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|
To test whether
comM is induced during competence
development, as would be expected based on the presence of the CRE, and
whether induction is prevented by the presence of Tn
916 in
the
mutant, total RNA from both KW20 (wild type) and RJ248 before
and
after competence induction was probed with a DNA fragment
from within
comM and with a DNA fragment from within HI1118. We
found
that in the wild type,
comM is strongly induced during
competence
development; however, in RJ248, there is no detectable
induction
of
comM (Fig.
3).
Expression of HI1118 diminishes in both backgrounds
and is unaffected
by the presence of Tn
916 (Fig.
3).
The
comM gene is clearly not induced in RJ248 during
competence development. This is due to the presence of
Tn
916, which disrupts
the putative promoter. In addition to
its transformation deficiency,
the only competence-associated defect we
have found in RJ248 is
a reduction in competence-dependent phage
recombination. This
phenotype and the ability of RJ248 to translocate
DNA normally
imply that
comM acts very late in
transformation, presumably at
a step in the recombination of the donor
DNA into the chromosome.
| |
ACKNOWLEDGMENTS |
This work was funded by grant NP838C from the American Cancer
Society and by grant GM48251 from the National Institutes of Health.
H. O. Smith is an American Cancer Society Research Professor.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: The Institute
for Genomic Research, 9712 Medical Center Dr., Rockville, MD 20850. Phone: (301) 315-2536. Fax: (301) 838-0208. E-mail:
mlgwinn{at}tigr.org.
Present address: The Institute for Genomic Research, Rockville, MD
20850.
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J Bacteriol, February 1998, p. 746-748, Vol. 180, No. 3
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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