Download Analysis of non‐polar deletion mutations in the genes of the spo0K

FEMS Microbiology Letters 153 (1997) 63^69
Analysis of non-polar deletion mutations in the genes of the spo0K
(opp) operon of Bacillus subtilis
John R. LeDeaux 1, Jonathan M. Solomon 2, Alan D. Grossman*
Department of Biology, Building 68-530, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Received 8 April 1997; revised 13 May 1997; accepted 13 May 1997
Abstract
The spo0K (opp) operon of Bacillus subtilis encodes an oligopeptide permease that is required for uptake of oligopeptides,
development of genetic competence, and initiation of sporulation. We made in-frame, non-polar deletion mutations in each of
the first four genes of the five-gene spo0K operon and tested effects on oligopeptide transport, sporulation, and expression of
competence genes. spo0KA, B, C, and D were required for sporulation, competence development, and oligopeptide transport.
Disruption of spo0KE caused a less severe phenotype than did disruption of any of the other genes of the operon.
Keywords :
Deletion mutant; spo0K operon; Bacillus subtilis
1. Introduction
The spo0K (opp) operon of Bacillus subtilis (Fig. 1)
encodes an oligopeptide permease [1,2] that is required for the initiation of sporulation and the development of genetic competence (reviewed in [3,4]).
Spo0K is a member of the large family of ATP-binding cassette (ABC) transporters that are involved in
the import or export of a wide variety of compounds
* Corresponding author. Tel.: +1 (617) 253-1515;
Fax: +1 (617) 253-8699; E-mail: [email protected]
1
Present address: USDA-ARS, North Carolina State
University, Department of Genetics, P.O. Box 7614, Raleigh,
NC 27695-7614, USA.
2
Present address: Department of Molecular Biology and
Microbiology, Tufts University School of Medicine,
136 Harrison Ave., Boston, MA 02111, USA.
in many di¡erent organisms [5]. The Spo0K oligopeptide permease in B. subtilis can import oligopeptides from 3^5 amino acids with apparently little, if
any, speci¢city, and is required for cells to utilize
oligopeptides as a source of amino acids (for example, see [6,7]). The function of each of the ¢ve gene
products of the spo0K operon is inferred based on
predicted protein sequence and analogy to many
ABC transporters [5]. Spo0KA is the ligand-binding
protein and is attached to the outside of the cell by a
lipid anchor [1]; Spo0KB and Spo0KC are membrane proteins that form a complex through which
the ligand is transported; Spo0KD and Spo0KE are
the ATPases that are thought to provide energy for
transport [1,2].
Previous work had shown that a variety of insertion mutations in each of the ¢rst four genes of the
operon cause defects in oligopeptide transport, competence, and sporulation [1,2,6,8], and that the de-
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 2 3 2 - 2
J.R. LeDeaux et al. / FEMS Microbiology Letters 153 (1997) 63^69
64
fects are similar to those caused by deletion of the
entire
operon.
All
mutations
reported
2. Materials and methods
(trans-
poson insertion, integrative plasmid), except for a
Strains are listed in Table 1 and plasmids in Table
spo0KD null [1], are polar on downstream genes,
2 and Fig. 1. Molecular biological procedures were
making it di¤cult to evaluate the necessity of each
according to standard protocols [9]. Cell growth,
of the genes individually. Null mutations in spo0KD
sporulation, and
cause
viously described [2,7,10,11].
a
phenotype
similar
to
that
caused
by
L-galactosidase
assays were as pre-
polar mutations in the upstream genes [1,2]. In contrast null mutations in spo0KE (the last gene of the
operon, Fig. 1) cause a much less severe phenotype
3. Results and discussion
[1,2].
To determine the relative contribution of each
3.1. Construction of non-polar deletion mutations
spo0K cistron to the phenotypes associated with
null alleles of the entire operon, we made in-frame,
To make each non-polar deletion, we ¢rst made a
non-polar deletion mutations in each of the ¢rst four
plasmid that contained an in-frame deletion in the
genes
1).
gene of interest within sequences that overlapped
Mutants were then tested for oligopeptide trans-
either the 5P or the 3P end of the operon (see Fig. 1
port,
and Table 2). Each deletion plasmid was integrated
genes.
in
the
operon
sporulation,
and
(Tables
1
and
expression
of
2;
Fig.
competence
into the spo0K locus in the chromosome by single
Fig. 1. Map of spo0K region and plasmids used to make spo0K mutations. Restriction site abbreviations : A, ApaI ; Bg, BglII ; C, ClaI ;
E, EcoRI ; Es, EspI ; N, NruI ; Nc, NcoI ; P, PvuII ; R, EcoRV ; Rs, RsrII ; S, SphI ; Sn, SnaBI ; Sp, SplI ; X, XcmI. The location of the
likely promoter site of spo0K is indicated by an arrow and P. The location of the Tn917lac [2] insertion is indicated by a triangle and Tn.
All clones are in the vector pJH101, except pJL28 (vector pDR63), pJL32 and pJL79 (vector pDR67).
65
J.R. LeDeaux et al. / FEMS Microbiology Letters 153 (1997) 63^69
crossover, selecting for plasmid-encoded chloramphenicol resistance.
Integration of the three plasmids that extend past
the 3P end of the operon, pJL34 for vspo0KC, pJL9
for vspo0KD, and pJL19 for vspo0KDE (Fig. 1),
gave rise to both Spo3 and Spo‡ transformants, indicating that these three deletions did indeed cause a
Spo3 phenotype. For these three plasmids, integration upstream of the deletion endpoint would place
the mutant gene under control of the operon promoter and separate the wild-type gene from the upstream promoter (Spo3 ). Integration downstream of
the deletion endpoint would leave the wild-type operon intact (Spo‡ ).
For plasmids with inserts that extend past the 5P
end of the operon, pJL18 for vspo0KA and pJL29
for vspo0KB (Fig. 1), all single crossover events
should result in transformants that are phenotypically Spo‡ , whether the integration occurred upstream or downstream of the deletion endpoints.
However, Spo3 transformants could result from
gene conversion events that replaced the wild-type
copy of the gene with the mutant allele from the
plasmid. For both vspo0KA (pJL18) and vspo0KB
Table 1
B. subtilis strains used
Strain
Genotypea
JH642
trpC2 pheA1
KI418
spo0KA : :Tn917lac
JRL43
JRL131
JRL135
JRL179
JRL189
JRL221
Commentsb or reference
vspo0KD43
vspo0KA131
vspo0KDE135
vspo0KA amyE : :(spo0KA‡
vspo0KB189
vspo0KE221 : : neo
[2]
JH642 trf to
JH642 trf to
JH642 trf to
cat)
vspo0KD with pJL9
vspo0KA with pJL18
vspo0KDE with pJL19
JRL131 trf to CmR with pJL28
JH642 trf to
vspo0KB
JRL275
amyE : :(comG-lacZ neo)
a.k.a. AG1046 [10]
JRL293
amyE : :(srfA-lacZ
[10,11]
61974 cat)
JRL358
vspo0KC322
vspo0K357 : : neo
vspo0K358 : : erm
JRL476
amyE : :(srfA-lacZ
JRL322
JRL357
JRL485
JRL486
JRL488
JRL489
JRL492
JRL493
JRL494
JRL584
JRL682
JRL685
JRL1082
JRL1102
JRL1103
JRL1104
JRL1105
JRL1114
JRL1115
JRL1117
JRL1118
with pJL29
JH642 trf to NeoR with pJL42
JH642 trf to
vspo0KC
with pJL34
[8]
61974 cat : :spc)
vspo0KA amyE : :(comG-lacZ neo)
vspo0KA amyE : :(srfA-lacZ spc)
vspo0KD amyE : :(comG-lacZ neo)
vspo0KD amyE : :(srfA-lacZ spc)
vspo0KDE amyE : :(srfA-lacZ spc)
vspo0K : : erm amyE : :(comG-lacZ neo)
vspo0K : : erm amyE : :(srfA-lacZ spc)
vspo0KE584 : : spc
vspo0KE : : spc amyE : :(comG-lacZ neo)
vspo0KE : : spc amyE : :(srfA-lacZ cat)
vspo0KC amyE : :(srfA-lacZ cat)
vspo0KB amyE : :(comG-lacZ neo)
vspo0KC amyE : :(comG-lacZ neo)
vspo0KDE amyE : :(comG-lacZ neo)
vspo0KB amyE : :(srfA-lacZ cat)
vspo0KB amyE : :(Pspac-spo0KB cat)
vspo0KB amyE : :(Pspac-spo0KBC cat)
vspo0KC amyE : :(Pspac-spo0KB cat)
vspo0KB amyE : :(Pspac-spo0KBC cat)
[8]
JRL293 converted to SpcR CmS with pJL62
JRL131 trf to NeoR
JRL131 trf to SpcR
JRL43 trf to NeoR
JRL43 trf to SpcR
JRL135 trf to SpcR
JRL358 trf to NeoR
JRL358 trf to SpcR
JH642 trf to SpcR with pJL75
JRL275 trf to SpcR
JRL293 trf to SpcR
JRL322 trf to CmR
JRL189 trf to NeoR
JRL322 trf to NeoR
JRL135 trf to NeoR
JRL189 trf to CmR
JRL489 trf to CmR with pJL79
JRL189 trf to CmR with pJL32
JRL322 trf to CmR with pJL79
JRL322 trf to CmR with pJL32
a
All strains are derived from JH642 and have the trp and phe mutations.
b
trf indicates transformation selecting for the indicated antibiotic resistance. Cm, Neo, and Spc refer to resistance to chloramphenicol,
neomycin, and spectinomycin, respectively.
J.R. LeDeaux et al. / FEMS Microbiology Letters 153 (1997) 63^69
66
Table 2
Plasmids
Plasmid
a
Comments, source, or reference
pAG58
Ap Cmp
JH101
Ap Tc Cm ; integrative vector
pGEMcat
Ap Cm ; integrative vector
pUC18erm
Ap MLS ; source of erm cassette
pBEST501
Ap Neo ; source of neo cassette
pUS19
pJL62
Ap Spc ; integrative vector, source of spc cassette
R
S
S
R
Ap Spc ; used to convert Cm Spc strains to Cm Spc [8]
pDR63
Ap Cm ; vector used to recombine fragments into the chromosome at amyE by double crossover, derived from
pDH32 [18] by deleting most of lacZ (from BamHI to BssHII) and deleting several restriction sites (from DraIII to SphI)
pDR67
Ap Cm ; vector used to place fragments under control of the IPTG-inducible promoter Pspac and recombine into the
pDR9
Ap Cm ; clone of spo0KB-E in pJH101 [2]
pDR18
Ap Cm ; clone of spo0K promoter region from PvuII site upstream of the promoter to EcoRI site in spo0KA, cloned
chromosome at amyE by double crossover [19]
into pJH101 [2]
a
Ap, Tc, Cm, Neo, Spc, MLS refer to resistance to ampicillin, tetracycline, chloramphenicol, neomycin, spectinomycin, and erythromycin +
lincomycin, respectively. Primary references for each of the ¢rst six plasmids are cited in [8].
3
‡
(pJL29), some transformants were Spo , indicating
(Fig. 1). The partial diploid was phenotypically Spo
that spo0KA and spo0KB are both required for spor-
as
ulation.
plates. A complementation test was also performed
Gene conversion events probably occurred in the
judged
on the
by
colony
vspo0KB
and
phenotype
vspo0KC
on
sporulation
mutations to deter-
transformants containing the plasmids that extend
mine if they were polar on downstream genes. Two
past
pJL19,
plasmids were made, one that had only spo0KB
pJL34). However, these would be phenotypically in-
under control of the IPTG-inducible promoter Pspac
distinguishable from recombinants that were the re-
(pJL79) and one that had both spo0KB and spo0KC
sult of a single crossover.
under the control of Pspac (pJL32). Both of the con-
the
3P
end
of
the
operon
(pJL9,
To make strains that were missing plasmid sequen-
structs were put at the nonessential amyE locus in
vspo0KB
vspo0KC strains. Both convspo0KB strain in an IPTG-deindicating that the vspo0KB dele-
ces and had a single mutant allele, we screened for a
both the
second recombination event that excised the plasmid
structs rescued the
and left behind the deletion mutation. One or more
pendent manner,
‡
and
recombinants from the transformation of each
tion is not polar on the downstream genes. In the
plasmid was isolated and grown under non-selective
presence of IPTG, the Pspac-spo0KB construct did
Spo
vspo0KC,
but the Pspac-spo0KBC
conditions (i.e. in the absence of chloramphenicol),
not rescue the
and plated on non-selective sporulation plates. Spo
construct did, indicating that the
colonies were chosen and then tested for sensitivity
was not polar on spo0KD and spo0KE, and that two
3
to chloramphenicol to indicate loss of the plasmid.
Strains
JRL135
JRL322
JRL43
v
( spo0KD),
v
(vspo0KC)
( spo0KDE),
JRL131
JRL189
v
v
( spo0KA),
( spo0KB),
and
were made in this way. The de-
letions in spo0KA, spo0KB, spo0KD, and spo0KDE
vspo0KC
deletion
copies of spo0KB could not substitute for a loss of
spo0KC. The
vspo0KD
deletion is not completely
polar on spo0KE because the
vspo0KD allele causes
vspo0KDE allele
a less severe phenotype than the
(see below).
were veri¢ed by PCR analysis (data not shown).
None of the deletions in the ¢rst four genes of the
3.2. Mutant phenotypes
spo0K operon were polar on downstream genes. A
complementation test was performed on the
vspo0-
We measured the e¡ects of the various spo0K mu-
KA mutation by putting spo0KA‡ (under control of
tations on transport (resistance to the toxic tripep-
the spo0K promoter) at the amyE locus using pJL28
tide bialophos), sporulation, and competence. E¡ects
J.R. LeDeaux et al. / FEMS Microbiology Letters 153 (1997) 63^69
67
Fig. 2. Expression of srfA-lacZ and comG-lacZ in di¡erent spo0K mutants. Strains were grown in S7 minimal medium with 1% glucose
and 0.1% glutamate and amino acids (40^50 Wg/ml) for auxotrophic requirements. Note the di¡erent scales on the y-axis. A: srfA-lacZ.
Wild-type (JRL293), ¢lled circles; vspo0KE : : spc (JRL685), open squares; vspo0KABCD : : erm (JRL494), open triangles. B: srfA-lacZ.
vspo0KD (JRL489), ¢lled diamonds; vspo0KA (JRL486), ¢lled triangles; vspo0KB (JRL1105), open diamonds; vspo0KC (JRL1082), open
circles; vspo0KDE (JRL492), ¢lled squares; vspo0KABCD : : erm (JRL494), open triangles (same data as in A). C: comG-lacZ. wild-type
(JRL275), ¢lled circles; vspo0KE : : spc, (JRL682), open squares; vspo0KABCD : : erm (JRL493), open triangles. D: comG-lacZ. vspo0KD
(JRL488), ¢lled diamonds; vspo0KA (JRL485), ¢lled triangles; vspo0KB (JRL1102), open diamonds; vspo0KC (JRL1103), open circles;
vspo0KDE (JRL1104), ¢lled squares; vspo0KABCD : : erm (JRL493), open triangles (same data as in C).
on competence were analyzed by measuring expression of srfA-lacZ and comG-lacZ. comG encodes
components required for DNA uptake and is expressed only in the sub-population of cells that actually develops competence ([3,4,12], and references
therein). Expression of comG (and other late com
genes) is an excellent indicator of competence development per se [13]. Expression of a small open reading frame (comS) in the srfA operon is required for
expression of the late com genes (comG) and development of competence [14,15]. srfA is expressed in virtually all of the cells, not just the sub-population
68
J.R. LeDeaux et al. / FEMS Microbiology Letters 153 (1997) 63^69
destined to become competent [12]. Its expression
depends, in part, on spo0K [10,11,16] and two peptide pheromones that accumulate in culture medium
as cells grow to high density [7,10,11].
3.2.1. spo0KA^D
The vspo0KA, vspo0KB, and vspo0KC non-polar
deletions and the vspo0KDE deletion caused phenotypes that were indistinguishable from those caused
by a vspo0KA^D : : erm mutation. The sporulation
frequency (Table 3), expression of srfA-lacZ (Fig.
2A,B) and comG-lacZ (Fig. 2C,D) were similar in
all of the mutants. In addition, transport of oligopeptides was defective in the mutants as all were
fully resistant to 50 Wg/ml of bialophos. vspo0KD
is virtually indistinguishable from spo0K null mutants in terms of sporulation and bialophos resistance consistent with results previously reported [1].
However, the vspo0KD mutation had a slightly less
severe e¡ect on expression of srfA-lacZ (Fig. 2B) and
comG-lacZ (Fig. 2D) than the other spo0K deletion
mutations.
3.2.2. spo0KE
In contrast to mutations in the rest of the operon,
null mutations cause only a slight defect in
sporulation (Table 3) [1,2].
A spo0KE insertion mutation was previously reported to cause a Com3 phenotype, where the competence of the cells was measured at a single time
point during growth [2]. A more extensive analysis
of competence gene expression and transformation
frequency at di¡erent time points in the growth
spo0KE
Table 3
Sporulation frequency of di¡erent
spo0K
curve indicated that spo0KE mutants have delayed
and somewhat reduced expression of srfA-lacZ
(Fig. 2A), comG-lacZ (Fig. 2B) and transformation
(data not shown).
As previously reported [1], spo0KE mutants are
able to transport peptides, as indicated by sensitivity
to bialophos. However, we found that the spo0KE
mutants were resistant to very low concentrations of
bialophos (0.02^0.1 Wg/ml) while wild-type cells were
sensitive to these concentrations. These results indicate that oligopeptide transport is slightly impaired
in the spo0KE mutants.
3.3. ATPases of ABC transporters
ABC transporters generally require two ATPase
domains or subunits [5]. Some importers have two
di¡erent ATPases (Opp), while others have two subunits of a single ATPase (His, Mal). We suspect that
in the spo0KE mutant another ATPase is substituting
for the missing subunit; perhaps a second subunit of
Spo0KD replaces Spo0KE. Alternatively, an ATPase
from a di¡erent transport system might substitute
for the Spo0KE ATPase. Preliminary experiments
indicate that the ATPases from app, which encodes
a cryptic oligopeptide permease similar to Spo0K [6],
are not substituting for Spo0KE.
There is precedence for one part of an ABC transporter to work with another ABC transporter. The
ligand-binding protein of the dipeptide permease
Dpp can work with the membrane proteins of
Spo0K to transport dipeptides: a mutation in dpp
which is not polar on the gene encoding the ligand-
mutants in DS medium
Strain
Genotype
Spores/mla
Percent sporulation
Frequency relative to wild-type
JH642
JRL131
JRL189
JRL322
JRL43
JRL221
JRL135
KI418
JRL358
WT
vspo0KA
vspo0KB
vspo0KC
vspo0KD
vspo0KE : : neo
vspo0KDE
spo0KA : :Tn917
vspo0K : : erm
2.4
2.5
2.3
2.5
1.5
2.9
7.3
5.9
3.7
51
1
0.9
0.8
0.6
11
0.25
0.24
1.3
1
2.0
1.8
1.6
1.1
2.1
5.0
4.8
2.5
U10
U10
U10
U10
U10
U10
U10
U10
U10
8
6
6
6
6
7
5
5
6
U1033
U103
U103
U103
U10
U1033
U103
U10
2
2
2
2
1
3
3
2
a
Data shown are from a single representative experiment, and similar results were obtained in at least three independent experiments. The
sporulation frequencies of the spo0K mutants (except spo0KE) were typically 0.5^3% of wild-type. The sporulation frequencies of the spo0KE
mutants were typically 20^50% of wild-type. The number of viable cells/ml ranged from 2.1 108 to 4.7 108 .
U
U
J.R. LeDeaux et al. / FEMS Microbiology Letters 153 (1997) 63^69
binding protein of Dpp will still allow transport of
dipeptides into the cell, but this transport is dependent on spo0K (F. Slack and A.L. Sonenshein, personal communication). Another example more pertinent
to these results is the ability of UgpC, the single
ATP-binding protein of the sn-glycerol-3-phosphate
ABC transporter Ugp, to substitute for MalK, the
single ATP-binding protein of the maltose ABC
transporter [17] and the ability of MalK to substitute
for UgpC [17]. The potential for mixing subunits
encoded by di¡erent transport operons increases
the possible variation in transport complexes. Transporters that were previously thought to be homogeneous might actually exist in di¡erent forms, each
with a potentially di¡erent function.
Acknowledgments
Bialophos was a generous gift from Meiji Seika
Kaisha, Ltd., Pharmaceutical Research Center, Yokohama, Japan. We are grateful to F. Slack and
A.L. Sonenshein for communicating unpublished results, and to members of our lab for useful discussions. J.M.S. was supported in part by a Howard
Hughes predoctoral fellowship. A.D.G. was a Lucille
P. Markey Scholar in Biomedical Sciences and this
work was supported in part by a grant from the
Lucille P. Markey Charitable Trust, and Public
Health Services Grants GM41934 and GM50895 to
A.D.G. from the NIH.
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