Department of Microbiology and Immunology,
The Medical School, University of Newcastle upon Tyne, Newcastle
upon Tyne, NE2 4HH, United Kingdom
YsxC is a member of a family of GTP-binding proteins carried by a
diverse range of organisms from bacteria to yeasts, plants, and humans.
To resolve the issue of whether ysxC of Bacillus
subtilis is essential for growth, we attempted to construct
mutants in which ysxC was either inactivated or placed
under the control of an inducible promoter. Viable mutants were
obtained only in the latter case, and these were inducer dependent,
demonstrating unambiguously that ysxC is an essential gene.
 |
TEXT |
GTP-binding proteins are frequently
involved in regulatory pathways as ubiquitous molecular switches
(6), operating at various stages of growth and life cycles.
The majority of these proteins are guanine nucleotide-binding proteins,
while others use GTP as a substrate for phosphorylation and/or
guanylation (10). In Bacillus subtilis,
GTP-binding proteins are involved in translation initiation and
elongation; cell division; protein secretion via the signal recognition
particle pathway; biosynthesis of flagella; the synthesis of
adenylosuccinate, pyrimidine, folic acid, and riboflavin; and the
oxidation of thiophene (7). The function of several small
putative GTP-binding proteins (e.g., Obg, Bex, EngA, YloQ, YsxC, and
YyaF) is, as yet, unknown. YloQ is essential for the growth of B. subtilis (2), while Bex has been shown to complement
the Escherichia coli essential gene era (EMBL
accession no. U18532, available at
http://www.embl-heidelberg.de/srs5/). Obg is essential
for both growth and sporulation of B. subtilis (18, 21) and is required for the activation of
B by stress (17).
The putative GTP-binding protein YsxC (also called OrfX
[13]) is encoded by a bicistronic operon that includes
lonA (Fig. 1A), which encodes
a cytosolic ATP-dependent serine endopeptidase (22).
ysxC is likely to be transcribed together with
lonA, since its start codon overlaps the lonA
coding sequence and no ysxC-specific promoter or
transcriptional initiation site has been detected. Furthermore,
lonA and ysxC showed similar transcription
patterns, including induction by heat and other stresses
(13).
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FIG. 1.
Construction of a fusion B. subtilis mutant
of ysxC. (A) Schematic representation of the ysxC
region of BFA2414 after integration of pYSXCF, which is a pMUTIN4-based
integration plasmid. Filled thick arrows indicate structural genes, and
putative  -independent terminators are shown as stem-loop structures.
Two promoters upstream of lonA
(PlonA) are marked with fine broken arrows.
Striped boxes show the tandem duplication of the RBS and 5' end of
ysxC. ysxC' is the 5' end of ysxC. Plasmid
pMUTIN4 is shown as a thick line. The lacZ reporter gene,
lacI, and ampicillin resistance (Apr) and
Emr genes are marked with fine arrows, and promoter
Pspac is marked with a fine broken arrow above that of
pMUTIN4. The region in pMUTIN4 used for replication in E. coli is labeled "ori," and three terminators
(t1t2t0) upstream of
Pspac are indicated as a stem-loop structure. The arrows
below the genes indicate the location and orientation of the primers,
while the dashed lines indicate their expected PCR products. The
positions of the primers specific for B. subtilis 168 in
respect to the entire genome (7, 11) are as follows: DS-REV,
2879355 to 2879372; FS-REV, 2879433 to 2879450; FS-FOR, 2879596 to
2879580; and US-FOR, 2879970 to 2879954. The 5' ends of the forward
(FOR) primers included a 10-bp linker with a HindIII
restriction site, while the reverse (REV) primers included a 9-bp
linker with a BamHI site. Positions of the primers specific
for pMUTIN4 were as follows: MUT-FOR, 147 to 165, and MUT-REV, 361 to
379. The numbers above the dashed lines correspond to the lanes of the
agarose electrophoresis gel shown in panel B. (B) Diagnostic PCR
confirming the correct integration of pYSXCF into ysxC in
mutant BFA2414. Lane 1, 100-bp ladder (Amersham Pharmacia Biotech Inc.,
Little Chalfont, United Kingdom). PCR was performed with BFA2414 (lanes
2 through 7) or B. subtilis 168 (lane 8) chromosomal DNA.
The primers (and expected product lengths) were as follows: lane 2, FS-FOR and DS-REV (261 bp); lane 3, MUT-FOR and DS-REV (399 bp); lane
4, US-FOR and FS-REV (557 bp); lane 5, US-FOR and MUT-REV (611 bp);
lane 6, MUT-FOR and MUT-REV (no PCR product expected since these
primers are oriented away from each other); lane 7, US-FOR and DS-REV
(no PCR product expected since the polymerization reaction time of
50 s is too short for the synthesis of the 9,409-bp fragment); and
lane 8, US-FOR and DS-REV (635 bp).
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|
The insertional inactivation of ysxC was previously reported
to have no effect on growth, nor did it result in any demonstrable phenotype (16). However, in a more recent genome-based
approach designed to identify essential genes in E. coli,
B. subtilis, and Saccharomyces cerevisiae,
ysxC of B. subtilis was reported to be essential
(2). In that study, which lacked experimental data, of six
genes of unknown function that were essential for the growth of
E. coli, five orthologs were essential for B. subtilis and one was essential for S. cerevisiae. Three
of the five essential B. subtilis genes, namely,
obg, yloQ, and ysxC, encode putative GTP-binding proteins, while yrrA (now called
trmU, SWISS-PROT accession no. O35020) encodes a
putative tRNA, (5-methylaminomethyl-2-thiouridylate)-methyltransferase, and ydiE encodes a putative metalloprotease (probably
o-sialoglycoprotein endopeptidase). These conserved
essential bacterial genes with nonessential orthologs in yeast
represent potential targets for novel broad-spectrum antimicrobial
agents (6).
Construction of integrational mutations in ysxC.
To
determine whether ysxC is essential for growth of B. subtilis 168, integrational mutants were constructed in which
ysxC was either inactivated (knockout mutant) or placed
under the control of a tightly regulated Pspac promoter
(23) (fusion mutant). In the case of the knockout mutant, a
303-bp internal fragment of ysxC (bp 2879356 to 2879054 [7]) was cloned into the integrational plasmid
pMUTIN4 (20), resulting in plasmid pYSXCK (Table
1). For the construction of the fusion
mutant, a 164-bp fragment from the 5' end of ysxC,
incorporating the ribosome binding site (RBS) and start codon of
ysxC, was also cloned into pMUTIN4, resulting in plasmid
pYSXCF (Table 1).
If ysxC is essential for the growth of B. subtilis 168, it should not be possible to isolate an
integrational mutant with pYSXCK, while integration of pYSXCF should
lead to isopropyl-
-D-thiogalactopyranoside (IPTG)-dependent growth. Repeated but unsuccessful attempts were made
to generate a pYSXCK-based knockout mutant, irrespective of the
presence of IPTG. In the case of the pYSXCF-based fusion mutant, about
100 erythromycin-resistant (Emr) and lincomycin-resistant
(Lmr) transformants per µg of pYSXCF DNA were
isolated in the presence of IPTG, while none were isolated in its
absence. These data indicate that ysxC is essential for
growth. The authenticity of the integration event in this
mutant (BFA2414) (Fig. 1A) was confirmed by PCR (Fig. 1B).
To ensure tight regulation of the Pspac promoter,
BFA2414 was transformed with plasmid p65, which provides multiple
copies of the E. coli lacI gene.
Expression of ysxC is essential for growth.
The
IPTG dependence of BFA2414(p65) was confirmed by growing the organism
to exponential phase (optical density at 600 nm [OD600] = ~0.3) in Luria-Bertani (LB) medium (15) containing 1 mM
IPTG and 0.3 µg of erythromycin and 10 µg of kanamycin per ml. The
cells were washed twice with prewarmed LB medium and diluted 10
5-fold into prewarmed LB media containing erythromycin,
kanamycin, and a range of concentrations of IPTG from 0 to 1 mM. As
shown in Fig. 2, growth was eventually
observed in each of the cultures. However, the time at which growth was
first observed was increasingly delayed with decreasing IPTG
concentrations. In the case of the culture with 0.1 mM IPTG, growth was
delayed by approximately 2 h with respect to the culture
containing 1 mM IPTG, while growth of the culture with no added IPTG
was delayed by more than 20 h. During exponential phase, the mean
generation times of cultures with smaller amounts (
0.1 mM) or no IPTG
was increased to ~50 min, compared to ~30 min for the culture with
1 mM IPTG.
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FIG. 2.
OD600 of BFA2414(p65) in LB media with the
following concentrations of IPTG: 1 mM ( ), 0.1 mM ( ), 0.01 mM
( ), 0.001 mM ( ), 0.0001 mM ( ), and 0 mM ( ).
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The transcription of ysxC was monitored (9) by
fusing the spoVG-lacZ reporter gene of pMUTIN4 to its native
promoter (Fig. 1A). Irrespective of the IPTG concentration,
-galactosidase production increased during exponential phase,
reaching a peak of ca. 22 nmol of ONP/min/OD600 unit at or
about the transition between exponential and stationary growth phase;
thereafter, the values declined.
To determine whether the delayed growth and decreased growth rate of
BFA2414(p65) at the lower (i.e., 0.1 mM) IPTG concentrations (Fig. 2)
were due to overgrowth by suppressor mutants, stationary-phase samples
were plated to determine the ratio of the IPTG-independent colonies
(putative suppressor mutants) to total CFU. There was a marked
difference in the plating efficiencies of the various cultures. In the
case of the culture with 1 mM IPTG, the plating efficiency was
(4.9 ± 1.2) × 107. The number of the
IPTG-independent colonies increased as the IPTG concentration
decreased, and cultures with 0.001 mM IPTG or less exhibited a plating
efficiency close to 1. These data suggest that the growth in the latter
cultures was due to the accumulation of derivatives with suppressor mutations.
To determine the location of putative suppressor mutation(s),
chromosomal DNA from IPTG-independent colonies [BFA2414SUP(p65)] was
used to transform B. subtilis 168. In each case, similar
numbers (ca. 103/µg of DNA) of Emr
Lmr transformants were observed, irrespective of the
presence of IPTG. In contrast, when chromosomal DNA from the
IPTG-dependent BFA2414 was used, transformants (ca. 9 × 102/µg of DNA) were obtained only in the presence of
IPTG. These results suggested that the observed suppression was linked
to the integrated pYSXCF.
One possibility was that the suppressor mutation(s) occurred in the
"oid" lac operator (14) of the
Pspac promoter, leading to its constitutive expression. The
oid, or ideal, lac operator has perfect symmetry and a
10-fold-higher affinity for the Lac repressor than the native
lac operator. Consequently, the native lac
operator associated with the Pspac promoter of
integrational vector pMUTIN2 was replaced by the oid operator in
pMUTIN4 to reduce the noninduced level of expression of this promoter
(20). The Pspac promoter regions from several
IPTG-independent BFA2414SUP(p65) mutants were PCR amplified using
primers MUT-FOR and DS-REV (Fig. 1A). Sequencing of the PCR products
revealed that all of the BFA2414SUP(p65) mutants contained a single
C
T transition at nucleotide 10 of the oid lac operator.
An identical base pair substitution at the same nucleotide of the
native lac operator has been shown to decrease its affinity
for the Lac repressor by 96% and to generate a constitutive phenotype
(3). We therefore concluded that the observed
IPTG-independent growth is due to the selection of clones with a
mutation in the integrated pYSXCF that severely reduces the capacity of
the lac operator upstream of the functional
ysxC gene to bind the lactose repressor. Since a single
spontaneous mutation in the lac operator of pMUTIN4 can
result in the loss of the IPTG dependence of target gene expression,
this may lead to an underestimation of the number of essential genes
when a gene fusion rather than a gene knockout strategy is initially
used to isolate such mutants. Our results indicated that
appropriate care needs to be taken when selective pressure is
applied to this controllable promoter system.
YsxC protein family.
YsxC is a member of a family of small
GTP-binding proteins that are carried by a diverse range of organisms
from bacteria to yeast, plants, and humans. An analysis of 29 members
of the YsxC protein family (Fig. 3) shows
that they can be classified into four distinct phylogenetic
groups. Group I includes YsxC orthologs from gram-negative
bacteria and Mycoplasma species, group II includes orthologs
from gram-positive bacteria (e.g., Bacillus,
Clostridium, and Staphylococcus), group III
includes orthologs from the Archaea, and group IV includes
orthologs from S. cerevisiae, Homo sapiens,
Arabidopsis thaliana, and, interestingly, two
hyperthermophilic bacteria, Aquifex aeolicus and
Thermotoga maritima.
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FIG. 3.
Radial phylogenetic tree of the YsxC protein
family. Multiple-sequence alignment and phylogenetic analysis were
performed using the web site of the Institut National de la Recherche
Agronomique (http://www.toulouse.inra.fr/multalin.html; see reference
4). The symbol comparison table was Blosum62, the
gap weight was 12, and the gap length weight was 2. The root of the
tree is marked with a triangle. Proteins with SWISS-PROT accession
numbers (in parentheses) are as follows: B. subtilis YsxC
(P38424), E. coli YihA (P24253), Haemophilus
influenzae HI1118 (P46453), Helicobacter pylori HP1567
(O26087), Mycoplasma genitalium MG335 (P47577), M. pneumoniae MP359 (P75303), Methanococcus jannaschii
MJ0320 (Q57768), Rickettsia prowazekii RP102 (Q9ZE46),
A. aeolicus AQ_1815 (O67679), and Caulobacter
crescentus CgpA (Q9ZG89). Proteins with GenBank accession number
are as follows: Archaeoglobus fulgidus AF1326 (AAB89919),
P. horikoshii PH0200 (BAA29269), T. maritima
TM1466 (AAD36534), L. lactis (AAF63739), Neisseria
meningitidis NMB1806 (AAF42143), Zymomonas mobilis CgpA
(AAD56911), S. cerevisiae Ydr336wp (AAB64772), H. sapiens HSPC135 (AAF29099), A. thaliana 219 is
At2g22870 (AAC32434) with a length of 219 amino acid residues, and
A. thaliana 318 is F15N18_70 (CAB87708) with a length of 318 amino acid residues. YsxC orthologs of B. anthracis,
B. stearothermophilus, C. acetobutylicum, C. difficile, E. faecalis, S. aureus, S. pneumoniae, and
S. pyogenes were obtained from unfinished genome sequencing
projects at TIGR, the Sanger Centre, Genome Therapeutics Corporation,
and the University of Oklahoma. In B. stearothermophilus,
the 130 amino acids at the N terminus of the YsxC homolog were deduced
from sequence contig 552. In B. brevis, the 162 amino acids
at the N terminus were deduced from the GenBank sequence (D00863) after
inserting one nucleotide between positions 2745 and 2746 (16). PAM, percent accepted mutation.
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In group II organisms, ysxC is located downstream of
lonA or clpX, both of which code for class III
ATP-dependent heat shock proteases. In the case of Bacillus
and Brevibacillus, ysxC is located downstream of
lonA, and its start codon overlaps the 3' end of the
lonA coding sequence by one nucleotide. In Clostridium difficile and Clostridium acetobutylicum, the coding
sequence of ysxC overlaps the 3' end of lonA by
eight nucleotides. In Streptococcus spp., Enterococcus
faecalis, Staphylococcus aureus, and Lactococcus lactis, ysxC is in a putative operon with
clpX. In L. lactis the start codon of
ysxC overlaps the 3' end of the clpX coding
sequence by one nucleotide. In the other three phylogenetic groups (I, III, and IV), lonA or clpX is located at a distal
site on the chromosome with respect to ysxC. Linkage between
an ATP-dependent protease and ysxC was also observed in
Pyrococcus horikoshii from group III, in which the
ysxC ortholog, PH0200, is located 59 bp downstream of a gene
encoding a putative regulatory subunit of the ATP-dependent 26S
protease. In addition to YsxC orthologs in eukaryotes, two
bacterial homologs from A. aeolicus and T. maritima belong to group IV. A. aeolicus is one
of the earliest diverging and most thermophilic bacteria known, and as
a chemolithoautotroph, it can grow on hydrogen, oxygen, carbon
dioxide, and mineral salts (5). T. maritima is
one of the deepest and most slowly evolving lineages in the
Eubacteria. Although the core of T. maritima may be eubacterial, almost one quarter of the genome is archaeal in nature
(12). A. thaliana has at least two homologs of
YsxC, one 219 and the other 318 amino acid residues in length, encoded on chromosomes II and V, respectively (8). The length of the smaller homolog is in the range of prokaryotic YsxC proteins (190 to
219 amino acids). The length of the longer YsxC homolog is similar to
that of the YsxC orthologs from S. cerevisiae and H. sapiens. With respect to YsxC of B. subtilis, these
proteins have a 100- to 110-amino-acid extension at their amino
terminus. Their N termini, which are highly conserved in S. cerevisiae and H. sapiens but not in A. thaliana, showed no homology to any other bacterial and archaeal
proteins. In the case of A. thaliana, the N terminus
contained a putative transmembrane helix (19) between amino
acid residues 11 and 29 and the protein is currently the only putative
membrane-bound member of the YsxC family.
No homologs of ysxC were observed on the complete genome
sequences of Borrelia burgdorferi, Chlamydia
trachomatis, Chlamydia pneumoniae, Deinococcus
radiodurans, Mycobacterium tubercolosis, and
Treponema pallidum using databases at The Institute for
Genomic Research (TIGR; http://www.tigr.org/tdb) and the Pasteur
Institute (http://genolist.pasteur.fr).
An alignment of YsxC and 28 homologs (data not shown) revealed four
regions of conservation: (i) (G/R)X(S/T)N(V/A)GKS(S/T), a putative
GTP-binding motif located toward the amino terminus; (ii) PGXTXXX(N/I),
located 15 to 23 residues downstream of the first region; (iii)
DXPG(Y/F)G(Y/F), a second putative GTP-binding motif located 10 to 20 residues downstream of the second region; and (iv) KXDK, located 56 to
74 residues downstream of the third motif. The second motif is shorter
in S. cerevisiae (GXTXXXN), and only the threonine residue
is conserved in the case of H. sapiens.
We were not able to generate a viable mutant of ysxC with
pYSXCK, which, after integration into the B. subtilis
chromosome, generates a strain carrying a YsxC protein that is
truncated at its C terminus by just 23 amino acids. Since this protein
includes the four conserved motifs described above, this indicates that the highly charged (5 K residues, 2 E residues, 1 D residue, 1 R
residue, and a serine dyad) C terminus is essential for function.
We thank S. D. Ehrlich for the gift of plasmids pMUTIN4 and
p65. We acknowledge the release of preliminary sequence data for the
YsxC orthologs in Bacillus anthracis, Bacillus
stearothermophilus, C. acetobutylicum, C. difficile, E. faecalis, S. aureus,
Streptococcus pneumoniae, and Streptococcus
pyogenes, which were obtained using the TIGR website
(http://www.tigr.org) from unfinished genome sequencing projects at
TIGR, the Sanger Centre, Genome Therapeutics Corporation, and the
University of Oklahoma.
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