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Journal of Bacteriology, May 1999, p. 3277-3280, Vol. 181, No. 10
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Histidinol Phosphate Phosphatase, Catalyzing the Penultimate Step
of the Histidine Biosynthesis Pathway, Is Encoded by
ytvP (hisJ) in Bacillus
subtilis
Dominique
le Coq,*
Sabine
Fillinger, and
Stéphane
Aymerich
Laboratoire de Génétique
Moléculaire et Cellulaire, Centre National de la Recherche
Scientifique ERS 567, Institut National de la Recherche
Agronomique, F-78850 Thiverval-Grignon, France
Received 5 November 1998/Accepted 23 February 1999
 |
ABSTRACT |
The deduced product of the Bacillus subtilis ytvP gene
is similar to that of ORF13, a gene of unknown function in
the Lactococcus lactis histidine biosynthesis operon. A
B. subtilis ytvP mutant was auxotrophic for histidine. The
only enzyme of the histidine biosynthesis pathway that remained
uncharacterized in B. subtilis was histidinol
phosphate phosphatase (HolPase), catalyzing the penultimate step of
this pathway. HolPase activity could not
be detected in crude extracts of the ytvP mutant, while
purified glutathione S-transferase-YtvP fusion
protein exhibited strong HolPase activity. These observations
demonstrated that HolPase is encoded by ytvP in
B. subtilis and led us to rename this gene hisJ. Together with the HolPase of Saccharomyces
cerevisiae and the presumed HolPases of
L. lactis and Schizosaccharomyces pombe, HisJ
constitutes a family of related enzymes that are not homologous to the
HolPases of Escherichia coli, Salmonella
typhimurium, and Haemophilus influenzae.
| |
TEXT |
In Bacillus subtilis, all
enzymes involved in the histidine biosynthesis pathway have been
characterized at the genetic level, except histidinol phosphate
phosphatase (HolPase) (10, 18). This enzyme
(L-histidinol-phosphate phosphohydrolase; EC 3.1.3.15) catalyzes dephosphorylation of histidinol phosphate to histidinol, the
direct precursor of histidine. In the well-studied prokaryotes Escherichia coli, Salmonella typhimurium, and
Haemophilus influenzae, HolPase activity is associated
with the N-terminal domain of the HisB bifunctional enzyme. The
C-terminal domain of HisB exhibits imidazoleglycerol phosphate
dehydratase activity (4, 5, 22). However, this does not
appear to be a common feature of these enzymes, as searches for
similarities with the E. coli HisB bifunctional enzyme
revealed the existence, in many organisms across all three kingdoms
(eubacteria, archaea, and eukaryotes), of imidazoleglycerol
phosphate dehydratases not fused to any other protein domain. For most
of these organisms, nothing is known about the HolPase. A search
for homologues to the N-terminal domain of the E. coli
enzyme does not reveal any well-conserved domain, except in the
S. typhimurium and H. influenzae bifunctional
enzymes, and only the more distantly related Streptomyces
lincolnensis LmbK protein has been associated with this family as
a putative HolPase (19). A HolPase has been
characterized at the genetic, physiological, and biochemical levels in
the yeast Saccharomyces cerevisiae (8, 14), but
its sequence has no similarity with the corresponding E. coli enzyme (12).
The complete genome of B. subtilis has been sequenced; among
the 4,100 open reading frames identified, about 70% (termed
y genes) correspond to putative genes for which the function
has not been ascertained (10). The goal of the B. subtilis systematic functional analysis program is to assign a
function to these genes. The applied strategy consists of constructing
a collection of mutants that correspond to each of these genes and
looking for a phenotype. We are currently testing whether the
utilization of various carbon sources is affected in these mutants.
In the work presented here, we demonstrate that the ytvP
gene encodes the B. subtilis HolPase, thus completing
our knowledge of the histidine biosynthesis pathway in this organism.
The B. subtilis ytvP mutant BFA1037 is devoid of
HolPase activity.
B. subtilis BFA1037
[trpC2 ytvP'::pBFA1037 (Eryr)] is a
ytvP mutant, obtained by single crossing-over
integration of plasmid pBFA1037 into the chromosome of the
reference strain 168 Marburg (trpC2). The integrative
plasmid pBFA1037 was derived from pMUTIN2mcs (21) by
insertion, between its unique HindIII and
BamHI sites, of a 218-bp
HindIII/BamHI ytvP internal
fragment amplified by PCR with B. subtilis 168 chromosomal
DNA as a template and primers ytvP-H
(5'-GCCGAAGCTTGGACAGCTTAGCTTAGCATACGG-3') and
ytvP-B (5'-CGCGGATCCACCGCTTCAATGCTTCC-3'). Correct integration of a single copy of plasmid pBFA1037
into the ytvP gene, confirmed by Southern blot analysis
(17), led to disruption of ytvP and created a
ytvp'-lacZ transcriptional fusion (Fig.
1).
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FIG. 1.
Structure of the ytvP chromosomal region of
mutant BFA1037. Integration of plasmid pBFA1037 led to disruption of
ytvP and created a ytvP'-lacZ transcriptional
fusion. The 218-bp ytvP internal fragment (symbolized by
hatching) used as integration platform is duplicated. In addition to
genes conferring resistance to ampicillin (amp) and
erythromycin (ery), plasmid pMUTIN2mcs and its pBFA
derivatives also carry a lacI gene and a
Pspac
isopropyl- -D-thiogalactopyranoside-inducible promoter
(broken arrow), which minimizes potential polar effects on expression
of genes downstream of the integration site (21). Genes are
symbolized by arrows indicating their orientation (not drawn to
scale).
|
|
During growth tests performed in chemically defined media, we observed
that strain BFA1037 was unable to grow in liquid MM medium
(1) containing succinate (15 mM) and glutamate (30 mM) as
carbon sources and supplemented with tryptophan (25 mg/liter), whereas the wild-type strain 168 could grow under these
conditions (Table 1). The same
observation was made with either glucose, glucitol, glycerol,
gluconate, asparagine, proline, or arginine as a carbon source, and in
all cases, growth of the ytvP mutant could be restored to
the wild-type level by adding Casamino Acids (data not shown). This
suggested that the mutant was auxotrophic for one or several amino
acids.
The ytvP gene (position 3030690 to 3031496 of the B. subtilis genome [10]) seems to be monocistronic.
It encodes a 268-amino-acid protein, which exhibits weak but
significant similarity (28% identity) to the product of
ORF13, the last gene of the Lactococcus lactis histidine biosynthesis operon (7) (Fig.
2). Although the function of this
L. lactis protein remains unknown, location of its
structural gene suggested that in B. subtilis YtvP could be
implicated in histidine biosynthesis. We observed that addition of
histidine could indeed restore the growth of strain BFA1037 to
wild-type level (Table 1). Growth could also be significantly
improved by the addition of histidinol, although not as efficiently
as with histidine (Table 1). These results showed that strain
BFA1037 is auxotrophic for histidine and suggested that it is affected in HolPase activity.
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FIG. 2.
Alignment of the B. subtilis YtvP (HisJ)
protein (BSUB; accession no. PIR:D70002), the S. cerevisiae
His9p HolPase (SCER; accession no. PIR:S56280), and the presumed
HolPases L. lactis ORF13 gene product (LLAC; accession
no. PIR:D47754) and S. pombe His9p homologue (SPOM;
accession no. SPTREMBLNEW:E1316727). Residues conserved (identity
and conservative change) in at least two of these proteins are against
a black background (residues conserved in YtvP) or a grey background
(residues not conserved in YtvP); groups of conservative change
considered are G and A; D and E; F and Y; I, L, V, and M; K and R; N
and Q; and S and T. Dashes indicate gaps introduced to maximize
alignment.
|
|
A 1,054-bp EcoRI/BamHI fragment carrying the
entire wild-type ytvP coding sequence and promoter region
was amplified by PCR with primers ptvpE
(5'-CCTGAATTCCAATGACAAACTCCATAATG-3') and
ftvpB (5'-CGCGGATCCGGACAGGAGACGTGG-3')
from B. subtilis strain 168 chromosomal DNA. This
fragment was cloned into plasmid pDG1662 and integrated into the
amyE locus of strain MO1099, following previously described methods (9). The amyE'::ytvP
chromosomal region of the resulting strain was then transferred into
BFA1037, which led to full complementation of the growth defect of this
strain (Table 1). This showed that the phenotype of BFA1037 was due to
the ytvP mutation.
We compared HolPase activity in extracts of the wild-type
strain 168 and of the mutant strain BFA1037. For this
purpose, B. subtilis cells were grown at 37°C with
shaking in liquid medium (MM containing succinate and glutamate plus
tryptophan, supplemented with histidine [50 mg/liter] when required),
harvested by centrifugation when the culture reached an
A600 of 0.5, and concentrated 10-fold in
triethanolamine-HCl buffer (0.1 M triethanolamine-HCl, 10 mM Na2EDTA [pH 7.5]). Glass beads (diameter, 0.10 to 0.11 mm; Braun Sciencetec) were added to the suspension (about 0.5 g/ml),
and the cells were broken by vigorous vortexing followed by sonication. After centrifugation, the supernatant was applied to a prepacked Sephadex G-50 column (Pharmacia, Uppsala, Sweden) pre-equilibrated with
triethanolamine-HCl buffer. The phosphate concentration of each
fraction was estimated with an ascorbate/ammonium molybdate color-developing reagent (13). Fractions containing
phosphate-free proteins were collected, and protein concentration was
determined by the Bradford method (3). HolPase activity
was assayed as previously described (13). No significant
HolPase activity (<0.4 U/mg of protein) could be detected in
extracts of the ytvP mutant strain BFA1037 grown in the
presence of histidine, whereas extracts of the wild-type strain grown
with and without histidine exhibited in both cases significant and
roughly equal levels of HolPase activity (6.4 and 6.9 U/mg of
protein, respectively).
From these results, we concluded that the ytvP gene encodes
either the B. subtilis HolPase or a positive regulator
of it.
ytvP encodes HolPase activity.
An 812-bp
BamHI/EcoRI fragment carrying the entire
ytvP sequence was amplified by PCR with primers 5'ytvP-Bam
(5'-CGCGGATCCATGCAAAAGCGAGACGGAC-3') and
3'ytvP-Eco (5'-CCGGAATTCTAGCACCGTTCCAAAAACG-3')
from B. subtilis 168 chromosomal DNA. This fragment
was cloned between the BamHI and EcoRI sites of
plasmid pGEX-2T (Pharmacia), generating pGST::YtvP, a plasmid
allowing overproduction of a glutathione S-transferase (GST)-YtvP fusion protein. Purification of GST and GST-YtvP proteins from E. coli TG1 [supE thi hsdD5
(lac-proAB) (F' traD36 proAB lacIq
ZM15)] transformed respectively with pGEX-2T and
pGST::YtvP was performed, following previously described
methods (20). As estimated by polyacrylamide gel
electrophoresis (11), the purity of both proteins was over
95%, and the apparent molecular mass of each purified product
corresponded to that expected (26 and 57 kDa for GST and GST-YtvP,
respectively) (results not shown). In vitro assays established tha the
GST-YtvP fusion protein possesses strong HolPase activity (840.0 U/mg of protein versus <0.1 U/mg of protein for GST), thus
demonstrating that in B. subtilis this activity is encoded
by ytvP. We therefore propose to rename this gene
hisJ.
As mentioned above, the protein most closely related to HisJ is the
product of ORF13 of the L. lactis his operon. The
structural gene encoding HolPase has not been identified in this
bacterium (18). It has been proposed that this activity
would be performed by the product of ORF8, based on its
homology with enzymes catalyzing phosphorylation of a hydroxyl group,
which is the opposite reaction to the dephosphorylation of such a group
as carried out by HolPase (7). From the work presented
here, it seems more likely that L. lactis HolPase
is encoded by ORF13. The B. subtilis HisJ protein is also related to the S. cerevisiae HolPase and its
Schizosaccharomyces pombe homologue, although more distantly
(24 and 23% identity, respectively) (Fig. 2). These four proteins seem
therefore to constitute a family of distantly related HolPases,
distinct from the E. coli-type HolPases. As recently
reported, this HisJ family belongs to a novel enzymatic superfamily
designated PHP, which also comprises DNA polymerase domains
(2). It has been suggested that these domains possess
intrinsic phosphatase activity that hydrolyzes the pyrophosphate
released during nucleotide polymerization. Among the PHP HolPases,
HolPase activity had been demonstrated only for His9p of S. cerevisiae. The PHP superfamily also comprises numerous proteins
of unknown function. While several of these seem to be clearly
related to DNA polymerases (e.g., the 570-amino-acid B. subtilis YshC protein), some, such as the 211-amino-acid
Archaeoglobus fulgidus AF1233 protein or the 246-amino-acid
Anaerocellum thermophilum YOR4 protein, might belong to the
His9p/HisJ HolPase family or even to other families of
phosphatases. Interestingly, the E. coli-type HolPases
belong to another superfamily of phosphohydrolases. This "DDDD"
family also includes several classes of nonspecific acid phosphatases,
phosphoglycolate phosphatases, phosphoserine phosphatases, and
trehalose-6-phosphatases, but no DNA polymerases (19). In
evolutionary terms, the existence of these two superfamilies could
reflect the existence of two different ancestral nonspecific phosphatases that have evolved through fusion to other domains having
specific properties, such as polymerase or binding of a particular substrate.
Identification of the hisJ HolPase structural gene in
B. subtilis completes our knowledge of the histidine
biosynthesis pathway in this bacterium. This also provides
strong evidence for the role of the L. lactis ORF13 gene and
will help with the identification of the functions of related gene
products in several other organisms.
Transcription of hisJ is repressed neither by histidine
nor by histidinol.
In L. lactis, the ORF13
gene belongs to the his operon, the
transcription of which is enhanced 15-fold in cultures grown in the
absence of histidine (6). The hisJ gene
characterized in this work does not belong to the B. subtilis
his operon and seems to be monocistronic. The two neighboring
genes (yttP and ytwP) are oriented in the
opposite direction (Fig. 1), and their inactivation does not confer
auxotrophy to histidine (data not shown). We did not observe any
significant difference in the level of HolPase activity in B. subtilis wild type grown in the absence or in the presence of 50 mg of histidine per liter (see above) or even 250 mg of histidine
per liter (data not shown). This suggested that neither HisJ enzymatic
activity nor hisJ transcription is regulated by this amino acid.
We also investigated potential regulation of hisJ
transcription in the mutant strain BFA1037, in which a hisJ
(ytvP)'-lacZ transcriptional fusion is under the
control of the hisJ promoter (Fig. 1). For these
-galactosidase assays, extracts were prepared and enzymatic activity
was determined as previously described (15, 16). The same
level of -galactosidase activity (about 20 to 25 Miller units/mg of
protein) was measured in extracts of the mutant grown in liquid MM
containing succinate and glutamate plus tryptophan, supplemented with
various concentrations of histidine or histidinol (-galactosidase
activity in extracts of the wild-type strain 168 grown in the same
conditions was <0.5 Miller units/mg of protein). In order to exclude
potential effects due to hyperaccumulation of histidinol phosphate (the
substrate for HolPase) in the mutant strain BFA1037, this
experiment was also performed with the complemented strain. As
mentioned above, this strain carries a wild-type hisJ gene
in the amyE ectopic locus and is prototrophic for histidine (Table 1). The same level of -galactosidase activity (about 15 to 20 Miller units/mg of protein) was measured in extracts of this strain
grown in liquid MM containing succinate and glutamate plus tryptophan
which was either not supplemented or supplemented with various
concentrations of histidine or histidinol (up to 1,250 mg/liter), or
even in rich medium (Luria broth). Thus, it appeared that transcription
of hisJ is not repressed by histidine or by histidinol.
| |
ACKNOWLEDGMENTS |
We thank Christine Delorme for helpful advice concerning the
HolPase assay, Matthieu Simon for technical assistance, and Josef Deutscher for comments on the manuscript.
This work was supported by funds from the Centre National de la
Recherche Scientifique and the Institut National de la Recherche Agronomique and was performed within the "Systematic Function Analysis of the Bacillus subtilis Genes" European Program
BIO4-CT95-0278.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
GénétiqueMoléculaire et Cellulaire, Centre National
de la Recherche Scientifique ERS 567, Institut National de la Recherche
Agronomique, F-78850 Thiverval-Grignon, France. Phone: (33)
1.30.81.54.45. Fax: (33) 1.30.81.54.57. E-mail:
lecoq{at}platon.grignon.inra.fr.
| |
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Journal of Bacteriology, May 1999, p. 3277-3280, Vol. 181, No. 10
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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