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Http://www.abbs.info e-mail:[email protected] ISSN 0582-9879
ACTA BIOCHIMICA et BIOPHYSICA SINICA 2001, 33(6):
607-614
CN 31-1300/Q |
Isolation
and Analysis of hupR Gene Required for the Expression of Hydrogenase in Rhodobacter
sphaeroides
( Institute of Plant Physiology and Ecology,
Shanghai Institute for Biological Science,
Liang
et al had screened the cosmid 1 containing hup cluster from the
gene library of R.sphaeroides in our lab. Complementary results showed
that it could restore the activity of H2 uptake hydrogenase of HupR-
mutant of R.capsulatus[7]. Here we report the cloning and
sequencing of the regulatory hupR gene, and demonstrate that R.sphaeroides
HupR activates the synthesis of hydrogenase in R.sphaeroides.
1.1
Material
Bacterial
strains and plasmids used are listed in Table 1.
1.2
Methods
1.2.1 Cell
culture Escherichia
coli strains were grown aerobically at 37 ℃
in Luria-Bertani (LB) medium[8]. Strains of R.capsulatus were
grown in standard minimal (RCV) medium[9], and R.sphaeroides
strains were grown in LB or RCV medium supplemented with vitamins and DL-malate
(39 mmol/L) and L-glutamate (7 mmol/L) as C and N sources, respectively.
Anaerobic cultures were grown in 20 ml screw-capped tubes filled fully and
incubated in light at 30 ℃.
Antibiotics were used at final concentrations of 50 mg/L for ampicilline (amp)
or rifampicin (rif), 25 mg/L for kanamycin (kan) and 1 mg/L for tetracycline
(tet) for Rhodobacter sp. strains or 20 mg/L for E. coli.
1.2.2 DNA
manipulation Plasmid
DNA was prepared by the alkaline method, restriction endonuclease and DNA
modification enzymes were obtained from Promega Company, and DNA digestion and
ligation were performed according to the instructions of the manufacturers. The
ligation, transformation, DNA hybridization and PCR amplification were
performed as described in reference[8]. Transformed E.coli cells were
selected on LB plates in the presence of the appropriate antibiotics.
1.2.3 Bacterial
mating Bacterial
matings were made with the triparental cross system of Ditta[10] by
using pRK2013 in E. coli as the mobilizing plasmid. E.coli and R.sphaeroides
strains, at exponential phase of aerobic growth were mixed and spread onto
solid RCV medium. After 24 h of aerobic growth at 30 ℃
in the dark, the transconjugants of R.sphaeroides were selected on RCV
plates in the presence of appropriate antibiotics.
1.2.4 Interruption
of the hupR gene The
kanamycin resistance gene cartridge was isolated from pUC4K by HincII
digestion and then inserted into the StuI site of pSER carryed hupR
gene to yield plasmid pRK1 and pRK6, which had the cartridge in opposite
orientations. Plasmid pRK202 was then modified by insertion of the 1.9 kb SalI-BglII
fragment of pRK1 into the SalI-BamHI site of the tetracycline
resistance gene in the suicide plasmid pSUP202. The mobilizable plasmid pRK202
was then introduced into DH5a, and transferred by triparental conjugation into
wild type R.sphaeroides 6001 strain with the helper plasmid pRK2013.
To
select the double recombinants, in which the chromosomal wild-type hupR
gene had been exchanged with the inactivated gene on the plasmid,
transconjugant cells were grown in the presence of kanamycin, the putative hupR
mutants were then obtained. The genomic constructions of hupR-
mutants were checked by Southern blot analyses with hupR gene and
kanamycin resistance gene DNA as probes.
1.2.5 Overproduction
and purification of HupR A
1.6 kb DNA fragment, containing the hupR gene amplified by PCR (using
primers 5'-CATATGTCGCGGCCGCTGGTC-3' and 5'-GAAGTGAACCTCGCCTGC-3'), was inserted
in the pGEMT-easy vector to produce plasmid pNER1. pNER1 was digested with NdeI
and EcoRI, the resulting 1.6 kb NdeI-EcoRI fragment was subcloned
into the expression vector pET28c digested with NdeI and EcoRI.
The resulting plasmid pNER2 was then introduced into E.coli strain BL21
to get the strain NER2. NER2 cells were grown at 37 ℃
in LB broth supplemented with kanamycin. The synthesis of the His-tagged fusion
HupR protein (His6-HupR) was induced at A600 of 1.0 by the
addition of IPTG to a final concentration of 0.5 mmol/L. After induction for 2
h, the cells were harvested, and suspended in the binding buffer (20 mmol/L
Tris-HCl, pH 7.9, 0.5 mol/L NaCl, 5 mmol/L imidazole and 1 mmol/L
phenylmethyl-sulphonide). The sample was centrifuged at 15 000 r/min for 30 min
at 4 ℃ after sonification. The pellet was
collected and dissolved in the binding buffer containing 8 mol/L urea. It was
purified with an Ni2+-NTA agarose column according to the pET system
manual of Novagen.
1.2.6 Site-directed
mutagenesis of hupR gene To
obatain mutanted HupR proteins, the hupR gene was amplified by PCR
amplification. The hupR gene in plasmid pSER was then mutageneized
according to the manufacturer's intructions by using the following
oligonucleotides as primers (changes in nucleotides are in bold and the
corresponding change of the amino acid is in brackets): DE11 5'-TCGACGAAGAGC-CGCAT-3'
(change D11E), DE53 5'-ATCATCTCGGAACAGAGG-3' (change D53E),
KH104 5'-GTTTCTGACCCATCCCTG-3' (change K104H), DR3
5'-CATATGCTCAATTCGACCGTCGAG-3' (N terminal was deleted from 1-690
bp). Four recombinant DNAs were constructed and verified by sequencing. The
produced fragments were cloned in pGEMT-easy vector (Promega). And HindIII-EcoRI
cartridges containing hupR from the hybride plasmids was fused to pfru
from pFRKII, and then to pRK415 to yield pDE11c, pDE53c, pKH104c. These hupR
mutations was then introduced into hupR mutants by triparental
conjugation.
1.2.7 Enzyme
assay Hydrogenase
activity in whole cells was assayed at pH 8 as previously described[19]
with 0.15 mmol/L methylene blue as the electron acceptor. Activity of
b-galactosidase was assayed according to Miller[20] with o-nitrophenyl-b-D-galactopyranoside
as the substrate. Whole cells in culture medium were made permeable with 1 drop
of 0.1% sodium dodecyl sulfate and 2 drops of chloroform; the reaction was
stopped with addition of 0.5 ml of 1 mol/L sodium carbonate (pH 10) and, after
sedimentation of the cell debris, the A420 was read with a
spectrophotometer (DU-70, Beckman Instruments, Inc.).
1.2.8 Nucleotide
and protein sequence studies DNA
sequencing was performed on both strands by the Shanghai Genecore Company by using
the dideoxy chain termination method. DNA sequence analyses were performed by
BioEdit. Protein alignment was done with BLAST2[21].
2.1
The isolation and cloning of hupR gene
It
had been identified that cosmid 1 could restored the hydrogenase activity and
autotrophic growth of the Hup- mutants JP91 (HupS-) and
RCC8 (HupR-) of R.capsulatus and also restored the repression
of hydrogenase gene expression in the R.capsulatus HupT-
mutant BSE8. It indicated that the insert region of R.sphaeroides DNA in
cosmid 1 contained a hup DNA including the hupR, hupS and hupT
genes. The size of the DNA insert, determined by enzyme digestion, was (28.8±1.6)
kb[7]. Cosmid 1 was digested with various endonucleases. The
restricted fragments were probed by Southern blotting with a 1.3 kb EcoRI
fragment isolated from plasmid pAC63[13], which contains the R.capsulatus
hupR gene. The 3.5 kb SmaI-EcoRI fragment was subcloned in
pBluescriptII KS+ to yield plasmid pSER (Fig.1). The insert of plasmid pSER was
then sequenced. It was found to contain the hupR gene of R.sphaeroides.
Nucleotide sequencing was also carried out upstream and downstream from hupR
on pSER, to get hupK, hypA and hypB genes, and hypC,
hypD and hypE genes, respectively. These genes were identified on
the basis of the known codon preference of previously sequenced genes from R.capsulatus
by BLAST.
Fig.1 Partial physical map of the
insertion cosmid I
B,
BamHI; E, EcoRI; S, SalI.
2.2
Nucleotide sequence of hupR
The
nucleotide sequences and the deduced amino acid sequences of the hupR
(EMBL accession number AJ243734) are presented in Fig.2. The gene encodes a
protein of 54.031 kD (492 amino acids).
Fig.2 Nucleotide sequences and deduced
amino acid sequence of hupR in R.sphaeroides
The
HupR protein of R.sphaeroides shares sequence similarity with several response
regulator proteins belonging to the superfamily of two-component regulatory
systerms, particularly, with HupR of R.capsulatus (73% identity) and
other four organisms[22-24]. The three asparatic acid residues (10,
11, 53), threonine (81) and lysine residue (104), highly conserved in the
response regulators of the superfamily, were also present in HupR of R.
sphaeroides 601 (Fig.3). The central domain of HupR seems to contain also
the motifs characteristic of a nucleotide-binding site, and at the C terminus a
helix-turn-helix structure present in DNA-binding proteins was also found as in
the R.capsulatus HupR[4].
Fig.3 Amino acid sequence
alignments of response regulator proteins in R.sphaeroides (deduced
HupR), R.capsulatus (HupR), Ralstonia eutrophus (HoxA)[22],
Treponema pallidum (HupR)[23], Thiocapsa roseopersicina
(Ato)[24]
The
HupR protein was produced in E. coli in the form of a His6-HupR fusion
protein. Most of the overproduced protein was found in inclusion bodies. The
molecular weight of HupR is about 54 kD, which is in accordance with the
molecular mass deduced from the amino acid sequence. Fig.4 shows the SDS-PAGE
analysis of HupR production and purification.
Fig.4 SDS-PAGE analysis of HupR
Samples were boiled with 2×sample
buffer, run on a 12% acrylamide separating gel and the protein was stained with
Coomassie brilliant blue. 1, soluble extract of NER2 before induction; 2,
soluble extract after IPTG induction; 3, purified HupR; 4, molecular mass
markers.
To
ascertain that the hupR gene was involved in hydrogenase synthesis, the
chromosomal hupR gene was insertionally inactivated by a kanR
gene cartridge. The sizes of the restricted fragments of total DNA digested
with both the SalI-BglII of the wild type strain and the mutants,
probed with the 1.9 kb SalI-BglII fragment having the hupR
gene [Fig.5(B)], confirmed that the kanR gene cartridge had
been inserted in hupR. The fragment containing hupR gene without insertion
was only 1.9 kb, while the DNA fragment with insertions was 3.3 kb. Other
hybridization data obtained by using the HincII fragment containing the kanR
gene cartridge as the probe [Fig.5(C)] showed that there was only one kanR
gene cartridge insertion and that it was exactly in the hupR gene. Thus,
the identified mutants, termed KR5 and KR7, are mutants of the hupR
gene.
Fig.5 Southern hybridization of the SalI-BglII
fragments, isolated from genomic DNA of the HupR- mutants KR5 and
KR7 and from the wild type 6001 strain
The
blots were probed with hupR (B) and the kamR gene
cartridge (C). 1, KR5; 2, markers; 3, KR7; 4, 6001 strain.
The
H2 uptake hydrogenase activity of the wild type B10 was 38 mmol/(h·mg)
when grown under anaerobic light conditions, while that of RCC8 was only 2
mmol/(h·mg)[7].
For comparision, the wild type R.sphaeroides 6001 had the H2-uptake
hydrogenase activity of 4.78 mmol/(h·mg)
in anaerobic and light condition, cells of R.sphaeroides carrying
inactivated hupR gene (KR5 and KR7) had no hydrogenase activities, which
indicated that the HupR protein participated in the activation of hydrogenase
expression. This is the same phenotype as hupR mutants of R.capsulatus[5,17].
A plasmid-borne hupS∷lacZ
fusion, pRSA15, could monitor the transcriptional expression of hydrogenase
synthesis in strain by measuring the b-galactosidase activity[17].
This plasmid was introduced into the wild-type strain 6001 and in the hupR
mutants KR5 and KR7 (Table 2). The lacZ gene expression of the mutants
KR5 or KR7, was very poor, and the b-galactosidase activity of the R.sphaeroides
wild type 6001, was about 7 to 9 fold that of the mutants, but very much lower
than that of R.capsulatus wild type B10. These data demonstrate that hupR
plays a role in the activation of the transcription of hydrogenase structural
genes, and indicate that there may be some differences in the transcription regulation
of hydrogenase between R.sphaeroides and R.capsulatus.
2.7
Activation of hupSL transcription by HupR and HupR mutations
The
plasmid pNRC3 was constructed by the insertion of 1.6 kb HindIII-EcoRI
cartridge containing hupR fused to pfru from pFRKII to pRK415.
The pNRC3 was then introduced into R.sphaeroides mutants KR5 and KR7 by
triparental conjugation. The hydrogenase activities of the transconjugants were
assayed (Table 3). This result confirm again that the hupR plays an
active role in the transcriptional regulation of structrual gene hupSL.
In
order to study the function of the conversed sites, HupR proteins bearing
various mutations were constructed in the putative phosphorylation site and
some other sites related with phosphorylation. Using site-directed mutagenesis,
the D11 residue related with the Mg2+ incorporation in
the reaction was changed for an amino acid with the same charge D11E,
the D53 residue related with phosphorylation was also change to D53E,
the K104 related with activity to K104H, and the
fragement containing sites related with phosphorylation was deleted by PCR.
These four mutative and the wild-type hupR genes were cloned downstream
from a pfru promotor which could be activated in the presence of
fructose.
To
assay the function of the mutated HupR proteins, these proteins were produced
in HupR- mutants cells (Table 3). The hydrogenase activities
indicated that hupR genes were correctly expressed from pfru
promotor, and the HupR mutants had regained hydrogenase activities. From these
results, it seems that no matter the sites related with phosphorylation or
phosphorylation domain exist, the HupR will interact with the promoter and
activate the hydrogenase activity. That may show that the HupR activate the hupSL
transcription in the non-phosphorylated from.
Several
kinds of hupR genes had been sequenced and identified in the former
studies[1,2]. It was the first time that the hupR gene was
cloned in the R.sphaeroides, and the partial physical map of cosmid 1
containing hup cluster was obtained. The cloning of hupR gene
makes it possible to understand how it regulates the structural gene and to
construct a good engineering strain for improving the efficiency of nitrogen
fixation. The locations of many hup genes of R.sphaeroides on the
hup cluster required for the expression of hydrogenase were also
identified in this study. It was found that the arrangement of these genes in
the hup cluster in R.sphaeroides was the same as that of in R.capsulatus.
By
sequence comparison, it was found that HupR belongs to the superfamily of
response regulator of two component systems, all conserved amino acids also
existed in HupR (Fig.3). Curiously, the gene encoding kinase hadn't been found
downstream or upstream near hupR gene. Dischert[5] had
brought forward that the HupT and HupR are the partners of the two-component
system regulating hydrogenase expression in R.capsulatus. But there were
still remaining questions for the cross-talk between regulator and kinase in
the two component systems.
Response
regulators are characterized by a conserved domain of approximately 125 amino
acids, usually attached via a linker sequence to a domain with an effector
function. The effector domains generally have DNA binding activities, and in
these cases, response regulator phosphorylation serves to modulate
transcription[25]. In this study, it was found that although the
conserved sites related with the phosphorylation of HupR were mutated, there
were still some hydrogenase activity, and a mutated HupR deleted the N-terminal
could also complement the HupR mutants and the mutants regained hydrogenase
activity. However, the level of hydrogenase activity was lower than that in the
wild-type 6001. This may be caused by a poor expression of the hupR gene
from the pfru promoter. These results show that the phosphorylation site
of R. sphaeroides HupR is not necessary for the regulation of the
hydrogenase expression. In non-phosphorylated form, the HupR could activate the
transcription of hydrogenase gene. Dischert et al[5] reached the
same conclusion for the HupR in R.capsulatus. They had replaced the
putative phosphorylation site (D54) of HupR protein with various
amino acids or by deleting it by using site-directed mutagenesis, and concluded
that the the HupR activates hupSL transcription in the unphosphorylated
form.
In
this study, it seems that the hydrogenase activity of R. sphaeroides is
rather low, while its arrangement of hup cluster is almost the same as
that of R.capsulatus. It arose our interests to learn more about that.
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Received: June 13, 2001 Accepted: July 25, 2001
This work was supported by a grant from
the National Natural Science Foundation of China, No.39870005
*Corresponding author: Tel,
86-21-64042090-2217; Fax, 86-21-64042385; e-mail, [email protected]