BACKGROUND
disulfide bonds contribute to the stability and function of many extracytoplasmic, soluble or membrane-bound proteins.
in gram-negative bacteria, the oxidative protein folding takes place in the periplasm and is controlled by proteins from the dsb family  <cit> .
in the highly oxidizing environment of the periplasm, there is also a need for selected proteins to be kept in a reduced form.
in the assembly of c-type cytochromes, essential for energy metabolism, for example, the cytochrome c maturation process requires ligation of heme  to reduced thiols of the cys-x-x-cys-his motif of apocytochrome  <cit> .
to date, there are two different systems for cytochrome-c biogenesis that have been found in bacteria: system i and system ii.
they comprise two kinds of proteins acting in a coordinated fashion: those involved in heme handling and heme ligation to the apocytochrome, and those contributing to reduction of a disulfide bond of the cxxch heme-binding motifs .
the cytochrome-c maturation machinery of the system i operating in many gram-negative bacteria consists of up to ten proteins .
the apocytochrome reduction is accomplished by the action of ccmg , ccmh, and dsbd or ccda.
dsbd/ccda transfers electrons from cytoplasmic thioredoxin to ccmg.
then ccmg is re-oxidized by shuttling its electrons to ccmh, which finally transfers them to apocytochrome c. however, the details concerning mixed disulfide complexes created during the process still remain unclear  <cit> .
the remaining proteins play a role in heme transport and its ligation to the apocytochrome  <cit> .
system ii is simpler and more widespread among bacterial species.
it is present in, for example, gram-positive bacillus subtilis.
it contains four proteins, resa, resb, resc and ccda, of which resa  is a functional counterpart of ccmg, and ccda is a functional counterpart of dsbd.
resb  and resc  form a complex to deliver the heme, and they function in the cytochrome c-heme ligation  <cit> .
while in most microorganisms, the ccsb and ccsa proteins are present as two separate polypeptides, there are a few species of bacteria, such as wolinella, bacteroides and some strains of helicobacter spp., whose genes encode ccsa and ccsb fused into one large orf   <cit> .
many studies have tried to decipher the cooperation among periplasmic dsb proteins in cytochrome-c biogenesis.
initially, it was thought that introduction of the disulfide bonds into the cxxch motif of the apocytochrome c, which happens just after its transport to the periplasm by the sec system, is an obligatory step of the process, as dsba and dsbb mutants in e. coli were unable to produce cytochrome c  <cit> .
however, recent data in the literature may contradict this scheme; the lack of the dsb proteins of the oxidative pathway in b. subtilis and r. capsulatus, for example, suppresses the cytochrome c deficiency of ccmg or ccda mutants .
however, it should be noted that the dsb oxidative pathway functioning is dependent on cell growth conditions  it has also been demonstrated that the heterologous expression of ccsba of h. pylori or ccsab from b. pertussis, both encoding cytochrome c synthetase, in an e. coli that lacks its own cytochrome c machinery results in c-type cytochrome formation.
this observed effect was significantly enhanced by addition of exogenous reductant  <cit> .
detailed analysis of the paracoccus denitrificans cytochrome maturation in an e. coli dsba, dsbd double-mutant led mavridou et al.
to the conclusion that apocytochrome is subjected to two competing reactions: either heme attachment to its cysteine thiols, or oxidation of those thiols  <cit> .
helicobacter pylori is a gram-negative spiral-shaped bacterium, a member of ε-proteobacteria that specifically colonizes the gastric epithelium of humans with severe consequences.
data on h. pylori cytochromes are not comprehensive.
analysis of its genome nucleotide sequence revealed that this microaerophilic microorganism possesses a rather simple respiratory chain consisting of three enzymes: quinol-cytochrome c reductase, cytochrome bc <dig> complex, cytochrome c <dig> and cb-type cytochrome c oxidase  <cit> .
further inspection of the h. pylori genome showed the existence of a second gene encoding a putative low molecular mass cytochrome c  named cycb.
both cyca and cycb are essential genes and cannot substitute for each other  <cit> .
cytochrome c <dig>  encoded by the cyca  gene, is a soluble periplasmic protein, a potential electron donor to the cb-type cytochrome c oxidase  <cit> .
to our knowledge, little is known about the c-type cytochrome maturation system of helicobacter pylori.
in the genome of h. pylori  <dig>  there are  <dig> proteins containing cxxc motifs characteristic of thiol:disulfide oxidoreductases identified to date.
only two of these proteins have a thioredoxin-like  fold  and are periplasm-located  <cit> .
this microorganism uses system ii, and hp <dig>  and hp <dig> , a homolog of resbc), whose products potentially play a central role in c-type cytochrome maturation, constitute one transcriptional unit  <cit> .
hp <dig> can, to some extent, complement for a lack of the e. coli cytochrome-c maturation machinery.
the recently determined crystal structure of hp <dig> shows similarity to the structure of b. subtilis resa.
however large structural differences between these two proteins were also observed  <cit> .
we have previously demonstrated that hp <dig> is a dimeric oxidoreductase that catalyzes disulfide bond formation in the periplasm.
in this work, to gain further insight into the helicobacter pylori dsb protein network, we analyzed hp <dig>  the second periplasmic oxidoreductase, using biochemical and genetic tools.
methods
bacterial strains, primers, plasmids, media and growth conditions
bacterial strains, plasmids and primers used in this study are listed in tables  <dig> and  <dig>  two h. pylori strains  were used in this study.
although the sequence of the h. pylori  <dig> genome is complete  <cit> , this strain is inconvenient for genetic manipulation.
h. pylori n <dig>  originally isolated from a patient with gastritis, is useful for complementation experiments, and it is highly motile  <cit> .
both h. pylori strains were grown on blood agar base  <dig>  plates  supplemented with 10 % horse blood and an antibiotic mixture consisting of vancomycin , polymyxin b , trimethoprim  and amphotericin b  at 37 °c under microaerobic conditions.
liquid cultures of h. pylori were grown in brain heart infusion  broth supplemented with 10 % fetal bovine serum .
for the selection of h. pylori mutants or complemented strains, kanamycin  or/and chloramphenicol  were added to the growth media.table  <dig> bacterial strains and plasmids used in this study
dsbi
+ in trans complementant of dsbi::aph
hp0231
+ in trans complementant of hp0231::cat
+
+
+
+
the e. coli strain tg <dig> was used as a host for the construction and preparation of recombinant plasmids.
the e. coli strain rosetta  was used to overexpress puwm <dig>  puwm <dig> and puwm <dig>  the e. coli strains jcb <dig> and jfc <dig> were employed for complementation experiments of e. coli dsba and dsbc mutants by hp <dig>  e. coli strains were grown at 37 °c on solid or liquid luria-bertani  medium or on m <dig> minimal medium  <cit> .
when needed, media were supplemented with antibiotics at the following concentrations:  <dig> μg ml− <dig> ampicillin,  <dig> μg ml− <dig> kanamycin and  <dig> μg ml− <dig> chloramphenicol.
general dna manipulations
standard dna manipulations were carried out as described earlier  <cit>  or according to the manufacturer’s instructions .
polymerase chain reactions  were performed with primestar hs dna polymerase  or hotstar hifidelity polymerase  under standard conditions.
synthetic oligonucleotides synthesis and dna sequencing were performed by genomed s.a., warsaw, poland.
natural transformation of h. pylori
the naturally competent h. pylori n <dig> was grown on ba plates for  <dig> h. subsequently, bacteria were plated onto fresh plates for  <dig> h. then  <dig> – <dig> μg of plasmid dna was added and plates were incubated for  <dig> h. afterwards, bacteria were transferred onto a plate supplemented with kanamycin or chloramphenicol/kanamycin, and transformants were grown for  <dig> days.
allelic exchange mutagenesis of the hp <dig> gene in h. pylori
to inactivate hp <dig>  a recombinant vector was constructed by a two-step pcr method  <cit> .
the upstream and downstream regions of the hp <dig> gene were amplified from h. pylori n <dig> genomic dna using two pairs of primers specific for hp <dig> and its flanking regions, n6_hp0265_up_bamhi-n6_hp0265_up_rev <dig> and n6_hp0265_dw_sali-n6_hp0265_dw_fwd, respectively.
the n6_hp0265_up_rev and n6_hp0265_dw_sali primers contained nucleotide sequences complementary to each other, respectively.
each pcr product was purified with a gel extraction kit .
next, a mixture of two purified products  was used as a template in a single pcr reaction, using the primers n6_hp0265_up_bamhi-n6_hp0265_dw_fwd.
the resulting pcr product contained a 274-nucleotide deletion of hp <dig>  the product was purified and cloned into pbluescript sk ii.
to add the aph gene between the two hp <dig> arms, smai restriction enzyme was used to yield suicide plasmid puwm <dig>
sequence analyses confirmed the correct construction of puwm <dig> and the recombinant plasmid was introduced into h. pylori n <dig> by natural transformation.
cells were plated and screened on plates containing kanamycin and kanamycin with  <dig> mm dtt to restore the reducing activity of hp <dig>
an hp0265::aph mutant was obtained by a double cross-over using puwm <dig>  and verified by pcr analysis, using the primer pair n6_hp0264_fwd1-n6_hp0265_rev <dig>
allelic exchange mutagenesis of the hp <dig> gene in h. pylori
to inactivate hp <dig>  two recombinant vectors, pumw <dig> and puwm <dig>  were constructed.
puwm <dig> was generated by a two-step pcr method  <cit> .
briefly, primers km1f–km2r, were used to amplify the aph gene from puwm <dig>  the upstream and downstream regions of the hp <dig> gene were amplified from h. pylori n <dig> genomic dna using two pairs of primers specific for hp <dig> and its flanking regions, hp0377i-hp0377iia and hp0377iiia-hp0377iv, respectively.
the hp0377iia and hp0377iiia primers contained 5′ leader nucleotide sequences complementary to km1f and km2r, respectively  <cit> .
each pcr product was purified with a gel extraction kit .
next a mixture of three purified products  was used as a template in a single pcr reaction, using primers: hp0377i-hp0377iv.
subsequently, the resulting pcr product, containing the aph gene inserted between the two hp <dig> arms in the same transcriptional orientation as the hp <dig> gene, was purified and cloned into pgem-t easy, generating the suicide plasmid puwm <dig>  sequence analyses confirmed the correct construction of puwm <dig>  and the recombinant plasmid was introduced into h. pylori n6/puwm <dig> by natural transformation.
selection was done on blood agar plates containing kanamycin and chloramphenicol, with or without isopropyl-β-d-thiogalactopyranoside.
the puwm <dig> plasmid used for insertional mutagenesis of the h. pylori n <dig> hp <dig> gene, was constructed by amplification of the hp <dig> gene and surrounding dna fragments by pcr, using the hp0377zew1-hp0377zew <dig> primers.
subsequently, the resulting pcr product was purified and cloned into pgem-t easy, creating puwm <dig>  the resulting construct was digested with bglii, to cut the restriction site in the middle of hp <dig> gene.
the sticky ends were blunted using klenow fragment.
the dna mixture was purified with a clean-up kit and ligated with a previously prepared kanamycin cassette, amplified from puwm <dig> by the pair of primers km1f–km2r.
the generated suicide plasmid puwm <dig>  containing the aph gene inserted between the two hp <dig> arms in the same transcriptional orientation as the hp <dig> gene, was confirmed by sequencing.
the recombinant plasmid was introduced into h. pylori n6/puwm <dig> by natural transformation.
selection was done on blood agar plates containing kanamycin and chloramphenicol, with or without isopropyl-β-d-thiogalactopyranoside.
to create a conditional mutant in the hp <dig> gene, puwm <dig> was constructed based on the shuttle e. coli/h.
pylori plasmid: pill <dig>  the hp <dig> coding nucleotide sequence was amplified from h. pylori  <dig> genomic dna without its own promoter by the primer pair hp0377mutf - hp0377mutr.
the purified pcr products, as well as the shuttle plasmid, were digested with ndei/bamhi and ligated together to form puwm <dig>  correct construction of puwm <dig> was confirmed by sequencing.
next, puwm <dig> was introduced into h. pylori n <dig>  and puwm529/puwm <dig> was used to inactivate hp <dig> gene.
the strategy for cloning into pill <dig> was identical.
site-directed mutagenesis of the hp <dig> gene
to obtain mutated hp <dig> proteins, a set of recombinant plasmids was constructed from puwm <dig>  which carries the hp <dig> gene without its promoter and signal sequence.
cys-to-ser point mutations were generated using the quick change site-directed mutagenesis kit  according to the manufacturer’s instructions, starting with  <dig> ng of puwm <dig> template and  <dig> ng of each primer .
to obtain c25a mutated hp <dig> protein, a recombinant plasmid was constructed from puwm <dig> carrying the hp <dig> gene with its promoter and signal sequence.
the cys-to-ala point mutation was generated as described above, using primers hp377_c25a_for and hp377_c25a_rev.
protein analysis
preparation of h. pylori and e. coli protein extracts, sds-page  and blotting procedures were performed by standard techniques  <cit> .
preparation of subcellular fractions
subcellular protein fractions were prepared from 48-h h. pylori cultures.
periplasmic proteins were released from the cells using an osmotic-shock procedure  <cit> .
after decanting the periplasmic fraction, bacterial pellets were resuspended in  <dig> mm tris–hcl, ph  <dig>  and sonicated to release the cell contents.
subsequently, cell wall debris was removed and the supernatants were ultracentrifuged  to separate the membrane and cytoplasmic fractions.
finally, the cell envelope was fractionated into inner and outer membranes by selective solubilization of the inner membrane with  <dig>  %  sodium lauryl sarcosine  <cit> .
overexpression and purification of apocytochrome c  and hp0231
hp <dig> was overexpressed by autoinduction from an e. coli rosetta/puwm <dig> strain and purified as previously described  <cit> .
hp <dig> expression vector was constructed by amplifying the region encoding the mature hp <dig> protein  from the chromosome of h. pylori  <dig>  with primers hp1227_ndei-for and hp1227_xhoi-rev.
the insert was cloned into pet24a with ndei and xhoi restriction enzymes, to yield plasmid puwm <dig>  expression was induced by  <dig> mm iptg  at od600 ~  <dig> .
after  <dig> h in 37 °c, cultures were centrifuged and the cell pellet was suspended in  <dig> mm sodium phosphate, ph  <dig> ,  <dig> mm nacl,  <dig> mm imidazole.
cells were disrupted by ultrasonication.
the cell lysate was centrifuged and the resulting supernatant was applied onto bio-scale mini profinity imac cartridges  containing ni-charged resin.
the protein was eluted with an imidazole gradient, using the ngc chromatography system .
overexpression and purification of hp0377
hp <dig> expression vector was constructed by amplifying the region encoding the mature hp <dig> protein  from the chromosome of h. pylori  <dig>  with primers hp377exi and hp377exii.
the insert was cloned into pet28a with ncoi and xhoi restriction enzymes, to yield plasmid puwm <dig>  the cytoplasm-located hp <dig> was overexpressed from puwm <dig> by autoinduction  <cit> , and then it was purified by affinity chromatography, dialyzed against phosphate buffered saline  and later used for rabbit immunization .
the anti-hp <dig> rabbit serum was specific and recognized native hp <dig>  as verified by western blot analysis.
the hp <dig> c89s and c92s expression vectors were constructed by cutting out hp0377-changed sequences from puwm <dig> and puwm <dig>  respectively, and cloning the inserts into pet28a as described above, resulting in formation of puwm <dig> and puwm <dig>  all plasmids carried the hp0377-his <dig> translation fusion.
for biochemical experiments, the protein was expressed and purified from e. coli rosetta harboring puwm <dig>  puwm <dig> and puwm <dig>  the proteins were overexpressed by autoinduction and then purified by affinity chromatography using ngc medium-pressure chromatography systems by bio-rad as described above.
biochemical assays
in vivo redox state of hp0377
the redox state of hp <dig> was visualized by alkylating the free cysteine residues using 4-acetamido-4′-maleimidylstilbene- <dig> ′-disulfonic acid .
this agent can only modify covalently free thiols, resulting in a molecular mass increase of  <dig> da  <cit> .
briefly, bacteria were harvested from ba plates after  <dig> or  <dig> h of incubation under microaerobic conditions.
samples were standardized using the od <dig> of the culture, and ice-cold trichloroacetic acid  was immediately added to the culture.
whole-cell proteins were precipitated and collected by centrifugation, washed with ice-cold acetone, and then dissolved in  <dig> mm tris–hcl ,  <dig> mm ethylenediaminetetraacetic acid ,  <dig>  % sds containing  <dig> mm ams by agitation for  <dig> min at 37 °c.
the proteins in non-reducing laemmli buffer were resolved by 14 % sds-page without reducing agent.
hp <dig> was then detected by an immunoblot analysis using an anti-hp <dig> antibody.
as controls, we used samples previously treated with  <dig> mm ditiotreitol  for  <dig> min at 30 °c before precipitation of the proteins with tca.
determination of the pka value
the ph-dependent ionization of the cys <dig> and cys <dig> was followed by the specific absorbance of the thiolate anion at  <dig> nm  <cit> .
measurements were carried out at 25 °c in a buffer consisting of 10 mm tris,  <dig> mm sodium citrate,  <dig> mm edta and  <dig> mm nacl, ph  <dig>  for ecdsba as a control, and for hp <dig> and its mutated forms, with an average initial protein concentration of  <dig> μm.
the ph of the protein solution was lowered to  <dig> by the stepwise addition of aliquots of  <dig>  m hcl.
absorbances at  <dig> and  <dig> nm were recorded on a spectrophotometer and corrected for the volume increase.
the ph dependence of the thiolate specific absorbance signal reduced/oxidized) was fitted according to the henderson-hasselbach equation.
determination of the redox potential of hp0377
the fractions of reduced and oxidized hp <dig> were determined using 4-acetamido-4′-maleimidylstilbene- <dig> ′-disulfonic acid  trapping  <cit> .
briefly, hp <dig>  were incubated overnight at room temperature in 50 mm kpi ph  <dig> ,  <dig>  mm edta and various glutathione /glutathione disulfide  ratios.
after incubation, proteins were precipitated with trichloroacetic acid  .
after 20-min incubation on ice, the samples were centrifuged , and the pellets were washed with cold acetone.
after a second centrifugation, pellets were dried and resuspended in a buffer containing 20 mm ams,  <dig>  % sds, 10 mm edta, and 50 mm tris–hcl .
after 45-min incubation at 37 °c, with  <dig>  rpm shaking samples were loaded onto 12 % sds-polyacrylamide gels under denaturing conditions.
fractions of reduced and oxidized protein were determined using imagej).
the redox potential was then calculated as described previously  <cit> .
alkaline phosphatase  assay
ap activity was measured as previously described  <cit> , with some modifications.
briefly, e. coli cell cultures were grown at 37 °c in minimal medium m <dig> until they reached mid-log phase.
then, the cells were pelleted by centrifugation, washed twice with tris–hcl , and resuspended in tris–hcl .
the od <dig> was measured spectrophotometrically.
samples  were equilibrated for  <dig> min in a water bath at 28 °c, then  <dig> μl p-nitrophenol phosphate  was added, and the time was recorded.
the reaction was allowed to proceed at 28 °c until development of yellow color was observed.
at this point the reaction was stopped by adding  <dig> μl  <dig> m kh2po <dig>  samples were centrifuged before the a <dig> and a <dig> measurements.
insulin reduction assay
the ability of hp <dig> and ecdsba to catalyze the reduction of insulin in the presence of dtt was determined as previously described  <cit> .
determination of oxidase and isomerase activities
oxidative folding of reduced rnasea
in vitro oxidative folding of reduced rnasea was performed for hp <dig> and ecdsba as described earlier, with a few modifications  <cit> .
proteins were oxidized with  <dig> mm oxidized glutathione  and incubated for  <dig> h at room temperature.
rnasea was reduced by overnight incubation at room temperature in  <dig> mm tris acetate ph  <dig>  containing  <dig> m guanidine hydrochloride and  <dig> mm dtt.
all proteins were then dialyzed on desalting columns  and concentrated in pbs.
native rnasea and ecdsba were used as positive controls.
the redox state of the thiols was confirmed by ellman’s assay, which exploits the colorimetric change at a <dig> when  <dig> ′-dithiobis-  is converted to 2-nitro- <dig> thiobenzoate upon cleavage of the disulfide bond by free thiols.
oxidase activity was measured by analyzing the cleavage of ccmp  at a <dig> by refolded rnasea in the presence of tested enzymes.
reactions  were carried out in  <dig> μl of pbs buffer containing  <dig> mm tris acetate ph  <dig> ,  <dig> mm edta,  <dig>  mm gssg,  <dig> mm gsh ,  <dig>  mm ccmp, rnasea  and analyzed enzyme .
the reaction mixtures were prepared in a 96-well plate format and read through  <dig> min at 27 °c in a sunrise™  plate reader.
three independent experiments were performed.
refolding of scrambled rnasea
in vitro refolding of scrambled rnasea was performed for hp <dig> and ecdsbc as described earlier, with a few modifications  <cit> .
proteins were reduced with  <dig> mm dtt and incubated overnight at 4 °c.
rnasea was first reduced by overnight incubation at room temperature in  <dig> mm tris acetate ph  <dig>  containing  <dig> m guanidine hydrochloride and  <dig> mm dtt.
then, in order to introduce incorrect disulfides, reduced rnasea was dialyzed against pbs buffer containing  <dig> m guanidine hydrochloride, sparged with oxygen and incubated for  <dig> days in the dark at room temperature.
finally,  <dig> mm hydrogen peroxide  was added for  <dig> min at 25 °c.
all proteins were then dialyzed on desalting columns  and concentrated in pbs.
ecdsbc was used as a positive control.
the redox state of the thiols was confirmed by ellman’s assay.
rnasea activity was measured by analyzing the cleavage of ccmp as described for the oxidative test, with a change in the reaction mixture:  <dig> mm tris acetate ph  <dig> ,  <dig> mm edta,  <dig> μm dtt,  <dig>  mm ccmp, rnasea  and analyzed enzyme .
three independent experiments were performed.
determination of the interaction between hp <dig> and hp1227
oxidized hp <dig> was prepared by incubating the purified hp <dig> with a  <dig> mm oxidized glutathione for  <dig> h at 37 °c.
reduced apocytochrome c was prepared by incubating the purified apocytochrome with  <dig> mm dtt for  <dig> h at 37 °c.
excess dtt and oxidized glutathione was removed using desalting columns that were previously equilibrated with  <dig> mm tris hcl ph  <dig> ,  <dig> mm nacl.
reduced apocytochrome c in  <dig> mm tris–hcl ph  <dig> ,  <dig> mm nacl was incubated for  <dig> h at 37 °c in the presence of a twofold excess of hp0231ox.
after incubation, the proteins were precipitated with trichloroacetic acid , washed three times with ice-cold acetone and then resuspended in  <dig> μl of a reaction buffer consisting of  <dig> mm tris–hcl ph  <dig> , 2 %  sds,  <dig> mm ams.
the oxidized and reduced forms of hp <dig> as well as reduced apocytochrome c were similarly treated with ams before sds–page under nonreducing conditions.
ams-treated protein samples were separated by nonreducing sds–page and visualized by coomassie staining.
size exclusion chromatography
to verify that hp <dig> exists as a monomeric protein, size exclusion chromatography was employed.
hp <dig> was loaded onto superdex <dig> hiload 16/ <dig> column  and eluted with  <dig> mm tris ph  <dig>   <dig> mm nacl.
determination of the oligomeric state of hp <dig> using glutaraldehyde
crosslinking of polypeptide chains with glutaraldehyde was performed essentially as described  <cit> .
hp <dig>  was incubated at room temperature with different concentration of glutaraldehyde  in  <dig>  m bicine–naoh buffer ,  <dig>  mm dtt,  <dig>  m nacl for  <dig> min and the reaction was quenched by adding ethanolamine–hcl  to a final concentration of  <dig>  m.
phenotype assays
dtt sensitivity assay
dtt sensitivity experiments were performed as previously described  <cit> .
a freshly prepared  <dig> m dtt  stock solution was dissolved in molten lb agar to the final concentration of  <dig> mm dtt.
the dtt agar plates were used within  <dig> min of pouring to prevent oxidation of dtt by air.
exponentially growing cultures were decimally diluted, and  <dig> μl aliquots were spotted on the plates.
the growth was observed after overnight incubation at 37 °c.
the experiments were conducted in triplicate.
motility assays
e. coli cells were grown in liquid culture in lb broth until the od <dig> value was close to  <dig>  then bacteria were inoculated on lb soft agar plates containing  <dig>  %  agar with a sterile toothpick and incubated for  <dig> h at 30 °c.
copper sensitivity assay
the copper sensitivity assay was performed as previously described  <cit> .
briefly, bacteria were grown in bhi media supplemented with  <dig> mm or  <dig> mm cucl <dig>  strains were grown at 34 °c.
ethics statement
all studies involving animals were performed in accordance with ethical standards, after approval from the local ethics committee no.
<dig>  warsaw, poland 966/ <dig>
RESULTS
determination of the in vivo redox state of hp0377
hp <dig> is an oxidoreductase, as it contains the trx domain with a cxxc  motif located at the n-terminus of the first helix in the trx fold.
the involvement of hp <dig> in the cytochrome c maturation process is indicated by its structure, its in vitro interaction with hp <dig>  and the genetic organization of the dna fragment containing the hp <dig> gene  <cit> .
to clearly define whether hp <dig> functions in the oxidizing pathway or the reducing pathway, we first decided to determine the redox state of that protein in vivo using the ams-trapping technique  <cit> .
the in vivo redox state of an oxidoreductase usually reflects its activity in the cell, i.e., proteins that function in the oxidizing pathway, such as ecdsba, are maintained in the oxidized state in vivo, whereas proteins that function in the reducing pathway, such as ccmg, are maintained predominantly in the reduced state.
we found that hp <dig> is present in the reduced form in wild-type cells, which suggests that hp <dig> functions as a reductase in h. pylori, which is in accord with its three dimensional structure  <cit> .
thus, we also determined the redox status of the hp <dig> in h. pylori lacking hp <dig> or dsbi .
both proteins are active in the dsb oxidative pathway.
as described earlier, hp <dig> introduces disulfide bonds and dsbi is partially responsible for hp <dig> re-oxidation  <cit> .
our results showed that a significant portion of hp <dig> is present in the oxidized form in both the hp <dig> and dsbi mutated cells .
also the overproduction of hp <dig> or dsbi from a moderate copy number plasmid disturbs the redox homeostasis and results in the presence of hp <dig> in both reduced and oxidized forms .
to clarify the link between hp <dig> and cytochrome c biogenesis, we decided to check whether apocytochrome c is a substrate of hp <dig>  we found that hp <dig> was able to oxidize the reduced apocytochrome c in vitro fig.
<dig> redox state of hp <dig> in wt and mutants: hp0231::cat, dsbi::aph and complemented strains of h. pylori strain n <dig>  bacterial cultures were treated with 10 % tca, followed by alkylation with ams.
cellular proteins including the reduced  and the oxidized  controls were separated by 14 % sds-page under non-reducing conditions, and western blot analysis antibodies against hp <dig> was performed.
each lane contains proteins isolated from the same amount of bacteria
fig.
<dig> oxidase assay of hp <dig> towards apocytochrome c .
two different sds pages were run to better visualize the shift between oxidized and reduced forms of proteins.
a hp0231-dependent oxidation of reduced apocytochrome c. lane  <dig>  oxidized hp <dig>  lane  <dig>  reduced hp <dig>  lane  <dig>  reduced apocytochrome c treated with oxidized hp <dig>  b hp0231-dependent oxidation of reduced apocytochrome c. lane  <dig>  oxidized apocytochrome c. lane  <dig>  reduced apocytochrome c, lane  <dig>  reduced apocytochrome c treated with oxidized hp <dig>  different redox forms were detected by nonreducing sds-page after ams treatment, which results in an increase in the molecular mass of reduced proteins by about  <dig>  kda per thiol group
.
biochemical characterization of hp0377
to clearly define whether hp <dig> functions as oxidoreductase, we analyzed the biochemical properties of the protein.
recombinant proteins used for biochemical analysis were produced as cytoplasmic proteins in e. coli cells and purified by affinity chromatography.
first, to gain insight into the role played by hp <dig>  we determined its redox potential by equilibrium incubation using gsh/gssg as a reference.
we found that the hp <dig> redox potential is –  <dig> mv , which makes it a rather weak reductant.
next we determined the ability of hp <dig> to reduce insulin in the presence of dtt.
the insulin reduction assay is commonly used to determine whether a protein can function as an oxidoreductase, regardless of its function in the reducing or the oxidizing pathway in vivo.
insulin contains two intramolecular disulfide bonds that connect the a and b chains: reduction of these disulfide bonds causes the precipitation of the b chain, which can be monitored by following the increase of turbidity at  <dig> nm  <cit> .
as shown in additional file 2: figure s <dig>  the reaction lag time with hp <dig> in the insulin reduction assay is almost as long as the control reaction , whereas the lag time with ecdsba is  <dig> min.
therefore, hp <dig> likely cooperates with a specific substrate, which is typical of ccmg proteins.
however, these data are in contrast to some previous data  <cit> .
to clarify this inconsistency, and because there is a similarity between the active site of hp <dig>  and the disulfide isomerase ecdsbc , we performed a disulfide isomerase assay by evaluating the ability of hp <dig> to reactivate oxidized, scrambled rnase .
scrnase was prepared as described in the methods section and did not contain free cysteines, as confirmed with ellman reagent.
in this assay, the refolding efficiency of hp <dig> was almost as high as ecdsbc .
to get a complete biochemical characterization of hp <dig>  we also investigated its ability to catalyze the refolding of reduced-unfolded rnasea.
as expected, hp <dig> did not show activity in this assay .
as hp <dig> revealed high isomerizing activity, and because dsb proteins involved in the isomerization pathway exist as dimers, we also evaluated the potential oligomerization of hp <dig> using two methods   hp <dig> that lacked its own signal sequence  and contained a c-terminal  <dig> his tag was purified from e. coli cytoplasm and used in both assays.
we found that exposure of hp <dig> to glutaraldehyde, which stabilizes oligomeric proteins by covalent crosslink formation, resulted in generation of a protein with a molecular weight of  <dig> kda.
this result clearly showed that hp <dig> can exist as a dimer.
the size exclusion experiment showed that hp <dig> eluted as two peaks, one with an estimated mass of  <dig> kda, consistent with the size of the monomer, and the second with estimated mass of  <dig> kda, consistent with the size of the homodimer.
thus, the presented data allowed us to conclude that at least a portion of hp <dig> exists as a dimer.fig.
<dig> isomerase activity assay.
the reaction contained  <dig> μm scrambled rnase in  <dig> mm potassium phosphate buffer, ph  <dig> , 2 mm edta,  <dig> μm dtt, and 9 mm ccmp.
the reaction was performed in the absence or presence of  <dig> μm ecdsbc,  <dig> μm hp <dig>  the cleavage of ccmp by refolded rnase was monitored continuously at  <dig> nm.
the changes in the absorbance at  <dig> nm as a function of time are presented.
three independent experiments were performed
fig.
<dig> glutaraldehyde crosslinking of hp <dig>  purified hp <dig> at  <dig>  mg/ml was cross-linked in the presence of different concentration of glutaraldehyde: lane 1–purified hp <dig> protein, lane 2– <dig>  %, lane 3– <dig>  %, lane 4– <dig>  %, lane 5– <dig>  %, lane 6– <dig>  % glutaraldehyde.
m–monomers, d–dimers
fig.
<dig> size exclusion profile of the purified hp <dig> separated on a superdex <dig> hiload 16/ <dig> column  and monitored by absorbance at  <dig> nm.
hp <dig> elutes as a two peaks at  <dig> min, with an estimated mass of  <dig> kda, consistent with the size of the homodimer and  <dig> min.
with estimated mass of  <dig> kda, consistent with the size of the monomer.
the column was calibrated with gel filtration standard : thyroglobulin , γ-globulin , ovalbumin , myoglobin , vitamin b <dig> .
the relative positions of the chosen standards are marked with arrows
the function of thiol-oxidoreductases depends on the pka values of their active-site cys residues.
the pka of the hp <dig> active site thiols was determined by observing the change in absorption of the thiolate anion at  <dig> nm as a function of ph in wt protein.
the ph titration of wt hp <dig> showed two transitions, one with pka =  <dig>  ±  <dig>  and the other with pka =  <dig>  ±  <dig>  fig.
6a.
because these data are atypical for ccmgs, we constructed two single-cys cxxc motif mutants of hp <dig> , which allowed independent measurement of the pka of each cys residue.
as shown in fig.
6b, c the c89s mutant had a pka =  <dig>  ±  <dig>  and the c92s mutant a pka =  <dig>  ±  <dig> .
these results agreed with the titrations of the wt protein.
the pka value of the solvent-exposed active site cysteine  is about  <dig> .
this is relatively acidic compared with the solvent-exposed active site cysteine of the e. coli ccmg protein, pka =  <dig>   <cit> , or the b. subtilis resa, pka =  <dig>   <cit> ; it is as acidic as e. coli dsba, which is a known oxidant whose pka of the solvent-exposed active site cysteine is  <dig>   <cit> , and it is close to the pka of ecdsbc,  <dig>  ±  <dig>   <cit> .
thus, hp <dig> is the first described ccmg protein having an acidic pka of the n-terminal cysteine of the cxxc motif, and at the same time, it presents a low redox potential.fig.
<dig> thiol ionization equilibrium in hp <dig> followed by uv absorbance at  <dig> nm.
panel a: hp <dig> wild type.
panel b: hp <dig> c89s.
panel c: hp <dig> c92s
hp <dig> is an essential protein for h. pylori
to investigate the function of hp <dig> in h. pylori we decided to construct an isogenic hp <dig> knock-out strain by allelic exchange.
initial attempts to knock out hp <dig> using kanamycin or chloramphenicol antibiotic cassettes and the allelic exchange methodology failed.
several laboratories have reported that loss of active ccmg can be compensated by the addition of reducing agents to the growth medium.
therefore, we tested whether the addition of the dtt into growth medium enable us to obtain an h. pylori n6/ <dig> hp <dig> mutated strain.
it did not.
moreover, our attempts at trans-complementation of a genomic deletion with a plasmid-borne copy of hp <dig>  based on the strategy using modified phel <dig> complementing plasmids of low and high copy number described by boneca et al.
were also unsuccessful  <cit> .
taken together, these studies, in combination with our observation that a h. pylori hp <dig> mutated strain can only be generated in the presence of a reducing agent, suggest that both hp <dig> and hp <dig> provide an essential function in h. pylori.
hp <dig> is a redox partner of hp0377
dsb proteins that play a role in a cytochrome-c biogenesis, in both system i and system ii, are kept in the reduced form by the integral membrane protein dsbd, or its shortened analogue ccda.
both proteins catalyze the transfer of electrons from cytoplasmic thioredoxin across the inner membrane to the periplasm; however the mechanism of the process in the case of ccda is still unknown  <cit> .
apart from hp <dig>  which was described as ccda, a search for dsb homologs in the h. pylori genome, employing the e. coli dsb sequence as a query sequence, revealed that h. pylori contains another dsbd homolog .
similar to rodobacter capsulatus ccda and bacillus subtilis ccda, hp <dig> is a six-transmembrane  protein containing two cysteines at the end of the first and the fourth tm segments  <cit> .
hp <dig>  however, has five cysteines with an unusual, and as yet uncharacterized, spatial arrangement, and it could be involved in a process other than the cytochrome c biogenesis.
thus, to establish whether hp <dig> is responsible for re-reducing hp <dig>  we created a h. pylori strain lacking hp <dig>  the recombinant plasmid puwm <dig>  was used for the mutagenesis.
the plasmid contains the hp <dig> gene disrupted by insertion of a kanamycin resistance cassette into the gene coding sequence.
the hp <dig> mutants were only produced in the presence of the reducing agent dtt, which indicates the importance of hp <dig> and its reducing activity for bacterial survival.
we then tested whether deletion of hp <dig> affects the redox state of hp <dig>  we noticed that growth of the hp <dig> mutated strain without dtt was much slower than growth of the wt strain .
therefore, to check the influence of hp <dig> on the hp <dig> redox state, the h. pylori n <dig> hp0265− strain was cultivated  <dig> h longer than the wt strain to achieve the same cell density.
as shown in fig.
8a, the knock-out of hp <dig> results in the accumulation of a noticeable amount of hp <dig> in the oxidized state, which indicates the role of hp <dig> in re-reducing hp <dig>  the addition of dtt to the medium resulted in the presence of hp <dig> in the reduced form, clearly verifying the role of hp <dig> .fig.
<dig> effect of dtt on helicobacter pylori hp0265
− growth.
cultures of h. pylori hp0265
− were grown in brain heart infusion  broth supplemented with 10 % fetal bovine serum and kanamycin.
one culture was additionally supplemented with  <dig> mm dtt.
as a control wild type of helicobacter pylori n <dig> was grown under the same conditions
fig.
<dig> redox state of hp <dig> in wt and mutant: hp0265::aph measured for cells grown in the absence  or presence  of dtt.
bacterial cultures were treated with 10 % tca, followed by alkylation with ams.
cellular proteins including the reduced  and the oxidized  controls were separated by 14 % sds-page under non-reducing conditions, and western blot analysis using antibodies against hp <dig> was performed.
each lane contains proteins isolated from the same amount of bacteria
localization of hp0377
most of the ccmg proteins are membrane-anchored via the n-terminal transmembrane domain.
in silico analysis  did not indicate the presence of the transmembrane n-terminal domain in hp <dig>  though it confirmed the occurrence of this kind of domain located in the n-terminus of b. subtilis resa.
at the same time, in silico analysis using the lipop  <dig>  server showed that the hp <dig> amino-acid sequence contains a putative signal sequence that can be processed by signal peptidase ii, which suggested that hp <dig> is an inner membrane lipoprotein.
to confirm the predicted localization of hp <dig> in h. pylori, a subcellular fractionation experiment was carried out.
detection of hp <dig> with specific rabbit antibody against rhp <dig> revealed that hp <dig> is present in the inner membrane proteins compartment .
as a control for the method of subcellular fractionation of h. pylori used, we traced the cellular location of dsbi , which is an inner-membrane protein .fig.
<dig> localization of hp <dig>  panel a: immunoblot analysis with subcellular fractions of h. pylori strain  <dig> using anti-hp <dig> antibody.
lanes:  <dig> - whole cell lysate;  <dig> - periplasmic proteins;  <dig> - protein ladder;  <dig> - cytoplasmic proteins,  <dig> - inner membrane proteins;  <dig> - outer membrane proteins;  <dig> - membranes protein.
panel b: immunoblot analysis with subcellular fractions of h. pylori strain  <dig> using anti-dsbi antibody.
lanes:  <dig> - whole cell lysate;  <dig> - periplasmic proteins;  <dig> - protein ladder;  <dig> - inner membrane proteins;  <dig> - outer membrane proteins;  <dig> - membrane proteins,  <dig> - cytoplasmic proteins
hp <dig> contains an lsac motif located in the c-terminal of its potential signal sequence.
it has been shown that the two main strategies commonly used to confirm lipid modification of a protein  have not worked in h. pylori  <cit> .
thus, to provide further insight into the role of lsac motif of hp <dig>  we carried out alanine mutagenesis of c <dig>  a gene encoding the c25a mutated protein was cloned into phel <dig> and introduced into h. pylori n <dig>  in this experiment, we used the wt strain as we had not been able to generate a knock-out of hp <dig>  we expected that the mutated form of the hp <dig> , overexpressed from the plasmid, would locate in the periplasm or cytoplasm.
we found that the overexpression of hp <dig> had no effect on cell growth and that the changing c <dig> into a in the lsac motif of hp <dig> did not influence its anchoring to the cytoplasmic membrane .
hp <dig> is not active in e. coli cells
to test whether it would be possible to study hp <dig> activity in e. coli cells, the hp <dig> gene was cloned with its own promoter into a shuttle plasmid, phel <dig> , and introduced by transformation into wild-type e. coli, and into e. coli dsba and e. coli dsbc mutated cells.
the presence of hp <dig> protein expressed from recombinant plasmid in e. coli cells was confirmed by western blot analysis using specific rabbit anti-hp <dig> serum .
we found that hp <dig> does not restore the dsba wild type phenotype, as measured by motility and dtt tests, as well as by the alkaline phosphatase assay .
moreover, hp <dig> is not able to complement the e. colidsbc mutant, as measured by the copper sensitivity assay .
to clarify the lack of complementation of the ecdsbc mutation by hp <dig>  we determined its redox state in e. coli cells.
in both the e. coli wild type and in dsbd mutated cells, hp <dig> exists in an oxidized form, in contrast to what was observed in h. pylori, where it was present in a reduced form .
these results suggest that hp <dig> does not cooperate with e. coli dsbd.fig.
<dig> redox state of hp <dig> in wt and dsbd mutant e. coli strain transformed with puwm <dig> carrying hp <dig>  bacterial cultures were treated with 10 % tca, followed by alkylation with ams.
cellular proteins including the reduced  and the oxidized  controls were separated by 14 % sds-page under non-reducing conditions, and western blot analysis using antibodies against hp <dig> was performed.
each lane contains proteins isolated from the same amount of bacteria
the h. pylori genome does not contain a dsbd gene, and hp <dig> is reduced by ccda .
thus, to start analysis of the hp <dig> function in an e. coli background, we attempted to introduce hp <dig> into e. coli.
we cloned hp <dig> into the low copy number plasmid pmpm-k6Ω, which has an arabinose inducible promoter.
after the correctness of the recombinant plasmid construction was verified by sequencing, the plasmid was introduced by transformation into wt e. coli and a dsbd mutant e. coli that harbored a plasmid expressing hp <dig>  when the expression of hp <dig> was induced by  <dig>  % arabinose, the production of hp <dig> was lethal for cells .
discussion
hp <dig> is a thioredoxin-fold protein containing the csyc motif, which indicates that it functions as a disulfide oxidoreductase.
although there is no evidence that hp <dig> is involved in cytochrome c assembly in vivo, that is the likely case because its resolved structure is similar to that of other ccmg proteins, and because it is able to reduce the oxidized form of apocytochrome c in vitro  <cit> .
additionally, it is co-transcribed with the ccsba  gene that is involved in heme transport and its ligation to apocytochrome c  <cit> .
in this work we have showed that hp <dig> is present in vivo in the reduced form, which is a characteristic feature of thiol oxidoreductases being reductants.
the hp <dig> redox state in vivo is consistent with its redox potential determined by us  and by others , which classifies hp <dig> as a mild reductant.
this value is in good accord with the standard redox potential determined for the most gram-negative ccmg proteins, such as ecccmg , bjccmg  or paccmg  .
additionally, we have shown that hp <dig>  like other ccmgs, does not reduce insulin.
this finding is consistent with a generally accepted view that ccmg proteins are specific thiol-oxidoreductases involved in only the cytochrome c maturation pathway.
so far, the only known exception to this rule is tlpa from b. japonicum.
this protein is a reductant for the copper metallochapherone scol, but it also acts in the cytochrome maturation process and catalyzes the insulin reduction  <cit> .
the tlpa structure, when compared to other ccmgs, revealed some unusual properties that potentially substantiate its activity in the insulin reduction assay  <cit> .
although hp <dig> is inactive in the insulin reduction assay, it confers a disulfide isomerase activity almost as high as that of ecdsbc.
this atypical hp <dig> attribute is in accord with its capability to generate a dimeric form, as shown by size exclusion and glutaradehyde crosslinking methods.
it should be noted, in contrast to our data, that yoon et al.
reported that hp <dig> is monomeric.
as we and yoon et al.
used almost identical recombinant hp <dig> proteins, the inconsistency may result from various experimental methods employed  <cit> .
furthermore, the pka of the n-terminal cysteine of the cxxc motif of hp <dig> appears to be similar to those observed for ecdsbc or ecdsba but not to those determined for most ccmgs.
this observation suggests that the activity of hp <dig> in h. pylori, which possesses only two proteins having a trx fold with the cxxc motif , is distinct from that described for the classical ccmgs.
taking into account that there is no classical dsbc protein in the h. pylori proteome and the dimeric hp <dig> does not catalyze the recovery of active rnase from scrambled rnase , we concluded that hp <dig>  in contrast to most ccmgs that are involved in only the cytochrome c biogenesis process, is at least a bifunctional reductase.
figure  <dig> presents a model of hp <dig> functioning.
as the gel filtration experiment indicated that hp <dig> exists as a mixture of monomeric and dimeric forms, its dual function may be regulated by the mutual ratio between the two forms.
this suggestion is also supported by our interesting finding that hp <dig> is essential for cell viability.
additionally, the in vivo experiments provide some intriguing data.
we have noticed that the in vivo redox state of hp <dig> is conditioned by the presence of hp <dig> or hp <dig>  previous experiments on the function of hp <dig> showed that it is a periplasmic oxidase, and that hp <dig> is partially responsible for hp <dig> reoxidation.
therefore, they create a redox pair playing a role in introduction of disulfide bonds  <cit> .
as h. pylori does not encode a classical dsba, hp <dig> may oxidize apocytochrome c just after its transport across the inner membrane.
by analogy to the function of b. subtilis resa, we suggest that lack of oxidized apocytochrome c in hp <dig> mutated cells prevents hp <dig> from reacting with its redox partner , so a part of hp <dig> remains in the oxidized form  <cit> .
however this hypothesis requires verification because hp <dig>  as indicated by biochemical characterization, may also play a role in processes other than the cytochrome c maturation process.
overproduction of the periplasmic thiol oxidases, hp <dig> and hp <dig>  results in changes to the mutual ratio of the reduced and oxidized forms of hp <dig>  as in the hp <dig> mutated cells, only part of hp <dig> is present in a reduced form.
since the presence of this form is necessary for hp <dig> to fulfill its function, we decided to compare the cytochrome c activity in hp <dig> mutated cells to that observed in wt cells, using the tmpd-oxidation assay.
tmpd  is an artificial substrate of cytochrome c oxidase  <cit> .
we did not observe any differences in bacterial cell pigmentation , indicating that when the main periplasmic oxidase, i.e., hp <dig>  is absent, full hp <dig> activity is probably dispensable for cytochrome c biogenesis.fig.
<dig> models representing the role of h. pylori hp <dig> in cytochrome c biogenesis and in oxidized protein folding.
following synthesis, apocytochrome c is translocated across the membrane by the sec protein complex, and there it is oxidized by hp <dig> .
subsequently, its cxxch motif is reduced by hp <dig>  and it is ligated to heme.
likely, as observed for other microorganisms, some molecules of the apocytochrome c are not oxidized and are directly ligated with heme .
lack of hp <dig> prevents hp <dig> reduction by hp <dig>  a small portion of hp <dig> is present in a dimeric form and potentially plays a role in the dsb isomerization/reduction pathway.
so far, the substrates of this pathway remains unknown
most of the gram-negative bacteria ccmgs are membrane-anchored, periplasm-facing proteins  <cit> .
we also tested the subcellular localization of hp <dig> by cell fractionation experiments, which showed that it is present in the inner membrane proteins fraction.
these data contradict those of yoon et al., who classified hp <dig> as periplasmic protein  <cit> .
hp <dig> contains a putative signal sequence with a lipobox  that can potentially be processed by signal peptidase ii, which is specific for lipoprotein precursors  <cit> .
it has been shown that a lipoprotein signal peptidase gene  is present in the h. pylori genome and that it is essential for this bacterium  <cit> .
however current knowledge about the mechanism for lipoproteins transport across the inner membrane and their sorting in h. pylori is still limited and comes mainly from bioinformatic studies  <cit> .
we hypothesize that hp <dig> is a lipoprotein similar to two well-characterized h. pylori lipoproteins, lpp <dig> and hpaa, which also contain a lipobox  and show inhibited processing, at least when expressed in e. coli cells, by a globomicin, a cyclic peptide antibiotic specific for signal peptidase ii  <cit> .
unexpectedly, changing the c of the lsac hp <dig> motif into a did not influence its cell localization.
understanding the role of the hp <dig> lipobox requires more investigation, as has been done for some legionella lipoproteins  <cit> .
ccmg proteins that play a role in a cytochrome c biogenesis, in both system i and system ii, are kept in the reduced form by the integral membrane protein dsbd or its shorter analogue, ccda.
both proteins catalyze the transfer of electrons from cytoplasmic thioredoxin across the inner membrane to the periplasm.
dsbd consists of eight transmembrane segments , an n-terminal  and a c-terminal domain .
both the n- and c-terminal domains face the periplasm  <cit> .
ccda, which is a shorter version of dsbd, consist of only the β transmembrane domain of dsbd.
in contrast to dsbd, which transfers reducing potential to a large number of periplasmic proteins, ccda was thought to be only involved in the cytochrome c maturation process  <cit> .
however, recently published data, have showed that in bacillus subtilis or b. anthracis ccda plays a role also in sporulation and virulence  <cit> .
recently a new class of dsbd proteins, named scsb – which have a domain organization similar to but not identical with that of dsbd – has been described.
however, their role in ccmg re-reduction has not yet been analyzed  <cit> .
we show that hp <dig> is kept in the reduced form by hp <dig> and does not cooperate with e. coli ecdsbd.
in contrast to our data, it was demonstrated by katzen et al.
that ccda from rhodobacter capsulatus complements an e. coli dsbd gene deletion, and ecdsbd complements the lack of rcccda  <cit> .
however, r. capsulatus is different from h. pylori and b. subtillius, in that it has a ccda and an scsb.
in the publication that describes the e. coli dsbd that reduces r. capsulatus ccmg in the absence of its ccda, the authors failed to knock out the scsb  <cit> .
due to the structural resemblance, it is possible that scsb complements the absence of ccda.
hence, the conclusion that e. coli dsbd transfers electrons to the r. capsulatus ccmg might have been misleading.
the two conserved cysteine residues of ccda are membrane-embedded, and ccda lacks the corresponding alpha and gamma domains of dsbd.
the mechanism of transmembrane electron transfer by ccda is still inexplicable.
it is likely that it adopts an hourglass-like structure, similar to the dsb β domain, that allows it to interact with cytoplasmic thioredoxin and its periplasmic partners  <cit> .
we suggest that any dsbd can only transfer electrons in a proteome that has either a dsbd or a scsb, but not in a proteome that has only a ccda.
the effective transmembrane electron transfer is also determined by the structure of the proteins that are substrates of the envelope dsbd or dsbd–like proteins.
the best example is the legionella pneumophila dsb system.
this bacterium produces two dsba-like proteins , two dsbbs and two dsbds.
the dsbd-like proteins differ in their structure: lpdsbd <dig> resembles ecdsbd, whereas lpdsbd <dig> lacks the α domain present in ecdsbd.
lpdsba <dig>  the bifunctional protein acting as an oxidase and isomerase, interacts with both lpdsbds and also with ecdsbd, whereas ecdsbc is not reduced by lpdsbd <dig> or by lpdsbd <dig>  <cit> .
therefore, e. coli dsbd reduces bordetella ccmg, and not hp <dig>  because the bordetella genome encodes a dsbd, while the h. pylori proteome has a ccda but no dsbd/scsb  <cit> .
similarly, b. subtilis has a ccda but no dsbd/scsb, and therefore e. coli dsbd will not reduce its ccmg.
CONCLUSIONS
the fact that h. pylori contains only two classical dsb proteins, and at the same time, its genome encodes many proteins containing consecutive and nonconsecutive disulfide bonds, implies the presence of an atypical dsb protein network.
the experimental results presented strongly suggest that hp <dig>  despite a structural similarity to other ccmgs, functions differently from them.
biochemical analysis leads us to conclude that hp <dig> is a multifunctional protein.
as h. pylori possesses several proteins containing nonconsecutive disulfide bonds, we postulate that the activity of hp <dig> complements the lack of a classical dsbc.
also, analysis of the hp <dig> redox state in wt or dsb mutant h. pylori cells supports this assumption, as it indicates cooperation between the dimeric oxidoreductase hp <dig> and hp <dig>  however, further biochemical or structural experiments are required to confirm the nature of potential interaction between hp <dig> and hp <dig>  and to identify substrates of hp <dig>  if any.
additional files
additional file 1: figure s <dig>  analysis of redox state of hp <dig>  analysis of redox state of 6xhis-hp <dig> in the buffer containing  <dig>  mm gssg  and an increasing concentration of gsh .
fractions were resolved by  sds page, followed by gel staining with coomassie brilliant blue.
the diagram represents determination of the redox potential by equilibration against glutathione.
the fraction  of reduced hp <dig> at equilibrium was measured by analysis of redox state, and the intensity of the bands corresponding to the reduced protein compared to intensity corresponding to a protein in both forms was measured by imagej program.
additional file 2: figure s <dig>  the insulin reduction assay.
the reaction contained  <dig> μm insulin in potassium phosphate buffer, ph  <dig>  and 2mm edta.
the reaction was performed in the absence or presence of  <dig> μm ecdsba,  <dig> μm hp <dig>  reactions started by adding dtt to the final concentration of  <dig> mm.
the changes in the absorbance at  <dig> nm as a function of time were measured.
three independent experiments were performed.
additional file 3: figure s <dig>  oxidase activity assay.
the reaction contained  <dig> μm reduced rnase in  <dig> mm tris acetate, ph  <dig> ,  <dig> mm edta,  <dig>  mm gssg,  <dig> mm gsh, and  <dig> mm ccmp.
the reaction was performed in the absence or presence of  <dig> μm ecdsba or  <dig> μm hp <dig>  the changes in absorbance at  <dig> nm were measured as a function of time.
three independent experiments were performed.
additional file 4: figure s <dig>  production of hp <dig> in e. coli.
plasmid puwm <dig> was introduced to e. coli.
the expression of the heterologous gene was confirmed by western blot using anti-hp <dig> antibody.
lanes:  <dig> – protein ladder,  <dig> – e. coli dsba::kan,  <dig> – e. coli dsba::kan <dig> with empty phel <dig>   <dig> - e. coli dsba::kan <dig> with puwm <dig>  unspecific signals recognized by anti-hp <dig> antibody.
additional file 5: figure s <dig>  hp <dig> doesn’t restore the e. coli dsba
− wild type phenotype in three independent functional assays.
as a negative control e. coli dsba::aph was transformed with an empty phel <dig> vector.
motility assay;  alkaline phosphatase assay.
dtt sensitivity assay.
additional file 6: figure s <dig>  hp <dig> does not restore the e. coli dsbc
− wild type phenotype in the copper sensitive assays.
as a negative control e. coli dsbc::aph was transformed with an empty phel <dig> vector.
the numbers indicate:  <dig> – wt,  <dig> - dsbc::kan, 3 – dsbc::kan/hp0231
+, 4 – dsbc::kan/hp <dig>  5 – dsbc::kan/phel <dig>
additional file 7: figure s <dig>  production of hp <dig> is lethal in e. coli.
the diagrams show growth curves of different strains of e. coli : pr <dig>  pr <dig> , pr <dig>  pr <dig>  or with addition of  <dig>  % arabinose  at the time indicated with a black arrow.
paula roszczenko and magdalena grzeszczuk contributed equally to this work.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
ekjk, pr and mg conceived and designed the study.
ew was responsible for in silico analysis.
pr, mg, pk, pu, pw, en carried out the laboratory work.
ekjk, pr, mg, pk and ew analyzed the data.
ekjk, pr and mg wrote the manuscript.
all authors read and approved the final manuscript.

 disulfide bonds contribute to the stability and function of many extracytoplasmic, soluble or membrane-bound proteins. in gram-negative bacteria, the oxidative protein folding takes place in the periplasm and is controlled by proteins from the dsb family. in the highly oxidizing environment of the periplasm, there is also a need for selected proteins to be kept in a reduced form. in the assembly of c-type cytochromes, essential for energy metabolism, for example, the cytochrome c maturation process requires ligation of heme to reduced thiols of the cys-x-x-cys-his motif of apocytochrome. to date, there are two different systems for cytochrome-c biogenesis that have been found in bacteria: system i and system ii. they comprise two kinds of proteins acting in a coordinated fashion: those involved in heme handling and heme ligation to the apocytochrome, and those contributing to reduction of a disulfide bond of the cxxch heme-binding motifs. the cytochrome-c maturation machinery of the system i operating in many gram-negative bacteria consists of up to ten proteins. the apocytochrome reduction is accomplished by the action of ccmg, ccmh, and dsbd or ccda. dsbd/ccda transfers electrons from cytoplasmic thioredoxin to ccmg. then ccmg is re-oxidized by shuttling its electrons to ccmh, which finally transfers them to apocytochrome c. however, the details concerning mixed disulfide complexes created during the process still remain unclear. the remaining proteins play a role in heme transport and its ligation to the apocytochrome. system ii is simpler and more widespread among bacterial species. it is present in, for example, gram-positive bacillus subtilis. it contains four proteins, resa, resb, resc and ccda, of which resa is a functional counterpart of ccmg, and ccda is a functional counterpart of dsbd. resb and resc form a complex to deliver the heme, and they function in the cytochrome c-heme ligation. while in most microorganisms, the ccsb and ccsa proteins are present as two separate polypeptides, there are a few species of bacteria, such as wolinella, bacteroides and some strains of helicobacter spp., whose genes encode ccsa and ccsb fused into one large orf. many studies have tried to decipher the cooperation among periplasmic dsb proteins in cytochrome-c biogenesis. initially, it was thought that introduction of the disulfide bonds into the cxxch motif of the apocytochrome c, which happens just after its transport to the periplasm by the sec system, is an obligatory step of the process, as dsba and dsbb mutants in e. coli were unable to produce cytochrome c. however, recent data in the literature may contradict this scheme; the lack of the dsb proteins of the oxidative pathway in b. subtilis and r. capsulatus, for example, suppresses the cytochrome c deficiency of ccmg or ccda mutants. however, it should be noted that the dsb oxidative pathway functioning is dependent on cell growth conditions it has also been demonstrated that the heterologous expression of ccsba of h. pylori or ccsab from b. pertussis, both encoding cytochrome c synthetase, in an e. coli that lacks its own cytochrome c machinery results in c-type cytochrome formation. this observed effect was significantly enhanced by addition of exogenous reductant. detailed analysis of the paracoccus denitrificans cytochrome maturation in an e. coli dsba, dsbd double-mutant led mavridou et al. to the conclusion that apocytochrome is subjected to two competing reactions: either heme attachment to its cysteine thiols, or oxidation of those thiols. helicobacter pylori is a gram-negative spiral-shaped bacterium, a member of ε-proteobacteria that specifically colonizes the gastric epithelium of humans with severe consequences. data on h. pylori cytochromes are not comprehensive. analysis of its genome nucleotide sequence revealed that this microaerophilic microorganism possesses a rather simple respiratory chain consisting of three enzymes: quinol-cytochrome c reductase, cytochrome bc complex, cytochrome c and cb-type cytochrome c oxidase. further inspection of the h. pylori genome showed the existence of a second gene encoding a putative low molecular mass cytochrome c named cycb. both cyca and cycb are essential genes and cannot substitute for each other. cytochrome c encoded by the cyca gene, is a soluble periplasmic protein, a potential electron donor to the cb-type cytochrome c oxidase. to our knowledge, little is known about the c-type cytochrome maturation system of helicobacter pylori. in the genome of h. pylori there are proteins containing cxxc motifs characteristic of thiol:disulfide oxidoreductases identified to date. only two of these proteins have a thioredoxin-like fold and are periplasm-located. this microorganism uses system ii, and hp and hp, a homolog of resbc), whose products potentially play a central role in c-type cytochrome maturation, constitute one transcriptional unit. hp can, to some extent, complement for a lack of the e. coli cytochrome-c maturation machinery. the recently determined crystal structure of hp shows similarity to the structure of b. subtilis resa. however large structural differences between these two proteins were also observed. we have previously demonstrated that hp is a dimeric oxidoreductase that catalyzes disulfide bond formation in the periplasm. in this work, to gain further insight into the helicobacter pylori dsb protein network, we analyzed hp the second periplasmic oxidoreductase, using biochemical and genetic tools. methods bacterial strains, primers, plasmids, media and growth conditions bacterial strains, plasmids and primers used in this study are listed in tables  and two h. pylori strains were used in this study. although the sequence of the h. pylori genome is complete, this strain is inconvenient for genetic manipulation. h. pylori n originally isolated from a patient with gastritis, is useful for complementation experiments, and it is highly motile. both h. pylori strains were grown on blood agar base plates supplemented with 10 % horse blood and an antibiotic mixture consisting of vancomycin, polymyxin b, trimethoprim and amphotericin b at 37 °c under microaerobic conditions. liquid cultures of h. pylori were grown in brain heart infusion broth supplemented with 10 % fetal bovine serum. for the selection of h. pylori mutants or complemented strains, kanamycin or/and chloramphenicol were added to the growth media.table bacterial strains and plasmids used in this study dsbi + in trans complementant of dsbi::aph hp0231 + in trans complementant of hp0231::cat + + + + the e. coli strain tg was used as a host for the construction and preparation of recombinant plasmids. the e. coli strain rosetta was used to overexpress puwm puwm and puwm the e. coli strains jcb and jfc were employed for complementation experiments of e. coli dsba and dsbc mutants by hp e. coli strains were grown at 37 °c on solid or liquid luria-bertani medium or on m minimal medium. when needed, media were supplemented with antibiotics at the following concentrations: μg ml− ampicillin, μg ml− kanamycin and μg ml− chloramphenicol. general dna manipulations standard dna manipulations were carried out as described earlier or according to the manufacturer’s instructions. polymerase chain reactions were performed with primestar hs dna polymerase or hotstar hifidelity polymerase under standard conditions. synthetic oligonucleotides synthesis and dna sequencing were performed by genomed s.a., warsaw, poland. natural transformation of h. pylori the naturally competent h. pylori n was grown on ba plates for h. subsequently, bacteria were plated onto fresh plates for h. then – μg of plasmid dna was added and plates were incubated for h. afterwards, bacteria were transferred onto a plate supplemented with kanamycin or chloramphenicol/kanamycin, and transformants were grown for days. allelic exchange mutagenesis of the hp gene in h. pylori to inactivate hp a recombinant vector was constructed by a two-step pcr method. the upstream and downstream regions of the hp gene were amplified from h. pylori n genomic dna using two pairs of primers specific for hp and its flanking regions, n6_hp0265_up_bamhi-n6_hp0265_up_rev and n6_hp0265_dw_sali-n6_hp0265_dw_fwd, respectively. the n6_hp0265_up_rev and n6_hp0265_dw_sali primers contained nucleotide sequences complementary to each other, respectively. each pcr product was purified with a gel extraction kit. next, a mixture of two purified products was used as a template in a single pcr reaction, using the primers n6_hp0265_up_bamhi-n6_hp0265_dw_fwd. the resulting pcr product contained a 274-nucleotide deletion of hp the product was purified and cloned into pbluescript sk ii. to add the aph gene between the two hp arms, smai restriction enzyme was used to yield suicide plasmid puwm sequence analyses confirmed the correct construction of puwm and the recombinant plasmid was introduced into h. pylori n by natural transformation. cells were plated and screened on plates containing kanamycin and kanamycin with mm dtt to restore the reducing activity of hp an hp0265::aph mutant was obtained by a double cross-over using puwm and verified by pcr analysis, using the primer pair n6_hp0264_fwd1-n6_hp0265_rev allelic exchange mutagenesis of the hp gene in h. pylori to inactivate hp two recombinant vectors, pumw and puwm were constructed. puwm was generated by a two-step pcr method. briefly, primers km1f–km2r, were used to amplify the aph gene from puwm the upstream and downstream regions of the hp gene were amplified from h. pylori n genomic dna using two pairs of primers specific for hp and its flanking regions, hp0377i-hp0377iia and hp0377iiia-hp0377iv, respectively. the hp0377iia and hp0377iiia primers contained 5′ leader nucleotide sequences complementary to km1f and km2r, respectively. each pcr product was purified with a gel extraction kit. next a mixture of three purified products was used as a template in a single pcr reaction, using primers: hp0377i-hp0377iv. subsequently, the resulting pcr product, containing the aph gene inserted between the two hp arms in the same transcriptional orientation as the hp gene, was purified and cloned into pgem-t easy, generating the suicide plasmid puwm sequence analyses confirmed the correct construction of puwm and the recombinant plasmid was introduced into h. pylori n6/puwm by natural transformation. selection was done on blood agar plates containing kanamycin and chloramphenicol, with or without isopropyl-β-d-thiogalactopyranoside. the puwm plasmid used for insertional mutagenesis of the h. pylori n hp gene, was constructed by amplification of the hp gene and surrounding dna fragments by pcr, using the hp0377zew1-hp0377zew primers. subsequently, the resulting pcr product was purified and cloned into pgem-t easy, creating puwm the resulting construct was digested with bglii, to cut the restriction site in the middle of hp gene. the sticky ends were blunted using klenow fragment. the dna mixture was purified with a clean-up kit and ligated with a previously prepared kanamycin cassette, amplified from puwm by the pair of primers km1f–km2r. the generated suicide plasmid puwm containing the aph gene inserted between the two hp arms in the same transcriptional orientation as the hp gene, was confirmed by sequencing. the recombinant plasmid was introduced into h. pylori n6/puwm by natural transformation. selection was done on blood agar plates containing kanamycin and chloramphenicol, with or without isopropyl-β-d-thiogalactopyranoside. to create a conditional mutant in the hp gene, puwm was constructed based on the shuttle e. coli/h. pylori plasmid: pill the hp coding nucleotide sequence was amplified from h. pylori genomic dna without its own promoter by the primer pair hp0377mutf - hp0377mutr. the purified pcr products, as well as the shuttle plasmid, were digested with ndei/bamhi and ligated together to form puwm correct construction of puwm was confirmed by sequencing. next, puwm was introduced into h. pylori n and puwm529/puwm was used to inactivate hp gene. the strategy for cloning into pill was identical. site-directed mutagenesis of the hp gene to obtain mutated hp proteins, a set of recombinant plasmids was constructed from puwm which carries the hp gene without its promoter and signal sequence. cys-to-ser point mutations were generated using the quick change site-directed mutagenesis kit according to the manufacturer’s instructions, starting with ng of puwm template and ng of each primer. to obtain c25a mutated hp protein, a recombinant plasmid was constructed from puwm carrying the hp gene with its promoter and signal sequence. the cys-to-ala point mutation was generated as described above, using primers hp377_c25a_for and hp377_c25a_rev. protein analysis preparation of h. pylori and e. coli protein extracts, sds-page and blotting procedures were performed by standard techniques. preparation of subcellular fractions subcellular protein fractions were prepared from 48-h h. pylori cultures. periplasmic proteins were released from the cells using an osmotic-shock procedure. after decanting the periplasmic fraction, bacterial pellets were resuspended in mm tris–hcl, ph and sonicated to release the cell contents. subsequently, cell wall debris was removed and the supernatants were ultracentrifuged to separate the membrane and cytoplasmic fractions. finally, the cell envelope was fractionated into inner and outer membranes by selective solubilization of the inner membrane with  % sodium lauryl sarcosine. overexpression and purification of apocytochrome c and hp0231 hp was overexpressed by autoinduction from an e. coli rosetta/puwm strain and purified as previously described. hp expression vector was constructed by amplifying the region encoding the mature hp protein from the chromosome of h. pylori with primers hp1227_ndei-for and hp1227_xhoi-rev. the insert was cloned into pet24a with ndei and xhoi restriction enzymes, to yield plasmid puwm expression was induced by mm iptg at od600 ~ . after h in 37 °c, cultures were centrifuged and the cell pellet was suspended in mm sodium phosphate, ph, mm nacl, mm imidazole. cells were disrupted by ultrasonication. the cell lysate was centrifuged and the resulting supernatant was applied onto bio-scale mini profinity imac cartridges containing ni-charged resin. the protein was eluted with an imidazole gradient, using the ngc chromatography system. overexpression and purification of hp0377 hp expression vector was constructed by amplifying the region encoding the mature hp protein from the chromosome of h. pylori with primers hp377exi and hp377exii. the insert was cloned into pet28a with ncoi and xhoi restriction enzymes, to yield plasmid puwm the cytoplasm-located hp was overexpressed from puwm by autoinduction, and then it was purified by affinity chromatography, dialyzed against phosphate buffered saline and later used for rabbit immunization. the anti-hp rabbit serum was specific and recognized native hp as verified by western blot analysis. the hp c89s and c92s expression vectors were constructed by cutting out hp0377-changed sequences from puwm and puwm respectively, and cloning the inserts into pet28a as described above, resulting in formation of puwm and puwm all plasmids carried the hp0377-his translation fusion. for biochemical experiments, the protein was expressed and purified from e. coli rosetta harboring puwm puwm and puwm the proteins were overexpressed by autoinduction and then purified by affinity chromatography using ngc medium-pressure chromatography systems by bio-rad as described above. biochemical assays in vivo redox state of hp0377 the redox state of hp was visualized by alkylating the free cysteine residues using 4-acetamido-4′-maleimidylstilbene- ′-disulfonic acid. this agent can only modify covalently free thiols, resulting in a molecular mass increase of da. briefly, bacteria were harvested from ba plates after or h of incubation under microaerobic conditions. samples were standardized using the od of the culture, and ice-cold trichloroacetic acid was immediately added to the culture. whole-cell proteins were precipitated and collected by centrifugation, washed with ice-cold acetone, and then dissolved in mm tris–hcl, mm ethylenediaminetetraacetic acid,  % sds containing mm ams by agitation for min at 37 °c. the proteins in non-reducing laemmli buffer were resolved by 14 % sds-page without reducing agent. hp was then detected by an immunoblot analysis using an anti-hp antibody. as controls, we used samples previously treated with mm ditiotreitol for min at 30 °c before precipitation of the proteins with tca. determination of the pka value the ph-dependent ionization of the cys and cys was followed by the specific absorbance of the thiolate anion at nm. measurements were carried out at 25 °c in a buffer consisting of 10 mm tris, mm sodium citrate, mm edta and mm nacl, ph for ecdsba as a control, and for hp and its mutated forms, with an average initial protein concentration of μm. the ph of the protein solution was lowered to by the stepwise addition of aliquots of m hcl. absorbances at and nm were recorded on a spectrophotometer and corrected for the volume increase. the ph dependence of the thiolate specific absorbance signal reduced/oxidized) was fitted according to the henderson-hasselbach equation. determination of the redox potential of hp0377 the fractions of reduced and oxidized hp were determined using 4-acetamido-4′-maleimidylstilbene- ′-disulfonic acid trapping. briefly, hp were incubated overnight at room temperature in 50 mm kpi ph ,  mm edta and various glutathione /glutathione disulfide ratios. after incubation, proteins were precipitated with trichloroacetic acid. after 20-min incubation on ice, the samples were centrifuged, and the pellets were washed with cold acetone. after a second centrifugation, pellets were dried and resuspended in a buffer containing 20 mm ams,  % sds, 10 mm edta, and 50 mm tris–hcl. after 45-min incubation at 37 °c, with  rpm shaking samples were loaded onto 12 % sds-polyacrylamide gels under denaturing conditions. fractions of reduced and oxidized protein were determined using imagej). the redox potential was then calculated as described previously. alkaline phosphatase assay ap activity was measured as previously described, with some modifications. briefly, e. coli cell cultures were grown at 37 °c in minimal medium m until they reached mid-log phase. then, the cells were pelleted by centrifugation, washed twice with tris–hcl, and resuspended in tris–hcl. the od was measured spectrophotometrically. samples were equilibrated for min in a water bath at 28 °c, then μl p-nitrophenol phosphate was added, and the time was recorded. the reaction was allowed to proceed at 28 °c until development of yellow color was observed. at this point the reaction was stopped by adding μl m kh2po samples were centrifuged before the a and a measurements. insulin reduction assay the ability of hp and ecdsba to catalyze the reduction of insulin in the presence of dtt was determined as previously described. determination of oxidase and isomerase activities oxidative folding of reduced rnasea in vitro oxidative folding of reduced rnasea was performed for hp and ecdsba as described earlier, with a few modifications. proteins were oxidized with mm oxidized glutathione and incubated for h at room temperature. rnasea was reduced by overnight incubation at room temperature in mm tris acetate ph containing m guanidine hydrochloride and mm dtt. all proteins were then dialyzed on desalting columns and concentrated in pbs. native rnasea and ecdsba were used as positive controls. the redox state of the thiols was confirmed by ellman’s assay, which exploits the colorimetric change at a when ′-dithiobis- is converted to 2-nitro- thiobenzoate upon cleavage of the disulfide bond by free thiols. oxidase activity was measured by analyzing the cleavage of ccmp at a by refolded rnasea in the presence of tested enzymes. reactions were carried out in μl of pbs buffer containing mm tris acetate ph, mm edta, mm gssg, mm gsh, mm ccmp, rnasea and analyzed enzyme. the reaction mixtures were prepared in a 96-well plate format and read through min at 27 °c in a sunrise™ plate reader. three independent experiments were performed. refolding of scrambled rnasea in vitro refolding of scrambled rnasea was performed for hp and ecdsbc as described earlier, with a few modifications. proteins were reduced with mm dtt and incubated overnight at 4 °c. rnasea was first reduced by overnight incubation at room temperature in mm tris acetate ph containing m guanidine hydrochloride and mm dtt. then, in order to introduce incorrect disulfides, reduced rnasea was dialyzed against pbs buffer containing m guanidine hydrochloride, sparged with oxygen and incubated for days in the dark at room temperature. finally, mm hydrogen peroxide was added for min at 25 °c. all proteins were then dialyzed on desalting columns and concentrated in pbs. ecdsbc was used as a positive control. the redox state of the thiols was confirmed by ellman’s assay. rnasea activity was measured by analyzing the cleavage of ccmp as described for the oxidative test, with a change in the reaction mixture: mm tris acetate ph, mm edta, μm dtt, mm ccmp, rnasea and analyzed enzyme. three independent experiments were performed. determination of the interaction between hp and hp1227 oxidized hp was prepared by incubating the purified hp with a mm oxidized glutathione for h at 37 °c. reduced apocytochrome c was prepared by incubating the purified apocytochrome with mm dtt for h at 37 °c. excess dtt and oxidized glutathione was removed using desalting columns that were previously equilibrated with mm tris hcl ph, mm nacl. reduced apocytochrome c in mm tris–hcl ph, mm nacl was incubated for h at 37 °c in the presence of a twofold excess of hp0231ox. after incubation, the proteins were precipitated with trichloroacetic acid, washed three times with ice-cold acetone and then resuspended in μl of a reaction buffer consisting of mm tris–hcl ph, 2 % sds, mm ams. the oxidized and reduced forms of hp as well as reduced apocytochrome c were similarly treated with ams before sds–page under nonreducing conditions. ams-treated protein samples were separated by nonreducing sds–page and visualized by coomassie staining. size exclusion chromatography to verify that hp exists as a monomeric protein, size exclusion chromatography was employed. hp was loaded onto superdex hiload 16/ column and eluted with mm tris ph mm nacl. determination of the oligomeric state of hp using glutaraldehyde crosslinking of polypeptide chains with glutaraldehyde was performed essentially as described. hp was incubated at room temperature with different concentration of glutaraldehyde in m bicine–naoh buffer, mm dtt, m nacl for min and the reaction was quenched by adding ethanolamine–hcl to a final concentration of m. phenotype assays dtt sensitivity assay dtt sensitivity experiments were performed as previously described. a freshly prepared m dtt stock solution was dissolved in molten lb agar to the final concentration of mm dtt. the dtt agar plates were used within min of pouring to prevent oxidation of dtt by air. exponentially growing cultures were decimally diluted, and μl aliquots were spotted on the plates. the growth was observed after overnight incubation at 37 °c. the experiments were conducted in triplicate. motility assays e. coli cells were grown in liquid culture in lb broth until the od value was close to then bacteria were inoculated on lb soft agar plates containing  % agar with a sterile toothpick and incubated for h at 30 °c. copper sensitivity assay the copper sensitivity assay was performed as previously described. briefly, bacteria were grown in bhi media supplemented with mm or mm cucl strains were grown at 34 °c. ethics statement all studies involving animals were performed in accordance with ethical standards, after approval from the local ethics committee no. warsaw, poland 966/  determination of the in vivo redox state of hp0377 hp is an oxidoreductase, as it contains the trx domain with a cxxc motif located at the n-terminus of the first helix in the trx fold. the involvement of hp in the cytochrome c maturation process is indicated by its structure, its in vitro interaction with hp and the genetic organization of the dna fragment containing the hp gene. to clearly define whether hp functions in the oxidizing pathway or the reducing pathway, we first decided to determine the redox state of that protein in vivo using the ams-trapping technique. the in vivo redox state of an oxidoreductase usually reflects its activity in the cell, i.e., proteins that function in the oxidizing pathway, such as ecdsba, are maintained in the oxidized state in vivo, whereas proteins that function in the reducing pathway, such as ccmg, are maintained predominantly in the reduced state. we found that hp is present in the reduced form in wild-type cells, which suggests that hp functions as a reductase in h. pylori, which is in accord with its three dimensional structure. thus, we also determined the redox status of the hp in h. pylori lacking hp or dsbi. both proteins are active in the dsb oxidative pathway. as described earlier, hp introduces disulfide bonds and dsbi is partially responsible for hp re-oxidation. our results showed that a significant portion of hp is present in the oxidized form in both the hp and dsbi mutated cells. also the overproduction of hp or dsbi from a moderate copy number plasmid disturbs the redox homeostasis and results in the presence of hp in both reduced and oxidized forms. to clarify the link between hp and cytochrome c biogenesis, we decided to check whether apocytochrome c is a substrate of hp we found that hp was able to oxidize the reduced apocytochrome c in vitro fig. redox state of hp in wt and mutants: hp0231::cat, dsbi::aph and complemented strains of h. pylori strain n bacterial cultures were treated with 10 % tca, followed by alkylation with ams. cellular proteins including the reduced and the oxidized controls were separated by 14 % sds-page under non-reducing conditions, and western blot analysis antibodies against hp was performed. each lane contains proteins isolated from the same amount of bacteria fig. oxidase assay of hp towards apocytochrome c. two different sds pages were run to better visualize the shift between oxidized and reduced forms of proteins. a hp0231-dependent oxidation of reduced apocytochrome c. lane oxidized hp lane reduced hp lane reduced apocytochrome c treated with oxidized hp b hp0231-dependent oxidation of reduced apocytochrome c. lane oxidized apocytochrome c. lane reduced apocytochrome c, lane reduced apocytochrome c treated with oxidized hp different redox forms were detected by nonreducing sds-page after ams treatment, which results in an increase in the molecular mass of reduced proteins by about kda per thiol group. biochemical characterization of hp0377 to clearly define whether hp functions as oxidoreductase, we analyzed the biochemical properties of the protein. recombinant proteins used for biochemical analysis were produced as cytoplasmic proteins in e. coli cells and purified by affinity chromatography. first, to gain insight into the role played by hp we determined its redox potential by equilibrium incubation using gsh/gssg as a reference. we found that the hp redox potential is – mv, which makes it a rather weak reductant. next we determined the ability of hp to reduce insulin in the presence of dtt. the insulin reduction assay is commonly used to determine whether a protein can function as an oxidoreductase, regardless of its function in the reducing or the oxidizing pathway in vivo. insulin contains two intramolecular disulfide bonds that connect the a and b chains: reduction of these disulfide bonds causes the precipitation of the b chain, which can be monitored by following the increase of turbidity at nm. as shown in additional file 2: figure s the reaction lag time with hp in the insulin reduction assay is almost as long as the control reaction, whereas the lag time with ecdsba is min. therefore, hp likely cooperates with a specific substrate, which is typical of ccmg proteins. however, these data are in contrast to some previous data. to clarify this inconsistency, and because there is a similarity between the active site of hp and the disulfide isomerase ecdsbc, we performed a disulfide isomerase assay by evaluating the ability of hp to reactivate oxidized, scrambled rnase. scrnase was prepared as described in the methods section and did not contain free cysteines, as confirmed with ellman reagent. in this assay, the refolding efficiency of hp was almost as high as ecdsbc. to get a complete biochemical characterization of hp we also investigated its ability to catalyze the refolding of reduced-unfolded rnasea. as expected, hp did not show activity in this assay. as hp revealed high isomerizing activity, and because dsb proteins involved in the isomerization pathway exist as dimers, we also evaluated the potential oligomerization of hp using two methods hp that lacked its own signal sequence and contained a c-terminal his tag was purified from e. coli cytoplasm and used in both assays. we found that exposure of hp to glutaraldehyde, which stabilizes oligomeric proteins by covalent crosslink formation, resulted in generation of a protein with a molecular weight of kda. this result clearly showed that hp can exist as a dimer. the size exclusion experiment showed that hp eluted as two peaks, one with an estimated mass of kda, consistent with the size of the monomer, and the second with estimated mass of kda, consistent with the size of the homodimer. thus, the presented data allowed us to conclude that at least a portion of hp exists as a dimer.fig. isomerase activity assay. the reaction contained μm scrambled rnase in mm potassium phosphate buffer, ph, 2 mm edta, μm dtt, and 9 mm ccmp. the reaction was performed in the absence or presence of μm ecdsbc, μm hp the cleavage of ccmp by refolded rnase was monitored continuously at nm. the changes in the absorbance at nm as a function of time are presented. three independent experiments were performed fig. glutaraldehyde crosslinking of hp purified hp at mg/ml was cross-linked in the presence of different concentration of glutaraldehyde: lane 1–purified hp protein, lane 2–  %, lane 3–  %, lane 4–  %, lane 5–  %, lane 6–  % glutaraldehyde. m–monomers, d–dimers fig. size exclusion profile of the purified hp separated on a superdex hiload 16/ column and monitored by absorbance at nm. hp elutes as a two peaks at min, with an estimated mass of kda, consistent with the size of the homodimer and min. with estimated mass of kda, consistent with the size of the monomer. the column was calibrated with gel filtration standard: thyroglobulin, γ-globulin, ovalbumin, myoglobin, vitamin b. the relative positions of the chosen standards are marked with arrows the function of thiol-oxidoreductases depends on the pka values of their active-site cys residues. the pka of the hp active site thiols was determined by observing the change in absorption of the thiolate anion at nm as a function of ph in wt protein. the ph titration of wt hp showed two transitions, one with pka =   ±  and the other with pka =   ±  fig. 6a. because these data are atypical for ccmgs, we constructed two single-cys cxxc motif mutants of hp, which allowed independent measurement of the pka of each cys residue. as shown in fig. 6b, c the c89s mutant had a pka =   ±  and the c92s mutant a pka =   ± . these results agreed with the titrations of the wt protein. the pka value of the solvent-exposed active site cysteine is about. this is relatively acidic compared with the solvent-exposed active site cysteine of the e. coli ccmg protein, pka = , or the b. subtilis resa, pka = ; it is as acidic as e. coli dsba, which is a known oxidant whose pka of the solvent-exposed active site cysteine is, and it is close to the pka of ecdsbc,  ± . thus, hp is the first described ccmg protein having an acidic pka of the n-terminal cysteine of the cxxc motif, and at the same time, it presents a low redox potential.fig. thiol ionization equilibrium in hp followed by uv absorbance at nm. panel a: hp wild type. panel b: hp c89s. panel c: hp c92s hp is an essential protein for h. pylori to investigate the function of hp in h. pylori we decided to construct an isogenic hp knock-out strain by allelic exchange. initial attempts to knock out hp using kanamycin or chloramphenicol antibiotic cassettes and the allelic exchange methodology failed. several laboratories have reported that loss of active ccmg can be compensated by the addition of reducing agents to the growth medium. therefore, we tested whether the addition of the dtt into growth medium enable us to obtain an h. pylori n6/ hp mutated strain. it did not. moreover, our attempts at trans-complementation of a genomic deletion with a plasmid-borne copy of hp based on the strategy using modified phel complementing plasmids of low and high copy number described by boneca et al. were also unsuccessful. taken together, these studies, in combination with our observation that a h. pylori hp mutated strain can only be generated in the presence of a reducing agent, suggest that both hp and hp provide an essential function in h. pylori. hp is a redox partner of hp0377 dsb proteins that play a role in a cytochrome-c biogenesis, in both system i and system ii, are kept in the reduced form by the integral membrane protein dsbd, or its shortened analogue ccda. both proteins catalyze the transfer of electrons from cytoplasmic thioredoxin across the inner membrane to the periplasm; however the mechanism of the process in the case of ccda is still unknown. apart from hp which was described as ccda, a search for dsb homologs in the h. pylori genome, employing the e. coli dsb sequence as a query sequence, revealed that h. pylori contains another dsbd homolog. similar to rodobacter capsulatus ccda and bacillus subtilis ccda, hp is a six-transmembrane protein containing two cysteines at the end of the first and the fourth tm segments. hp however, has five cysteines with an unusual, and as yet uncharacterized, spatial arrangement, and it could be involved in a process other than the cytochrome c biogenesis. thus, to establish whether hp is responsible for re-reducing hp we created a h. pylori strain lacking hp the recombinant plasmid puwm was used for the mutagenesis. the plasmid contains the hp gene disrupted by insertion of a kanamycin resistance cassette into the gene coding sequence. the hp mutants were only produced in the presence of the reducing agent dtt, which indicates the importance of hp and its reducing activity for bacterial survival. we then tested whether deletion of hp affects the redox state of hp we noticed that growth of the hp mutated strain without dtt was much slower than growth of the wt strain. therefore, to check the influence of hp on the hp redox state, the h. pylori n hp0265− strain was cultivated h longer than the wt strain to achieve the same cell density. as shown in fig. 8a, the knock-out of hp results in the accumulation of a noticeable amount of hp in the oxidized state, which indicates the role of hp in re-reducing hp the addition of dtt to the medium resulted in the presence of hp in the reduced form, clearly verifying the role of hp.fig. effect of dtt on helicobacter pylori hp0265 − growth. cultures of h. pylori hp0265 − were grown in brain heart infusion broth supplemented with 10 % fetal bovine serum and kanamycin. one culture was additionally supplemented with mm dtt. as a control wild type of helicobacter pylori n was grown under the same conditions fig. redox state of hp in wt and mutant: hp0265::aph measured for cells grown in the absence or presence of dtt. bacterial cultures were treated with 10 % tca, followed by alkylation with ams. cellular proteins including the reduced and the oxidized controls were separated by 14 % sds-page under non-reducing conditions, and western blot analysis using antibodies against hp was performed. each lane contains proteins isolated from the same amount of bacteria localization of hp0377 most of the ccmg proteins are membrane-anchored via the n-terminal transmembrane domain. in silico analysis did not indicate the presence of the transmembrane n-terminal domain in hp though it confirmed the occurrence of this kind of domain located in the n-terminus of b. subtilis resa. at the same time, in silico analysis using the lipop server showed that the hp amino-acid sequence contains a putative signal sequence that can be processed by signal peptidase ii, which suggested that hp is an inner membrane lipoprotein. to confirm the predicted localization of hp in h. pylori, a subcellular fractionation experiment was carried out. detection of hp with specific rabbit antibody against rhp revealed that hp is present in the inner membrane proteins compartment. as a control for the method of subcellular fractionation of h. pylori used, we traced the cellular location of dsbi, which is an inner-membrane protein.fig. localization of hp panel a: immunoblot analysis with subcellular fractions of h. pylori strain using anti-hp antibody. lanes: - whole cell lysate; - periplasmic proteins; - protein ladder; - cytoplasmic proteins, - inner membrane proteins; - outer membrane proteins; - membranes protein. panel b: immunoblot analysis with subcellular fractions of h. pylori strain using anti-dsbi antibody. lanes: - whole cell lysate; - periplasmic proteins; - protein ladder; - inner membrane proteins; - outer membrane proteins; - membrane proteins, - cytoplasmic proteins hp contains an lsac motif located in the c-terminal of its potential signal sequence. it has been shown that the two main strategies commonly used to confirm lipid modification of a protein have not worked in h. pylori. thus, to provide further insight into the role of lsac motif of hp we carried out alanine mutagenesis of c a gene encoding the c25a mutated protein was cloned into phel and introduced into h. pylori n in this experiment, we used the wt strain as we had not been able to generate a knock-out of hp we expected that the mutated form of the hp, overexpressed from the plasmid, would locate in the periplasm or cytoplasm. we found that the overexpression of hp had no effect on cell growth and that the changing c into a in the lsac motif of hp did not influence its anchoring to the cytoplasmic membrane. hp is not active in e. coli cells to test whether it would be possible to study hp activity in e. coli cells, the hp gene was cloned with its own promoter into a shuttle plasmid, phel, and introduced by transformation into wild-type e. coli, and into e. coli dsba and e. coli dsbc mutated cells. the presence of hp protein expressed from recombinant plasmid in e. coli cells was confirmed by western blot analysis using specific rabbit anti-hp serum. we found that hp does not restore the dsba wild type phenotype, as measured by motility and dtt tests, as well as by the alkaline phosphatase assay. moreover, hp is not able to complement the e. colidsbc mutant, as measured by the copper sensitivity assay. to clarify the lack of complementation of the ecdsbc mutation by hp we determined its redox state in e. coli cells. in both the e. coli wild type and in dsbd mutated cells, hp exists in an oxidized form, in contrast to what was observed in h. pylori, where it was present in a reduced form. these results suggest that hp does not cooperate with e. coli dsbd.fig. redox state of hp in wt and dsbd mutant e. coli strain transformed with puwm carrying hp bacterial cultures were treated with 10 % tca, followed by alkylation with ams. cellular proteins including the reduced and the oxidized controls were separated by 14 % sds-page under non-reducing conditions, and western blot analysis using antibodies against hp was performed. each lane contains proteins isolated from the same amount of bacteria the h. pylori genome does not contain a dsbd gene, and hp is reduced by ccda. thus, to start analysis of the hp function in an e. coli background, we attempted to introduce hp into e. coli. we cloned hp into the low copy number plasmid pmpm-k6Ω, which has an arabinose inducible promoter. after the correctness of the recombinant plasmid construction was verified by sequencing, the plasmid was introduced by transformation into wt e. coli and a dsbd mutant e. coli that harbored a plasmid expressing hp when the expression of hp was induced by  % arabinose, the production of hp was lethal for cells. discussion hp is a thioredoxin-fold protein containing the csyc motif, which indicates that it functions as a disulfide oxidoreductase. although there is no evidence that hp is involved in cytochrome c assembly in vivo, that is the likely case because its resolved structure is similar to that of other ccmg proteins, and because it is able to reduce the oxidized form of apocytochrome c in vitro. additionally, it is co-transcribed with the ccsba gene that is involved in heme transport and its ligation to apocytochrome c. in this work we have showed that hp is present in vivo in the reduced form, which is a characteristic feature of thiol oxidoreductases being reductants. the hp redox state in vivo is consistent with its redox potential determined by us and by others, which classifies hp as a mild reductant. this value is in good accord with the standard redox potential determined for the most gram-negative ccmg proteins, such as ecccmg, bjccmg or paccmg. additionally, we have shown that hp like other ccmgs, does not reduce insulin. this finding is consistent with a generally accepted view that ccmg proteins are specific thiol-oxidoreductases involved in only the cytochrome c maturation pathway. so far, the only known exception to this rule is tlpa from b. japonicum. this protein is a reductant for the copper metallochapherone scol, but it also acts in the cytochrome maturation process and catalyzes the insulin reduction. the tlpa structure, when compared to other ccmgs, revealed some unusual properties that potentially substantiate its activity in the insulin reduction assay. although hp is inactive in the insulin reduction assay, it confers a disulfide isomerase activity almost as high as that of ecdsbc. this atypical hp attribute is in accord with its capability to generate a dimeric form, as shown by size exclusion and glutaradehyde crosslinking methods. it should be noted, in contrast to our data, that yoon et al. reported that hp is monomeric. as we and yoon et al. used almost identical recombinant hp proteins, the inconsistency may result from various experimental methods employed. furthermore, the pka of the n-terminal cysteine of the cxxc motif of hp appears to be similar to those observed for ecdsbc or ecdsba but not to those determined for most ccmgs. this observation suggests that the activity of hp in h. pylori, which possesses only two proteins having a trx fold with the cxxc motif, is distinct from that described for the classical ccmgs. taking into account that there is no classical dsbc protein in the h. pylori proteome and the dimeric hp does not catalyze the recovery of active rnase from scrambled rnase, we concluded that hp in contrast to most ccmgs that are involved in only the cytochrome c biogenesis process, is at least a bifunctional reductase. figure  presents a model of hp functioning. as the gel filtration experiment indicated that hp exists as a mixture of monomeric and dimeric forms, its dual function may be regulated by the mutual ratio between the two forms. this suggestion is also supported by our interesting finding that hp is essential for cell viability. additionally, the in vivo experiments provide some intriguing data. we have noticed that the in vivo redox state of hp is conditioned by the presence of hp or hp previous experiments on the function of hp showed that it is a periplasmic oxidase, and that hp is partially responsible for hp reoxidation. therefore, they create a redox pair playing a role in introduction of disulfide bonds. as h. pylori does not encode a classical dsba, hp may oxidize apocytochrome c just after its transport across the inner membrane. by analogy to the function of b. subtilis resa, we suggest that lack of oxidized apocytochrome c in hp mutated cells prevents hp from reacting with its redox partner, so a part of hp remains in the oxidized form. however this hypothesis requires verification because hp as indicated by biochemical characterization, may also play a role in processes other than the cytochrome c maturation process. overproduction of the periplasmic thiol oxidases, hp and hp results in changes to the mutual ratio of the reduced and oxidized forms of hp as in the hp mutated cells, only part of hp is present in a reduced form. since the presence of this form is necessary for hp to fulfill its function, we decided to compare the cytochrome c activity in hp mutated cells to that observed in wt cells, using the tmpd-oxidation assay. tmpd is an artificial substrate of cytochrome c oxidase. we did not observe any differences in bacterial cell pigmentation, indicating that when the main periplasmic oxidase, i.e., hp is absent, full hp activity is probably dispensable for cytochrome c biogenesis.fig. models representing the role of h. pylori hp in cytochrome c biogenesis and in oxidized protein folding. following synthesis, apocytochrome c is translocated across the membrane by the sec protein complex, and there it is oxidized by hp. subsequently, its cxxch motif is reduced by hp and it is ligated to heme. likely, as observed for other microorganisms, some molecules of the apocytochrome c are not oxidized and are directly ligated with heme. lack of hp prevents hp reduction by hp a small portion of hp is present in a dimeric form and potentially plays a role in the dsb isomerization/reduction pathway. so far, the substrates of this pathway remains unknown most of the gram-negative bacteria ccmgs are membrane-anchored, periplasm-facing proteins. we also tested the subcellular localization of hp by cell fractionation experiments, which showed that it is present in the inner membrane proteins fraction. these data contradict those of yoon et al., who classified hp as periplasmic protein. hp contains a putative signal sequence with a lipobox that can potentially be processed by signal peptidase ii, which is specific for lipoprotein precursors. it has been shown that a lipoprotein signal peptidase gene is present in the h. pylori genome and that it is essential for this bacterium. however current knowledge about the mechanism for lipoproteins transport across the inner membrane and their sorting in h. pylori is still limited and comes mainly from bioinformatic studies. we hypothesize that hp is a lipoprotein similar to two well-characterized h. pylori lipoproteins, lpp and hpaa, which also contain a lipobox and show inhibited processing, at least when expressed in e. coli cells, by a globomicin, a cyclic peptide antibiotic specific for signal peptidase ii. unexpectedly, changing the c of the lsac hp motif into a did not influence its cell localization. understanding the role of the hp lipobox requires more investigation, as has been done for some legionella lipoproteins. ccmg proteins that play a role in a cytochrome c biogenesis, in both system i and system ii, are kept in the reduced form by the integral membrane protein dsbd or its shorter analogue, ccda. both proteins catalyze the transfer of electrons from cytoplasmic thioredoxin across the inner membrane to the periplasm. dsbd consists of eight transmembrane segments, an n-terminal and a c-terminal domain. both the n- and c-terminal domains face the periplasm. ccda, which is a shorter version of dsbd, consist of only the β transmembrane domain of dsbd. in contrast to dsbd, which transfers reducing potential to a large number of periplasmic proteins, ccda was thought to be only involved in the cytochrome c maturation process. however, recently published data, have showed that in bacillus subtilis or b. anthracis ccda plays a role also in sporulation and virulence. recently a new class of dsbd proteins, named scsb – which have a domain organization similar to but not identical with that of dsbd – has been described. however, their role in ccmg re-reduction has not yet been analyzed. we show that hp is kept in the reduced form by hp and does not cooperate with e. coli ecdsbd. in contrast to our data, it was demonstrated by katzen et al. that ccda from rhodobacter capsulatus complements an e. coli dsbd gene deletion, and ecdsbd complements the lack of rcccda. however, r. capsulatus is different from h. pylori and b. subtillius, in that it has a ccda and an scsb. in the publication that describes the e. coli dsbd that reduces r. capsulatus ccmg in the absence of its ccda, the authors failed to knock out the scsb. due to the structural resemblance, it is possible that scsb complements the absence of ccda. hence, the conclusion that e. coli dsbd transfers electrons to the r. capsulatus ccmg might have been misleading. the two conserved cysteine residues of ccda are membrane-embedded, and ccda lacks the corresponding alpha and gamma domains of dsbd. the mechanism of transmembrane electron transfer by ccda is still inexplicable. it is likely that it adopts an hourglass-like structure, similar to the dsb β domain, that allows it to interact with cytoplasmic thioredoxin and its periplasmic partners. we suggest that any dsbd can only transfer electrons in a proteome that has either a dsbd or a scsb, but not in a proteome that has only a ccda. the effective transmembrane electron transfer is also determined by the structure of the proteins that are substrates of the envelope dsbd or dsbd–like proteins. the best example is the legionella pneumophila dsb system. this bacterium produces two dsba-like proteins, two dsbbs and two dsbds. the dsbd-like proteins differ in their structure: lpdsbd resembles ecdsbd, whereas lpdsbd lacks the α domain present in ecdsbd. lpdsba the bifunctional protein acting as an oxidase and isomerase, interacts with both lpdsbds and also with ecdsbd, whereas ecdsbc is not reduced by lpdsbd or by lpdsbd. therefore, e. coli dsbd reduces bordetella ccmg, and not hp because the bordetella genome encodes a dsbd, while the h. pylori proteome has a ccda but no dsbd/scsb. similarly, b. subtilis has a ccda but no dsbd/scsb, and therefore e. coli dsbd will not reduce its ccmg.  the fact that h. pylori contains only two classical dsb proteins, and at the same time, its genome encodes many proteins containing consecutive and nonconsecutive disulfide bonds, implies the presence of an atypical dsb protein network. the experimental results presented strongly suggest that hp despite a structural similarity to other ccmgs, functions differently from them. biochemical analysis leads us to conclude that hp is a multifunctional protein. as h. pylori possesses several proteins containing nonconsecutive disulfide bonds, we postulate that the activity of hp complements the lack of a classical dsbc. also, analysis of the hp redox state in wt or dsb mutant h. pylori cells supports this assumption, as it indicates cooperation between the dimeric oxidoreductase hp and hp however, further biochemical or structural experiments are required to confirm the nature of potential interaction between hp and hp and to identify substrates of hp if any. additional files additional file 1: figure s analysis of redox state of hp analysis of redox state of 6xhis-hp in the buffer containing mm gssg and an increasing concentration of gsh. fractions were resolved by sds page, followed by gel staining with coomassie brilliant blue. the diagram represents determination of the redox potential by equilibration against glutathione. the fraction of reduced hp at equilibrium was measured by analysis of redox state, and the intensity of the bands corresponding to the reduced protein compared to intensity corresponding to a protein in both forms was measured by imagej program. additional file 2: figure s the insulin reduction assay. the reaction contained μm insulin in potassium phosphate buffer, ph and 2mm edta. the reaction was performed in the absence or presence of μm ecdsba, μm hp reactions started by adding dtt to the final concentration of mm. the changes in the absorbance at nm as a function of time were measured. three independent experiments were performed. additional file 3: figure s oxidase activity assay. the reaction contained μm reduced rnase in mm tris acetate, ph, mm edta, mm gssg, mm gsh, and mm ccmp. the reaction was performed in the absence or presence of μm ecdsba or μm hp the changes in absorbance at nm were measured as a function of time. three independent experiments were performed. additional file 4: figure s production of hp in e. coli. plasmid puwm was introduced to e. coli. the expression of the heterologous gene was confirmed by western blot using anti-hp antibody. lanes: – protein ladder, – e. coli dsba::kan, – e. coli dsba::kan with empty phel - e. coli dsba::kan with puwm unspecific signals recognized by anti-hp antibody. additional file 5: figure s hp doesn’t restore the e. coli dsba − wild type phenotype in three independent functional assays. as a negative control e. coli dsba::aph was transformed with an empty phel vector. motility assay; alkaline phosphatase assay. dtt sensitivity assay. additional file 6: figure s hp does not restore the e. coli dsbc − wild type phenotype in the copper sensitive assays. as a negative control e. coli dsbc::aph was transformed with an empty phel vector. the numbers indicate: – wt, - dsbc::kan, 3 – dsbc::kan/hp0231 +, 4 – dsbc::kan/hp 5 – dsbc::kan/phel additional file 7: figure s production of hp is lethal in e. coli. the diagrams show growth curves of different strains of e. coli: pr pr, pr pr or with addition of  % arabinose at the time indicated with a black arrow. paula roszczenko and magdalena grzeszczuk contributed equally to this work. competing interests the authors declare that they have no competing interests. authors’ contributions ekjk, pr and mg conceived and designed the study. ew was responsible for in silico analysis. pr, mg, pk, pu, pw, en carried out the laboratory work. ekjk, pr, mg, pk and ew analyzed the data. ekjk, pr and mg wrote the manuscript. all authors read and approved the final manuscript. 
in the genome of h. pylori proteins containing the cxxc motif characteristic of thioldisulfide oxidoreductases have been identified to date. additionally, although its standard redox potential is - mv, it is the first described ccmg protein having an acidic pka of the n-terminal cysteine of the cxxc motif, similar to e. coli dsba or e. coli dsbc. the ccmg proteins that play a role in a cytochrome c-maturation, both in system i and system ii, are kept in the reduced form by an integral membrane protein dsbd or its analogue, ccda. in h. pylori hp is re-reduced by ccda; however in e. coli it remains in the oxidized state as it does not interact with e. coli dsbd. the present work shows that hp is present in h. pylori cells only in a reduced form and that absence of the main periplasmic oxidase hp influences its redox state. however, only two of these proteins have a thioredoxin-like fold and are periplasm-located. our in vivo work also suggests that both hp which plays a role in apocytochrome reduction, and hp which is involved in heme transport and its ligation into apocytochrome, provide essential functions in h. pylori. our biochemical analysis indicates that hp is a specific reductase, as it does not reduce insulin. the present data, in combination with the resolved three-dimensional structure of the hp suggest that hp is an unusual, multifunctional ccmg protein. we have previously shown that hp is a dimeric oxidoreductase that catalyzes disulfide bond formation in the periplasm.
 in the genome of h. pylori proteins containing the cxxc motif characteristic of thioldisulfide oxidoreductases have been identified to date. however, only two of these proteins have a thioredoxin-like fold and are periplasm-located. we have previously shown that hp is a dimeric oxidoreductase that catalyzes disulfide bond formation in the periplasm. although hp was originally described as dsbc homologue, its resolved structure and location of the hp gene in the genome indicate that it is a counterpart of ccmg/dsbe.  the present work shows that hp is present in h. pylori cells only in a reduced form and that absence of the main periplasmic oxidase hp influences its redox state. our biochemical analysis indicates that hp is a specific reductase, as it does not reduce insulin. however, it possesses disulfide isomerase activity, as it catalyzes the refolding of scrambled rnase. additionally, although its standard redox potential is - mv, it is the first described ccmg protein having an acidic pka of the n-terminal cysteine of the cxxc motif, similar to e. coli dsba or e. coli dsbc. the ccmg proteins that play a role in a cytochrome c-maturation, both in system i and system ii, are kept in the reduced form by an integral membrane protein dsbd or its analogue, ccda. in h. pylori hp is re-reduced by ccda; however in e. coli it remains in the oxidized state as it does not interact with e. coli dsbd. our in vivo work also suggests that both hp which plays a role in apocytochrome reduction, and hp which is involved in heme transport and its ligation into apocytochrome, provide essential functions in h. pylori.  the present data, in combination with the resolved three-dimensional structure of the hp suggest that hp is an unusual, multifunctional ccmg protein. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords dsbhelicobacter pyloricytochrome c biogenesisisomerase activityissue-copyright-statement© the author 2015 