BACKGROUND
iron is the second most abundant metal in the earth’s crust; however, ferrous iron, the useable form, is scarce, due to an oxidative environment .
it is an essential element for most bacteria with irreplaceable functions in many basic biological processes, including the fe3+ cofactor of metabolic respiratory chain reactions.
these include photosynthesis, n <dig> fixation, methanogenesis, respiration, tricarboxylic acid  cycle, and dna biosynthesis  <cit> .
to prevent the invasion of bacteria, hosts, including humans, have evolved to minimize the concentration of free iron by secreting transferrin and lactoferrin, which have high affinity for iron  <cit> .
therefore, bacteria have evolved effective ways to survive in this harsh environment.
under this process, cell envelope proteins in gram-negative bacteria, including periplasmic, outer and inner membrane proteins, are essential in the transport of iron related compounds into the intracellular environment.
the cell envelope proteins related to iron homeostasis mechanisms are well studied in escherichia coli, in which outer membrane proteins , such as cira, feca, fepa, btub, fhua, fhue, and ybil, serve as receptors of siderophores or heme to transport iron into the periplasmic space  <cit> .
some other omps, such as ompw, tsx and ompx function as either iron exporters or receivers  <cit> .
during this process, the periplasmic protein tonb and inner membrane proteins  exbb and exbd form a protein complex to mediate the transport of iron compounds from the outer membrane to periplasm  <cit> .
the periplasmic binding proteins, such as fepb, fhub, fhud and fecb in escherichia coli deliver iron compounds to atp-binding cassette  transporters in the cytosolic membrane and then into the cytosol  <cit> .
meanwhile, iron starvation triggers some complex networks and metabolic pathways via the fur gene family which regulates iron homeostasis  <cit> .
it is well known that the aquatic pathogen, aeromonas hydrophila, causes serious disease outbreaks in numerous farmed fish populations, and leads to big economic losses in the aquaculture and fishery industry, annually .
however, knowledge is limited on the iron scavenging mechanism of this pathogen, especially for the role of the cell envelope proteins during the competition for iron resources with the host.
in this study, the differential expressions of the envelop proteins of a. hydrophila were compared in the presence/absence of the iron chelating medium by a dimethyl labelling based quantitative proteomic method.
bioinformatics analysis found some important biological processes involved.
furthermore, some of the altered proteins were validated by quantitative polymerase chain reaction  analysis and subjected to related functional validation.
we provide the first report, to our knowledge, on the iron homeostasis functions of cell envelope proteins in a. hydrophila.
RESULTS
characterization and identification of the envelope protein fraction in a. hydrophila
according to the growth curve of a. hydrophila, the strain treated with 150 μm  <dig>  2′-dipyridyl  grew slower than the control strain, especially after the middle exponential phase .
to investigate the biological functions of envelope proteins in iron homeostasis, bacterial envelope proteins in presence/absence of dip with the concentration of 150 μm were extracted.
sds-page separation showed some significant differences between dip treatment and the control, and the whole cell lysates of a. hydrophila were made to probe the cell envelope preparation enrichment in the preparations .
after comparing with these differences between dip treatment and non-treatment, the extracted cell envelope proteins of a. hydrophila were then in-solution digested and labeled by stable isotope dimethyl.
further lc ms/ms analysis identified  <dig> proteins with at least two peptides required for identification and a false discovery rate  less than 1 % filtered in the total number of  <dig> proteins .fig.
<dig> comparative characteristics of cell envelope protein of a. hydrophila in iron limited medium a growth curve of a. hydrophila atcc  <dig> with and without 150 μm dip in lb medium; b cbb-stained sds page map of the cell envelope of a. hydrophila with and without 150 μm dip and whole cell lysates of a. hydrophila as the comparison.
lane m contained molecular mass standards; c the subcellular localization of the identified proteins from ms results predicted by online software gneg-mploc; d blastp top-hit species distribution using local blast2go.
numbers of top hit sequences from blastp were calculated for each species; e and f gene ontology categories for the differentially expressed proteins of a. hydrophila in iron starvation using local blast2go analysis and classified into biological processes  and molecular functions .
the red and blue bars indicate up-regulation  and down-regulation  differential ratios of genes, respectively.
each related gene numbers are showed on the right of bars
note: pm., om., im., ex.
and cm.
mean the location of periplasm, outer membrane, inner membrane, extracell and cytoplasm, respectively
according to the online location prediction software, we found that these identified proteins included  <dig> cytoplasmic proteins ,  <dig> inner membrane proteins ,  <dig> periplasmic proteins ,  <dig> outer membrane proteins ,  <dig> extracellular proteins , and  <dig> fimbrium proteins  with some overlapping proteins in the predicted locations .
differential expression of envelope proteins in a. hydrophila in iron-limited medium
to further investigate the biological behavior of a. hydrophila under iron-limiting conditions, dimethyl labeling based quantitative proteomic technology was used to analyze the differential expression of envelope proteins.
in the  <dig> identified proteins,  <dig> membrane envelope proteins were found to be differentially expressed.
taking overlapping locations into account,  <dig> proteins including  <dig> omps,  <dig> imps,  <dig> periplasmic proteins and three fimbrial proteins were up-regulated, while  <dig> proteins including seven omps,  <dig> imps and  <dig> periplasmic proteins decreased in abundance in the iron starvation medium.
of these altered proteins, seven outer membrane proteins, a0kjp <dig>  r4w0j <dig>  r4vc <dig>  a0kjn <dig>  a0kgw <dig>  r4va <dig>  and k1jld <dig>  which serve as ferrienterobactin, ferrichrome, hemin, siderophore and heme receptors, and at less five periplasmic abc transporters  and exbb family protein  increased in abundance.
however, ompw  of a. hydrophila presented a down-regulated trend, although its biological function is still unknown.
interestingly, besides the outer membrane siderophore receptors, there were more outer membrane proteins altered, including r4vaf <dig> , a0kk <dig> , a0klx <dig> , r4vch <dig> , r4vrx <dig> , a0kny <dig> , r4vnf <dig> , a0khf <dig>  and r4vr <dig> .
meanwhile, seven c-type cytochromes  decreased in abundance.
these proteins contained iron ion binding domains suggesting they reduce the use of iron and lead to the decrease of the respiratory electron transfer chain under iron starvation.
on the contrary, atp synthase subunits , the final terminal point of cellular respiration, were found to be up-regulated.
while the purpose of this opposite phenomenon is unknown, we hypothesize that it provides an atp source for ferrous iron scavenging.
proteomics analysis also showed that almost all of the altered proteins located in the extracellular space were up-regulated, such as hemolysin  and fimbrial protein  could be explained as an attempt by the cell to strengthen the acquisition of scarce iron ions from outside and try to escape from the threatening surroundings.
in addition, many proteases such as q49ka <dig> , a0kiu <dig> , a0kgx <dig> , r4vu <dig>  reductase) and a0kfk <dig>  were found to increase in extracellular space.
investigation of the classification and functional annotation analysis of altered proteins
to further characterize the altered envelope proteins, a biological functional classification was clustered using bioinformatic analysis.
the local blast2go software was chosen to maximize homologous gene/protein annotations because of the limitation of gene ontology  annotations of a. hydrophila.
the blastp program was run against the swiss-prot database, producing  <dig>  matches from a total of  <dig> candidates with blast expected values ≤10− <dig> using blast2go.
figure 1d shows the basic local alignment search tool  hit distribution across various species of organisms for all altered sequences in the dataset.
further analysis identified the most enriched go terms comparing the up- and down-regulated proteins clusters including transport , macromolecule metabolic process , oxidation reduction process , small molecule metabolic process , generation of precursor metabolites and energy  in biological process category and cation binding , tetrapyrrole binding , iron-sulfur cluster binding , active transmembrane transporter activity  and iron chelating transmembrane transporter activity  in molecular function category .
validation of selected altered proteins at the mrna level using q-pcr
according to the proteomic results and data analysis, up-regulated transport processes, down-regulated redox and energy generation process were the major response characteristics of envelope proteins in iron starvation.
to confirm this, we investigated the mrna levels of a total of  <dig> selected genes found to be altered in the proteomic analysis, using a q-pcr method.
of these  <dig> genes, five genes  were h+-atpase subunits, seven genes  were related with the transport process, six genes  were involved in the redox process and three genes  were involved in the energy generation process.
results showed that most of the transcription levels of these  <dig> related genes were consistent with our proteomics conclusions .
in the selected transport-process related genes, the mrna level of a0kjp <dig>  a tonb-dependent siderophore receptor, displayed a very significant up-regulation  under iron starvation, suggesting its crucial role in iron uptake.
on the other side, the transcription of other candidate genes increased from  <dig>  to  <dig>  fold, except for r4vlv <dig> and r4vj <dig>  which only increased slightly, with a ratio of  <dig>  and  <dig> , respectively.fig.
<dig> the validation of relative expression behaviors of transport, redox, and energy generation processes were selected and verified by q-pcr the mrna level of  <dig> transport related genes which were up-regulated at the protein level ,  <dig> oxidation-reduction and  <dig> energy generation process related genes which were down-regulated at the protein level , were analyzed by q-pcr
in the redox process and energy generation process, the down-regulated mrna levels of r4v7y <dig>  r4vnp <dig>  r4vf <dig>  a0kl <dig>  and r4vs <dig> were in agreement with protein levels, while k1jgn <dig>  a0kqv <dig> only changed slightly.
interestingly, the transcription of r4vnl <dig>  and r4vh <dig>  increased, contradictory to the proteomic results.
in summary, up to 81 %  of genes displayed the same directional change in both protein and mrna levels, 19 %  showed no correlation, while only 10 %  were dysregulated in the opposite direction.
tca related exogenous metabolites elevate susceptibility of a. hydrophila to iron starvation
in this study, we found the down-regulation of the tca related energy generation processes under iron starvation, using a proteomics approach.
we assume that the down-regulation of the energy generation process takes part in the iron homeostasis mechanism rather than simply stimulation from iron depletion.
to validate this, serial dilutions of the tca related exogenous metabolites were added in lb medium with or without dip to stimulate the tca process.
as shown in fig.
<dig>  low of concentration exogenous metabolites alone did not affect bacterial growth while high levels of metabolites, such as oxaloacetic acid , α-ketoglutarate , succinic acid  and citric acid , did depress the bacterial growth rate.
however, when the exogenous metabolites were added in lb medium with 300 μm dip, most of them elevated susceptibility of a. hydrophila to iron starvation, especially α-ka and ca, which both depressed bacterial growth significantly at 8 mm .fig.
<dig> effect of exogenous metabolites on susceptibility of a. hydrophila to iron starvation.
a–d the growth curve of a. hydrophila strain in 300 μm dip mm oxaloacetic acid , α-ketoglutarate , succinic acid  and citric acid  treatment, respectively.
the concentration of these four reagents are in the gradient of  <dig>   <dig>   <dig> and 8 mm.
the growth kinetics of treated cells were recorded by absorbance measurements at od <dig> nm, at 30 °c with the multi-mode detection platform
h+-atp synthase activity was increased in the iron starvation environment
we further confirmed the behavior of h+-atp synthase under iron limited conditions.
h+-atpase activity was measured with a serial dilution of dip treatment.
compared to control, the h+-atpase activities of a. hydrophila were increased in response to iron starvation , which are consistent with our proteomics results.
we then investigated the biological characteristics of h+-atp synthase in iron starvation using n, n’-dicyclohexylcarbodiimide , an inhibitor of atp synthase.
when treated with 20 μg/ml dccd, the bacterial survival rate was significantly decreased by  <dig> ,  <dig>  and  <dig>  fold with the increase of dip concentrations 150 μm, 200 μm and 300 μm, respectively .
to further investigate the extent of functional differences under iron-limitation, an orthogonal test was performed by using serials of dip and dccd concentrations and then displayed as color grading according to their survival colony accounting .
results showed that, with the increasing of dip plus dccd treatments, the survival rates of a. hydrophila, were dramatically decreased.fig.
<dig> the functional validation of atp synthase under iron starvation conditions.
a the h+-atp synthase activity of a. hydrophila in a serial of dip concentrations in  <dig>   <dig>   <dig> and 300 μm were measured spectrophotometrically at 340 nm using the h+-atpase assay kit b the survival ratio of a. hydrophila when treated with 20 μg/ml dccd in a serial dilution of dip concentrations in  <dig>   <dig>   <dig> and 300 μm for 12 h and observed by colony counting, respectively.
c color grading of the effect of a serial of concentrations of dip from 0 μm to 300 μm and dccd from 0 μg/ml to 40 μg/ml.
the colony amounts of bacteria without treatment were normalized to  <dig> as control
discussion
aquatic pathogenic bacteria a. hydrophila cause a fulminant epidemic in aquaculture, including some extremely severe bacterial diseases, such as hemolytic ascitic disease, bacterial septicemia, and fulminant hemorrhage  <cit> .
so far, the response mechanism of a. hydrophila under some metal ion deficient conditions is not clear.
other bacteria, in order to respond to physiological iron-deficient conditions, have evolved a series of strategies to compete with the host for the uptake of life-essential iron .
during this process, the cell envelope has an important effect on the extracellular and intrinsic communications due to its specific position, especially for the competition of essential nutrients in harsh environments  <cit> .
in order to systematically study the iron acquisition mechanism of the bacterial cell envelope system under the external pressure of iron limiting conditions, a dimethyl labeling based quantitative proteomic strategy was used to compare the differential expression of cell envelope proteins in a. hydrophila.
about 57 % of cell envelope proteins were extracted and identified in the current study.
based on the fact that cytoplasmic proteins contributed to the major contamination in all reported extraction methods, the tse  extraction used in this study was documented to produce the cleanest extraction of envelope proteins up to date  <cit> .
compared to the 52 % enrichment ratio in e. coli, using the same method in the mentioned reference, the 57 % of cell envelope proteins enriched in a. hydrophila, should be acceptable  <cit> .
thus, in this study we acquired an acceptable cell envelope protein enrichment in a. hydrophila.
in the present study, according to the final quantitative proteomic analysis, which can be summarized into the following statements: firstly, the expression of iron source receptors, which are located in the bacterial outer membrane, induced iron scavenging.
these are high-affinity iron chelators and competitively bind iron from iron sources.
actually, under iron deprivation, there are many reports about the upregulation of proteins involved in regular channel forming proteins, such as siderophore receptor channels, cira, feca, fepa, fhua, fhue, and ybil in e coli  <cit> , and other channel proteins, such as ompx in e.coli  <cit> .
those results demonstrated the competitive uptake system of iron in a.hydrophila under iron-limiting conditions.
secondly, after the ferri-siderophores and other iron sources specifically bind to outer membrane receptors, an energy generation system for transport is required.
because there was no atp energy supply in the outer membrane, the exbb–exbd–tonb system assisted ferri-siderophores translocation from the outer membrane to the periplasm in a. hydrophila as well as in other bacteria .
generally, it is provided the energy by the energy-transduction tonb-exbb-exbd protein complex, such like in e.coli  <cit> .
although we could not identify tonb and exbd proteins in the current study, an exbb family protein, r4w2t <dig> was up-regulated three fold, suggesting that tonb-exbb-exbd might participate in iron protein uptake in a. hydrophila as well.
besides, periplasmic iron binding proteins, such as the heme- and vitamin b12- binding proteins, and the associated cytoplasmic membrane abc permease transporters mediated the transport of ferri-siderophore complexes across the periplasm and cytoplasm similar to the other fish pathogenic bacteria edwardsiella ictaluri  <cit> .
thirdly, besides the siderophore receptors are located in outer membrane, there were many omps differentially expressed under iron starvation.
in our previous study, we found that the outer membrane protein r4vij <dig>  was down-regulated in e.coli in iron-limited medium  <cit> .
some other channel proteins were in the identical tread of down-regulated, such as and a0klx <dig> , and also many found up-regulated channel proteins, such as r4vrk <dig> , a0kmi <dig> , r4vnf <dig> , q4zhs <dig>  and a0kny <dig>  in iron starvation in this study as well.
the biological functions related to iron depletion are largely unknown for these proteins.
according to the fact that most of these proteins are the channels on outer membrane and control the influx or efflux of small molecular substrates, they might have an important effect on the controlling and monitoring of the cellular free iron concentration  <cit> .
because of the channel characteristics of omps, the down-regulation of omps might influence the influx of iron related substrates, whereas the up-regulation of omps might affect the efflux function  <cit> .
additionally, the peptidoglycan-associated lipoprotein, pal, and periplasmic tolb increased under iron starvation.
the tol–pal system was energized by the proton motive force and contributed to maintain the cell envelope integrity  <cit> .
since tol–pal system related proteins are homologous with the tonb-exbb-exbd system, they may participate in the iron uptake for bacteria survival  <cit> .
fourthly, bacteria have to reduce their intracellular redox reaction rates because many functional related enzymes are iron-cluster proteins.
thus, the energy generation system and electron respiration chain were depressed under iron starvation.
we assume that the down-regulation of the energy generation process takes part in the iron homeostasis mechanism rather than simply stimulation from iron depletion.
the down-regulation of tca cycle and its related proteins under iron starvation was reported in other bacteria species, such as frda, frdb, frdc and frdc in e. coli  <cit> , sdha, citg, citb, citc and pcka in staphylococcus aureus  <cit> .
smaldone et al., proposed the derepression of fur-regulated fsra may repress expression of glutamate synthase , and finally down-regulate tca cycle intermediate α-ketoglutarate, and dctp which imports succinate and fumarate  <cit> .
thus, the down-regulation of tca cycle under iron-deficient condition demonstrated that the stimulation of the tca process depresses bacterial growth in iron starvation environment and plays an important role in iron homeostasis.
finally, the reduction of the electron respiration chain, to some extent, might relieve the danger of bacterial reactive oxygen species  and help for survival  <cit> .
on the other hand, cells require iron resources such as siderophores or heme urgently for iron homeostasis by transporting iron compounds from the periplasm to cytosol and this process consumes atp  <cit> .
during this process, atpase should be activated for atp support.
in this study, the down-regulation of the tca cycle, cellular respiration and the increasing of atpase were identified by proteomics and validated in mrna level by q-pcr.
this outcome was consistent with our proteomics result confirms the previous report that mrna and protein levels do not correlate perfectly and suggests that protein post-translational modifications  <cit> .
the intrinsic reason of this phenomenon remains largely unknown.
to our understanding, atpase subunits do not have iron-sulfur clusters and would not affect the use of limited iron resource  <cit> .
the increasing expression of atpase may be a compensation mechanism to produce atp resource for abc transport as much as possible in the environment where the proton motive force  is resulting in a decrease.
to confirm this hypothesis, we investigated the atp synthase function under iron limiting conditions using an enzyme activity assay and atp synthase inhibitor.
our results validated that the activity of h+-atp synthase was activated in iron starvation and the inhibition of its activity would increase the susceptibility of a. hydrophila to iron starvation.
according to the proteomic results of cell envelope proteins involved in iron starvation, the scheme of iron uptake systems in a. hydrophila is represented graphically in the additional file 2: figure s <dig>
CONCLUSIONS
the aim of this study was to investigate the iron homeostasis mechanism in the aquatic pathogen, a. hydrophila.
to our knowledge, this is the first report on the iron homeostasis functions of cell envelope proteins in a. hydrophila by using a dimethyl labeling based quantitative proteomic method.
the transmembrane transporter activity and atp synthase increased significantly in abundance to enhance iron transport and maintain cellular iron homeostasis, while redox processes, precursor metabolites, energy generation processes and other iron-sulfur cluster binding processes decreased significantly in abundance to alleviate the pressure of the iron-limitation, by reducing iron use.
in general, this mechanism plays a crucial role in the signal transmission and regulation of the pathogenic bacteria under metal ion limited conditions.
a deep understanding of the iron acquisition mechanism of bacteria in the metal ion deficient conditions provided a theoretical support for the invasion mechanism of a. hydrophila disease.

 iron is the second most abundant metal in the earth’s crust; however, ferrous iron, the useable form, is scarce, due to an oxidative environment. it is an essential element for most bacteria with irreplaceable functions in many basic biological processes, including the fe3+ cofactor of metabolic respiratory chain reactions. these include photosynthesis, n fixation, methanogenesis, respiration, tricarboxylic acid cycle, and dna biosynthesis. to prevent the invasion of bacteria, hosts, including humans, have evolved to minimize the concentration of free iron by secreting transferrin and lactoferrin, which have high affinity for iron. therefore, bacteria have evolved effective ways to survive in this harsh environment. under this process, cell envelope proteins in gram-negative bacteria, including periplasmic, outer and inner membrane proteins, are essential in the transport of iron related compounds into the intracellular environment. the cell envelope proteins related to iron homeostasis mechanisms are well studied in escherichia coli, in which outer membrane proteins, such as cira, feca, fepa, btub, fhua, fhue, and ybil, serve as receptors of siderophores or heme to transport iron into the periplasmic space. some other omps, such as ompw, tsx and ompx function as either iron exporters or receivers. during this process, the periplasmic protein tonb and inner membrane proteins exbb and exbd form a protein complex to mediate the transport of iron compounds from the outer membrane to periplasm. the periplasmic binding proteins, such as fepb, fhub, fhud and fecb in escherichia coli deliver iron compounds to atp-binding cassette transporters in the cytosolic membrane and then into the cytosol. meanwhile, iron starvation triggers some complex networks and metabolic pathways via the fur gene family which regulates iron homeostasis. it is well known that the aquatic pathogen, aeromonas hydrophila, causes serious disease outbreaks in numerous farmed fish populations, and leads to big economic losses in the aquaculture and fishery industry, annually. however, knowledge is limited on the iron scavenging mechanism of this pathogen, especially for the role of the cell envelope proteins during the competition for iron resources with the host. in this study, the differential expressions of the envelop proteins of a. hydrophila were compared in the presence/absence of the iron chelating medium by a dimethyl labelling based quantitative proteomic method. bioinformatics analysis found some important biological processes involved. furthermore, some of the altered proteins were validated by quantitative polymerase chain reaction analysis and subjected to related functional validation. we provide the first report, to our knowledge, on the iron homeostasis functions of cell envelope proteins in a. hydrophila.  characterization and identification of the envelope protein fraction in a. hydrophila according to the growth curve of a. hydrophila, the strain treated with 150 μm 2′-dipyridyl grew slower than the control strain, especially after the middle exponential phase. to investigate the biological functions of envelope proteins in iron homeostasis, bacterial envelope proteins in presence/absence of dip with the concentration of 150 μm were extracted. sds-page separation showed some significant differences between dip treatment and the control, and the whole cell lysates of a. hydrophila were made to probe the cell envelope preparation enrichment in the preparations. after comparing with these differences between dip treatment and non-treatment, the extracted cell envelope proteins of a. hydrophila were then in-solution digested and labeled by stable isotope dimethyl. further lc ms/ms analysis identified proteins with at least two peptides required for identification and a false discovery rate less than 1 % filtered in the total number of proteins.fig. comparative characteristics of cell envelope protein of a. hydrophila in iron limited medium a growth curve of a. hydrophila atcc with and without 150 μm dip in lb medium; b cbb-stained sds page map of the cell envelope of a. hydrophila with and without 150 μm dip and whole cell lysates of a. hydrophila as the comparison. lane m contained molecular mass standards; c the subcellular localization of the identified proteins from ms results predicted by online software gneg-mploc; d blastp top-hit species distribution using local blast2go. numbers of top hit sequences from blastp were calculated for each species; e and f gene ontology categories for the differentially expressed proteins of a. hydrophila in iron starvation using local blast2go analysis and classified into biological processes and molecular functions. the red and blue bars indicate up-regulation and down-regulation differential ratios of genes, respectively. each related gene numbers are showed on the right of bars note: pm., om., im., ex. and cm. mean the location of periplasm, outer membrane, inner membrane, extracell and cytoplasm, respectively according to the online location prediction software, we found that these identified proteins included cytoplasmic proteins, inner membrane proteins, periplasmic proteins, outer membrane proteins, extracellular proteins, and fimbrium proteins with some overlapping proteins in the predicted locations. differential expression of envelope proteins in a. hydrophila in iron-limited medium to further investigate the biological behavior of a. hydrophila under iron-limiting conditions, dimethyl labeling based quantitative proteomic technology was used to analyze the differential expression of envelope proteins. in the identified proteins, membrane envelope proteins were found to be differentially expressed. taking overlapping locations into account, proteins including omps, imps, periplasmic proteins and three fimbrial proteins were up-regulated, while proteins including seven omps, imps and periplasmic proteins decreased in abundance in the iron starvation medium. of these altered proteins, seven outer membrane proteins, a0kjp r4w0j r4vc a0kjn a0kgw r4va and k1jld which serve as ferrienterobactin, ferrichrome, hemin, siderophore and heme receptors, and at less five periplasmic abc transporters and exbb family protein increased in abundance. however, ompw of a. hydrophila presented a down-regulated trend, although its biological function is still unknown. interestingly, besides the outer membrane siderophore receptors, there were more outer membrane proteins altered, including r4vaf, a0kk, a0klx, r4vch, r4vrx, a0kny, r4vnf, a0khf and r4vr. meanwhile, seven c-type cytochromes decreased in abundance. these proteins contained iron ion binding domains suggesting they reduce the use of iron and lead to the decrease of the respiratory electron transfer chain under iron starvation. on the contrary, atp synthase subunits, the final terminal point of cellular respiration, were found to be up-regulated. while the purpose of this opposite phenomenon is unknown, we hypothesize that it provides an atp source for ferrous iron scavenging. proteomics analysis also showed that almost all of the altered proteins located in the extracellular space were up-regulated, such as hemolysin and fimbrial protein could be explained as an attempt by the cell to strengthen the acquisition of scarce iron ions from outside and try to escape from the threatening surroundings. in addition, many proteases such as q49ka, a0kiu, a0kgx, r4vu reductase) and a0kfk were found to increase in extracellular space. investigation of the classification and functional annotation analysis of altered proteins to further characterize the altered envelope proteins, a biological functional classification was clustered using bioinformatic analysis. the local blast2go software was chosen to maximize homologous gene/protein annotations because of the limitation of gene ontology annotations of a. hydrophila. the blastp program was run against the swiss-prot database, producing matches from a total of candidates with blast expected values ≤10− using blast2go. figure 1d shows the basic local alignment search tool hit distribution across various species of organisms for all altered sequences in the dataset. further analysis identified the most enriched go terms comparing the up- and down-regulated proteins clusters including transport, macromolecule metabolic process, oxidation reduction process, small molecule metabolic process, generation of precursor metabolites and energy in biological process category and cation binding, tetrapyrrole binding, iron-sulfur cluster binding, active transmembrane transporter activity and iron chelating transmembrane transporter activity in molecular function category. validation of selected altered proteins at the mrna level using q-pcr according to the proteomic results and data analysis, up-regulated transport processes, down-regulated redox and energy generation process were the major response characteristics of envelope proteins in iron starvation. to confirm this, we investigated the mrna levels of a total of selected genes found to be altered in the proteomic analysis, using a q-pcr method. of these genes, five genes were h+-atpase subunits, seven genes were related with the transport process, six genes were involved in the redox process and three genes were involved in the energy generation process. results showed that most of the transcription levels of these related genes were consistent with our proteomics conclusions. in the selected transport-process related genes, the mrna level of a0kjp a tonb-dependent siderophore receptor, displayed a very significant up-regulation under iron starvation, suggesting its crucial role in iron uptake. on the other side, the transcription of other candidate genes increased from to fold, except for r4vlv and r4vj which only increased slightly, with a ratio of and, respectively.fig. the validation of relative expression behaviors of transport, redox, and energy generation processes were selected and verified by q-pcr the mrna level of transport related genes which were up-regulated at the protein level, oxidation-reduction and energy generation process related genes which were down-regulated at the protein level, were analyzed by q-pcr in the redox process and energy generation process, the down-regulated mrna levels of r4v7y r4vnp r4vf a0kl and r4vs were in agreement with protein levels, while k1jgn a0kqv only changed slightly. interestingly, the transcription of r4vnl and r4vh increased, contradictory to the proteomic results. in summary, up to 81 % of genes displayed the same directional change in both protein and mrna levels, 19 % showed no correlation, while only 10 % were dysregulated in the opposite direction. tca related exogenous metabolites elevate susceptibility of a. hydrophila to iron starvation in this study, we found the down-regulation of the tca related energy generation processes under iron starvation, using a proteomics approach. we assume that the down-regulation of the energy generation process takes part in the iron homeostasis mechanism rather than simply stimulation from iron depletion. to validate this, serial dilutions of the tca related exogenous metabolites were added in lb medium with or without dip to stimulate the tca process. as shown in fig.  low of concentration exogenous metabolites alone did not affect bacterial growth while high levels of metabolites, such as oxaloacetic acid, α-ketoglutarate, succinic acid and citric acid, did depress the bacterial growth rate. however, when the exogenous metabolites were added in lb medium with 300 μm dip, most of them elevated susceptibility of a. hydrophila to iron starvation, especially α-ka and ca, which both depressed bacterial growth significantly at 8 mm.fig. effect of exogenous metabolites on susceptibility of a. hydrophila to iron starvation. a–d the growth curve of a. hydrophila strain in 300 μm dip mm oxaloacetic acid, α-ketoglutarate, succinic acid and citric acid treatment, respectively. the concentration of these four reagents are in the gradient of and 8 mm. the growth kinetics of treated cells were recorded by absorbance measurements at od nm, at 30 °c with the multi-mode detection platform h+-atp synthase activity was increased in the iron starvation environment we further confirmed the behavior of h+-atp synthase under iron limited conditions. h+-atpase activity was measured with a serial dilution of dip treatment. compared to control, the h+-atpase activities of a. hydrophila were increased in response to iron starvation, which are consistent with our proteomics results. we then investigated the biological characteristics of h+-atp synthase in iron starvation using n, n’-dicyclohexylcarbodiimide, an inhibitor of atp synthase. when treated with 20 μg/ml dccd, the bacterial survival rate was significantly decreased by, and fold with the increase of dip concentrations 150 μm, 200 μm and 300 μm, respectively. to further investigate the extent of functional differences under iron-limitation, an orthogonal test was performed by using serials of dip and dccd concentrations and then displayed as color grading according to their survival colony accounting. results showed that, with the increasing of dip plus dccd treatments, the survival rates of a. hydrophila, were dramatically decreased.fig. the functional validation of atp synthase under iron starvation conditions. a the h+-atp synthase activity of a. hydrophila in a serial of dip concentrations in and 300 μm were measured spectrophotometrically at 340 nm using the h+-atpase assay kit b the survival ratio of a. hydrophila when treated with 20 μg/ml dccd in a serial dilution of dip concentrations in and 300 μm for 12 h and observed by colony counting, respectively. c color grading of the effect of a serial of concentrations of dip from 0 μm to 300 μm and dccd from 0 μg/ml to 40 μg/ml. the colony amounts of bacteria without treatment were normalized to as control discussion aquatic pathogenic bacteria a. hydrophila cause a fulminant epidemic in aquaculture, including some extremely severe bacterial diseases, such as hemolytic ascitic disease, bacterial septicemia, and fulminant hemorrhage. so far, the response mechanism of a. hydrophila under some metal ion deficient conditions is not clear. other bacteria, in order to respond to physiological iron-deficient conditions, have evolved a series of strategies to compete with the host for the uptake of life-essential iron. during this process, the cell envelope has an important effect on the extracellular and intrinsic communications due to its specific position, especially for the competition of essential nutrients in harsh environments. in order to systematically study the iron acquisition mechanism of the bacterial cell envelope system under the external pressure of iron limiting conditions, a dimethyl labeling based quantitative proteomic strategy was used to compare the differential expression of cell envelope proteins in a. hydrophila. about 57 % of cell envelope proteins were extracted and identified in the current study. based on the fact that cytoplasmic proteins contributed to the major contamination in all reported extraction methods, the tse extraction used in this study was documented to produce the cleanest extraction of envelope proteins up to date. compared to the 52 % enrichment ratio in e. coli, using the same method in the mentioned reference, the 57 % of cell envelope proteins enriched in a. hydrophila, should be acceptable. thus, in this study we acquired an acceptable cell envelope protein enrichment in a. hydrophila. in the present study, according to the final quantitative proteomic analysis, which can be summarized into the following statements: firstly, the expression of iron source receptors, which are located in the bacterial outer membrane, induced iron scavenging. these are high-affinity iron chelators and competitively bind iron from iron sources. actually, under iron deprivation, there are many reports about the upregulation of proteins involved in regular channel forming proteins, such as siderophore receptor channels, cira, feca, fepa, fhua, fhue, and ybil in e coli, and other channel proteins, such as ompx in e.coli. those results demonstrated the competitive uptake system of iron in a.hydrophila under iron-limiting conditions. secondly, after the ferri-siderophores and other iron sources specifically bind to outer membrane receptors, an energy generation system for transport is required. because there was no atp energy supply in the outer membrane, the exbb–exbd–tonb system assisted ferri-siderophores translocation from the outer membrane to the periplasm in a. hydrophila as well as in other bacteria. generally, it is provided the energy by the energy-transduction tonb-exbb-exbd protein complex, such like in e.coli. although we could not identify tonb and exbd proteins in the current study, an exbb family protein, r4w2t was up-regulated three fold, suggesting that tonb-exbb-exbd might participate in iron protein uptake in a. hydrophila as well. besides, periplasmic iron binding proteins, such as the heme- and vitamin b12- binding proteins, and the associated cytoplasmic membrane abc permease transporters mediated the transport of ferri-siderophore complexes across the periplasm and cytoplasm similar to the other fish pathogenic bacteria edwardsiella ictaluri. thirdly, besides the siderophore receptors are located in outer membrane, there were many omps differentially expressed under iron starvation. in our previous study, we found that the outer membrane protein r4vij was down-regulated in e.coli in iron-limited medium. some other channel proteins were in the identical tread of down-regulated, such as and a0klx, and also many found up-regulated channel proteins, such as r4vrk, a0kmi, r4vnf, q4zhs and a0kny in iron starvation in this study as well. the biological functions related to iron depletion are largely unknown for these proteins. according to the fact that most of these proteins are the channels on outer membrane and control the influx or efflux of small molecular substrates, they might have an important effect on the controlling and monitoring of the cellular free iron concentration. because of the channel characteristics of omps, the down-regulation of omps might influence the influx of iron related substrates, whereas the up-regulation of omps might affect the efflux function. additionally, the peptidoglycan-associated lipoprotein, pal, and periplasmic tolb increased under iron starvation. the tol–pal system was energized by the proton motive force and contributed to maintain the cell envelope integrity. since tol–pal system related proteins are homologous with the tonb-exbb-exbd system, they may participate in the iron uptake for bacteria survival. fourthly, bacteria have to reduce their intracellular redox reaction rates because many functional related enzymes are iron-cluster proteins. thus, the energy generation system and electron respiration chain were depressed under iron starvation. we assume that the down-regulation of the energy generation process takes part in the iron homeostasis mechanism rather than simply stimulation from iron depletion. the down-regulation of tca cycle and its related proteins under iron starvation was reported in other bacteria species, such as frda, frdb, frdc and frdc in e. coli, sdha, citg, citb, citc and pcka in staphylococcus aureus. smaldone et al., proposed the derepression of fur-regulated fsra may repress expression of glutamate synthase, and finally down-regulate tca cycle intermediate α-ketoglutarate, and dctp which imports succinate and fumarate. thus, the down-regulation of tca cycle under iron-deficient condition demonstrated that the stimulation of the tca process depresses bacterial growth in iron starvation environment and plays an important role in iron homeostasis. finally, the reduction of the electron respiration chain, to some extent, might relieve the danger of bacterial reactive oxygen species and help for survival. on the other hand, cells require iron resources such as siderophores or heme urgently for iron homeostasis by transporting iron compounds from the periplasm to cytosol and this process consumes atp. during this process, atpase should be activated for atp support. in this study, the down-regulation of the tca cycle, cellular respiration and the increasing of atpase were identified by proteomics and validated in mrna level by q-pcr. this outcome was consistent with our proteomics result confirms the previous report that mrna and protein levels do not correlate perfectly and suggests that protein post-translational modifications. the intrinsic reason of this phenomenon remains largely unknown. to our understanding, atpase subunits do not have iron-sulfur clusters and would not affect the use of limited iron resource. the increasing expression of atpase may be a compensation mechanism to produce atp resource for abc transport as much as possible in the environment where the proton motive force is resulting in a decrease. to confirm this hypothesis, we investigated the atp synthase function under iron limiting conditions using an enzyme activity assay and atp synthase inhibitor. our results validated that the activity of h+-atp synthase was activated in iron starvation and the inhibition of its activity would increase the susceptibility of a. hydrophila to iron starvation. according to the proteomic results of cell envelope proteins involved in iron starvation, the scheme of iron uptake systems in a. hydrophila is represented graphically in the additional file 2: figure s  the aim of this study was to investigate the iron homeostasis mechanism in the aquatic pathogen, a. hydrophila. to our knowledge, this is the first report on the iron homeostasis functions of cell envelope proteins in a. hydrophila by using a dimethyl labeling based quantitative proteomic method. the transmembrane transporter activity and atp synthase increased significantly in abundance to enhance iron transport and maintain cellular iron homeostasis, while redox processes, precursor metabolites, energy generation processes and other iron-sulfur cluster binding processes decreased significantly in abundance to alleviate the pressure of the iron-limitation, by reducing iron use. in general, this mechanism plays a crucial role in the signal transmission and regulation of the pathogenic bacteria under metal ion limited conditions. a deep understanding of the iron acquisition mechanism of bacteria in the metal ion deficient conditions provided a theoretical support for the invasion mechanism of a. hydrophila disease. 
bioinformatics analysis showed that outer membrane siderophores, heme and iron receptors, periplasmic iron binding proteins, inner membrane abc transporters and h+-atp synthase subunits increased in abundance while iron-cluster proteins, electron transport chain and redox proteins were down-regulated. further q-pcr validation, in vivo addition of exogenous metabolites, and an enzyme inhibition assay revealed that redox, the energy generation process, and atp synthase elevated the susceptibility of a. hydrophila to iron starvation. our study demonstrates that the redox and energy generation process, and atp synthase in a. hydrophila may play critical roles in iron acquisition under conditions of iron-stress. to understand the iron homeostasis mechanism in a. hydrophila, a dimethyl labelling based quantitative proteomics method was used to compare the differential expression of cell envelope proteins under iron starvation. an understanding of the iron scavenging mechanism may be helpful for the development of strategies for preventing and treating a. hydrophila infection. currently, knowledge is limited on the iron scavenging mechanism of the cell envelope in the aquatic pathogen, aeromonas hydrophila. a total of cell envelope proteins were identified by lc-ms/ms, with down-regulated and up-regulated proteins. aeromonas hydrophilacell envelopedimethyl labelingiron homeostasishttp://dx.doi.org/ /501100001809national natural science foundation of china 3120010531470238lin xiangmin the fujian agricultural and forestry university foundation for distinguished young scholarsxjq201201lin xiangmin issue-copyright-statement© the author 2016
 iron homeostasis is an essential process over the entire lives of both hosts and bacterial pathogens, and also plays roles in many other metabolic functions. currently, knowledge is limited on the iron scavenging mechanism of the cell envelope in the aquatic pathogen, aeromonas hydrophila. to understand the iron homeostasis mechanism in a. hydrophila, a dimethyl labelling based quantitative proteomics method was used to compare the differential expression of cell envelope proteins under iron starvation.  a total of cell envelope proteins were identified by lc-ms/ms, with down-regulated and up-regulated proteins. bioinformatics analysis showed that outer membrane siderophores, heme and iron receptors, periplasmic iron binding proteins, inner membrane abc transporters and h+-atp synthase subunits increased in abundance while iron-cluster proteins, electron transport chain and redox proteins were down-regulated. further q-pcr validation, in vivo addition of exogenous metabolites, and an enzyme inhibition assay revealed that redox, the energy generation process, and atp synthase elevated the susceptibility of a. hydrophila to iron starvation.  our study demonstrates that the redox and energy generation process, and atp synthase in a. hydrophila may play critical roles in iron acquisition under conditions of iron-stress. an understanding of the iron scavenging mechanism may be helpful for the development of strategies for preventing and treating a. hydrophila infection. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords aeromonas hydrophilacell envelopedimethyl labelingiron homeostasishttp://dx.doi.org/ /501100001809national natural science foundation of china 3120010531470238lin xiangmin the fujian agricultural and forestry university foundation for distinguished young scholarsxjq201201lin xiangmin issue-copyright-statement© the author 2016 