BACKGROUND
cysteine is an extraordinary amino acid residue because of the chemical reactivity of its thiol group .
in contact with oxidative compounds, the thiol group can oxidize itself into sulfenic acids  or form a disulfide bond  with another cysteine residue, either from the same or from a different protein.
these oxidized states can revert back to the reduced form  via the action of an electron donor  <cit> .
in contrast, autooxidation into sulfinic  and sulfonic acid  leads to irreversible states of cysteine.
besides cysteine also enables the sequestration of redox metals alone or in complex structure like iron-sulfur cluster that can modulate activity of the protein  <cit> .
this amino acid can also bind non-redox metals such as zinc ion, for example in the zinc finger complex, thus enabling the correct folding of proteins.
it is worth noting this latter complex is mainly found in eukaryotic cells  <cit> .
due to the redox properties of cysteine, the oxidation of this amino acid can activate transcriptional factors directly, as has been described in e. coli  and bacillus subtilis , as well as indirectly, via the oxidation of a  cluster as described in soxr and fnr in escherichia coli.
soxr detects superoxide anions via oxidation of its  cluster, while fnr detects oxygen via oxidation-destruction of its   <cit> .
in addition, cysteine residues have also been associated with enzymatic activities, such as those involving glyceraldehyde dehydrogenase , heat shock chaperone proteins , and thioredoxins and glutaredoxins .
thus, because of their involvement in multiple key aspects of cellular metabolism, cysteine residues in proteins should be widely distributed across all organisms, from viruses to complex forms like the eukaryotic cells.
unexpectedly, recent studies on extracytoplasmic proteins whose activation is mediated by intramolecular disulfide bonds revealed that proteins in gram-positive bacteria are proportionately poorer in cysteine residues than proteins found in other bacteria, a phenomenon termed “cysteine exclusion”  <cit> .
one proposed explanation was that, unlike gram-negative bacteria, gram-positive bacteria have neither a periplasmic space nor outer membrane.
thus, exported cysteine-rich proteins would be subject to oxidation, which, in the absence of repair machinery such as the dsb system in e. coli  <cit> , would lead to their inactivation.
in gram-positive bacteria, some systems have been described: bdb system in b. subtilis  <cit>  and more recently in mycobacterium tuberculosis  <cit>  and in streptococcus gordonii  <cit> .
compared to e. coli or b. subtilis, in lactococcus lactis, a facultative aerobic bacterium of high technological interest, such system of repair has not yet been reported although this organism is widely used to overproduce recombinant proteins like cytokine il- <dig> containing disulfide bridge for its activity  <cit> .
unexpectedly, this organism has an even lower amount of cysteine residue in its proteins  <cit> .
thus far, investigations of the proteins of this bacterium have characterized only a few whose cysteine residues interact with an iron-sulfur cluster or catalyze reduction of substrates, and all of these are cytosolic proteins.
in the activase protein  of the anaerobic ribonucleotide reductase complex , these residues are predicted to sequestrate a  cluster  <cit>  and only three cysteines might be engaged to complex it as reported in e. coli nrdg  <cit> .
in the dihydroorotate dehydrogenase , four cysteines sequester a  redox cluster  <cit> .
both rnr and pyrk are connected to dna synthesis.
in the alkylhydroperoxide reductase   <cit>  and thioredoxin reductase/thioredoxin system   <cit> , cysteines directly catalyze the reduction of substrates.
ahpc/ahpf is one of the few enzymes described so far that play a role in oxidative stress resistance.
tr/trx maintains the intracellular redox state to protect intracellular proteins  against oxidation  <cit> .
finally, clp atpase  was reported to contribute to the degradation of misfolded or truncated intracellular proteins  <cit> , and its activity could depend on a putative cysteine-containing zinc finger  <cit> .
in this work, we aimed to understand role of cysteines in l. lactis proteins and role of these proteins when cells are exposed to stressful conditions.
using a bioinformatic approach, we selected cysteine-rich proteins located in the membrane as they might constitute a first signaling pathway to detect environmental stress, or a defense line, through cysteine oxidation.
among a few proteins found in l. lactis strain mg <dig>  we focused on llmg_ <dig> and llmg_ <dig>  of which respective genes form an operon including also a small gene, llmg_ <dig>  this operon was transiently induced at very early log growth phase.
in the proteins, cysteine residues are organized in a cx2cx10cx2c motif involved in zinc coordination.
finally, through different constructions in llmg_ <dig> we found that this protein had a severe impact on growth, affected resistance to cumene hydroperoxide and lysozyme suggesting this protein is linked to cell envelope integrity.
RESULTS
in silico analysis of the cysteine-rich membrane proteins
to identify the predicted membrane proteins that contained the cx2c motif in l. lactis strain mg <dig>  we used a bioinformatic approach.
from the genome database we found that proteins harboring the potential redox cx2c motif represented only  <dig>  % of total proteins  .
this score is the lowest when we compared it to  <dig>  % found in b. subtilis  or  <dig>  % in e. coli .
these data are in agreement with the theory of “cysteine exclusion” and is also strengthened by the capacity of l. lactis strain to grow in presence of large amount of reducing agents like dithiothreitol   <cit> .
when we used the prediction program  to extract only membrane proteins, this number dropped to seven against  <dig> found in b. subtlilis or  <dig> in e. coli.
three of these proteins had a predicted function, whereas the remaining four did not .
llmg_ <dig>  and llmg_ <dig>  have been linked to metal  homeostasis in bacteria .
the former protein is particularly likely to serve an essential function, as iron is necessary in l. lactis not only for dna synthesis  but also for heme synthesis from protoporphyrinogen ix  <cit> .
heme enables l. lactis to undergo respiration, and this growth condition  translates into increased biomass yield and an increase in long-term survival versus fermentation  <cit> .
llmg_ <dig>  contained six cysteines, including two cx2c motifs separated by  <dig> amino acid residues.
this protein is similar to pild , a peptidase involved in pili synthesis.
in pseudomonas species, the cysteine residues of pild should complex a zinc ion.
however, studies revealed that the cysteine-zinc complex may be unstable in vitro as, in some protein preparations, a disulfide bond was formed instead  <cit> .
llmg_ <dig> and llmg_ <dig> each contained four cysteines, in a cx2cx10cx2c motif in the n-terminal extremity  , while llmg_ <dig> harbored a cx2c motif in its c-terminal extremity .
llmg_ <dig> contained four target motifs in its nter domain, which were organized into two cx2cx14cx2c motifs.
with the exception of the cysteine motif, no other signatures were detected that predicted the function of the latter four proteins.table  <dig> putative membrane proteins containing cx2c motif in l. lactis strain mg1363
tmd, transmembrane domain
to understand the role of the cysteine motif in l. lactis proteins, we continued our investigation by focusing on two cysteine-rich proteins, llmg_ <dig> and llmg_ <dig>  which might sense stressful environments via cysteine oxidation and their putative membrane localization.
we aimed to determine: i) the genetic organization and expression of these proteins; ii) the localization and properties of the cysteine motif within the proteins; and iii) the role of these proteins.
temporal expression of the llmg_0524-0525- <dig> operon
we found a stem loop upstream of the llmg_ <dig> orf  and another one downstream of the llmg_ <dig> orf , suggesting a potential operonic structure for the three genes: the llmg_ <dig> orf; the llmg_ <dig> orf, which encodes a small  hydrophobic protein free of cysteines; and the llmg_ <dig> orf .
to test our hypothesis, we performed rt-pcr assays with different primer pairs and total rna that had been extracted from cells harvested in the exponential growth phase .
the results showed that an mrna transcript overlapped two adjacent genes .
the pcr product had the same size of that obtained from genomic dna  and was absent when rna preparation was pretreated with a rnase .
we also performed pcr with primers designed inside the llmg_ <dig> and llmg_ <dig> orfs; the transcripts of this pcr product covered all three genes.
no pcr amplification was obtained with primer pairs designed for the llmg_ <dig> and llmg_ <dig> orfs , a result that was consistent with the presence of stem loops .
finally, we cloned a dna fragment that covered the region from the middle of the llmg_ <dig> orf to the beginning of the llmg_ <dig> orf ; this fragment was inserted upstream of the promoterless lacz gene of the ptcv-lac plasmid  <cit> , generating plasmid p0526-ptcv-lac.
after we introduced this plasmid into l. lactis strain mg <dig>  we did not detect any β-galactosidase  activity under the tested conditions , suggesting the absence of a promoter, specifically the one upstream of the llmg_ <dig> orf.
taken together, these results indicate that the three genes are clustered in an operon and under the control of a promoter located upstream of the llmg_ <dig> orf.fig.
<dig>
llmg_ <dig>  llmg_ <dig>  and llmg_ <dig> form an operon.
a schematic representation of the locus; bp - base pair.
b rt-pcr analysis of the locus.
total rna was translated into cdna, which in turn was used as the template for pcr with different primer pairs .
pos control, positive control with genomic dna as a template; neg control, negative control with treatment of rna preparation with a rnase
to study the expression of the operon, we cloned the promoter region of llmg_ <dig> orf into the ptcv-lac plasmid, creating plasmid p0524-ptcv-lac, and introduced this into l. lactis strain mg <dig>  with ortho-nitrophenyl galactoside as the growth substrate and glucose as the energy source, we did not detect any β-gal activity.
however, using a more sensitive test with a fluorescent substrate , we managed to detect some β-gal activity, indicating that the operon was poorly expressed.
under these conditions, we observed β-gal induction an hour after inoculation , with a peak around od <dig>  <dig> .
beyond this point, β-gal activity decreased progressively with further growth.
when glucose was replaced by galactose as the energy source, β-gal expression increased but appeared later  in growth than was observed with glucose.
moreover, instead of having a peak, transcript quantity reached a stable plateau with minor changes until od <dig>  <dig>  and then it went down.
to validate gene expression we performed semi-quantitative rt-pcr assays , which revealed relative transcript amounts that were concordant with the β-gal activity patterns.
none of the other tested growth conditions  resulted in changes to the expression of the operon.
in addition, deletion of llmg_ <dig> or llmg_ <dig>  decreased two-fold expression of the operon .
we thus conclude that operon is transiently transcribed and occurs only during the early log growth phase.fig.
<dig> temporal expression of the operon.
a expression kinetics of the operon.
cells were grown in m17glucose  or m17galactose .
at regular intervals, aliquots were collected to assess β-gal activity.
values, plus standard deviations, are the means of three independent experiments.
b semi-quantification of the llmg_ <dig> transcript.
cells were grown on glucose  or on galactose  and collected at certain growth phases.
total rna was prepared for semi-quantitative rt-pcr determination.
control - ahpc.
dil.
- dilution
membrane localization of llmg_ <dig> and llmg_0526
analyses of these proteins with topology prediction programs suggested that llmg_ <dig> contained two transmembrane domains  but the programs yielded no information about the localization of the nter domain .
llmg_ <dig> contained a transmembrane domain with the nter region, indicating that this end is probably inside the cell.
to obtain direct experimental evidence regarding the membrane topology of these proteins, we performed an alkaline phosphatase  assay  <cit> .
phoa is activated via the oxidation of its cysteine residue to form a disulfide bridge, a reaction which occurs mainly in the periplasm of e. coli cells.
the phoa assay thus consists of fusing the signal-sequence-less phoa gene to a target dna sequence and then monitoring for phoa activity; such activity should only be detectable if the target dna addresses the resulting protein into the periplasmic space.
we therefore constructed a set of plasmids that each contained a different portion of the llmg_ <dig> and the llmg_ <dig> orfs fused to phoa , and then established the plasmids in the e. coli phoa mutant.
bacteria were grown on lb agar supplemented with arabinose for the production of fusion proteins and with a bcip compound for the detection of phoa activity.
cells that produced the phoa <dig> hybrid proteins  generated a strong blue color corresponding to the degradation of bcip, while those that produced the phoa <dig> constructs  displayed only weak coloration indicating that phoa was addressed out of cytosol with phoa <dig> construction .
quantification of the phoa activity confirmed these observations, revealing the strongest activity in cells with the phoa <dig> fusions .
we therefore conclude the two proteins are located in the membrane, with the nter ends located in the cytoplasm.fig.
<dig> nter end of llmg_ <dig> and llmg_ <dig> is intracellular.
a schematic representation of the proteins with position of transmembrane domain  and protein fusion with phoa.
b phosphatase activity of cells that overproduced the protein fusions.
fusion proteins were overproduced in a e. coli phoa mutant, which were then treated with arabinose.
phosphatase activity was revealed in presence of bcip compound present in agar plates
llmg_ <dig> and llmg_ <dig> are zinc metalloproteins
as the cx2cx10cx2c motif in llmg_ <dig> and llmg_ <dig> was identical to that of the zinc metalloprotein dnaj  <cit> , we hypothesized that our target proteins also bound a metal.
to test this, we purified the nter domain of llmg_ <dig> and llmg_ <dig>  as a hybrid protein to the maltose binding protein male, creating male-0524nter and male-0526nter, respectively.
the protein male-laczα corresponding to the plasmid without insert  was used as a control.
after purification, the hybrid proteins were analyzed by inductively coupled plasma mass spectrometry  to determine nature of metal ion if present in protein .
we found magnesium ion was present in all preparation at relative similar concentration but this ion has not been reported to bind with cysteine residues.
male-laczα did not contain iron or zinc metal in significant amount in contrast to other proteins.
in the presence of male-0524nter, zinc was far more abundant than iron.
in preparations of male-0526nter, although zinc was still the most abundant, there was also a quantity of iron ion.
however, this protein  was colorless  and its uv-visible spectrum did not display any absorption bands in visible wavelengths, which are characteristic of iron-cysteine proteins  <cit> .
furthermore, when the medium was supplemented with zinc salt, the level of iron in the preparation decreased dramatically, indicating that the affinity of male-0526nter to iron was not specific.
altogether these results indicate that llmg_ <dig> and llmg_ <dig> are zinc metalloproteins.table  <dig> llmg_ <dig> and llmg_ <dig> are zinc metalloproteins
male-0524nter
male- <dig> nter
male- <dig> nter
male- <dig> nter
all buffers were treated with chelex.
values are the mean of three independent protein purifications.
+ zinc, cultures were supplemented with  <dig>  mm of zinc salt.
male-laczα  is used as a control, nd: not determined
the cx2cx10cx2c motif binds zinc metal directly
the icp-ms analysis allowed us to determine the stoichiometry ratio of zinc to protein:  <dig> : <dig> for male-0524nter and  <dig> : <dig> for male-0526nter .
these ratios did not change when we supplemented the media with zinc salt indicating the lb broth contained enough metals to load synthesized proteins.
these ratios were further confirmed by the titration of zinc ions with the chelator par,  resorcinol .
to demonstrate that this metal was coordinated in the proteins via the thiol group of the cysteine, we treated the proteins with p-hydroxy mercury-phenylsulfonate agent .
pmps specifically disrupts the cysteine-zinc bond by forming a cysteine-mercury and the released zinc ion can be titrated with par  <cit> .
evidence of zinc-par <dig> complex formation appeared progressively with increasing amounts of pmps in the mixture .
for male-0524nter, two equivalents of pmps versus protein were required to release around 90 % of the zinc ions, and adding more pmps  did not significantly change the absorbance.
as there were no cysteines in the male portion of the fusion protein, this result meant that two cysteine residues of llmg_0524nter were involved in binding the zinc metal.
if this were the case, the protein should therefore also contain free thiols, which we determined via titration with  <dig> -dithiobis , a compound which reacts to free thiol groups  <cit> .
we observed that the ratio of free cysteine:protein was close to one .
the second residue was not titrated in our assays probably because it was not accessible to dtnb due for instance to folding of llmg_ <dig> fragment.
in male-0526nter, instead, four equivalents of pmps were required to release most of the zinc from the protein, indicating a stoichiometry of 4: <dig> of pmps:zinc and thus the presence of a zinc <dig> complex.
in agreement with these observations, titration with dtnb indicated a low amount of free thiol groups in our preparations .
we thus conclude that each of the two proteins harbors a zinc-cysteine module but in different coordination spheres: zinc <dig>  in llmg_ <dig> and zinc <dig> in llmg_ <dig> fig.
<dig> presence of a cysteine-zinc module in proteins.
the cysteine-zinc bond was characterized by pmps-par assays with 20 μm of proteins.
male-0524nter  or male-0526nter  were purified from cultures  and from those that were supplemented with zinc salt  values, plus standard deviations, are the means of three independent experiments
20 μm of protein  was used for thiol group determination by dtnb assays
in addition, we tested the stability of the zinc module in the presence of oxidative agents  and a temperature of 37 °c, which affects protein folding in l. lactis.
in the presence of ten equivalents of oxidant or 37 °c, no released zinc ions were detected by par , indicating that the modules in these proteins are very stable in tested conditions.
llmg_ <dig> is required for resistance against cumene hydroperoxide and cell wall integrity
to investigate the function of the llmg_0524-0525- <dig> operon in vivo, an in frame-markerless deletion of the llmg_ <dig> or llmg_ <dig> gene was constructed in the chromosome of strain mg <dig>  as our in silico analyses did not yield any information about the potential functions of these proteins, we screened the mutants in several growth conditions.
from these we found no clear phenotypic changes in the ∆llmg_ <dig> mutant compared to its isogenic wild-type  strain .
in the ∆llmg_ <dig> mutant, instead, we found one growth condition in which the ∆llmg_ <dig> mutant was more sensitive than the wt strain: growth in the presence of cumene hydroperoxide , an oxidative agent  <cit> , and with galactose as an energy source .
in contrast, we did not observe any difference between the two strains treated hydrogen peroxide .
resistance to chp was completely restored when we transformed the ∆llmg_ <dig> mutant with a plasmid expressing the wt operon .
interestingly, the plasmid pllmg_0524c,∆cyst, which carried a version of the operon in which the dna region encoding the cysteine motif of llmg_ <dig> had been deleted, was unable to complement the mutant .
in fact, the mutant producing the truncated llmg_ <dig> grew very poorly even in the absence of stress, and on glucose as energy sources as well .
then, we asked whether the modifications of lipid or cell wall synthesis were responsible of that growth defect.
addition of tween- <dig>  a donor of long chain fatty acids, did not have any effect on the growth.
in contrast, we found that the mutant, carrying the plasmid pllmg_0524c,∆cyst, became more resistant to lysozyme, an hydrolase of peptidoglycan, than the other strains  indicating that the growth defect was due to cell wall modifications.
we conclude the llmg_ <dig> is linked to cell wall integrity and resistance to stressful conditions.fig.
<dig> role of llmg_ <dig> mutant in cumene hydroperoxide resistance and growth.
cells were grown in m17galactose in static conditions at 30 °c.
when cell density reached od <dig>  <dig> ,  <dig>  mm of cumene hydroperoxide  was added to the culture.
wt - black line, ∆llmg_ <dig> mutant - gray line, ∆llmg_ <dig> mutant carrying the plasmid encoding the full operon  - gray dashed line.
values, plus standard deviations, are the means of three independent experiments
fig.
<dig> role of llmg_ <dig> in the cell wall integrity.
a effect on the growth of the deletion of the cysteine-rich motif in llmg_ <dig>  cells were grown in m17glucose in static conditions at 30 °c.
the ∆llmg_ <dig> mutant carrying the plasmid pllmg_0524
c,∆cyst is represented by a dark gray dashed line.
the plasmid encoded the full operon, but lacked the region in the llmg_ <dig> orf corresponding to the cysteine motif.
values, plus standard deviations, are the means of three independent experiments .
b effect of lysozyme resistance of the deletion of the cysteine-rich motif in llmg_ <dig>  after overnight cultures, strains were washed in pbs buffer and recovered at the same od <dig>  after 60 min of incubation in absence  or in presence of lysozyme , sds was added at  <dig>  % final concentration and measured od <dig>  values, plus standard deviation, are the means of three independent experiments.
they are expressed in percent of od <dig> measured in samples incubated 60 min in absence of lysozyme and sds
discussion
in this study we characterized llmg_ <dig> and llmg_ <dig>  two proteins of unknown functions with the same cx2cx10cx2c motif.
this motif bound a zinc ion.
we found that the cysteine-zinc complex was stable when subjected to stress, suggesting it plays a role in protein stability rather than as a sensor.
this hypothesis is supported by our observation that deletion of this motif in llmg_ <dig> had serious impacts on cell growth.
in addition, we did find that llmg_ <dig> also increased resistance against the oxidative agent cumene hydroperoxide.
a cysteine motif to chelate metal
in l. lactis strain, we demonstrated that the cx2cx10cx2c motif bound zinc ion.
this by itself is not novel, as this motif is also found in other proteins , and in some of these, it has been experimentally demonstrated to bind this metal.
for example, the dnaj protein of e. coli binds zinc ion using all four cysteines of the motif, and formation of the zinc-cysteine complex is required for protein activation  <cit> .
more recently, the same motif was found in e. coli ycim, a membrane protein, in which the four cysteine residues were involved in metal coordination.
however, ycim is proposed to bind iron ion instead of zinc  <cit> .
from these observations alone, it was clear from this study and others  that the motif is used for trapping metal, but the question remained: is it possible to predict which metal, and the individual cysteine residues’ contributions to sphere coordination, based only on the motif?
in this study and dnaj, zinc ion is the major metal found suggesting that this motif may have a bigger affinity to this metal than iron.
in l. lactis, this motif may contain zinc since in strain mg <dig> level of zinc ion is 25-fold higher than that of iron whereas in e. coli strain zinc is present in an amount similar as iron  <cit> .
however, there are few examples describing the complex cysteine-metal to make this hypothesis true to proteins having this motif in other bacteria.
with the male-0526nter fusion protein, pmps-par assays demonstrated the presence of a zinc <dig> module in llmg_ <dig>  clearly indicating that all cysteine residues were engaged in zinc coordination.
unexpectedly, however, similar tests using the fusion male-0524nter revealed that only two residues bound zinc.
the two other cysteines of the protein were free, although we detected only one residue.
that observation also indicated that male-0524nter lacked a disulfide bond, suggesting that the fusion protein was not redox reactive in vitro.
however, in vivo these two free cysteine residues could play a role in the intact protein by forming intra- or intermolecular disulfide bridge between two llmg_ <dig> or llmg_ <dig> and another protein.
to our knowledge, zinc usually binds four ligands, with cysteine and histidine residues as the most common ligands by far  <cit> .
however, no histidine is present in the cytoplasmic portion of llmg_ <dig>  which suggests that the metal coordination of this protein is unusual.
additional experiments will be needed to determine the other ligands of zinc ion in llmg_ <dig>  as well as the parameters that could govern the specificity of metal.
role of the llmg_0524-0525- <dig> operon
unlike cysteine, zinc is not redox reactive in proteins.
as the bound modules in llmg_ <dig> and llmg_ <dig> were very stable in the presence of oxidative agents or 37 °c, which affects the protein folding in l. lactis, it is unlikely that the module is used as a direct sensor of stress, as has been described, e.g., for the chaperone hps <dig>  <cit> .
instead, for llmg_ <dig> at least, we think that the zn module serves to stabilize the folded structure of the protein as required for its function, perhaps for interaction with other proteins.
this hypothesis is supported by the dramatic growth differences we observed between the wild-type and the truncated-llmg_ <dig> mutant strain, which lacked the cysteine motif .
even in the absence of stress, this strain displayed a severe growth defect.
similarly, in e. coli, ycim mutants lacking this motif were unstable and formed aggregates  <cit> .
in addition, our experiments with deletion mutants suggest that this operon plays a role in cell wall integrity in particular with peptidoglycan synthesis since that mutant, producing the truncated llmg_ <dig>  was more resistant to lysozyme than other strains.
a modification of cell wall may explain the light resistance to cumene hydroperoxide observed in the ∆llmg_ <dig> mutant.
studies are in progress to characterize the modification of cell wall linked to ∆llmg_ <dig> deletion.
our experiments with the ∆llmg_ <dig> mutant did not produce any reproducible phenotypes even against the cumene hydroperoxide toxicity.
in a protein blast search, this protein displays similarity to s. aureus tcaa, which has a cysteine motif inside, which might bind metal although this has not been confirmed  <cit> .
downstream of the motif, we found a transmembrane domain followed by a long extracellular chain  <cit> .
in s. aureus, tcaa is induced by envelope stress and resistant to teicoplanin, an antibiotic of glycopeptide family  <cit> .
although they share a similar primary structure, llmg_ <dig> does probably not play the same role of tcaa.
in contrast to tcaa, l. lactis, llmg_0524-0525- <dig> operon is not induced by the envelope stress response  <cit> .
CONCLUSIONS
cysteine motifs in proteins are often associated to redox activity  or iron-sulfur cluster  in bacteria, but less is known about cysteine motif in chelating zinc ion.
in this work, we characterized in lactococcus lactis, two membrane proteins  carrying a cx2cx10cx2c motif.
the motif bound zinc ion although the sphere of coordination of zinc was remarkably different between the two proteins.
our works suggest that llmg_ <dig>  and maybe other proteins of operon, is linked to cell wall integrity.
the function of this protein in the machinery of cell wall synthesis remains to uncover now, as well as its zinc module.

 cysteine is an extraordinary amino acid residue because of the chemical reactivity of its thiol group. in contact with oxidative compounds, the thiol group can oxidize itself into sulfenic acids or form a disulfide bond with another cysteine residue, either from the same or from a different protein. these oxidized states can revert back to the reduced form via the action of an electron donor. in contrast, autooxidation into sulfinic and sulfonic acid leads to irreversible states of cysteine. besides cysteine also enables the sequestration of redox metals alone or in complex structure like iron-sulfur cluster that can modulate activity of the protein. this amino acid can also bind non-redox metals such as zinc ion, for example in the zinc finger complex, thus enabling the correct folding of proteins. it is worth noting this latter complex is mainly found in eukaryotic cells. due to the redox properties of cysteine, the oxidation of this amino acid can activate transcriptional factors directly, as has been described in e. coli and bacillus subtilis, as well as indirectly, via the oxidation of a cluster as described in soxr and fnr in escherichia coli. soxr detects superoxide anions via oxidation of its cluster, while fnr detects oxygen via oxidation-destruction of its. in addition, cysteine residues have also been associated with enzymatic activities, such as those involving glyceraldehyde dehydrogenase, heat shock chaperone proteins, and thioredoxins and glutaredoxins. thus, because of their involvement in multiple key aspects of cellular metabolism, cysteine residues in proteins should be widely distributed across all organisms, from viruses to complex forms like the eukaryotic cells. unexpectedly, recent studies on extracytoplasmic proteins whose activation is mediated by intramolecular disulfide bonds revealed that proteins in gram-positive bacteria are proportionately poorer in cysteine residues than proteins found in other bacteria, a phenomenon termed “cysteine exclusion”. one proposed explanation was that, unlike gram-negative bacteria, gram-positive bacteria have neither a periplasmic space nor outer membrane. thus, exported cysteine-rich proteins would be subject to oxidation, which, in the absence of repair machinery such as the dsb system in e. coli, would lead to their inactivation. in gram-positive bacteria, some systems have been described: bdb system in b. subtilis and more recently in mycobacterium tuberculosis and in streptococcus gordonii. compared to e. coli or b. subtilis, in lactococcus lactis, a facultative aerobic bacterium of high technological interest, such system of repair has not yet been reported although this organism is widely used to overproduce recombinant proteins like cytokine il- containing disulfide bridge for its activity. unexpectedly, this organism has an even lower amount of cysteine residue in its proteins. thus far, investigations of the proteins of this bacterium have characterized only a few whose cysteine residues interact with an iron-sulfur cluster or catalyze reduction of substrates, and all of these are cytosolic proteins. in the activase protein of the anaerobic ribonucleotide reductase complex, these residues are predicted to sequestrate a cluster and only three cysteines might be engaged to complex it as reported in e. coli nrdg. in the dihydroorotate dehydrogenase, four cysteines sequester a redox cluster. both rnr and pyrk are connected to dna synthesis. in the alkylhydroperoxide reductase and thioredoxin reductase/thioredoxin system, cysteines directly catalyze the reduction of substrates. ahpc/ahpf is one of the few enzymes described so far that play a role in oxidative stress resistance. tr/trx maintains the intracellular redox state to protect intracellular proteins against oxidation. finally, clp atpase was reported to contribute to the degradation of misfolded or truncated intracellular proteins, and its activity could depend on a putative cysteine-containing zinc finger. in this work, we aimed to understand role of cysteines in l. lactis proteins and role of these proteins when cells are exposed to stressful conditions. using a bioinformatic approach, we selected cysteine-rich proteins located in the membrane as they might constitute a first signaling pathway to detect environmental stress, or a defense line, through cysteine oxidation. among a few proteins found in l. lactis strain mg we focused on llmg_ and llmg_ of which respective genes form an operon including also a small gene, llmg_ this operon was transiently induced at very early log growth phase. in the proteins, cysteine residues are organized in a cx2cx10cx2c motif involved in zinc coordination. finally, through different constructions in llmg_ we found that this protein had a severe impact on growth, affected resistance to cumene hydroperoxide and lysozyme suggesting this protein is linked to cell envelope integrity.  in silico analysis of the cysteine-rich membrane proteins to identify the predicted membrane proteins that contained the cx2c motif in l. lactis strain mg we used a bioinformatic approach. from the genome database we found that proteins harboring the potential redox cx2c motif represented only  % of total proteins. this score is the lowest when we compared it to  % found in b. subtilis or  % in e. coli. these data are in agreement with the theory of “cysteine exclusion” and is also strengthened by the capacity of l. lactis strain to grow in presence of large amount of reducing agents like dithiothreitol. when we used the prediction program to extract only membrane proteins, this number dropped to seven against found in b. subtlilis or in e. coli. three of these proteins had a predicted function, whereas the remaining four did not. llmg_ and llmg_ have been linked to metal homeostasis in bacteria. the former protein is particularly likely to serve an essential function, as iron is necessary in l. lactis not only for dna synthesis but also for heme synthesis from protoporphyrinogen ix. heme enables l. lactis to undergo respiration, and this growth condition translates into increased biomass yield and an increase in long-term survival versus fermentation. llmg_ contained six cysteines, including two cx2c motifs separated by amino acid residues. this protein is similar to pild, a peptidase involved in pili synthesis. in pseudomonas species, the cysteine residues of pild should complex a zinc ion. however, studies revealed that the cysteine-zinc complex may be unstable in vitro as, in some protein preparations, a disulfide bond was formed instead. llmg_ and llmg_ each contained four cysteines, in a cx2cx10cx2c motif in the n-terminal extremity, while llmg_ harbored a cx2c motif in its c-terminal extremity. llmg_ contained four target motifs in its nter domain, which were organized into two cx2cx14cx2c motifs. with the exception of the cysteine motif, no other signatures were detected that predicted the function of the latter four proteins.table putative membrane proteins containing cx2c motif in l. lactis strain mg1363 tmd, transmembrane domain to understand the role of the cysteine motif in l. lactis proteins, we continued our investigation by focusing on two cysteine-rich proteins, llmg_ and llmg_ which might sense stressful environments via cysteine oxidation and their putative membrane localization. we aimed to determine: i) the genetic organization and expression of these proteins; ii) the localization and properties of the cysteine motif within the proteins; and iii) the role of these proteins. temporal expression of the llmg_0524-0525- operon we found a stem loop upstream of the llmg_ orf and another one downstream of the llmg_ orf, suggesting a potential operonic structure for the three genes: the llmg_ orf; the llmg_ orf, which encodes a small hydrophobic protein free of cysteines; and the llmg_ orf. to test our hypothesis, we performed rt-pcr assays with different primer pairs and total rna that had been extracted from cells harvested in the exponential growth phase. the results showed that an mrna transcript overlapped two adjacent genes. the pcr product had the same size of that obtained from genomic dna and was absent when rna preparation was pretreated with a rnase. we also performed pcr with primers designed inside the llmg_ and llmg_ orfs; the transcripts of this pcr product covered all three genes. no pcr amplification was obtained with primer pairs designed for the llmg_ and llmg_ orfs, a result that was consistent with the presence of stem loops. finally, we cloned a dna fragment that covered the region from the middle of the llmg_ orf to the beginning of the llmg_ orf; this fragment was inserted upstream of the promoterless lacz gene of the ptcv-lac plasmid, generating plasmid p0526-ptcv-lac. after we introduced this plasmid into l. lactis strain mg we did not detect any β-galactosidase activity under the tested conditions, suggesting the absence of a promoter, specifically the one upstream of the llmg_ orf. taken together, these results indicate that the three genes are clustered in an operon and under the control of a promoter located upstream of the llmg_ orf.fig. llmg_ llmg_ and llmg_ form an operon. a schematic representation of the locus; bp - base pair. b rt-pcr analysis of the locus. total rna was translated into cdna, which in turn was used as the template for pcr with different primer pairs. pos control, positive control with genomic dna as a template; neg control, negative control with treatment of rna preparation with a rnase to study the expression of the operon, we cloned the promoter region of llmg_ orf into the ptcv-lac plasmid, creating plasmid p0524-ptcv-lac, and introduced this into l. lactis strain mg with ortho-nitrophenyl galactoside as the growth substrate and glucose as the energy source, we did not detect any β-gal activity. however, using a more sensitive test with a fluorescent substrate, we managed to detect some β-gal activity, indicating that the operon was poorly expressed. under these conditions, we observed β-gal induction an hour after inoculation, with a peak around od. beyond this point, β-gal activity decreased progressively with further growth. when glucose was replaced by galactose as the energy source, β-gal expression increased but appeared later in growth than was observed with glucose. moreover, instead of having a peak, transcript quantity reached a stable plateau with minor changes until od and then it went down. to validate gene expression we performed semi-quantitative rt-pcr assays, which revealed relative transcript amounts that were concordant with the β-gal activity patterns. none of the other tested growth conditions resulted in changes to the expression of the operon. in addition, deletion of llmg_ or llmg_ decreased two-fold expression of the operon. we thus conclude that operon is transiently transcribed and occurs only during the early log growth phase.fig. temporal expression of the operon. a expression kinetics of the operon. cells were grown in m17glucose or m17galactose. at regular intervals, aliquots were collected to assess β-gal activity. values, plus standard deviations, are the means of three independent experiments. b semi-quantification of the llmg_ transcript. cells were grown on glucose or on galactose and collected at certain growth phases. total rna was prepared for semi-quantitative rt-pcr determination. control - ahpc. dil. - dilution membrane localization of llmg_ and llmg_0526 analyses of these proteins with topology prediction programs suggested that llmg_ contained two transmembrane domains but the programs yielded no information about the localization of the nter domain. llmg_ contained a transmembrane domain with the nter region, indicating that this end is probably inside the cell. to obtain direct experimental evidence regarding the membrane topology of these proteins, we performed an alkaline phosphatase assay. phoa is activated via the oxidation of its cysteine residue to form a disulfide bridge, a reaction which occurs mainly in the periplasm of e. coli cells. the phoa assay thus consists of fusing the signal-sequence-less phoa gene to a target dna sequence and then monitoring for phoa activity; such activity should only be detectable if the target dna addresses the resulting protein into the periplasmic space. we therefore constructed a set of plasmids that each contained a different portion of the llmg_ and the llmg_ orfs fused to phoa, and then established the plasmids in the e. coli phoa mutant. bacteria were grown on lb agar supplemented with arabinose for the production of fusion proteins and with a bcip compound for the detection of phoa activity. cells that produced the phoa hybrid proteins generated a strong blue color corresponding to the degradation of bcip, while those that produced the phoa constructs displayed only weak coloration indicating that phoa was addressed out of cytosol with phoa construction. quantification of the phoa activity confirmed these observations, revealing the strongest activity in cells with the phoa fusions. we therefore conclude the two proteins are located in the membrane, with the nter ends located in the cytoplasm.fig. nter end of llmg_ and llmg_ is intracellular. a schematic representation of the proteins with position of transmembrane domain and protein fusion with phoa. b phosphatase activity of cells that overproduced the protein fusions. fusion proteins were overproduced in a e. coli phoa mutant, which were then treated with arabinose. phosphatase activity was revealed in presence of bcip compound present in agar plates llmg_ and llmg_ are zinc metalloproteins as the cx2cx10cx2c motif in llmg_ and llmg_ was identical to that of the zinc metalloprotein dnaj, we hypothesized that our target proteins also bound a metal. to test this, we purified the nter domain of llmg_ and llmg_ as a hybrid protein to the maltose binding protein male, creating male-0524nter and male-0526nter, respectively. the protein male-laczα corresponding to the plasmid without insert was used as a control. after purification, the hybrid proteins were analyzed by inductively coupled plasma mass spectrometry to determine nature of metal ion if present in protein. we found magnesium ion was present in all preparation at relative similar concentration but this ion has not been reported to bind with cysteine residues. male-laczα did not contain iron or zinc metal in significant amount in contrast to other proteins. in the presence of male-0524nter, zinc was far more abundant than iron. in preparations of male-0526nter, although zinc was still the most abundant, there was also a quantity of iron ion. however, this protein was colorless and its uv-visible spectrum did not display any absorption bands in visible wavelengths, which are characteristic of iron-cysteine proteins. furthermore, when the medium was supplemented with zinc salt, the level of iron in the preparation decreased dramatically, indicating that the affinity of male-0526nter to iron was not specific. altogether these results indicate that llmg_ and llmg_ are zinc metalloproteins.table llmg_ and llmg_ are zinc metalloproteins male-0524nter male- nter male- nter male- nter all buffers were treated with chelex. values are the mean of three independent protein purifications. + zinc, cultures were supplemented with  mm of zinc salt. male-laczα is used as a control, nd: not determined the cx2cx10cx2c motif binds zinc metal directly the icp-ms analysis allowed us to determine the stoichiometry ratio of zinc to protein:: for male-0524nter and: for male-0526nter. these ratios did not change when we supplemented the media with zinc salt indicating the lb broth contained enough metals to load synthesized proteins. these ratios were further confirmed by the titration of zinc ions with the chelator par, resorcinol. to demonstrate that this metal was coordinated in the proteins via the thiol group of the cysteine, we treated the proteins with p-hydroxy mercury-phenylsulfonate agent. pmps specifically disrupts the cysteine-zinc bond by forming a cysteine-mercury and the released zinc ion can be titrated with par. evidence of zinc-par complex formation appeared progressively with increasing amounts of pmps in the mixture. for male-0524nter, two equivalents of pmps versus protein were required to release around 90 % of the zinc ions, and adding more pmps did not significantly change the absorbance. as there were no cysteines in the male portion of the fusion protein, this result meant that two cysteine residues of llmg_0524nter were involved in binding the zinc metal. if this were the case, the protein should therefore also contain free thiols, which we determined via titration with -dithiobis, a compound which reacts to free thiol groups. we observed that the ratio of free cysteine:protein was close to one. the second residue was not titrated in our assays probably because it was not accessible to dtnb due for instance to folding of llmg_ fragment. in male-0526nter, instead, four equivalents of pmps were required to release most of the zinc from the protein, indicating a stoichiometry of 4: of pmps:zinc and thus the presence of a zinc complex. in agreement with these observations, titration with dtnb indicated a low amount of free thiol groups in our preparations. we thus conclude that each of the two proteins harbors a zinc-cysteine module but in different coordination spheres: zinc in llmg_ and zinc in llmg_ fig. presence of a cysteine-zinc module in proteins. the cysteine-zinc bond was characterized by pmps-par assays with 20 μm of proteins. male-0524nter or male-0526nter were purified from cultures and from those that were supplemented with zinc salt values, plus standard deviations, are the means of three independent experiments 20 μm of protein was used for thiol group determination by dtnb assays in addition, we tested the stability of the zinc module in the presence of oxidative agents and a temperature of 37 °c, which affects protein folding in l. lactis. in the presence of ten equivalents of oxidant or 37 °c, no released zinc ions were detected by par, indicating that the modules in these proteins are very stable in tested conditions. llmg_ is required for resistance against cumene hydroperoxide and cell wall integrity to investigate the function of the llmg_0524-0525- operon in vivo, an in frame-markerless deletion of the llmg_ or llmg_ gene was constructed in the chromosome of strain mg as our in silico analyses did not yield any information about the potential functions of these proteins, we screened the mutants in several growth conditions. from these we found no clear phenotypic changes in the ∆llmg_ mutant compared to its isogenic wild-type strain. in the ∆llmg_ mutant, instead, we found one growth condition in which the ∆llmg_ mutant was more sensitive than the wt strain: growth in the presence of cumene hydroperoxide, an oxidative agent, and with galactose as an energy source. in contrast, we did not observe any difference between the two strains treated hydrogen peroxide. resistance to chp was completely restored when we transformed the ∆llmg_ mutant with a plasmid expressing the wt operon. interestingly, the plasmid pllmg_0524c,∆cyst, which carried a version of the operon in which the dna region encoding the cysteine motif of llmg_ had been deleted, was unable to complement the mutant. in fact, the mutant producing the truncated llmg_ grew very poorly even in the absence of stress, and on glucose as energy sources as well. then, we asked whether the modifications of lipid or cell wall synthesis were responsible of that growth defect. addition of tween- a donor of long chain fatty acids, did not have any effect on the growth. in contrast, we found that the mutant, carrying the plasmid pllmg_0524c,∆cyst, became more resistant to lysozyme, an hydrolase of peptidoglycan, than the other strains indicating that the growth defect was due to cell wall modifications. we conclude the llmg_ is linked to cell wall integrity and resistance to stressful conditions.fig. role of llmg_ mutant in cumene hydroperoxide resistance and growth. cells were grown in m17galactose in static conditions at 30 °c. when cell density reached od,  mm of cumene hydroperoxide was added to the culture. wt - black line, ∆llmg_ mutant - gray line, ∆llmg_ mutant carrying the plasmid encoding the full operon - gray dashed line. values, plus standard deviations, are the means of three independent experiments fig. role of llmg_ in the cell wall integrity. a effect on the growth of the deletion of the cysteine-rich motif in llmg_ cells were grown in m17glucose in static conditions at 30 °c. the ∆llmg_ mutant carrying the plasmid pllmg_0524 c,∆cyst is represented by a dark gray dashed line. the plasmid encoded the full operon, but lacked the region in the llmg_ orf corresponding to the cysteine motif. values, plus standard deviations, are the means of three independent experiments. b effect of lysozyme resistance of the deletion of the cysteine-rich motif in llmg_ after overnight cultures, strains were washed in pbs buffer and recovered at the same od after 60 min of incubation in absence or in presence of lysozyme, sds was added at  % final concentration and measured od values, plus standard deviation, are the means of three independent experiments. they are expressed in percent of od measured in samples incubated 60 min in absence of lysozyme and sds discussion in this study we characterized llmg_ and llmg_ two proteins of unknown functions with the same cx2cx10cx2c motif. this motif bound a zinc ion. we found that the cysteine-zinc complex was stable when subjected to stress, suggesting it plays a role in protein stability rather than as a sensor. this hypothesis is supported by our observation that deletion of this motif in llmg_ had serious impacts on cell growth. in addition, we did find that llmg_ also increased resistance against the oxidative agent cumene hydroperoxide. a cysteine motif to chelate metal in l. lactis strain, we demonstrated that the cx2cx10cx2c motif bound zinc ion. this by itself is not novel, as this motif is also found in other proteins, and in some of these, it has been experimentally demonstrated to bind this metal. for example, the dnaj protein of e. coli binds zinc ion using all four cysteines of the motif, and formation of the zinc-cysteine complex is required for protein activation. more recently, the same motif was found in e. coli ycim, a membrane protein, in which the four cysteine residues were involved in metal coordination. however, ycim is proposed to bind iron ion instead of zinc. from these observations alone, it was clear from this study and others that the motif is used for trapping metal, but the question remained: is it possible to predict which metal, and the individual cysteine residues’ contributions to sphere coordination, based only on the motif? in this study and dnaj, zinc ion is the major metal found suggesting that this motif may have a bigger affinity to this metal than iron. in l. lactis, this motif may contain zinc since in strain mg level of zinc ion is 25-fold higher than that of iron whereas in e. coli strain zinc is present in an amount similar as iron. however, there are few examples describing the complex cysteine-metal to make this hypothesis true to proteins having this motif in other bacteria. with the male-0526nter fusion protein, pmps-par assays demonstrated the presence of a zinc module in llmg_ clearly indicating that all cysteine residues were engaged in zinc coordination. unexpectedly, however, similar tests using the fusion male-0524nter revealed that only two residues bound zinc. the two other cysteines of the protein were free, although we detected only one residue. that observation also indicated that male-0524nter lacked a disulfide bond, suggesting that the fusion protein was not redox reactive in vitro. however, in vivo these two free cysteine residues could play a role in the intact protein by forming intra- or intermolecular disulfide bridge between two llmg_ or llmg_ and another protein. to our knowledge, zinc usually binds four ligands, with cysteine and histidine residues as the most common ligands by far. however, no histidine is present in the cytoplasmic portion of llmg_ which suggests that the metal coordination of this protein is unusual. additional experiments will be needed to determine the other ligands of zinc ion in llmg_ as well as the parameters that could govern the specificity of metal. role of the llmg_0524-0525- operon unlike cysteine, zinc is not redox reactive in proteins. as the bound modules in llmg_ and llmg_ were very stable in the presence of oxidative agents or 37 °c, which affects the protein folding in l. lactis, it is unlikely that the module is used as a direct sensor of stress, as has been described, e.g., for the chaperone hps. instead, for llmg_ at least, we think that the zn module serves to stabilize the folded structure of the protein as required for its function, perhaps for interaction with other proteins. this hypothesis is supported by the dramatic growth differences we observed between the wild-type and the truncated-llmg_ mutant strain, which lacked the cysteine motif. even in the absence of stress, this strain displayed a severe growth defect. similarly, in e. coli, ycim mutants lacking this motif were unstable and formed aggregates. in addition, our experiments with deletion mutants suggest that this operon plays a role in cell wall integrity in particular with peptidoglycan synthesis since that mutant, producing the truncated llmg_ was more resistant to lysozyme than other strains. a modification of cell wall may explain the light resistance to cumene hydroperoxide observed in the ∆llmg_ mutant. studies are in progress to characterize the modification of cell wall linked to ∆llmg_ deletion. our experiments with the ∆llmg_ mutant did not produce any reproducible phenotypes even against the cumene hydroperoxide toxicity. in a protein blast search, this protein displays similarity to s. aureus tcaa, which has a cysteine motif inside, which might bind metal although this has not been confirmed. downstream of the motif, we found a transmembrane domain followed by a long extracellular chain. in s. aureus, tcaa is induced by envelope stress and resistant to teicoplanin, an antibiotic of glycopeptide family. although they share a similar primary structure, llmg_ does probably not play the same role of tcaa. in contrast to tcaa, l. lactis, llmg_0524-0525- operon is not induced by the envelope stress response.  cysteine motifs in proteins are often associated to redox activity or iron-sulfur cluster in bacteria, but less is known about cysteine motif in chelating zinc ion. in this work, we characterized in lactococcus lactis, two membrane proteins carrying a cx2cx10cx2c motif. the motif bound zinc ion although the sphere of coordination of zinc was remarkably different between the two proteins. our works suggest that llmg_ and maybe other proteins of operon, is linked to cell wall integrity. the function of this protein in the machinery of cell wall synthesis remains to uncover now, as well as its zinc module. 
in order to understand the role played by this motif we focused our work on two membrane proteins of unknown function: llmg_ and llmg_ each of these proteins has two cx2c motifs separated by ten amino-acid residues. cysteinezincmembrane proteinsgrowthcumene hydroperoxidelysozymeissue-copyright-statement© the author 2015 in both proteins, the cysteine-zinc complex was very stable at 37 °c or in the presence of oxidative agents, suggesting a probable role in protein stability. in both proteins, we found that the cx2cx10cx2c motif chelated a zinc ion via its cysteine residues, but the sphere of coordination was remarkably different in each case. lactococcus lactis, a gram-positive bacterium, houses around one hundred cysteine-rich proteins in the cytoplasm, but only a few in the membrane. in the case of llmg_ two of the four cysteines were ligands of a zinc ion whereas in llmg_ all four residues were involved in binding zinc. our data suggest that the cx2cx10cx2c motif is used to chelate a zinc ion but we cannot predict the number of cysteine residue involved as ligand of metal. although no other motif is present in sequence to identify roles played by these proteins, our results indicate that llmg_ contributes to the cell wall integrity. we found that the complete deletion of llmg_ increased the sensitivity of the mutant to cumene hydroperoxide whereas the deletion of the cysteine motif in llmg_ resulted in a growth defect.
 due to its extraordinary chemical properties, the cysteine amino acid residue is often involved in protein folding, electron driving, sensing stress, and binding metals such as iron or zinc. lactococcus lactis, a gram-positive bacterium, houses around one hundred cysteine-rich proteins in the cytoplasm, but only a few in the membrane.  in order to understand the role played by this motif we focused our work on two membrane proteins of unknown function: llmg_ and llmg_ each of these proteins has two cx2c motifs separated by ten amino-acid residues. together with a short intervening gene, the genes of these two proteins form an operon, which is induced only during the early log growth phase. in both proteins, we found that the cx2cx10cx2c motif chelated a zinc ion via its cysteine residues, but the sphere of coordination was remarkably different in each case. in the case of llmg_ two of the four cysteines were ligands of a zinc ion whereas in llmg_ all four residues were involved in binding zinc. in both proteins, the cysteine-zinc complex was very stable at 37 °c or in the presence of oxidative agents, suggesting a probable role in protein stability. we found that the complete deletion of llmg_ increased the sensitivity of the mutant to cumene hydroperoxide whereas the deletion of the cysteine motif in llmg_ resulted in a growth defect. the latter mutant was much more resistant to lysozyme than other strains.  our data suggest that the cx2cx10cx2c motif is used to chelate a zinc ion but we cannot predict the number of cysteine residue involved as ligand of metal. although no other motif is present in sequence to identify roles played by these proteins, our results indicate that llmg_ contributes to the cell wall integrity. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords cysteinezincmembrane proteinsgrowthcumene hydroperoxidelysozymeissue-copyright-statement© the author 2015 