BACKGROUND
members of the genus lactobacillus are widely recognized as the hallmark of the normal or healthy vagina and are thought to play a major role in protecting the vaginal environment from non-indigenous and potentially harmful microorganisms .
lactobacillus gasseri is a human autochthonous microorganism  <cit>  and one of the dominant lactobacillus species in the vaginal ecosystem, together with lactobacillus jensenii, lactobacillus iners and lactobacillus crispatus .
thus, decreasing of lactobacilli titer in the vagina, results in bacterial vaginosis , where the normal flora is replaced by undesirable bacteria, including bacterial pathogens such gardnerella vaginalis, atopobium vaginae, mobiluncus curtisii, prevotella bivia, leptotrichia amnionii, eggerthella spp,, sneathia spp and megasphaera type i spp.
.
l. gasseri has been well documented as a commensal of the vaginal mucosa and exhibits a negative correlation with bv .
some strains of this species have a high potential for being used as probiotics in order to maintain vaginal homeostasis .
l. gasseri may confer colonization resistance against pathogens in the vagina by displacing them through competitive adhesion  <cit>  or by direct inhibition through production of antimicrobial compounds, including lactic acid, hydrogen peroxide and bacteriocins  <cit> .
bacteriocins could play a major role to control non-indigenous or pathogenic organisms in the female genitourinary tract  <cit> .
bacteriocins are ribosomally-synthesized antimicrobial peptides of bacterial origin that are active against a variable spectrum  of bacteria.
production of these antimicrobial peptides plays an important role in bacterial competition, allowing survival advantages to the producing strain  <cit> .
bacteriocins from gram-positive bacteria are nowadays classified into two major classes  <cit> : the lantibiotics  and the non-lantibiotics .
class ii is divided into four subgroups: class iia , iib , iic , and iid .
currently, four different bacteriocins from l. gasseri have been purified and genetically characterized: gassericin t from l. gasseri sbt <dig>  <cit> , acidocin lf221a  and acidocin lf221b  from l. gasseri lf <dig>  <cit> , and gassericin a from l. gasseri la <dig>  <cit> .
while gassericin a is a class iic circular bacteriocin, gassericin t and acidocins lf221a and lf221b have been proposed to belong to class iib  bacteriocins, although no experimental data supporting this hypothesis has been published, yet.
in this work we describe the biochemical and genetic characterization of gassericin e, a novel bacteriocin produced by a l. gasseri strain isolated from the vagina of a healthy woman.
production of this bacteriocin was shown to be promoted by the presence of certain specific bacteria in co-culture with the producer strain.
the genetic background showed the presence of a three-component regulatory operon that may be involved in the regulation of the production of this bacteriocin.
methods
bacterial strains and growth conditions
lactobacillus gasseri ev <dig> was isolated from the vagina of a healthy woman , while we were searching for bacteriocin-producing lactobacilli species isolated from different human sources.
the volunteer gave written informed consent to the protocol , which had been approved by the ethical committee of clinical research of hospital clínico san carlos madrid .
most of the bacterial strains used in this study  were grown routinely in mrs medium  at 37 °c in aerobic conditions.
actinomyces neuii, escherichia coli, staphylococcus spp.
and streptococcus spp., were grown in brain heart infusion  medium  at the same conditions described above.
fastidious organisms were grown in different culture media at 37 °c in anaerobic atmosphere  as follows.
gardnerella vaginalis was grown in casman’s medium base  with 5 % v/v rabbit blood; porphyromonas gingivalis, atopobium vaginae, anaerococcus vaginalis and leptotrichia amnionii were grown in tryptic soy broth agar ; prevotella bivia was grown in modified reinforced clostridial agar broth medium ; mobiluncus spp.
were grown in enriched tryptic soy agar  medium .
unless indicated, strains from human sources belonged to our own collection, while strains from other sources belonged to different bacterial collections .
they were maintained as frozen stocks at –80 °c in their respective culture medium plus 20 %  glycerol.table  <dig> inhibitory spectrum of lactobacillus gasseri ev <dig> against bacterial strains from human origin
actynomyces neuii
actinomyces turicensis
anaerococcus vaginalis
atopobium vaginae
bifidobacterium longum
candida albicans
corynebacterium amycolatun/xerosis
corynebacterium aurimucosum
corynebacterium freneyi
corynebacterium glucurunolyticum
corynebacterium tuberculostearicum
corynebacterium simulans
corynebacterium striatum
enterococcus faecalis
enterococcus faecium
enterococcus gallinarum
enterococcus saccharolyticus
escherichia coli
facklamia hominis
gardnerella vaginalis
lactobacillus fermentum
lactobacillus gasseri
lactobacillus paracasei
lactobacillus salivarius
leuconostoc mesenteroides
leptotrichia amnionii
mobiluncus mulieris
mobiluncus curtisii
subsp.
curtisii
subsp.
holmesii
porphyromonas gingivalis
prevotella bivia
propionibacterium acnes
propionibacterium avium
propionibacterium granulosum
staphylococcus aureus
staphylococcus caprae
staphylococcus epidermidis
-
-
-
streptococcus agalactiae
streptococcus anginosus
streptococcus intermedius
streptococcus parasanguinis
a, b
abbreviations:
c colostrums, h faeces, ev, o, ps and v vaginal exudates, fr rectal frotis, m meconium, ms semen, mp glans, l breast milk, la amniotic liquid, p skin, sc umbilical cord blood, fvm facultad de veterinaria , dsmz german collection of microorganisms and cell cultures
csensitivity to cell-free supernatants of l. gasseri ev <dig>  assayed by the agar drop diffusion test method.
sensitivity scale: + , ++ , +++ , and ++++ , reflecting the degree of sensitivity according to the diameter of the inhibition halo ; -, resistant
bacillus cereus
enterococcus faecium
enterococcus faecalis
lactobacillus acidophilus
lactobacillus bulgaricus
lactobacillus casei
lactobacillus coryniformis
lactobacillus curvatus
lactobacillus fermentum
lactobacillus helveticus
lactobacillus pentosus
lactobacillus plantarum
lactobacillus salivarius
lactobacillus sakei
lactococcus lactis
subsp.
cremoris
subsp.
lactis
leuconostoc cremoris
pediococcus pentosaceus
staphylococcus carnosus
streptococcus thermophilus
a
abbreviations:
cect colección española de cultivos tipo , cig colección instituto de la grasa , cit cranfield institute of technology , fvm facultad de veterinaria, universidad complutense , inra institut national de la recherche agronomique , ncdo national collection of dairy organisms , tno nutrition and food research
bsensitivity to cell-free supernatants of l. gasseri ev <dig>  assayed by the agar drop diffusion test method.
sensitivity scale: + , ++ , +++ , and ++++ , reflecting the degree of sensitivity according to the diameter of the inhibition halo ; -, resistant
bacteriocin assays
to check for bacteriocin production on solid medium, a pointed sterile inoculating handle was soaked in overnight broth cultures of the producing strain and then punctured in mrs agar plates.
the plates were incubated at 37 °c for 6 h and then were overlaid with  <dig>  ml soft agar  inoculated with ca.
105 cfu/ml of the indicator strains.
plates were further incubated at 37 °c for 16–18 h, and examined for clear halos of inhibition around the punctured cultures.
bacteriocin activity in cell-free supernatants  from stationary-phase broth cultures  of l. gasseri ev <dig> was assayed by using the agar drop diffusion test as described previously  <cit> , using the strains listed in tables  <dig> and  <dig> as indicator microorganisms.
bacteriocin activity was quantified by a microtiter plate assay, as described previously  <cit>  using l. paracasei c <dig> as the indicator strain.
one bacteriocin unit  was defined as the amount of bacteriocin active cfs that inhibited the growth of the indicator strain by 50 %, using as a reference the turbidity of control cultures without cfs added.
this was expressed as the reciprocal of the highest dilution exhibiting 50 % inhibition of the indicator strain per milliliter .
conditional bacteriocin production of l. gasseri ev1461
two or three  and  <dig> to  <dig> colonies  were inoculated into fresh 10-ml mrs broth tubes and incubated at 37 °c.
cfs was tested for antimicrobial activity at  <dig> and 16 h of growth as described above.
the minimum number of cells that were necessary to obtain a bac+ or a bac- culture was determined by serial dilutions of a 16 h culture of l. gasseri ev <dig>
bac+ cfss from l. gasseri ev <dig> were tested for their ability to autoinduce bacteriocin production in l. gasseri ev <dig> bac- cultures.
for autoinduction, 20 μl of bac+ cfs  were added to fresh mrs broth  containing ca.
102 cfu/ml of an overnight culture of l. gasseri ev <dig> , and incubated at 37 °c for 16 h. in control experiments, the same amount of bac+ cfs was added to 1 ml mrs broth and then the antimicrobial activity was assayed.
production of bacteriocins by l. gasseri ev <dig> in co-cultures with the inducer strains l. gasseri lc <dig>  lactobacillus pentosus 128/ <dig>  lactobacillus plantarum ce <dig> and propionibacterium avium h <dig> was determined as described previously  <cit>  with some modifications.
briefly, fresh mrs broth was inoculated with an overnight bac-culture of l. gasseri ev <dig>  plus an overnight culture  of each inducer strain.
the mixed cultures were held at 37 °c for 6 h, centrifuged, and the cfss adjusted to ph  <dig>  with 5 n naoh, filter-sterilized, and finally their inhibitory activity assayed by the agar drop diffusion test, using lactobacillus paracasei c <dig> as the indicator strain.
in control assays, all of the strains used in the mixed cultures were propagated as pure cultures in their respective media and then assayed for antimicrobial activity as described above.
purification of gassericin e
all of the purification steps were carried out at room temperature, and all of the chromatographic equipment and media were purchased from amersham biosciences europe gmbh, freiburg, germany.
gassericin e  was purified from 2-litre cultures as follows.
two 10 ml mrs tubes were inoculated at high cellular densities by picking ca.
<dig> isolated colonies of a 48-h plate culture of l. gasseri ev <dig> and incubated at 37 °c for 16 h. then, two 200-ml mrs bottles were inoculated each one with the 16 h 10 ml l. gasseri ev <dig> broth cultures, and further incubated at 37 °c for 10 h. finally, a 2-litre mrs flask was inoculated with the two 200-ml broth cultures of l. gasseri ev <dig> and further incubated for 16 h. the cells were removed by centrifugation at  <dig>  × g for 10 min at 4 °c and, then, the bacteriocin was purified from the cfs by the same method described for plantaricin nc <dig>  <cit> .
briefly, the cfs was precipitated with ammonium sulfate , desalted through pd- <dig> columns, and consecutively applied to cation-exchange  and hydrophobic-interaction  columns.
fractions showing bacteriocin activity were applied to reverse-phase chromatography  in a c2/c <dig> rpc column  coupled to a fast protein liquid chromatography  system.
the bacteriocin was eluted from the rpc column with a linear gradient of 2-propanol  in aqueous  <dig>  %  trifluoroacetic acid.
fractions showing inhibitory activity after the rpc-fplc were pooled and rechromatographed to obtain pure bacteriocin.
purity of bacteriocin fractions were checked by sds-page as described below.
sds-page
during the purification process, the rpc-fplc eluted fractions of gase were analyzed in duplicate by tris-tricine sds-page, using an 18 % acrylamide resolving gel  <cit> .
after electrophoresis at 100 mv for 2 h, one gel was silver stained while the other was used to detect the inhibitory activity in an overlay assay as described previously  <cit> .
l. paracasei c <dig> was used as the indicator strain.
the precision plus protein dual xtra  was used as molecular weight standards.
n-terminal amino acid sequence and mass spectrometry
the n-terminal amino acid sequences of purified gase peptide was determined by automated edman degradation with a beckman lf <dig> sequencer/phenylthiohydantoin amino acid analyzer .
molecular mass of the peptides was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry .
these analysis were performed by dr. silvia bronsoms .
pcr sequencing and location of the gassericin e locus
the primers g1-f, gatx-r, gata-f and gata-r were designed on the basis of the locus encoding gassericin t of l. gasseri sbt <dig>  .
the primers gt1-f, gt1-r, gt2-f, gt3-f,gt4-f,gt5-f,gt6-f,gt7-f and gt8-r were designed on the basis of the locus encoding gassericin t of l. gasseri la <dig>  .
total dna was extracted from l. gasseri ev <dig> colonies as described previously  <cit> .table  <dig> oligonucleotides used in this study
amplification with the primer pair g1-f/gatx-r was carried in 50 μl reaction mixtures containing  <dig>  mm mg cl <dig>  1× reaction buffer, 200 μm concentrations of each of the deoxynucleotides triphosphates , 1 μm of each of the primers,  <dig>  u of taq dna polymerase  and 5-μl of template dna.
amplification included denaturation at 94 °c for 4 min, followed by 30 cycles of denaturation at 94 °c for 30 s, annealing at 56 °c for 1 min, polymerization at 72 °c for 1 min, and a final polymerization step at 72 °c for 5 min.
amplification with the primer pair gt1-f/gt8-r was carried out in 50-μl reaction mixtures containing  <dig>  mm mg cl <dig>  1× reaction buffer, 200 μm concentrations of each of the dntps, 1 μm of each of the primers,  <dig>  u of taq dna polymerase  and 5 μl of template dna.
amplification included denaturation at 94 °c for 2 min, followed by 10 cycles of denaturation at 94 °c for 10 s, annealing at 57 °c for 30 s, and polymerization at 68 °c for 3:30 min, 20 cycles of denaturation at 94 °c for 10 s, annealing at 57 °c for 30 s and polymerization at 68 °c for 3:30 min plus 20 s/cycle, and a final polymerization step at 68 °c for 7 min.
the amplified fragments were excised from  <dig>  % agarose gels, purified using the nucleospin® extract ii kit , and sequenced using the primers described in table  <dig> at the genomics unit of the universidad complutense .
dna sequences were assembled using the seqman software in the dnastar package and deposited in the genbank database .
dna and amino acid sequence analysis
searches for dna and amino acid similarities in nucleotide and protein databases were done using the basic local alignment search tool   <cit> .
searches for promoter sequences were done with the neural network promoter prediction web interface   <cit> .
for detection of rho-independent terminators, the arnold web server  was used  <cit> .
for alignment of the amino acid sequences of the leader and mature peptides of the bacteriocins, the clustalw <dig> multiple sequence alignment program was used   <cit> .
for physico-chemical analysis of peptides  the winpep program was used   <cit> .
RESULTS
antimicrobial spectrum of l. gasseri ev1461
bac+ cfss from l. gasseri ev <dig> broth cultures showed antimicrobial activity against many indicator strains used in this study, including species both related and non-related to the producing strain as shown in tables  <dig> and  <dig>
conditional bacteriocin production
this strain showed antimicrobial activity  when grown on solid medium.
however, after the inoculation of two or three colonies into fresh mrs broth , l. gasseri ev <dig> cultures do no display antimicrobial activity .
by contrast, after the inoculation of a larger number of colonies , l. gasseri ev <dig> showed bacteriocin activity  after at least 6 h of growth, maintaining this activity up to 16 h of growth.
thus, at low cellular densities  the antimicrobial activity produced by l. gasseri ev <dig> was low or no detectable  .
in these diluted bac- cultures, the bac+ phenotype was restored when the strain was grown on solid medium as isolated colonies or in broth cultures after the addition of a bac+ cfs from a previous bacteriocin-producing culture .
thus, the addition of a bac+ cfs from l. gasseri ev <dig> to l. gasseri ev <dig>  broth cultures resulted in bacteriocin production  , indicating the existence of an autoinducing factor in the supernatant.
in control experiments, the addition of a bac+ cfs from l. gasseri ev <dig> to mrs broth showed no antimicrobial activity.table  <dig> conditional bacteriocin production of l. gasseri ev1461
l. gasseri ev <dig> b
l. gasseri ev <dig> c
l. gasseri ev <dig> + cfs ev <dig> d
abacteriocin units per mililiter
bbacteriocin non-producing l. gasseri ev <dig>
cbacteriocin producing l. gasseri ev <dig>
dcfs: cell free supernatant from a bac+ l. gasseri ev <dig> culture containing  <dig> bu/ml
in addition, when highly diluted l. gasseri ev <dig> cultures were co-cultured with certain specific gram-positive bacteria, production of bacteriocin was notably increased  with respect to that displayed by the ev <dig> pure cultures  .
thus, mixed cultures of l. gasseri ev <dig> with propionibacterium avium h1544b, lactobacillus plantarum ce <dig>  lactobacillus pentosus 128/ <dig> or lactobacillus gasseri lc <dig> induced  bacteriocin production in l. gasseri ev <dig> .
none of the inducing strains exhibited any antimicrobial activity when they were cultivated as single, pure cultures.
the ability of these species to induce bacteriocin production in l. gasseri ev <dig> was independent of their resistance or sensitivity to the bacteriocin produced .
purification of gassericin e
gase was isolated from the bac+ cfs of a 2-litre broth culture of l. gasseri ev <dig> as described in the methods section.
the behaviour of gase throughout the purification process was that of a cationic and hydrophobic peptide.
two runs on rpc-fplc were necessary to obtain fractions containing pure bacteriocin .
sds-page analysis showed a single peptide band with an apparent molecular size of 5-kda  which presented inhibitory activity against l. paracasei c <dig> in the corresponding sds-page activity gel .
the inhibitory activity of pure gase had a titer of  <dig> bu ml− <dig> against l. paracasei c <dig> fig.
<dig> maldi-tof mass spectra of purified gassericin e .
+, monoisotopic peak of gase.
a.u., absorbance units.
inset panel: sds-page  and bioassay  of purified gase.
l. paracasei c <dig> was used as the indicator strain.
mwm, molecular weight marker
mass spectrometry and n-terminal amino acid sequencing
maldi-tof ms analysis of purified gase indicated that a monoisotopic peak  of the bacteriocin was present, suggesting that the molecular mass of the peptide is  <dig> .0 da .
partial amino acid sequencing of gase showed that the 18 n-terminal amino acids shared high similarity with the first 18 n-terminal amino acids of mature gassericin t from l. gasseri sbt <dig> .fig.
<dig> alignment of the amino acid sequences of double-glycine leader peptides and mature peptides of gassericin e and other similar gassericin bacteriocins.
the sequences were aligned with the clustalw <dig> software at the embl-ebi online server.
the arrow indicates the gly-gly cleavage site of the peptide.
asterisks, dots and double dots mean fully, strongly and weakly conserved residues, respectively.
the first  <dig> aa obtained by edman sequencing of the peptide gase are underlined.
the two deduced amino acid sequences deposited in databases from l. gasseri k <dig>  differed in one amino acid at position  <dig> of mature peptide .
theoretical molecular weights  of the mature bacteriocins are shown.
the genebank accession numbers are baa <dig> for gassericin t, aap <dig> for acidocin 221b, efj <dig> for lactacin-f subunit lafa, aap <dig> and kda99085for gassericin k <dig> b complemental factor  and , respectively
dna and deduced protein sequence analysis of the gase locus
two dna fragments of  <dig>  and  <dig>  kbp were amplified with the primer pairs g1-f/gatx-r and gt1-f/gt8-r, respectively.
dna sequencing of the gase locus revealed the presence of up to nine open reading frames  which seemed to be organized into two putative operons , which could be involved in biosynthesis and immunity of this bacteriocin , and in regulation, transport and processing  .
the arrangement of these putative operons was similar to other gene clusters previously described in other bacteriocin-producing lactobacillus gasseri strains .fig.
<dig> schematic representation of the locus for gassericin e  production  and detailed analysis of dna sequences of putative promoters and rho-independent terminators .
a p <dig>  p <dig>  p3a and p3b are putative promoter sequences, and t <dig> is a putative rho-independent transcription terminator.
genes encoding gassericin e , the putative complement peptide gaex  and their putative immunity protein , could form an transcriptional unit, driven by two putative alternative promoters .
genes encoding the three-component regulatory system formed by the autoinducer peptide , the histidine protein kinase  and the response regulator , as well as the abc-transporter  and the accessory protein , seem to form one transcriptional unit; however two different transcripts could be formed, one larger driven by promoter p <dig> involving gaepkrtc and one shorter driven by promoter p <dig> involving gaetc.
a, b, c, d the putative promoters p <dig>  p <dig>  p3a and p3b were detected with the neural network promoter prediction online server  <cit> , with a promoter score cut-off of  <dig>  .
the typical - <dig> and - <dig> boxes and the ribosome binding sites  are shown; the + <dig> indicates the putative transcription start.
putative regulatory dna sequences  are in italics.
e rho-independent terminator; base-pairs are in boldface and apical loop in italics.
f alignment of the direct dna repeats  found upstream of the gase and gaep putative promoters  and the consensus l- and r- repeats involved in quorum-sensing regulation of blp bacteriocins in streptococcus thermophilus  <cit>
gase biosynthesis and immunity operon
detailed analysis of the  <dig> kbp dna sequencing of the putative gase biosynthesis and immunity operon showed that gaea of ev <dig> encodes for a peptide of  <dig> aa which presents an  <dig> aa double-glycine leader peptide that, upon processing, gives rise to a mature peptide of  <dig> aa which has a theoretical molecular mass of  <dig> .42 da .
this deduced molecular weight  coincided with the mw of the gase peptide determined by maldi-tof ms .
in addition, the partial 18-aa sequence of gase obtained by edman degradation was 100 % identical to the sequence of the first  <dig> aa deduced of the mature peptide gase encoded by gaea of l. gasseri ev <dig>  the genes gata from l. gasseri sbt <dig> and l. gasseri la <dig>  as well as orfb <dig> from l. gasseri lf <dig> and l. gasseri k <dig>  encode for a peptide of  <dig> aa, which presents a typical double-glycine leader peptide that, once processed, results in a  <dig> aa mature peptide  with a theoretical molecular mass of  <dig> .34 da .
both the amino acid sequence deduced for mature gase as well as the first  <dig> aa determined experimentally of purified peptide gase differed in one amino acid with that deduced for mature gast.
more specifically, gase contains a leucine residue in position  <dig>  instead of the tryptophan residue of the peptide gast at the same position  .
the leader peptides of gase of l. gasseri ev <dig>  gast of l. gasseri sbt <dig> and l. gasseri la <dig>  acd221β of l. gasseri lf <dig>  gask7bα of l. gasseri, lafa of l. gasseri jv-v <dig> and lactacin f precursor of l. gasseri k <dig> were 100 % identical .
however, the hypothetical mature peptide lafa and lactacin f differed in one amino acid with that of gassericin t and gassericin e .
immediately downstream of gaea, there were two orfs .
the first one  encodes a putative 65-aa peptide which presents a double-glycine leader peptide that, upon processing, gives rise to a mature peptide of  <dig> aa and a theoretical molecular mass of  <dig> .41 da.
this putative peptide bacteriocin was 100 % identical to gatx of l. gasseri sbt <dig>  la <dig>  k <dig>  jv-v <dig> and g <dig>  and 98 % identical to acidocin lf221b and gassericin k <dig> b of l. gasseri lf <dig> and l. gasseri k <dig>  respectively .
downstream of gaex, gaei encodes a putative incomplete protein of  <dig> aa that showed a 100 % of identity with the putative immunity protein to gassericin t of l. gasseri sbt <dig>  la <dig>  jv-v <dig>  k <dig>  g <dig> and lf <dig> .
two putative promoter sequences  containing the typical - <dig> and - <dig> regions were found upstream of gaea thus suggesting that the genes gaea-gaex-gaei are produced on the same transcript .
in addition, upstream of p3b and overlapping p3a, two imperfect direct repeats of 9 bp separated by a rich at region were found .
these putative regulatory elements showed a high similarity with those of streptococcus thermophilus involved in quorum-sensing regulation of blp bacteriocins  <cit>  .
regulatory operon
in l. gasseri ev <dig>  the regulatory operon for bacteriocin production appears to involve three orfs: gaep, gaek and gaer .
the gene gaep encodes a putative peptide of  <dig> aa residues showing all the features previously described for inducer peptides, known also as autoinducing peptides or peptide pheromones  <cit> .
as with bacteriocin-like peptides, the product of gaep possesses a leader sequence of the double-glycine type that, once processed, gives rise to a mature peptide of  <dig> aa residues, with a theoretical pi of  <dig>  and a mw of  <dig> .31 da.
this putative inducer peptide was 100 % identical to that harboured by l. gasseri la <dig>  jv-v <dig> and k <dig> .
the gene gaek, which encodes a putative protein showing significant homology with the family of the histidine protein kinases , was located 2 bp downstream of gaep.
the highest similarity for gaek  was obtained with gatk, the hpk of the gassericin t locus of l. gasseri la <dig>
immediately downstream of gaek, the gene gaer was found.
the putative protein encoded by gaer was 100 % identical to the response regulators previously found in l. gasseri la <dig>  jv-v <dig> and k <dig> .
therefore, based on homology and relative position, the gaep-gaek-gaer gene cluster seems to form a regulatory operon of the so-called three-component type involved in regulation of bacteriocin production.
a similar cluster was found in the genomes of l. gasseri la <dig> , jv-v <dig>  and k <dig> .
a putative promoter sequence  containing the typical - <dig> and - <dig> regions was found upstream of gaep .
in addition, upstream of p <dig>  two imperfect direct repeats of 10 bp separated by a rich at region were found.
these regulatory elements were similar to those found upstream of promoter p3a .
transport and processing operon
downstrean of the gene cluster gaepkr, the orf gaet encodes a  <dig> aa putative protein with 100 % identity with the putative abc-transporter of gassericin t encoded by gatt of l. gasseri la <dig>  jv-v <dig> and k <dig> .
just 1 bp overlapping gaet, gaec encodes a putative  <dig> aa protein with a 99 % identity to the accessory protein for the abc-transporter of l. gasseri jv-v <dig>  lf <dig> and k <dig> .
a putative promoter sequence  was found upstream of gaet, just overlapping gaer .
in addition, two inverted repeats of 10 bp  which may function as a rho-independent transcription terminator were found downstream of gaec .
the existence of two promoter sequences, p <dig> and p <dig>  and a terminator  downstream of gaec indicates that two putative transcripts could be formed, one driven by the p <dig> promoter that would include co-transcription of the gaep-gaek-gaer-gaet-gaec genes, and one driven by the promoter p <dig>  that would include co-transcription of gaet-gaec genes .
discussion
in this work, we describe the purification and genetic characterization of gassericin e, a novel bacteriocin produced by l. gasseri ev <dig>  a strain isolated from the vagina of a healthy woman.
gassericin e is very similar to gassericin t, a bacteriocin purified from l. gasseri sbt <dig>  a strain isolated from human feces  <cit> .
in fact, the amino acid sequence of the mature active peptide gase differs only in one amino acid from that of gast .
in addition, close to the structural gene encoding gase , there was a second structural gene , which encodes a bacteriocin-like peptide  100 % identical to the putative peptide gatx of l. gasseri sbt <dig> and the gassericin k <dig> b peptide of l. gasseri k <dig>  in contrast, the amino acid sequence of the bacteriocin acidocin lf <dig> b from l. gasseri lf <dig> is 98 % identical to that of gatx of l. gasseri sbt2055; l. gasseri lf <dig> also produces the acd221β peptide, which is 100 % identical to gassericin t. it is important to note that the putative peptides encoded by orfb <dig> from l. gasseri lf <dig>  and l. gasseri k <dig>  are 100 % identical to gassericin t but, surprisingly, they have received different names  <cit> .
recently, the genome sequence of l. gasseri k <dig>  has shown that the deduced amino acid sequences of gask7bα differed in one amino with that previously described  <cit> .
interestingly, while gase and gast were purified from supernatants of l. gasseri ev <dig> and sbt <dig>  respectively, the peptides acd221β and gask7_α were not detected as active components in the cfss of l. gasseri lf <dig> and l. gasseri k <dig>  respectively.
on the other hand, active acidocin lf <dig> b and gask <dig> b were isolated from l. gasseri lf <dig> and l. gasseri k <dig> cultures, respectively, while the gatx and gaex peptides were not detected in l. gasseri sbt <dig> and l. gasseri ev <dig> supernatants, respectively.
however, both biochemical and genetic data indicate that the peptide pairs gase/gaex, gast/gatx, acd221b/acd221β and gask <dig> a/gask <dig> b are bacteriocins of the class iib .
class iib two-peptide bacteriocins consists of two different peptides whose genes are next to each other in the same operon and whose optimal antibacterial activity requires the presence of both peptides  <cit> .
thus, on the basis of the genetic organization of the genes involved in gase biosynthesis this bacteriocin can be considered as belonging to the class iib two-peptide bacteriocins, being the first component of the bacteriocin.
peptide gaex is most probably the second component of this two-peptide bacteriocin, although assays of complementary activity between gaee and gaex are necessary to confirm this hypothesis.
interestingly, the inhibitory spectrum of gassericin e appears to be slightly different to that of gassericin k <dig>  a putative two-peptide bacteriocin composed of the peptides k <dig> a and k <dig> b, which are virtually identical to gastt and gatx of l. gasseri sbt <dig>  respectively.
more specifically, the cfss of l. gasseri ev <dig> presented a high inhibitory activity against l. curvatus ncfb  <dig> and p. pentosaceus fbb <dig>  while those of l. gasseri k <dig> had no inhibitory activity against such strains  <cit> .
it has been shown that a single amino acid change in one of the peptides which compose a two-peptide bacteriocin can be responsible for determining the specificity for the target strains, as it is the case of enterocin c   <cit> .
the aa sequence of peptide entc <dig> was identical to that of ent1071a, while entc <dig> differed only in one amino acid from ent1071b .
this difference was sufficient to explain the different inhibitory spectrum of the bacteriocins enterocin c  and enterocin  <dig>   <cit> .
in fact, although gaex from cfss of l. gasseri ev <dig> has not been purified yet, we observed the existence of putative complementary activity during bacteriocin purification since halos of antimicrobial activity were detected between adjacent drops of assayed fractions, which probably resulted from diffusion and mixing of complementary peptides .
recently, during purification of gassericin t from l. gasseri la <dig>  both peptides  were detected in active fractions by maldi-tof-ms analysis  <cit> .
production of gase was lost in highly diluted cultures but it could be restored by the addition of a cfs from a previous bac+ culture or by the co-culture with other specific gram-positive bacteria.
this fact has been previously observed in other class ii bacteriocins whose production is regulated by a quorum sensing mechanism involving a three-component regulatory system  consisting of an autoinducer peptide , a histidine protein kinase  and a response regulator   <cit> .
the aip is secreted to the medium, being sensed by the membrane-associate hpk, which activates by phosphorylation the rr, which then activates expression of all operons necessary for bacteriocin synthesis, transport, and regulation  <cit> .
in these systems, the quorum sensing mechanism is not sufficient to maintain bacteriocin production in highly diluted cultures , since concentration of the autoinducers  or peptide pheromones produced is not sufficient to trigger full bacteriocin production, suggesting the involvement of other  factors.
previously, we have shown that cell-to-cell contact with specific bacteria acts as environmental signals to switch on bacteriocin production in lactobacillus plantarum nc <dig> through the activation of a quorum sensing mechanism involving the three component regulatory ipnc8-hknc8-plnd  <cit> .
this quorum sensing mechanism served to sense not only cell density of the nc <dig> population but, also, that of the competitor bacteria.
this interspecies bacterial-bacterial phenomenon was shown to be widely distributed among l. plantarum strains  <cit>  and, in fact, the number of studies covering this subject has increased rapidly.
thus, to date, induction of bacteriocin production by specific bacteria has been described in l. plantarum, , lactobacillus acidophilus  <cit> , lactobacillus helveticus  <cit> , carnobacterium divergens  <cit> , leuconostoc citreum  <cit>  and enterococcus faecium  <cit> .
the analysis of the loci involved in gase production showed the presence of a putative tcrs formed by the inducer peptide gaep, the histidine protein kinase gaek and the response regulator gaer.
the promoter sequences in front of the gene clusters involved in regulatory and biosynthesis of this bacteriocin has conserved regulatory sequences where the response regulator  would bind to activate the expression of these regulated genes  <cit> .
these putative regulatory elements are similar to those involved in quorum-sensing regulation of blp bacteriocins in streptococcus thermophilus  <cit> .
as in other regulated class ii bacteriocins, the aip  must be present in the environment at a certain threshold in order to be recognized by their cognate histidine kinase , a process leading to the activation of the response regulator  which, in turn, will activate expression of the proper regulatory operon  and the operon involved in biosynthesis of gase  <cit> .
based on phenotypic experiments and in the genetic data, we suggest that bacteriocin production by l. gasseri ev <dig> is regulated by a quorum sensing mechanism involving the tcrs operon gaep-gaek-gaer, and that specific bacteria induce bacteriocin production in l. gasseri ev <dig> through the activation of such regulatory system, as it has been demonstrated in l. plantarum  <cit> .
although the regulatory operon found in l. gasseri ev <dig>  was very similar to those described in other l. gasseri strains, such as jv-v <dig> , k <dig>  <cit>  and la <dig>  <cit> , bacteriocin induction in these strains has not been described yet.
bacterial vaginosis  is the most common disorder of the female reproductive tract, characterized by the displacement of commensal vaginal lactobacilli and the overgrowth of mixed pathogenic bacterial populations  <cit> .
interestingly, l. gasseri ev <dig> has shown to possess inhibitory activity against the main pathogenic species of the bacterial vaginosis-associated bacteria  , such atopobium vaginae, gardnerella vaginalis, porphyromonas gingivalis, and prevotella bivia.
CONCLUSIONS
presently, gassericin-producing l. gasseri strains had been isolated from either adult  or infant feces ; in contrast, l. gasseri ev <dig> was isolated from the vagina of healthy woman.
this origin and the ability to produce bacteriocins that inhibit the grown of pathogenic bvab, may be an advantage for using l. gasseri ev <dig> as a probiotic strain to fight and/or prevent bacterial infections as bv, since it could be better adapted to live and compete into the vaginal environment.
the use of probiotic lactobacilli to prevent vaginal infection has a good rationale, and an excellent safety record, but so far only a few strains have been clinically proven to be effective, in particular to prevent bv  <cit> .
more studies are required to elucidate the potential of l. gasseri ev <dig> as a vaginal probiotic but its origin and its peculiar mechanism for bacteriocin production makes it a good candidate for such use in the future.
additional files
additional file 1: figure s <dig>  schematic representation of the locus for gassericin e  production of l. gasseri ev <dig> and comparison with the locus involved in the production of other similar l. gasseri bacteriocins.
incomplete orfs are indicated with an asterisk.
in l. gasseri jv- <dig> and k <dig>  the putative genes associated to a locus tag were shown in brackets.
genebank accession numbers and references for these strains are: l. gasseri sbt <dig> ; l. gasseri la <dig> ; l. gasseri lf <dig> ; l. gasseri k <dig> ; l. gasseri k <dig> genome ; l. gasseri jv- <dig> ; l. gasseri g <dig> .
additional file 2: table s <dig>  comparison between deduced amino sequences of the gassericin e locus against databases .
additional file 3: figure s <dig>  bacteriocin activity of fractions obtained during first purification steps of gassericin e through c2/c <dig> reverse-phase chromatography.
the numbers in the plate indicate the fractions assayed.
the black arrow indicates the putative complementary activity between different fractions, i.e, between fractions 16– <dig> and 24– <dig>  which is most probably due to the diffusion and mixing of complementary active peptides.
l. gasseri lc <dig> was used as the indicator strain.
abbreviations
bvbacterial vaginosis
gasegassericin e
cfsscell-free supernatants
pcrpolymerase chain reaction
bhibrain heart infusion
tsbtryptic soy broth agar
rcareinforced clostridial agar broth medium
etsaenriched tryptic soy agar
rpc-fplcreverse-phase chromatography-fast protein liquid chromatography
sds-pagesodium dodecyl sulfate polyacrylamide gel electrophoresis
maldi-tof-msmatrix-assisted laser desorption/ionization time of flight mass spectrometry
dntpsdeoxynucleotides triphosphates
blastbasic local alignment search tool
bacbacteriocin
cfucolony forming units
bubacteriocin units
orfsopen reading frames
aipautoinducer peptide
hpkhistidine protein kinases
rrresponse regulator
tcrsthree-component regulatory system
bvabbacterial vaginosis associated bacteria
competing interests
the authors declare that they have no competing interests.
authors’ contributions
amb carried out the antimicrobial spectrum of activity, co-cultures, bacteriocin purification, assembling and analysis of the dna sequences and the writing of the manuscript.
bcg carried out the conditional bacteriocin studies and pcr reactions.
vm carried out the antimicrobial spectrum of activity against vaginal, semen and glans isolates.
jlrb and jmr participated in the coordination of the study and the critical reading of the manuscript.
all authors read and approved the final version of the manuscript.
this work was supported by the spanish ministry of economy and competitiveness  through the project agl2012- <dig> and agl2013-41980-p, and by the junta de andalucía excellence project agr- <dig>  these projects included feder funds.
amb was the recipient of a post-doctoral grant awarded by the junta de andalucía as part of the project agr- <dig>  we would like to express our gratitude to esther jiménez for kindly provide us the strain lactobacillus gasseri ev <dig>  who has provided her approval to be mentioned by name in this section.

 members of the genus lactobacillus are widely recognized as the hallmark of the normal or healthy vagina and are thought to play a major role in protecting the vaginal environment from non-indigenous and potentially harmful microorganisms. lactobacillus gasseri is a human autochthonous microorganism and one of the dominant lactobacillus species in the vaginal ecosystem, together with lactobacillus jensenii, lactobacillus iners and lactobacillus crispatus. thus, decreasing of lactobacilli titer in the vagina, results in bacterial vaginosis, where the normal flora is replaced by undesirable bacteria, including bacterial pathogens such gardnerella vaginalis, atopobium vaginae, mobiluncus curtisii, prevotella bivia, leptotrichia amnionii, eggerthella spp,, sneathia spp and megasphaera type i spp.. l. gasseri has been well documented as a commensal of the vaginal mucosa and exhibits a negative correlation with bv. some strains of this species have a high potential for being used as probiotics in order to maintain vaginal homeostasis. l. gasseri may confer colonization resistance against pathogens in the vagina by displacing them through competitive adhesion or by direct inhibition through production of antimicrobial compounds, including lactic acid, hydrogen peroxide and bacteriocins. bacteriocins could play a major role to control non-indigenous or pathogenic organisms in the female genitourinary tract. bacteriocins are ribosomally-synthesized antimicrobial peptides of bacterial origin that are active against a variable spectrum of bacteria. production of these antimicrobial peptides plays an important role in bacterial competition, allowing survival advantages to the producing strain. bacteriocins from gram-positive bacteria are nowadays classified into two major classes: the lantibiotics and the non-lantibiotics. class ii is divided into four subgroups: class iia, iib, iic, and iid. currently, four different bacteriocins from l. gasseri have been purified and genetically characterized: gassericin t from l. gasseri sbt, acidocin lf221a and acidocin lf221b from l. gasseri lf, and gassericin a from l. gasseri la. while gassericin a is a class iic circular bacteriocin, gassericin t and acidocins lf221a and lf221b have been proposed to belong to class iib bacteriocins, although no experimental data supporting this hypothesis has been published, yet. in this work we describe the biochemical and genetic characterization of gassericin e, a novel bacteriocin produced by a l. gasseri strain isolated from the vagina of a healthy woman. production of this bacteriocin was shown to be promoted by the presence of certain specific bacteria in co-culture with the producer strain. the genetic background showed the presence of a three-component regulatory operon that may be involved in the regulation of the production of this bacteriocin. methods bacterial strains and growth conditions lactobacillus gasseri ev was isolated from the vagina of a healthy woman, while we were searching for bacteriocin-producing lactobacilli species isolated from different human sources. the volunteer gave written informed consent to the protocol, which had been approved by the ethical committee of clinical research of hospital clínico san carlos madrid. most of the bacterial strains used in this study were grown routinely in mrs medium at 37 °c in aerobic conditions. actinomyces neuii, escherichia coli, staphylococcus spp. and streptococcus spp., were grown in brain heart infusion medium at the same conditions described above. fastidious organisms were grown in different culture media at 37 °c in anaerobic atmosphere as follows. gardnerella vaginalis was grown in casman’s medium base with 5 % v/v rabbit blood; porphyromonas gingivalis, atopobium vaginae, anaerococcus vaginalis and leptotrichia amnionii were grown in tryptic soy broth agar; prevotella bivia was grown in modified reinforced clostridial agar broth medium; mobiluncus spp. were grown in enriched tryptic soy agar medium. unless indicated, strains from human sources belonged to our own collection, while strains from other sources belonged to different bacterial collections. they were maintained as frozen stocks at –80 °c in their respective culture medium plus 20 % glycerol.table inhibitory spectrum of lactobacillus gasseri ev against bacterial strains from human origin actynomyces neuii actinomyces turicensis anaerococcus vaginalis atopobium vaginae bifidobacterium longum candida albicans corynebacterium amycolatun/xerosis corynebacterium aurimucosum corynebacterium freneyi corynebacterium glucurunolyticum corynebacterium tuberculostearicum corynebacterium simulans corynebacterium striatum enterococcus faecalis enterococcus faecium enterococcus gallinarum enterococcus saccharolyticus escherichia coli facklamia hominis gardnerella vaginalis lactobacillus fermentum lactobacillus gasseri lactobacillus paracasei lactobacillus salivarius leuconostoc mesenteroides leptotrichia amnionii mobiluncus mulieris mobiluncus curtisii  subsp. curtisii  subsp. holmesii porphyromonas gingivalis prevotella bivia propionibacterium acnes propionibacterium avium propionibacterium granulosum staphylococcus aureus staphylococcus caprae staphylococcus epidermidis - - - streptococcus agalactiae streptococcus anginosus streptococcus intermedius streptococcus parasanguinis a, b abbreviations: c colostrums, h faeces, ev, o, ps and v vaginal exudates, fr rectal frotis, m meconium, ms semen, mp glans, l breast milk, la amniotic liquid, p skin, sc umbilical cord blood, fvm facultad de veterinaria, dsmz german collection of microorganisms and cell cultures csensitivity to cell-free supernatants of l. gasseri ev assayed by the agar drop diffusion test method. sensitivity scale: +, ++, +++, and ++++, reflecting the degree of sensitivity according to the diameter of the inhibition halo; -, resistant bacillus cereus enterococcus faecium enterococcus faecalis lactobacillus acidophilus lactobacillus bulgaricus lactobacillus casei lactobacillus coryniformis lactobacillus curvatus lactobacillus fermentum lactobacillus helveticus lactobacillus pentosus lactobacillus plantarum lactobacillus salivarius lactobacillus sakei lactococcus lactis   subsp. cremoris   subsp. lactis leuconostoc cremoris pediococcus pentosaceus staphylococcus carnosus streptococcus thermophilus a abbreviations: cect colección española de cultivos tipo, cig colección instituto de la grasa, cit cranfield institute of technology, fvm facultad de veterinaria, universidad complutense, inra institut national de la recherche agronomique, ncdo national collection of dairy organisms, tno nutrition and food research bsensitivity to cell-free supernatants of l. gasseri ev assayed by the agar drop diffusion test method. sensitivity scale: +, ++, +++, and ++++, reflecting the degree of sensitivity according to the diameter of the inhibition halo; -, resistant bacteriocin assays to check for bacteriocin production on solid medium, a pointed sterile inoculating handle was soaked in overnight broth cultures of the producing strain and then punctured in mrs agar plates. the plates were incubated at 37 °c for 6 h and then were overlaid with  ml soft agar inoculated with ca. 105 cfu/ml of the indicator strains. plates were further incubated at 37 °c for 16–18 h, and examined for clear halos of inhibition around the punctured cultures. bacteriocin activity in cell-free supernatants from stationary-phase broth cultures of l. gasseri ev was assayed by using the agar drop diffusion test as described previously, using the strains listed in tables  and as indicator microorganisms. bacteriocin activity was quantified by a microtiter plate assay, as described previously using l. paracasei c as the indicator strain. one bacteriocin unit was defined as the amount of bacteriocin active cfs that inhibited the growth of the indicator strain by 50 %, using as a reference the turbidity of control cultures without cfs added. this was expressed as the reciprocal of the highest dilution exhibiting 50 % inhibition of the indicator strain per milliliter. conditional bacteriocin production of l. gasseri ev1461 two or three and to colonies were inoculated into fresh 10-ml mrs broth tubes and incubated at 37 °c. cfs was tested for antimicrobial activity at and 16 h of growth as described above. the minimum number of cells that were necessary to obtain a bac+ or a bac- culture was determined by serial dilutions of a 16 h culture of l. gasseri ev bac+ cfss from l. gasseri ev were tested for their ability to autoinduce bacteriocin production in l. gasseri ev bac- cultures. for autoinduction, 20 μl of bac+ cfs were added to fresh mrs broth containing ca. 102 cfu/ml of an overnight culture of l. gasseri ev, and incubated at 37 °c for 16 h. in control experiments, the same amount of bac+ cfs was added to 1 ml mrs broth and then the antimicrobial activity was assayed. production of bacteriocins by l. gasseri ev in co-cultures with the inducer strains l. gasseri lc lactobacillus pentosus 128/ lactobacillus plantarum ce and propionibacterium avium h was determined as described previously with some modifications. briefly, fresh mrs broth was inoculated with an overnight bac-culture of l. gasseri ev plus an overnight culture of each inducer strain. the mixed cultures were held at 37 °c for 6 h, centrifuged, and the cfss adjusted to ph  with 5 n naoh, filter-sterilized, and finally their inhibitory activity assayed by the agar drop diffusion test, using lactobacillus paracasei c as the indicator strain. in control assays, all of the strains used in the mixed cultures were propagated as pure cultures in their respective media and then assayed for antimicrobial activity as described above. purification of gassericin e all of the purification steps were carried out at room temperature, and all of the chromatographic equipment and media were purchased from amersham biosciences europe gmbh, freiburg, germany. gassericin e was purified from 2-litre cultures as follows. two 10 ml mrs tubes were inoculated at high cellular densities by picking ca. isolated colonies of a 48-h plate culture of l. gasseri ev and incubated at 37 °c for 16 h. then, two 200-ml mrs bottles were inoculated each one with the 16 h 10 ml l. gasseri ev broth cultures, and further incubated at 37 °c for 10 h. finally, a 2-litre mrs flask was inoculated with the two 200-ml broth cultures of l. gasseri ev and further incubated for 16 h. the cells were removed by centrifugation at  × g for 10 min at 4 °c and, then, the bacteriocin was purified from the cfs by the same method described for plantaricin nc. briefly, the cfs was precipitated with ammonium sulfate, desalted through pd- columns, and consecutively applied to cation-exchange and hydrophobic-interaction columns. fractions showing bacteriocin activity were applied to reverse-phase chromatography in a c2/c rpc column coupled to a fast protein liquid chromatography system. the bacteriocin was eluted from the rpc column with a linear gradient of 2-propanol in aqueous  % trifluoroacetic acid. fractions showing inhibitory activity after the rpc-fplc were pooled and rechromatographed to obtain pure bacteriocin. purity of bacteriocin fractions were checked by sds-page as described below. sds-page during the purification process, the rpc-fplc eluted fractions of gase were analyzed in duplicate by tris-tricine sds-page, using an 18 % acrylamide resolving gel. after electrophoresis at 100 mv for 2 h, one gel was silver stained while the other was used to detect the inhibitory activity in an overlay assay as described previously. l. paracasei c was used as the indicator strain. the precision plus protein dual xtra was used as molecular weight standards. n-terminal amino acid sequence and mass spectrometry the n-terminal amino acid sequences of purified gase peptide was determined by automated edman degradation with a beckman lf sequencer/phenylthiohydantoin amino acid analyzer. molecular mass of the peptides was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. these analysis were performed by dr. silvia bronsoms. pcr sequencing and location of the gassericin e locus the primers g1-f, gatx-r, gata-f and gata-r were designed on the basis of the locus encoding gassericin t of l. gasseri sbt. the primers gt1-f, gt1-r, gt2-f, gt3-f,gt4-f,gt5-f,gt6-f,gt7-f and gt8-r were designed on the basis of the locus encoding gassericin t of l. gasseri la. total dna was extracted from l. gasseri ev colonies as described previously.table oligonucleotides used in this study amplification with the primer pair g1-f/gatx-r was carried in 50 μl reaction mixtures containing  mm mg cl 1× reaction buffer, 200 μm concentrations of each of the deoxynucleotides triphosphates, 1 μm of each of the primers, u of taq dna polymerase and 5-μl of template dna. amplification included denaturation at 94 °c for 4 min, followed by 30 cycles of denaturation at 94 °c for 30 s, annealing at 56 °c for 1 min, polymerization at 72 °c for 1 min, and a final polymerization step at 72 °c for 5 min. amplification with the primer pair gt1-f/gt8-r was carried out in 50-μl reaction mixtures containing  mm mg cl 1× reaction buffer, 200 μm concentrations of each of the dntps, 1 μm of each of the primers, u of taq dna polymerase and 5 μl of template dna. amplification included denaturation at 94 °c for 2 min, followed by 10 cycles of denaturation at 94 °c for 10 s, annealing at 57 °c for 30 s, and polymerization at 68 °c for 3:30 min, 20 cycles of denaturation at 94 °c for 10 s, annealing at 57 °c for 30 s and polymerization at 68 °c for 3:30 min plus 20 s/cycle, and a final polymerization step at 68 °c for 7 min. the amplified fragments were excised from  % agarose gels, purified using the nucleospin® extract ii kit, and sequenced using the primers described in table  at the genomics unit of the universidad complutense. dna sequences were assembled using the seqman software in the dnastar package and deposited in the genbank database. dna and amino acid sequence analysis searches for dna and amino acid similarities in nucleotide and protein databases were done using the basic local alignment search tool. searches for promoter sequences were done with the neural network promoter prediction web interface. for detection of rho-independent terminators, the arnold web server was used. for alignment of the amino acid sequences of the leader and mature peptides of the bacteriocins, the clustalw multiple sequence alignment program was used. for physico-chemical analysis of peptides the winpep program was used.  antimicrobial spectrum of l. gasseri ev1461 bac+ cfss from l. gasseri ev broth cultures showed antimicrobial activity against many indicator strains used in this study, including species both related and non-related to the producing strain as shown in tables  and conditional bacteriocin production this strain showed antimicrobial activity when grown on solid medium. however, after the inoculation of two or three colonies into fresh mrs broth, l. gasseri ev cultures do no display antimicrobial activity. by contrast, after the inoculation of a larger number of colonies, l. gasseri ev showed bacteriocin activity after at least 6 h of growth, maintaining this activity up to 16 h of growth. thus, at low cellular densities the antimicrobial activity produced by l. gasseri ev was low or no detectable. in these diluted bac- cultures, the bac+ phenotype was restored when the strain was grown on solid medium as isolated colonies or in broth cultures after the addition of a bac+ cfs from a previous bacteriocin-producing culture. thus, the addition of a bac+ cfs from l. gasseri ev to l. gasseri ev broth cultures resulted in bacteriocin production, indicating the existence of an autoinducing factor in the supernatant. in control experiments, the addition of a bac+ cfs from l. gasseri ev to mrs broth showed no antimicrobial activity.table conditional bacteriocin production of l. gasseri ev1461 l. gasseri ev b l. gasseri ev c l. gasseri ev + cfs ev d abacteriocin units per mililiter bbacteriocin non-producing l. gasseri ev cbacteriocin producing l. gasseri ev dcfs: cell free supernatant from a bac+ l. gasseri ev culture containing bu/ml in addition, when highly diluted l. gasseri ev cultures were co-cultured with certain specific gram-positive bacteria, production of bacteriocin was notably increased with respect to that displayed by the ev pure cultures. thus, mixed cultures of l. gasseri ev with propionibacterium avium h1544b, lactobacillus plantarum ce lactobacillus pentosus 128/ or lactobacillus gasseri lc induced bacteriocin production in l. gasseri ev. none of the inducing strains exhibited any antimicrobial activity when they were cultivated as single, pure cultures. the ability of these species to induce bacteriocin production in l. gasseri ev was independent of their resistance or sensitivity to the bacteriocin produced. purification of gassericin e gase was isolated from the bac+ cfs of a 2-litre broth culture of l. gasseri ev as described in the methods section. the behaviour of gase throughout the purification process was that of a cationic and hydrophobic peptide. two runs on rpc-fplc were necessary to obtain fractions containing pure bacteriocin. sds-page analysis showed a single peptide band with an apparent molecular size of 5-kda which presented inhibitory activity against l. paracasei c in the corresponding sds-page activity gel. the inhibitory activity of pure gase had a titer of bu ml− against l. paracasei c fig. maldi-tof mass spectra of purified gassericin e. +, monoisotopic peak of gase. a.u., absorbance units. inset panel: sds-page and bioassay of purified gase. l. paracasei c was used as the indicator strain. mwm, molecular weight marker mass spectrometry and n-terminal amino acid sequencing maldi-tof ms analysis of purified gase indicated that a monoisotopic peak of the bacteriocin was present, suggesting that the molecular mass of the peptide is.0 da. partial amino acid sequencing of gase showed that the 18 n-terminal amino acids shared high similarity with the first 18 n-terminal amino acids of mature gassericin t from l. gasseri sbt.fig. alignment of the amino acid sequences of double-glycine leader peptides and mature peptides of gassericin e and other similar gassericin bacteriocins. the sequences were aligned with the clustalw software at the embl-ebi online server. the arrow indicates the gly-gly cleavage site of the peptide. asterisks, dots and double dots mean fully, strongly and weakly conserved residues, respectively. the first aa obtained by edman sequencing of the peptide gase are underlined. the two deduced amino acid sequences deposited in databases from l. gasseri k differed in one amino acid at position of mature peptide. theoretical molecular weights of the mature bacteriocins are shown. the genebank accession numbers are baa for gassericin t, aap for acidocin 221b, efj for lactacin-f subunit lafa, aap and kda99085for gassericin k b complemental factor and, respectively dna and deduced protein sequence analysis of the gase locus two dna fragments of and kbp were amplified with the primer pairs g1-f/gatx-r and gt1-f/gt8-r, respectively. dna sequencing of the gase locus revealed the presence of up to nine open reading frames which seemed to be organized into two putative operons, which could be involved in biosynthesis and immunity of this bacteriocin, and in regulation, transport and processing. the arrangement of these putative operons was similar to other gene clusters previously described in other bacteriocin-producing lactobacillus gasseri strains.fig. schematic representation of the locus for gassericin e production and detailed analysis of dna sequences of putative promoters and rho-independent terminators. a p p p3a and p3b are putative promoter sequences, and t is a putative rho-independent transcription terminator. genes encoding gassericin e, the putative complement peptide gaex and their putative immunity protein, could form an transcriptional unit, driven by two putative alternative promoters. genes encoding the three-component regulatory system formed by the autoinducer peptide, the histidine protein kinase and the response regulator, as well as the abc-transporter and the accessory protein, seem to form one transcriptional unit; however two different transcripts could be formed, one larger driven by promoter p involving gaepkrtc and one shorter driven by promoter p involving gaetc. a, b, c, d the putative promoters p p p3a and p3b were detected with the neural network promoter prediction online server, with a promoter score cut-off of. the typical - and - boxes and the ribosome binding sites are shown; the + indicates the putative transcription start. putative regulatory dna sequences are in italics. e rho-independent terminator; base-pairs are in boldface and apical loop in italics. f alignment of the direct dna repeats found upstream of the gase and gaep putative promoters and the consensus l- and r- repeats involved in quorum-sensing regulation of blp bacteriocins in streptococcus thermophilus gase biosynthesis and immunity operon detailed analysis of the kbp dna sequencing of the putative gase biosynthesis and immunity operon showed that gaea of ev encodes for a peptide of aa which presents an aa double-glycine leader peptide that, upon processing, gives rise to a mature peptide of aa which has a theoretical molecular mass of.42 da. this deduced molecular weight coincided with the mw of the gase peptide determined by maldi-tof ms. in addition, the partial 18-aa sequence of gase obtained by edman degradation was 100 % identical to the sequence of the first aa deduced of the mature peptide gase encoded by gaea of l. gasseri ev the genes gata from l. gasseri sbt and l. gasseri la as well as orfb from l. gasseri lf and l. gasseri k encode for a peptide of aa, which presents a typical double-glycine leader peptide that, once processed, results in a aa mature peptide with a theoretical molecular mass of.34 da. both the amino acid sequence deduced for mature gase as well as the first aa determined experimentally of purified peptide gase differed in one amino acid with that deduced for mature gast. more specifically, gase contains a leucine residue in position instead of the tryptophan residue of the peptide gast at the same position. the leader peptides of gase of l. gasseri ev gast of l. gasseri sbt and l. gasseri la acd221β of l. gasseri lf gask7bα of l. gasseri, lafa of l. gasseri jv-v and lactacin f precursor of l. gasseri k were 100 % identical. however, the hypothetical mature peptide lafa and lactacin f differed in one amino acid with that of gassericin t and gassericin e. immediately downstream of gaea, there were two orfs. the first one encodes a putative 65-aa peptide which presents a double-glycine leader peptide that, upon processing, gives rise to a mature peptide of aa and a theoretical molecular mass of.41 da. this putative peptide bacteriocin was 100 % identical to gatx of l. gasseri sbt la k jv-v and g and 98 % identical to acidocin lf221b and gassericin k b of l. gasseri lf and l. gasseri k respectively. downstream of gaex, gaei encodes a putative incomplete protein of aa that showed a 100 % of identity with the putative immunity protein to gassericin t of l. gasseri sbt la jv-v k g and lf. two putative promoter sequences containing the typical - and - regions were found upstream of gaea thus suggesting that the genes gaea-gaex-gaei are produced on the same transcript. in addition, upstream of p3b and overlapping p3a, two imperfect direct repeats of 9 bp separated by a rich at region were found. these putative regulatory elements showed a high similarity with those of streptococcus thermophilus involved in quorum-sensing regulation of blp bacteriocins. regulatory operon in l. gasseri ev the regulatory operon for bacteriocin production appears to involve three orfs: gaep, gaek and gaer. the gene gaep encodes a putative peptide of aa residues showing all the features previously described for inducer peptides, known also as autoinducing peptides or peptide pheromones. as with bacteriocin-like peptides, the product of gaep possesses a leader sequence of the double-glycine type that, once processed, gives rise to a mature peptide of aa residues, with a theoretical pi of and a mw of.31 da. this putative inducer peptide was 100 % identical to that harboured by l. gasseri la jv-v and k. the gene gaek, which encodes a putative protein showing significant homology with the family of the histidine protein kinases, was located 2 bp downstream of gaep. the highest similarity for gaek was obtained with gatk, the hpk of the gassericin t locus of l. gasseri la immediately downstream of gaek, the gene gaer was found. the putative protein encoded by gaer was 100 % identical to the response regulators previously found in l. gasseri la jv-v and k. therefore, based on homology and relative position, the gaep-gaek-gaer gene cluster seems to form a regulatory operon of the so-called three-component type involved in regulation of bacteriocin production. a similar cluster was found in the genomes of l. gasseri la, jv-v and k. a putative promoter sequence containing the typical - and - regions was found upstream of gaep. in addition, upstream of p two imperfect direct repeats of 10 bp separated by a rich at region were found. these regulatory elements were similar to those found upstream of promoter p3a. transport and processing operon downstrean of the gene cluster gaepkr, the orf gaet encodes a aa putative protein with 100 % identity with the putative abc-transporter of gassericin t encoded by gatt of l. gasseri la jv-v and k. just 1 bp overlapping gaet, gaec encodes a putative aa protein with a 99 % identity to the accessory protein for the abc-transporter of l. gasseri jv-v lf and k. a putative promoter sequence was found upstream of gaet, just overlapping gaer. in addition, two inverted repeats of 10 bp which may function as a rho-independent transcription terminator were found downstream of gaec. the existence of two promoter sequences, p and p and a terminator downstream of gaec indicates that two putative transcripts could be formed, one driven by the p promoter that would include co-transcription of the gaep-gaek-gaer-gaet-gaec genes, and one driven by the promoter p that would include co-transcription of gaet-gaec genes. discussion in this work, we describe the purification and genetic characterization of gassericin e, a novel bacteriocin produced by l. gasseri ev a strain isolated from the vagina of a healthy woman. gassericin e is very similar to gassericin t, a bacteriocin purified from l. gasseri sbt a strain isolated from human feces. in fact, the amino acid sequence of the mature active peptide gase differs only in one amino acid from that of gast. in addition, close to the structural gene encoding gase, there was a second structural gene, which encodes a bacteriocin-like peptide 100 % identical to the putative peptide gatx of l. gasseri sbt and the gassericin k b peptide of l. gasseri k in contrast, the amino acid sequence of the bacteriocin acidocin lf b from l. gasseri lf is 98 % identical to that of gatx of l. gasseri sbt2055; l. gasseri lf also produces the acd221β peptide, which is 100 % identical to gassericin t. it is important to note that the putative peptides encoded by orfb from l. gasseri lf and l. gasseri k are 100 % identical to gassericin t but, surprisingly, they have received different names. recently, the genome sequence of l. gasseri k has shown that the deduced amino acid sequences of gask7bα differed in one amino with that previously described. interestingly, while gase and gast were purified from supernatants of l. gasseri ev and sbt respectively, the peptides acd221β and gask7_α were not detected as active components in the cfss of l. gasseri lf and l. gasseri k respectively. on the other hand, active acidocin lf b and gask b were isolated from l. gasseri lf and l. gasseri k cultures, respectively, while the gatx and gaex peptides were not detected in l. gasseri sbt and l. gasseri ev supernatants, respectively. however, both biochemical and genetic data indicate that the peptide pairs gase/gaex, gast/gatx, acd221b/acd221β and gask a/gask b are bacteriocins of the class iib. class iib two-peptide bacteriocins consists of two different peptides whose genes are next to each other in the same operon and whose optimal antibacterial activity requires the presence of both peptides. thus, on the basis of the genetic organization of the genes involved in gase biosynthesis this bacteriocin can be considered as belonging to the class iib two-peptide bacteriocins, being the first component of the bacteriocin. peptide gaex is most probably the second component of this two-peptide bacteriocin, although assays of complementary activity between gaee and gaex are necessary to confirm this hypothesis. interestingly, the inhibitory spectrum of gassericin e appears to be slightly different to that of gassericin k a putative two-peptide bacteriocin composed of the peptides k a and k b, which are virtually identical to gastt and gatx of l. gasseri sbt respectively. more specifically, the cfss of l. gasseri ev presented a high inhibitory activity against l. curvatus ncfb and p. pentosaceus fbb while those of l. gasseri k had no inhibitory activity against such strains. it has been shown that a single amino acid change in one of the peptides which compose a two-peptide bacteriocin can be responsible for determining the specificity for the target strains, as it is the case of enterocin c. the aa sequence of peptide entc was identical to that of ent1071a, while entc differed only in one amino acid from ent1071b. this difference was sufficient to explain the different inhibitory spectrum of the bacteriocins enterocin c and enterocin. in fact, although gaex from cfss of l. gasseri ev has not been purified yet, we observed the existence of putative complementary activity during bacteriocin purification since halos of antimicrobial activity were detected between adjacent drops of assayed fractions, which probably resulted from diffusion and mixing of complementary peptides. recently, during purification of gassericin t from l. gasseri la both peptides were detected in active fractions by maldi-tof-ms analysis. production of gase was lost in highly diluted cultures but it could be restored by the addition of a cfs from a previous bac+ culture or by the co-culture with other specific gram-positive bacteria. this fact has been previously observed in other class ii bacteriocins whose production is regulated by a quorum sensing mechanism involving a three-component regulatory system consisting of an autoinducer peptide, a histidine protein kinase and a response regulator. the aip is secreted to the medium, being sensed by the membrane-associate hpk, which activates by phosphorylation the rr, which then activates expression of all operons necessary for bacteriocin synthesis, transport, and regulation. in these systems, the quorum sensing mechanism is not sufficient to maintain bacteriocin production in highly diluted cultures, since concentration of the autoinducers or peptide pheromones produced is not sufficient to trigger full bacteriocin production, suggesting the involvement of other factors. previously, we have shown that cell-to-cell contact with specific bacteria acts as environmental signals to switch on bacteriocin production in lactobacillus plantarum nc through the activation of a quorum sensing mechanism involving the three component regulatory ipnc8-hknc8-plnd. this quorum sensing mechanism served to sense not only cell density of the nc population but, also, that of the competitor bacteria. this interspecies bacterial-bacterial phenomenon was shown to be widely distributed among l. plantarum strains and, in fact, the number of studies covering this subject has increased rapidly. thus, to date, induction of bacteriocin production by specific bacteria has been described in l. plantarum,, lactobacillus acidophilus, lactobacillus helveticus, carnobacterium divergens, leuconostoc citreum and enterococcus faecium. the analysis of the loci involved in gase production showed the presence of a putative tcrs formed by the inducer peptide gaep, the histidine protein kinase gaek and the response regulator gaer. the promoter sequences in front of the gene clusters involved in regulatory and biosynthesis of this bacteriocin has conserved regulatory sequences where the response regulator would bind to activate the expression of these regulated genes. these putative regulatory elements are similar to those involved in quorum-sensing regulation of blp bacteriocins in streptococcus thermophilus. as in other regulated class ii bacteriocins, the aip must be present in the environment at a certain threshold in order to be recognized by their cognate histidine kinase, a process leading to the activation of the response regulator which, in turn, will activate expression of the proper regulatory operon and the operon involved in biosynthesis of gase. based on phenotypic experiments and in the genetic data, we suggest that bacteriocin production by l. gasseri ev is regulated by a quorum sensing mechanism involving the tcrs operon gaep-gaek-gaer, and that specific bacteria induce bacteriocin production in l. gasseri ev through the activation of such regulatory system, as it has been demonstrated in l. plantarum. although the regulatory operon found in l. gasseri ev was very similar to those described in other l. gasseri strains, such as jv-v, k and la, bacteriocin induction in these strains has not been described yet. bacterial vaginosis is the most common disorder of the female reproductive tract, characterized by the displacement of commensal vaginal lactobacilli and the overgrowth of mixed pathogenic bacterial populations. interestingly, l. gasseri ev has shown to possess inhibitory activity against the main pathogenic species of the bacterial vaginosis-associated bacteria, such atopobium vaginae, gardnerella vaginalis, porphyromonas gingivalis, and prevotella bivia.  presently, gassericin-producing l. gasseri strains had been isolated from either adult or infant feces; in contrast, l. gasseri ev was isolated from the vagina of healthy woman. this origin and the ability to produce bacteriocins that inhibit the grown of pathogenic bvab, may be an advantage for using l. gasseri ev as a probiotic strain to fight and/or prevent bacterial infections as bv, since it could be better adapted to live and compete into the vaginal environment. the use of probiotic lactobacilli to prevent vaginal infection has a good rationale, and an excellent safety record, but so far only a few strains have been clinically proven to be effective, in particular to prevent bv. more studies are required to elucidate the potential of l. gasseri ev as a vaginal probiotic but its origin and its peculiar mechanism for bacteriocin production makes it a good candidate for such use in the future. additional files additional file 1: figure s schematic representation of the locus for gassericin e production of l. gasseri ev and comparison with the locus involved in the production of other similar l. gasseri bacteriocins. incomplete orfs are indicated with an asterisk. in l. gasseri jv- and k the putative genes associated to a locus tag were shown in brackets. genebank accession numbers and references for these strains are: l. gasseri sbt; l. gasseri la; l. gasseri lf; l. gasseri k; l. gasseri k genome; l. gasseri jv-; l. gasseri g. additional file 2: table s comparison between deduced amino sequences of the gassericin e locus against databases. additional file 3: figure s bacteriocin activity of fractions obtained during first purification steps of gassericin e through c2/c reverse-phase chromatography. the numbers in the plate indicate the fractions assayed. the black arrow indicates the putative complementary activity between different fractions, i.e, between fractions 16– and 24– which is most probably due to the diffusion and mixing of complementary active peptides. l. gasseri lc was used as the indicator strain. abbreviations bvbacterial vaginosis gasegassericin e cfsscell-free supernatants pcrpolymerase chain reaction bhibrain heart infusion tsbtryptic soy broth agar rcareinforced clostridial agar broth medium etsaenriched tryptic soy agar rpc-fplcreverse-phase chromatography-fast protein liquid chromatography sds-pagesodium dodecyl sulfate polyacrylamide gel electrophoresis maldi-tof-msmatrix-assisted laser desorption/ionization time of flight mass spectrometry dntpsdeoxynucleotides triphosphates blastbasic local alignment search tool bacbacteriocin cfucolony forming units bubacteriocin units orfsopen reading frames aipautoinducer peptide hpkhistidine protein kinases rrresponse regulator tcrsthree-component regulatory system bvabbacterial vaginosis associated bacteria competing interests the authors declare that they have no competing interests. authors’ contributions amb carried out the antimicrobial spectrum of activity, co-cultures, bacteriocin purification, assembling and analysis of the dna sequences and the writing of the manuscript. bcg carried out the conditional bacteriocin studies and pcr reactions. vm carried out the antimicrobial spectrum of activity against vaginal, semen and glans isolates. jlrb and jmr participated in the coordination of the study and the critical reading of the manuscript. all authors read and approved the final version of the manuscript. this work was supported by the spanish ministry of economy and competitiveness through the project agl2012- and agl2013-41980-p, and by the junta de andalucía excellence project agr- these projects included feder funds. amb was the recipient of a post-doctoral grant awarded by the junta de andalucía as part of the project agr- we would like to express our gratitude to esther jiménez for kindly provide us the strain lactobacillus gasseri ev who has provided her approval to be mentioned by name in this section.
some strains of this species have a high potential for being used as probiotics in order to maintain vaginal homeostasis, since they may confer colonization resistance against pathogens in the vagina by direct inhibition through production of antimicrobial compounds, as bacteriocins. in this work we have studied bacteriocin production of gassericin e, a novel bacteriocin produced by l. gasseri ev a strain isolated from the vagina of a healthy woman, and whose production was shown to be promoted by the presence of certain specific bacteria in co-culture. biochemical and genetic characterization of this novel bacteriocin are addressed. the antimicrobial activity was due to the production of a novel bacteriocin, gassericin e. at low cell densities, bacteriocin production ceased and only was restored after the addition of a supernatant from a previous bacteriocin-producing ev culture, or through co-cultivation with several other gram-positive strains. the gaeaxi, encodes for the bacteriocin gassericin e, a putative peptide bacteriocin, and their immunity protein. lactobacillus gasseri is one of the dominant lactobacillus species in the vaginal ecosystem. the gaepkr encodes a putative three-component regulatory system, involving an autoinducer peptide, a histidine protein kinase and a response regulator, while the gaetc encodes for an abc transporter and their accessory protein, involved in transport and processing of the bacteriocin. the origin of the strain and its ability to produce bacteriocins with inhibitory activity against vaginal pathogens may be an advantage for using l. gasseri ev as a probiotic strain to fight and/or prevent bacterial infections as bacterial vaginosis, since it could be better adapted to live and compete into the vaginal environment. we found that the inhibitory spectrum of l. gasseri ev was broad, being directed to species both related and non-related to the producing strain. dna sequence of the gase locus revealed the presence of two putative operons which could be involved in biosynthesis and immunity of this bacteriocin, and in regulation, transport and processing. interestingly, l. gasseri ev inhibited the grown of pathogens usually associated with bacterial vaginosis.
 lactobacillus gasseri is one of the dominant lactobacillus species in the vaginal ecosystem. some strains of this species have a high potential for being used as probiotics in order to maintain vaginal homeostasis, since they may confer colonization resistance against pathogens in the vagina by direct inhibition through production of antimicrobial compounds, as bacteriocins. in this work we have studied bacteriocin production of gassericin e, a novel bacteriocin produced by l. gasseri ev a strain isolated from the vagina of a healthy woman, and whose production was shown to be promoted by the presence of certain specific bacteria in co-culture. biochemical and genetic characterization of this novel bacteriocin are addressed.  we found that the inhibitory spectrum of l. gasseri ev was broad, being directed to species both related and non-related to the producing strain. interestingly, l. gasseri ev inhibited the grown of pathogens usually associated with bacterial vaginosis. the antimicrobial activity was due to the production of a novel bacteriocin, gassericin e. production of this bacteriocin in broth medium only was achieved at high cell densities. at low cell densities, bacteriocin production ceased and only was restored after the addition of a supernatant from a previous bacteriocin-producing ev culture, or through co-cultivation with several other gram-positive strains. dna sequence of the gase locus revealed the presence of two putative operons which could be involved in biosynthesis and immunity of this bacteriocin, and in regulation, transport and processing. the gaepkr encodes a putative three-component regulatory system, involving an autoinducer peptide, a histidine protein kinase and a response regulator, while the gaetc encodes for an abc transporter and their accessory protein, involved in transport and processing of the bacteriocin. the gaeaxi, encodes for the bacteriocin gassericin e, a putative peptide bacteriocin, and their immunity protein.  the origin of the strain and its ability to produce bacteriocins with inhibitory activity against vaginal pathogens may be an advantage for using l. gasseri ev as a probiotic strain to fight and/or prevent bacterial infections as bacterial vaginosis, since it could be better adapted to live and compete into the vaginal environment. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords bacteriocinquorum sensinggassericinthree-component regulatory systemspanish ministry of economy and competitivenessagl2012-33400agl2013-41980-pruiz-barba josé luis junta de andalucía excellence projectagr-07345ruiz-barba josé luis issue-copyright-statement© the author 2016 