Enteric YaiW Is a Surface-Exposed Outer Membrane
Lipoprotein That Affects Sensitivity to an Antimicrobial
Peptide
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Citation
Arnold, M. F. F., P. Caro-Hernandez, K. Tan, G. Runti, S.
Wehmeier, M. Scocchi, W. T. Doerrler, G. C. Walker, and G. P.
Ferguson. “Enteric YaiW Is a Surface-Exposed Outer Membrane
Lipoprotein That Affects Sensitivity to an Antimicrobial Peptide.”
Journal of Bacteriology 196, no. 2 (January 15, 2014): 436–444.
As Published
http://dx.doi.org/10.1128/JB.01179-13
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American Society for Microbiology
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Author's final manuscript
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Thu May 26 05:46:14 EDT 2016
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http://hdl.handle.net/1721.1/86392
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1
Enteric YaiW Is a Surface Exposed Outer Membrane Lipoprotein that Affects Sensitivity to
2
an Antimicrobial Peptide
3
Markus F.F. Arnold1, Paola Caro-Hernandez1ψ, Karen Tan2ψ, Giulia Runti3, Silvia Wehmeier1,
4
Marco Scocchi3, William T. Doerrler4, Graham C. Walker5#, and Gail P. Ferguson1†
5
1
6
AB25 2ZD, UK, 2Institute of Cell Biology and Centre for Science at Extreme Conditions, School of
7
Biological Sciences, King’s Buildings, University of Edinburgh, Edinburgh EH9 3JR, UK,
8
3
Department of Life Sciences, Via Giorgieri, 5 University of Trieste 34127 Trieste ITALY,
9
4
Complex Carbohydrate Research Centre, University of Georgia, Athens, 5Biology Department,
10
School of Medicine & Dentistry, Institute of Medical Sciences, University of Aberdeen,
Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139
11
12
#
13
Email: gwalker@mit.edu
14
ψ
These authors contributed equally to this work
15
†
Deceased, December 27, 2011
16
Running title: Enteric YaiW is a surface exposed lipoprotein
17
Keywords: E. coli, Salmonella enterica, lipoproteins, Antimicrobial peptides, protein localization
18
Abbreviations: VLCFA, Very long chain fatty acids, AMP, antimicrobial peptide
Corresponding Author: Graham C. Walker, Phone: (617)-253-6716 Fax: (617)-253-2643
19
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1
1
ABSTRACT
2
yaiW is a previously uncharacterized gene found in enteric bacteria that is of particular interest
3
because it is located adjacent to the sbmA gene, whose bacA ortholog is required for Sinorhizboium
4
meliloti symbiosis and Brucella abortus pathogenesis. We show that yaiW is co-transcribed with
5
sbmA in Escherichia coli and Salmonella enterica serovars Typhi and Typhimurium strains. We
6
present evidence that the YaiW is a palmitate-modified surface exposed outer membrane
7
lipoprotein. Since BacA function affects the very long chain fatty acid (VLCFA) modification of S.
8
meliloti and B. abortus lipid A, we tested whether SbmA function might affect either the fatty acid
9
modification of the YaiW lipoprotein or the fatty acid modification of enteric lipid A, but found that
10
it did not. Interestingly, we did observe that E. coli SbmA suppresses deficiencies in the VLCFA
11
modification of the LPS of an S. meliloti bacA mutant despite the absence of VLCFA in E. coli.
12
Finally, we found that both YaiW and SbmA positively affect the uptake of proline-rich Bac7
13
peptides, suggesting a possible connection between their cellular functions.
14
2
1
INTRODUCTION
2
The yaiW gene is closely linked to the sbmA gene in the sequenced genomes of enteric
3
bacteria (Fig. 1A) (1). These include E. coli, Salmonella enterica and other pathogenic species. In
4
E. coli and S. enterica serovar Typhimurium, sbmA and yaiW could be shown to be part of the
5
sigma E regulon, which is involved in the response to envelope stresses (2, 3). The close proximity
6
of the sbmA and yaiW genes suggests that there could be a functional relationship between the
7
SbmA and YaiW proteins, a possibility of particular interest because the molecular mechanism(s) of
8
action of SbmA and its orthologs is still incompletely understood.
9
SbmA is an integral inner membrane protein whose function plays a positive role in the
10
uptake of certain peptide substrates across the inner membrane (4, 5). These peptide substrates
11
include microcins B17 and J25 (6, 7), bleomycin (8) and truncated, proline-rich Bac7 peptides (7,
12
9). Moreover, the Sinorhizobium meliloti and Brucella abortus orthologs of SbmA, BacA (10), are
13
required for S. meliloti symbiosis (11) and B. abortus pathogenesis (12), while the Mycobacterium
14
tuberculosis SbmA/BacA homolog, which also includes an ATPase domain (13), is involved in
15
maintenance of M. tuberculosis chronic murine infections (14). Similarly to enteric SbmA, the S.
16
meliloti and B. abortus BacA proteins mediate the uptake of bleomycin and truncated Bac7 proteins
17
(15, 16). In addition, S. meliloti and B. abortus BacA deficient mutants have ~50% reduction in
18
their LPS very-long chain fatty acids (VLCFA) content, demonstrating that BacA proteins play a
19
key role in ensuring the complete modification of their LPS species with this unusual lipid (17, 18).
20
In S. meliloti, lipid A VLCFA biosynthesis is dependent upon the acpXL-lpxXL cluster, which is
21
absent in the genomes of enteric bacterial species (19). S. meliloti and B. abortus BacA-deficient
22
mutants exhibit an increased detergent sensitivity relative to their parent strains, consistent with
23
their altered LPS structures (18). In contrast, no detergent sensitivity phenotype was observed for
24
E. coli mutants lacking SbmA (20), suggesting that they do not possess LPS alterations. BacA
25
protein also protects S. meliloti against the toxic effects of a cysteine-rich nodule-specific (NCR)
26
peptide produced during the legume symbiosis (21). NCR peptides have been shown to be essential
3
1
for bacterial differentiation into nitrogen fixing bacteroids (22). In addition, it was also suggested
2
that the Mycobacterium tuberculosis BacA protein protects against human β-defensin 2 and that
3
these human defensins might direct human pathogens towards a chronic infection state rather than
4
leaving them in a potentially life threatening acute state (13). The molecular mechanism(s)
5
underlying these complex effects of the SbmA/BacA family members remains unknown.
6
We therefore undertook an investigation of the hitherto uncharacterized yaiW gene with the
7
long term goal of gaining insights into YaiW that would explain its function in enteric bacteria and
8
might also inform our understanding of SbmA/BacA. In this paper, we report that yaiW is co-
9
transcribed with sbmA, that its gene product YaiW is a palmitate-modified surface exposed outer
10
membrane lipoprotein, and that both YaiW and SbmA positively affect the uptake of proline-rich
11
Bac7 peptides, suggesting a possible connection between their cellular functions.
12
13
4
1
METHODS
2
Bacterial strains and growth conditions. All bacterial strains and plasmids used in this
3
study are described in Table S1 and S2, respectively. Unless stated otherwise, all E. coli strains
4
were grown in either Lysogeny broth (LB) (23) prepared with 10 g l-1 NaCl or Müller-Hilton (MH)
5
(24) medium or on LB or MH with 1.5 % (w/v) agar at 37°C. When required, for E. coli strains the
6
antibiotics were added at the following concentrations: ampicillin (Ap, 100 µg ml-1), kanamycin
7
(Ka, 50 µg ml-1) and chloramphenicol (Cp, 34 µg ml-1). For all experiments, S. meliloti strains were
8
grown in either LB prepared with 10 g l-1 NaCl or LB supplemented with 2.5 mM CaCl2 and 2.5
9
mM MgSO4 (LB/MC) for 48 hours at 30ºC. When required, for S. meliloti strains the antibiotics
10
were added at the following concentrations: streptomycin (Sm, 500 µg ml-1), spectinomycin (Spc,
11
100 µg ml-1) and tetracycline (Tc, 5 µg ml-1).
12
Construction of mutants. A list of all plasmids and primers used can be found in Tables S2
13
and S3, respectively. The sbmA::kan and yaiW::kan deletions from the E. coli Keio collection (25)
14
were transduced using bacteriophage P1 into MG1655. The kanamycin resistance cassettes were
15
flipped out using pCP20 following a previously published method (25). The deletion mutants were
16
confirmed by PCR using EcolisbmAprom_154 and EcolisbmA_R for ΔsbmA, EcoliMG1655yaiW_F
17
and EcoliMG1655yaiW_R for ΔyaiW and EcolisbmAprom_154 and EcoliMG1655yaiW_R primers
18
for the ΔsbmA/ΔyaiW double mutant.
19
RNA isolation and RT-PCR. RNA from S. Typhi and S. Typhimurium was extracted from
20
25 ml of exponential phase cultures (OD600 = 0.4). Cultures were rapidly microcentrifuged, and
21
pellets were frozen for 20 minutes in liquid nitrogen. Total RNA was extracted using the RNeasy
22
minikit (Qiagen). Cells were lysed in RLT buffer (supplied with the kit) using Fast Protein tubes
23
(Qbiogen) and the FastPrep FP120 cell disrupter (30 s at 6.5 m/s2). The RNA was then purified
24
using the RNeasy minikit protocol. RT-PCR was performed using the SuperScript One-Step RT-
25
PCR kit (Invitrogen) according to the manufacturer’s protocol. Co-transcription of sbmA and yaiW
26
was analyzed using primers sbmA_F+930 und yaiW_R+74 (Table S2). To assess the quality of the
5
1
extracted RNA, the universal ribosomal RNA primers 8F and 1492R were also used. For all primer
2
pairs, PCRs were also set up using the extracted RNA or S. Typhi or S. Typhimurium genomic
3
DNA as a template using Taq DNA polymerase. RNA from E. coli HB101 was extracted from a
4
bacterial cell sample of 109 cells harvested at OD600 = 0.4 according to the User Manual of the Pure
5
Link RNA Mini Kit (Invitrogen) and then treated with RNAse-free DNAse (Promega) according to
6
the manufacturer’s instructions. cDNA was synthesized from approximately 1 µg of isolated RNA,
7
using random hexamer primers (Promega). PCR based on the cDNA template (7 µl) was performed
8
using sbmA-yaiW fw and sbmA-yaiW rev primers to amplify the intergenic region between sbmA
9
and yaiW. Specific primer pairs to amplify the end of sbmA and the start of yaiW were also used as
10
a control (Table S2). As for the RNA extracted from S. Typhi or S. Typhimurium also for E. coli
11
HB101 a positive control was set up with genomic DNA as a template using Taq DNA polymerase.
12
Cloning of the yaiW genes. The S. enterica serovar Typhimurium MC2 yaiW gene was
13
amplified by PCR using StmyaiW+1F_NdeI and StmyaiW-1092R_XhoI.
The yaiW gene was
14
digested with NdeI and XhoI, ligated into plasmid pET22b and transformed into E. coli DH5α. The
15
insert in pSTyaiW was then confirmed by sequencing and then the plasmid was transformed into the
16
E. coli expression strains BL21(DE3) and BL21(DE3)pLysS. Expression from the T7lac promoter
17
in pSTyaiW creates a C-terminal 6x His-tagged fusion protein.
18
amplified by PCR using the yaiW-66_PstI and yaiW_rev_EcoRI primers. After digestion of the
19
gene with PstI and EcoRI, yaiW was ligated directly into plasmid pUT18 and transformed into
20
E. coli DH5α. The insert was then confirmed by sequencing.
The E. coli yaiW gene was
21
[3H]-Palmitate labeling. To demonstrate that YaiW is a lipoprotein, [3H]-palmitate labeling
22
was performed following a modification of a previously published method (26). Stationary phase
23
cultures of either BL21(DE3) with pET22b or pSTyaiW were diluted 10-fold into minimal medium
24
[spizizen salts, 0.2% (w/v) glucose and 1 µg ml-1] containing a protease inhibitor cocktail (Roche).
25
The cultures were then grown until OD600 = 0.6 and then plasmid gene expression was induced by
26
the addition of 1 mM IPTG for 45 minutes. E. coli host gene expression was then inhibited by the
6
1
addition of rifampicin (200 µg ml-1) for 1 hour. To inhibit lipoprotein processing, cultures were
2
then treated with and without globomycin (200 µg ml-1 dissolved in DMSO) for 20 minutes. The
3
lipoproteins were then labeled by the addition of [3H]-palmitate (50 µgCi ml-1) and incubated for a
4
further 20 minutes. The cells were then pelleted in a microcentrifuge and washed 3 times with T-C
5
buffer (10mM Tris-HCl pH 7.3 and 8 mM CaCl2). The labeled lipoproteins were solubilized by
6
boiling in protein loading buffer [2% (w/v) SDS, 10% (v/v) glycerol, 20 mM DTT, 0.02% (w/v)
7
bromophenol blue, 63mM Tris-HCl pH 6.8] for 15 minutes and resolved by SDS-PAGE. After
8
separation, the gels were fixed for 30 minutes in isopropanol:water:aceticacid (25:65:10) and then
9
immersed in Amplify Solution (Amersham). The gel was dried and the radioactive bands detected
10
by autoradiography.
11
Membrane extraction and separation. Overnight cultures of the defined strains were
12
diluted into fresh LB and grown until an OD600 = 0.8. Plasmid gene expression was induced by the
13
addition of 1 mM IPTG cultures and then the cells were incubated for a further 3 hours at 25°C.
14
The cells were collected by centrifugation (32000 x g, 5 minutes) and re-suspended in 10 ml 25%
15
sucrose (w/w) prepared in Tris Acetate (pH 7.8). One ml of lysozyme (2 mg ml-1) was added on ice
16
and after 2 minutes, 16 ml of 1.5 mM EDTA (pH 7.8) was added gradually over a time period of 6
17
minutes. Conversion of cells into spheroplasts was confirmed by microscopy. Cells were disrupted
18
using a French Press (500 bar) and lysed cells were centrifuged at 9000 x g at 4°C for 15 minutes.
19
The total membrane extraction in the supernatant was collected by ultracentrifugation at 100
20
000 x g at 4°C for 1 hour. The membrane pellet (~0.3 g) was homogenized in 2 ml of 25% sucrose
21
(w/w) prepared in 10 mM Tris Acetate (pH 7.8).
22
The inner and outer membranes were separated at 4°C on a 30-60% (w/w) sucrose gradient
23
prepared by layering from the bottom of a ultra-centrifuge tube (Beckman) 0.4 ml of 60%, 0.9 ml
24
55%, 2.2 ml of 50%, 45%, 40%, 1.3 ml of 35% and 0.4 ml of 30% (w/w) sucrose solutions prepared
25
in 10 mM Tris Acetate and 0.5 mM EDTA (w/w) pH 7.8. The total membrane suspension was then
26
layered onto the sucrose gradient and ultracentrifugation performed at 120,000 x g (SW41 rotor) at
7
1
4°C for 18 hours. Fractions (0.5 ml) were collected from the bottom of the tube and the OD595
2
recorded and protein content determined by the Bradford assay. Ten µl aliquots of each fraction
3
were mixed with 5 µl of Laemmli Buffer (27), denatured at 90°C for 10 minutes and then samples
4
were resolved using a 10% (w/v) SDS-PAGE gel. The molecular weight of the proteins was
5
determined using protein standards (Biorad). Separation of the membrane fractions was confirmed
6
by using anti-OmpA and anti-SecA (28) antibodies, which will recognize proteins in the outer and
7
inner membrane fractions, respectively.
8
Western blots. After being resolved by SDS-PAGE, the proteins were transferred to a
9
nitrocellulose membrane using a semi-dry transfer apparatus (Biorad) at 12 V for 1 hour and the
10
membranes stained with Ponceau Red. Western blot analysis was performed according to the
11
QIAexpress Detection and Assay Handbook (Qiagen). The rabbit anti-OmpA antibody and anti-
12
SecA antibodies were used at titers of 1:80,000 and 1:10,000, respectively. A goat anti-rabbit
13
secondary antibody conjugated to HRP (Imagenex) was used at a titer of 1:5,000. For detection of
14
His-tagged proteins, the penta-His HRP conjugate (Qiagen) was used at a titer of 1:100,000. In all
15
cases, the ECL plus kit (Amersham Bioscience) was used for the detection of HRP.
16
Surface labeling of cells using NHS-LC-LC-Biotin. Cell surface proteins of E. coli were
17
labeled with NHS-LC-LC biotin as described previously (29). In brief, the desired strains were
18
grown in LB with 100 µg ml-1 Ap to an OD600 ~ 0.8 and then expression of YaiW was induced by
19
the supplementation of the growth medium with 1 mM IPTG. Then the cultures were continued to
20
grow for 3 hours at 25°C, then washed 3 x in phosphate buffered saline (PBS) and finally
21
resuspended to a final OD600 = 10 in PBS. Then NHS-LC-LC-Biotin (Pierce) was added to a final
22
concentration of 2% and the reaction was stopped after 20 minutes by the addition of Tris (pH 7.5)
23
to a final concentration of 250 mM. Whole cells lysates were produced using the French press at
24
20,000 PSI and then the protein concentration was determined using a Bio-Rad Bradford assay
25
reagent (Bio-Rad).
8
1
Bac7 sensitivity assay. Bac7(1-16), Bac7(1-35) and the BODIPY fluorescently labeled
2
derivative Bac7(1-35)-BY have been prepared as described in (30). To monitor bacterial growth
3
inhibition, a suspension of 1x106 CFU ml-1 E. coli cells were grown in microtiter plates with
4
periodic shaking at 37°C in the presence of 0.25 µM Bac7(1-16) or Bac7(1-35). The OD620 was
5
measured every 10 minutes on a microtiter plate reader (Tecan Trading AG, Switzerland).
6
Flow cytometry assays. Uptake of BODIPY-labeled Bac7(1-35) in E. coli cells was
7
determined by flow cytometry using a Cytomics FC500 instrument (Beckman-Coulter, Inc.)
8
equipped as previously described (9). Cultures of mid-log phase bacteria were harvested, diluted to
9
106 CFU ml-1in MH broth, incubated with 0,25 µM Bac7(1-35)-BODIPY® at 37°C for 10 minutes
10
and analyzed immediately. All experiments were conducted in triplicate and data were expressed as
11
Mean Fluorescence Intensity (MFI) ± S.D. Data analysis was performed with the FCS Express V3
12
software (De Novo Software, CA).
13
Statistical analysis. The significance of differences among bacterial strains was assessed
14
using GraphPad Prism using ANOVA analysis followed by Bonferroni's Multiple Comparison Test.
15
Bioinformatics. Bioinformatic analysis of sbmA-yaiW gene clusters in the different strains
16
was performed using the Absynthe tool (http://archaea.u-psud.fr/absynte) using the full length
17
S. Typhimurium LT2 yaiW gene sequence (Pubmed gene ID: 1251896) (31).
18
9
1
RESULTS
2
The yaiW gene is co-transcribed with sbmA. In the genomes of enteric bacteria the YaiW
3
gene is located in close proximity to the sbmA gene, which encodes an inner membrane transport
4
protein (Fig. 1A). To investigate whether the sbmA and yaiW genes are co-transcribed, RNA was
5
extracted from the pathologically important bacterial strains S. Typhimurium MC2, and S. Typhi
6
Ty2 and then RT-PCR was performed using forward and reverse primers internal to the sbmA and
7
yaiW genes, respectively (Fig. 1B). In both cases, the RT-PCR reactions generated a product of
8
~0.6 kb (Fig. 1B, lanes II), which were sequenced and shown to be correct (data not shown). No
9
PCR product was observed for the RNA preparations using the same primer pair in the absence of
10
reverse transcriptase (Fig. 1B, lanes III). A 0.45 kb transcript of the two genes was also obtained in
11
E. coli (Fig. 1C) confirming that the sbmA and yaiW genes are co-transcribed.
12
The yaiW gene encodes a lipoprotein. In the genomes of S. Typhimurium and S. Typhi the
13
yaiW gene is annotated to encode a putative lipoprotein (NCBI reference No. NP_459372.1 and
14
NP_806215.1, respectively) because it possesses a conserved lipobox sequence at its N-terminus
15
(Fig. S1). To experimentally verify that YaiW is a lipoprotein, we constructed an S. Typhimurium
16
MC2 ΔyaiW::kan mutant and then grew this mutant strain and its parent strain in the presence of
17
[3H]-palmitate, which labels all lipoproteins (32). The [3H]-palmitate-labeled proteins were then
18
resolved by SDS-PAGE. However, due to the large number of lipoproteins present in enteric
19
bacteria, we were unable to detect any differences in the radioactive protein bands observed
20
between the parent and ΔyaiW mutant using this approach (data not shown).
21
To overcome this technical difficulty, the S. Typhimurium MC2 yaiW gene was cloned into
22
the E. coli expression vector, pET22B under control of the T7 lac promoter to create pSTyaiW.
23
Expression of yaiW from pET22B creates a C-terminal His-tagged protein, enabling detection of
24
YaiW by Western blotting. To enable us to determine if YaiW is a lipoprotein, we grew E. coli
25
BL21 (DE3) with either pSTyaiW or the control vector, pET22B in the presence of IPTG to induce
26
yaiW expression, and included [3H]-palmitate to radio-label lipoproteins. To prevent E. coli host
10
1
gene expression, the cultures were also treated with rifampicin, which inhibits E. coli RNA
2
polymerases but not T7 RNA polymerase (33). Using this approach, we identified a radioactive
3
band of ~39 kDa, corresponding to the mature YaiW C-terminal His-tagged protein, which was
4
present in the extract from BL21 (DE3) with pSTyaiW but not with the control plasmid without
5
insert (Fig. 2B). The antibiotic globomycin inhibits lipoprotein processing by interfering with the
6
pro-lipoprotein signal peptidase (LspA), which results in the retention of the pro-lipoprotein in the
7
inner membrane fused to its signal peptide (34, 35). We found that growth of E. coli BL21 (DE3)
8
with pSTyaiW in the presence of globomycin inhibited the processing of the YaiW C-terminal His-
9
tagged protein resulting in a larger, unprocessed YaiW protein (Fig. 2B). Therefore, since we
10
determined that the S. Typhimurium YaiW protein is labeled with [3H]-palmitate and the processing
11
of this protein is inhibited by globomycin, our findings clearly demonstrate that YaiW is a
12
lipoprotein.
13
SbmA is not involved in the lipid modifications of YaiW or enteric LPS but
14
complements the LPS lipid alteration of an S. meliloti BacA-deficient mutant. Since S. meliloti
15
and B. abortus BacA affect the VLCFA modification of lipid A, we examined whether its enteric
16
homolog SbmA might be required for either the lipid modifications of YaiW or of enteric LPS. To
17
investigate this, an sbmA::Tn5 insertion was transduced into E. coli BL23 (DE3) with pSTyaiW and
18
then the ability of [3H]-palmitate to label the YaiW-His tagged protein was investigated after IPTG
19
induction. We found no reduction in the amount of [3H]-palmitate labeling of YaiW and no
20
difference in the mobility of the purified YaiW His-tagged protein by PAGE in the absence of the
21
E. coli SbmA protein (Fig. 2C). In addition, we found no difference in the LPS species produced in
22
the E. coli sbmA and yaiW mutants relative to the parent strain as determined by PAGE analysis
23
(data not shown). Nor did we find any differences in the analyzed LPS fatty acid composition
24
(C12:0, C14:0, 3-OH C14:0 and C16:0) of an S. Typhimurium sbmA mutant as determined by GC-
25
MS (Fig. S2) following established procedures (17). In addition, we found no difference in the LPS
26
species produced in the E. coli sbmA and yaiW mutants relative to the parent strain as determined by
11
1
PAGE analysis (data not shown). However, despite the fact that enteric bacterial species lack the
2
LPS VLCFA biosynthesis cluster (19), we found that the plasmid-encoded E. coli sbmA gene
3
(pAI351) was able to restore the LPS VLCFA content to the same extent as the plasmid-encoded
4
S. meliloti bacA gene (pJG51A) in an S. meliloti BacA-deficient mutant in which the VLCFA
5
biosynthesis is reduced (Table 1) (11).
6
YaiW is an outer membrane lipoprotein. In Gram-negative bacterial species, lipoproteins
7
can be attached to either the inner or outer membrane (35). Inner membrane lipoproteins have an
8
aspartic acid at residue 2 in the mature lipoprotein, which serves as an inner membrane retention
9
sequence (35). In contrast, the S. Typhimurium YaiW protein has a serine residue in this position
10
(Fig. 2A), suggesting that YaiW is an outer membrane lipoprotein (Pubmed gene ID: 1251896)
11
(35). Creating an active antibody that targets YaiW proved to be very difficult and therefore we
12
introduced the plasmid pSTyaiW, which encodes YaiW carrying a His tag, into E. coli to facilitate
13
our investigation of the membrane localization of YaiW. Total membrane fractions from E. coli
14
BL21 (DE3) with pSTyaiW grown in the presence of IPTG were isolated and the inner and outer
15
membranes were then separated by sucrose-density gradient (30 – 60%) centrifugation (Fig. 3A).
16
To ensure that effective separation of the inner and outer membranes had occurred, Western blots
17
using antibodies against the outer membrane OmpA protein and the inner membrane SecA protein,
18
were performed (36). We found the highest amount of the outer membrane protein OmpA in
19
fraction 6 of the sucrose gradient (Fig. 3B) whereas the greatest amount of the inner membrane
20
SecA protein was found in fractions 16 and 18 (Fig. 3C). Having demonstrated that we had
21
achieved separation of the outer and inner membrane fractions, using a penta-His HRP conjugate
22
we identified a band of the expected size for the mature YaiW-His tagged protein (39 kDa) in the
23
membrane fractions obtained from E. coli BL23 (DE3) with pSTyaiW. This band was absent in the
24
total membrane fraction prepared from BL21 (DE3) with the control plasmid, pET22B (labeled as
25
negative control) (Fig. 3D). Since the same band was also observed in the lane containing purified
26
YaiW-His tagged protein (labeled as positive control) (Fig. 3D), these findings show that the penta 12
1
his HRP conjugate was specifically recognizing the YaiW C-terminal His-tagged protein in the
2
membrane fractions. The highest amount of YaiW-His tagged protein was found in fraction 6 (Fig.
3
3D), which also contained the greatest amount of the outer membrane OmpA protein (Fig. 3B).
4
Therefore, these findings demonstrate that yaiW encodes an outer membrane lipoprotein. 5
The YaiW lipoprotein is surface exposed. A previous study described an effective way to
6
detect surface exposed outer membrane proteins in E. coli using the NHS-LC-LC-Biotin (Pierce)
7
compound (Fig. 4A) (29). This compound forms stable bonds with primary amine groups (-NH2) of
8
amino acids (especially Lys) (Fig. 4A). Very detailed analysis demonstrated that NHS-LC-LC-
9
Biotin labeled only surface exposed proteins and did not cross the E. coli cell membrane to interact
10
with any cytoplasmic or periplasmic proteins (29). Initially, we surface-labeled the surface proteins
11
of the parent, the yaiW::kan mutant and the ompA::kan mutant strains from the single deletion
12
mutant Keio strain collection with the NHS-LC-LC-Biotin compound (25). Streptavidin, which
13
forms a very strong non-covalent bond with the vitamin biotin, was used to detect the protein-bound
14
NHS-LC-LC-Biotin compound. The ompA::kan mutant was used as a negative control for the
15
Biotin labeling as it lacks the outer membrane protein OmpA (29). We found that a range of
16
proteins of different sizes were surface exposed in all three strains. The outer membrane profiles of
17
surface exposed proteins of the parent and yaiW::kan mutant strains appeared to look very similar
18
and no band that could be clearly attributed to YaiW (39 kDa) seemed to be missing for the
19
yaiW::kan mutant strain compared to the parent strain (Fig. 4B). However, OmpA (37.3 kDa),
20
which is known to be surface exposed in E. coli, is absent in the lane loaded with ompA::kan mutant
21
lysate (Fig. 4B) (29). Due to the presence of a large amount of surface proteins in the size range
22
where YaiW (39 kDa) would be expected and the possibility that YaiW is only weakly expressed,
23
S. Typhimurium YaiW was over-expressed in E. coli BL21(DE3) from pSTyaiW and purified after
24
surface labeling. The YaiW proteins of E. coli K12 and S. Typhimurium LT2 are 92% similar (85%
25
identical) (data not shown). In order to ensure that the His-tag itself was not recognized by
26
Streptavidin-HRP, YaiW-His was purified after induction of its expression without prior surface
13
1
labeling by NHS-LC-LC-Biotin. A penta anti-His antibody (αHis) detected the purified YaiW-His
2
protein (Fig. 4C) but the biotin specific Streptavidin-HRP did not (Fig. 4C). This finding proved
3
that the His-tag does not interfere with Streptavidin-HRP and that Streptavidin-HRP detection was
4
exclusive to biotin-labeled proteins. Over-expression of YaiW (39 kDa) in an E. coli BL21(DE3)
5
parent strain and purification of His-tagged proteins subsequent to surface labeling resulted in a
6
clean band detected by a penta anti-His antibody (Fig. 4D). No band was observed with pET22b
7
only. The same was found when YaiW was expressed from pSTyaiW in a ΔsbmA mutant (Fig. 4D).
8
Streptavidin-HRP detected biotin labeled proteins of the same size for the parent and the ΔsbmA
9
mutant with pSTyaiW (Fig. 4E). No bands corresponding to the size of YaiW were observed for the
10
parent strain with pET22b only (Fig. 4D and E). We therefore demonstrated that YaiW is surface
11
exposed in both E. coli and S. Typhimurium.
12
YaiW is involved in the uptake of proline-rich Bac7 peptides. The finding that sbmA and
13
yaiW are co-transcribed suggests that they might possibly be involved in a related process in enteric
14
bacteria. It was shown previously that mutations in sbmA decrease the susceptibility of enteric
15
bacterial species towards different types of antimicrobial peptides (AMPs) such as bleomycin,
16
microcin B17 and truncated Bac7 peptides (6, 9, 20). Therefore, to investigate whether YaiW is
17
involved in the sensitization of E. coli towards AMPs, we compared the sensitivity of isogenic
18
deletion mutants of sbmA and yaiW towards two fragments of Bac7 that differed in length. In the
19
presence of sub-lethal concentrations of both Bac7(1-35) (data not shown) and Bac7(1-16) the
20
deletion of yaiW conferred a moderate increase in the ability of the strain to grow (Fig. 5A). To
21
investigate whether the increased viability of the ΔyaiW mutant in the presence of the peptide could
22
be due to a decreased internalization of the peptide, we measured the uptake of fluorescently labeled
23
Bac7(1-35)-BY in both the ΔsbmA and ΔyaiW mutants. We found that, similarly to the effect of
24
deleting sbmA, deletion of yaiW resulted in a decrease of almost 50% of peptide internalization
25
(Fig. 5B). In addition the sensitivity of isogenic deletion mutants of sbmA, yaiW and sbmA/yaiW
26
towards bleomycin sulfate and microcin B17 was also tested. We also found that deletion of sbmA
14
1
conferred decreased sensitivity of E. coli towards these two AMPs but deletion of yaiW had no
2
effect. The ΔsbmA/ΔyaiW double mutant had the same sensitivity to bleomycin and microcin B17
3
as the ΔsbmA single mutant (data not shown).
4
In order to investigate whether the YaiW and SbmA proteins might interact with each other
5
we expressed yaiW and sbmA fused to the two subunits of the adenylate cyclase of Bordetella
6
pertussis. Since this two-hybrid system detects only interactions occurring at the cytoplasm or inner
7
membrane level (37), a truncated form of YaiW in which the lipobox signature was removed was
8
used. No detectable interaction between SbmA and YaiW was revealed in these experimental
9
conditions, suggesting either that the two proteins may not be associated at the inner membrane
10
level or that interaction of SbmA and YaiW could occur in the periplasm, which would be out of the
11
context of this two hybrid system (data not shown).
12
15
1
DISCUSSION
2
In this study, we demonstrate that enteric yaiW is in an operon with sbmA and that YaiW is a
3
surface-exposed lipoprotein. To our knowledge only a few lipoproteins have been proven to be
4
surface exposed in E. coli. These include Wza(K30), a lipoprotein required for surface
5
polysaccharide polymerisation (38), TraT, a lipoprotein involved in F-sex factor conjugation (39)
6
and CsgG, a lipoprotein important for the assembly of curli fibers (40, 41). Recently, the most
7
abundant lipoprotein in E. coli, Lpp has also been demonstrated to be exposed at the cells surface
8
using the NHS-LC-LC-Biotin compound we employed in our study (29).
9
We also show that, like SbmA, YaiW, affects the internalization of the proline-rich peptide
10
Bac7(1-35). Until now, such functional relationship between both proteins could only be suggested
11
based on their location near each other in bacterial genomes. A plausible explanation for this
12
relationship is that YaiW contributes positively to the proline-rich peptide crossing the outer
13
membrane, while SbmA contributes positively to it crossing the cytoplasmic membrane. Although a
14
direct interaction of the SbmA and YaiW proteins was not observed in a bacterial two-hybrid
15
system (data not shown), which detects interactions in the cytoplasm, it may still be possible that
16
both proteins indirectly interact in the periplasm via a periplasmic protein. Additionally, there
17
appears to be some specificity to the functional relationship of SbmA and YaiW since we did not
18
observe any effects of YaiW on the sensitivity to microcin B17 or bleomycin. Further studies will
19
be required to clarify the physiological role of this relationship. Finally, in the course of our study,
20
we made the interesting observation that, although VLCFA’s are not present in enteric bacteria and
21
SbmA does not affect the lipid modifications of E. coli LPS, when introduced into S. meliloti SbmA
22
can substitute for BacA’s role in the VLCFA modification of S. meliloti LPS.
23
24
16
1
ACKNOWLEDGEMENTS
2
This paper is dedicated to Gail P. Ferguson who passed away during the preparation of this
3
manuscript. We are grateful to Prof. Renato Gennaro for carefully reading the manuscript. Thanks
4
to Masatoshi Inukai for the kind gift of globomycin and to Monica Benincasa for the flow
5
cytometry assays. This work was supported by MRC New Investigator (G0501107) and BBSRC
6
(BB/D000564/1) grants to G.P.F. and by National Institute of Health grant GM31030 to G.C.W.,
7
who is an American Cancer Society Professor. The MFFA was a SULSA funded Ph.D. student.
8
P.C. and K.T. were funded by the MRC grant and S.W. was funded by the BBSRC grant. This
9
study was also supported by grants from the Italian Ministry for University and Research (PRIN
10
2008) and from the Regione Friuli Venezia Giulia grant under the LR 26/2005, art. 23 for the R3A2
11
network.
12
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17
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49
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26
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1
TABLES AND FIGURES
2
Table 1. SbmA complements the lipid A alteration of an S. meliloti BacA-deficient null mutant
Amount in mol/2mol GlcN
S. meliloti bacA-deficient mutant
Fatty acid
pAI351
pRK404
pJG51A
13:0(3-OH)
0.02
0.02
0.03
14:0(3-OH)
1.46
1.52
1.43
15:0(3-OH)
0.10
0.12
0.10
16:0(3-OH)
0.45
0.52
0.45
17:0(3-OH)
0.10
0.12
0.10
18:0(3-OH)
1.06
1.20
1.08
18:0(3-OH)
0.11
0.14
0.11
19:0(3-OH)
0.08
0.10
0.07
20:0(3-OH)
0.08
0.12
0.09
28:0(27-OH)
0.79
0.39
0.81 
30:0(29-OH)
0.33
0.19
0.35
3
21
yaiZ
ddl
t2485
ddl
yaiY
yaiW
sbmA
yaiV
ampH
A
t2486
yaiW
sbmA
t2490
ampH
S. Typhimurium LT2
ddlA
ddl
ddlA
11190
0366
20996
11185
0246
yaiZ
yaiY
0367
0247
11180
yaiW
0248
11175
11170
20998
20997
yaiW
sbmA
sbmA
21000
21001
map2
Pseudomonas aeruginosa DK2
11165
11160
Shigella flexneri 2002017
sbmA
ampH
0251
Shigella dysenteriae Sd197
yaiH
0371
E. coli K12 substrain MG1655
bacA
yaiV
ampH
S. Typhi Ty2
S. meliloti Sm1021
B S. enterica
C E. coli
S. Typhi
kb
kb
1.0
S. Typhimurium
1.6
1.0
negative RT-­PCR positive
0.5
0.25
0.5
M
I II III
sbmA
II
1
2
3
4
5
M M 1 2 3 1 2 3 1 2 3
C
A
I II III
yaiW
0.6 kb
sbmA
(1)
(2)
(3)
B
yaiW
D
320 bp
121 bp
451 bp
Figure 1. The sbmA gene is co-transcribed with yaiW. (A) Schematic representation of the sbmA-yaiW gene
regions in the genomes of selected Gram negative bacteria. Genomic data was imported and analyzed by the
Absynthe tool (http://archaea.u-psud.fr/absynte). (B) Agarose gel analysis of DNA products generated by RT-PCR
Figure 1.
The sbmA gene is co-transcribed with yaiW. (A) Schematic representation of the
using either primers internal to the 16S rRNA gene (rrsH) (lanes I) or a forward primer internal to sbmA and a reverse
internal to yaiW (lane II). The expected products of 1.5 and 0.6 kb for lanes I and II were obtained, respectively.
A negative
was also
using the same
primers as inGram
lane 1 but
only Taq polymerase
was added
in the data was
sbmA-yaiW
genecontrol
regions
inperformed
the genomes
of selected
negative
bacteria.
Genomic
absence of reverse transcriptase (lane III). M=Invitrogen 1kb ladder. (C) Agarose gel analysis of DNA products generated
by RT-PCR from E. coli HB101 using either primers specific for the tail of sbmA (lane 1) or for the start of yaiW (lane 2) or a
internalby
to sbmA
a reverse internal
yaiW (lane 3). The expected products of 0.32, 0.12 and
kb
importedforward
and primer
analyzed
theand
Absynthe
toolto(http://archaea.u-psud.fr/absynte).
(B)0.45
Agarose
gel
for lanes 1, 2 and 3 were obtained, respectively. A negative and positive control were also performed using the same
primers as in each lane but adding no template or gDNA respectively.
analysis of DNA products generated by RT-PCR using either primers internal to the 16S rRNA
6
gene (rrsH) (lanes I) or a forward primer internal to sbmA and a reverse internal to yaiW (lane II).
7
The expected products of 1.5 and 0.6 kb for lanes I and II were obtained, respectively. A negative
8
control was also performed using the same primers as in lane 1 but only Taq polymerase was added
9
in the absence of reverse transcriptase (lane III). M=Invitrogen 1kb ladder. (C) Agarose gel
10
analysis of DNA products generated by RT-PCR from E. coli HB101 using either primers specific
11
for the tail of sbmA (lane 1) or for the start of yaiW (lane 2) or a forward primer internal to sbmA
12
and a reverse internal to yaiW (lane 3). The expected products of 0.32, 0.12 and 0.45 kb for lanes 1,
22
1
2 and 3 were obtained, respectively. A negative and positive control were also performed using the
2
same primers as in each lane but adding no template or gDNA respectively.
A N-­terminus of S. Typhimurium YaiW Residue 2
MSAASRLYPLPFLAVAILAGCSSQSGQ
Lipobox
B [3H]-­palmitate labeling
-­
Globomycin
+
-­
+
kDa
47.5
32.5
pET22B
pSTyaiW
E. coli BL21 (DE3) C [3H]-­palmitate labeling
E. coli BL21 (DE3) WT
sbmA::Tn5
WT
pET22b
pSTyaiW
pSTyaiW
kDa
47.5
32.5
3
gure 2. YaiW is a lipoprotein. (A) The N-terminus sequence of the S. enterica serovar Typhimurium YaiW
otein is shown
andis
predicted
second residue (A)
in theThe
matureN-terminus
protein. (B) E. coli
BL21(DE3)of the S. enterica serovar
4 indicating
Figure the
2. lipobox
YaiW
a lipoprotein.
sequence
ith either the control vector (pET-22B) or pSTyaiW (as indicated) were grown in the presence of 1mM IPTG,
00 μg ml-1 rifampicin and [3H]-palmitate and the lipoproteins present detected as described in the materials
5 To inhibit
Typhimurium
YaiW protein
shown
indicating
theml-1
lipobox
andaspredicted second residue in the
nd methods.
lipoprotein processing,
cultures is
were
also treated
with 200 μg
globomycin
efined. The predicted molecular masses of the full length and truncated YaiW his tagged protein are 41.2
nd 39.2 kDa, respectively. (C) Cultures of either BL21 (DE3) or BL21 (DE3) sbmA::Tn5 with either pT22B or
6
mature protein. (B) E. coli BL21 (DE3) with either the control vector (pET-22B) or pSTyaiW (as
STyaiW (as defined) were grown and the lipoproteins labeled as described in Figure 2B. Experiments were
nly performed in the absence of globomycin.
7
indicated) were grown in the presence of 1mM IPTG, 200 µg ml-1 rifampicin and [3H]-palmitate
8
and the lipoproteins present detected as described in the materials and methods. To inhibit
9
lipoprotein processing, cultures were also treated with 200 µg ml-1 globomycin as defined. The
10
predicted molecular masses of the full length and truncated YaiW his tagged protein are 41.2 and
11
39.2 kDa, respectively. (C) Cultures of either BL21 (DE3) or BL21 (DE3) sbmA::Tn5 with either
12
pT22B or pSTyaiW (as defined) were grown and the lipoproteins labeled as described in Figure 2B.
13
Experiments were only performed in the absence of globomycin.
23
OM
1.2
IM
2000
Turbidity (OD595)
1800
1.0
1600
1400
0.8
1200
1000
0.6
800
0.4
600
400
0.2
200
protein contenent (µg ml-­1)
A
0
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Fraction number B
OmpA (37.2 KDa)
2
4
6
8
10
12
14
16
18
2
4
6
8
10
12
14
16
18
Positive 4
control 6
8
10
12
14
16
18 Negative control C
SecA (102 KDa)
D
YaiW (39 KDa)
Fraction number
1
Figure 3. The YaiW-his tagged protein is localized to the outer membrane. Cultures of E. coli BL21 (DE3)
with pSTyaiW were grown in the presence of 1 mM IPTG. The total membranes were then isolated, the inner and
2
Figure 3. The YaiW-his tagged protein is localized to the outer membrane. Cultures of E. coli
outer membranes separated and resolved by sucrose-gradient density centrifugation. (A) The turbidity (solid line)
and protein concentration (dashed line) of the different membrane fractions obtained from the sucrose density
gradient 3
centrifugation.
(B-D) Western
using 10 μl
aliquots
of thein
different
membrane of
fractions
were
then The total membranes were
BL21 (DE3)
withblots
pSTyaiW
were
grown
the presence
1 mM
IPTG.
performed using antibodies against either the known outer membrane OmpA protein (B), the known inner
membrane SecA protein (C) or the His tag (D). The total membrane fraction (10 μl) from BL21 (DE3) with the
4
then
the inner
outer
membranes
and resolved
by sucrose-gradient density
control plasmid
(-YaiW)isolated,
and the purified
YaiW-hisand
tagged
protein
(3 μl; YaiW-his)separated
were also included
in (D) as
negative and positive controls, respectively.
5
centrifugation. (A) The turbidity (solid line) and protein concentration (dashed line) of the different
6
membrane fractions obtained from the sucrose density gradient centrifugation. (B-D) Western blots
7
using 10 µl aliquots of the different membrane fractions were then performed using antibodies
8
against either the known outer membrane OmpA protein (B), the known inner membrane SecA
9
protein (C) or the His tag (D). The total membrane fraction (10 µl) from BL21 (DE3) with the
10
control plasmid (-YaiW) and the purified YaiW-his tagged protein (3 µl; YaiW-his) were also
11
included in (D) as negative and positive controls, respectively.
12
13
14
15
16
24
B Streptavidin
A
E. coli K12 (Keio collection)
kDa
WT
yaiW
ompA
46
Protein interaction
OmpA
Biotin
30
C
D ĮHis
E. coli BL21 (DE3) pSTyaiW kDa
ĮHis
E. coli BL21 (DE3) kDa
Streptavidin
46
pSTyaiW
WT
pET22b
sbmA::Tn5
pSTyaiW
46
46
30
YaiW
30
E Streptavidin
E. coli BL21 (DE3) kDa
pSTyaiW
WT
pET22b
sbmA::Tn5
pSTyaiW
46
YaiW
30
1
Figure 4. A plasmid encoded His-tagged Salmonella YaiW protein is surface exposed in E. coli. (A) Chemical structure of
NHS-LC-LC-Biotin. (B) The defined Keio collection parent and mutant strains were treated with NHS-LC-LC-Biotin. After cell
lysis 2
10 µg ofFigure
whole cell 4.
lysateA
wasplasmid
separated using
SDS-PAGE and
probed using Salmonella
Streptavidin-HRP. YaiW
(C) E. coli BL21(DE3)
encoded
His-tagged
proteinharbouring
is surface exposed in
pSTyaiW was grown with 1mM IPTG. After cell lysis the whole cell lysate was run through a Ni-NTA agarose bead loaded gravity
column and the elution fractions were separated using SDS-PAGE and probed using a penta anti-his antibody or Streptavidin-HRP.
(D and
defined
grown in the
presence ofof
1 mM
IPTG and then treated with NHS-LC-LC-Biotin.
After cell
lysiscollection
the
3 E) TheE.
coli.strains
(A)were
Chemical
structure
NHS-LC-LC-Biotin.
(B) The defined
Keio
parent and
whole cell lysates were run through a Ni-NTA agarose bead loaded gravity column and the elution fractions were separated using
SDS-PAGE and probed using a penta anti-his antibody (D) or Streptavidin-HRP (E). All data shown are representative of at least two
independent
experiments.
4
mutant
strains were treated with NHS-LC-LC-Biotin. After cell lysis 10 µg of whole cell lysate
5
was separated using SDS-PAGE and probed using Streptavidin-HRP. (C) E. coli BL21(DE3)
6
harbouring pSTyaiW was grown with 1mM IPTG. After cell lysis the whole cell lysate was run
7
through a Ni-NTA agarose bead loaded gravity column and the elution fractions were separated
8
using SDS-PAGE and probed using a penta anti-his antibody or Streptavidin-HRP. (D and E) The
9
defined strains were grown in the presence of 1 mM IPTG and then treated with NHS-LC-LC-
10
Biotin. After cell lysis the whole cell lysates were run through a Ni-NTA agarose bead loaded
11
gravity column and the elution fractions were separated using SDS-PAGE and probed using a penta
12
anti-his antibody (D) or Streptavidin-HRP (E). All data shown are representative of at least two
13
independent experiments.
14
15
25
1
2
3
0.5
0.4
0.2
0.1
0
20
40 60 80 100 120 140 160 180 200 220 240
time (min)
Bac7(1-­16) 0,25 µM 0.6
0.5
0.4
**
**
***
***
400
200
CTRL 0
20
10 min ǻyaiW
ǻsbmA
BW25113
ǻyaiW
ǻsbmA
BW25113
ǻyaiW
ǻyaiW
ǻsbmA
BW25113
0.2
0.1
ǻsbmA
BW25113
0
0.3
0
Bac7(1-­35) 0,25 µM 600
ǻyaiW
ǻsbmA
BW25113
0.3
0
B E. coli BW25113
MH Broth
0.6
Peptide uptake
Fluorescence (M.F.I.) Cell density (OD620)
Cell density (OD620)
A E. coli BW25113
60 min 40 60 80 100 120 140 160 180 200 220 240
time (min)
Figure 5. E. coli YaiW is important for peptide stress resistance. (A) Growth kinetics of E. coli BW25113, ΔsbmA and ΔyaiW strains
in MH Broth in absence (upper panel) or in presence of 0.25 µM Bac7(1-16). Bacterial suspensions of 1 × 106 cells/ml have been
grown for5.
4 hours
and the
OD at 620
has been measured
every 10 minutes.
are the mean
of three
independent
Figure
E. coli
YaiW
is nm
important
for peptide
stressResults
resistance.
(A)
Growth
kinetics of E. coli
experiments. (B) Uptake of Bac7(1-35)-BY in E. coli BW25113, ΔsbmA and ΔyaiW strains measured as medium fluorescence intensity
(M.F.I.). Results are the mean of more than three independent experiments. The significant values *** p≤0.0004 and ** p≤0.0015 were
determined using
ANOVA and
followed
by Bonferroni’s
datasetsin
represent
trends(upper
observedpanel)
in at leastor
twoin
independent
BW25113,
ΔsbmA
ΔyaiW
strainspost
intest.
MHAllBroth
absence
presence of 0.25
experiments.
4
µM Bac7(1-16). Bacterial suspensions of 1 × 106 cells/ml have been grown for 4 hours and the OD
5
at 620 nm has been measured every 10 minutes. Results are the mean of three independent
6
experiments. (B) Uptake of Bac7(1-35)-BY in E. coli BW25113, ΔsbmA and ΔyaiW strains
7
measured as medium fluorescence intensity (M.F.I.). Results are the mean of more than three
8
independent experiments. The significant values *** p≤0.0004 and ** p≤0.0015 were determined
9
using ANOVA followed by Bonferroni’s post test. All datasets represent trends observed in at least
10
two independent experiments.
11
12
13
14
15
26