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| Salmonella Typhimurium |
www.pnas.org/cgi/doi/10.1073/pnas.1608858113
prokaryotic cells, or both (10). For example, the Vibrio cholerae Vas
and the Pseudomonas aeruginosa H2-T6SS target and kill bacteria
but also inject toxins into host cells to prevent phagocytosis or to
facilitate invasion, respectively (1318). The T6SS is a multiprotein
machine that uses a contractile mechanism for toxin secretion (19).
In short, the T6SS comprises a transenvelope complex that docks a
contractile tail composed of an inner tube, made of stacked Hcp
protein hexamers, tipped by the VgrG syringe, and surrounded by a
sheath, comprising polymerized TssB and TssC subunits (2022).
Sheath contraction provides the energy necessary for the injection
of the toxins that are confined within the Hcp tube or bound to
VgrG (23). After injection, the ClpV ATPase recycles the contracted sheath to permit a new assembly/injection step to occur (24).
Salmonella enterica serovar Typhimurium is a leading cause of
human gastroenteritis worldwide and causes a typhoid-like disease
in mice. Because this pathogen is transmitted by the fecaloral
route, it spends a significant part of its life cycle within intestinal microbial communities. Although absence of the microbiota
allows the pathogen to multiply to high densities (25), a highcomplexity microbiota facilitates S. Typhimurium clearance (26).
This not only illustrates the critical role of the intestinal microbiota in modulating Salmonella infection, but also suggests that
Salmonella must modulate its interactions with the microbiota (27).
Several reports have shown that acute inflammation triggered
by S. Typhimurium modifies the gut bacterial community to facilitate pathogen colonization in a mouse model (2832). However, it is currently unknown whether Salmonella can directly
target commensal bacteria with an antibacterial activity.
Significance
Gram-negative bacteria use the type VI secretion system (T6SS) to
deliver effectors into adjacent cells. Salmonella Typhimurium is
an enteric pathogen that causes disease in millions of individuals
each year. Its ability to infect the mammalian gut is a key factor
that contributes to its virulence and transmission to new hosts.
However, many of the details on how Salmonella successfully
colonizes the gut and persists among members of the gut
microbiota remain to be deciphered. In this work, we provide
evidence that Salmonella uses an antibacterial weapon, the
type VI secretion system, to establish infection in the gut. In
addition, our results suggest that S. Typhimurium selectively
targets specific members of the microbiota to invade the
gastrointestinal tract.
Author contributions: T.G.S., E.C., and D.M.M. designed research; T.G.S., N.F., K.A.L., L.H.L.,
A.J., V.B., and E.D. performed research; T.G.S., N.F., K.A.L., E.D., L.J., E.C., and D.M.M. analyzed data; and T.G.S., E.C., and D.M.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1
MICROBIOLOGY
Edited by Scott J. Hultgren, Washington University School of Medicine, St. Louis, MO, and approved June 30, 2016 (received for review June 2, 2016)
Fig. 1. SPI-6 is important for colonization of the gut. (A) The 129x1/SvJ Mice
were infected orally with 108 WT or SPI-6 mutant S. Typhimurium bacterial
strains. Feces were collected and plated over time on media that were selective for Salmonella. Every data point represents one mouse, and the bar
represents the geometric mean. (B) The 129x1/SvJ mice were orally infected
with 108 WT or SPI-6 Salmonella Typhimurium bacterial strains, and tissues
were harvested at day 7 postinfection and plated on selective media. Data
for mesenteric lymph nodes (MLNs), Peyers patches (PPs), liver (L), and
spleen (S) are shown. Statistical significance is shown based on Mann
Whitney U test (NS, not significant; **P < 0.01; ***P < 0.001).
Hcp1, but Not Hcp2, Binds Tae4 and Is Required for Interbacterial
Antagonism. T6SS effectors are delivered into target cells by a
Fig. 2. SPI-6 is activity is enhanced by bile salts and provides competitive advantage against members of the microbiota in vitro. (A) WT and mutant
S. Typhimurium strains were incubated with E. coli K-12 DH5 to assess T6SS-dependent killing. The indicated S. Typhimurium attacker strain was mixed with
E. coli prey at a 1:1 ratio and incubated for 48 h on an LB agar plate supplemented or not with 0.05% porcine bile salts. Recovered mixtures were plated onto
selective media. (B) WT and mutant S. Typhimurium strains were incubated with the indicated commensal strains (K. oxytoca, E. cloacae, K. variicola, and
E. coli JB2) for 48 h on an LB agar plate supplemented with 0.05% porcine bile salts. Recovered mixtures were plated onto selective media. Statistical significance
is shown based a Students t test corresponding to the values of the WT strain (NS, not significant; *P < 0.05; **P < 0.01).
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MICROBIOLOGY
Fig. 3. Hcp1 binds the Tae4 amidase effector and mediates K. oxytoca
killing. (A) WT and mutant S. Typhimurium strains were incubated with
K. oxytoca for 48 h on an LB agar plate supplemented with 0.05% porcine
bile salts. Recovered mixtures were plated onto selective media. Statistical
significance is shown based on a Students t test corresponding to the values
of the WT strain (NS, not significant; *P < 0.05; **P < 0.01; ****P < 0.0001).
(B) Bacterial two-hybrid analyses. BTH101 reporter cells producing the Hcp1
or Hcp2 proteins fused to the T18 domain of the Bordetella adenylate cyclase and the Tae4 protein fused to the T25 domain were spotted on plates
supplemented with IPTG and the chromogenic substrate X-Gal. Interaction
between the two fusion proteins is attested by the blue color of the colony.
The TolB-Pal interaction serves as a positive control. (C) Coprecipitation assay. Purified and biotinylated Tae4 was mixed with 6His-tagged Hcp1 (Left)
or Hcp2 (Right) and subjected to ion metal affinity chromatography on Ni2+
resin. The total (T), unbound (U), wash (W), and eluted (E) fractions were
collected and subjected to SDS/PAGE and Western blot analyses using antiHis antibody (Upper) and streptavidin-conjugated alkaline phosphatase
(Lower). The position of the proteins and the molecular mass markers (in
kDa) are indicated.
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Discussion
In nature or within hosts, microbes often exist in complex communities, and therefore they must compete with other species for
limited resources. Many Gram-negative bacteria encode a molecular machine called the T6SS that is dedicated to target and
kill other bacteria and sometimes to inject effector proteins into
eukaryotic cells (10). For example, the human pathogen V. cholerae
employs the T6SS to kill E. coli or to disable the amoebae
Dictyostelium discoideum and macrophage cell lines (14, 48, 49).
Recent studies demonstrated that T6SS organelles in V. cholerae,
P. aeruginosa, and entero-aggregative E. coli cells are very dynamic and likely expel their T6SS spike/tube VgrG/Hcp complex
toward prokaryotic cells and cause target cell lysis by delivering
antibacterial toxins (50, 51). A broad diversity of antibacterial
toxins have been described so far (10, 42). In this work, we show
that the SPI-6 T6SS is required for efficient Salmonella establishment in the gut and that it is the antibacterial activity of the
T6SS that contributes to this phenotype, suggesting a direct interaction between the pathogen and the indigenous microbiota. In
vitro competition assays demonstrated a SPI-6 T6SS-dependent
killing of members of the gut microbiota in vitro, which was
confirmed by in vivo survival assays. Finally, we provide details
on the molecular mechanism underlying SPI-6mediated competition by showing that the Tae4 amidase interacts with Hcp1,
facilitating its delivery and activity within prey bacterial cells.
This report demonstrates that the antibacterial activity of a T6SS
is important for a pathogen to establish within the host gut and
opens new exciting perspectives.
In addition to the core set of 13 genes required for T6SS assembly and function, the S. Typhimurium SPI-6 locus contains
two copies of the hcp gene, hcp1 and hcp2 (33). Hcp proteins
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Hcp1 and Hcp2 Structure Modeling. The homology models of Hcp1 were built
using Coot (69) according to a Multalin alignment with the closest homolog,
the enteroaggregative E. coli (EAEC) Hcp protein (PDB ID code 4HKH) (70).
In Vitro Competition. Overnight cultures of bacteria were spun down and
washed with PBS before resuspension at 1010 bacteria per milliliter. Then, 10 L
of each resuspension were spotted on an LB plate supplemented with 0.05%
of porcine bile salts (Sigma-Aldrich) and grow at 37 C for 48 h. Spots were
serially diluted and plated on LB supplemented with the appropriate antibiotic, or MacConkey agar (Fisher Scientific). Bait recovered was calculated as the
ratio of total bait cfus divided by total Salmonella cfus recovered after scraping
and counting on selective plates. For in vitro competition in anaerobic conditions, a similar protocol was followed, with the exception that bacteria were
grown at 37 C in an anaerobic chamber for 96 h on blood plates [horse blood
from Sigma-Aldrich at 10% in BrainHeart Infusion (BHI) agar medium].
Monitoring Fecal Shedding of S. Typhimurium. Individual mice were identified
by distinct tail markings and tracked throughout the duration of infection.
Between two and three fresh fecal pellets were collected directly into
Eppendorf tubes and weighed at the indicated time points. Pellets were
resuspended in 500 mL of PBS, and colony-forming units per gram of feces
were determined by plating serial dilutions on LB agar plates with the
appropriate antibiotics.
Statistical Analyses. Prism (GraphPad) was used to perform all statistical
analyses. Differences in cfus and strain composition between groups were
examined by unpaired nonparametric MannWhitney U tests. Differences
for in vitro competition were examined by Student t test. Differences in
competitive index were examined by Dunnett tests. Significance was defined
by P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****).
1. Leatham MP, et al. (2009) Precolonized human commensal Escherichia coli strains
serve as a barrier to E. coli O157:H7 growth in the streptomycin-treated mouse intestine. Infect Immun 77(7):28762886.
2. van der Waaij D, Berghuis-de Vries JM, Lekkerkerk Lekkerkerk-v (1971) Colonization
resistance of the digestive tract in conventional and antibiotic-treated mice. J Hyg
(Lond) 69(3):405411.
3. Ivanov II, et al. (2009) Induction of intestinal Th17 cells by segmented filamentous
bacteria. Cell 139(3):485498.
4. Mazmanian SK, Round JL, Kasper DL (2008) A microbial symbiosis factor prevents
intestinal inflammatory disease. Nature 453(7195):620625.
5. Round JL, Mazmanian SK (2009) The gut microbiota shapes intestinal immune responses during health and disease. Nat Rev Immunol 9(5):313323.
6. Hooper LV (2009) Do symbiotic bacteria subvert host immunity? Nat Rev Microbiol
7(5):367374.
7. Hibbing ME, Fuqua C, Parsek MR, Peterson SB (2010) Bacterial competition: Surviving
and thriving in the microbial jungle. Nat Rev Microbiol 8(1):1525.
8. Ferreyra JA, Ng KM, Sonnenburg JL (2014) The enteric two-step: Nutritional strategies
of bacterial pathogens within the gut. Cell Microbiol 16(7):9931003.
9. Hood RD, et al. (2010) A type VI secretion system of Pseudomonas aeruginosa targets
a toxin to bacteria. Cell Host Microbe 7(1):2537.
10. Alcoforado Diniz J, Liu YC, Coulthurst SJ (2015) Molecular weaponry: Diverse effectors
delivered by the Type VI secretion system. Cell Microbiol 17(12):17421751.
11. Cascales E (2008) The type VI secretion toolkit. EMBO Rep 9(8):735741.
12. Bingle LE, Bailey CM, Pallen MJ (2008) Type VI secretion: A beginners guide. Curr
Opin Microbiol 11(1):38.
13. Jiang F, Waterfield NR, Yang J, Yang G, Jin Q (2014) A Pseudomonas aeruginosa type
VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells.
Cell Host Microbe 15(5):600610.
14. MacIntyre DL, Miyata ST, Kitaoka M, Pukatzki S (2010) The Vibrio cholerae type VI
secretion system displays antimicrobial properties. Proc Natl Acad Sci USA 107(45):
1952019524.
15. Pukatzki S, Ma AT, Revel AT, Sturtevant D, Mekalanos JJ (2007) Type VI secretion
system translocates a phage tail spike-like protein into target cells where it cross-links
actin. Proc Natl Acad Sci USA 104(39):1550815513.
16. Bleves S, Sana TG, Voulhoux R (2014) The target cell genus does not matter. Trends
Microbiol 22(6):304306.
17. Sana TG, et al. (2015) Internalization of Pseudomonas aeruginosa strain PAO1 into
epithelial cells is promoted by interaction of a T6SS effector with the microtubule
network. MBio 6(3):e00712.
18. Sana TG, et al. (2012) The second type VI secretion system of Pseudomonas aeruginosa strain PAO1 is regulated by quorum sensing and Fur and modulates internalization in epithelial cells. J Biol Chem 287(32):2709527105.
19. Zoued A, et al. (2014) Architecture and assembly of the type VI secretion system.
Biochim Biophys Acta 1843(8):16641673.
20. Basler M, Pilhofer M, Henderson GP, Jensen GJ, Mekalanos JJ (2012) Type VI secretion
requires a dynamic contractile phage tail-like structure. Nature 483(7388):182186.
21. Durand E, et al. (2015) Biogenesis and structure of a type VI secretion membrane core
complex. Nature 523(7562):555560.
22. Leiman PG, et al. (2009) Type VI secretion apparatus and phage tail-associated protein
complexes share a common evolutionary origin. Proc Natl Acad Sci USA 106(11):
41544159.
23. Silverman JM, et al. (2013) Haemolysin coregulated protein is an exported receptor
and chaperone of type VI secretion substrates. Mol Cell 51(5):584593.
24. Kapitein N, et al. (2013) ClpV recycles VipA/VipB tubules and prevents non-productive
tubule formation to ensure efficient type VI protein secretion. Mol Microbiol 87(5):
10131028.
25. Collins FM, Carter PB (1978) Growth of salmonellae in orally infected germfree mice.
Infect Immun 21(1):4147.
26. Endt K, et al. (2010) The microbiota mediates pathogen clearance from the gut lumen
after non-typhoidal Salmonella diarrhea. PLoS Pathog 6(9):e1001097.
27. Thiennimitr P, Winter SE, Bumler AJ (2012) Salmonella, the host and its microbiota.
Curr Opin Microbiol 15(1):108114.
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resolved by SDS/PAGE. After transfer onto nitrocellulose, the Hcp and Tae4
proteins were detected using monoclonal anti-5His antibody (QIAGEN) and
streptavidin-coupled to alkaline phosphatase (Molecular Probes, Life Technologies), respectively.
MICROBIOLOGY
Hcp1, Hcp2, and Tae4 Protein Purification. Plasmids encoding the Hcp proteins
fused to an N-terminal 6His tag or the Tae4 protein fused to a 6His-TRXTEV tag were obtained by restriction-free cloning as previously published
(68) using pairs ECO2217/ECO2219 (Hcp1), ECO2218/ECO2219 (Hcp2), and
ECO2220/ECO2221 (Tae4). The 6His-tagged Hcp proteins were purified by
ion metal affinity chromatography (IMAC) from 0.5 L of culture of E. coli
BL21(DE3) cells bearing the pRSF-1 plasmid derivatives grown at 37 C to an
OD600 = 0.6 and gene induction with 500 M IPTG for 18 h at 16 C. Bacteria
were harvested, resuspended to an OD600 = 80 in buffer A (50 mM TrisHCl,
pH 8.0, 150 mM NaCl) supplemented with 1 mM EDTA, 100 g/mL lysozyme,
100 g/mL DNase, 10 mM MgCl2, and protease inhibitors (Complete; Roche).
Bacteria were broken using an Emulsiflex apparatus, and the insoluble
material was discarded by centrifugation for 30 min at 55,000 g. All of the
subsequent purification steps were performed using an AKTA FPLC system.
First, the soluble fraction was loaded into a 5-mL HisTrap column (GE Health
Sciences). After extensive washing with a 0- to 20-mM gradient of imidazole,
the Hcp proteins were eluted using 500 mM imidazole in buffer A. The
pooled fractions were dialyzed overnight at 4 C on 3,500-Da pore membrane tubing (Spectra/Por; Spectrumlabs) in buffer A supplemented with
10 mM imidazole. The 6His-TRX-TEVtagged Tae4 protein was purified
using an identical protocol, except that purified 6His-tagged tobacco etch
virus (TEV) protease and 1.2 mM dithiotreitol were added in the membrane
tubing during dialysis. The cleaved, untagged Tae4 protein was obtained in
the flow-through of a second IMAC.
28. Behnsen J, et al. (2014) The cytokine IL-22 promotes pathogen colonization by suppressing related commensal bacteria. Immunity 40(2):262273.
29. Liu JZ, et al. (2012) Zinc sequestration by the neutrophil protein calprotectin enhances
Salmonella growth in the inflamed gut. Cell Host Microbe 11(3):227239.
30. Raffatellu M, et al. (2009) Lipocalin-2 resistance confers an advantage to Salmonella
enterica serotype Typhimurium for growth and survival in the inflamed intestine. Cell
Host Microbe 5(5):476486.
31. Stecher B, et al. (2012) Gut inflammation can boost horizontal gene transfer between
pathogenic and commensal Enterobacteriaceae. Proc Natl Acad Sci USA 109(4):
12691274.
32. Winter SE, et al. (2010) Gut inflammation provides a respiratory electron acceptor for
Salmonella. Nature 467(7314):426429.
33. Blondel CJ, Jimnez JC, Contreras I, Santiviago CA (2009) Comparative genomic
analysis uncovers 3 novel loci encoding type six secretion systems differentially distributed in Salmonella serotypes. BMC Genomics 10:354.
34. Russell AB, et al. (2012) A widespread bacterial type VI secretion effector superfamily
identified using a heuristic approach. Cell Host Microbe 11(5):538549.
35. Benz J, Reinstein J, Meinhart A (2013) Structural insights into the effector-immunity
system Tae4/Tai4 from Salmonella typhimurium. PLoS One 8(6):e67362.
36. Brunet YR, et al. (2015) H-NS silencing of the Salmonella pathogenicity island
6-encoded type VI secretion system limits Salmonella enterica serovar Typhimurium
interbacterial killing. Infect Immun 83(7):27382750.
37. Lam LH, Monack DM (2014) Intraspecies competition for niches in the distal gut
dictate transmission during persistent Salmonella infection. PLoS Pathog 10(12):
e1004527.
38. Miyata ST, Bachmann V, Pukatzki S (2013) Type VI secretion system regulation as a
consequence of evolutionary pressure. J Med Microbiol 62(Pt 5):663676.
39. Silverman JM, Brunet YR, Cascales E, Mougous JD (2012) Structure and regulation of
the type VI secretion system. Annu Rev Microbiol 66:453472.
40. Sana TG, Soscia C, Tonglet CM, Garvis S, Bleves S (2013) Divergent control of two type
VI secretion systems by RpoN in Pseudomonas aeruginosa. PLoS One 8(10):e76030.
41. Bachmann V, et al. (2015) Bile salts modulate the mucin-activated type VI secretion
system of pandemic Vibrio cholerae. PLoS Negl Trop Dis 9(8):e0004031.
42. Durand E, Cambillau C, Cascales E, Journet L (2014) VgrG, Tae, Tle, and beyond: The
versatile arsenal of type VI secretion effectors. Trends Microbiol 22(9):498507.
43. Shneider MM, et al. (2013) PAAR-repeat proteins sharpen and diversify the type VI
secretion system spike. Nature 500(7462):350353.
44. Unterweger D, et al. (2015) Chimeric adaptor proteins translocate diverse type VI
secretion system effectors in Vibrio cholerae. EMBO J 34(16):21982210.
45. Flaugnatti N, et al. (2016) A phospholipase A1 antibacterial Type VI secretion effector
interacts directly with the C-terminal domain of the VgrG spike protein for delivery.
Mol Microbiol 99(6):10991118.
46. Gulati AS, et al. (2012) Mouse background strain profoundly influences Paneth cell
function and intestinal microbial composition. PLoS One 7(2):e32403.
47. Willing BP, Russell SL, Finlay BB (2011) Shifting the balance: Antibiotic effects on hostmicrobiota mutualism. Nat Rev Microbiol 9(4):233243.
48. Ma AT, Mekalanos JJ (2010) In vivo actin cross-linking induced by Vibrio cholerae type
VI secretion system is associated with intestinal inflammation. Proc Natl Acad Sci USA
107(9):43654370.
49. Pukatzki S, et al. (2006) Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci
USA 103(5):15281533.
50. Basler M, Ho BT, Mekalanos JJ (2013) Tit-for-tat: Type VI secretion system counterattack during bacterial cell-cell interactions. Cell 152(4):884894.
51. Brunet YR, Espinosa L, Harchouni S, Mignot T, Cascales E (2013) Imaging type VI
secretion-mediated bacterial killing. Cell Reports 3(1):3641.
8 of 8 | www.pnas.org/cgi/doi/10.1073/pnas.1608858113
52. Brunet YR, Hnin J, Celia H, Cascales E (2014) Type VI secretion and bacteriophage tail
tubes share a common assembly pathway. EMBO Rep 15(3):315321.
53. Mougous JD, et al. (2006) A virulence locus of Pseudomonas aeruginosa encodes a
protein secretion apparatus. Science 312(5779):15261530.
54. Zhou Y, et al. (2012) Hcp family proteins secreted via the type VI secretion system
coordinately regulate Escherichia coli K1 interaction with human brain microvascular
endothelial cells. Infect Immun 80(3):12431251.
55. Gopinath S, Lichtman JS, Bouley DM, Elias JE, Monack DM (2014) Role of diseaseassociated tolerance in infectious superspreaders. Proc Natl Acad Sci USA 111(44):
1578015785.
56. Faber F, et al. (2016) Host-mediated sugar oxidation promotes post-antibiotic pathogen expansion. Nature 534(7609):697699.
57. Zhang H, et al. (2013) Structure of the type VI effector-immunity complex (Tae4-Tai4)
provides novel insights into the inhibition mechanism of the effector by its immunity
protein. J Biol Chem 288(8):59285939.
58. Chow J, Mazmanian SK (2010) A pathobiont of the microbiota balances host colonization and intestinal inflammation. Cell Host Microbe 7(4):265276.
59. Lertpiriyapong K, et al. (2012) Campylobacter jejuni type VI secretion system: Roles in
adaptation to deoxycholic acid, host cell adherence, invasion, and in vivo colonization. PLoS One 7(8):e42842.
60. Fu Y, Waldor MK, Mekalanos JJ (2013) Tn-Seq analysis of Vibrio cholerae intestinal
colonization reveals a role for T6SS-mediated antibacterial activity in the host. Cell
Host Microbe 14(6):652663.
61. Russell AB, et al. (2014) A type VI secretion-related pathway in Bacteroidetes mediates
interbacterial antagonism. Cell Host Microbe 16(2):227236.
62. Coyne MJ, Roelofs KG, Comstock LE (2016) Type VI secretion systems of human gut
Bacteroidales segregate into three genetic architectures, two of which are contained
on mobile genetic elements. BMC Genomics 17(1):58.
63. Chatzidaki-Livanis M, Geva-Zatorsky N, Comstock LE (2016) Bacteroides fragilis type VI
secretion systems use novel effector and immunity proteins to antagonize human gut
Bacteroidales species. Proc Natl Acad Sci USA 113(13):36273632.
64. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97(12):66406645.
65. Maier L, et al. (2013) Microbiota-derived hydrogen fuels Salmonella typhimurium
invasion of the gut ecosystem. Cell Host Microbe 14(6):641651.
66. Karimova G, Pidoux J, Ullmann A, Ladant D (1998) A bacterial two-hybrid system
based on a reconstituted signal transduction pathway. Proc Natl Acad Sci USA 95(10):
57525756.
67. Battesti A, Bouveret E (2012) The bacterial two-hybrid system based on adenylate
cyclase reconstitution in Escherichia coli. Methods 58(4):325334.
68. Aschtgen MS, Bernard CS, De Bentzmann S, Lloubs R, Cascales E (2008) SciN is an
outer membrane lipoprotein required for type VI secretion in enteroaggregative
Escherichia coli. J Bacteriol 190(22):75237531.
69. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot.
Acta Crystallogr D Biol Crystallogr 66(Pt 4):486501.
70. Douzi B, et al. (2014) Crystal structure and self-interaction of the type VI secretion tailtube protein from enteroaggregative Escherichia coli. PLoS One 9(2):e86918.
71. Gully D, Bouveret E (2006) A protein network for phospholipid synthesis uncovered by
a variant of the tandem affinity purification method in Escherichia coli. Proteomics
6(1):282293.
72. Helaine S, et al. (2010) Dynamics of intracellular bacterial replication at the single cell
level. Proc Natl Acad Sci USA 107(8):37463751.
73. Marchesi JR, et al. (1998) Design and evaluation of useful bacterium-specific PCR
primers that amplify genes coding for bacterial 16S rRNA. Appl Environ Microbiol
64(2):795799.
Sana et al.