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Bacterial surface appendages strongly impact nanomechanical and electrokinetic properties of Escherichia coli cells subjected to osmotic stress.

Francius G, Polyakov P, Merlin J, Abe Y, Ghigo JM, Merlin C, Beloin C, Duval JF - PLoS ONE (2011)

Bottom Line: Additionally, for a given surface appendage, the magnitude of the nanomechanical parameters decreases significantly when increasing bulk salt concentration.This effect is ascribed to a bacterial exoosmotic water loss resulting in a combined contraction of bacterial cytoplasm together with an electrostatically-driven shrinkage of the surface appendages.Altogether, AFM and electrokinetic results clearly demonstrate the intimate relationship between structure/flexibility and charge of bacterial envelope and propensity of bacterium and surface appendages to contract under hypertonic conditions.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, Nancy Université, CNRS UMR7564, Villers-lès-Nancy, France. gregory.francius@lcpme.cnrs-nancy.fr

ABSTRACT
The physicochemical properties and dynamics of bacterial envelope, play a major role in bacterial activity. In this study, the morphological, nanomechanical and electrohydrodynamic properties of Escherichia coli K-12 mutant cells were thoroughly investigated as a function of bulk medium ionic strength using atomic force microscopy (AFM) and electrokinetics (electrophoresis). Bacteria were differing according to genetic alterations controlling the production of different surface appendages (short and rigid Ag43 adhesins, longer and more flexible type 1 fimbriae and F pilus). From the analysis of the spatially resolved force curves, it is shown that cells elasticity and turgor pressure are not only depending on bulk salt concentration but also on the presence/absence and nature of surface appendage. In 1 mM KNO(3), cells without appendages or cells surrounded by Ag43 exhibit large Young moduli and turgor pressures (∼700-900 kPa and ∼100-300 kPa respectively). Under similar ionic strength condition, a dramatic ∼50% to ∼70% decrease of these nanomechanical parameters was evidenced for cells with appendages. Qualitatively, such dependence of nanomechanical behavior on surface organization remains when increasing medium salt content to 100 mM, even though, quantitatively, differences are marked to a much smaller extent. Additionally, for a given surface appendage, the magnitude of the nanomechanical parameters decreases significantly when increasing bulk salt concentration. This effect is ascribed to a bacterial exoosmotic water loss resulting in a combined contraction of bacterial cytoplasm together with an electrostatically-driven shrinkage of the surface appendages. The former process is demonstrated upon AFM analysis, while the latter, inaccessible upon AFM imaging, is inferred from electrophoretic data interpreted according to advanced soft particle electrokinetic theory. Altogether, AFM and electrokinetic results clearly demonstrate the intimate relationship between structure/flexibility and charge of bacterial envelope and propensity of bacterium and surface appendages to contract under hypertonic conditions.

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Spatial organization of the external structures for the various E coli strains investigated.Pili and fimbriae are structures with reported total lengths of about ∼10-100 µm (E2302) and ∼1-10 µm (E2146), respectively, while antigen Ag43 protein layer is a short and rigid structure ∼10 nm thick (E2498).
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pone-0020066-g001: Spatial organization of the external structures for the various E coli strains investigated.Pili and fimbriae are structures with reported total lengths of about ∼10-100 µm (E2302) and ∼1-10 µm (E2146), respectively, while antigen Ag43 protein layer is a short and rigid structure ∼10 nm thick (E2498).

Mentions: The E. coli K-12 strains used in this study are listed in Table 1 and Table 2 where relevant information on their respective construction, antibiotic resistance, genotype and surface appendages expression can be found. These isogenic strains were constructed from Escherichia coli MG1655 (E. coli genetic stock center CGSC#6300) by transformation and λ red linear DNA gene inactivation method using the pKOBEG plasmid [49], [50], followed by P1vir transduction into a fresh E. coli background when possible. Alternatively, strains were constructed by P1vir transduction of previously constructed and characterized mutation or insertion. All strains used in this study contain the gfpmut3 gene linked to the bla ampicillin resistance gene (ampR, 100 µg/ml) that makes them fluorescent, and a deletion of the fliE to fliR genes replaced by the cat chloramphenicol resistance gene (cmR, 25 µg/ml), which ensures the absence of flagella. Absence of flagella was verified by absence of motility using motility assay on low agar motility plates (data not shown). Our reference strain (E2152) has been constructed by creation and P1vir transduction of mutations previously shown to i/abolish type 1 fimbriae production (deletion of the type 1 fimbriae encoding operon, ΔfimA-H: zeo, zeoR 50 µg/ml), or ii/abolish adhesin Ag43 production (deletion of the flu gene, Δflu: km, kmR 100 µg/ml or Δflu: zeo, zeoR 50 µg/ml). Primers used to construct the ΔfliE-R: cat, Δflu: km, Δflu: zeo and the ΔfimA-H: zeo deletions are listed in Table 1. The strain E2152, devoid of these 3 surface appendages, was selected as a reference for comparing AFM data and electrokinetic results with those obtained for the strains E2146, E2498 and E2302 which constitutively produces the external ultrastructure type 1 fimbriae, the Ag43 protein and the type-F pili, respectively (Fig 1). Constitutive expression of the Ag43 protein was previously obtained by placing a constitutive promoter in front of the flu gene [12] whereas constitutive expression of the type 1 fimbriae was previously obtained by placing a constitutive promoter in front of the fimA-H operon [51]. Production of the F pili was obtained by introducing the F'tet plasmid into the reference strain E2152 by conjugation, creating E2302. All constructions were verified by PCR. The E2152, E2146, E2302 and E2498 strains deleted for- or selectively expressing- the three different appendages listed above were phenotypically checked before proceeding with the experiments reported in this study. All three strains displayed phenotypes in line with previous description [12], [47], [52]. In details, we systematically verified that i/absence and constitutive production of type 1 fimbriae were respectively verified by deficiency in- or enhancement of- yeast agglutination and biofilm formation; ii/absence and constitutive production of the Ag43 adhesin were respectively verified by deficiency in- or enhancement of- autoaggregation as well as by Western Blot immunodetection; iii/presence of the F pili was verified by sensitivity to phage M13 and increased biofilm formation (Table S1).10.1371/journal.pone.0020066.g001Figure 1Spatial organization of the external structures for the various E coli strains investigated.


Bacterial surface appendages strongly impact nanomechanical and electrokinetic properties of Escherichia coli cells subjected to osmotic stress.

Francius G, Polyakov P, Merlin J, Abe Y, Ghigo JM, Merlin C, Beloin C, Duval JF - PLoS ONE (2011)

Spatial organization of the external structures for the various E coli strains investigated.Pili and fimbriae are structures with reported total lengths of about ∼10-100 µm (E2302) and ∼1-10 µm (E2146), respectively, while antigen Ag43 protein layer is a short and rigid structure ∼10 nm thick (E2498).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105017&req=5

pone-0020066-g001: Spatial organization of the external structures for the various E coli strains investigated.Pili and fimbriae are structures with reported total lengths of about ∼10-100 µm (E2302) and ∼1-10 µm (E2146), respectively, while antigen Ag43 protein layer is a short and rigid structure ∼10 nm thick (E2498).
Mentions: The E. coli K-12 strains used in this study are listed in Table 1 and Table 2 where relevant information on their respective construction, antibiotic resistance, genotype and surface appendages expression can be found. These isogenic strains were constructed from Escherichia coli MG1655 (E. coli genetic stock center CGSC#6300) by transformation and λ red linear DNA gene inactivation method using the pKOBEG plasmid [49], [50], followed by P1vir transduction into a fresh E. coli background when possible. Alternatively, strains were constructed by P1vir transduction of previously constructed and characterized mutation or insertion. All strains used in this study contain the gfpmut3 gene linked to the bla ampicillin resistance gene (ampR, 100 µg/ml) that makes them fluorescent, and a deletion of the fliE to fliR genes replaced by the cat chloramphenicol resistance gene (cmR, 25 µg/ml), which ensures the absence of flagella. Absence of flagella was verified by absence of motility using motility assay on low agar motility plates (data not shown). Our reference strain (E2152) has been constructed by creation and P1vir transduction of mutations previously shown to i/abolish type 1 fimbriae production (deletion of the type 1 fimbriae encoding operon, ΔfimA-H: zeo, zeoR 50 µg/ml), or ii/abolish adhesin Ag43 production (deletion of the flu gene, Δflu: km, kmR 100 µg/ml or Δflu: zeo, zeoR 50 µg/ml). Primers used to construct the ΔfliE-R: cat, Δflu: km, Δflu: zeo and the ΔfimA-H: zeo deletions are listed in Table 1. The strain E2152, devoid of these 3 surface appendages, was selected as a reference for comparing AFM data and electrokinetic results with those obtained for the strains E2146, E2498 and E2302 which constitutively produces the external ultrastructure type 1 fimbriae, the Ag43 protein and the type-F pili, respectively (Fig 1). Constitutive expression of the Ag43 protein was previously obtained by placing a constitutive promoter in front of the flu gene [12] whereas constitutive expression of the type 1 fimbriae was previously obtained by placing a constitutive promoter in front of the fimA-H operon [51]. Production of the F pili was obtained by introducing the F'tet plasmid into the reference strain E2152 by conjugation, creating E2302. All constructions were verified by PCR. The E2152, E2146, E2302 and E2498 strains deleted for- or selectively expressing- the three different appendages listed above were phenotypically checked before proceeding with the experiments reported in this study. All three strains displayed phenotypes in line with previous description [12], [47], [52]. In details, we systematically verified that i/absence and constitutive production of type 1 fimbriae were respectively verified by deficiency in- or enhancement of- yeast agglutination and biofilm formation; ii/absence and constitutive production of the Ag43 adhesin were respectively verified by deficiency in- or enhancement of- autoaggregation as well as by Western Blot immunodetection; iii/presence of the F pili was verified by sensitivity to phage M13 and increased biofilm formation (Table S1).10.1371/journal.pone.0020066.g001Figure 1Spatial organization of the external structures for the various E coli strains investigated.

Bottom Line: Additionally, for a given surface appendage, the magnitude of the nanomechanical parameters decreases significantly when increasing bulk salt concentration.This effect is ascribed to a bacterial exoosmotic water loss resulting in a combined contraction of bacterial cytoplasm together with an electrostatically-driven shrinkage of the surface appendages.Altogether, AFM and electrokinetic results clearly demonstrate the intimate relationship between structure/flexibility and charge of bacterial envelope and propensity of bacterium and surface appendages to contract under hypertonic conditions.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, Nancy Université, CNRS UMR7564, Villers-lès-Nancy, France. gregory.francius@lcpme.cnrs-nancy.fr

ABSTRACT
The physicochemical properties and dynamics of bacterial envelope, play a major role in bacterial activity. In this study, the morphological, nanomechanical and electrohydrodynamic properties of Escherichia coli K-12 mutant cells were thoroughly investigated as a function of bulk medium ionic strength using atomic force microscopy (AFM) and electrokinetics (electrophoresis). Bacteria were differing according to genetic alterations controlling the production of different surface appendages (short and rigid Ag43 adhesins, longer and more flexible type 1 fimbriae and F pilus). From the analysis of the spatially resolved force curves, it is shown that cells elasticity and turgor pressure are not only depending on bulk salt concentration but also on the presence/absence and nature of surface appendage. In 1 mM KNO(3), cells without appendages or cells surrounded by Ag43 exhibit large Young moduli and turgor pressures (∼700-900 kPa and ∼100-300 kPa respectively). Under similar ionic strength condition, a dramatic ∼50% to ∼70% decrease of these nanomechanical parameters was evidenced for cells with appendages. Qualitatively, such dependence of nanomechanical behavior on surface organization remains when increasing medium salt content to 100 mM, even though, quantitatively, differences are marked to a much smaller extent. Additionally, for a given surface appendage, the magnitude of the nanomechanical parameters decreases significantly when increasing bulk salt concentration. This effect is ascribed to a bacterial exoosmotic water loss resulting in a combined contraction of bacterial cytoplasm together with an electrostatically-driven shrinkage of the surface appendages. The former process is demonstrated upon AFM analysis, while the latter, inaccessible upon AFM imaging, is inferred from electrophoretic data interpreted according to advanced soft particle electrokinetic theory. Altogether, AFM and electrokinetic results clearly demonstrate the intimate relationship between structure/flexibility and charge of bacterial envelope and propensity of bacterium and surface appendages to contract under hypertonic conditions.

Show MeSH
Related in: MedlinePlus