Escherichia coli tol-pal Mutants Form Outer Membrane Vesicles

ABSTRACT Mutations in the tol-pal genes induce pleiotropic effects such as release of periplasmic proteins into the extracellular medium and hypersensitivity to drugs and detergents. Other outer membrane defective strains such as tolC, lpp, and rfa mutations are also altered in their outer membrane permeability. In this study, electron microscopy and Western blot analyses were used to show that strains with mutations in each of thetol-pal genes formed outer membrane vesicles after growth in standard liquid or solid media. This phenotype was not observed intolC and rfaD cells in the same conditions. AtolA deletion in three different Escherichia coli strains was shown to lead to elevated amounts of vesicles. These results, together with plasmid complementation experiments, indicated that the formation of vesicles resulted from the defect of any of the Tol-Pal proteins. The vesicles contained outer membrane trimeric porins correctly exposed at the cell surface. Pal outer membrane lipoprotein was also immunodetected in the vesicle fraction oftol strains. The results are discussed in view of the role of the Tol-Pal transenvelope proteins in maintaining outer membrane integrity by contributing to target or integrate newly synthesized components of this structure.

[1]  R. E. Webster,et al.  TolA: a membrane protein involved in colicin uptake contains an extended helical region. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[2]  J. Hearst,et al.  Molecular cloning and characterization of acrA and acrE genes of Escherichia coli , 1993, Journal of bacteriology.

[3]  K. Postle,et al.  TonB protein appears to transduce energy by shuttling between the cytoplasmic membrane and the outer membrane in Escherichia coli , 1997, Molecular microbiology.

[4]  T. Beveridge,et al.  Virulence factors are released from Pseudomonas aeruginosa in association with membrane vesicles during normal growth and exposure to gentamicin: a novel mechanism of enzyme secretion , 1995, Journal of bacteriology.

[5]  M H Saier,et al.  A family of extracytoplasmic proteins that allow transport of large molecules across the outer membranes of gram-negative bacteria , 1994, Journal of bacteriology.

[6]  T. Ogura,et al.  Chromosome partitioning in Escherichia coli: novel mutants producing anucleate cells , 1989, Journal of bacteriology.

[7]  R. Lloubès,et al.  Binding of colicins A and El to purified TolA domains. , 1997, Microbiology.

[8]  J. Lazzaroni,et al.  The excC gene of Escherichia coli K‐12 required for cell envelope integrity encodes the peptidoglycan‐associated lipoprotein (PAL) , 1992, Molecular microbiology.

[9]  E. Bouveret,et al.  Peptidoglycan-associated lipoprotein-TolB interaction. A possible key to explaining the formation of contact sites between the inner and outer membranes of Escherichia coli , 1995, The Journal of Biological Chemistry.

[10]  V. Braun.  Energy-coupled transport and signal transduction through the gram-negative outer membrane via TonB-ExbB-ExbD-dependent receptor proteins. , 1995, FEMS microbiology reviews.

[11]  E. Bouveret,et al.  Distinct regions of the colicin A translocation domain are involved in the interaction with TolA and TolB proteins upon import into Escherichia coli , 1998, Molecular microbiology.

[12]  R. Hancock,et al.  The bacterial outer membrane as a drug barrier. , 1997, Trends in microbiology.

[13]  M. Hofnung,et al.  Further genetic analysis of the C-terminal external loop region in Escherichia coli maltoporin. , 1997, Research in microbiology.

[14]  J. Popot,et al.  Transmembrane alpha-helix interactions are required for the functional assembly of the Escherichia coli Tol complex. , 1995, Journal of molecular biology.

[15]  K.,et al.  Genetics of resistance to colicins in Escherichia coli K-12: cross-resistance among colicins of group A , 1975, Journal of bacteriology.

[16]  J. Lazzaroni,et al.  Protein complex within Escherichia coli inner membrane. TolA N-terminal domain interacts with TolQ and TolR proteins , 1995, The Journal of Biological Chemistry.

[17]  R. E. Webster,et al.  Characterization of the tol-pal region of Escherichia coli K-12: translational control of tolR expression by TolQ and identification of a new open reading frame downstream of pal encoding a periplasmic protein , 1996, Journal of bacteriology.

[18]  R. E. Webster,et al.  Membrane topology and mutational analysis of the TolQ protein of Escherichia coli required for the uptake of macromolecules and cell envelope integrity , 1994, Journal of bacteriology.

[19]  L. Rothfield,et al.  Role of murein lipoprotein in morphogenesis of the bacterial division septum: phenotypic similarity of lkyD and lpo mutants , 1978, Journal of bacteriology.

[20]  P. Delepelaire,et al.  TolC, an Escherichia coli outer membrane protein required for hemolysin secretion. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[21]  P. Reeves,et al.  Genetics of resistance to colicins in Escherichia coli K-12: cross-resistance among colicins of group B , 1975, Journal of bacteriology.

[22]  S. Létoffé,et al.  Involvement of lipopolysaccharide in the secretion of Escherichia coli α haemolysin and Erwinia chrysanthemi proteases , 1993, Molecular microbiology.

[23]  C. Georgopoulos,et al.  The htrM gene, whose product is essential for Escherichia coli viability only at elevated temperatures, is identical to the rfaD gene. , 1991, Nucleic acids research.

[24]  E. Bouveret,et al.  The TolB protein interacts with the porins of Escherichia coli , 1997, Journal of bacteriology.

[25]  R. E. Webster,et al.  fii, a bacterial locus required for filamentous phage infection and its relation to colicin-tolerant tolA and tolB , 1986, Journal of bacteriology.

[26]  V. Braun,et al.  Import of biopolymers into Escherichia coli: nucleotide sequences of the exbB and exbD genes are homologous to those of the tolQ and tolR genes, respectively , 1989, Journal of bacteriology.

[27]  B. Ahmer,et al.  Energy transduction between membranes. TonB, a cytoplasmic membrane protein, can be chemically cross-linked in vivo to the outer membrane receptor FepA. , 1993, The Journal of biological chemistry.

[28]  R. E. Webster,et al.  Role of the carboxyl-terminal domain of TolA in protein import and integrity of the outer membrane , 1993, Journal of bacteriology.

[29]  V. Braun,et al.  Membrane topologies of the TolQ and TolR proteins of Escherichia coli: inactivation of TolQ by a missense mutation in the proposed first transmembrane segment , 1993, Journal of bacteriology.

[30]  P. Reeves,et al.  Role of micF in the tolC-mediated regulation of OmpF, a major outer membrane protein of Escherichia coli K-12 , 1987, Journal of bacteriology.

[31]  C. Georgopoulos,et al.  The rpoE gene encoding the sigma E (sigma 24) heat shock sigma factor of Escherichia coli. , 1995, The EMBO journal.

[32]  G. Storz,et al.  Small RNAs in Escherichia coli. , 1999, Trends in microbiology.

[33]  H. W. Veen,et al.  Handbook of Biological Physics , 1996 .

[34]  J. Lazzaroni,et al.  TolB protein of Escherichia coli K‐12 interacts with the outer membrane peptidoglycan‐associated proteins Pal, Lpp and OmpA , 1998, Molecular microbiology.

[35]  C. Higgins,et al.  A sequence‐specific function for the N‐terminal signal‐like sequence of the TonB protein , 1993, Molecular microbiology.

[36]  R. E. Webster.  The tol gene products and the import of macronmolecules into Escherichia coli , 1991, Molecular microbiology.

[37]  J. Lazzaroni,et al.  Membrane topology of the Escherichia coli TolR protein required for cell envelope integrity , 1993, Journal of bacteriology.

[38]  J. Klena,et al.  Genetics of lipopolysaccharide biosynthesis in enteric bacteria. , 1993, Microbiological reviews.

[39]  U. Henning,et al.  Cell envelope and shape of Escherichia coli: multiple mutants missing the outer membrane lipoprotein and other major outer membrane proteins , 1978, Journal of bacteriology.

[40]  C. Lazdunski,et al.  Protein import into Escherichia coli: colicins A and E1 interact with a component of their translocation system. , 1991, The EMBO journal.

[41]  M. Gavioli,et al.  TolA central domain interacts with Escherichia coli porins. , 1996, The EMBO journal.

[42]  S. Matsuyama,et al.  A novel periplasmic carrier protein involved in the sorting and transport of Escherichia coli lipoproteins destined for the outer membrane. , 1995, The EMBO journal.

[43]  R. Pettit,et al.  Characterization of naturally elaborated blebs from serum‐susceptible and serum‐resistant strains of Neisseria gonorrhoeae , 1992, Molecular microbiology.

[44]  S. Falkow,et al.  Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. , 1977, Gene.

[45]  J. Lazzaroni,et al.  Maturation and localization of the TolB protein required for colicin import , 1994, Journal of bacteriology.

[46]  P. Boulanger,et al.  Colicin A and the Tol proteins involved in its translocation are preferentially located in the contact sites between the inner and outer membranes of Escherichia coli cells. , 1994, The Journal of biological chemistry.

[47]  B. Witholt,et al.  Release of outer membrane fragments from wild-type Escherichia coli and from several E. coli lipopolysaccharide mutants by EDTA and heat shock treatments , 1989, Journal of bacteriology.

[48]  V. Géli,et al.  Chapter 29 colicin transport, channel formation and inhibition , 1996 .

[49]  J. Lazzaroni,et al.  Effects of lkyB mutations on the expression of ompF, ompC and lamB porin structural genes in Escherichia coli K‐12 , 1986 .

[50]  J. Hearst,et al.  Efflux pumps and drug resistance in gram-negative bacteria. , 1994, Trends in microbiology.

[51]  J. Betton,et al.  New components of protein folding in extracytoplasmic compartments of Escherichia coli SurA, FkpA and Skp/OmpH , 1996, Molecular microbiology.

[52]  J. Beckwith,et al.  An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[53]  H. C. Wu,et al.  Physiological characterization of an Escherichia coli mutant altered in the structure of murein lipoprotein , 1978, Journal of bacteriology.

[54]  N. Yokota,et al.  A novel outer membrane lipoprotein, LolB (HemM), involved in the LolA (p20)‐dependent localization of lipoproteins to the outer membrane of Escherichia coli , 1997, The EMBO journal.

[55]  J. Lazzaroni,et al.  Expression of the tolQRA genes of Escherichia coli K‐12 is controlled by the RcsC sensor protein involved in capsule synthesis , 1996, Molecular microbiology.

[56]  T. Mizuno,et al.  A novel peptidoglycan-associated lipoprotein found in the cell envelope of Pseudomonas aeruginosa and Escherichia coli. , 1979, Journal of biochemistry.

[57]  Joe A. FRALICKl,et al.  Additive effect of tolC and rfa mutations on the hydrophobic barrier of the outer membrane of Escherichia coli K-12 , 1994, Journal of bacteriology.

[58]  J. Fralick.  Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli , 1996, Journal of bacteriology.

[59]  R. E. Webster,et al.  Nucleotide sequence of a gene cluster involved in entry of E colicins and single-stranded DNA of infecting filamentous bacteriophages into Escherichia coli , 1987, Journal of bacteriology.