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Enteropathogenic Escherichia coli: identification of a gene cluster coding for bundle-forming pilus morphogenesis.

ABSTRACT: Sequence flanking the bfpA locus on the enteroadherent factor plasmid of the enteropathogenic Escherichia coli (EPEC) strain B171-8 (O111:NM) was obtained to identify genes that might be required for bundle-forming pilus (BFP) biosynthesis. Deletion experiments led to the identification of a contiguous cluster of at least 12 open reading frames, including bfpA, that could direct the synthesis of a morphologically normal BFP filament. Within the bfp gene cluster, we identified open reading frames that share homology with other type IV pilus accessory genes and with genes required for transformation competence and protein secretion. Immediately upstream of the bfp gene cluster, we identified a potential replication origin including genes that are predicted to encode proteins homologous with replicase and resolvase. Restriction fragment length polymorphism analysis of DNA from six additional EPEC serotypes showed that the organization of the bfp gene cluster and its juxtaposition with a potential plasmid origin of replication are highly conserved features of the EPEC biotype.

SUBMITTER: Sohel I

PROVIDER: S-EPMC177987 | biostudies-other | 1996 May

REPOSITORIES: biostudies-other

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Enteropathogenic Escherichia coli: identification of a gene cluster coding for bundle-forming pilus morphogenesis.

Sohel I I Puente J L JL Ramer S W SW Bieber D D Wu C Y CY Schoolnik G K GK

Journal of bacteriology 19960501 9

Sequence flanking the bfpA locus on the enteroadherent factor plasmid of the enteropathogenic Escherichia coli (EPEC) strain B171-8 (O111:NM) was obtained to identify genes that might be required for bundle-forming pilus (BFP) biosynthesis. Deletion experiments led to the identification of a contiguous cluster of at least 12 open reading frames, including bfpA, that could direct the synthesis of a morphologically normal BFP filament. Within the bfp gene cluster, we identified open reading frames ...[more]

PMID: 8626330

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Project description:Although the bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) mediates microcolony formation on epithelial cells, the adherence of BFP-deficient mutants is significantly abrogated, but the mutants are still adherent due to the presence of intimin and possibly other adhesins. In this study we investigated the contribution of the recently described E. coli common pilus (ECP) to the overall adherence properties of EPEC. We found that ECP and BFP structures can be simultaneously observed in the course (between zero time and 7 h during infection) of formation of localized adherence on cultured epithelial cells. These two pilus types colocalized at different levels of the microcolony topology, tethering the adhering bacteria. No evidence of BFP disappearance was found after prolonged infection. When expressed from a plasmid present in nonadherent E. coli HB101, ECP rendered this organism highly adherent at levels comparable to those of HB101 expressing the BFP. Purified ECP bound in a dose-dependent manner to epithelial cells, and the binding was blocked with anti-ECP antibodies, confirming that the pili possess adhesin properties. An ECP mutant showed only a modest reduction in adherence to cultured cells due to background expression levels of BFP and intimin. However, isogenic mutants not expressing EspA or BFP were significantly less adherent when the ecpA gene was also deleted. Furthermore, a DeltaespA DeltaecpA double mutant (unable to translocate Tir and to establish intimate adhesion) was at least 10-fold less adherent than the DeltaespA and DeltaecpA single mutants, even in the presence of BFP. A Delta bfp DeltaespA DeltaecpA triple mutant showed the least adherence compared to the wild type and all the isogenic mutant strains tested, suggesting that ECP plays a synergistic role in adherence. Our data indicate that ECP is an accessory factor that, in association with BFP and other adhesins, contributes to the multifactorial complex interaction of EPEC with host epithelial cells.

Project description:Type 4 pili (T4P) are retractable surface appendages found on numerous bacteria and archaea that play essential roles in various microbial functions, including host colonization by pathogens. An ATPase is required for T4P extension, but the mechanism by which chemical energy is transduced to mechanical energy for pilus extension has not been elucidated. Here, we report the cryo-electron microscopy (cryo-EM) structure of the BfpD ATPase from enteropathogenic Escherichia coli (EPEC) in the presence of either ADP or a mixture of ADP and AMP-PNP. Both structures, solved at 3 Å resolution, reveal the typical toroid shape of AAA+ ATPases and unambiguous 6-fold symmetry. This 6-fold symmetry contrasts with the 2-fold symmetry previously reported for other T4P extension ATPase structures, all of which were from thermophiles and solved by crystallography. In the presence of the nucleotide mixture, BfpD bound exclusively AMP-PNP, and this binding resulted in a modest outward expansion in comparison to the structure in the presence of ADP, suggesting a concerted model for hydrolysis. De novo molecular models reveal a partially open configuration of all subunits where the nucleotide binding site may not be optimally positioned for catalysis. ATPase functional studies reveal modest activity similar to that of other extension ATPases, while calculations indicate that this activity is insufficient to power pilus extension. Our results reveal that, despite similarities in primary sequence and tertiary structure, T4P extension ATPases exhibit divergent quaternary configurations. Our data raise new possibilities regarding the mechanism by which T4P extension ATPases power pilus formation. IMPORTANCE Type 4 pili are hairlike surface appendages on many bacteria and archaea that can be extended and retracted with tremendous force. They play a critical role in disease caused by several deadly human pathogens. Pilus extension is made possible by an enzyme that converts chemical energy to mechanical energy. Here, we describe the three-dimensional structure of such an enzyme from a human pathogen in unprecedented detail, which reveals a mechanism of action that has not been seen previously among enzymes that power type 4 pilus extension.

Dataset Information

Enteropathogenic Escherichia coli: identification of a gene cluster coding for bundle-forming pilus morphogenesis.

Publications

Enteropathogenic Escherichia coli: identification of a gene cluster coding for bundle-forming pilus morphogenesis.

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