Oliver and Beckwith

Escherichia coli Mutant Pleiotropically Defective in the Export of Secreted Proteins

Donald B. Oliver and Jon Beckwith

Background information

There are 6 main secretary pathways in gram-negative bacteria:

  • Type I; Chaperone-dependent secretion system, the process begins as a leader sequence HlyA is recognized and binds HlyB on the membrane. This signal sequence is extremely specific for the ABC (ATP-binding cassette) transporter. It is also called ISP( type I secretory pathway)
  • Type II; Proteins secreted through the type II system, or main terminal branch of the general secretory pathway (SEC), depend on the Sec or Tat system for initial transport into the periplasm. It is also called IISP( type II secretory pathway)
  • Type III; It is homologous to the basal body in bacterial flagella. It is like a molecular syringe through which a bacterium can inject proteins into eukaryotic cells. It is also called IIISP( type III secretory pathway.
  • Type IV; it is homologous to conjugation machinery of bacteria. It is capable of transporting both DNA and proteins. It is also called IVSP( type IV secretory pathway)
  • Type V; also called the autotransporter system, type V secretion involves the use of the Sec system for crossing the inner membrane. . It is also called VSP( type V secretory pathway)
  • Type VI; identified in 2006

There are many outer and inner membrane proteins that cause channel-forming in gram-negative bacteria. Gram-negative bacterial inner membrane channel-forming translocases

  1. ATP-binding cassette translocase (ATP(ISP))
  2. General secretary translocase (SEC(IISP))
  3. Flagellum/ virulence-related translocase (Fla/ Path (IIISP))
  4. Conjugation related translocase (Conj(IVSP))
  5. Twin-arginine targeting translocase (Tat (IISP))
  6. Cytochrome oxidase biogenesis family (Oxa1(YidC))
  7. Large conductance mechanosensitive channel family (MscL)
  8. Holing functional superfamily (Holins)

Gram-negative bacterial outer membrane channel- forming translocases;

  1. Main terminal branch of the general secretory translocase (MTB(IISP))
  2. Fimbrial usher protein autotransporter-1 (FUP)
  3. Autotransporter –2 (AT-2)
  4. Outer membrane factor (OMF (ISP))
  5. Two-partner secretion (TPS)
  6. Secretin (IISP and IIISP)
  7. Outer membrane insertion porin (OmpIP)

Sec-dependent protein secretion (also called general secretion pathway)

The Sec machinery is an ensemble of proteins that facilitates the translocation of proteins and pre-proteins into and across biological membranes. This pathway is also similar in eukaryotes and archaea. This pathway translocates the proteins from the cytoplasm across or into the plasma membrane. Secreted proteins are initially synthesized as “preprotein” which contain an amino-terminal signal peptide. The translocase pathway comprises 7 proteins;

  • SecA; a motor protein that uses ATP as an energy source and threads the unfolded polypeptide through the channel.
  • SecB; chaperone protein, a highly acidic protein that exists in the bacterial cytoplasm. SecB maintains preproteins in an unfolded state after translation and targets these to the peripheral membrane protein ATPase SecA for secretion.
  • SecY, SecE, and SecG; integral membrane complex, the structure of the Escherichia coli SecYEG assembly revealed a sandwich of two membranes interacting through the extensive cytoplasmic domains.
  • SecD and SecF; additional membrane proteins that promote the release of the mature peptide into the periplasm.

Image result for sec pathway

Oliver and Beckwith research

Back at the 1970s most of the studies were about the proteins in the cytoplasm and inner membrane of E.coli. The further information was unknown about outer membrane or periplasm proteins and their translocation. According to the signal hypothesis proposed by Blobel; there was an N-terminal sequence at the end of the periplasmic and outer membrane proteins’ sequence which determine whether the protein will be secret or not. Later by this hypothesis was supported by Smith et al.

The study of genetic and protein translocation began in the late 1970s with the Beckwith group. These studies played an important role to identify the protein components that comprise the machinery and providing that the information for export is located within the signal sequence. This signal sequence was important for protein secretion but Oliver and Beckwith wanted to go further and see whether other regions of the peptide chain has a role in the  localization of secreted proteins. Briefly, they wanted to isolate and characterize the mutants that change the cell’s secretion machinery and result in pleiotropic effects on protein localization. They used an E.coli which harbors a hybrid β-galactosidase protein localized in the cytoplasmic membrane. This hybrid protein had different enzymatic properties when compared with the same protein which is located in the cytoplasm of E. coli. Beckwith and friends exploited fusion of the preprotein maltose binding protein (MBP) with cytosolic reporter β- galactosidase encoded by the lacZ gene which, when active, allows the cell to use lactose as a carbon source. PreMBP encoded by the malE gene is a periplasmic protein and is needed for the cell to use maltose as a carbon source. The MalE-LacZ fusion protein is toxic when expressed by the addition of maltose in E.coli; therefore, the cells were maltose sensitive. They used this as a discriminative method.

Isolation of mutants impaired in protein localization

They used E.coli strain (MM18) which harbors malE gene (this gene encodes maltose binding proteins) and fused to a lacZ gene which encodes β -galactosidase. This strain made a hybrid protein which changed the N-terminus of β-galactosidase to the large N terminus of maltose binding protein (MBP) but still retained the β -galactosidase activity. β -galactosidase was normally cytoplasmic and MBP was periplasmic. This hybrid protein was found in the cytoplasmic membrane. They assumed that this hybrid protein was bigger to pass through the cytoplasmic membrane and somehow blocked the secretion process which caused the cytoplasmic accumulation of the hybrid protein.

They found a significant enzymatic difference of the hybrid β-galactosidase when they are cytoplasm (MM7) or membrane associated (MM18). To discriminate β-galactosidase activity of such mutants (MM7 and MM18) they used lactose tetrazolium agar and MM7 gave red (Lac+), MM18 (Lac -) gave colorless cells. Red MM7 cells were picked, purified and tested for their phenotypes on various media at 30°C and 40°C. Two mutants out of 80 were screened MM49 and MM51 were temperature sensitive (ts). They applied malE-lacZ fusion, all derivatives kept being ts. So, they concluded that being ts was not related to the fusion. By several maltose-containing agar growth experiments for all mutants, they concluded that the mutants maintained their sensitivity to maltose while having intermediate β- galactosidase activities. They also determined whether the ts and β-galactosidase activity were due to a single mutation.

A defect in secretion of maltose-binding protein

They tasted the old and newly synthesized MBP by radioactively labeled (pulse labeled) methionine at different temperatures (30°C, 37°C and 42°C) and time intervals (fig.1 and fig.2). By the several experiments they run, they found that MM52 codes for a ts processing enzyme worked slower or persistent in reduced amount of permissive temp which cause the conclusion of MBP precursor present after a short pulse of 37°C is stable for up to 20 min during which time of the half of the newly synthesized MBP precursor was matured during a 20 sec pulse (fig.3) MM52 synthesized some stable MBP which is not subsequently processed. Moreover, MM51 was found in the cytoplasm after pulse-labelling of the protein at 37°C.

A defect of secretion of other exported proteins; they identified a precursor of another outer membrane protein (an ompF gene product) accumulating in MM52. Since MM52 accumulates several periplasmic and outer membrane proteins the thought it seemed possible it was defective in the transport of all secreted proteins. To understand this, they pulse labeled periplasmic fraction of mutant and wild type by cold osmotic shock method and EDTA-lysozyme method. They saw that many proteins were in common for both types except 3-4 proteins were only found in wildtype especially the amount of MBP was more than founded the mutant type.

Genetic mapping of the TS locus; they used Hfr strains to determine the location of ts51 mutation. After determining its locus, they wanted to map it by using Tn5 and Tn10 insertions. As a result, of this experiment, they concluded that this mutant gene was in the 2 to 4 min region of E. coli chromosome. At that suspected location there were ftsQ, ftsA, ftsZ and envA genes with similar phenotypes. However the one that they found was lambda specialized transducing phage which is a complement the ts51 mutation, and this locus was not previously described. They called this gene as secA.


Gizem Levent

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