Also: Science (Aug. 30, 1996) Vol. 273 pages 1183-1184; 1231-1236. Role of cell-to-cell contact and Type III secretion apparatus in pathogenesis

Gram-Negative Bacteria have 5 cellular compartments to assemble (G+ have 3):

1. Cytoplasm

2. Cytoplasmic Membrane

3. Periplasmic Space/Peptidoglycan

4. Outer Membrane

5. Exterior

How are proteins, lipids, and polysaccharides directed to their proper location?

Secretion: 3 systems.

Type I = GEP (General Export Pathway = Sec system) + GSP (General Secretory Pathway)

Type II = ABC TRANSPORTERS (not Sec dependent)

Type III = Flagella Export System and related pathogenesis systems (not Sec dependent)

Cytoplasmic membrane contains several types of phospholipids and ~200-300 proteins

Phospholipid insertion takes place at inner leaflet of cytoplasmic membrane; translocation then occurs to outer leaflet. May not require energy (Energy IS required in eucaryotes, where an ATPase is used to establish lipid asymmetry).

Primary structure (i.e., sequence) of protein determines its location (cytoplasm, membrane, etc.).

Lipid insertion and distribution to daughter cells not well understood. Most evidence suggests that lipids and proteins are inserted at multiple sites in the membrane. Protein-protein and protein-lipid interactions can lead to differentiation, however. Example: ICM/thylakoid formation in phototrophs.

All membrane proteins have hydrophobic domains inserted in to the lipid bilayer. The most common structural motif is trans-membrane a-helices that pass through the membrane perpendicular to its plane (or at a slight angle of up to 30° relative to the plane). One helix is sufficient to anchor a protein to the membrane; likewise, attachement of a single fatty acyl group can anchor a protein to a membrane.

As for other proteins, can be single protein or multiprotein complexes in membrane.

Insertion typically requires energy (protonmotive force and/or ATP).

Most proteins targeted for compartments outside the cytoplasmic membrane are made with a "LEADER SEQUENCE" or "SIGNAL PEPTIDE/SEQUENCE."

Sequences are not highly conserved, but characteristics are conserved:

1. Length: 18-30 AA, 20-25 AA most common

2. N-terminal region has a hydrophilic, usually basic character (Lysines, Arginines)

3. Hydrophobic region follows; often contains 1 or 2 glycine or proline residues

4. Hydrophobic region is followed by a hydrophilic region making a "reverse turn;" cleavage site often adjacent to a small amino acid (Ala, Gly, Ser).

Function of the signal sequence? Still not known. It retards folding; can interact with membrane, SecA, SecB, and SecY;

Stable, folded proteins do not seem to be substrates for transport across membrane. Growing peptide complexes with components of the secretion apparatus (in eucaryotes, peptide complexes with SIGNAL RECOGNITION PARTICLE (SRP).

Eubacteria and Archaea have homologs, but may perform somewhat different function?

E. coli protein Ffh = SRP54; 4.5 S RNA = 7 S RNA.

SecA 102 kDa peripheral membrane protein; it is an ATPase when bound with preprotein to membrane Sec components (SecE-SecY)

SecB 17 kDa cytoplasmic protein required for some but not all secreted proteins; appears to act as a chaperone to maintain secretion-competent conformation of protein

SecY 49 kDa membrane protein; interacts with SecE and SecA; has 10 transmembrane a-helices and plays critical role (pore?) in secretion

SecE 14 kDa integral membrane protein; 3 transmembrane a-helices and may interact with signal sequences

SecD 67 kDa integral membrane protein; function unknown

SecF 35 kDa membrane protein; function unknown

SecG 11 kDa membrane protein; function unknown but part of core secretion apparatus

LepB Leader peptidase for most envelope proteins; 36 kDa integral membrane serine proteinase

LspA 18 kDa integral membrane serine proteinase; specific for prolipoproteins

Ffh Part of signal recognition particle that affects efficiency of elongation

Ffs 4.5 S RNA; part of signal recognition particle that affects efficiency of elongation

Why remove the leader signal sequence/leader peptide? Translocation occurs even when leader peptidase is inactive. Probably allows native conformation to be attained.

After SecA-dependent ATPase driven initiation of translocation and formation of SecA-SecY-SecE-SecG complex with pro-protein, energy is provided by protonmotive force.

Proteins that reach the periplasm can have several different fates.

1. They may remain bound to cytoplasmic membrane by internal or C-terminal hydrophobic regions.

2. They can become soluble periplasmic proteins.

3. They can insert into outer membrane.

4. They can interact with other proteins to pass the outer membrane. Assisting complexes can be simple (one protein) or complex.

GSP can have up to 12-14 proteins, and are used to secrete proteins beyond outer membrane. Secretes cellulases, pectate lyases, lipases toxins, proteases, some types of pilin subunits. Signals not clear; probably 3-dimensional in nature. In contrast to situation for Sec transport, protein folding seems to be required.

1. ABC TRANSPORTERS

ABC = ATP-Binding Cassette

No signal sequence, and protein crosses both membranes at once with no periplasmic intermediate. Involved in both uptake of substances (binding protein mediated uptake) as well as protein and small molecule secretion. Each transporter is specific to a single molecule or very closely related molecules

2. "Type III" Secretion system

YOP PATHWAY(YERSINIA SP. )

YOP= Yersinia sp. virulence protein

Similar systems found in a wide variety of pathogens. Transporter is a complex of at least 20 proteins. Clearly related to secretion system used in FLAGELLA biogenesis.

In Yersinia sp., YOP secretion is inhibitied by Ca²⁺; cell-cell adhesion blocks Ca and allows YOP export via Type III secretion system. Yersinia sp. proteins allow secreted proteins to be delivered to cytoplasm of phagocytic cells, which are inactivated by YOPs

Contains 50-100 proteins, including wall biosynthetic enzymes; binding proteins for ions/nutrients; degradative enzymes; resistance proteins

Signal sequence is part of the story, but not all. Gene fusion experiments demonstrate that simply adding a signal peptide is not always sufficient to allow secretion of a protein.

Precursors are synthesized in the cytoplasm, but polymerization takes place outside the cell. Several wall biosynthetic enzymes bind penicillin and are irreversibly inactivated by it and cephalosporins (both are b-lactam antibiotics).

How are precursors moved from inside the cell to the outside?

Lipid-soluble carrier, Undecaprenol (C55 Isoprenoid) (Bactoprenol) is employed. Same carrier is used for teichoic acid and lipopolysaccharide precursors.

1. N-Ac-Muramic Acid-Pentapeptide is synthesized in cytoplasm and covalently joined to Undecaprenol-Pi

2. N-Ac-Glucosamine is added to form M-G disaccharide/pentapeptide

3. Bactoprenol flips orientation moving disaccharide to outer surface of CM

4. Transglycosylase transfers disaccharide to the growing glycan chains at growth point; Bactoprenol-PPi

5. Bactoprenol-PPi flips back to inside and Pi is cleaved

6. As transglycosylation occurs, transpeptidation is also performed.

7. Precursor chains appear to be inserted as rings or "hoops" surrouding the cylindrical portion of the cell. About 1100 hoops required for E. coli cell, and it is estimated that about 200 growth points must exist. It takes about 8 minutes to synthesize a ring of PG.

8. Septum formation requires another penicillin binding protein (PBP 3) that is a distinct transglycosylase. One notion is that rings of decreasing diameter are synthesized.

In G+ bacteria: basically the same process with a couple of twists. Firstly, G+ bacteria usually have peptide cross-bridges in peptidoglycan; these are added to AA3 of the peptide chain before translocation of disaccharide outside cell. Secondly, PG grows by adding a layer proximal to the CM while sloughing off a layer at the outside of the cell (thickness remains constant). Similar to the sloughing of skin cells. Material that is lost from outer surface is recycled. Thirdly, teichoic acid synthesis resembles that of PG--precursors are transported outside by Bactoprenol and added to growing polymer by transfer enzyme.

Wall synthesis in Streptococcus sp. occurs in a zonal fashion. Material inserted along mid-line of cell (see p. 119, text). Growth doesn't seem to occur at the poles, but 2 new poles must be synthesized for each cell division process. Not the case in E. coli where grow occurs at many sites. Streptococcus sp. behaves as if "all poles, while E. coli has 2 distinct synthesis regions: poles, longitudinal regions.

Lipopolysaccharides/Endotoxin forms the outer leaflet of the outer membrane. Assembly of LPS occurs in two parts: Lipid A-Core and O-antigen chains. O-antigen sugars are added sequentially to bactoprenol carrier to form one repeat unit that is transported to outer surface of CM as for murein synthesis. Multiple repeating units are built up on the carrier.

Lipid A is synthesized at cytoplasmic membrane. INNER CORE sugars (KDO and heptose) are added, then OUTER CORE hexoses are added. Lipid A acts as transporter and flips to outside (requires protonmotive force, not ATP). O-antigen chain is transferred to sugar of outer core.

Phospholipid transfer from CM to OM also requires protonmotive force; mechanism not well understood but may occur at Bayer's junctions.

OM is not highly complex; 3-4 major proteins and perhaps 50 minor ones. Most proteins of the OM differ from those of the CM--more hydrophilic. Predominant structural feature is NOT transmembrane a-helices, but amphipathic b-sheet structures. Outer membrane proteins pass through the general export pathway (signal peptides removed) and reach the periplasmic space. Final assembly and possibly refolding occurs as proteins insert into the OM. Sites of insertion appear near Bayer's junctions; assembly of OM proteins seems to require interaction with LPS; lipids can promote FOLDING of OM proteins

Braun lipoprotein is the most abundant (numerically) protein in E. coli

Synthesized with 20-aa leader sequence (exported to periplasmic space).

Cleavage of signal sequence reveals new N-terminal Cys residue. The Cys side chain is modified by addition of diacylglycerol (2 fatty acids + glyc.)

NH2 terminus is modified by N-acylation (fatty acid addition) as well.

Transport protein may be involved in periplasm for translocation to OM

Some LPP molecules (1/3) are covalently attached to peptidoglycan via the e-NH2 group of the C-terminal Lysine

Lipoprotein and all modification enzymes are essential to viability of E. coli

Outstanding recent Reviews: R. MacNab in Escherichia coli and Salmonella, 2nd Edition (1996). Neidhardt et al. pp. 123-145. Also: R. Macnab (1992), Annu. Rev. Genetics 26: 131-158.

Assembly proceeds from basal body to hook to filament--largely in that order.

See in-class handout for general scheme.

1. M ring inserts in CM, then S ring is added; then switching complex ("C-ring") is added.

2. Export apparatus is constructed--Type III secretion apparatus--has ATPase.

3. The rod is added and capped--makes the "rivet" structure

4. P-ring (PG) is added, then L-ring (OM). Sec-dependent secretion; may require chaperone

5. Hook is added after all rings assembled. Length carefully regulated. How? "Yardstick or scaffolding proteins?"

6. Hook is capped--adaptors for attachment of filament

7. Filament is first capped, then grows by FliC addition.

8. Motility proteins added to complete the assembly--probably could be added shortly after M-S-rings assembled; however, gene regulation controls cause this to occur late in assembly

All axial structure proteins are secreted by a Type III secretion system specific for flagellum proteins: includes the rod, hook, hook cap, hook-filament junction, filament, and filament cap. The proteins DO NOT have a signal sequence, hence NOT Sec mediated. Assembly takes place in order from distal end of growing structure; subunits must be delivered in the correct order. Channel exists through the basal body, the rod, the hook, and the filament--forced through a straw-like structure. An ATPase, FliI, related to b subunit of ATP synthase, is part of the secretion apparatus.

Interesting case of structure regulating gene expression. Gene encoding hook cap, filament, and filament cap are expressed very late (Class 3 genes), and require a specific sigma factor, FliA. FliA is inactive due to anti-sigma factor FliM until export apparatus is completed. FliM is then exported from cell, activating FliA, which allows transcription of Class 3 genes. Filament is then assembled.

FlhA, FlhB, FliH, FliI, FliO, FliP, FliQ, FliR are probable components of the Type III secretion system. Present thinking is that some structural motif is recognized by system allowing only proteins with that structural motif to pass through the channel.

Nice review article: S. J. Hultgren and C. H. Jones (1995) ASM News 61: 457-464

Book says pilus assembly not well understood--actually this is reasonably well understood now.

Quite different from flagellar assembly, and more simple.

P (Pathogenesis) pili are involved in acute urinary tract infections: "pyelonephritis"

P pili are encoded/regulated by 11 pap genes

PapA Major component of pilus rod

PapB Regulation

PapC Outer Membrane "Usher chaperone"

PapD Periplasmic Chaperone

PapE Major protein of fibrillar tip

PapF Tip Adaptor for Adhesin

PapG Gal-binding "adhesin"

PapH Pilus Anchor in outer membrane

PapI Regulation

PapJ Unknown

PapK Fibrillar tip adaptor/initiator (Pilus shaft to Tip Fibrillum adapter). Also regulates length of Fibrillar tip

Pilus has 4 main structural features:

1. Anchor to OM

2. Pilus shaft

3. Tip Fibrillum

4. Adhesin

Pilus components are secreted by Sec system into the periplasmic space, where they are bound by PapD. PapD promotes proper folding and maintains pilus subunits in assembly-inactive form.

Pilis has hollow core, but it is too narrow to allow passage of proteins through. Assembly takes place at OM, with growth occuring at the OM itself.

PapC "Usher" chaperone of OM promotes release of PapD from assembling subunits and controls tip fibrillum assembly. PapG adhesin is attached to PapF and then PapE fibrillar tip assembly is constructed with assistance of PapC/PapD. Adapter/length controller PapK is added with PapC/PapD assistance. The major PapA pilus subunits are added with PapD but not PapC assistance. Finally, addition of anchor PapH terminates elongation process. The general picture seems clear, although not all details completely understood.

If PapD or PapC not present, pili do not form. In absence of PapD, pilus subunits are degraded in periplasmic space by DegP protease. Finally, proper structures of several pilus proteins requires DsbA function to introduce appropriate disulfide bonds.

Capsules are important in pathogenesis (resistance to host defense, especially phagocytosis) and in nature (resistance to dessication)

Assembly of some polysaccharide capsules is similar to the O-antigen synthesis for LPS.

Bactoprenol/undecaprenol carrier is employed to move precursors to the outside for polymerization. In some cases, a phospholipid is attached to anchor the polymer to the outer membrane.

In other cases, sugars or amino acids are polymerized by transferase activities from activated (disaccharide) precursors outside cell (e.g., dextrans, levans).