Lecture #11: The Enterics, Cross-Membrane Transport I

Question 1

Typhoid Fever

Answer 1

From contaminated food, water (fecal contamination). Salmonella typhi. Some people are immune and may become carriers.

Early 1900’s Typhoid Fever caused many deaths in the U.S. Poor handwashing by food handlers one way bacteria spread*.

Mary Mallon came to New York from Ireland, house cook for wealthy families. Carrier. Tied to 53 cases, 3 deaths.

*Hand to mouth.

Question 2

Proteobacteria

Answer 2

Largest group of bacteria; over 500 known genera. May be phylogenetically related, but are still very diverse.

Morphology of these gram- bacteria range from simple rods and cocci to genera with prosthecae, buds, and even fruiting bodies.

Physiologically, they include photoautotrophs, chemolithotrophs, and chemoheterotrophs.

No obvious overall pattern in metabolism, morphology, or reproductive strategy characterizes proteobacteria.

Comparison of 16S rRNA sequences has revealed five lineages of descent within the phylum Proteobacteria: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, and Epsilonproteobacteria.

Question 3

Pseudomonas

Answer 3

Pseudomonas most important genus in the order Pseudomonadales, the family Pseudomonaceae.

These bacteria are straight or slightly curved rods, and are motile by one or several polar flagella.

They’re chemoheterotrophs that usually carry out aerobic respiration. Sometimes nitrate is used as the terminal electron acceptor in anaerobic respiration.

All pseudomonads have a functional tricarboxylic acid cycle and can oxidize substrates completely to CO2.

The genus pseudomonas is an exceptionally heterogenous taxon currently composed of about 60 species. Many can be placed in one of seven rRNA homology groups. The 3 best characterized groups are subdivided according to properties such as the presence of poly-beta-hydroxybutyrate (PHB), the production of of a fluorescent pigment, pathogenicity, the presence of arginine dihydrolase, and glucose utilization.

Use oxygen as an electron acceptor, and fully combust sugar (36 ATP), some reduce nitrate, 70 plus species, common in soil and water.

Metabolically diverse—use a wide variety of foods. Large, complex genome. UTI’s and food spoilage.

Question 4

Order Vibrionales

Answer 4

The order Vibrionales contains only one family, the Vibrionaceae. Members of this family are straight or curved, flagellated rods. Most are oxidase positive, and all use D-glucose as their sole or primary carbon and energy source. The majority are aquatic microbes, widespread in freshwater and the sea. The family has eight genera.

Several vibrios are important pathogens. Vibrio cholerae causes chlorea. Its genome contains about 3800 open reading frames distributed between two circular chromosomes. The larger chromosome has genes for essential cell functions like DNA replication, transcription, and protein synthesis. It also has most virulence genes. The other chromosome has essential genes like transport genes and ribosomal protein genes.

Vibrio. Straight to curved rods (‘commas’). Many aquatic. V. cholerae is infamous. Waterborn illness. Adhere to intestinal lining and secrete a toxin that stimulates hypersecretion of water – watery diarrhea, cramps, dehydration, vomiting. Can lead to death.

Question 5

Order Neisseriales

Answer 5

Has one family, Neisseriaceae, with 15 genera. Best known and most intensely studied genus is Neisseria.

Members of this genus are nonmotile, aerobic, gram- cocci that most often occur in pairs with adjacent sides flattened.

They may have capsules and fimbriae (often referred to as pili).

The genus is chemoorganotrophic and produces the enzymes oxidase and catalase.

They’re inhabitants of the mucous membranes of mammals, and some are human pathogens. Neisseria meningitidis is responsible for some cases of bacterial meningitis.

Cocci in pairs. Aerobic. Mucous membranes of vertebrates. Neisseria meningitidis is reason for college vaccination. Genus causes meningitis, gonorrhea. Rare combination indeed of Gram – and coccus!!!

Question 6

Protein Translocation and Secretion in Bacteria

Answer 6

Almost 1/3 of the proteins synthesized by cells leave the cytoplasm and reside in membranes, the periplasmic space of bacterial and archaeal cells, or the external environment. It’s not surprising then that over 15 different systems for moving proteins out of the cytoplasm have evolved. Some of these systems are found in all domains in life, others more rare.

When proteins are moved from the cytoplasm to the membrane or the periplasmic space, the movement is called translocation.

Protein secretion refers to the movement of proteins from the cytoplasm to the external environment.

Many important proteins are located in membranes. These include transport proteins that bring needed materials into cell and wastes out of cell. Also proteins involved in electron transport.

The protein subunits of external structures such as flagella and fimbriae must also be moved out of the cell and assembled onto its external surface.

Question 7

Common Translocation and Secretion Systems

Answer 7

In bacteria, the major pathway for translocating proteins across the plasma membrane is the Sec (secretion) pathway.

In gram- bacteria, proteins can be transported across the outer membrane by several different mechanisms, some of which bypass the Sec system moving proteins directly from cytoplasm to outside of cell.

All protein translocation and secretion pathways described here require the expenditure of energy at some step in the process. The energy is usually supplied by the hydrolysis of high-energy molecules such as ATP and GTP. However, the proton motive force also sometimes plays a role.

Question 8

Sec System

Answer 8

Sometimes called the general secretion pathway. It’s highly conserved having been identified in all 3 domains of life. It translocates unfolded proteins across the plasma membrane or integrates them into the membrane itself.

Proteins to be transported across the plasma membrane by this pathway are synthesized as presecretory proteins called preproteins. The amino-terminus of the preprotein has a signal peptide, which is recognized by the Sec machinery. Soon after the signal peptide is synthesized, chaperone proteins (e.g. SecB) bind it. This helps delay protein folding, thereby heping the preprotein reach the Sec transport machinery in the conformation needed for transport.

Evidence exists that translocation of proteins can begin before the completion of their synthesis by ribosomes. Certain Sec proteins (SecY, SecE, and SecG) are thought to form a channel in the membrane through which the preprotein passes. Another protein (SecA) binds to the SecYEG proteins and the SecB-preprotein complex. SecA acts as a motor, using the energy released from ATP hydrolysis to translocate the preprotein through the plasma membrane. When the preprotein emerges from the plasma membrane, free from chaperones, an enzyme called signal peptidase removes the signal peptide. The protein then folds into the proper shape.

Question 9

Type I Secretion Systems

Answer 9

Ubiquitous in gram+ and gram- bacteria as well as Archaea. They’re evolutionarily related to ABC transport systems. In pathogenic gram- bacteria, type I secretion systems are involved in the secretion of toxins (alpha-hemolysin), as well as proteases, lipases, and specific peptides. Secreted proteins usually contain C-terminal secretion signals that help direct the newly synthesized protein to the type I machinery.

In gram- bacteria, the type I machinery spans the plasma membrane, the periplasmic space, and the outer membrane. These systems move proteins in one step across both membranes, bypassing the Sec system.

Gram+ bacteria use a modified version of a type I system to translocate proteins across the plasma membrane. Analysis of the Bacillus subtilis genome has identified 77 type I secretion systems. This may reflect the fact that ABC transporters move a wide variety of solutes in addition to proteins, including sugars and amino acids, as well as exporting antibiotics from the cell interior.

Question 10

Type IV Secretion Systems

Answer 10

Unique in that they are used to secrete proteins as well as to transfer DNA from a donor bacterium to a recipient during bacterial conjugation. These systems observed in both gram+ and - bacteria.

Type IV injects materials from one bacterium to another during conjugation (proteins, DNA). Sexual pilus.

Question 11

Sec Dependent vs. Independent

Answer 11

Gram- bacteria use the type II and type V systems to transport proteins across the outer membrane after the protein has first been translocated across the plasma membrane by the Sec system.

The type I, III, and VI systems don’t transport proteins that are first translocated by the Sec system, so they are Sec-independent.

The type IV pathway for secretion sometimes is linked to the Sec pathway but usually functions on its own.

Question 12

Type III Secretion System

Answer 12

Form a needlelike structure that is sometimes called an injectisome. It extends beyond the outer membrane and can make contact with other cells. It injects virulence factors into the plant and animal host cells that these pathogens attack.

Type III systems also transport other proteins:

1) some of the proteins from which the system is built
2) proteins that regulate the secretion process
3) proteins that aid in the insertion of secreted proteins into target cells.

Type III is basically a hypodermic to inject toxins. Salmonella and Pseudomonas. Inject chemicals to disable host systems, poison the cell, or harness host cell machinery.

Question 13

Type V Secretion System

Answer 13

They employ proteins called autotransporters because after being translocated across the membrane by the Sec pathway, the proteins are able to transport themselves across the outer membrane. Autotransporters have two domans. One domain is thought to form a pore in the outer membrane through which the other domain (virulence factor) is transported.

Autotransporters.

Proteins that use a Sec-dependent channel to get into the periplasm and then transport themselves through the outer membrane. Build own channel.

Question 14

Type VI Secretion System

Answer 14

Injects toxins that may kill prey, disable a competitor, or allow infection of a host cell. Structurally different than Type III.

Question 15

Additional Facts on Secretion Systems

Answer 15

Secretion of proteins, especially enzymes, is vital to a bacterium as there is no exocytosis.

Type II and IV only occur in GN bacteria. Type I is universal.

Typically, there is large number of different transporters in a single cell. Some species have more than others.

Type III and VI inject virulence factors. Important in Salmonella, Yersinia, Escherichia and Pseudomonas –GN’s.

Type V is a sexual pilus.

Question 16

Uptake of Nutrients

Answer 16

The first step in nutrient use is their uptake by the microbial cell. Microbes can only take in dissolved molecules. Uptake mechanisms must be specific-that is, the necessary substances, and not others, must be acquired. It does a cell nothing to take in a substance it can’t use.

Because microbes often live in nutrient-poor habitats, they must be able to transport nutrients from dilute solutions into the cell against a concentration gradient.

Finally, nutrient molecules must pass through a selectively permeable plasma membrane that prevents the free passage of most substances.

Microbes use several different transport mechanisms. Bacteria and Archaea use facilitated diffusion, active transport, and group translocation for nutrient uptake.

Bacterial cells need to get substances in. Some substances enter the cell through simple diffusion.

If more concentrated outside the cell, and can freely penetrate the membrane*, they move in to equalize concentrations inside and outside. With a concentration gradient.

Question 17

Facilitated Diffusion

Answer 17

Rate of diffusion across selective membranes is greatly increased using carrier proteins, sometimes called permeases, which are embedded in the membrane and create channels through which the substance passes. Diffusion involving carrier proteins is called facilitated diffusion.

The rate of facilitated diffusion increases with the concentration gradient much more rapidly and at lower concentrations of the diffusing molecule than that of passive diffusion. Note that the diffusion rate reaches a plateau above a specific gradient value because the carrier protein is saturated (it is transporting as many solute molecules as possible).

Permeases also resemble enzymes in their specificity for the substance to be transported; each permease is selective and transports only closely related solutes.

Although a carrier protein is involved, faciliated d is truly diffusion. A concentration gradient spanning the membrane drives the movement of molecules, and no metabolic energy input is required. If the concentration gradient disappears, net inward movement ceases. The gradient is maintained by transforming the transport nutrient to another compound.

After the solute molecule binds to the outside, the carrier is thought to change conformation and release the molecule on the cell interior. The carrier subsequently changes back to its original shape and is ready to pick up another molecule. The net effect is that a hydrophilic molecule can enter the cell in response to its concentration gradient. Remember that the mechanism is driven by gradients and therefore is reversible. If the solute’s concentration is greater inside, it will move outward. However, because the cell metabolizes nutrients upon entry, influx is favored.

Question 18

Active Transport

Answer 18

Microbes must have transport mechanisms that can move solutes against a concentration gradient. Active transport is the transport of solute molecules to higher concentrations with the input of metabolic energy.

Because active transport involves transport proteins, it resembles facilitated diffusion in ways. The transport protein bind particular solutes with great specificity. Similar solute molecules can compete for the same carrier protein in both facilitated diffusion and active transport.

Active transport is also characterized by the carrier saturation effect at high solute concentrations. Nevertheless, active transport differs from facilitated diffusion in its use of metabolic energy and its ability to concentrate substances. Metabolic inhibitors that block energy production inhibit active transport but don’t immediately affect facilitated diffusion.

Active transport proteins are divided into two types: primary and secondary transporters. Primary use energy provided by ATP hydrolysis to move substances against a concentration gradient. Secondary couple the potential energy of ion gradients to transport of substances.

Typically what bacteria need. Often cells are in nutrient poor media, and solutes tend to be more concentrated in the cells.

Question 19

ATP-binding Cassette Transporter (ABC Transporter)

Answer 19

Important primary active transporter. Some are used for import of substances and others are used for export of substances.

Usually ABC transporters consist of two hydrophobic membrane-spanning domains associated on their cytoplasmic surfaces with two ATP-binding domains. The membrane-spanning domains form a pore in the membrane, and the ATP-binding domains bind and hydrolyze ATP to drive uptake.

ABC transporters employ substrate-binding proteins, which are located in the periplasmic space of gram- bacteria or are attached to membrane lipids on the external face of the plasma membrane of gram+ bacteria. These proteins bind the molecule to be transported and then interact with the transporter proteins to move the molecule into the cell. Because a single molecule is transported, this is termed uniport transport.

Recall that gram- bacteria have an outer membrane in addition to the plasma membrane. Thus substances entering must pass through the outer membrane before ABC transporters can take action. This is accomplished in several ways. When the substance is small, a porin protein such as OmpF (outer membrane protein) can be used. The transport of larger molecules requires use of specialized, high-affinity outer membrane receptors that function is association with specific transporters in the plasma membrane.

Question 20

Symport

Answer 20

Linked transport of two substances in the same direction.

Question 21

Antiport

Answer 21

Linked transport in which the transported substances move in opposite directions.

Question 22

Group Translocation (Lecture)

Answer 22

Group of proteins in the cell that work as a series to bring a substance in, then modify it to keep more coming in.

Think of a conveyor belt. If finished items are removed and packaged at the end, don’t pile up and movement of more items continues (production and delivery). If do, production slows.

Sugar uptake. A group of proteins acts to deliver a phosphate group to the membrane proteins, it phosphorylates the sugar (adds phosphate) to keep the entry going.

Question 23

Types of Proteins Translocated or Secreted

Answer 23

In gram- bacteria, the periplasmic space is loaded with proteins such as chemotaxis proteins, enzymes involved in cell wall synthesis, and periplasmic components of nutrient uptake systems.

Many organisms secrete hydrolytic enzymes into the external environment. These enzymes break down macromolecules into monomers more easily brought into the cell.

Pathogenic microbes often release toxins that are important in the infection process.

Question 24

Secretion of Proteins

Answer 24

Protein secretions poses different difficulties, depending on the structure of the microbial cell envelope. For gram+ bacteria to secrete proteins, the proteins must be translocated across the plasma membrane. Once across the membrane, the protein either passes through the relatively porous peptidoglycan into the external environment or becomes embedded in or attached to the peptidoglycan.

Likewise, secreted archaeal proteins must be moved across or into their unique cell walls.

Gram- bacteria have more hurdles to jump when they secrete proteins. They too must transport proteins across membrane, but to complete the secretion process, the proteins must be transported across the outer membrane.