Reading: Chapters 6 and 7, pages 174-225

Transport mechanisms must operate efficiently, since growth is extremely rapid. Moreover, since growth rate is constant until some nutrient becomes limiting, the cell must be able to maintain constant intracellular concentration of metabolites.

In G- bacteria, small molecules must pass the OM (primarily via porins), the peptidoglycan (porous, not really a problem), and then must pass the true permeability barrier, the CM. Concentration of solute in the periplasmic space will typically be even lower than in solution, so permeases must be able to transport when external concentration is very low.

1. Simple diffusion

2. Facilitated diffusion

3. Active transport

a. secondary active transport

b. shock-sensitive transport systems

4. Group translocation

In E. coli, redundant systems for uptake exist. These systems typically have different apparent affinities for substrates--often one is for high affinity uptake, one for low affinity uptake. A general rule is that high affinity systems have lower transport rates, while lower affinity systems have higher transport rates.

Gases (O₂, CO₂, NH₂, H₂S) and H₂O pass by diffusion. Non-polar (hydrophobic) compounds can pass the CM but not the OM, due to LPS layer. Hydrophilic molecules pass the porins of the OM, but do not diffuse across the CM. There must be a concentration gradient for molecule to diffuse into the periplasm. Periplasmic concentrations of solutes maintained at low level by binding proteins and by active transport systems.

Stereospecific, carrier-mediated process by which a molecule is assisted in diffusing across other impermeable membrane. No energy utilized, and only speeds the attainment of equilibrium (equal concentrations inside and out)

More common in eucaryotes than in procaryotes. Glycerol and propanediol are transported by carriers, AKA permeases or "facilitators." The GlpF protein, the glycerol facilitator, has 6 trans-membrane a-helices and resembles several eucaryotic channel proteins. Facilitators work quite well for waste secretion in aqueous environments--equilibrium hard to achieve!

Permease/carrier is required, and compound can be concentrated against a gradient in a stereospecific manner. Concentration of substrate against a gradient can be achieved. 10³ to 10⁶-fold concentration is possible. Can only be achieved by input of energy.

H⁺ extrusion during respiration or photosynthesis, or by ATP hydrolysis by ATP synthase, creates protonmotive force. Sometimes called "primary active transport." Primary active transport can be used by cell to perform work: synthesize ATP, swim, transport nutrients. Coupling of H⁺ gradient to solute transport to establish ion gradient is called "secondary active transport."

1. Symport: single carrier moves to substrates in the same direction; one must flow downhill in concentration. Most common type. See Figure 16.

Classical example: E. coli lactose permease

Most of these proteins have 10-12 (LacY has 12) trans-membrane a-helices. Very hydrophobic.

2. Antiport: one material moves inside, the other outside via one carrier. One must move downhill versus concentration gradient. Classical example: H⁺/Na⁺ antiporter

3. Active Uniport. Ion flow driven directly by the concentration of that ion (similar to facilitated diffusion, but involves charge compound). Least common class.

Osmotic shock releases proteins of the periplasmic space, including BINDING PROTEINS for a wide variety of sugars, amino acids, short peptides, vitamin B₁₂, Fe-hydroxamate, Fe-dicitrate, Fe-enterobactin, sulfate, phosphate, molybdate, polyamines, etc. BPD transporters are ABC transporters.

Bind substrates tightly (10^-7 to 10^-6 M binding constants), and are free in the periplasm.

In the example in the text on p. 179: Also see Figure 17 on Web.

HisJ is the binding protein; HisM and HisQ are the CM proteins that form the pore; and HisP (should probably be present in two copies per complex) is the ATPase that drives the important of histidine from the periplasm. It is clear now that ATP provides the driving force for all ABC transporters.

Transported substrate is chemically modified to an impermeable derivative during transport across CM. NOT active transport, since concentration gradient is not established (different chemical species formed). However, same net effect, since modified substrate conc. can be higher than unmodified substrate outside the cell. Effectively there is a net savings of energy, since transport and activation performed at the same time (e.g., glucose converted to glucose-6-P). Very common in strict and facultative anaerobes, since energy is more limiting for these organisms.

Used to translocate a variety of sugars and poly-alcohols. Phosphorelay transfers phosphate group from phosphoenolpyruvate (PEP) as ultimate donor (equal to 1 ATP) to a Histidine of Enzyme 1 and from there to a Histidine on a small, heat-stable protein (HPr). Enzyme 1 and HPr are common to all PTS chains.

Minimally, a third cytoplasmic membrane protein, Enzyme II, specific for the transported compound, is required. In the case of mannitol, this protein has three domains: A, B, and C, with C forming the CM-spanning domain. Phosphate is transferred from a Histidine in domain A to a Cysteine in domain B to mannitol, forming mannitol-1-P. Other arrangements of domains A, B, and C occur: for glucose, domain A is soluble, and domains B + C are membrane-bound (see Figure 18). For mannose, there are two different membrane subunits required to form Enzyme II, and domains A + B are soluble.

IMPORTANT NOTE: Phosphorylated Enz. IIAGlu is a positive effector (stimulates activity) of adenylate cylase, which in turn controls catabolite repression by forming cAMP. If there is no glucose, then Enz. IIAGlu is more highly phosphorylated, thus cAMP levels rise, and operons for utilization of other sugars such as lactose can be transcribed IF those sugars are present to release sugar-specific repressors such as the Lac repressor). When Enz. IIAGlu is not phosphorylated, it can also directly inhibit the activities of some other Enz. II's, so-called "inducer exclusion."

Other group translocation systems exist: Acyl-CoA synthases modify fatty acids for uptake; phosphoribosyltransferases ribosylate purines or pyrimidines to form nucleotide monophosphates.

About 1% of cells energy budget used for swimming in E. coli--pretty small component (not terribly different from DNA synthesis for that matter, however). In peritrichous (uniformly distributed) and lophotrichous (tufts) organisms, counter-clockwise (CCW) rotation of flagella allows bundle formation of all flagella (due to handedness of the flagella helices) and smooth swimming. Clockwise (CW) rotation causes bundles to tangle, fly apart, and cell tumbles. Rates are typically 10-20 mm sec^-1 which is equivalent to about 40 mph for humans--not bad!! Motors generally use H⁺ gradient as energy source (some bacteria us Na⁺ gradient).

Swimming is characterized by periods of smooth swimming and tumbling--it is the interval between these events that is important. Inhibition of tumbling allows the bacteria "to make progress." Tumbling is random--the swimming direction adopted after tumbling is random as well. Bacteria move towards attractants (away from repellents) by a BIASED RANDOM WALK process.

Bacteria with only a single, polar flagellum cannot behave the same way, since there is no bundle to fly apart. In Rhodobacter sphaeroides, a well studied case, motors alternate between turning and not turning. When the motor stops, the flagellum coils up very tightly, and the cells tumble until motor starts again. The flagellum uncoils as torsional stress increases as the motors starts to turn again (see p. 191 of text).

Axial filaments (endoflagella or periplasmic flagella) are seen in spirochetes--really just flagella that are located in the periplasmic space. Spirochetes are well adapted to swim in viscous liquids.

Gliding Motility: Mechanism not known, but appears to use proton gradient. Unusual sulfonolipids found in some gliding bacteria, such as Cytophaga sp. Some gliding bacteria can move at rates that resemble swimming. There is some evidence that the outer membrane may move relative to the pepidoglycan, like the treads on a tank? Both gliding and swimming bacteria can exhibit specific, tactic responses.

Bacteria are too small to sense a SPATIAL gradient of a chemical, but they do respond to TEMPORAL gradients of chemicals, either attractants or repellents. Movement in response to chemicals is "chemotaxis." Bacteria can also move in response to oxygen (aerotaxis), magnetic field (magnetotaxis), light (phototaxis). Bacteria move in response to a change in nutrient concentration, not to the absolute concentration level. Hence, cells must adapt to prevailing conditions as a function of time.

CheA: Protein kinase that can autophos-phorylate itself on a histidine residue, and can transfer that phosphate to either CheY or CheB.

CheB: Methylesterase that removes -CH₃ groups from methylated glutamate residues of MCPs

CheR: Methyltransferase that transfers -CH₃ group from S-adenosylmethionine to glutamate residues of MCPs

CheW: Required for formation of ternary complex between CheA and MCP protein.

CheY: When phosphorylated, CheY can interact with FliM of the basal body motors to promote CW rotation and hence promote tumbling.

CheZ: Phosphatase that removes phosphate from CheY

E. coli additionally has 4 CM-Proteins, MCPs = Methyl-accepting Chemotaxis Protein

All MCPs are trans-CM proteins with periplasmic domain for SENSING and cytoplasmic domain for SIGNALING AND ADAPTATION. The MCPs are arranged in a patch of CM near one end of the E. coli cell, forming a "sensing organelle" reminiscent of a "nose."

Tar: Binds the attractants aspartate, glutamate, and maltose-binding protein; binds repellants Ni⁺² and Co⁺²

Tap: Binds the attractant dipeptide-binding-protein

Tsr: Binds the attractants serine, alanine, glycine, aminoisobutyrate

Trg: Binds ribose and galactose binding proteins

Salmonella typhimurium has 5 MCP proteins, including a protein (Tcp) that binds citrate (attractant) and phenol (repellant).

Attractant binding to MCPs causes conformational change in MCP protein that is transmitted to the signalling domain in cytoplasm. This inhibits kinase reaction of CheA, lowers phosphorylation of CheY, and promotes CCW rotation of motors and smooth swimming. Kinase activity is also controlled by the level of methylation of the MCPs

In addition to signals from MCPs, Enzymes II of PTS system and a signal(s) from electron transport chain (perhaps cytochrome oxidase) senses oxygen (can be both an attractant (low O₂) or a repellent (high O₂)).

Bacteria sense external environment through "SENSOR KINASES" that span the cytoplasmic membrane. Binding of some molecule(s) alters conformation and promotes a change in the ability of the protein to autophosphorylate itself on specific histidine residues. Transfer of the phosphate group to an aspartate residue on a "RESPONSE REGULATOR" allows the organism to elicit a response to the chemical signal. In many cases, the response regulator is a transcription factor, controlling gene expression. Response regulator may catalyze auto-dephosphorylation, or a separate phosphatase may be required.

Sensing in chemotaxis is closely related to 2-component sensor-response regulation systems: CheA is the "sensor kinase" and CheY is the "response regulator." MCPs are "transducers," transducing information from outside to CheA.

Attractant binding to MCP decreases the rate of phosphorylation of CheA, whereas repellent binding to MCP increases the rate of CheA phosphorylation. CheY is an "integrator", since it can receive inputs from all 4 MCPs found in E. coli (see Figure 19).

IF this was the full story, the system could only signal that an attractant or repellent had bound but could tell the cell whether binding was increasing or decreasing as a function of time.

ADAPTATION is the 2nd part of the story. MCPs can be methylated on specific glutamate residues by CheR using S-adenosylmethionine as methyl donor. Phosphorylated CheA can also phosphorylate CheB; phosphorylation of CheB activates its demethylase activity. Methylation status of MCPs determines their ability to stimulate kinase activity of CheA. When fully methylated, the MCPs no longer respond at all to bound attractant and CheA becomes fully phosphorylated, causing phosphorylation of CheY and CheB. Tumbling results, and bacterium stays put--it has sufficient nutrient. CheB-P_i will act to remove some methyl groups, however, and system can be adjusted to respond again.