Trecerea moleculelor hidrofile prin membrana bacteriilor gram negative.pdf

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EBS 1988

Review

Permeation of hydrophilic molecu les through the outer membrane

of

gram-negative bacteria

Review

on

bacterial porins

Roland

BENZ

a nd K a tha r ina BA U ER

Lehrstuhl fur Biotechnologie, Universitat Wiirzburg

(Received February 8 /Ma y 10, 1988) EJB 88 0165

The cell envelope of gram-negative bacteria such as

Escherichia coti,

Salmonella

typhimurium

and Pseudomonas

aeruginosa consists of three distinct layers : the outer mem-

brane, the peptidoglycan (murein) layer, and the inner mem-

brane. The inner membrane acts as a real diffusion barrier

and contains, in addition to the respiratory chain and the

H+-

ATPase, a large number

of

uptake systems for hydrophilic

substrates

[l] .

All these hydrophilic solutes have to pass the

outer membrane on their way into the cell. This means that

this membrane must have very special permeability properties

because these substrates have various structures and can all

pass through the same transport pathways.

On the other hand, the outer membrane makes the gram-

negative bacteria resistant to host-defense hctors such as

lysozyme, fl-lysin, and different leukocyte proteins

[2 -41.

Furthermore, in enteric bacteria living in the intestinal tract

of animals, the outer membrane represents a very effective

barrier, which protects the bacterial cells from the action of

bile acid detergents and degradation by digestive enzymes

[5]

At the same time the outer membrane of enteric and some

other gram-negative bacteria represents a strong permeability

barrier to many antibiotics that are effective against gram-

positive bacteria

[5 ,

61.

The outer membrane of gram-negative bacteria acts as a

molecular filter for hydrophilic compounds. The active

components of these molecular sieving properties are a major

class

of

proteins called porins

[7].

The porins have molecular

masses between 30 kDa and 50 kDa and normally form

oligomers in the outer membrane that are, in many but not

all cases, stable

in

sodium dodecyl sulfate. Dependent on their

permeability properties for hydrophobic solutes, the porins

can be divided into two classes. Most porins form, in the

outer membrane and reconstituted systems, large water-filled

channels and are generally diffusion porins because they sort

solutes mostly according to their molecular mass. Other

porins have a certain solute specificity and contain binding

sites for sugar

[8, 91,

phosphate [lo,

1 ],

and nucleosides

I121

inside the pores. These specific porins represent a considerable

advantage for the diffusion of defined substrates through the

outer membrane but they do not act as a general diffusion

pore.

Correspondence

tu

R . Benz, Lehrstuhl fiir Biotechnologie der

Universitat Wiirzburg, Rontgenring 11, D-8700 Wiirzburg, Federal

Republic

of

G e rma ny

T H E O U T E R M E M B R A N E O F G R A M - N E G AT I V E B AC T ER IA

The cell envelope of gram-negative bacteria consists

of

two membranes separated by the peptidoglycan (murein) layer

and the periplasmic space. This additional cellular compart-

ment contains a variety of soluble proteins, some of which

function as processing enzymes and convert nontransportdble

metabolites to transport substrates

[l].

Furthermore, it con-

tains a variety of binding proteins essential for the uptake of

nutrients into the cells and the chemotaxis of the organisms

[l].

The periplasmic space appears to be iso-osmolar to the

cell interior which means that the high osmotic pressure across

the cell envelope in very dilute solutions is maintained across

the outer membrane and the attached murein and not across

the inner membrane [13, 141. The peptidoglycan layer is re-

sponsible for the maintenance of cell shape and for the ability

of the cell to withstand the very high internal osmotic pressure

in dilute environments. It consists of a network of amino

sugars and amino acids (see

[I51

for a review). The amino

sugars N-acetylglucosaminyl-N-acetylmuramoyl dimers)

form long linear strands which are covalently linked together

between two muramoyl residues by short tetrapeptides

[I

51

Components of the outer membrane such as the lipoprotein

or the porins are either covalently bound to the murein or

interact with this macromolecule via ion bridges

[16, 171.

Whereas the basic structure

of

inner membrane is formed

by a normal phospholipid bilayer similar to most biological

membranes and contains a variety of different proteins, the

outer membrane has an unusual lipid composition and a

relatively simple protein composition. The outer monolayer

contains lipopolysaccharide as its major or exclusive (in

enteric bacteria) lipid, while the inner leaflet contains

phospholipids (mostly

phosphatidylethanolamine

and small

amounts of phosphatidylglycerol and cardiolipin

[5 ,

16,

181.

Interestingly, this asymmetry is dependent on the presence

oi

an intact peptidoglycan layer in the periplasm, since degra-

dation of peptidoglycan with lysozyme results in a redistri-

bution of lipopolysaccharide within both leaflets

[39 ] .

Lipopolysaccharide (see [20] for a review) is an amphi-

philic molecule containing a hydrophobic region (lipid A, also

known as endotoxin) that has five or six fatty acids linked to

diglucosamine phosphate. Covalently attached to this is the

rough oligosaccharide core containing in its proximal portion

an unusual sugar, 3-deoxy-~-rnanno-octulos0nic cid (dOclA,

previously known as

KDO),

as well as a variety of heptose

and hexose residues

[16,

201. The rough oligosaccharide core


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2

may be substituted with a variable number of repeated tri- to

pentasaccharide units called the 0-antigen [20

-

21.

Lipopolysaccharides carry net negative charge resulting in a

strong negative surface potential of gram-negative bacteria

[20]. They have attracted special interest because of their role

in assembly and maintenance of the outer membrane per-

meability barrier [16,22], their importance for biological func-

tion of most outer membrane proteins [23]. Their function as

phage receptors [24] and their endotoxic effects [25] are also

important.

Lipopolysaccharides appear to be anchored in the outer

membrane by binding to outer membrane proteins [26, 271,

possibly through hydrophobic interactions with lipid A [27],

and by noncovalent cross-bridging of adjacent lipopolysac-

charide molecules with divalent cations [28- 01. Thus treat-

ment of gram-negative cells with EDTA generally results in

removal, by chelation, of divalent cations and consequent

disruption of the outer membrane [29]. In the absence of

such chelators, however, the ion bridges formed by negatively

charged groups and divalent cations are responsible for many

of the properties of the outer membrane of gram-negative

bacteria, including resistance to hydrophobic antibiotics, bile

salts, detergents, proteases, lipases, and lysozyme

[ 5 ,

16, 30,

311.

The outer membrane also contains, besides the porins, a

small number of so-called ‘major’ proteins present in high

copy number (OmpA and lipoprotein,

lo5

and 7 x

lo5

copies

per cell, respectively

[5 ,

14, 161). These proteins have definitely

no channel-forming activity but are important for the struc-

ture and the stability of the outer membrane. One third of the

lipoprotein (Braun’s lipoprotein,

M ,

7200 in E .

coli

[32]) is

covalently bound to the peptidoglycan, whereas the rest and

OmpA ( M , 35000, ‘heat-modifiable protein’ [33, 341) interact

with the murein via ion bridges. Little is known about the

function of many minor outer membrane proteins with the

exception of proteins important for the uptake of iron [35]

and vitamin

B 1 2

[36].

EXPRESSION OF PORINS

Major outer membrane proteins

Besides the most abundant outer membrane protein, the

lipoprotein, the outer membrane of wild-type cells of E . coli

K 1 2

contains several other major proteins (Omp proteins)

which are expressed in large amounts under standard labora-

tory conditions. The nomenclature for these proteins has been

different in different laboratories mostly because of their un-

usual behavior in

SDS

gel electrophoresis [37

-

01. At pre-

sent, it is agreed that the proteins are named according to

their structural genes (i.e. OmpF is the gene product of the

ompF gene [41]). The relative amounts of the OmpF and

OmpC proteins (both forming general diffusion pores) are

dependent on the growth conditions of the organisms, es-

pecially the osmolarity of the growth media and their tempera-

ture [42- 71. Three genetic loci are known to be involved in

their expression. The loci ompC and ompF mapping at

47.1 min and 20.7 min, respectively, on the E . coli

chromosome [48] are structural genes [49- 11. Mutations of

ompC and ompF result in the absence of OmpC and OmpF,

respectively in the outer membrane. A third genetic locus

mapping at 74 min, ompB, plays a central role in the regulation

of OmpC and OmpF. ompB contains two genes, envZ (coding

for a ‘sensor’ and located in the cell envelope) and ompR

(coding for a cytoplasmic ‘regulator’). The ompB locus affects

transcriptional expression of the ompF and ompC genes, both

absolutely and relatively [52, 531.

Mutations in ompB result in many different combinations

of porin phenotypes: OmpC- OmpF’, OmpC’ OmpF- or

OmpC- OmpF- [54,

551.

Several factors affect the expression

of the porin proteins. The relative level at which the two

proteins are expressed is determined by the growth medium.

OmpC is preferentially expressed in media of high osmolarity

[50,

511, while OmpF

is

preferentially incorporated into the

outer membrane at low osmolarity. The expression of OmpC

is also favored in the presence of a fermentable carbon source.

Other cultural conditions lead also to different relative levels

in the expression of OmpC and Omp F [56]. The synthesis of

the porins and other outer membrane proteins (especially of

OmpA) is further modulated in such a way that the total

number of copies of these proteins remains constant [33, 44,

571. In addition, alterations of the lipopolysaccharide core

sugars [44] and of the lipid structure [58, 591 can affect ex-

pression of these proteins.

The construction of strains in which the lac operon is fused

to the

ompC

[49] and the ompF [52] genes, respectively, have

established that the ompB gene product has a dual role in

regulating both the absolute and the relative transcriptional

levels of expression of the ompF and ompC genes. Deletions

and base substitutions of the ompR gene and lacZ fusions

with this gene [60- 41 and subsequent mutant analysis re-

vealed that the OmpR protein activates the expression of

ompF and ompC genes most likely by binding to the upstream

regions of their promoters [60- 651. These studies, especially

those of the osmoregulation of such mutants also supported

the hypothesis that the OmpR protein has a two-domain

structure. Each domain plays a different role in the activation

of the

ompC

and

ompF

genes. Studies with the purified OmpR

showed that the interaction between this protein and the pro-

spective modulator EnvZ occurs at the N-terminal portion of

the protein, while the C-terminus is responsible for its binding

to the ompC and ompFpromotors [60-641. This is in accor-

dance with the fact that there is extensive conservation of

N-terminal regions between products of E .

coli

ompR and

other two-component regulatory systems like the phosphate-

starvation-dependent regulatory gene phoB and the nitrogen-

assimilation regulatory gene ntrC. There is also extensive con-

servation of C-terminal regions between the products of E .

coli envZandphoR, ntrB, and others [66]. It was proposed that

these regulatory genes comprise two-component regulatory

systems that evolved from a common ancestral system involv-

ing transduction of information about the status of the en-

vironment by one protein domain (C-terminal region

of

EnvZ) to a second one (N-terminal region of OmpR) [66].

Also, the fact that under certain conditions OmpR can acti-

vate the expression of OmpC or OmpF in the complete ab-

sence of envZ gives further evidence that EnvZ may function

as a transducer protein, which is not directely involved in

promotor recognition and DNA-binding [67]. Furthermore,

some exists evidence that the RNA polymerase is also involved

in the activation of porin genes [60- 4, 681.

The

micF

gene may serve as a third regulatory gene for

the expression of ompC and ompF. It codes for an RNA that

is complementary to the

5’

end of the ompF mRNA [69] and

seems to function as an antisense RNA in expression of the

ompF gene during osmoregulation. In this respect, tole,

coding for a minor outer membrane protein, may also play

an important role: to le mutants show reduced levels in OmpF

and overproduction of OmpC protein [70]. The properties of

micF mutants led to the hypothesis that the micF and the


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3

+maltose

malA (74 ) malB(91 )

Fig. 1 . The maltose regulon qf E. coli. The lower part of the figure showes the genetic organization and regulation of the maltose regulon,

whereas the upper half of the figure indicates the molecular masses (given in kDa in circles) and cellular locations of the gene products. The

abbreviations I M, OM, PPS represent inner membrane, outer membrane and periplasmic space, respectively. Positive control is exerted by

the

malT

gene product and by the cyclic-AMP

atabolite-activator-protein

complex (CAMP-cap). The wavy line represents the direction of

transcription. This figure is adapted from

[256]

ompC

genes are probably coregulated [71]. It was proposed

furthermore, that

tolC

mutation leads to overexpression of

the coregulated micF and ompC genes. The increased level of

the micF product would then lead subsequently to a reduction

of OmpF.

Also the growth temperature seems to play an important

role in the expression of the major porins. Decreasing growth

temperatures result in increased amounts of OmpF and de-

creased quantities of OmpC. The regulatory gene responsible

for these effects was named

e n v y

[72]. It seems to map at

12.9 rnin on the

E . coli

linkage map

[48]

and codes for an

envelope protein which appears at low temperatures in

minicells [72]. How Envy functions and interacts with the

other porin regulatory components is not known.

Inducible major outer m embr ane proteins

Lam B prote in.

LamB is the product of the

lamB

gene

located at 91 rnin on the

E .

coli chromosome [48] and it is part

of the maltose regulon. This regulon consists of two regions,

malA

at 74 rnin

[73,

741 and

m a l B

at 90 rnin [75 -771. It con-

tains a number of genes coding for proteins involved in the

uptake of maltose and maltodextrins into the cell and their

degradation. LamB protein is involved in the permeation of

sugars across the outer membrane (see paragraph about

LamB under section on specific porins below). Maltose bind-

ing protein

( m a l E

gene product) is a periplasmic recognition

protein and is essential for chemotaxis. MalF, MalG, MalK

are inner membrane proteins responsible for the transport of

the sugars through the cytoplasmic membrane. Maltodextrin

phosphorylase

(malP

gene product) and amylomaltase male

gene product) are metabolic enzymes located in the cytoplasm

(see Fig. 1). The

ma1

system

is

positively regulated by the

malT

gene product [78], which is activated if maltose or

maltodextrins are added to the growth media. Furthermore,

it

is sensitive to catabolite repression: the complex of CAMP

and catabolite activator protein (cap) stimulates the ex-

pression of the regulator m al T gene and of the malE,F,G and

of the

malK-lam B

operons directly, whereas the stimulation

of the malP,Q expression may only be controlled via malT

V91.

PhoE protein.

The synthesis of the PhoE protein can be

induced in wild-type strains by growth under the conditions

of Pi starvation

[46, 801.

This process is coregulated with the

expression of serveral other Pi-starvation-inducible proteins

in a single regulon, the

pho

regulon [81]. The

p h o E

structural

gene is localized at 6 min of the chromosome of E. coli [81].

The other structural genes of the operon include

p h o A

(alka-

line phosphatase),

y h a S

(Pi binding protein), and

ugpB

(glycerol-3-phosphate binding protein). The gene products of

at least three regulatory genes are involved in this regulation:

p h o B ,p h o R ,

and

phoM

[82], together with a phosphate-specific

transport system for the uptake of Pi across the inner mem-

brane. The

p h o B

gene product is an activator required for

transcription of the structural genes, whereas transcription of

p h o B

is regulated by the

p h o R

and

p h o M

gene products (see

Fig. 2). Evidence for the model of Fig. 2 was obtained from

the cloning of the regulatory genes and the identification of

their products. The p h o R gene product represses the synthesis

of the PhoB protein in minicells

[83].

Furthermore, by using

p h o B - b e 2

[84] and

phoB-cat

[85]

gene fusions, it has been

shown that the synthesis of t h ep h o B product is induced under

Pi limitation. I t has to be noted, however, that Pi is probably

not the molecule that directly influences the state of the p h o R

gene product. In mutants with a defective phosphate-specific

transport system, a high intracellular Pi level can also be

maintained by another uptake system, the phosphate inor-

ganic transport system [86], while these mutants are still

derepressed for the

pho

regulon. It was recently shown that

the intracellular nucleotide pools are changed upon Pi limi-

tation and that the

phoU

gene is involved in these alterations

[87].These nucleotides are most likely the actual effectors of

thephoR gene product [88].

BIOSYNTHESIS OF THE PORINS

Common to all porins is their synthesis as a larger precur-

sor form with an N-terminal leader extension (see [89] for


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4

I

Fig.

2.

The pho regulon

qf

E. coli.

B,

R, and M are the products of

the

genesphoB, p h o R , and phoM respectively.B is an activator which

is essential for transcription of the structural genes of thepho regulon

including phoE. The presence

of

sufficient Pi leads R to be in the

repressor form

RR,

which prevents transcription ofphoB. Pi starvation

converts R

to

the activator form RA. Both RA and M stimulate

transcription of phoB. The figure is adapted from

[88]

a recent review on export of protein). The leader sequence

contains between 21 and 22 amino acids in the case of the

three porins OmpF [90], OnipC [91], and PhoE [92] ofE.

oli,

whereas OmpA [93] and Braun’s lipoprotein [94] have leader

sequences of 21 and 20 amino acids, respectively. The existence

of leader sequences with approximately the same length and

a similar composition suggests that the major outer membrane

proteins (with the exception of the lipoprotein, see [95] for a

review) may share a common pathway for their biosynthesis

and their assembly. The genes most likely to be involved in

the ‘export machinery’ across the inner membrane are the sec

genes of the A, B,

D,

and

Y

(pr lA)

classes. The

secY ( p r lA )

gene product is an integral component of the cytoplasmic

membrane [96]and it is known to be essential for the translo-

cation of exported proteins across the cytoplasmic membrane

[97].

Other important genes are

secA

[98] and

secB

which are

also involved in protein secretion.

The biosynthesis and assembly of outer membrane pro-

teins have been studied using gene fusion products of outer

membrane protein genes with

lacZ [99

- 1021. So far it is not

clear

if

the results are meaningful because the gene fusion

products may be located in the cytoplasmic membrane and in

the periplasmic space and not in the outer membrane [103,

1041. The overproduction of OmpC using a multicopy plasmid

blocked the assembly of OmpA into the outer membrane but

not

of

a LacZ-LamB chimeric protein which is obviously

located in the periplasmic space [104].

The biosynthesis and assembly of OmpA was studied in

detail [105, 1061. As this protein probably shares the same

pathway of synthesis and assembly with the porins and LamB,

its description may serve as a model for porin biosynthesis.

OmpA protein is synthesized on free and membrane-bound

ribosomes

in

the cell as a water-soluble precursor which ac-

cumulates inside the cell

if

the membrane potential is partially

inhibited by the addition

of

an uncpupler of oxidative

phosphorylation [106]. At normal membrane potential, the

pro-OmpA protein is translocated through the cytoplasmic

membrane and processed by a membrane-bound leader

peptidase [107]. This leader peptidase also cleaves pro-mem-

brane proteins of the cytoplasmic membrane, proteins se-

creted into the periplasmic space, and most likely also the

porins, but it is not identical to the leader peptidase which

processes the lipoprotein [108].

So far, it is not clear in all aspects of processing and

assembly if the pro-porins and the pro-LamB share the ident-

ical mechanism to the pro-OmpA, i.e. if a membrane potential

across the cytoplasmic membrane is needed for the translo-

cation of the pro-porins across this membrane, although it

has been found that the presence of the FIF O s indispensable

for the export of LamB [109]. However, also in the case of the

OmpF porin, it is likely that the chain is almost, if not entirely,

complete before the processing star ts [110]. On the other hand,

some evidence also exists for a co-translational processing of

periplasmic proteins in

E .

coli, i.e. translocation of the nascent

chain after completion of approximately

80%

of its entire

length. When inner and outer membrane are separated, the

pro-OmpF is localized in the inner membranes, which indi-

cates its processing at the inner membrane by a membrane-

bound leader peptidase [110] (see above). The processing of

the pro-OmpF into the mature protein occurs very rapidly

with an apparent lifetime of the precursor of 30 s [110].

The mechanism of the assembly of the trimers in the outer

membrane from three mature monomers is still completely

unknown, although some indication exists for a somewhat

delayed formation of stable trimers from metastable trimers

in the case of LamB [ l l l ] . In any case, it is obvious from the

study of the mutant OmpC and the overexpression of OmpC

that OmpA, LamB and the porins share the same machinery

for the assembly into the outer membrane [104, 1121. This

could be shown by the use of an OmpC mutant of

E .

coli, in

which only the amino acids Val-300 and Gly-301 were re-

moved [112]. The expression of this protein caused a dramatic

decrease of the expression of OmpA, of LamB, and of the

other porins by blocking the assembly machinery into the

outer membrane but it did not effect the assembly of the

lipoprotein

[

1121.

ISOLATION AND PURIFICATION

OF

PORINS

The general diffusion pores of different gram-negative

bacteria were found to be tightly associated with the

peptidoglycan layer [113, 1141. Their isolation is relatively

simple because only a few outer membrane proteins are associ-

ated with the murein, while the others are lost during the

washing of the cell envelope fraction with SDS-containing

solutions. Other pore-forming proteins, such as the

Tsx

chan-

nel, are not murein-associated [12, 1151. In this case the outer

membrane has to be separated from the inner membrane by

procedures similar to that used first by Miura and Mizushima

[116].

Isolation of the outer membrane

The first step is always the disruption of the cells. This can

be done by French pressure cell treatment, by ultrasonication,

by shaking the cells in the presence of glass beads, or by

osmotic lysis of the spheroplasts. The disruption followed

by differential centrifugation leads to a crude cell envelope

fraction which is composed of outer membrane, cytoplasmatic

membrane, and peptidoglycan layer. The separation between

outer membrane and inner membrane can be done in the

following way [12,116]. The crude envelope fraction is layered

on a two-step sucrose gradient

(70%

sucrose and 54% su-

crose) and is centrifuged for 10 h at

80000 xg.

The outer

membrane accumulates at the interface between 54% and

70

sucrose, because of its higher density which may be

caused by the larger content of carbohydrates of the outer

membrane. Another method for the isolation of the outer


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5

membrane uses its relatively low solubility in nonionic deter-

gents. The cytoplasmic membrane is removed in this case by

a washing procedure with Triton X-100 or sodium lauryl

sarcosinate in the presence of MgZf [40]. It has to be noted,

however, that in both cases a considerable fraction of

phospholipids and lipopolysaccharides may be lost from the

outer membrane during the washing procedure with deter-

gents.

Enzym atic degradation

o f

the porin-murein com plex

Porins, tightly associated with the peptidoglycan layer, can

easily be isolated by the method proposed first by Rosenbusch

11171. After breaking the cells, the cell envelope fraction is

solubilized in 2% SDS at 60°C. The insoluble material con-

tains the peptidoglycan with the covalently bound lipoprotein

and the associated porins. The porin can either be separated

from the peptidoglycan by digestion of the peptidoglycan

layer using lysozyme and trypsin [7, 1141 or in a more elegant

way by a salt extraction method 1118- 1201 (see below). Boil-

ing of the porin-peptidoglycan complex for 5 min in SDS

leads also to the dissociation but it denatures the protein.

Salt extraction method

A more recent method uses a salt extraction method for

the preparation of trimers first proposed by Nakamura and

Mizushima [118]. The method was extended by Tokunaga et

al. [119] and a recent excellent description has been given by

Nikaido [120]. A short scheme of the procedure is as follows.

The gram-negative bacteria are grown under normal culture

conditions at 37°C; the addition of 0.5% glucose represses

the expression of the maltoporin (LamB); after washing the

cells they are resuspended in a small volume and passed three

times through a French pressure cell; the cell envelope con-

taining the cytoplasmic membrane, the peptidoglycan, and

the outer membrane are pelleted by cenrifugation. The SDS-

soluble components

of

the outer and the inner membrane

are removed by washing the pellet with a buffer solution

containing 2% SDS at elevated temperature (up to 60°C).

After centrifugation the pellet should contain only peptido-

glycan and the associated protein. Considerable amounts of

non-porin proteins in the pellet can be removed by a repetition

of the SDS-wash step followed by centrifugation. The

peptidoglycan-associated protein preparation is resuspended

at 37°C in a buffer which contains as essential components,

besides 1 SDS, 0.4 M NaCl and 5 mM EDTA. The super-

natant of a subsequent centrifugation step is applied to a

Sepharose 4B or a Sephacryl S-200 superfine column. The

porin trimers are eluted from the column with the same buffer.

Ion-exchange columns (for example DEAE-Sephacel) can

also be used for the purification of porins because the pore-

forming complexes adsorb to the column material according

to their net surface charge [12]. For this, the large amount of

salts introduced by the salt extraction have first to be removed

by dialysis against low-ionic-strength buffer. Also, if SDS was

used for extraction, it is exchanged with non-ionic detergents

such as Triton X-100. The column is first washed with low-

ionic-strength detergent buffer. The porins are then eluted

from the column with linear salt gradients ranging over 50-

500 mM a t a given pH. The salt and detergent content of the

eluted protein can be decreased by another dialysis procedure

but this is not essential for the pore-forming activity which

remains constant for several months at 4°C in a refrigarator

or frozen in a freezer. The protein solutions can also be

lyophilized in many cases without any loss of the pore-forming

activity. In lypophilized form the protein remains active for

at least a year stored in a freezer at -20°C.

Saline extraction of whole cells

This method utilizes the susceptibility of the outer mem-

brane of photosynthetic bacteria towards 150 mM NaCl

solu-

tion [121, 1221. Freshly harvested cells are shaken for 2 h

in 150 mM NaCl at 37°C. The supernatant obtained after

centrifugation is dialysed against distilled water and lyo-

philized. Part of the material obtained is porin. The rest is

composed of lipid and lipopolysaccharide and occasionally of

other outer membrane proteins [121, 1221. The porin may be

purified as described above. It has the same pore-forming

properties as porin isolated by the salt extraction method

described above [123].

MODEL

M E M B R A N E

STUDIES W I T H P O R I N PORES

The permeability properties of porin pores for hydrophilic

molecules can either be studied

in vivo

or

in vitro. In vivo

experiments allow the study of the pores in their natural

environment which means that the substrates can interact

with the whole uptake system including periplasmic binding

proteins and periplasmic enzymes. The properties of the porin

pores were studied in vivo using the p-lactamase activity locat-

ed in the periplasmic space [124, 1251 and the uptake of

radioactively labeled solutes into the cells [126]. Both methods

only yield precise information on outer membrane per-

meability if the flow of the p-lactam antibiotics across the

outer membrane is rate-limiting [127], i.e. the p-lactamase

activity is

so

high that the enzyme is not saturated and the

gradient of the p-lactam antibiotics is established across the

outer membrane. Similar considerations apply to the uptake

studies. In these cases, it is necessary that the solutes are

actively taken up into the cell and that their gradient is again

established across the outer membrane which is

only

possible

at very low solute concentration because of the high affinity

of most inner membrane transport systems [127].

In

vitro

studies allow much better control of the experimental con-

ditions. On the other hand, the possibility of artifacts exists

(see below).

The vesicle permeability assay

This method was the first to be used for the identification

of the pore-forming proteins of the outer membrane of

E .

coli

and S. typhimurium 17, 113, 1141. The basic principle of the

vesicle permeability assay is as follows. Liposomes are formed

in a buffer solution containing two radiolabeled solutes (for

example [14C]sucrose and [3H]dextran) from bacterial or

other lipids in the presence of protein fractions or purified

porin. In the first investigations lipopolysaccharide was also

added at a molar ratio of 1 8 to the lipid. However, its pres-

ence is not essential for the pore-forming activity of the porins

and the formation of the liposomes [120]. Vesicles or lipo-

somes are formed by shaking the suspension, followed by mild

sonication in a water-bath-type sonicator. During liposome

formation both radiolabeled substances are entrapped. Sub-

sequently, the liposomes are passed through a Sepharose 4B

column and elute just after the void volume. The [3H]dextran

is retained in the liposomes, whereas the low-molecular-mass

['4C]sucrose may leave the liposomes through the pores dur-

ing the chromatography. In this case, it is retarded on the


6/19

6

column and the initial

1 4 C/3 H

atio is decreased and indicates

the permeability of the pores for the low-molecular-mass sol-

ute. Similar experiments can be performed with 14C-labeled

solutes of different molecular masses. The 14C /3H atio mea-

sured as a function of the molecular mass of the solutes allows

the evaluation of the exclusion molecular mass and of the

effective diameter of the pore [7, 113, 114, 1281.

Experiments of this type have been used to measure the

exclusion molecular mass of porins of

E .

coli, of

S .

t y p h i m u r i u m , of P . aeruginosa and of Haemophilus i n f l ue nza

[7, 113, 114, 128, 1291. The exclusion limits of the porins of

E. col i and of

S .

t y p h i m u r i u m were between 550-800 Da

which would be consistent with diameters around 1.1-

1.3 nm. The diameter of the protein F pore of P. aeruginosa

outer membrane appears to be much larger because solutes

with molecular masses of more than 5000 Da could permeate

the pore [130] (Fig. 3). All these diameters were found to be

consistent with in vivo studies, although the exclusion limit of

the outer membrane of P. aeruginosa was a matter of debate

[131, 1321. Furthermore, the role of protein F as a porin was

questioned [132].

The vesicle permeability assay can be termed an all-or-

none method. This means that it does ngt allow the measure-

ment of the kinetics of the permeation of different substrates

relative to one another. The time needed for the elution of the

column is so long that it is not possible to see any difference

between the rates of permeation of different solutes or any

specificity of the pore for one class of solutes. This all-or-none

assay may also lead to artificial results if more than one porin

is present in the protein preparation. In this case only the

larger pore can be detected even if it was only a small fraction

of the total number of pores. The presence of only one larger

pore per liposome would be enough to swamp the effects of

many small pores. This effect presumably played a role in the

first characterization of LamB incorporated into liposomes

[129]. Another possible artifact may arise if the permeability

of only one solute is measured by means of the assay described

above. Denaturated pore-forming complexes could in this

case cause a certain permeability of the liposomes for

hydrophilic solutes without the formation of a defined dif-

fusion pathway. However, the existence of a defined exclusion

limit may be questionable in this case.

The

liposome

swe l l i ng m e thod

This method was introduced in the study of the per-

meability properties of porin pores by Nikaido and coworkers

[120, 133- 361 following a method established by Bangham

et al. [137, 1381. Lipid and protein are dispersed in a buffer

first by shaking and then by sonication in a bath-type

sonicator. The buffer contains a large-molecular-mass dextran

or stachyose to maintain a certain osmolarity (around

40 mOsmol). The stachyose or the large-molecular-mass

dextran are not rendered permeable by the porin and are

entrapped inside the liposomes. The liposomes are added

under rapid mixing to an isotonic solution of a test solute. If

this solute can penetrate the pores, the tptal concentration of

solutes inside the liposomes increases because stachyose or

dextran are retained. Liposomes behave like an ideal

osmometer which means that water molecules permeate into

the liposomes according to the osmotic gradient. This swelling

process can be detected by a decrease of the average refractive

index of the liposome suspension, i.e. by a measurement of

the absorbance (see Fig. 4). The initial swelling rate can be

used as a measure of the penetration rate of the test solute

0.3 1.0 10

50

Molecular ma s s

k Da)

Fig.

3.

Exclusion limit fo r saccharides in vesicles reconstituted f ro m

outer membranes of P. aeruginosa

a)

and S . typhimurium LT2

M I

0 ) s obtained fr o m the vesicle permeability assay. The results imply

that porin of P. aeruginosa outer membrane has a larger exclusion

limit than porin from S . typhimurium. (From [258] with permission)

0.51

0 1 2 3 0 1 2 3 4

Time after mixing (min)

Fig.

4.

Absorbance tracings in a liposome-swelling experimen t recorded

at a wavelength

of 450

nm with liposomes reconstituted fr om lipid and

porin from E. coli

B.

The figure shows the results obtained after

dilution into L-arabinose (Ara), N-acetyl-D-glucosamine (GlcNAc),

sucrose (SUC),and buffer (5 m M Tris/HCl, pH

7.4) with

liposomes

reconstituted in the presence (right) and in the absence (left) of

porin.

(From [ I 331 by copyright permission of

the

Rockefeller

University

Press)

100

50

g 20

150

200

250 3 0 0

350

M o le cu la r m a ss

Fig. 5. Relative rates of permeation of different sugars into phospho-

lipid-porin liposomes. Different

sugars

(from top to bottom) of

150 kDa (1,-arabinose),

180

kDa

(D-galactose,

D-fructose,D-mannOSe,

D-glucose), 221 kDa (N-acetyl-D-glucosamine),

262

kDa

(2,3-

diacetamido-2,3-dideoxy-D-glucose)nd

342 kDa (sucrose,

meli-

biose, maltose, lactose). (From [133]

by

copyright permission of the

Rockefeller University Press)


7/19

7

Table 1. Physical properties and pare cha racteristics

of

general dif fusion po rin pores

The corresponding references are given after each result. The complete names of the organisms are: Aeromonas salmonicida, Anabaena

variabilis, Brucella a bortu s, Campylo bact er,fetu s, Cam pylob acter ejun i, Haemo philu s influenza, Klebsiella pneu mon iae, Legionella pneumophilia,

Neisseria gonorrhoae, Paracoccus denitrificans, Proteus mirabilis. Pseudomonas aeruginosa, Rhodobacter capsulatus (formerly

Rhodopseudomonas capsulata [255]), Rhodobacter sphaeroides (formerly Rhodopseudomonas sphaeroides [255]), Salmonella typhimurium,

Spirochaeata aurantia, Yersinia pestis

Porin Bacterial species Molecular mass Diameter derived from

exclusion liposome single-channel

limit swelling- conductance

General diffusion porins

OmpF

OmpC

PhoE

Lc

Lc (HK 253)

NmpC

K

OmpF

OmpC

OmpD

PhoE

37-kDa

40-kDa

PhoE

PhoE

I

F

E

MOMP

43-kDa

46-kDa

32-kDa

40-kDa

Group

2

33-kDa

43-kDa

47-kDa

Porin

42-kDa

40-kDa

Escherichia coli

Salmonella tyohimurium

typhimurium

Enterohacter cloacae

K. pneumoniae

N . gonorrhoae

P. aeruginosa

Y.

pestis

L. pneumophila

C.

ejuni

S. aurantia

A .

salmonicida

P. mirabilis

H . influenza

B. abortus

P. denitrgicans

R

.

capsula tus

R

.

sphaero ides

A. variabilis

C . e t u s

kDa

32.7 [90, 2421

36.0 [91]

36.8

[9]

36.5 [21 I]

40 [242]

40 [243

39.3 [149, 1191

39.8 [149, 1191

38.0 [149, 1191

36 [245]

37 [246]

40 [246]

39.5 [211]

37 [212]

37 [212]

34 [141 , 2241

39 [215]

33 [247]

-25 [258]

43 [248]

46 [248]

32 [249]

42 [250]

40 [251]

40 [128]

40 [I471

33 [252]

43 [143]

47 [I221

nm

600

[I141

-

-

-

-

-

800

[113]

800 [I 131

800 [I131

-

-

-

-

-

5000 [130]

-

-

-

-

-

1400 [128]

700 [I471

-

-

-

1.2 [134]

1.1 [I341

1.1 [I341

-

-

-

-

-

[257]

- [257]

-

1.2 [241]

1.6 [241]

-

1.0 [141]

2.0 [144]

-

-

-

-

-

1.4 [128]

1.2 [I471

1.7 [252]

1.6 [I431

1.2 [I221

-

1.2 [I581

1.1 [158]

1.1 [I581

1.3 [242]

1.0 [158], 1.4 [I501

1.1 1243, 2441

1.2 [I481

1.3 [I481

1.3 [148]

1.1 [240]

-

1.1 [170]

1.1 [I701

1.1 [156]

2.0 [155]

1.1 [I581

1.0 [258]

0.9 [248]

0.9 [248]

2.3 [249]

I O

[250]

1 O”

1.0 [I571

1.7h

1.6 [I231

1.6 [253]

-

Specific porins Specificity

LamB

E. coli

Tsx

E. coli

D1 P. aeruginosa

P P. aeruginosa

LamB

S. typhimurium

48.0 [I721

31.4‘

specific for nucleosides

[12]

46

[115, 1961 specific for sugarsd [I151

4 8 [ l l ]

specific for anions

[lo , 11, 1591

44 [254] specific for sugars (?)

specific for sugars [9, 135, 145, 154, 1611

a Schmid,

A, ,

Bauer,

K.,

Benz, R., unpublished results.

‘

Bremer,

E.,

Martinussen,

J.,

Valentin-Hansen, P., unpublished results.

Benz,

R. ,

Schmid, A. , Rosenberg, E. Y., Nikaido,

H.,

unpublished results.

Nikaido, H., personal communication.

through the porin pore. Using the same liposome preparation,

the pentration rate of different solutes can be compared

striking of the pore edge and by the friction of the pore

interior) is given by

-

(Fig: 5). For a general diffusion pore, i.e. a water-filledcyl in-

der, the logarithm of the relative rate of permeation is a linear

A /

=

[l

(a /r)]’[1-2 .104(u /r)

+

2.09

-

.95 (u/r)’]

,”,

function of the molecular mass of the solutes, which allows

1)

where A is the effective area of the pore with the total area

A,, r and a are the radii of the pore and of the solute respec-

tively. The use of the liposome swelling assay allowed a mean-

an estimation of the effective diameter of the pore according

to the theory of Renkin [139].According to this theory the

total hindrance to diffusion of the solutes (caused by the


8/19

8

ingful comparison to be made between different porin pores

of

E .

coli outer membrane [I341. Furthermore, this method

was used to demonstrate the specificity of the LamB channel

for maltose and maltodextrins compared to other saccharides

[9, 1351 and to characterize the propert ies of different LamB

mutants [140]. The properties of a variety of porin pores

of

different gram-negative bacteria were also investigated using

this method [128, 141 -1441. The pore diameter varied be-

tween 1 I - .2 nm (see Table 1).

Although the liposome swelling assay is far more precise

than the vesicle permeability method, i t may possibly have a

number of complications. The theoretical evaluation

of

the

Renkin equation as used for the calculation of the pore diame-

ter is based on a number

of

simplifications [139]. In the case

of the experiments with porin pores, these may not be justified.

A second problem is the poor time resolution which is limited

by the stirring that is necessary after the addition of the

liposomes to the test solute [133]. Information may be lost

because the early decay of the absorbance cannot be recorded.

This may be the reason for the non-linear dependence of the

swelling rate on porin concentration which was observed for

OmpC of E . coli K12 [134]. A final difficulty is estimation of

the effective diameter of the test solutes. Disaccharides of

identical molecular mass caused a substantial variability of

the swelling rates (3 -7-fold) [134]. This variability may be

caused by differences in the hydration shell of the solutes. The

large-molecular-mass solute (dextran or stachyose) may also

create problems. The osmolarity of the large-molecular-mass

dextran was difficult to adjust in the experiments with LamB

[9].

Stachyose, on the other hand, which was also used in these

experiments [140], binds to the binding site inside the LamB

channel [145]. This means that the relative rate of permeation

of different sugars through LamB mutants may be influenced

by the binding of stachyose.

Lipid hiluyer

experiments

The swelling experiments with reconstituted liposomes

provide excellent information about the size of porin pores.

Less detailed information is available on the selectivity of

the pores. This is because membrane potentials occur in the

liposome swelling assay. These are created by the use of

charged test solutes which may greatly complicate the analysis

of

the experimental data [134].

More detailed informat ion about the pore interior and the

pore selectivity can be obtained from lipid bilayer exper-

iments. Three different method have been used successfully

to reconstitute porin pores into lipid bilayer membranes. In

the first

of

these, detergent-solubilized porin was added di-

rectly to the aqueous-phase-bathing solvent containing

(‘painted’) lipid bilayer membranes [146]. In the second

method, solvent-free (‘folded’) membranes were formed from

vesicles reconstituted from lipid and protein according to the

Montal-Mueller method [147]. The third method inserted the

porin pores into lipid bilayer via fusion of reconstituted ves-

icles with membranes [148]. This methodhas only occasionally

been used. It will not be further described here.

The simplest method consists of the addit ion of detergent-

solubilized porin, in very small concentration

(1

- 00

nglml),

to the aqueous phase bathing a black lipid bilayer membrane.

After an initial lag of some minutes, presumably caused by

the diffusion of the protein through unstirred layers, the con-

ductance (i.e. membrane current per unit voltage) of the mem-

brane increased by many orders of magnitude within about

20-30 min [14, 149,

1501.

Only a slight additional increase

400

p s

A

1 *in

Fig.

6 .

Single-channel recording

of

a

diphytanoylglycerophospho-

cholineln-decane

in

the presence of

20

nglmlprote in

P ojP.

aeruginosa

a n d 3 0

mM

KCI

in the ayueousphase.

A

voltage

of 50 m V

was applied

through calomel electrodes with salt bridges; temperature

= 25‘ C

(as compared with the initial one) occurred after that time.

The conductance increase was found to be dependent on the

type of porin used for the reconstitution and on the

membrane-forming material. In membranes from oxidized

cholesterol, a completely unphysiological lipid, porins usually

showed about 100-fold more pore-forming activity than in

membranes f rom normal phospholipids [14,148]. The reason

for the dependence of the reconstitution rate on the

membrane-forming lipid is not clear. It may arise from small

structural differences between the membranes. Using this

method, the surface area of the membrane is in principle not

limited. Multi-channel experiments (i.e. experiments with a

large number

of

channels) were usually performed with sur-

face areas between 1 and 2 mm2, whereas the membrane areas

were below 0.1 mmz in the case of the single-channel record-

ings [146, 1511. A maximum of between l o 6 - lo8 pores/cm’

could be incorporated into lipid bilayer membranes using this

method. This means that the reconstitution of porin pores is

not a rare event.

On

the other hand, it is obvious that this

number is far below the pore density in the outer membrane

of gram-negative bacteria (1012 pores/cm2 [149]).

A n

alternative method for the reconstitution of porin

pores was used by Schindler and Rosenbusch [147, 1521. Ves-

icles from lipid and protein were spread on the surface of the

aqueous phase on both sides of thin teflon foil which has a

small circular hole (about 100 pm diameter) above the initial

water levels. The surfaces of both aqueous compartments were

covered by monolayers. The water levels on both sides of the

membrane are now raised and a folded lipid bilayer membrane

is formed across the small hole.

So

far it is not clear how the

porin pores are incorporated into the lipid bilayers. In the

early investigations the pores were always activated as mul-

tiples of the single conductance unit at large voltages, presum-

ably because of the fusion of porin-containing vesicles with

the membrane [147]. More recently, especially in the case of

PhoE [153] and LamB [154], the pores were activated as single

conductive units. The diameter of folded membranes is limited

to about 100-200 pm (area about 0.07 mm’).

Both experimental approaches allow the resolution of

single channels [14, 1521. This is one of the main advantages


9/19

9

Table 2. Average single-channel conductance of Rhodobacter capsu-

latus porin in differen t salt solutions

The solution contained

10

ng/ml porin and less than

0.1

Fg/ml SDS

or Triton

X-100.

The pH of the aqueous salt solutions was around 6

unless otherwise indicated. The membranes were formed from

diphytanoyl glycerophosphocholine/n-decane. Temperature

=

25°C;

V , =

25 mV. (Taken from [123])

Salt c A A / .

KCI

RbCl

NaCl

LiCl

K z S 0 4

MgClz

CaCI,

KCH3C02 (PH

7)

Tris/HCI

K/Hepes (pH

8)

Tris/Hepes (pH 8)

M

0.003

0.01

0.03

0.1

0.3

1

o

3.0

1

o

1

o

1 o

0.5

0.5

0.5

1 o

0.5

0.5

0.5

nS

0.012

0.038

0.12

0.35

1.1

3.3

9.5

3.4

1.8

1.4

2.1

1.3

I

.8

2.3

0.52

0.83

0.12

nm

0.29

0.29

0.29

0.29

0.29

0.30

0.35

0.30

0.21

0.20

0.28

0.20

0.23

0.33

0.17

0.28

0.18

of the lipid bilayer assay. The experiments were performed in

the following way. Small amounts of porin

(1

-

0

ng/ml) were

added to the aqueous phase bathing a black membrane of

small surface. Subsequently, a step-wise increase of the current

through the membrane was observed. Fig.

6

shows an exper-

iment of this type with protein P of P.

aeruginosa [159].

All

conductance steps were directed upward. Closing events were

in

general only rarely observed. This means that the lifetime

of porin pores from gram-negative bacteria usually exceeded

1

min. The only pores with a much shorter lifetime (around

50

-

00

ns) were those observed in the presence of total outer

membrane of

P.

aeruginosa [155],

although the absolute level

of the pore conductance (i.e. pore current per unit voltage)

was the same as with purified protein F.

The single-channel conductance, A , of many but not all

porin pores was a linear function of the specific conductance

ci

of

the bulk aqueous phase. This means that, despite a large

variation of the average single-channel conductance, the ratio

A / a

varied

on ly a

little (see Table

2).

The single-channel con-

ductance

of

these general difusion pores (see the next section)

can be used to calculate the effective diameter of the porin

pores. Assuming that the pores are filled with a solution of

the same specific conductivity as the external solution and

assuming a cylindrical pore with a length

1

of

6

nm, the effec-

tive pore diameter

d (

=

2r)

and the cross-section can be calcu-

lated according to the equation

[146]:

A

=

m r 2 / 1 .

The diameter of the porin pore of Rhodob'acter

capsulatus

(compare Table

1)

may be calculated to be about

1.6

nm from

the single channel conductance

(0.40

nS) in 0.1

M KC1

and

the corresponding specific conductance of the aqueous phase

(ci

= 14

mS/cm)

[123].

As already pointed out, the liposome swelling method

cannot be used to obtain detailed information on the selec-

tivity properties of porin pores. Experiments with lipid bilayer

Table 3. Zero-current membrane potentials

V,

for diflerent porins

in

the presence oJ u tenfold KCl gradient

Species Porin

V P , / P a

Reference

mV

E.

coli OmpF (B)

27

3.9

OmpF (K12) 28 3.8

OmpC

50

26

K

46

1.6

PhoE -24 0.30

NmpC -26 0.21

ti 581

OmpC 54 41 11581

OmpD

0

4 23 .54

~ 5 8 1

245]

S . ~ ~ p ~ i ~ u r j u ~mpF 46 14

PhoE

P . aeruginosa P -59


10/19

10

The basic advantage of the lipid bilayer method is the

measurement of molecular events. Every step of Fig. 6 reflects

the conductance due to insertion of one conductive unit, i.e.

of one protein

P

trimer of

P.

aeruginosa

into the membrane.

This allows an easy check

of

the purity of the porin prep-

aration and of the integrity of the pore-forming units after

the isolation and purification of the porin [161]. The interpre-

tation of single-channel studies requires a statistically signifi-

cant number of measurements. This means that the measure-

ment of only ten channels is not sufficient to characterize a

porin pore. Instead, hundreds of pores must be studied in

order to avoid identifying random fluctuations, which may

occur in a limited number of events in single-channel measure-

ments, as characteristic properties of the pore [162].

GENERAL DIFFUSION PORINS

Most porin pores of gram-negative bactrial outer mem-

brane that have been studied in vivo or in vitro are general

diffusion pores. Table 1 summarises the properties of all the

general diffusion pores that have been characterised to date

using one of the three methods described in the previous

section. The diameter of the different porin pores varies f rom

about

1 0

nm to 2.2 nm. This means that the diameter of the

porin pores is much larger than that of the ‘ion’ channels in

nerve and muscle membranes. Reconstitution experiments

have shown that the porins of

E.

coli outer membrane are

permeable to trisaccharides and tetrapeptides, i.e. the pores

allow the penetration of nutrients and ions through the outer

membrane

[ 5 ,

14, 17, 1601. Lipid bilayer experiments have

demonstrated that the ions move inside the porin pores similar

to the way in which they move in the aqueous phase. This

finding allowed the calculation of an effective radius on the

basis of Eqn (2). It is interesting to note that the radii as

calculated from the different methods described in the preced-

ing section show satisfactory agreement (compare Table 1).

Only in the case of the major outer membrane protein of

Huemophilus influenza did the radii estimated f rom the differ-

ent methods not coincide [157]. The reason for this is not

completely clear. It has to mentioned, however, that the

unheated porin did not run as oligomers on SDS/

polyacrylamide electrophoretograms and that different lipids

were used for the reconstitution experiments. On the other

hand, it cannot be excluded that the explanation proposed by

the authors (i.e. that the branching of the channel is respon-

sible for the difference) may be valid in this case [157, 1601.

I t

has already been mentioned above that in vivo and

certain in vitro studies give only vague information about the

selectivity of the porin pores [134, 1631. The swelling rate of

PhoE-containing liposomes was only a factor of two larger,

in

the case of negatively charged solutes than that of a neutral

solute with a similar molecular mass [134]. The lipid bilayer

assay allows good access to both sides of the membrane and

it is easy to establish a salt gradient across the membranes

and to perform zero-current potential. measurements [151,

1581. The use of salts with equally mobile ions like K + and

CI-

(limiting molar conductivities 73.5 mS/M and 76.4 mS/

M [164]) yields precise information about the selectivity of the

porin pores. Other salts such as LiCl and KC H3 CO0 can be

used to test whether the pore is a general diffusion pore

or not. Li’ and acetate- have similar aqueous mobilities

(38.7 mS/M and 40.9 mS/M [164]) but smaller mobilities than

K + and C1- (see above). This means that a general diffusion

pore must have a larger cation selectivity in KCI than in LiCl

and a smaller cation selectivity in KCI than in KCH3CO0.

Table 3 shows that these considerations are correct for the

general diffusion pores but not for the anion-specific porin

protein

P

from

P.

aeruginosa

outer membrane.

Is

PhoE

a specific porin?

PhoE has been shown to be effective for the uptake of

phosphate, polyphosphate and other negatively charged

sol-

utes [126, 163, 165-1681, The general diffusion pores listed

in Tables 1 and 3 have been accepted as such, except in the

case of PhoE pore of E. coli outer membrane. In this case some

authors have proposed the existence of specific phosphate or

polyphosphate binding site, because in in vivo and in in vitro

experiments, polyphosphate could block the pore [126, 153,

1691. However, more recent investigation of this ‘specific’

inhibition effect showed that it only occurred if the divalent

cation Mg2 and polyphosphate (or other polyvalent anions

like citrate) were present in the aqueous phase [170, 1711. The

inhibition

of

the pore function in the presence

of

both ions

can be explained by complex formation inside the pore, which

reduces the flow

of

solutes. Polyphosphate alone is not able

to reduce the permeability of the PhoE pore in the in vivo or

in vitro

experiments but changed its selectivity from anion to

cation selectivity, because the excess of positively charged

groups inside the channel

is

converted into an excess of nega-

tively charged groups by the presence of the polyphosphates

[171].

SPECIFIC PORINS

L am B o f E. coli

Transport of maltose and maltodextrins in E . coli is me-

diated by a transport system that is composed of several

components including LamB (see above). The

lumB

gene

codes for the receptor of phage /z in the outer membrane

(molecular mass 48 kDa [172]) which was found to be

important for the maltose uptake because

lamB

mutants were

found to be impaired in maltose transport when the concen-

tration of this sugar in the growth media was less than 0.1 mM

[173]. This result indicated an important role of the LamB

protein in the maltose uptake machinery. Early reconstitution

experiments with purified LamB suggested that

it

formed a

general diffusion pathway similar to the general diffusion

pores but with a larger diameter [129,174]. More recent inves-

tigations have shown that LamB (also known as ‘maltoporin’)

has a defined substrate specificity [9, 135, 145, 154, 161, 1751

and that i t is able

to

discriminate between disaccharides of

identical molecular mass, for example between sucrose and

maltose [9, 1351 (compare Table 4). Furthermoe, the per-

meation of glucose through LamB could be blocked by

maltodextrins [I 351, and LamB was identified as a cell-surface-

binding site for sugars in vivo [8, 1761. LamB is obviously not

a general diffusion pore which means that the early reconsti-

tution data and the finding that LamB acts as a general dif-

fusion pore in vivo may be caused by an unidentified general

diffusion pore. However, the permeation of small solutes with

molecular masses less than 300 Da through LamB seems to

be possible.

The exact function of the MalE protein (the maltose bind-

ing protein) is not completely clear at present although it is

essential for maltose uptake [177, 1781. It has been reported

that MalE binds to LamB [179] and that this is important for

voltage-dependent pore function [1621. However, more recent

investigations

of

the interaction between MalE and LamB in


11/19

11

Table

4. Stability constants.

K ,

(half-saturation constant K,

= l / K )

, fbr

fh e binding of different sugars to the La m B channel, and relative

ra te q fpermeation , P,

of

the sugars through L am B relative to that

01

maltose

The data for the relative rate of permeation (adjusted to

100

for

maltose) were taken from

[9].

The L amB-containing liposomes were

added

to

buffer solutions containing

40

m M of the corresponding test

sugars. The da ta for the sugar binding were taken fro m [145]. K (=

1

K m )was calculated from titration experiments similar to tha t shown

in Fig. 8 for the

Tsx

channel

Sugar K S P

M- ' mmol / l

Maltose 100 10 I00

Maltotriose

2 500

0.40 66

Maltotetraose 10000

0.10 19

M

altopentaose

17000 0.059 -

Maltohexaose 15000 0.067 -

M altoheptaose

5000 0.067 2.5

Trehalose

Lactose

Sucrose

Gentibiose

Melibiose

Cello biose

D-Glucose

L-Glucose

D-Galactose

D-Fructose

D-Mannose

46 22 76

I 8 56 9

67 15 2.5

250

4.0

42

180 5.5

33

6.7 150 13

9.5 110 290

22 46

24 42 225

1.7 600 135

6.3 160 160

-

Stachyose

20 50 < I

Raffinose

46 22

vivo

[180] and

in

vitro [154, 1611 have shown that this modu-

lation of the pore function is likely to be an artifact.

Lipid bilayer experiments in the presence of purified LamB

resulted in a strong conductance increase caused by the forma-

tion of small ion-permeable pores with a 10-times-smaller

single-channel conductance (160 pS) than OmpF under other-

wise identical conditions [145, 154, 1611. The ion permeation

through the pores could be blocked completely by the addition

of increasing concentrations of sugars, i.e. by titration exper-

iments [145, 154, 1611. Because of the absence of any interac-

tion between binding site and ions, the half-saturation con-

stant could be calculated from the t itration experiments. The

structure of the binding site was studied by the investigation

of

the binding of large variety of sugars (included in Table 4).

The data for the binding of the sugars allowed the deduction

of a rough steric model of the binding site [145]. The half-

saturation of the binding of the maltodextrins decreased with

their chain length, but was saturated after five residues

(Table 4, see also Fig. 7). This result makes it very likely that

the binding site has a length of approximately five glucose

residues, corresponding to a maximum length of 3 nm. Mu-

tations of LamB [181] can change the half-saturation constant

of the binding between the sugars and the site in the channel.

This is shown for a mutant (BW 1333,Trp74 Arg; R. Benz,

A .

Schmid, T. Ferenci and T. Nakase, unpublished results) in

Fig.

7.

The effect of the mutation may be explained by a

reduction of the number of binding sites inside the channel.

The maltodextrins apparently move through the channel in

single file and block the channel for the passage

of

ions. From

this it can be concluded that the channel is only 0.7 nm wide,

0

2

4

6

8

glucose

res idues

Fig. 7. Binding constants of glucose and diffe rent maltooligosaccharides

to the binding site inside the La m B channel

[I451

and inside La mB

m u ta nt ( B W 1333 Trp74 Arg ) as derived fr o m titration of ion

conductance with increasing concentrations of sugars.

wt, wild-type

La mB; BW

1333,

mu tant (Benz, R., Schmid,

A

Ferenci, T., Nakae,

T., unpublished results). Note that the half-saturation constant

of

Lam B for maltohexaose is identical to th at derived by a completely

different m ethod

[I761

whereas the diameter of the general diffusion pores of E .

coli outer membrane is of the order of 1.1 nm (see Table 1).

Further studies on mutant LamB and of the investigation of

the influence of the mutations on the sugar binding may give

further insight into the three-dimensional structure of the

binding site and may help to decide the question of which

amino acids are involved in the binding of the sugars [140,

181

-

841. The cation selectivity of LamB makes it very likely

that carbonyls are present inside the channel [145]. This means

that hydrogen bonds are presumably responsible for the bind-

ing of the sugars. It is interesting to note that the data of

Table 4, i.e. the combination of the relative swelling rate and

the half-saturation constant, can be used for the evaluation

of mechanism of sugar permeation through the LamB channel

on the basis of a one-site, two-barrier model [145]. The pro-

cedure is not discussed here and the interested reader is refered

to the original literature [145]. The result of the analysis is

similar to that shown in the next paragraph for Tsx protein

from

E.

coli

outer membrane. The important advantage

of

the binding site is the high turnover of molecules at very low

substrate concentration, although the pore saturates at high

substrate concentration. In contrast to this, the

flow

of mol-

ecules through a general diffusion pore is always a linear

function of the substrate concentration.

T sx

o E. coli

The pore function of another outer membrane protein,

the Tsx protein, was recently established [12]. This protein

was shown to serve as the receptor of colicin K and bacterio-

phage T6 [186]. It is known that Tsx protein

is

part of the

nucleoside uptake system because mutants lacking this protein


12/19

12

1.0

I

1

-

[Adenos ine] mM)

10

rnin

Fig. 8. Titrution o the Tsx-induced membrane conductance with increasing concentrations

of

adenosine. The membrane was formed from

diphytanoyl

glycoerophosphocholine/n-decane

in 1

M

KCI

and 200 ng/ml Tsx of

E. coli .

V

=

50 mV;

temperature

=

25°C

are impaired in the uptake of several nucleosides [187-1891.

The specificity of the nucleoside-specific pathway in the outer

membrane seems to be very high because the rate of uptake

of adenosine and thymidine was strongly reduced in these

tsx-

mutants whereas the uptake of cytidine was similar in

tsx’ and t s x - strains. Similar to the situation described above

for LamB and maltose uptake, transport by Tsx protein be-

comes rate-limiting at nucleoside concentrations below about

Tsx protein is not murein-associated like the ‘normal’

porins OmpF and OmpC and

i t

is only a minor outer mem-

brane protein under normal growth conditions. Furthermore,

it was found that the presence of SDS is deleterious for the

pore-forming activity in reconstitution experiments and for

the phage inactivation [12]. Tsx was isolated and purified from

outer membranes of an overproducing E.

coli

strain using the

detergent Triton X-100. It is interesting to note that, no matter

what the solubilization temperature, Tsx has the same molec-

ular mass of 28 kDa on SDS/polyacrylamide electropho-

retograms [12] whereas its correct molecular mass is

31.418 kDa

(E.

Bremer,

J .

Martinusson, P. Valentin-Hanson,

unpublished results). This observation is completely different

to the behavior of outer membrane porins of

E. coli

which

show temperature-dependent mobility on SDS gels as a conse-

quence of their SDS-resistant trimeric structure.

So

far it

remains unclear if the active Tsx channel is also an oligomer.

Reconstitution experiments with purified Tsx showed that it

is able to increase the conductance of lipid bilayer membranes

by many orders of magnitude [12]. This conductance could

be inhibited by the addition of nucleosides to the membranes

(Fig. 8) and a half-saturation constant could be calculated

for the binding of the nucleoside to the membrane from a

Lineweaver-Burke plot of the data of Fig.

8

(Fig. 9). This

result indicated that Tsx protein formed a channel which

contained a binding site for nucleosides. Surprisingly, the

single-channel conductance in 1 M KC1 was extremely small.

It was only 10 pS which can be compared with the 160 pS of

LamB and 1.9

nS

(1900 pS) of OmpF of E. coli K12 [158].

1 PM [187- 1891.

I

llbinding

I

I

llbinding

,

Adenos ine :

K =

0.5

rnM

’

-

5 -

1

3 -

2 -

I I

I

-l/fts 0 2

6

8

10

Fig.

9.

Lineweaver-Burke

plot

qf the data given in Fig.

5 .

The half-

saturation constant for the binding of adenosine to the binding site

inside the Tsx channel was calculated using the assumption that the

channel does not conduct ions when it is occupied by a nucleoside

The titration experiments could be used for the evaluation

of the half-saturation constant for the binding of different

nucleosides to the binding site inside the pore (Table

5)

[190].

As can be seen from Table 5, the stability constant for

adenosine binding was approximately the same as for

thymidine (corresponding to a similar half-saturation con-

stant). Surprisingly, the binding of the deoxynucleosides to

the channel interior was much stronger than that of the

nucleosides which may indicate that the binding of the mol-

ecules occurs via hydrophobic interaction between site and

nucleosides. Accordingly, a saturation of the flux of

nucleosides through the Tsx channel may be expected for

increasing concentrations of nucleosides [12, 1901.

If

we as-


13/19

13

Table 5. Stability constants K,, fo r the binding ofnucleosides to th e Tsx

channel (holf'saturation constant K,

=

1 j K )

The data were taken from [190]. K was calculated from titration

experiments similar to that given in Fig. 8

Nucleoside K K ,

M - ' m M

Adenine

500

2.0

Adenosine

2 000 0.50

Deoxyadenine

7100

0.14

Thymine

112

5.8

5 -Deoxyth ymidine 20000

0.050

Thymidine 5

000 0.20

l

0.01 0.1

1.0

10

100

=-.n./mM

Fig.

10. Flow

ofadenosine through Tsx and through a general diffusion

pore us

function qf

rhe

nucleoside concentration

on one

side

o j

the

pores.

The concentration on the othe r side of the pores is set to

zero.

The flow through Tsx,

4 ,

is given relative the maximum flux

For the flux of adenosine through the general diffusion pore, it was

assumed thai, at 0.1 m M , only

1%

of the flux was throu gh Tsx

sume that the concentration of adenosine on one side of the

Tsx channel is zero and that on the other side is c , the flux

4

of the substrate through Tsx as a function of

c

is given by the

following equation

:

4

=

4rnaxKc./(2+ Kc)

(4)

where is the maximum flux at very high substrate concen-

trations and

K

= l/ Km s the stability constant for the binding

of adenosine

to

the binding site (Table

5).

The flux saturates

at high substrate concentrations as shown in Fig.

10

for

adenosine. On the other hand, the flux of adenosine through

a general diffusion pore is linearly dependent on the concen-

tration (Fig. 10). This means that the relative throughputs of

the two types of pores is dependent on the substrate concen-

tration. Fig.

10

clearly shows that the flux of adenosine at

high concentrations through a general diffusion pore can ex-

ceed that through the specific Tsx channel. This is in spite of

the fact that in 0.1 mM adenosine, the flux through the general

diffusion pore is only 1 of that through a Tsx pore. This

result is consistent with the in

vivo

situation, in which the

general diffusion pores have an insignificant role at low

adenosine concentration. However, according to the data

given in Fig.

10,

we would expect the crossover of the two

throughputs curves to occur in response to an adenosine con-

centration between 10- 100pM, and not a below 0.1 pM [12].

It has been reported that

Tsx

also allows the flux of amino

acids [191]. Titration experiments with lipid bilayer mem-

branes showed that no binding site for amino acids or sugars

exists inside the channel [190]. This means that if Tsx acted as

pore for these substrates, it is a general diffusion pore for

them.

Porin

P

of Pseudomonas aeruginosa

The outer membrane of

P .

aeruginosa has very special

sieving properties which make this organism very resistant to

most antibiotics [192, 1931. Part of these sieving properties

could arise from the limited permeability of the large protein

F channel [136, 144, 1551. On the other hand, the role of

protein F as a porin has recently been questioned and the low

outer membrane permeability of P. aeruginosa was explained

by the presence of an as yet unidentified porin with a small

exclusion limit [131, 1321. Under conditions of phosphate

limitation, another porin (protein P) is induced together with

an alkaline phosphatase, a phosphate binding protein, and

some inner membrane proteins [lo, 11, 1941. The induced

system therefore has some similarities with the

p h o

system

of the Enterobacteriaceae and immunological cross-reactivity

exists between protein P and PhoE [195]. Protein P forms

highly anion-selective channels in lipid bilayer membranes

which a re the only

in vitro

system for the study of this channel.

Zero-current membrane potentials have a Nernstian slope

(corresponding to P,/Pa

<

I), and they indicated that the

protein P channel was at least

1000

times more permeable for

chloride than for potassium [lo,

11,

1591.

The conductance steps of the porin P channel were found

to be fairly homogeneous in size as compared with the steps

measured in the presence of the general diffusion pores [I591

(compare Fig. 6). Current-voltage curves linear up to 200 mV

were observed for the protein P channel. The cation present

in the aqueous salt solutions had no influence on the single-

channel conductance. In

100

mM chloride solutions this con-

ductance was always about 160 pS irrespective of the size and

charge of the cations. The change of the anion had a more

substantial influence on the single-channel conductance and

anion size could be used to probe the diameter of the protein

P channel [159].

Fig. 11 shows the dependence of the single-channel con-

ductance on the radii of different anions in

0.1

M salt solu-

tions. The diameter of the selectivity filter of the channel

was approximately between

0.5-0.7

nm, as estimated from

Fig. 11 and the observation that chlorate and bromate could

traverse the channel, whereas perchlorate and iodate could

not. The selectivity among the halides followed the Eisenmdn

sequence

AVI

which also indicated a closely spaced selectivity

filter [159]. The binding of anions to this filter accounts for

the concentration dependence of the single-channel conduc-

tance as shown in Fig. 12 for chloride and monovalent phos-

phate (pH 6) [lo] (and

R.

Benz, R. E.

W .

Hancock, unpub-

lished). The data

of

Fig. 12 could easily be fitted to the follow-

ing equation, which assumes single occupancy of the pore

[I591 (i.e. a single binding site inside the pore):

A c ) =

A,,,Kc/(I +KC).

(5)

The binding constant K (corresponding to the inverse half

saturation) and the maximum single-channel conduatance,

A,,, can be used to calculate the kinetics for the movement

of the different ions through the protein P channel. Fig. 12


14/19

14

200

100

A

PS

-

5 0 -

20

10

5 -

-

-

-

-

0.10

aa QZO

rlnm25

Fig. 11. Single-channel conductance o the protein P channel in 0.1 M

salt solutions as a func tion o the ionic radius o the anions. The

posi-

tively charged counterion was in all cases potassium

A l p s

10’

10

1

K I

I

I

I

1 10 10’ 10’ c l m M

Fig. 12.

Average single-channel conductance o protein P of

P.

aeruginosa given as a function

o

the KCI

(

x

)

and

o

the K H 2 P 0 4

concentration

0

in the aqueous phase. pH 6, temperature

=

25 “C

shows that this channel is not efficient at high phosphate

concentrations. However, under phosphate starvation (i.e.

phosphate concentrations below 1 niM) the channel conducts

phosphate better than chloride because of its smaller half-

saturation constant for phosphate (150 pM . This is another

clear demonstration of the advantage to be gained by the

possession of a binding site inside the channel for specific

substrates.

Protein

DI

of

P. aeruginosa

The addition of glucose to the grpwth medium of

P .

aeruginosa

resulted in the synthesis of an outer membrane

protein D1 with a molecular mass of 46 kDa [115, 1961. Pro-

tein D1 could be considered as a glucose-specific channel

because its presence resulted in a release of glucose from

liposomes [115]. The rate of penetration of different sugars

was measured using the liposome swelling assay (H. Nikaido,

personal communication). Comparison between protein F

and protein D1 clearly indicated that D1 is sugar-specific. The

diffusion of a variety of different sugars was facilitated by

protein D1 including that of L-glucose which binds also to

LamB [145] (and H. Nikaido, personal communication). This

indicated that sugar transport through protein D1 and

through LamB cannot be compared with that in epithelia

[197, 1981. This is because L-glucose is not transported in

epithelial cells but it binds to LamB [145] and is transported

through D1 (H. Nikaido, personal communication). The use

of a variety of different glucose analogs allowed the identifi-

cation of the groups responsible for the interaction with the

pore interior

(H.

Nikaido, personal communication).

As

in

the case of LamB, it seems that the interaction between the

sugars and the channel interior occurs with carbonyl groups

by means of hydrogen bonds.

ARE

T H E

PORIN PORES VOLTAGE-REGULATED?

Neither the vesicle permeability assay nor the liposome

swelling method allow the rapid application and measurement

of the effects of a transmembrane voltage. This is the unique

advantage of the lipid bilayer instrumentation. The first two

lipid bilayer studies of porin pores appeared as early as 1978

[146, 1471. These studies agreed on the size and selectivity of

the pores, but they disagreed about the voltage-dependence

of the pores because porin pores require a considerable voltage

for activation and inactivation when they are in folded mem-

branes [147, 1521. As a consequence, a voltage-control of the

outer membrane of gram-negative bacteria was postulated

which resulted in considerable confusion because many

microbiologists found the idea of voltage control of the outer

membrane permeability very attractive. However, in painted

membranes, porin pores were not found to be voltage-depen-

dent [146]. In addition, they become active in single steps and

not in large bursts as seen in folded membranes. It is generally

believed today that voltage control of outer membrane per-

meability in bacteria does not exist [17]. This is based on the

consideration of the physiology of the cells and on experimen-

tal results over the last ten years. Firstly,

in vivo

experiments

have demonstrated that the Donnan potential (up to 80-

100 mV [199, 2001) across the outer membrane has no influ-

ence on its permeability [17, 2001 and other potentials are

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