(Advances in microbial physiology 9) a.h. rose and d.w. tempest (eds.) academic press (1973)

Advances in
MICROBIAL
PHYSIOLOGY
Edited by
A. H. ROSE
School of Biological Sciences
Bath University
England
and
D. W. TEMPEST
Laboratorium voor Mikrobiologie,
Universiteitvan Amsterdam,
Amsterdam-C
The Netherlands
VOLUME 9
1973
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PRINTED IN GREAT BRITAIN BY
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Contributors to Volume 9
M. DWORKIN,Department of Microbiology, University of Minnesota,
D. J. RSHER,Long Ashton Research Station, University of Bristol, Bristol,
Minneapolis,Minnesota 55455, U.8.A.
Enaland
L.N.ORNSTON,Departmentof Biology,YaleUniversity,NewHaven,Connecticut,
U.S.A.
D. V. RICHMOND,Long Ashton Research Xtation, University of Bristol,
M. H. RICHMOND,Department of Bacteriology, Universityof Bristol, University
R. Y. STANIER,Service de Physiologie Microbienne, Institut Pasteur, Paris,
5.Z. SUDO,Department of Microbiology, Universityof Minnesota,Minneapolis,
R. B. SYKES,Department of Bacteriology, University of Bristol, University
Bristol, England
Walk,Bristol, England
Prance
Minnesota 55455 U.S.A.
Walk, Bristol, England
V

The Electrophoretic Mobility of Micro-Organisms
D. V. RICHMONDAND D. J. FISHER
Long Ashton Research Xtation, Universityof Bristol, Bristol BX18 9AP,
England
I. Introduction .
11. Theory .
111. Methods .
A. Measurement of Electrophoretic Mobility
B. Apparatus .
C. Related Techniques .
A. ModelSystems .
B. SpecificChemical Treatments .
V. Results .
A. Viruses .
B. Bacteria .
C. Trypanosomes .
D. Cellular SlimeMoulds .
E. Fungi .
F. Algae .
References .
IV. Identification of Surface Components .
. 1
. 2
. 3
. 3
. 3
. 7
. 9
. 9
. 10
. 11
. 11
. 11
. 20
. 20
. 21
. 26
. 27
I. Introduction
Electrophoretic mobility is a measure of the movement of a particle
in a solution when subjected to an externally applied electric field. The
direction and rate of this movement depends on the polarity and density
of the surface charges. Sincemany surfaces acquire a charge in aqueous
media, measurements of electrophoretic mobility can give useful
information regarding the composition of surfaces and the physical
behaviour of suspended particles. Phenomena in which surface charge
may be involved include flocculation, aggregation, self-recognition,
antigen-antibody reactions and the binding of some drugs to surface
receptor sites.
Surface properties may play an important role in the behaviour of
micro-organisms, and gene expression may be modified by responses of
the surface to changes in environment. The external surfaces of micro-
organisms,varywidely in structure and composition. Fungal zoospores
1

2 D. V. RICHMOND AND D. J. FISHER
and the L-forms of bacteria are more or less naked protoplasts but
other micro-organismsare surrounded by walls of varying complexity.
Bacterial and fungal spores often have a layered wall structure and a
complexsurfacemorphology. ApH-mobility curveis often characteristic
of a particular species but sometimes may be altered by a change in
growth conditions. Ionic surface groups may be identified by studying
pH-mobility curves of cells before and after treatment with specific
chemical reagents or enzymes.
The theoretical background and general principles of micro-electro-
phoresis have been authoritatively reviewed (James, 1957 ;Brinton and
Lauffer, 1959; Shaw, 1969); this review will therefore be concerned
mainly with the practical applications of the technique to the study of
various micro-organisms. The electrophoretic behaviour of animal cells
is reviewed by Ambrose (1966) and will not be described here.
II. Theory
Most particles acquire an electric charge in aqueous suspension due
to the ionization of their surface groups and adsorption of ions. The
surfacecharge attracts ions of opposite chargein the medium and results
in the formation of an electric double layer. If a tangential electric field
is applied along the charged surface the particle tends to move in one
direction while the ions in the mobile part of the double layer show an
equivalent motion in the opposite direction carrying solvent with them.
Thus, when carried out in a closed system, electrophoresis and electro-
osmosis at the chamber wall take place simultaneously. The electro-
phoretic mobility of a particle depends on the zeta potential at the
plane of shear between the charged surface and the electrolyte solution.
Smoluchowski (1914) regarded electrophoresis as the opposite of
electro-osmosisand derived the equation:
where m is the electrophoretic mobility of the particle, D is the dielectric
constant of the medium, q is the viscosity of the medium, and 5 is the
potential at the surface of shear. This equation is applicableto a particle
of any size, shape or orientation provided it is of “easy” shape and the
radius of curvature of the surface is at all points much greater than the
thickness of the double layer. More precise treatments have been dis-
cussed by James (1957) and by Shaw (1969) but, because of theoretical
difficulties and ambiguities, most workers have preferred to express
their results as electrophoretic mobilities,the measured values.

THE ELECTROPHORETIC MOBILITY OF MICRO-ORGANISMS 3
111. Methods
A. MEASUREMENTOF ELECTROPHORETICMOBILITY
Microelectrophoresis involves the direct observation under the
microscopeof visibleparticles asthey migrate in an electricfield.Soluble
material can also be examined by this technique if it is first adsorbed on
to carrier particles. One of the great advantages of the method is that it
is possible to make determinations on living cells without causing any
permanent damage. Individual particles can be selected for measure-
ment, their sizeand shape can be observedand photographic records can
be made. Very dilute dispersions can be studied and under these con-
ditions interaction between particles is negligible. The particles, sus-
pended in buffer, are placed in a transparent cell through which an
electric current is passed. The time required for a particle to cover a
given distance, as measured by a micrometer eyepiece, is noted. The
results are expressedas mobility per unit field strength. As zeta potential
is sensitive to changes in ionic strength of the suspending medium the
ionic strength must be rigidly defined (Barry and James, 1952).
The electrophoretic mobility may be influenced by diffusion of ions
through the cell membrane (James,Loveday and Plummer, 1964) or by
the presence of capsules, mucilage or fimbriae (James and List, 1966).
Also difficultiesmay be encountered in interpreting the electrophoretic
mobility of motile flagellates. Moreover, the plane of shear may not
necessarily coincidewith the cell surface as observed by light or electron
microscopy.
B. APPARATUS
A microelectrophoresis apparatus consists essentially of a cell into
which a microscope can be focused, electrodes, and an arrangement for
filling and emptying the cell. Provision must be made for the efficient
control of temperature since mobility is dependent on the viscosity of
the medium. Convection currents also must be avoided. The numerous
electrophoresis cells which have been described fall into two main
categories-rectangular and cylindrical-the rectangular cell being
preferred for larger particles, such as fungal spores, which tend to
sediment. The walls of the electrophoresis cell assume a charge relative
to the suspensionmedium and thereby cause electro-osmoticstreaming.
Liquid is caused to flow along the walls and back through the centre of
the cell. Thus, the true mobility of particles only can be observed at the
two stationary levels, and electrophoretic measurements must therefore
be made at one of these.

1. Rectungulur Cell
The early work was carried out using a flat rectangular cell mounted
horizontallyon a microscopestage. Alaterally mounted cellispreferable,
however, for larger particles which sediment under gravity. For very
large particles the cell may be mounted vertically and the electric field
applied parallel to the direction of sedimentation under gravity (see
Fig. 1).
The apparatus designed by Sachtleben et ad. (1961)is widely used for
the examination of blood cells. With this apparatus, the laterally
mounted cell is surrounded by a water jacket and a water immersion
objective penetrates the jacket through a flexible membrane. Particles
can be examined by transmitted or phase-contrast illumination. The
Loferal
FIG.1. Possible orientations of a rectangular microeIectrophoresiscell.
apparatus has reversible non-polarizable copper-copper sulphate
electrodesseparated from the electrophoresiscell by gelatin and plaster
plugsenclosedby two semi-permeablemembranes.Usingthis equipment
it has been found possible to examine leucocytes in their own sera
(Puhrmann and Ruhenstroth-Bauer, 1965).
For the examination of fungal spores we have used a modification
of the apparatus of Gittens and James (1960).The cell was mounted in
the lateral position (Hartman et ul., 1952)and the x20 water-immersion
objective focused on the stationary layer through a close fitting rubber
sheet (Figs.2 and 3).Theposition of the stationary levelswas determined
by means of a dial gauge attached to the microscope. In this apparatus,
silver-silver chloride electrodes were used and sintered glass discs
prevented contamination of the electrophoresis cell by material from
the electrode chamber (Loveday and James, 1957).Dry batteries were

THE ICLECTROPHORETIC MOBILITY OF MICRO-ORGANISMS 5
FIG.2. Microelectrophoresis apparatus showing the laterally mounted water
jacketed ccll. Dial gauge and microscope removed. (Modified from Gittens and
James, 1960.)
replaced by a D.C. power supply. The conductivity of buffered particle
suspensions was measured in a stoppered conductivity cell, at 25", using
a Wayne-Kerr B221 bridge. The cross-sectional area of the cell may be
calculated by measuring the velocity of standard particles at the
stationary levels. A suspension of human red blood cells in 0.067 M -
phosphate buffer (pH 7.4), at 2 5 O , which has been shown to have a
2

6 D . V. RICHMOND AND D. J. FISHER
FIG.3. Muxoelectrophoresis apparatus showlng the dial gauge and microscope in
poSition. (Modifiedfrom Gittcns and James, 1960.)
mobility of 1.31 x lo-* m' v-' s-' (Abramson, 1929; Seaman, 1965),pro-
vicled a suitable standard. The cell symmetry may be checked by deter-
mining the electrophoretic mobility of bacteria, or other cells, at
different depths. The results should form a parabolic velocity profile
(James, 1957).
2,similar type of apparatus has been described by Neihof (1969) who

THE ELECTROPHORETIC MOBILITY O F MICRO-ORGANISMS 7
used palladium electrodes charged with atomic hydrogen by cathodic
electrolysis. These electrodes permit high current densities without
evolving gas and there is no danger of contamination of suspensions by
heavy metal ions. Marshall (1966) described a simple rectangular cell
constructed from glass microscope cover slips.
2. Cylindrical Cell
The apparatus of Bangham et al. (1958a) employs a cylindrical
capillary tube and has been widely used, particularly for studies of
animal cells. Using this apparatus, measurements on as little as 0.1 ml
of suspension have been made. A magnetic stirrer syst'em can be incor-
porated to resuspend cells which have sedimented (Ambrose, 1966).
3. Vertical Cell
Lukiewicz and Korohoda (1961)have described an apparatus to study
the electrophoretic behaviour of large plant and animal cells over
extended periods of time. The flat microelectrophoresis cell had a
cylindrical return tube through which electro-osmotic flow occurred.
Consequently no streaming occurred in the flat tube. Measurements were
carried out on particles migrating in t'he centre of the flat tube.
C. RELATEDTECHNIQUES
Sher and Schwan (1965) showed that the thermal and gravitational
driftsthat affect particles in conventional microelectrophoresis chambers
could be overcome if an alternating electric current was used instead of
a direct current. With this technique, the amplitude of oscillatory
motion of a particle is measured from a photographic record. Under
these conditions
WA
E '
m=-
where m is the mobility, A the amplitude of oscillatory migration,
w the angular frequency of the applied sinusoidal electric field, and
E the amplitude of the applied electric field. The frequency of the
applied voltage can be adjusted so that the particle oscillates rapidly
about the origin and at the same time gives a large easily measured
amplitude.
Three other related techniques, although strictly outside the subject
of this review deserve brief mention here. They are the use of electro-
phoresis in water treatment, the preparative separation of cells and

organelles by continuous electrophoresis and lastly the technique of
dielectrophoresis.
1. Water Treatment
The flocculation of suspensions by the addition of electrolytes is
frequently used in water purification. Colloidal material can be removed
if the zeta potential of the particles is decreased to zero. I n practice an
inorganic coagulant and an organic polyelectrolyte are added simul-
taneously. A mass-transport cell has been developed for measuring
electrophoretic mobility (ROSSand Long, 1969). On applying a known
potential gradient to a suspension some of the suspended material moves
into a collection chamber surrounding one of the electrodes. The weight
that moves in a given time is determined by weighing the chamber or
analysing its contents.
2. Continuous Electrophoresis
The technique of continuous free-flow electrophoresis described by
Hannig (1964) can be used for the preparative separation of cells and
subcellular components. I n this technique the sample flows vertically
down a rectangular chamber in the presence of buffer. An electric field
is applied across the chamber and the continuously injected sample
divides into bands containing particles of equal mobility. The bands are
isolated at the bottom of the chamber in 50-100 collection tubes. Blood
cells can be separated into erythrocytes, granulocytes and lymphocytes
(Hannig and Krussmann, 1968; Ganser et al., 1968). Preparations of
synaptosomes and synaptic vesicles obtained from crude guinea pig
brain extracts have been found to be at least as pure as those obtained
by ultracentrifugation (Ryan et al., 1971).Mandel (1971) has used the
method to show that type 1 polio virus has two isoelectric points, at
pH 7.0 and 4.5. A similar apparatus known as a “continuous particle
electrophoresis device” was designed by Strickler (1967) and has been
used to study the mobility of proteins adsorbed on polystyrene latex
particles, and also of bacteria. Mixtures of four different bacterial species
were resolved into their separate viable components (Lemp et al., 1971).
3. Bielectrophoresis
Dielectrophoresis is defined as the motion of a neutral particle due
to the action of a non-uniform electric field on its permanent or induced
dipole movement (Crane and Pohl, 1968). Using this technique it has
been found possible to separate living and dead yeast cells (Pohl and
Hawk, 1866; Pohl and Crane, 1971) and washed yeast cells previously

grown in different media (Mason and Townsley, 1971). The yield
spectrum of spinach chloroplasts stabilized by 3-(3,4-dichlorophenyI)-
1,l-dimethylurea (DCMU)showed three peaks (Ting et al., 1971).
IV. Identificationof Surface Components
Microelectrophoresis is a useful technique for giving information
about the outermost surface layers of micro-organisms. The treatment
causes little or no damage to the cells and unless subjected to chemical
treatments they remain viable. Since most biological constituents have
a characteristic charge behaviour the surface components of cells can be
identified by studying the effect of various treatments on the electro-
phoretic mobility. Comparisons with the behaviour of model particles
of known surface composition can also give useful information.
A. MODEJ,SYSTEMS
Many compounds such as proteins, phospholipids, carbohydrates and
nucleic acids can be adsorbed on the surface of microscopically visible
particles. These carrier particles which may be hydrocarbon droplets,
quartz, carbon, aluminium oxide or silica gel all assume the properties
of the added film surface (Overbeek and Bungenberg de Jong, 1949).
Polystyrene latex particles (diameter I a099 pm) are very convenient as
carrier particles since their mobility is independent of their concentration
and their density is similar to the density of biological particles (Lemp
et al., 1971). Carrier particles are coated by allowing them to remain in
contact with a buffer solution containing an excess of the coating
material which must be highly purified. The mobility of the particles is
independent of their size and shape. The mobilities of dissolved proteins
as measured by the moving boundary method are often identical with
mobilities of the same protein adsorbed on a carrier surface. Comparisons
of the shape of the pH-mobility curve and the value of the isoelectric
point of a micro-organism with that of a model particle can help in
identifying surface components.
Another technique devised by Bungenberg de Jong (1949) is to
determine the concentrations of various metal cations required to
reverse the direction of the electrophoretic mobility of colloidal particles
containing carboxyl, phosphate or sulphate groups. These “cation
charge reversal spectra” are then compared with those given by micro-
organisms. The reversal of charge concentrations of Th3+,Ce3+,La3+
and UOi+ are particularly valuable. Douglas and Parker (1957) used
these four cations, together with Pb2+,Ba2+and Mg2+,in comparing
charge reversal spectra of model particles with those of bacterial spores
and cells.

The information given by model particle studies must be confirmed
by specific chemical and enzymic treatments. The information given by
charge reversal spectra suggested that the negative mobility of erythro-
cytes was due to the ionization of phosphate groups (Bangham et al.,
1958b).It is now established that the negative mobility was due to the
presence of AT-acylatedneuraminic acids. Treatment with neuraminidase
reduced the mobility and sialic acids were released into the medium
(Seaman and Cook, 1965).
B. SPECIFICCHEMICALTREATMENTS
1. Effect of Xurface Active Agents
Dyar (1948) showed that lipid could be detected on the surface of
bacteria by the increase in mobility produced in the presence of anionic
surfaceactive agents such as dodecyl, tetradecyl andhexadecyl sulphonic
acids. He suggested that the hydrocarbon end of the molecule was
specificallyadsorbed to the surface lipid and consequently the negatively
charged end increased the negative charge on the organism. The mobility
of hydrocarbon and lipid droplets also increased in the presence of the
surface active agents. Polysaccharide particles showed no increase at
any pH; protein particles were unaffected if the pH was above the
isoelectric point but the mobility was altered at more acid pH owing to
association of the surface active anions with NH3+groups. The effect
of surface active agents on the electrokinetic properties of bacteria has
been reviewed by James (1965).
2. Modi$cation of Surface Xtructures
Surface groups can be identified by comparing pH-mobility curves of
untreated cells with curves of cells altered by specific chemical or
enzymic treatments. This method was first used by Cohen (1945) who
treated cells of Bacillus proteus with benzenesulphonyl chloride. The
treated cells had a higher negative charge than untreated cells and he
suggested that imidazole and amino groups had been substituted.
Douglas (1959) showed that p-toluenesulphonyl chloride was a more
effective reagent for amino groups. Dyar (1948)found that treatment of
Xicrococcus aureus cellswith lipase altered the electrophoreticbehaviour
of the cells and abolished the effect of anionic surface active agents thus
providing additional evidence of the presence of surface lipid.
Capsular material may be removed from Xtreptococcus pyogenes cells
by treatment with hyaluronidase ; the protein antigens can then be
removed by trypsin or pepsin (Hill et al., 1963~).Amino groups on the
bacterial surface can be detected by treatment with an ethanolic solution

THE ELECTROPHORETIC B'IOBILITY OF MICRO-ORGANISMS 11
of fluoro-2,4-dinitrobenzene,and carbosyl groups by treatment of acid-
washed cells with ethanolic diazomethane (Gittens and James, 1963a).
Some C-terminal groups at the bacterial surface can be detected by
treatment with specific amino acid decarboxylases followed by electro-
phoresis (Hill et al., 1963b).Surface phosphate groups may be identified
by the reduction of mobility produced in the presence of UOj+
(McQuillen, 1950) or Ca2+(Forrester et al., 1965),or by pretreatment
with alkaline phosphatase (Hill et al., 1963~).
V. Results
9.VIRUSES
Microelectrophoresis has usually been applied to cells in the size
range 1-10 pm. However the method has now been used to study pox
and vaccinia viruses which measure only about 0.25 pm (Douglas et al.,
1966, 1969). A micro-apparatus of rectangular channel section was used
(Douglas, 1955) and the particles were detected by dark field illumina-
tion. Electrophoresis was carried out in molar sucrose to increase vis-
cosity and hence reduce Brownian movement. The pH mobility curves
were all similar in shape although there were some differences in slope
and isopotential point. The results suggested that the surfaces were
protein or lipoprotein. After treatment with p-toluenesulphonyl
chloride, to eliminate NH,+ groups, cowpox had an acidic surface
consistent with the presence of carboxyl groups.
B. BACTERIA
Bacterial cell walls are complex structures which vary widely in
organization and composition. Studies on the chemical composition of
the walls are summarized by Salton (1964) and Rogers and Perkins
(1968); the physical structure and arrangement of the wall layers are
described by Glauert and Thornley (1969).
The mechanical strength of the wall is seemingly due mainly to
mucopepticle; in addition, Gram-negative bacteria usually contain
protein, lipid,lipoprotein andlipopolysaccharide. The lipopolysaccharide
has important endotoxin and antigen properties. Gram-positive walls
may contain teichoic acids, polysaccharides and proteins, all of which
may have antigenic properties. Most Gram-positive walls have little
lipid but the walls of mycobacteria contain complex lipids and glyco-
lipids. Outside the walls, a capsular or slime layer may occur and flagella
may be present.
It is usually necessary to wash bacteria by at least three successive

cycles of centrifugation and resuspension in the buffer solution to be
used for niobility measurements. Adsorbed metabolites may be present
on the surfaces of unwashed spores. On the other hand, washing may
remove capsular material from the bacteria ;therefore washed organisms
always should be carefully examined in the electron microscope before
making mobility measurements. The mobility of bacteria may vary
with the nature of the growth medium and the age ofthe culture.
1. Escherichia coli
Studies of pH-mobility curves and charge reversal spectra suggest
that the cell exterior of Escherichia coli had a polysaccharide composition
(Davies et al., 1956). Anionic surface active agents had little effect on
the negative mobility of the cells. The cell surface was therefore con-
sidered to be hydrophilic and to contain little lipid. Brinton et al. (1954)
removed fimbriae from the slower moving S-form by shaking in a high
speed mixer and found that a mobility similar to the R-form was
obtained. When the filaments were allowed to regrow the mobility
returned to the lower value. The effect of fimbriae on electrophoretic
mobility has been further studied by James and List (1966)who investi-
gated two strains of E. coli and seven of Klebsiella aerogenes. The
electrophoretic mobility of capsular organisms was independent of the
presence or absence of fimbriae. Capsulate organisms have a high
negative charge due to the presence of glucuronic acids in the capsular
material and this completely overrides any effect the fimbriae may have
on mobility. The mobility of non-capsular, non-fimbriate organisms was
higher than that of the fimbriate ones due to a difference in the charge
density of the cells. The fimbriae increase the surface area of the cells
and hence reduce the charge density, they do not, however. reduce
mobility by exerting a viscous drag.
Gittens and James (1963a) further examined the surface of E. coli.
Treatment with fluoro-2,4-dinitrobenzenehad no effect on mobility but
p-toluenesulphonyl chloride treatment showed the presence of secondary
amino groups. Treatment with diazomethane was unsatisfactory for
cells of E. coli since the ethanol solvent removed the lipoprotein surface
and revealed the rnucopeptide layer.
8. Aerobacter aeroyenes
The electrophoretic mobility of suspensions of Aerobacter aerogenes
has been shown to be constant over a period of 6 months, and to be
independent of the growth medium. Cells killed wit'h formaldehyde had
the same mobility as untreated cells but those that were heat-killed had

a different mobility. The mobility was insensitive to pH over a wide
range indicating the absence of protein. The charge density of A.
aerogenes was found to be sensitive to changes in ionic strength, and
hence this factor had to be rigidly controlled when determining bacterial
mobilities (Barry and James, 1952, 1953).The mobility of A. aerogenes
increased to a maximum early in the period of logarithmic growth and
attained a constant value from the beginning of the stationary phase.
The changes in mobility could be correlated with changes in capsule
size (Plummer and James, 1961). The behaviour of the capsulated
organisms indicated a simple carboxyl surface, amino groups and lipid
being absent, and the pK values of old and young cells were identical.
The observed variation in mobility during growth therefore was not due
to changes in the nature of surface components but to a variation in
their relative amounts. The mobility was found to increase asthe capsule
size increased, thereafter both decreased until the mobility reached a
constant value (at 5 hr) while the capsule further decreased in size up
to 24 hr. During the early stages of growth the capsule may consist of
an open meshwork and as the capsule shrinks some of the carboxyl
groups may be dragged beneath the electrokinetic surface thus producing
the observed reduction in mobility.
Gittens and James (l963a)treated cells of A. aerogenes with a variety
of compounds to find a simple method of completely modifying the
surface carboxyl groups preferably in aqueous solution. The most
satisfactory reagents were diazomethane and methanolic HC1 but both
methylations had to be carried out in ethanolic or ethereal solutions for
complete reaction. Cells treated with diazomethane or with methanolic
HC1 had a zero mobility, independent of ionic strength and pH over
the range where no ester hydrolysis occurred. Hence only carboxyl
groups were present on the surface and no adsorption of anions or cations
from the solution occurred.
Gittens and James (196310) have studied the effect of surface con-
ductance on the zeta potential and surface charge density of A.aerogenes.
The surface conductance correction to the zeta potential is important
for ionogenic surfaces at low ionic strengths. Most of the observed surface
conductance appears to arise in the Stern layer or the region inside the
shearing plane.
Spheroplasts are produced by growing A. aerogenes cells in a medium
containing penicillin and sucrose. Morphological studies suggest that
the spheroplasts still have cell wall components outside the protoplasmic
membrane (Gebickiand James, 1960)and the electrophoretic properties
of the spheroplasts were identical with those of normal cells, and quite
distinct from the lipoprotein surface of the plasma membrane (Gebicki
and James, 1962).

14 D. V. RICHMOND AND D. J. PISHER
3. Bacillus subtilis and B. megaterium
The electrokinetic behaviour of resting spores of Bacillus subtilis and
B. megaterium suggested the presence on the surface of a hexosamine
peptide which is liberated into the medium on germination (Douglas,
1957).Carboxyl and amino groups occurred in equal amounts on resting
spores of B. subtilis (Douglas, 1959).Treatments with lysozyme, trypsin
and lipase confirmed the presence of hexosamine peptide on the resting
spores (Douglas and Parker, 1958).
4. Streptococcus pyogenes and X . faecalis
The negative mobility of Xtreptococcus pyogenes was found to increase
during growth and to reach a maximum at the end of the logarithmic
phase; the mobility then decreased to a constant value. The changes in
mobility were found to be due to a hyaluronic acid-containing capsule
which is formed during logarithmic growth but disappears during the
stationary phase (Plummer et al., 1962).Hill et ak. (1963a, c, d) studied
a number of strains of S.pyogenes and found that after treatment with
hyaluronidase all cells had similar structures. The pH-mobility curves
indicated the presence of carboxyl groups, amino groups and the imida-
zole group of histidine. The carboxyl groups were identified (by the
use of specific amino acid decarboxylases) as alanine and the a- and
y-carboxyl groups of glutamic acid. The outer wall layers are probably
composed of a polysaccharide-protein complex. Trypsin was found to
remove the T antigen more readily from matt than from glossy variants
of S.pyogenes. Electrokinetic studies show that after removal of antigens
by proteolytic enzymes all strains have a similar surface.
The lipid content of the cell wall of some strains ofS.pyogenes can be
increased by repeatedly subculturing the organism in the presence of
glycerol, sodium oleate or sodium acetate (Hill et al., 196310). Cells
grown in the presence of glycerol or acetate had normal lipase activity.
The increase in the lipid content of walls of cells grown in the presence
of oleate was due to the inhibition of extracellular lipase. The lipid
content of the cell walls of organisms grown in normal Todd-Hewitt
medium was about 1%,but when grown in the presence of glycerol or
sodium acetate it rose to 20-25%. The presence of surface lipid was
demonstrated by the increase in mobility produced in the presence of
sodium dodecyl sulphate. Some tetracycline resistant strains have a
high lipid content even when grown in the absence of glycerol, acetate
or oleate. The extracellular lipase in the medium of all these strains had
normal activity. Strains of Streptococcus isolated from impetigo lesions
had a high lipid content when grown in the absence of glycerol, acetate

or oleate but produced no lipase and resembled normal cells grown in
the presence of sodium oleate.
A further number of tetracycline sensitive and resistant stsains were
examined by Norrington and James (1970).Surface lipid as detected by
the effect of sodium dodecyl snlphate on the mobility was not always
accompanied by an increase in total cell wall lipid. Tetracycline sensitive
strains isolated before 1935 had about 32% saponifiable lipid, similar
strains isolated after 1953had 42% lipid.
James et al. (1965) have studied the bacterial cell wall, protoplasts
and L-form of S.pyogenes. The L-form envelope differed in composition
from both cell wall and protoplast membrane. All structures had surface
protein but surface lipid was absent from L-forms and protoplasts.
Hill et al. (1964) have shown that microelectrophoresis can be used to
detect antibody bound to cells ofS. pyogenes. The technique is suitable
for the detection of antibody in relatively small amounts.
Schott and Young (1972) studied the electrophoretic mobility of
S.faecalis. All the surface acidic groups were carboxyl. A smaller number
of basic groups was present. There was little change in mobility with
increase in culture age between 29 and 96 hr.
5. Micrococcus lysodeikticus
The electrophoretic behaviour of whole cells, protoplasts and proto-
plsst membranes of Micrococcus lysodeikticus have been studied by
Few et al. (1960). All the materials examined had surface amino and
carboxyl groups. Surface lipid was seemingly absent from protoplasts
since intact protoplasts were electrophoretically similar to defatted
protoplast membranes.
Einolf and Carstensen (1967) investigated the conductivity of an
unknown species of Micrococcus. The bacterial conductivity must be
considered in calculating the zeta potential and surface charge density
of bacteria at low ionic strengths.
6. Staphylococcus aureus
The pH-mobility curve of Staphylococcus aureus was found to be of a
non-sigmoid shape and had a maximum value at pH 4-5 (James and
Brewer, 1968a). The maximum in the curve was due neither to in-
complete removal of growth medium from the surface, nor to adsorption
of buffer components, nor to irreversible surface denaturation. When
teichoic acid was removed from the surface by mild oxidation with
sodium metaperiodate the maximum of the curve was eliminated. The
unusual shape of the curve may be due to a pH-dependent change in the
configuration of surface teichoic acid molecules.

Some strains of X.aureus such as Cowan 1 carry a surface protein
which includes the Jensen protein A (Lofkvist and Sjoquist, 1962).
Other strains such as Wood 46 have no surface protein component.
Treatment of Cowan 1 cells with trypsin resulted in a large increase in
negative mobility particularly in the pH range 6-9. Trypsin treatment
had no effect on the mobility of Wood 46. Some strainsof S. aureus were
electrokinetically heterogeneous suggesting that the protein may be
distributed in discrete patches rather than as a continuous layer. The
microelectrophoretic technique identifies the teichoic acid and protein
overlying the glycopeptide layer (James and Brewer, 1968b).
7. iVycobacteriumphlei
Adams and Rideal (1959) examined the electrophoretic properties of
two strains of Nycobacteriuna phlei. One strain had a phospholipid and
protein surface while the other was almost entirely covered by phospho-
lipid.
8. Halobacterium cutirubrum
Halobacterium cutirubrum is a red pigmented psychrophilic Gram-
negative marine bacterium which can grow only within the temperature
range 0-19" in tryptone-supplemented seawater. The organism lyses if
the temperature is raised above 21" or if it is transferred to distilled
water. Lysed cells became heterogeneous in mobility due to adsorption
of intracellular material of high negative charge onto the cell surface.
Cellslysed in water or at acid pH reverted to control values after repeated
washing. Temperature-lysed cells acquire a permanent high negative
mobility due to an irreversible surface change (Madeley et al., 1967).
9. Actinomycete Xpores
Douglas et al. (1970) examined spores of the actinomycete genera
Micromonospora, Nocardia, Streptomyces and Thermoactinomyces. The
electrophoretic results suggested the presence of hexosamine-peptide
polymers in all strains. Micromonospora and Streptomyces spores had
surface amino and carboxyl groups. Thermoactinomyces spores and the
bacillary or coccoid elements of Nocardia bad carboxyl, but not amino,
groups. All species showed changes in mobility after treatment with
lysozyme. Treatment with sodium dodecyl sulphate showed lipid to be
present on Thermoactinomyces and Nocardia but absent from Micro-
monospora and Xtreptomyces. There was no correlation between water-
repellant properties and the presence of surface lipid. Of the genera
which have surface lipid, Thermoactinomyces has hydrophobic spores
while LVocardiaspores are easily wetted.

10. Soil Bacteria
The Rhixohium species are of great scientific and agricultural im-
portance because of their ability to form a symbiotic association with
legumes in which molecular nitrogen is fixed. Marshall (1967) examined
the electrophoretic mobility of fast and slow growing Rhizobium species
and compared the mobilities of normal strains of Rhixobium trifolii with
those of mutants unable to form nodules on clover roots. The slow-
growing bacteria had an entirely carboxyl surface whereas fast-growing
strains had some amino groups also present. Contrary to the findings of
Tittsler et ul. (1932)for R. meliloti, there was no relationship between
surface charge density and the ability of R.trifolii strains to form nodules
on clover. Loss of nitrogen-fixing ability was not accompanied by any
change in electrophoretic mobility of the cells. The electrophoretic
mobility of the cells at a given pH was a constant and characteristic
property of the bacterial strain. The mobility of two strains of R. trifolii
was similar to that of their isolated lipopolysaccharide antigens. The
surface charge of the two strains was presumably determined by the
structure of the somatic antigen (Humphreyand Vincent, 1969).
Cells of R. trifolii grown in the absence of Ca2+formed swollen, but
osmotically stable, spheres. The electrophoretic mobility of these cells
was the same as normal cells. Since calcium was found to be contained
entirely in the wall of calcium deficient cells, most of this element must
be located in an electrophoretically inaccessible region of the cell wall
(Humphrey et ul., 1965).
Soil bacteria may be enveloped in a protective layer of colloidal clay
particles which enhances survival under adverse conditions. Lahav (1962)
found that the electrophoretic mobility of Bacillus subtilis cells could
be markedly altered by the presence of montmorillonite particles.
Adsorption was a reversible process dependent on the concentration of
unadsorbed clay and was greatest at low pH. Marshall (1968)examined
the interaction between colloidal montmorillonite and cells of seven
Rhixobium strains having different ionogenic surfaces. Montmorillonite
increased the mobility of all strains to a value similar to montmorillonite
itself. The clay presumably covered the cell surface completely. Cells
with entirely carboxyl surface groups adsorbed more clay per unit surface
area than cells with an amino-carboxyl surface. Treatment of Rhizobium
isoIates with illite confirmed that the cell mobilities observed at high
clay concentrations were a reflection of the mobility of the surface-
adsorbed clay (Marshall, 1969a). Electron micrographs of shadowed
and unshadowed preparations confirmed the presence of an envelope of
clay round the cells. Most clay particles were adsorbed to the cells in an
edge to face rather than face to face manner. Cells were treated with

is D. V. RICHMOND AND D. J. FISHER
montmorillonite or illite which had been suspended in sodium hexa-
inetaphosphate. These clays were not adsorbed onto cells with entirely
carboxyl surfaces since the edgewise orientation is prevented by sodium
hexametaphosphate blocking the positive groups at the edges of the clay
particles (Marshall, 1969b).
11. Eflect of BacteriostaticAgents on Mobility
(a) fiulphanilamide. Bradbury and Jordan (1942) studied the effect of
sulphanilamide, p-aminobenzoic acid and other chemically related
compounds on the electrophoretic mobility of Escherichia coli. The
effect of sulphanilamide 011 mobility was similar to that of p-amino-
benzoic acid and quite different from that of inactive substances. The
authors concluded that the association of the drug with the organism
was a function of the amino group.
(b) Phenols and substitutedphenols. Haydon (1956) found that the zeta
potential of E. coli decreased with time when suspended in phenol. The
results suggested a closerelationship between Iysis and death of the cells.
When cells of Aerobacter aerogmhes were treated with phenol, p-alkyl-
phenols orp-halogenophenols the electrophoreticmobilit'yincreased with
increasing phenol concentration. The substituted phenols were more
active than phenol itself (James et al., 1964). The increased mobility of
young cells may have been due to the presence of phenoxy ions on the
surface. The decrease in the mobility of treated cells which occurred
above pH 7 may have been due to the combination of amino acids
(released from the cell in the presence of phenols) with the surface
phenoxy groups. An alternative explanation of the increased mobility
is that phenol caused a contraction of the capsule thus increasing the
surface charge density of the cells.
Hugo and Franklin (1968) studied the effect of cellular lipid on the
antistaphylococcal activity of phenols. Staphylococcus aureus grown in
the presence or absence of glycerol was treated with a homologous series
of 4-alkylphenols. As the side-chain was made to increase, solubility in
water decreased and solubility in lipid increased. The molecule also
tended to become polar and consequently surface active. As lipid
solubility increased phenol became attached to the surface with the
alkyl side chain attached to the cell lipid and the phenolic hydroxy
group projecting into the solution. The difference in response between
iiormal and fattened cells became apparent with n-butylphenol. Phenols
at an equivalent concentration cause an increase in the mobility of
fattened cells as compared with normal cells. With fattened cells the
drug was adsorbed by the surface lipid and hence did not penetrate the

THE ELECTROPHORFTIC MOBILITY OF MICRO-ORGANISMS 19
cell. The authors considered electrophoresis to be the most sensitive of
the methods they used to study drug/cell interaction.
(c) Chlorhexidine. Chlorhexidine decreased the electrophoretic mobility
of Escherichia coli and Xtaphylococcus aureus. With 8.aureus the charge
was not reversed. Cells of E. coli became positively charged at 600 pg/ml.
-4s the maximum amount of chlorhexidine which can be bound in a
monolayer at the surface is 85.5 pg chlorhexidine diacetatelmg dry
weight of cells a complete layer of the drug cannot be formed at the cell
surface. The electrophoreticevidence suggeststhat the drug accumulated
in aggregates at the cell surface. This has been confirmed by electron
microscopy (Hugo and Longworth, 1966).
(d) Proflavine. James and Barry (1954) found that proflavine caused
a linear decrease in rnobi1it)y of Aerobacter aerogenes with increasing
concentration. Very high concentrations caused flocculation. Cells
trained to grow in gradually increasing concentrations of proflavine
showed a normal mobility distribution up to 40 mg/l proflavine. Above
78 mg/l the distribution of mobilities was heterogeneous.
(e) Crystal violet. Resting cells of Aerobacter aerogenes have a lower
mobility in the presence of crystal violet. At very high crystal violet
concentrations the cellsflocculate. When cellswere grown in the presence
of crystal violet they behaved electrophoretically in three different ways
depending on the age of the parent inocculum. The change in electro-
phoretic behaviour occurred only during cell division in the presence of
crystal violet. The altered electrokinetic properties were transmitted to
subsequent generations in the presence of low drug concentrations
(Lowick and James, 1955). The different electrophoretic properties
were due to alterations in the nature of the cell surface (Lowick and
James, 1957).The parent strain of A. aerogenes has a polysaccharide sur-
face but the pH-mobility curve of the trained strain suggested a protein
surface. Treatment with lipase, extraction with solvents and mobility
measurements in the presence of sodium dodecyl sulphate all showed
that the trained cells had a lipid surface. Lowick and James (1957)
considered that while lipid was present in the walls of both trained
and untrained cells, the lipid in the trained cells was exposed, whereas
it occurred in the deeper layers of the walls of untrained cells.
(f) Methieillin. The relationship between methicillin resistance and
surface properties in Xtaphylococcus aureus was studied by Marshall and
James (1971) and Marshall et al. (1971). There was found to be no
relationship between antibiotic resistance and the amount of surface

lipid. The pH mobility curve of S. aureus had a plateau between pH 6
and 8 and reached a maximum between pH 4 and 5 . James and Brewer
(1968b) defined the percentage increase in the maximum value of the
mobility above the value on the plateau as the H-value, and this value
was found to be characteristic of the strain. Strains of S.aureus resistant
to methicillin all had low H-values and were electrophoretically homo-
geneous. Methicillin sensitive cells (with a high H-value) when adapted
to increasing concentrations of methicillin showed a progressive decrease
in H -value to a very low level.
C. TRYPANOSOMES
Hollingshead et al.(1963) have studied the electrophoretic mobility
of cultureformsof Trypanosoma rhodesiense and of intravascularformsof
T . rhodesiense, T . vivax, T . equinum, T . congolense and T . lewisi.
Nobilities were determined at 5"asthe inherent mobility of the organisms
at higher temperatures made electrophoretic measurements difficult.
The organisms were oriented randomly in the electric field and hence
there was no charge localization at either tip of the organism.
The intravascular form of T . rhodesiense was found to have an iso-
electric point between pH 5.8 and 7.0, and at physiological pH values
was capable of circulating in an uncharged state. The culture form of
T . rhodesiense had a different pH mobility curve with a highly negative
surface and an isoelectric point at 3.0. The culture form of T .rhodesiense
resembled the form found in the insect vector, the tsetse fly.Adaptation
to life in the insect thus resulted in a change in metabolism and a change
in the nature of the cell surface. Unmasking of inner surface components
may occur on introduction into the vector. The surface of T. lewisi was
electrophoretically similar to that of freely-circulating cells such as
lymphocytes and tumour cells. Trypanosidal drugs such as Ethidium
bromide and Prothididium decreased the negative mobility of the blood
form of T . lewisi and the culture form of 1'. rhodesiense but had little
effect on the blood form of T . rhodesiense.
D. CELLULARSLIMEMOULDS
The cellular slime moulds and particuIarly Dictyostelium discoideum
have been extensively used in studies of developmental biology. They
grow and divide as independent cells, then aggregate into cell masses
and finally differentiate into stalk cells and spores (Bonner, 1971).
Garrod and Gingell (1970) have investigated the surface properties
of preaggregation cells of D.discoideum by cell electrophoresis. Cell
mobility decreased with time as the cells approached the spontaneous

aggregation stage at 22”. Cells did not aggregate at 3’ and there was
no change in mobility after 6 hr at this temperature. The reduction in
mobility is under metabolic control. Cells did not aggregate in the
presence of EDTA, but this effect was not due to the removal of divalent
cations since cells in buffer elone had the same electrophoretic mobility
as cellsin buffer plus EDTA (Gingelland Garrod, 1969).It is not known
how EDTA prevents cell aggregation.
E. FUNGI
I. Fungal Xpores
The electrophoretic mobility of asexual spores of four fungal species;
Nucor ramannianus, Fusarium lini, Penicillium cyclopium and P.
spinulosum was studied by Douglas et al. (1959). Each species had a
different pH-mobility curve; P. cyclopium and P . spinulosum were
considered to have a lipid surface with possibly some carbohydrate on
P. spinulosum. The curves for B. lini and N . ramannianus suggested a
polysaccharide surface. Hannan (1961) investigated the electrokinetic
properties of Aspergillus niger spores and concluded that lipid and
protein were absent and the surface was mainly polysaccharide.
Fisher and Richmond (1969) examined spores of eight fungal species
and identified surface groups by treatment with specific chemical re-
agents. The sporesallhad cha,racteristicand distinct pH-mobility curves.
The zero mobility of Phytoph,thorainfestanssporangia over the pH range
2-1 1 suggested a surface, probably of carbohydrate, free from ionizable
groups. The mobility of basidiospores of Stereum purpureum depended
entirely on the presence of carboxyl groups. The pH-mobility curve of
Alternaria tenuis (Fig. 4) was characteristic of a mixed aminocarboxyl
surface. Treatment with alkaline phosphatase had no effect on the
mobility showingthe absence of phosphate groups. After treatment with
fluoro-2,4-dinitrobenzene the positive mobility at low pH was replaced
by a negative mobility throughout the pH range 3-11. Methylation with
diazomethane decreased the negative mobility by removing the charge
on the carboxyl groups. The positive mobility below pH 6-0 is due to
the remaining amino groups. The surface amino groups were identified
chromatographically after treatment with fluoro-2,4-dinitrobenzeneas
e-lysine, histidine, leucine and tyrosine. Conidia of Botrytis fabae had
surface amino and carboxyl groups. Isolated spore walls of B. fabae had
similar electrophoretic properties to the intact spores. “Protoplasts”
isolated from mycelium of A . tenuis and Neurospora crassa and from
conidia of B. fabae had pH-mobility curves characteristic of a protein
surface
3

The electrophoretictechnique can detect surfacelipid onfungal spores
(Fisher and Richmond, 1969; Fisher et al., 1972). The mobility of A .
tenuis, B. fabae, N . crassa and Rhizopus stolonifer rose progressively in
increasing concentrations of sodium dodecyl sulphate indicating the
presence of surface lipid (Table 1). Mobilities of isolated spore walls
I I I I I I I L
2 3 4 5 6 7 8 91011
pH value
FIG.4. pH-Mobility curves of conidia of Ahernaria tenuis. Untreated, 0-0;
phosphatase-treated, 0-0 ; fluoro-2,4-dinitrobenzene-treated,H-W ; diazo-
methane-treated, x-x. (Fisher and Richmond, 1969.)
confirmed the results obtained with whoIe spores. The spores with
surface lipid are airborne and difficult to wet. The absence of surface
lipid from water dispersed spores such as Verticillium albo-atrum and
Nectria galligena is to be expected but other spores such as Erysiphe
cichoracearum, E. graminis and Penicillium expansum lack surface lipid
and are nevertheless hydrophobic. Douglas et al. (1970) found a similar
lack of correlation between water-repellent properties and the presence
of surface lipid in actinomycete spores. The physical conformation of

TABLE1. The Effect of Sodium Dodecyl Sulphate (SDS)on the Electrophoretic
Mobility of Spores and Isolated Spore Walls
Electrophoretic % Increasein negative mobility with:
Material (10-8m2V-' S-l) M-SDS M-SDS M-SDS
mobility
Intact spores of:
Alternaria tenuis
Botrytisfabae
Erysiphe cichoracearum
Erysiphe graminis
Mucor rouxii
Nectria galligena
Neurospora crassa
Penicillium expansum
Rhizopus stolonifer
Verticilliumalbo-atrum
Spore walls of:
Alternaria tenuis
Botrytisfabae
Neurospora crassa
Penicillium expansum
-2.62
-1.54
-0.80
-3.54
-1.72
-3.75
-0.45
-1.54
-1.78
-1.27
-1.55
-1.41
-0.69
-1.52
15
5
0
0
6
0
7
17
3
0
2
3
16
6
0
1
6
0
9
14
5
0
5
6
78
3
24
15
0
2
4
0
24
14
33
0
112
9
130
3
- = not determined.
the surface may be sufficient to account for the hydrophobic properties
of spores.
Fisher et al. (1972) examined the fatty acids and hydrocarbons in the
surfacelipidsby gas-liquid chromatography.The fatty acidswere found
to be mainly straight-chain compounds of even carbon number, and
palmitic and stearic acids predominated ; polyunsaturated acids were
absent. Surface hydrocarbons consisted almost entirely of n-alkanes.
The compositions of the surface and wall lipids from the same species
were different.
(a) Penicillium conidia. The spore surface of Penicillium expansum
when grown on malt agar has amino, carboxyl and phosphate groups.
The phosphate groups were missing from washed cell walls (Fisher and
Richmond, 1969). The nature of this easily removable phosphate and
its effect on the mobility of the Penicillium conidium was studied by
Fisher and Richmond (1970). The pH-mobility curves of conidia from
five species of Penicillium were all different and characteristic (Fig. 5 ) .
The curves suggested amino-carboxyl surfaces containing varying
amounts of phosphate. The phosphate was identified by thin-layer
chromatography and metachromasy as polyphosphate containing less

than ten phosphorus atoms. The composition of the polyphosphate layer
which appeared 2 days after conidial initiation was dependent on the
phosphate content of the growth medium. The function of the surface
polyphosphate is unknown.
pH value
FIG.5. pH-Mobility curves of Penicillium expoansum, 9-0; P. thomii, U-U;
P. roquuefortii,0-0 ; P. digitatum, A-A ; and P. notatum, Q-Q, 7 day conidia
from malt agar. (Fisher and Richmond, 1970.)
2. Yeasts
(a) Yeast cells. Eddy and Rudin (1958a) studied the electrophoretic
mobility of various strains of Xaccharomyces cerevisiae and X. carls-
bergensis. The pH-mobility curves suggested the presence of surface
phosphate andprotein. A strainofS.carlsbergensisproducedaphosphate-
free surfacewhen grown in the absence of phosphate. Briley et al. (1970)
examinedthe ascosporeof S.cerevisiae. The pH-mobilitycurveindicated
an aminocarboxylsurfaceprobably of protein. Sodium dodecylsulphate
had no effect onthe mobility but treatment with pepsin or chymotrypsin
removed the positive mobility at low pH. The ascospore surface is free
of lipid but may be covered with a hydrophobic protein.
(b) Yeast jlocculation. Flocculation is the agglomeration of yeast cells
that generally occurs at the end of fermentation (Geilenkotten and

Nyns, 1971). Flocculation is undoubtedly a complex phenomenon and
no definite biochemical difference between flocculent and non-flocculent
yeasts has been found so far. One of the factors involved in flocculation
may be the mutual attractions induced by negative and positive iono-
genic groups on the cell surface (Lindquist, 1953). Eddy and Rudin
(1958b)examined the electrophoretic properties of a number of strains
of top and bottom yeasts. They concluded that most of the charged
groups at the cell surface played no direct part in flocculation and that
flocculation was not connected in any simple way with surface charge.
3. Reaction of Fungal Xpores with Toxicants
Most toxicants act within the cytoplasmic membrane but to reach the
cytoplasm a fungicide or antibiotic must first penetrate the cell wall.
0
FIG.6. Effect of dodine on the electrophoretic mobility of Neurosporu crasm
conidia and cell walls. 0-0, conidia; A-A, cell walls. (Somersand Fisher, 1967.)
The charged surface surrounding many fungal spores may play a role in
cation uptake. Cationic fungicides may bind to surface sites before
transfer across the membrane.
(a) Dodine. Somers and Fisher (1967) have studied the effect of the
cationic surface active agent dodine (n-dodecylguanidine acetate) on
the electrophoretic properties of Neurospora crassa conidia. The surface
of N . crassa conidia has amino, carboxyl and phosphate groups. Treat-
ment with increasing dodine concentrations gradually decreased the
negative charge on the conidia to zero and with increasing concentration
finally reversed the mobility (Fig. 6). Dodine lowered the mobility of
sucrose-stabilized N . crussaprotoplasts very rapidly (Fig. 7). The anionic

26 D. V. RICHMOND AND D. J. FISHEB
40
12
x -0.2 -
n
-c
.-.-
0
-
+0.2 I I I I I
0 0.1 0.2 0.3 0.4 0.5 0.6
Dodine (pM)
FIG.7. Effect of dodine on the electrophoretic mobility of Neurospora craaaa
protoplasts. (Somersand Fisher, 1967).
charges were neutralized by lower concentrations of dodine than those
required to kill conidia and hence cell wall binding may have the effect
of detoxifying the fungicide.
(b) Streptomycin. Streptomycin controls some diseases caused by
Oomycetes but is ineffective against all other fungi. Sporangia of
Pseudoperonospora humuli were found to have a pH-mobility curve
typical of an amino carboxyl surface. A marked reduction of mobility
occurred at pH 5.6 in the presence of 1 mg streptomycin/ml showing
binding of the antibiotic to surface ionic groups (Fisher, unpublished
observation).
F. ALGAE
1. Chlorella
Lukiewicz and Korohoda (1963, 1965a, b) have studied the electro-
phoretic properties of synchronized Chlorella cells in an apparatus of
their own design. The rate of growth of D-form cells in light slows down
as the cells become transformed into L-stage cells and this change was
foundto be accompaniedby a considerableloweringof theelectrophoretic
mobility. During the subsequent period of darkness a rapid increase in
mobility occurred asthe cellsdivided, and the high mobility characteris-
tic of the D-form cells was reached. The decrease in mobility during
growth in light was dueto somedevelopmental changein the cellsurface.
Shcherbakova (1970) found that the isoelectric point of Chlorella
vulgaris was at pH 0.85 and of C. pyrenoidosa at pH 1.25. Under
unfavourable growth conditionsthe zeta potential becamevery variable.

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The /I-Lactamases of Gram-Negative Bacteria and their
Possible Physiological Role
M. H. RICHMONDAND R. B. SYKES
Department of Bacteriology, Universityof Bristol,
University Walk,Bristol BS8 ITD, England
I. Introduction .
A. Basic Properties of @-Lactamases
B. Methods of Assay .
C. Enzymeunits .
D. Turnover Number and “Physiological Efficiency”
E. SpecificEnzyme Activity .
F. Substrate Profile .
A. @-Lactamasesfrom Gram-Positive Species .
B. p-Lactamases from Mycobacteria .
C. /%Lactamasesfrom Gram-Negative Species
111. Genetic Basis of @-LactamaseFormation .
IV. Physiology of p-Lactamases in Gram-Negative Species
A. Expression of 8-Lactamase Activity .
B. Location of @-Lactamasesin the Bacterial Cell
A. Role of @-Lactamases .
B. Intrinsic Resistance .
.
.
11. TheEnzymes .
.
.
.
V. Resistance of Gram-NegativeBacteria to ,&Lactam Antibiotics .
. 31
. 31
. 36
. 37
. 38
. 39
. 40
. 40
. 40
. 41
. 41
. 61
. 63
. 63
. 69
. 72
. 72
. 75
C. Possible Interactions Between @-Lactamases and “Intrinsic”
Resistance Mechanisms . . 79
D. Physiological Role and Evolutionary Origin of @-Lactamases . 82
VI. Acknowledgements . . 85
References . . 85
I. Introduction
A. BASICPROPERTIESOF /3-LACTAMASES
Enzymes which destroy penicillin have been known almost as long as
penicillin has been available for therapy. Abraham and Chain detected
penicillin-destroyingactivity in extracts of Escherichia coli in 1940, but
called the enzyme “penicillinase”, largely because the cephalosporins
were unknown at that time and the enzymeswere thought to be specific
for the /3-lactam bond of the penicillin nucleus (Abraham and Chain,
31

32 M. H. RICHMOND AND R. B. SYXES
1940; Abraham et al., 1949). Subsequently the Enzyme Commission
perpetuated this partial view of p-lactamases when they described
their enzyme E.C. 3.5.2.6. as penicillin-amido-,8-lactam-hydrolase,even
though cephalosporins were known at that time (Newton and Abraham,
1955, 1956). This error has proved particularly unfortunate since a
number of p-lactamases exist whose activity is confined almost ex-
clusively to cephalosporins. Throughout this review, therefore, we will
use the term p-lactamase for the enzyme capable of hydrolysing the
p-lactam bond of penicillins and cephalosporins, and refer to “peni-
cillinase” and “cephalosporinase))only when specific manifestations of
lactamase activity are involved.
The reaction catalysed by ,!3-lactamaseswith penicillins as substrates
is the rupture of the p-lactam bond to form the corresponding penicilloic
acid (Fig. 1).With the penicillins this product is normally stable and
there is a stoicheiometric conversion of the penicillin to the anti-
0
I/
0
v-,:::R-C-HN
I/
R-C-HN
F“Z _3
c-o-
0
0
N
OH H II
c-o-
0
0
II
(i) (ii)
FIG. 1. Generalized reactions catalysed by p-lactamases with penicillins as
substrates. (i)basic penicillin structure ; (ii)basic penicilloic acid structure.
biotically inactive “oic))acid. With cephalosporins the picture is more
complex.As with penicillinsthe primary target once againisthep-lactam
bond, but hydrolysis of this link is accompanied by a series of further
changes in the molecule, many of which have not been elucidated in
detail (Fig. 2). Furthermore, the exact sequence of changes depends to
some extent on the particular cephalosporin involved. Probably the first
change is the expulsion, if this is chemically possible, of the substituent
at the %position of the dihydrothiazine ring (acetate in the case of
cephalosporin C and pyridine in the case of cephaloridine; Sabath et al.,
1965). Subsequent,ly the residual 7-substituted cephalosporanic acid
breaks down further to a number of fragments of unknown structure
(Newton and Hamilton-Miller, 1967). This series of reactions can be
independent of the expulsion of the 3-substituent since it occurs in
cephalosporins in which such an expulsion is impossible; for example,
in cephalexin (Table 1).One consequence of the complexity of break-
down of the cephalosporins after ,!3-lactamaseaction is that there is no
stoicheiometric relationship between the destruction of the cephalo-

/3-LACTAMASESOF GRAM-NEGATIVE BACTERIA 33
sporin and the formation of any single product, a fact that invalidates
the iodometric assay of “cephalosporinase” for absolute measurement
(seep. 36).
Even when the substituent at the 3-position in a cephalosporin is
unable to leave, the opening of the /3-lactambond may cause changes
I
II +CH,CO;Nn+
-C N, Hvoothetical
0 I
0
R-C-HN
/I
7’’Fragments
p’
Na+O--C
11 -HN-
0
C-OH
0
FIG.2. Possible reaction sequence catalysed by P-lactamaseswith cephalosporins
as substrates.
ll
in the electron configuration of the molecule. When the 3-substituent
is 2,4.-dinitrostyryl,for example, opening the lactam bond produces a
change in resonance and a shift in the absorption due to the nitro
substituents of the styryl residue from a maximum at 325nm to 485 nm
(O’Callaghanet aZ., 1972);that isthe yellow solutionof the cephalosporin
goes red on openingthe lactam bond, a fact that may be used to assay
“cephalosporinase” activity.

34 M. H. RICHMOND AND R. B. SYKES
Although showing a wide and varying specificity among penicillins
and cephalosporins, p-lactamases seem to require the 4-membered
azetidinone ring to be condensed either with a thiazolidine (penicillins)
or a dihydrothiazine nucleus (cephalosporins). Even a shift in the
position of the double bond in the dihydrothiazine residue of cephalori-
dine from the 3 :4 to the 2 :3 position makes the molecule insusceptible
to p-lactamase (O’Callaghan et al., 1968).
TABLE1. Structuresof Penicillins and Cephalosporins
a Penicillins: Nucleus
R CONSTITUENTS 0
Penicillin G @CH2-C- ll
Ampicillin
Cloxacillin
Mcthicillin
W
Carbenicillin

/~-LACTAMASES OF GRAM-NEGATIVE BACTERIA 35
TABLE1-continued
b. Cephalosporim:
CephalosporinC
Cephaloridine
Cephalexin
~-
Nucleus
R CONSTITUENTS
Rl R2
0
-H
Reports by Saz and his colleagues (Saz and Lowery, 1964, 1965) that
certain p-lactamases could hydrolyse peptides have not been further
substantiated, nor repeated (seePollock, 1967).
Not all penicillins and cephalosporins are susceptible to all p-
lactamases even though they may contain the appropriate nucleus.
A largenumber of substrateprofilesarefound amongnaturally occurring
bacterial enzymes, ranging from extreme “cephalosporinases” on the
one hand to extreme “penicillinases” on the other, and with a number
of intermediates. Certain penicillins may even act as irreversible inhi-
bitors of some lactamases, notably methicillin and cloxacillin acting on
staphylococcalpenicillinase (Gourevitch et al., 1962).
B. METHODSOF ASSAY
A large number of different assay techniques have been used for
p-lactamases and it is worth considering them briefly since no one

technique is ideal for all situations, and the limitations implicit in the
various methods must be taken into account when assessing published
work on this group of enzymes.
All of the early studies on p-lactamases were carried out mano-
metrically, using the appearance of the carboxyl group on the rupture
of the lactam bond as a source of hydrogen ions to liberate carbon
dioxide from bicarbonate buffer (Henry and Housewright, 1947;
Pollock, 1952). This method is now largely obsolete and has not been
used in any recent papers on p-lactamases from Gram-negative bacteria.
By far the most widely used assay is the iodometric method. The
method was originally published by Perret (1954)but since then it has
undergone a number of modifications and refinements. The method is
ideal for the assay of the majority of penicillins, since it relies on the
reaction of eight equivalents of iodine with the penicilloic acid produced
by p-lactamase action (Alicino, 1946). Normally the amount of iodine
that has reacted with the penicilloicacid is determined by back titration
with sodium thiosulphate but, for more precise or sensitive measure-
ments, spectrophotometric estimation, either of the 1,- ion present in
IJKI mixtures (Ferreri et al., 1959; Goodall and Davies, 1961))or of
the blue starchliodine complex, has been used (Novick, 1962b; Sykes
and Nordstrom, 1972).
Iodometric assay of cephalosporins has proved less reliable than with
penicillins, largely because the breakdown pattern of these molecules
is less clear cut (see Newton and Hamilton-Miller, 1967). About four
equivalents of iodine will react with the products of P-lactamase action
on cephalosporin C (Alicino, 1961)but the stoicheiometry of the reaction
varies somewhat depending on the nature of the cephalosporin and the
exact conditions of the assay. In general iodometric assay of cephalo-
sporinsisuseful onlyfor comparative studiesinvolving a singlesubstrate,
and it is unsatisfactory for absolute measurements. A further minor
disadvantage of the iodometric method is that certain penicillins and
cephalosporins react with iodine before the lactam bond is open. Such
molecules are usually those with an iodine-reacting substituent in the
azetidinone ring, either in the 6-position of penicillins or the 7-position
of cephalosporins, but certain substituents in the 3-position of cephalo-
sporins also cause trouble. Examples are penicillins with unsaturated
aliphatic6-substituents(GroveandRandale, 1955)andp-hydroxybenzyl-
penicillin (Sneath and Collins, 1961).
Rupture of the p-lactam bond of cephalosporins (but not of peni-
cillins) causes a change in the absorption spectrum, the band in the
260 nm region being replaced by one at higher wavelength. The exact
position of this band varies from one cephalosporin to another, but is
always in the region 255 to 270 nm. With cephaloridine, for example, it

8-LACTAMASESOF CRAM-NEGATIVE BACTERIA 37
is at 265 nm (O’Callaghanet al., 1968).The rate of destruction of cephalo-
sporins may therefore be followed spectrophotometrically by measuring
the change in absorption at 260 nm, the only exception being cephalo-
sporins in which the 7-substituent has a large absorption maximum in
the same region. The advantage of this method of assay, apart from its
convenience and its ease of adaption to autoanalysers (Lindstrom and
Nordstrom, 1972), is its relative sensitivity when compared with the
standard iodometrictechnique. Its disadvantage is that it cannot be used
€or penicillinase assay. The method is however particularly useful for
studying the inhibitory effects of penicillins, which do not have a
masking ultraviolet absorption on cephalosporin hydrolysis (O’Callaghan
et al., 1968).
Molecules containing p-lactam bonds react spontaneously with strong
hydroxylamine solutions at pH 7.0 to give the relevant hydroxamate
(Boxer and Everett, 1949), and this in turn can be assayed by the colour
produced with Fe3+ions. Although often described in the literature as
purple” the colour obtained with ferric chloride is nearer to a muddy
brown and has a very broad absorption maximum. This fact, together
with the fugitive nature of the colour reaction (Henstock, 1949) and its
great sensitivityto the oxido-reduction potential in the reaction mixture
makes the method somewhat unreliable. Furthermore, compared with
the spectrophotometric and iodometric methods, the hydroxamate
assay is relatively insensitive (Hamilton-Miller et al., 1963). As an assay
method it is now largely passing into disuse. However, it has been widely
used in some of the early papers on p-lactamases from Gram-negative
species, a fact that creates considerable difficulty in relating those
papers, and the enzymes described in them, to more recent work.
Some workers used microbiological assay techniques of various kinds
to assay penicillins and cephalosporins. These methods are inconvenient
unless the technique is being run routinely each day with a large number
of samples. In practice, this means that its use is confined almost
exclusively to the pharmaceutical industry, and since the method has
not been used widely for the characterization of enzymes we will not
consider it further here.
<<
C. ENZYMEUNITS
The most commonly used unit in the early publications on /3-lacta-
mases was the one defined by Pollock and Torriani (1952);namely that
onepenicillinaseunit wasequivalent to one micromol of benzylpenicillin
destroyed per hour at 30” C and at pH 7.0. This unit, as modified by
Richmond (1963), is used in this review. Subsequently the Enzyme
Commission recommended that enzyme units should, if possible, be
based on a time factor of 1min, and consequently a number of workers

have used micromols/min rather than micromols/h as the basis of
the /3-lactamaseunit. A complication that introduces more than simple
calculation is caused by those that have used 35”C or 37”C as assay tem-
peratures in place of 30” C, since the relationship between the rate of
hydrolysis of penicillins and cephalosporins by /3-lactamaseis not linear
nor identicalforallj?-lactamases(SmithandHamilton-Miller, 1963).This
means that it is difficult to relate absolute measurements made at differ-
ent temperatures with any real confidence.
Another complication is introduced by the pH value of the assay
mixture. Pollock and Torriani (1952)were concerned with the assay of
benzylpenicillin by penicillinase from Bacillus cereus and, since pH 7.0
was close to the optimum for that enzyme acting on that substrate, the
choiceof conditions was logical. But the pH optimum of staphylococcal
penicillinase acting on benzylpenicillin is about 5.9 and, accordingly,
the Pollock/Torriani unit was modified for use with that enzyme (Rich-
mond, 1963, 1965).Novick’s decision to use pH 5.9 and 35” C was less
defensible (Novick, 1962a).Ideally the activity of an enzyme against its
substrate should always be measured at the pH optimum, but with
/3-lactamases this has rarely been done. Thus all of the comparative
studies carried out on enzymes from Gram-negative bacteria in this
laboratory (Jackand Richmond, 1970;Richmond et al., 1971)have been
done at pH 5.9, even though the optimum for some of the enzymes
against some of the substrates involved is rather far from this value.
However, although in many ways unsatisfactory, this approach does
greatly decrease the number and nature of the buffer solutions needed
for work on @-lactamasesand does not invalidate comparative studies
so long as the values obtained are not regarded as absolute.
D. TURNOVERNUMBERAND “PHYSIOLOGICALEFFICIENCY”
The wide range of assay conditions used for /3-lactamases leads to
difficultiesover the turnover number (molesof substratehydrolysed/mol
enzymelmin) of purified enzymes. Quite apart from the problems
associated with temperature and pH value, there is no simple solution
to the question of which substrate to use. In the early days this problem
was less acute since a much smaller range of substrates was available
and their properties were reasonably similar. Nowadays, however, apart
from the fact that many of the enzymes are active against both peni-
cillins and cephalosporins, a wide range of different structures with
different properties is available.
Benzylpenicillin has often been used to determine turnover numbers
for /3-lactamases with a predominant “penicillinase” activity. This is
partly because the compound is readily available and also because it

has been widely used for as long as penicillins have been available; and
this is convenientfor comparative purposes. Yet, even with the common
R-factor-mediated enzyme found in many Gram-negative species, the
rate of hydrolysis of ampicillin is almost twice as great as benzyl-
penicillin;yet turnover numbers have been quoted for benzylpenicillin
as substrate.
This difficulty becomes more acute when enzymes that are pre-
dominantly “cephalosporinases” are studied. In certain cases, the rate
of hydrolysisof benzylpenicillin by these enzymes can be measured, but
may be only one-hundredth of the rate of cephaloridine. Does one
therefore quote turnover number in terms of cephaloridine, as would
seem logical in view of the enzymes specificity, or in terms of benzyl-
penicillin? In practice workers in this field seem undecided and ex-
amples of both courses of action are available. The best compromise
in this admittedly unsatisfactory situation is to take benzylpenicillin
as a reference substrate for enzymes with either a predominant “peni-
cillinase” profile or those with a broad specificity covering both peni-
cillins and cephalosporins, and cephaloridine for “cephalosporinases”
despite the fact that the enzymemay not quite exhibit its full hydrolytic
powers on these substrates. Although arbitrary, this compromise can be
justified on the grounds of availability and cheapnessof material, which
is needed in large quantities if systematic enzyme assays are to be
carried out in large numbers. Furthermore, the two antibiotics are used
relatively widely in clinical medicine, and it is useful to have such
compounds when one comes to try to apply /3-lactamase studies to
antibiotic use in a clinicalsituation.
Pollock (1965)introduced the term “physiological efficiency”,defined
as Vmax/Km,as a measure of the behaviour of a /3-lactamase under
“physiologicalconditions”. In many waysthisisa more usefulparameter
of enzyme performance than turnover number, if only because one can
consider the value in relation to substrates of practical importance.
The numerical value of “physiological efficiency” is still, however, at
the mercy of the choice of assay conditions, such as temperature and pH
value. Furthermore, there is a formal objection to studying the “physio-
logical efficiency” of any enzyme out of its context in the cell; but this
point will be discussed more fully later (seep. 79).
E. SPECIFICENZYMEACTIVITY
This term has two, sometimes confusing, uses as applied to P-lacta-
mases. The first (usually quoted as enzyme units/unit mass of enzyme
protein) is a variant of turnover number and is used when the molecular
weight of the enzyme is unknown. The second (usually expressed as

40 M. H . RICHMOND AND R . B. SYKES
enzyme units/mg dry wt bacteria or enzymeunitslunit mass of bacterial
protein) is used to express the amount of enzyme activity expressed by
bacterial culture. Values expressed as units/mg dry wt can be converted
without too much error to unitslunit mass of bacterial protein using a
protein content of about 50% of bacterial dry weight. Rarely, specific
activities are quoted as enzyme unitslnumber of organisms present in
the preparation. Conversion to the other units in this instance can be
achieved if one assumes that lo9bacteria are equivalent to about 1 mg
dry weight.
F. SUBSTRATEPROFILE
The term “substrate profile” has been evolved for strictly com-
parative purposes since it escapes the difficulties inherent in the very
wide differencesin the level of p-lactamase expression in bacterial cells.
Normally, profiles are expressed in terms of ratios related to a value for
one chosen substrate (usually benzylpenicillin) of 100. Thus a profile
of Pen l00:Amp 175:CER 150 indicates an enzyme with a rate of
ampicillin hydrolysis at 1-75times, and of cephaloridine hydrolysis at
1.50 times, that of benzylpenicillin. Although this method of quoting
relative enzyme activities has great advantages, it tends to fall down in
two circumstances. It can be misleading if the rate of hydrolysis of one
particular penicillin or cephalosporin is proportionately very great ;
and secondlyit is difficult to use the ratios for a meaningful comparison
of enzymes that are predominately penicillinases with those that are
predominately cephalosporinases.Furthermore, the values used in the
calculation of the ratios are subject to all of the inherent difficulties of
p-lactamase assay, a fact that may be somewhat obscured by the series
of apparently firm values.
11. The Enzymes
A. p-LACTAMASESFROM GRAM-POSITIVESPECIES
Although this review is primarily concerned with p-lactamases from
Gram-negative species, it is important to refer briefly to enzymes from
other bacteria because their nature and properties are important for any
consideration of the evolution and physiological role of penicillin- and
cephalosporin-destroying enzymes in general. In fact nearly all of the
early work on penicillinases and cephalosporinases concerned Gram-
positive species,and it was only following the introduction of ampicillin
as the first p-lactam antibiotic with a significant activity against Gram-
negative species that attention turned from Bacillus cereus, B. licheni-
formis andXtaphylococcusaureus to the enteric bacteria and Pseudomonas
aeruginosa. Citri and Pollock (1966)reviewed the information available

up to 1965 in great detail, and there isno need to add to that information.
Since then only two major pieces of work have concerned p-lactamases
from Gram-positivespecies.The first isthe elucidation of the amino-acid
sequence of the single polypeptide chain of staphylococcal penicillinase
type A (Ambler and Meadway, 1969). The other is the discovery that
strains of B.cereus569, already known to produce an activepenicillinase,
also synthesize a separate cephalosporinase, but only under certain
environmental conditions. The cephalosporinase is closely related in
structure to the penicillinase made by the same strain, but seems to be
abnormal among ,C?-lactamasesexamined so far in containing Zn2”and
some inucopolysaccharide (Kuwabara, 1970; Kuwabara et al., 1970).
The exact molecular relationship between these two /3-lactamases in
B. cereus 569, together with the molecular basis of their genetic and
regulatory co-ordination, remain to be elucidated.
B. P-LACTAMASESFROM MYCOBACTERIA
Kasic has described the properties of p-lactamase from three species
of mycobacteria in some detail (Kasicet al., 1966; Kasic and Peacham,
1968). The substrate profiles of these three enzymes are summarized in
Table 2, together with those of Bacillus cereus, B. licheniformis and
Staphylococcus aureus for comparison. Unlike the enzymes from the
non acid-fast species, however, the p-lactamases from the mycobacteria
were all constitutive and cell bound. Furthermore, the specific activity
of the enzymes in the cultures increased sharply on disruption of the
cells, the greatest effect being found with Mycobacterium smegmatis
NCTC 8158 where the disrupted bacteria showed about ten times the
activity of the intact cultures (Kasic and Peacham, 1968). In most of
their characteristics, therefore, p-lactamases from mycobacteria show
greater similarity to the enzymesfrom Gram-negative than from Gram-
positive species (see p. 44). It is interesting to notice that the specific
activity of whole cells is larger, and consequent increase in activity on
breakage smaller with the M . smegmatis enzyme when cephaloridine is
used as substrate than is the case with benzylpenicillin or cephalosporin
C (seealso Table 2).
C. /3-LACTAMASESFROM GRAM-NEGATIVESPECIES
The first attempts to classify the p-lactamases from Gram-negative
bacteria were made by Ayliffe (1963) soon after ampicillin was first
introduced into clinical use. In this case, both enzymes studied were
penicillinases” but, soon after, Fleming and his colleagues described a
fblactamase predominantly active against cephalosporins (Fleming et
<<

TABLE2. Comparison of the Substrate Profilesof ,6-Lactamases from Mycobacterium smegmatis, M.jortuitum and M . phlei with
Enzymes from Bacillus cereus, B. lichenijomisand Staphylococcus aureus
Substrateprofile
Temperature
Assay pH value ("C) Penicillin Ampicillin Cloxacillin Cephalosporin C Cephaloridine
Mycobacterium phlei M 7.0 30 100 NT NT 109 285
Mycobacterium srnegmatis M 7.0 30 100 68 0 12 77
Mycobacteriumjortuitum M 7.0 30 100 NT 0 74 91
Bacillus cereus I 7.0 30 100 120 0.5 0 3
Bacillus lichen+wmis I 7.0 30 100 64 0 15 20
Staphylococcus aureus I 5.9 30 100 120 0 0.5 10
Abbreviations:M, manometric assay; I, iodometric assay;NT, not determined.

)B-LACTAMASES OF GRAM-NEGATIVE BACTERIA 43
al., 1963). Since that time such a wide variety of different /3-lactamase
profiles have been detected in various species of enteric bacteria and
pseudomonads that a simple classification into “penicillinases” and
“cephalosporinases” is no longer of much value. Indeed, there seems
to be an almost continuous spectrum of properties extending from
extreme cephalosporinaseson the one hand to enzymes predominately
active against penicillins on the other.
Againstthis diffusebackground, there have been a number of attempts
to group the enzymes in various categoriessince there appears to be an
instinctive feeling among workers in this field that the evolutionary
pattern of a group of organisms will have given rise to a number of
different categories of ,$-lactamase which should be reflected in their
detailed properties, evenif the evolutionary sourceof all of the molecules
is ultimately the same. This may, however, be pure illusion. In the last
analysis the only information that gives any reliable indication as to
the absolute similarity of the proteins is their polypeptide sequences,
and we are still some way from obtaining this information for more than
one of the enzymes concerned. But a number of arbitrary tests have
been used to aid classification; the only rationale behind the choice of
those used being that they do in fact give some sort of pattern within
the whole range encountered so far.
Undoubtedly the ease of identification, and thereby of classification,
has been much aided by the increasing use throughout the world of a
few generally agreed techniques for /3-lactamase assay. Already the
laboratories in Japan (Sawaiet al., 1968), Bristol (Jack and Richmond,
1970; Richmond et al., 1971), Ume&(Lindstrom et al., 1970) and, with
reservations, The School of Pharmacy, London (Dale and Smith, 1971)
are all using similar techniques.
From the information published by these groups over the last few
years, it is possible to identify 15 types of /3-lactamasewith some con-
fidence, several of which have been described in more than one of the
laboratories. The substrate profiles of these enzymes are shown in
Table 3. The properties of the enzymesrange from the extreme cephalo-
sporinase activity of the enzyme from Enterobacter cloacae (Type Ia,
Table 3) to the enzyme almost exclusively active against penicillins
(Type IIa, Table 3). In between these extremes are a range of inter-
mediate, or general purpose profiles. In the following sections we will
classifytheseenzymesby anextensionoftheschemesuggestedpreviously
(Jack et al., 1970; Richmond et al., 1971). This scheme originally recog-
nized four main classes of /3-lactamases:
Class I: Enzymes predominantly active against cephalosporins.
Class I1: Enzymes predominantly active against penicillins.

TABLE3. Overall Classification of fi-Lactamases from Gram-Negative Bacteria on the Basis of their
Substrate Profiles and Relative Activities
Substrate profile
Enzyme Enzyme
class type Penicillin Ampicillin Carbenicillin Cloxacillin Cephaloridine Cephalexin
I a 100 0 0 ND 8000 620
b 100 0 0 ND 350 80
C 100 150 ND ND 2000 ND
d 100 10 0 0 600 80
I1 a 100 180 45 ND t 2 0 0
b 100 160 ND 0 120 0
I11 a 100 180 10 0 140 <10
I V a 100 120 10 1 1 0 150 0
b 100 125 45 20 50 t 1 0
c 100 170 50 20 70 0
V a 100 950 ND 200 120 ND
b 100 300 ND 200 50 ND
C 100 100 60 0 20 <10
d 100 180 80 0 40 t 1 0
ND indicates not determined.

Class I11: Enzymes with approximately equal activity against
penicillins and cephalosporins,but which are sensitive to cloxacillin
inhibition and resistant to p-chloromercuribenzoate.
Class IV: Enzymes of similar substrate profile to those of Class 111,
but which are resistant to cloxacillin and sensitive to p-chloro-
mercuribenzoate. Some, at least, of the enzymes in this group
hydrolyse cloxacillin.
To these four Classes we now propose to add a fifth: Enzymes that
have a penicillinase profile which includes cloxacillin and which are
resistant to sulphydryl agents. This scheme is undoubtedly arbitrary
(like many classification schemes)but does have the advantage that it
reflects the antibiotics currently used for therapy and also that certain
other parameters of enzyme function and nature (sensitivity to inhi-
bitors, sensitivity to antisera, general electrophoretic properties)
correlate with the distribution of the various enzymes in the groups.
One must admit, however, that the complexity of P-lactamase classi-
fication will soon require the full treatment by the formal techniques of
numerical taxonomy.
1. Relationship Between ThisClassi.cationand Those Used Previously
Other classifications have been used in the early stages of the work
summarized in this review and some confusion has arisen as a result.
Strains were “grouped” in Fig. 2 of Jack and Richmond (1970),and the
TABLE4. Correlation of the Jack and Richmond (1970) Classification with
the SchemeUsed in this Review and in Richmond et al. (1971)
Classification in Jack and Richmond (1970)
Grouping in Fig. 2 Type in Table 5
Classification in this review
and in Richmond et al.
(1971)(seaTable 3)
I
I11
I
I
IV
I1
I1
I
Class IIIa
Ia
IVa
IVb
Ib
IIb
IIa
IVC
relationship between the strains shown in that figure and the classifica-
tion used in this review is shown in Table 4.When further parameters
were considered, in addition to substrate profile, a further subdivision,

which cuts across the groups shown in Fig. 2 of Jack and Richmond
(1970),emerged.Eight enzyme types were shownin Table 5 of Jack and
Richmond (1970) and the relationship between these types and the
classificationused now is also shown in Table 4.
Class I Enzymes (seeTable 5 )
All of the enzymes in Class I share a profile in which the rate of
cephaloridine hydrolysis is markedly greater than that of benzyl-
penicillin and ampicillin. Cephalexin is always more resistant to
hydrolysis than cephaloridine. Where examined, the enzymes also
share a number of other characters: (1) powerful competitive inhibition
by cloxacillinand by carbenicillin;(2) positive electrophoretic mobility
at pH 8.5; and (3) a molecular weight close to 29,000. The most widely
examined of these enzymes are the Type Ia enzyme (synthesized by
Aerobacter cloacae strain P99; Goldner et al., 1968: and by Enterobacter
cloacae, 214; Hennessey, 1967 ; Hennessey and Richmond, 1968) and
Type Id, the inducible enzyme from Pseudomonas aeruginosa (Sabath
et al., 1965).
Detailed molecular characteristics of some purified Class I enzymes
are also available. The molecular weight of Type Ia enzyme was
originallyreported to be about 15,000 on thebasis of the rate of filtration
through Sephadex (Hennessey and Richmond, 1968). However, this
value now seems too low since all other estimates for both Ia and Ib
enzyme give values of about 29,000 (Lindstromet al., 1970; R. B. Sykes
and M. H. Richmond, unpublished data).Amino-acidanalyses have also
been published for Type Ia (Hennessey and Richmond, 1968) and
Type Ib (Lindstromet al., 1970) enzymes. These show striking similari-
ties, both to one another and also to the analyses published for some
Class I11 and IV enzymes (seeTables 8, p. 51 and 11, p. 53). This point
is discussed further on p. 60.
A range of enzymes studied by other workers are likely to be members
of this class, and in some cases identification with specific types is
possible. This information is summarized in Table 12 (p. 55). There is
some confusion over the identity of the enzyme synthesized by E. coli
strain D31 (Lindstrom et al., 1970). The Swedish workers quote a
substrate profile of (penicillinG, 100; ampicillin,-0; cephaloridine, 110)
but, when the same enzyme was examined in this laboratory, the profile
was found to be (100:-0: 380) which is typical of Type Ib enzyme (Jack
and Richmond, 1970). It has therefore been included as an example of
this type in Table 5. This is an important point since detailed molecular
characteristics are available for this enzymeand it is the onefl-lactamase
whose synthesis has been proved to be mediated by a chromosomal gene
(Eriksson-Grennberg,1968).

TABLE5. Properties of Class I Enzymes
Inhibited by
Relative activity against ~ Electro-
p-Chloro- phoretic
Enzyme Host Peni- Ampi- Carbeni- Cloxa- Cepha- Cepha- Cloxa- mercuri- mobility Jlolecular Strain
type species cillin cillin cillin cillin loridine lexin cillin benzoate (cm/h) weight number Reference
a Aerobaeter 100 0 0 0 8000 620 S R +0.1 29,000 P99 Jack and Richmond
CIoaeae (1970)
freudii
Escherkhia 100 10 0 0 3300 ND ND ND ND ND 6N324 Sawaiet al. (1968)
b Escherichia 100 0 0
Wli
0 350 80 S R +0.7 ND 719 Jack and Richmond
(1970)
Klebsi5lk~ 100 0 0 0 370 80 s R +07 ND D535
Eseherichh 100 0 0 0 350 ND S R ND ND D31' Lindstrom e6 al.
aeropems
Wli (1970)
c Proteua 100 139 ND 10 1780 ND ND ND ND ND GN76 Sawai et al. (1968)
vulgaris
eulgaris
aeruginosa Sykes and Richmond
Proteus 100 174 ND 10 1700 ND ND ND ND ND GN104
d Pseudmnonas 100 < 10 0 0 600 80 s R +0.3 29,000 All Sabath et al. (1965);
(1971)
a For allocation of this enzyme to Class I,see p. 46.
Inhibition is recorded as enzymesensitivity (S);no inhibition is recorded as enzymeresistance (R). ND indicates not determined.
2

(Advances in microbial physiology 9) a.h. rose and d.w. tempest (eds.) academic press (1973)

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