FEMS Microbiology Letters 10 (1981) 17-20
© Copyright Federation of European Microbiological Societies
Published by Elsevier/North-Holland Biomedical Press
INHIBITION OF SWARMING IN PROTEUS
17
MIRABILIS
BY C A L C I U M I O N S
SIMON A. COLE and DAVID G. SMITH
Department of Botany and Microbiology, University College London, Gower Street, London WC1E 6BT, U.K.
Received and accepted 24 September 1980
1. Introduction
Swarming in Proteus spp. is associated with the
development of greatly elongated, highly flagellate
swarm cells which can spread on the surface of welldried media [1,2]. Many treatments have been shown
to inhibit this swarming [3] ; some swarm-inhibiting
media act by preventing the development of swarmers,
while others provide conditions under which swarmers, although formed, cannot move.
It has long been known that certain cations inhibit
the growth of some bacteria [4], and evidence points
to a central role for divalent cations in microstructures
[ 5 - 9 ] . Calcium and magnesium ions are involved in
the structural integrity of the outer membrane of
Gram-negative bacteria, although details of their function are unclear. Chelation of divalent cations with
EDTA and EGTA allows previously excluded hydrophobic molecules to permeate the cell [8,10,11], so
that Ca 2÷ ions are probably involved in cross-linking
the lipopolysaccharide (LPS) within the outer membrane; loss of these ions releases LPS (but not covalently bound LPS); possibly exposing regions of lipid
bilayer in the membrane and allowing access to the
peptidoglycan and plasma membrane.
Proteus mirabilis is unaffected by lysozyme/EDTA
lysis procedures, because O-acetylation o f Proteus
peptidoglycan renders it insensitive to lysozyme [12]
and possibly because the sensitive sites on the cell are
inaccessible to EDTA; however, EDTA does inhibit
swarming of Proteus [6]. EDTA prevents swarmer
differentiation but not the progress of an established
swarm [6], perhaps by removing a small proportion
of the divalent cations from the LPS.
It is reported here that Ca 2÷ prevents swarming on
solid medium, an event apparently contrary to the
effects of EDTA.
2. Materials and Methods
2.1. Organisms
The strain o f Proteus mirabilis used was a hospital
isolate designated P11.23 other swarming strains of
Proteus sp. were also tested, and a swarming strain of
Clostridium tetani was used to compare anaerobic
swarmers with Proteus grown aerobically.
2.2. Media and culture conditions
Liquid growth medium was Oxoid Nutrient Broth
No. 2; solid media were made by adding 2% (w/v)
Difco-Bacto agar, or from Oxoid MacConkey agar
No. 2. Autoclaved agar was cooled to 50°C before
pouring and plates were dried at 60°C for 60 min.
When used, 7% (v/v) defibrinated horse blood (Oxoid
(w) HDO3) was added at 50-55°C.
Calcium ions were added to broth, or to molten
agar before pouring as 10× concentrated autoclaved
stock solutions of anhydrous CaC12 or the dihydric
salt Ca (NO3)2.4H20 was also tried.
Ca 2÷ ions (as the chloride salt) were added to nutrient agar to final concentrations from 0.001 M to
0.2 M. Concentrations above 0.1 M produced a slight
haziness in the agar.
Plates were inoculated from slope cultures and cultures were incubated at 30°C.
CI. tetani was cultured anaerobically using "Gas
Pak" anaerobic generators.
18
2.3. [ 14C] Uracil incorporation
Rifampicin (25/lg/ml), [2J4C] uracil (0.1 #Ci/ml;
Radiochemical Centre, Amersham) and Ca 2+ ions at
concentrations of 0.05 M and 0.125 M were added to
exponential nutrient broth grown cells. The cultures
were shaken at 30°C and 0.1 ml samples were taken
every 20 rain for 3 h onto 2 cm discs of Whatman
3MM filter paper. The papers were kept overnight at
4°C in 5% (w/v) trichloroacetic acid (TCA) containing
100 g/ml [X2C]uracil" They were then washed twice
in cold 5% TCA, and once each in 50 : 50 (v/v)
ether : ethanol and ether. The dry papers were put
into scintillation vials with 10 ml of toluene containing 6 g/1 butyl-PBD scintillant, and counted in a
Tracerlab Coru/matic Liquid Scintillation Spectrometer.
3. Results
Increasing the level of Ca 2÷ ions from zero caused
a decrease in the size of swarm zones ofP. mirabilis
P11 (Fig. 1) and greater definition of the edge of each
zone. 24 strains of Proteus were tested and complete
/
control
0'0020005
02
Zone
diametel
{cm)
4
3
,
1
Zone
i
2
number
i
3
i
4
Fig. 1. The effect of Ca2+ on the swarming of Proteus mirabilis
P11. For each concentration of Ca2+ zone diameter is plotted
against zone number. A fall in gradient shows inhibition of
swarming, due to reduction in the diameter of swarm zones.
inhibition of swarming occurred at concentrations
varying between 0.09 M and 0.15 M Ca 2+ in nutrient
agar, the value being strain-dependent. In general
0.125 M Ca 2+ was sufficient to inhibit most strains,
but three required 0.15 M. The inhibitory concentrations were unaffected by the addition of horse blood,
and at lower Ca 2÷ concentrations the blood did not
affect the size of swarm zones. In a test using a haemolytic strain of Streptococcus faecalis (H69D5) it
was shown that the presence o f Ca 2+ ions in blood
agar did not affect haemolysis although growth was
reduced. Ca(NO3)2 was slightly more inhibitory to
swarming than CaCI2, being effective from 0.75 M to
0.1 M, probably due to the nitrate ions.
The Proteus strains were also tested on MacConkey
agar, where the level of Ca 2÷ needed to inhibit swarming was 0.005 M (less than 5% of the level needed in
nutrient agar) for all but one strain, which required
0.02 M (this strain also required a high Ca ~÷ concentration on nutrient agar). Half the strains did not
swarm on MacConkey agar control plates without
Ca 2÷, and swarming of the others was less than on
nutrient agar.
Cells from non-swarming colonies on inhibitory
Ca 2+ media were subcultured onto fresh nutrient agar
without added Ca 2÷, and all strains were able to
swarm, showing that the effect of calcium ions was
reversible.
Ca 2+ ions were added to liquid medium at final
concentrations between 0.1 M to 0.2 M, and growth
ofP. mirabilis P11 at 30°C was followed by measuring absorbance at 490 nm in a Hilger colorimeter.
There was a fall in the growth rateunder these conditions, but the stationary population was not greatly
affected.
In a separate experiment, 50 mg/ml of rifampicin
was added to liquid medium containing 0.2 M Ca ~÷
ions and growth was followed as above; by comparison with controls growth was greatly reduced, showing
that Ca 2+ ions permitted rifampicin to enter previously
impermeable cells.
The permeation of rifampicin into Ca2+-treated
cells was also followed by studying the inhibition of
[14C]uracil incorporation into RNA (Fig. 2). Rifampicin inhibited lac incorporation in the presence o f
CaC12 and Ca(NO3)2 equally, with the degree of
inhibition being dependent on Ca 2+ concentrations.
Using "half-plates" containing EDTA-agar and nu-
19
o
o
o
./
7
CPM
(x 1000)
5
i Oi
I
1
i
i
i
2
i
from plate counts, each method gave consistent results
and there was no difference in counts between 0.125 M
Ca 2÷ agar and CLED. However, moisture added to the
plates in the sample allowed Proteus to produce small
(0.5 cm) swarms on CLED; these did not prevent
counting but enumeration of the colonies was easier
on Ca2+-agar where no such swarms were produced.
As a comparison with Proteus, a swarming strain
of the anaerobe Clostridium tetani was tested for
growth and swarming ability on blood agar containing
0.125 M Ca2+; growth o f Proteus on this agar was as
good as on a control without added Ca 2÷, while that
o f C/. tetani was poor by comparison with the control.
Proteus grown on Ca 2÷ agar was observed on agar
by phase contrast microscopy. The outer edge of a
swarming colony o f Proteus consists of long cells up
to 6 0 - 8 0 wn in length. At concentrations of Ca :÷
causing swarm reduction, the cells at the swarming
edge were motile but not as elongated as normal
swarmers; non-swarming colonies showed only short
cells. In a hanging-drop preparation, cells grown on
Ca 2÷ were non-motile, and electron microscopy
showed that this was due to Ca2÷-agar grown cells
being devoid of flagella.
i
Time(h)
Fig. 2. The incorporation of [ 14C] uracil into RNA in P. mirabilis P11, and the effect on incorporation of Ca~" and rifarr.picin (o, cc.ntrol; o, 25 ~g/nd rifampicin; [], 0.05 M Ca~;
*, 0.125 M Ca~'; A, 0.05 M Ca~" and 25 ~g/ml rifampicin;
A, 0.125 M Ca~ and 25/~g/ml rifampicin).
trient agar, Weiser et al. [6] showed that EDTA
inhibits swarm initiation but not established swarmhag. Using the same technique with Ca 2÷ agar "halfplates", it was shown that Ca :÷ inhibited both swarm
initiation and established swarming, suggesting that
the effect of Ca :÷ is more far-reaching than that of
EDTA. Inhibition occurred before the swarm reached
the Ca:+-agar, due to diffusion of Ca 2+ into the nutrient agar.
Viable counts ofP. mirabilis P11 were carried out
on 0.125 M Ca :+ agar and Cystein-Lactose Electrolyte
Deficient Medium (CLED; Oxoid), using either the
plate count method with a glass spreader or the Miles
and Misra [ 13 ] technique. While counts from the
Miles and Misra technique were lower than those
4. Discussion
The function of individual components in the
Gram-negative outer cell wall membrane is largely unclear.
Molecules of LPS seem to be held in position by
two mechanisms. The first, hydrophobic and ionic
interactions with other molecules; the second involves
divalent cation binding through a monophosphate at
the end unit of Lipid A. The hydrophilic "picket
fence" representation of Leive [8] and Costerton et
al. [14] requires the presence of divalent cations, and
adding EDTA releases a fraction of the LPS; the
remaining LPS is probably bound by the first mechanism. Leive [8] suggests high divalent cation concentrations increase LPS-LPS binding, and evidence [ 16,
17] indicates re-arrangement of the outer membrane.
Newly synthesised LPS is added to existing LPS at
insertion sites [18,19], and excess Ca 2÷ here would
prevent new LPS bridging gaps, allowing previously
excluded molecules to pass through the outer membrane.
20
Flagella development during swarmer differentiation occurs before cell elongation [20] and may act
as a trigger for the latter. LPS, cross-linked by Ca 2÷
or Mg2÷ ions, may be involved in holding the flagella
and allowing the shaft to rotate without damaging the
membrane. Re-arrangement of LPS round the flagella
may expose areas o f phospholipid and alter permeability [21]. Armitage et al. [22] showed that swarm
cells are more permeable than short cells to rifampicin (normally excluded), suggesting LPS aggregation
around the flagella; it was shown here that Ca 2÷ ions
allow rifampicin to permeate P. mirabilis P11, perhaps
due to heterogeneity in the outer membrane as a
result of locally increased LPS-LPS linking. Cells
grown in Ca 2÷ were non-flagellate, due to inadequate
retention of the flagella by LPS, or to an effect on
their assembly.
EDTA inhibits swarming initiation but not established swarming [6], while it is reported here that
Ca 2÷ inhibits both events. EDTA may crosslink firmly
held Ca ions in the LPS, securing the LPS and preventing re-arrangement round the flagella, thus halting
flagellar development. However, established swarmers
would not be affected, and swarming would continue
to the end of the phase. EDTA inhibition was reversed
by Ca 2÷ ions [6], possibly by exchange with EDTAbound Ca 2÷ in the LPS or saturation of the chelating
sites, which allowed movement of the LPS. Presumably
in swarm inhibition by Ca 2+, the Ca 2÷ ions cross-linked
the LPS more securely and flagella development was
prevented. But, in addition, Ca 2÷ ions may have pulled
the LPS away from the flagella, releasing them so that
established swarming is also prevented.
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