Journal of General Microbiology (1 985), 131, 23 13-23 18. Printed in Great Britain
2313
Three Immunity Types of Klebicins which Use the Cloacin DF13 Receptor
of Klebsiella pneumoniae
By P E A R L C . C O O P E R * A N D R I C H A R D J A M E S
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U K
(Received I1 March I985 ;revised 24 May 1985)
We have investigated a group of bacteriocinogenic strains used in the epidemiological
investigation of Klebsiella infections. Transfer of plasmids from these strains to laboratory
strains allowed the identification of three klebicins which use the cloacin DF13 receptor in
Klebsiella, but are of three distinct immunity types. These klebicins use the ferric-aerobactin
receptor determined by ColV-K30 in Escherichia coli, which is also used by cloacin DF13. We
propose to call them group A klebicins, of immunity types A l , A2 and A3. On the basis of
immunity, cloacin DF13 also belongs to the klebicin A1 group.
INTRODUCTION
Klebsiella species are common causes of hospital-acquired infections of the lower respiratory
tract, urinary tract, surgical wounds and blood (Cross et al., 1983). Several bacteriocin typing
schemes have been proposed for the epidemiological investigation of Klebsiella infections
(Slopek & Maresz-Babczyszyn, 1967; Hall, 1971 ; Buffenmeyer et al., 1976; Heddell & Mitchell,
1978;Edmondson & Cooke, 1979;Bauernfeind et al., 1981 ;Israil, 198l), but none has come into
widespread clinical use. Each scheme, with various modifications, involves testing isolates for
resistance or sensitivity to the bacteriocins (klebicins) produced by a set of producer strains. The
choice of producer strains, different in most of the schemes, is empirical. The klebicins used are
uncharacterized as regards the number, titre and type, whilst the interpretation of results is
subjective, especially the ‘weak sensitive’ response. We believe that the use of stable, high-titre
preparations of receptor-specific klebicins will increase the reliability of klebicin typing. We
have analysed a number of producer strains in order to characterize their klebicins. Here we
describe three klebicins which use the same cell surface receptor, but which can be
differentiated by immunity tests. Immunity, a plasmid-determined function, protects a
bacteriocinogenic cell from the killing effect of the bacteriocin it produces. Cross-immunity
between bacteriocinogenic strains indicates that their bacteriocins are very similar or identical,
whereas lack of cross-immunity indicates that they are different. The receptor used by the three
klebicins described here is apparently the same as that for cloacin DF13.
METHODS
Bacterial strains undplasmids. The strains o f Klebsiellapneumoniae and Escherichia coli are listed in Table 1. The
addresses of workers who supplied the strains are: N . Carbonetti, Department of Genetics, University of
Leicester, UK ; E. M. Cooke, Public Health Laboratory Service, Colindale, U K ; E. Lederberg, Plasmid Reference
Center, Stanford University, Calif., USA; M. Merrick, Unit of Nitrogen Fixation, University of Sussex, U K ; J.
Miller, Department of Molecular Biology, University of Geneva, Switzerland; B. Oudega, Department of
Microbiology, Vrije Universiteit, Amsterdam, The Netherlands; P. Williams, Department of Genetics,
University of Leicester, UK.
Media. LB and M9 media were used with the appropriate supplements (Miller, 1972) and solidified with 1 %
Davis agar for plates. All incubations were at 37°C.
Conjugation. The R-plasmid R64- 1 1 was used to mobilize klebicin plasmids to genetically marked laboratory
strains. R+ (tetracycline-resistant) transconjugants were purified and screened for klebicin production. For liquid
0001-2556 0 1985 SGM
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P. C . C O O P E R A N D R . J A M E S
Table 1. Bacterial strains and plasmids
Strain
K . pneumoniae
M5al
UNF801
UNF 1785
UNF5023
PC901
PC902
P1 to P15
PC903
PC904
PC905
PC906
PC907
PC908
PC909
PC910
PC911
E. coli K12
W3110
LG 1740
LG1741
CSHSO
PC663
PC664
PC258
F205
Plasmid
R64-11
pJN73
ColV-K30
ColV-K30 : :Tn801
Relevant properties
Source or derivation
Wild-type
his hsdR nif: :TnS
trp leu lys rpsL
his hsdR rpsL
UNF1785 spontaneous cloacin DF13
resistant mutant
U NF5023(pJN73)
Klebicin producers
P3(R64-11)
P5(R64-11)
U N F5023(pP5a)
UNF5023(R64-1 l)(pPSa)
UN F801(R64-1 l)(pPSa)
UNF80 1(R64-1 l)(pPSb)
U NF5023(pP5b)
UNF5023(R64-1 l)(pP3)
U N F5023(R64- I l)(pPSa)(pPSb)
M. Merrick
M. Merrick
M. Merrick
M. Merrick
This laboratory
Prototrophic, plasmid-free
W3110 (ColV-K30); cloacin DF13
sensitive
W3110 (ColV-K30 : :Tn801); lacks
ferric-aerobactin receptor
Auxotrophic, plasmid-free
CSHSO (ColV-K30); cloacin DF13
sensitive
CSHSO (ColV-K30 : :Tn801); lacks
ferric-aerobactin receptor
R64-11 donor
pJN73; produces cloacin DF13
E. Lederberg
P. Williams
Tet', Tra+
pClo-DF13 : :Tn901; cloacin production
and immunity, Amp'
ColV+, cloacin sensitivity
Cloacin production and immunity;
lacks ferric-aerobactin receptor gene
F205 x UNF5023 by transformation
E. M. Cooke
PC258 x P3 by conjugation
PC258 x P5 by conjugation
P5 x UNF5023 by transformation
PC258 x PC905 by conjugation
PC906 x UNF801 by conjugation
PC904 x UNF801 by conjugation
PC908 x UNF5023 by transformation
PC903 x UNF5023 by conjugation
PC907 x PC909 by conjugation
N. Carbonetti
J. Miller
LG 1740 x CSH5O by conjugation
LG1741 x CSHSO by conjugation
This laboratory
B. Oudega
This laboratory
B. Oudega
P. Williams
N. Carbonetti
matings, 5 pl of overnight broth cultures of donor and recipient strains were inoculated into 5 ml LB medium and
incubated overnight without shaking, and then plated on selective medium. For plate matings, a streak plate of
donor colonies was replicated onto a lawn of recipient cells on LB agar, which was incubated overnight and then
replicated onto selective medium.
Transjormation. Cells were transformed as previously described (Chak & James, 1984). After time for
expression, transformed cells were diluted into broth consisting of 1 vol. sterile klebicin extract and 2 vols LB
medium, grown overnight with shaking, then plated out to obtain single colonies for klebicin testing.
Klebicin production. The stab/overlay method described for colicins (Ozeki et al., 1962) was used to detect
klebicin-producing transformants and transconjugants.
Preparation of klebicin extracts. Klebicinogenic strains were grown to ODss0 0.15 4 . 2 with shaking, and then
mitomycin C was added to a final concentration of 0.5 yg ml-' . After 4 h of further incubation, chloroform was
added and the cultures were shaken for an additional 30 min. After centrifugation at 20000g for 10 min the pellets
were discarded. The supernatants, with klebicin activity, were stored at 4 "C and retained activity for at least 4
weeks. They gave clear zones on M5a1, the sensitive control strain, when used undiluted or at a lo-' dilution.
Klebicin sensitiuity testing. A 0.1 ml sample of an overnight culture of indicator bacteria in 2.5 mlO*6%Davis agar
was overlaid on an LB agar plate. Drops ( 5 yl) of undiluted klebicin extracts were then spotted on the plate. Clear
zones of inhibition after overnight incubation were scored as sensitivity to the klebicin extract. Sensitivity testing
was repeated at least three times.
Plasmid preparation and agarose gel electrophoresis. The methods for the preparation of plasmid DNA and
electrophoresis have been previously described (Chak & James, 1984).
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Three immunity types ojklebicins
Table 2. Sensitivity and resistance of uarious strains to klebicin extracts
+,
The plasmid present in the indicator strains is given in brackets:
clear zone of inhibition; -, no
zone of inhibition; (+), turbid zone of inhibition probably due to separate colicin activity.
Reaction to extract of:
Indicator
strain
f
M5al
W3110
LG1740[ColV-K30]
UNF1785
PC90 1
UNF5023
PC902 [pJN73]
PC905 [pPSa]
PC907 [pPSa]
PC908 [pPSb]
A
P1
+
+
++-
+
P2
P3
(+)
-
+
+
++-
+
+
+
++
-
-
+
+
-
+
P4
+
+
+
++
-
+
+
P5
P8
+
+
+
++
+
++
+
+
++-
+
-
+
+-
+
-
-
-
+
+
\
PC902
-
-
Table 3. Immunity tests between representatiue klebicin-producing strains
The Klb+ plasmid present in the strains from which extracts were made is shown in brackets :
zone of inhibition; -, no zone of inhibition; NT, not tested.
+ , clear
Reaction to extract of:
Indicator
strain
PC905
PC907
PC902
PC908
PC910
PC663
PC664
PC905
[pPSaI
PC907
[pPSaI
PC902
ipJN731
PC908
-
-
-
-
-
-
-
-
-
+
+
+
-
+
+
+
-
+
+
+
NT
-
+
+
+
+
-
PC910
[PP~I
-
+
+
+
+
-
-
1
P5
P3
-
-
NT
NT
+
+
+
+
+
+
+
+
+
RESULTS
Identification of klebicins which use the cloacin DF13 receptor
K . pneumoniae strain M5al and derivatives were sensitive to cloacin DF13 determined by
pJN73 suggesting that, like Enterobacter cloacae H478 or E . coli K 12 strains carrying ColV-K30,
sensitivity was due to the presence of an outer membrane protein which serves as the ferricaerobactin receptor (van Tiel-Menkveld et al., 1981; Bindereif et al., 1982; Grewal et al., 1982).
To identify klebicins which use this receptor, the 15 extracts of the Edmondson & Cooke (1979)
producer panel were screened for those active on UNF1785 and inactive on cloacin DF13resistant mutants such as PC901. Six extracts fell into this category (Table 2), all of which were
inactive against E. coli K12 strains, inherently cloacin-resistant due to lack of a receptor, but
were active on E. coli K12 strains carrying ColV-K30. Extracts of P1 and P2 were inactive on
PC902 which carries pJN73, suggesting that these klebicins are of the same immunity type as
cloacin DF13. The extracts of P3, P4, P5 and P8 were active on strain PC902, showing that they
contained klebicins of an immunity type different from cloacin DFl3. Klebicinogenic (Klb+)
plasmids were transferred from strains P5 and P3 to laboratory strains of K . pneumoniae for
further analysis.
Isolation ojpP5a
Plasmid DNA isolated from strain P5 was used to transform UNF5023. One type of Klb+
transformant only was isolated, characterized by PC905 (Table 3). Like P5, its klebicin was
active against UNF1785 but not PC901. However, its klebicin was also inactive against PC902,
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P. C. COOPER A N D R . JAMES
Fig. 1. Agarose gel electrophoresis of DNA isolated from klebicin-producing strains : (a) PC903 ; (b)
PC908; (c) P5; ( d ) PC905; (e) PC909; (j)PC911. The positions and approximate sizes of plasmids
present in these strains are shown on the left-hand side of the figure. The faint, large plasmid present in
P5 is a native plasmid and not R64-11.The chromosomal DNA band (chr) contaminating the plasmid
preparations is indicated.
indicating that pJN73 carries immunity to it. This result was unexpected because PC902 was
sensitive to P5 extract. As an indicator PC905 was resistant to all six klebicin extracts and
cloacin DF13 and presumably lacked the appropriate receptor. Agarose gel electrophoresis of
plasmid DNA isolated from PC905 showed that it carried a 10 kb plasmid, pP5a (Fig. 1). R64-11
mobilized pP5a from the transformant to UNF801 and transconjugants, like PC907, were
obtained which were cross-immune with PC902, confirming that their bacteriocins were of the
same immunity type. Sensitivity of both strains to P5 extract showed that this extract contained
a second klebicin of a different immunity type. Surprisingly, a plasmid band equivalent to pP5a
was not seen on agarose gels of DNA from P5 (Fig. l), despite several attempts. However, two
other plasmids of 7-5kb and 110 kb were visible, one of which presumably coded for the second
klebicin.
Isolation of pP5b
It was considered likely that the plasmid coding for the second klebicin was the 7.5 kb
plasmid. The R-plasmid R64-11 was introduced into strain P5 to try to mobilize this small
plasmid. (Although P5 has a large and possibly transmissible plasmid, it carried no obvious
selectable marker such as antibiotic resistance.) The resulting strain PC904 was mated with
UNF8Ol. Several Tcr transconjugants, such as PC908, were obtained, all of which were Klb+
and their klebicin was active on the cloacin-immune strains PC902 and PC907. They were
sensitive to cloacin and the pP5a klebicin produced by PC902 and PC907 respectively (Table 2).
Agarose gel electrophoresis of plasmid DNA isolated from strain PC908 showed that this strain
contained the 7.5 kb plasmid of strain P5, which we call pPSb, together with R64-11 (Fig. 1).
Strain PC908 was sensitive to extracts prepared from strains PI, P2, P3, P4 and P5 (Table 2). It
was immune to P8 extract, suggesting that the P8 klebicin belonged to the same immunity group
as p5b.
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Three immunity types of klebicins
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Isolation of pP3
An extract of strain P3 was active on UNF5023 strains carrying pP5a or pPSb, or both,
indicating that strain P3 produced a klebicin of a third immunity type. In agarose gels of DNA
isolated from this strain only one plasmid band, of 13 kb, was visible. R64-11 was used to
mobilize this plasmid from PC903 into UNF5023 and Klb+ transconjugants were isolated, i.e.
strain PC910. The klebicin produced by PC910 was active on PC902, PC907 and PC908, whilst
PC910 was sensitive to the klebicins produced by these strains (Table 3). Agarose gel
electrophoresis showed that PC910, like PC903 (Fig. l), carried R64-11 and the 13 kb plasmid
pP3 which presumably codes for the klebicin of the third immunity type.
Requirement for the ferric-aerobactin receptor of’ E. coli (ColV-K30)
The activity of the three klebicins determined by pPSa, pP5b and pP3 was tested against an E.
coli K12 strain, PC663, carrying ColV-K30 and PC664 carrying the mutant ColV-K30 plasmid,
in which the gene for the ferric-aerobactin receptor is inactivated by Tn801 (Grewal et al., 1982).
All three extracts, active on the former, were inactive on the latter (Table 3), providing further
evidence that this receptor is required presumably for adsorption into sensitive cells.
DISCUSSION
The results presented here show that there is a group of klebicins which apparently require the
same receptor as cloacin DF 13. They are inactive on cloacin-resistant mutants of K . pneumoniae
which presumably lack this receptor. They are active on E. coli K12 strains carrying ColV-K30,
but not if the structural gene for the ferric-aerobactin receptor on this plasmid has been
inactivated by transposon mutagenesis. The cloacin (ferric-aerobactin) receptor of Enterobacter
cloacae is a 90 kDal outer membrane protein (Oudega et al., 1979) and that determined by ColVK30 in E. coli K12 is 74 kDal (Grewal et al., 1982). As yet, we have not identified the relevant
receptor of the K . pneumoniae strains used in this study. We predict that it is the ferric-aerobactin
receptor in these strains as well.
We have established that there are three immunity types in the group of klebicins we studied,
and further immunity types may occur. In the case of the E colicins which use the vitamin B12
receptor of E. coli for adsorption, nine immunity types have been reported (Fredericq, 1956;
Males & Stocker, 1982; Cooper & James, 1984). We will refer to the klebicins reported here as
group A klebicins, with A l , A2 and A3 as the immunity types. Thus the klebicin encoded by
pP5a is klebicin A1-P5, that by pP5b is klebicin A2-P5, and that by pP3 is klebicin A3-P3. By
this convention cloacin DF13 could be referred to as klebicin A1-DF13. The results in Table 2
show that the klebicins produced by strains P1 and P2 belong to the A1 group, and that of P8
belongs to the A2 group. Analysis of strain P4 showed that it is similar to strain P5, having
klebicin A 1 and A2 activities (J. Schneider, personal communication). These results imply
considerable redundancy in the original typing panel. Transfer of plasmids to laboratory strains,
which has proved invaluable in the classification and identification of colicins, has allowed a
detailed analysis which is essential in rationalizing the choice of klebicins for a typing panel.
These klebicin plasmids were introduced into M5al derivatives by transformation or by Rplasmid mobilization. The reason why plasmid pP5a is not visible in agarose gels of DNA from
strain P5, but is visible in K . pneumoniue M5al derivatives carrying pP5a alone or with pP5b and
R64-11 (Fig. l), is not clear. Perhaps it is present in lower copy number in the genetic
background of P5. Transformants with a plasmid preparation of P5 were selected on the basis of
immunity to P5 extract, which was higher in klebicin Al-P5 activity than in klebicin A2-P5
activity, and would thus favour pP5a transformants. R-plasmid mobilization of pP5b from
PC904 may be favoured over pP5a as it is a smaller plasmid and/or may be present in higher
copy number. We have not explored the mobilization frequencies of the two plasmids further.
Plasmid pP5a appears to be similar to pClo-DF13 on the basis of restriction mapping. Plasmid
pClo-DF13 confers colicin E6 immunity on E. coli K12 (Males & Stocker, 1982) and we have
found that pP5a also confers E6 immunity on E. coli K 12. The colicin E6 immunity gene on this
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P . C . COOPER A N D R . JAMES
plasmid is distinct from the klebicin immunity gene. In contrast, pP5b and pP3 confer colicin E3
immunity but not E6 immunity on E. coli K12 (unpublished observation). The relationship
between E colicins and A klebicins is intriguing and may be significant in understanding their
evolutionary origins.
We thank Drs Carbonetti, Cooke, Lederberg, Merrick, Oudega and Williams for bacterial strains and useful
information, Kin-Fu Chak, Mark Lawrence and Jorg Schneider for helpful discussions of this work and Jill
Debbage for excellent technical assistance. This research was funded by an MRC project grant.
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