Download Colicins produced by the Escherichia fergusonii strains closely

FEMS Microbiology Letters 208 (2002) 259^262
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Colicins produced by the Escherichia fergusonii strains closely
resemble colicins encoded by Escherichia coli
David Símajs
b
a;b
, Sandor E. Karpathy
a;b
, Jan Símarda
c;
, George M. Weinstock
a;b;d
a
Department of Microbiology and Molecular Genetics, University of Texas-Houston Medical School, Houston, TX, USA
Center for the Study of Emerging and Re-emerging Pathogens, University of Texas-Houston Medical School, Houston, TX, USA
c
Department of Biology, Faculty of Medicine, Masaryk University, Jos›tova 10, CZ 662 43 Brno, Czech Republic
d
Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA
Received 7 December 2001; accepted 8 January 2002
First published online 6 February 2002
Abstract
Plasmid DNA of six Escherichia fergusonii colicinogenic strains (three producers of colicin E1, two of Ib and one of Ia) was isolated and
the colicin-encoding regions of the corresponding Col plasmids were sequenced. Two new variants of colicin E1, one of colicin Ib, and one
of colicin Ia were identified as well as new variants of the colicin E1 and colicin Ib immunity proteins and the colicin E1 lysis polypeptide.
The recombinant Escherichia coli producer harboring pColE1 from E. fergusonii strain EF36 (pColE1-EF36) was found to be only
partially immune to E1 colicins produced by two other E. fergusonii strains suggesting that pColE1-EF36 may represent an ancestor
ColE1 plasmid. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Col plasmid ; Colicin; Immunity ; Escherichia fergusonii
1. Introduction
Colicinogeny (i.e. the ability of bacterial strains to produce colicins) is one of the frequent characteristics of Escherichia coli strains isolated from both natural and clinical isolates. Approximately 40% of E. coli strains of
human origin have previously been shown to be colicinogenic [1].
Within the group of 50 Escherichia fergusonii strains
mostly of human origin, six strains (12%) were identi¢ed
as colicinogenic (J. Símarda, unpublished results). E. fergusonii was established as a new species of the genus Escherichia in 1985 and is most closely related to E. coli and
Shigella sp. and more distantly related to other bacterial
species of the family Enterobacteriaceae [2].
To test whether the close relatedness of E. fergusonii
strains to strains of E. coli and Shigella sp. also re£ects
similarities in colicin types produced by E. fergusonii we
characterized six individual Col plasmids isolated from
E. fergusonii colicinogenic strains. In this communication
we describe colicin-encoding regions of E. fergusonii-derived Col plasmids and compare the corresponding colicin,
immunity and lysis proteins to those described in E. coli.
2. Materials and methods
2.1. Bacterial strains and growth conditions
All E. fergusonii colicinogenic strains were provided by
the National Reference Laboratory for E. coli and Shigellae, Center of Epidemiology and Microbiology, National
Institute of Public Health, Prague, Czech Republic. All
colicinogenic E. fergusonii strains were of human origin.
E. coli TOP10FP (Invitrogen, San Diego, CA, USA) was
used as a colicin indicator. Bacterial strains were grown at
37‡C in TY medium containing 8 g Bacto-tryptone (Difco
Laboratories, Sparks, MD, USA), 5 g yeast extract and 5 g
NaCl per liter (pH 7). For selection and maintenance of
plasmids, 25 Wg of chloramphenicol or 25 Wg of kanamycin
per ml of liquid medium and 1.5% TY agar (w/v) were
added.
* Corresponding author. Tel. : +420 (5) 42126-259;
Fax : +420 (5) 42126-200. E-mail address: [email protected]
(J. Símarda).
0378-1097 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 4 6 8 - 8
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2.2. Crude colicin preparations and colicin activity assays
2.5. Nucleotide sequence accession numbers
Cells from the TY cultures of colicinogenic E. fergusonii
or recombinant E. coli strains (producers of colicins E1, Ib
and Ia) induced by mitomycin C (Sigma, St. Louis, MO,
USA ; 0.5 Wg ml31 ) were harvested, resuspended in distilled water, washed and sonicated. The sonicates were
used as crude colicins. Colicin activity was tested by spotting 10-fold dilutions of colicin-containing crude cell lysates on agar plates seeded with sensitive bacteria ; TY
agar plates were overlaid by 3 ml of 0.75% (w/v) TY
agar with 100 Wl of an overnight culture of indicator bacteria. Each experiment was performed at least three times
and the data represent the average of three independent
measurements.
The nucleotide sequences reported in this study were
deposited in the GenBank under the accession numbers
AF453410^AF453415.
3. Results and discussion
Among 50 strains of E. fergusonii investigated, six colicinogenic strains were identi¢ed (J. Símarda, unpublished
results). Plasmid DNA from all six colicinogenic strains
was isolated and colicin-encoding regions were identi¢ed
and sequenced using in vitro transposon insertions.
3.1. E. fergusonii EF43
2.3. In vitro transposition
To isolate the plasmid governing colicin synthesis and to
sequence the colicin-encoding region, two subsequent in
vitro transposition steps were performed with Tn5- and
Tn7-based in vitro transposition systems. For Tn7 plasmid
mutagenesis, an in vitro Tn7 transposition system
(GPS1-1 Genome Priming System, New England Biolabs)
was used according to the manufacturer’s recommendations. An EZ: :TN1 pMOD1-2 Transposon Construction
Vector (Epicentre Technologies, Madison, WI, USA) with
a cloned kanamycin resistance gene served as the DNA
template for ampli¢cation of the Tn5 transposon, and
transposition was performed according to the EZ: :TN1
TET-1 Insertion Kit (Epicentre Technologies) protocol.
The DNA sequencing reactions were directed from both
ends of the Tn5 transposon using pMODFseq (5P-GCCAACGACTACGCACTAGCCAAC-3P) and pMODRseq (5P-GAGCCAATATGCGAGAACACCCGAGAA3P) primers. Alternatively, Tn7RN and Tn7LS primers recognizing the ends of Tn7 transposon were used for sequencing (Tn7RN: 5P-ACTTTATTGTCATAGTTTAGATCTATTTTG-3P; Tn7LS: 5P-TATTAGGAATTTTTGAGGTAAAGGTGGGGA-3P).
Tn5 insertions into plasmid DNA isolated from the
E. fergusonii EF43 strain and screening for colicinogenic
transformants resulted in an E. coli TOP10FP strain harboring colicinogenic plasmid pDS455. The original colicinogenic plasmid of E. fergusonii EF43 strain was named
pColE1-EF43. The activity and immunity genes encoding
colicin E1 (cea) and immunity protein (imm) encoded by
pColE1-EF43 were completely identical (Fig. 1) to cea and
imm reported previously [4]. However, the kil gene on
pColE1-EF43 had one nucleotide change (C71G) resulting
in a one-amino acid replacement (P24R) in the corresponding polypeptide when compared to the peptide encoded by kil on pColE1 [4]. Restriction digestion of
pColE1-EF43 revealed its molecular size to be 6.7 kb
(data not shown), which is close to the size of the sequenced pColE1 (6646 bp; [4]), indicating that those two
ColE1 plasmids are closely related.
3.2. E. fergusonii EF3
Plasmid pColE1 from strain E. fergusonii EF3 (pColE1-
2.4. Isolation of plasmid DNA, restriction analysis and
sequencing
Standard methods were used for plasmid isolation, restriction endonuclease analysis and agarose gel electrophoresis [3]. DNA was sequenced using the Taq Dye-deoxy
Terminator method and a model 377 DNA sequencing
system (Applied Biosystems, Foster City, CA, USA).
The complete sequence of colicin activity, immunity and
lysis genes was ¢nished using speci¢cally designed synthetic oligonucleotides. Computer-assisted sequence analysis was performed using the LASERGENE program
package (DNASTAR, Madison, WI, USA).
Fig. 1. A dendrogram of sequence homology among colicin E1 proteins.
E. fergusonii proteins are shown in bold. Protein sequences were aligned
using the CLUSTAL method and protein sequences were taken from [4]
(pColE1-JC411), [6] (pColE1-EC31, -EC71, -K30, -EC50, -EC40, -EC12,
and -EC39) and [5] (pColE1-pKY-1). The length of each pair of
branches indicates the distance between protein pairs, the scale shows
distance among sequences in numbers of substitutions.
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Table 1
The cross-immunity among colicin E1 producers
E. coli strain
Relevant genotype
TPO10FP
TPO10FP pDS300
TPO10FP pDS503
TPO10FP pDS455
^
pColE1-EF3
pColE1-EF36
pColE1-EF43
Colicin E1 from E.
fergusonii strain
EF3
EF36
EF43
2(3)a
ib
0(1)
i
2(3)
i
i
i
2(3)
i
0(1)
i
a
The numbers indicate the exponents of the highest colicin dilution that
resulted in a clear zone of growth inhibition and the last dilution that
resulted in turbid zones (in parentheses) on the lawn of sensitive bacteria, e.g. 3 = 103 .
b
Immune strain.
EF3) was isolated after insertion of Tn7 into the plasmid
backbone, resulting in the colicinogenic plasmid DS300.
The colicin E1 cea gene encoded by this plasmid had a
one-nucleotide replacement (G211A) when compared to
cea of pColE1-EF43, resulting in a one-amino acid change
(A71T) in colicin E1 protein (Fig. 1). pColE1-EF3 imm
and kil gene sequences were identical to those of
pColE1-EF43. Consistent with this ¢nding, E. coli strain
TOP10FP with pDS455 was immune to colicin E1 produced by EF3 strain and vice versa (Table 1). The size
of pColE1-EF3 was determined to be approximately 9 kb
(data not shown), which is considerably more than for
pColE1-EF43.
3.3. E. fergusonii EF36
pDS503, a result of Tn7 insertion into pColE1 from
strain EF36 (pColE1-EF36), encoded a cea gene that
was most closely related to cea of Shigella sonnei pKY-1
plasmid (97.2% identity; [5]), di¡ering by 16 nucleotide
changes and insertion of 7 bp and 1-bp deletion. However,
the EF36 colicin E1 protein sequence was most closely
related (96.6% identity) to colicin E1 encoded by
pColE1-EC39 isolated from E. coli EC39 [6], di¡ering in
12 amino acid replacements and insertion of two amino
acid residues. Cea of pColE1-EF36 is more distantly related to Cea encoded by pKY-1 (92.9% identity; Fig. 1).
This discrepancy was because the 90-bp region between bp
342 and 432 of pKY-1 cea is translated from a di¡erent
reading frame. Cea of EF36 di¡ers in 68 amino acid residues from Cea encoded on pColE1-EF43 (corresponding
genes di¡er in 205 nucleotides) and the amino acid replacements are preferably localized in the middle third of
the colicin E1 molecule. The pColE1-EF36 imm gene was
most closely related to the imm from E. coli EC39 strain
[6], di¡ering in four nucleotides that resulted in a oneamino acid replacement (E81V). The Imm proteins from
EF36 and EF43 strains were di¡erent in eight amino acid
residues (92.9% identity). The E. coli TOP10FP pDS455
producer of colicin E1 was completely immune against
colicin from the EF36 strain, but the protection of the
261
E. coli TOP10FP pDS503 producer is at least two orders
of magnitude lower (Table 1). These data indicate that the
immunity proteins encoded on pColE1-EF36 and pColE1EF43 di¡er in the recognition of colicin E1 variants. In the
interaction between colicin E1 and its cognate immunity
protein, four critical amino acids were identi¢ed [7]. Since
colicins E1 encoded by pColE1-EF43 and pColE1-EF36
di¡er in one of these four amino acid residues (A472 and
V473 in colicin E1 of EF43 and EF36, respectively), it is
possible that this residue is involved in di¡erent recognition by immunity protein encoded on pColE1-EF36. The
kil genes encoded on both pColE1-EF36 and pColE1EC39 were identical [6] and the Kil of EF36 di¡ered
from the EF43 Kil in two amino acid residues (I31V,
A43S). The plasmid size of pColE1-EF36 was estimated
to be 7.4 kb (data not shown).
Sequences of colicin E1-encoding regions for strains
EF43, EF3 and EF36 were deposited in the GenBank
under accession numbers AF453410, AF453411,
AF453412, respectively.
3.4. E. fergusonii EF6 and EF24
E. fergusonii producers of colicin Ib, strains EF6
(pColIb-EF6) and EF24 (pColIb-EF24), were found to
have identical sequences in colicin Ib activity and immunity genes on ColIb plasmids. When compared to the sequence of the colicin Ib structural gene encoded on E. coli
plasmid pColIb-P9 [8], 16 nucleotide changes were detected out of 1881 nucleotides. The colicin Ib proteins
encoded by pColIb-EF6 and pColIb-EF24 di¡ered in eight
amino acid residues (98.7% identity) from the colicin Ib
encoded by pColIb-P9 (Fig. 2). Five out of the eight substitutions are localized within the C-terminal 200-amino
acid pore-forming domain. The colicin Ib immunity genes
on pColIb-EF6 and pColIb-EF24 strains di¡ered in four
nucleotides from that encoded on pColIb-P9, and the corresponding proteins di¡ered in three amino acid residues
(97.4% identity; I17S, N31S, L108F). Strains E. coli pColIb-P9 and TOP10FP pColIb-EF6 were cross-immune. Despite the identical sequence of colicin activity and immun-
Fig. 2. Sequence homology among colicin Ia and Ib proteins. E. fergusonii proteins are shown in bold. Protein sequences were taken from [6]
(pColIa-EC3, -EC34, -EC28, -EC15, -IHE3113, -EC14, and -pABZ106),
and [8] (pColIa-CA53; pColIb-P9). The length of each pair of branches
indicates the distance between protein pairs, the scale shows distance
among sequences in numbers of substitutions.
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ity genes on pColIb-EF6 and pColIb-EF24, the HindIII
restriction pattern for both plasmids di¡ered (data not
shown) indicating that both strains harbor unique pColIb
plasmids. Sequences of the colicin Ib-encoding region for
pColIb-EF6 and pColIb-EF24 were deposited in the GenBank under the accession numbers AF453413 and
AF453414, respectively.
against E. fergusonii indicator strains (data not shown)
as the original E. fergusonii producers. The di¡erences in
activity spectra of E. fergusonii producers thus seem to
re£ect di¡erent levels of colicin synthesis as a result of
Col plasmid di¡erences together with a di¡erent degree
of sensitivity of E. fergusonii indicators to colicins.
3.5. E. fergusonii EF31
Acknowledgements
E. fergusonii strain EF31 was identi¢ed as a producer of
colicin Ia. The colicin Ia structural gene was most related
to that encoded by pColIa-EC3 [6] di¡ering only in one
nucleotide (G910C), resulting in one amino acid replacement (Fig. 2) in the resulting colicin Ia protein (D304H,
99.8% identity). The same degree of sequence homology
was found between colicin Ia from the EF31 strain when
compared to that encoded by pColIa-CA53 [8] and colicin
Ia from EF31 showed one amino acid substitution when
compared to colicin Ia from CA53 (R351Q). The immunity gene encoded by strain EF31 was found to be identical
to those encoded by pColIa-EC3 and pColIa-EC34 [6].
Sequences of the colicin Ia-encoding region of EF31
were deposited in the GenBank under accession number
AF453415.
The colicin-encoding regions of the Col plasmids isolated from E. fergusonii closely resembled the coding determinants of those isolated from E. coli. However, six of
the E. fergusonii Col plasmids yielded four new colicin
variants, two new colicin immunity protein variants, and
one new variant of the colicin lysis protein.
The di¡erent spectra of inhibitory e¡ects of colicin E1
and colicin Ib variants produced by E. fergusonii strains
(J. Símarda, unpublished results) on E. fergusonii indicator
strains cannot be explained solely by the di¡erences in the
colicin protein sequences. The E. coli TOP10FP strain with
introduced colicinogenic plasmids encoding E. fergusonii
variants of colicins E1 and Ib showed identical spectra
This work was partly supported by Grant no. 310/98/
0083 from the Grant Agency of the Czech Republic.
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