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Tools for Safety Assessment
Identification and monitoring of Escherichia coli K-12 safety
Peter Kuhnert and Joachim Frey
Institute of Veterinary-Bacteriology, University of Bern,
Länggass-Str. 122, CH-3012 Bern
Agency for Biosafety Research and Assessment of
Technology Impacts of the
Swiss Priority Programme Biotechnology
Scope of the bulletin
The bulletin 1/96 appears in the series "Tools for safety assessment".
The reader is offered a summary on special subjects with relevance for
safety assessment of biotechnology applications. The series would like to
meet the need for a more intensive transfer of "first hand" informations in
a structured and issue focused synthesis of available knowledge.
The bulletin 1/96 has been assembled in co-operation with Dr. P. Kuhnert
and Dr. J. Frey of the Institute of Veterinary-Bacteriology, University of Bern.
The collaboration was initiated because an unmistakable basis for
decision-making in the categorisation of E. coli strains was not available.
The research project financed by the Swiss Priority Programme Biotechnology
yielded molecular tools which allow an unambiguous classification of
E. coli strains. The report which is based on the paper in reference 4,
gives a summary of the theoretical background and methodical details.
Table of Contents
1.1 Identification of Escherichia coli K-12
1.2 Common genetic marker for E. coli K- 12 derivatives
2 Test system for the identification of Escherichia coli K-12 safety
2.1 Sample preparation for PCR
2.2 Oligonucleotide primers
2.3 Premix for PCR assay
2.4 PCR reaction
2.7 Identification of E. coli K-12 derivatives
Bacterial safety strains are requested for many biotechnological
processes, in particular for those involving expression of recombinant
genes. Such strains must be non-pathogenic for human, animal and plant.
In addition they should exhibit an impaired survival under non-laboratory
The best known bacterial safety strains fulfilling these criteria are the
Escherichia coli K-12 derived strains. These strains show the following
advantages: i) they represent one of the genetically best understood
living organisms; ii) they are classified in all major national and
international safety guidelines as biologically safe vehicles for the
propagation of a broad range of efficient gene cloning and expression
vectors; iii) they are easily modified by many genetic methods, and vi)
they exist as a large variety of genetically and phenotypically different
mutant variants selected for many specialized scientific and technological
While very little is known about the pathogenicity of the wild-type
E. coli K-12 strain, which was isolated from a convalescent diphteria
patient at Stanford University in 1922, derivatives of E. coli K-12 which
were the first time reported in 1944 (ref. 3) seem to be entirely
apathogenic. Derivatives of E. coli K-12 were obtained through intensive
mutagenesis and extended growth for many passages under laboratory
conditions which probably resulted in the loss of all currently known
virulence genes (Fig. 1). Moreover, E. coli K-12 derivatives are rough,
lacking the 0-antigen which is part of the lipopolysaccharide (Fig. 1). No
case of disease, caused by E. coli K-12 derivatives has ever been
reported since 1944 in spite of very frequent use of these strains in
teaching and research. It was also shown that K-12 strains are unable
to colonize the human gut (ref. 8). The K-12 lineage is therefore
considered to be a prototype of safe and non-pathogenic bacterial
strains. No virulence genes have been found so far in E. coli K-12
derivatives (ref. 7) in contrast to pathogenic E. coli strains which possess
normally several chromosomal or plasmid borne virulence genes, which
are often located as gene clusters on "pathogenicity islands" (Fig. 1)
(ref. 2; 6). This confirms the non-pathogenic phenotype of K-12
derivatives. The absence of virulence genes gives K-12 derivatives
further the advantage that no sudden reversion of a mutated gene
which might result in the reemergence of pathogenic revertants could
Fig. 1. Comparison of an E. coli K-12 derivative with a pathogenic
E. coli strain with regard to virulence attributes and their genes (ref. 7).
Shown are two E. coli bacteria and their genomes. E. coli K-12
derivatives do not contain virulence genes. A specific insertion
sequence IS5 is contained in the genes encoding the lipopolysaccharide
(LPS) pathway genes, rendering these strains rough colony type. The
pathogenic E. coli possesses virulence genes a-e encoding the
variouse virulence attributes A-E.
1.1. Identification of Escherichia coli K-12
The broad range of genetic and phenotypic varieties deduced from
K-1 2 and other E. coli strains cause major problems in identifying their
correct origins. Entire pedigrees would therefore be required for clear
identification of strains. Complete description of bacterial lineages,
however, are only available for a limited number of ancestral K-12 strains
(ref. 1). This often causes severe problems in the interpretation of
experimental data and in particular in biosafety assessments. A
particular difficulty is the lack of a common character to E. coli K-12
derivatives which would enable to differentiate them easily from other,
apparently very similar E. coli strains. Hence, an accurate and rapid
method to discriminate E. coli K-12 from other E. coli strains is needed
as a standard tool in biological safety procedures.
1.2. Common genetic marker for E. coli K-1 2 derivatives
E. coli K-12 strains are rough, apparently lacking the 0-antigen which is
part of the lipopolysaccharide (LPS) and which is encoded by the rfb-gene
cluster. Liu and Reeves (ref. 5) recently showed that the lack of 0-antigen
in some K-12 derivative strains is due to a mutation (rfb-50) within the
rfb-cluster which inactivates the rhamnose transferase, a key enzyme in
the 0-antigen biosynthesis. The rfb-50 mutation is characterized by an
IS5 insertion that is located within the last gene (orf264 also named orf11
or orf5) of the rfb-cluster which encodes the rhamnose transferase (Fig.2)
(ref. 5,9). This mutation was shown to be specific to K-12 derivatives. It
can be identified by an appropriately designed ploymerase chain reaction
(PCR) (ref. 4). The analysis of 37 K-12 derivatives including strains of all
the main branches of the pedigree as well as newly emerged K-12
derivatives showed that they all seem to carry the rfb-50 mutation
(ref. 4). In contrast over 70 other E. coli or closely related strains (Shigella
and Salmonella) isolated from human, animal, water and soil were devoid
of IS5 in the rfb gene. The herein described PCR reaction is therefore
considered to be a reliable method for the rapid and accurate identification
of E. coli K-12 derivatives (ref. 4).
Fig. 2. Map of the rfb-cluster and region analyzed in this study. The
rfb-cluster is located 5' to the 6-phosphogluconate dehydrogenase
gene (gnd). Boxes represent genes. The entire rfb-cluster extends
over more than 10 kb and contains 11 genes. The gene located at
the 3' end of the cluster, orf264 (shaded box), encodes the rhamnose
transferase. It harbors an IS5 sequence in most K-12 derivative
strains specifying the rfb-50 mutation. Arrows indicate the location
of primers used for PCR.
2. Test system for the identification of Escherichia coli KA 2
2.1. Sample preparation for PCR
A) By lysate preparation:
E. coli strains are grown over night on LB-plates containing, per liter:
10 g Bacto tryptone (Difco)
5 g yeast extract (Difco)
5 g NaCI
2 ml 1 M NaOH and 15 g agar (Difco).
Three to five colonies are lysed in 450 ml lysis-buffer (100 mM Tris-HCI
pH 8.5, 0.05% Tween20 and 240 µg/ml proteinase K).
Samples are incubated 1h at 60 °C and then heated to 97 °C
for 15 min in order to inactivate proteinase K.
Alternatively, take 1 µl bacterial liquid culture or few bacteria from a
colony taken by a toothpick from a plate, and place them directly in the
premix containing Taq-polymerase.
2.2. Oligonucleotide primers
|| 5'-CGCGATGGAAGATGCTCTGTA-3' (nt 293-313 on IS5)
||5'-ATCCTGCGCACCAATCAACAA-3' (nt 508-488 on orf264)
||5'-GGCAATTGCGGCATGTTCTTCC-3' (nt 50-71 on pal)
||5'-CCGCGTGACCTTCTACGGTGAC-3' (nt 328-307 on pal)
Primer synthesis was done at: Microsynth, Schützenstr.15, CH-9436,
The primer pair K121S-L and K12-R is specific to K-12 and will amplify a
970 bp fragment only in Escherichia coli K-12. The primer pair ECPAL-L
and ECPAL-R is specific for all Escherichia coli and some related enteric
bacteria and will amplify a control fragment of 280 bp from the exeC
gene (PAL protein) (ref. 4).
2.3. Premix for PCR assay
100 µl 10 x reaction buffer (Boehringer Mannheim)
10 µl 100 mMol dNTP (Boehringer Mannheim)
400 pMol KR
400 pMol KL
400 pMol CR
400 pMol CL
up to 1 ml with ddH2O
2.4. PCR reaction
Prepare in a MicroAmpTM (Perkin Elmer Cetus, Norwalk, CT, USA) tube
50 ml premix and add.
1 µl bacterial lysate
+2.5 U Taq polymerase (Boehringer Mannheim)
Place PCR reaction tube in a PE9600 (Perkin Elmer Cetus, Norwalk, CT,
USA) or equivalent cycling machine and run the following program:
3 min 94 °C, followed by 35 cycles:
30 sec 94 °C
30 sec 60 °C
1 min 72 °C
Load 5 µl of the cycled PCR reaction on a 1% agarose gel containing
500 ng/ml ethidium bromide. Run at 110V for 2h or according to other
specifications in TBE-buffer (1 l: 5.4 g Tris, 0.4 g EDTA, 2.75 g Boric acid).
2.7. Identification of E. coli K-12 derivatives
E. coli K-12 will produce a specific 970 bp band in addition to the 280 bp
control band. If only the 280 bp control band shows up, the strain
investigated is not a K-12 derivative. If no band at all shows up, either the
reaction was not working properly or the strain investigated is not E. coli
or a related species.
Fig. 3: PCR results showing the K-12 specific band at 0.97 kb resulting
from amplification with K121S-L and K12-1R. A natural isolate of E. coli
from water does not show the specific band. Control amplifications for
E. coli and related enteric species show a band at 0.28 kb resulting from
amplification with primers ECPAL-L and ECIDAL-R. Lower bands represent
primer-dimer artifacts. A Gram-positive control shows no amplification
product at all. Marker: HindIll digested λ DNA with the sizes
The herein described specific PCR assay for the identification of
E. coli K-12 derivatives constitutes a useful method in research and quality
control for laboratories involved in recombinant genetechnology. It is also
a rapid and convenient method to monitor the presence of K-12 derivatives
used in biotechnological production processes, thereby providing a basic
instrument to contribute to the safety of such processes.
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