Presentation of the article "Identification of Yersinia
enterocolitica genes affecting survival in an animal
host using signature-tagged transposon Mutagenesis. "
Andrew J. Darwin & Virginia L. Miller, 1999.
Introduction
Here I will review the main points from
The above article from the reputable
journal Molecular Microbiology.
First I give the background to
survey was conducted, what the authors
purpose and hypothesis was how they did what
they found out and finally I will comment
implications of the findings.
Background
Three species of the genus Yersinia, a Gram-negative
rod-shaped facultative anaerobic Enterobacteriaceae,
pathogenic to humans. These three species are Y.
pestis, Y. pseudotuberculosis and Y. enterocolitica
and are associated with both local and systemic
infections. It is the last of these bacteria
authors here deal with, but
results are nevertheless significant for all three species. Mon
have previously identified a 70KB virulensplasmid
with Yersinia, called pYV that can be found in all three
species. Genes on this plasmid codes for many
virulence factors such as type III secreting
(TTSS) (discussed later) and effektorproteinerne
to secretion. However, it is shown that pYV is not alone
on virulence, but there are also chromosomal
genes involved. Mix Secondly, genes
coding for adhesion and penetration into the host cell,
binding of iron (siderophorersyntese) and
synthesis of lipopolysakkarid O-antigen.
Purpose and hypothesis
As the sequencing of entire genomes is
easier, it has been genetically mapped several
pathogenic bacteria. However, shortcomings still
much insight into which genes coding for the
proteins, and particularly what genes are
involved in bacterial virulensmønster.
Due to difficulties with the need to test each
single mutants virulensegenskaber and associated
genes in a testdyr had it at that
date not earlier been possible to make
Large-scale trials that would identify this particular
for the whole genomes. We can now through
signature-tagged Mutagenesis (STM). Here,
authors make a variety of mutants, and a part
these will be with impaired virulensegenskaber,
simultaneously test multiple strains in a
single host, and then find out which genes
in the given case is disturbed.
They expect from study start to a series of
their mutants will be mutated in pYV and that these
would have sharply reduced virulence. In addition,
they hope to discover the genes on the chromosome which
the virulence and survival in the host.
Method
A number of signature-tagged mutants were
manufactured by PUT vector containing
mini-Tn5 km2 transposons were inserted into E. coli
strain CC118, and then transferred to Y.
enterocolitica strain JB580v via conjugation.
These mutants were screened for insertion of the
said transposon by ampicilin Selection
(Different colonization pattern with and without
insert) and were performed only at 18 Southern
random samples which were screened for
kanamycinresistens who sat on transposons.
Preliminary studies showed that the best
infection method on test animals (6-7 weeks old
BALB / c mice) were intraperitoneal (IP) injection
(Direct injection into abdomen), with approx. 107 colonyforming
units (cfu), followed by capture from
spleen after 48 hours.
In order to avoid injecting the weakened auxotrofe
bacteria were selected transposonmutanter
grown on minimal medium.
STM
Method to screen for attenuated bacteria
was referred done with InDesign tours-tagged
Mutagenesis (STM). STM is a negative
selection method where this experiment uses
their pool of mutants (21 pools of 96 mutants)
to infect groups of three mice. What is special about
STM is that the inserted DNA has a special
signature trademark, a signature tag that is a little
familiar sequence which can subsequently be found by
Southern blot hybridicering. During the initial
screening infected mice and bacteria recovered
from spleen. These are mixed and transferred to 96
chamber micro titter plate. Then screener Mon
with proper original bacteria (input - all
original mutants) and those who were found in the spleen
(Output - the survivors). That way you can
find the attenuated bacteria by their lack
presence in the spleen accumulated
bacteria (negative selection), which is seen as
missing or weak hybridicering compared to the
from input (the first part of Fig. 1 from the article). Then
was taken to the mutants that demonstrated a lack
hybridicering - so attenuated virulence, and drove
secondary screening with the same procedure as
before but with two conditions (2x3 mice).
This should help reduce the risk to take
mutants actually was not attenuated (second part
fig. One from the article).
Subsequently, attenuated mutants studied
and classified phenotypic and by cloning
followed by sequencing and was performed
competition and vækstassays.
Results
Based on the two initial STM screening
they found up to 68 mutants with attenuated
virulence. As expected, a proportion of these being
mutated in pYV and it was indeed found that 29
were pYV mutants. This was examined by
growth pattern on LBMOX plates and kanamycinsensitivitet.
This means that out of the original 68
mutants were 39 who had mutations in
chromosomal virulensgener. Out of these were in
16 cases observed the same phenotype
(Aggregation in liquid medium, and turbid growth
LB agar plates) due to a defect in LPS
synthesis and thus synthesis of O antigen, which
is essential for Y. enterocolitica virulence. So
there were now 23 mutants back where mutation
sat somewhere else than in pYV or LPS synthesis
genes. To be confirmed whether they were really
weakened, it was checked every 68 in a competition
assay in mice, which measured about bacteria
fared worse than wild-type Y.
Enterocolitica. For this test it was found that 10 of
the 23 were actually weakened and were overall 55 of the
68 fact impaired. This in their eyes, good result
attribute the dual STM screenings
procedure.
By pYV mutants was especially type III
sekretionsgenerne that the insert was to identify and
especially in lcrV and yscL, this could indicate a
'Hot spot' for insertion of transposons mini-Tn5
Km2 just here (Fig. 2 from the article).
Of the 16 with suspected mutation in LPS / Oantigen
synthesis apparatus, this was verified
by sequencing (Fig. 2 from the article).
The remaining 10 chromosomal mutants were also
by cloning and sequencing studied. Here was
Mon insert, and thus the disruption of genes
coded outer membrane synthesis (yifH and nlpD)
nutrient uptake / acquisition (pstC and irp1) and stress
response (dnaJ). The last five had mutations in
genes homologous to E. coli genes coding for DNA
topoisomerase I (topaz), phage shock protein
operon (pspC) and two were found with
mutations in yibP, a gene of unknown function in E.
coli. Finally found a mutation in a sequence
there was inconclusive in Genbank database.
(Fig. 2 from the article).
NB. The article makes the reconstruction of pspC
mutant for further study. This, I
explain in question 3 later.
Discussion
They were in this study demonstrated that STM technique can
used to identify a number of genes in Y.
enterocolitica with more or less important
for its virulence. They were reidentificeret a series
Essential virulensgener in pYV (type III
secreting) and in LPS synthesis genes in
chromosome. The results from the chromosomal
mutants recalled earlier STM results
the pathogenic Gram-negative bacteria Salmonella
typhimurium and Vibrio cholera which also
found that bacteria with mutations in genes
involved in O-anti-reunion theory, stress response and
nutrient uptake / acquisition had weakened
virulence. The most sensational they found out
of was to pspC gene, homologous to the gene in E.
coli is part of the phage shock protein operon,
had significance for Y. enterocolitica's virulence.
This mutant exhibited very low virulensgrad in
test animal, but grew well in vitro. This gene has
in E. coli no known function, but we see an increased
expression by overexpression of sekretinproteiner,
inter alia used in Type III secreting. So
a suspected link to important virulensgener.
There must be investigated further in this
context for understanding the importance of pspC
gene and its virulensfunktion.
The article has the sense that it shows that a long
number virulensgener be identified quite
fast and relatively simple in STM method.
This means that other pathogenic bacteria, Gram-positive
as well as Gram-negative, rapidly
screened virulensgener, which can have significant
important for disease treatment, drug development
and our general knowledge of
pathogenic bacteria.
It is also useful in connection with that
more and more organisms (particularly bacteria) genome
sequences, and the need to understand
importance of individual genes in the studied
organism.
General works article vellavet over
trials building, reproducibility and
general structure of the article.
Specific questions
Question 1:
Signature-Tagged Mutagenesis (STM) is a technique
used to examine genes function in
vivo. You make a transposon with a reputation
signature sequence, which then transferred to the
organism to be studied mostly by E. coli.
Transposons, with signature tag, sits a
random location in the genome or a plasmid, and will
thus very likely disturb and
destroy the gene and thus the translated
protein. In STM, each transposon its own
unique signature, and can subsequently
found by DNA probehybridicering (Southern
only). Unlike ordinary random transposon
Mutagenesis (RTM) where each
mutation need a testdyr to negative control
here you can use a whole pot (in case the article
using the 96 per. testdyr) and find a specific
attenuated mutant. In the original design of the STM as
was designed by Hensel a al.1 used, as in
experiment here hybridiseringsprober, now a
Another method of PCR detection, developed by
Lehoux a al.2 used. Here is the idea that each
roof amplified with tag-specific primers.
The presence of a PCR product from a roof in
input pool and the absence of the same PCR product from
output pool shows the loss of the specific mutant
during the negative selection screening.
The advantages of STM with both signature-tag
detection methods are so clear, since it both
saves time (and thus money) and testdyr. Outright
disadvantages compared to RTM, I have a hard time
find.
Question 2:
The article dobbeltscreener their authors
mutants. This is done by making probes are
designed to hybridize to the inserted transposon
(Base pairing principle). When one acquires bacteria
from mouse spleen, the bacteria are finding that,
be those who are able to infect the mouse, so
the bacteria we do not want to investigate. But if
found the bacteria there, so to speak, is missing
can you find the gene is disrupted by
look at the input pool. This makes the order that the
primarily attenuated bacteria they inject, and
Following this double screening obtains the a
result which states that 55 of the original 68
mutants were effectively attenuated, which corresponds to
81%. This is an improvement over previous
results, where only 45% of those screened
mutants were effectively attenuated (here screened Mon
Vibrio cholera).
1 Hensel M, Shea JE, Gleeson C, Jones MD, Dalton E, Holden DW:
Simultaneous identification of bacterial virulence genes by negative
selection. Science 1995, 269:400-403.
2 Lehoux DE, Sanschagrin F, Levesque RC: Defined oligonucleotide tag
pools and PCR screening in signaturetagged Mutagenesis of essentialism
genes from bacteria. Biotechniques 1999, 26:473-478, 480th
Question 3:
The trial will determine whether the phenotypic
changes caused by transposon mutagenesis or
whether there is a spontaneous mutation. In all of
near a single mutant, they may exclude
spontaneous mutation. The exception is gay gallery to
E. coli gene pspC. Therefore, the authors construct
three new pspC mutants in the same strain of Y.
enterocolitica. One new mutant would be a
copy of the mutant from the original trial
while the other two would be mutants with a
insertion-deletion in the gene, with a kanamycinresistens
cassette. The copy was made by the
inserted fragment from the original was propagated
by PCR and equality into a vector and transferred to Y.
enterocolitica by conjugation. The other two were
made by kanamycin resistance cassette was
inserted in two different orientations of the ring. Of
these three new bacteria were again conducted a
konkurrenceassay. The results of this assay
with the reconstructed mutants found in Table 3 in
article and can be seen in comparison with pspC
results from the first konkurrenceassay (Table
2) the results are very similar - mutants
infect mice poorly, but grows well in
Vitro. The finds therefore that mutations in pspC
gay gallery Y. enterocolitica substantially weakens
its virulence.
Question 4:
Type III secreting (TTSS) is a complex
trans-membrane protein structure found in many
Gram-negative bacteria, pathogens and nonpatogene.
In the pathogenic used it mainly to
secrete proteins that help bacteria to
infect its host, and therefore there is strong
correlation between virulence and the presence
of TTSS. Hall Marketplace in the system is the needle.
It sits anchored only to the inner membrane in
the inner membrane ring, via a connector over
peptidoglykanlaget to the outer membrane ring
after which it has crossed the entire bacterial
wall system. The actual needle hole with a diameter
around. 3 nm, which means that most effector
proteins to be secreted in the unfolded state. (See fig.
1 below)
Figure 1 Schematic drawing of the TTSS. From Wikimedia Commons
Previously it was thought that the needle was able to
perforating the host cells, but this picture is
now changed. Now the theory that it emits
proteins called translocators, which form a pore
in the host cell membrane through which other
effectors can flow.
The article will transposons in one of the mutants
inserted into a sycT-yopM gene region, and this
suppose they can affectation genes involved
in TTSS. They examine whether it is due to errors in
secretion of proteins called yop (Yersinia
outer proteins), which helps to perforate
host cells that are the cause of the weakened
virulence. Therefore examined the protein composition
in growth medium for the said
mutants, and compared with wild type (Fig. 2).
In the experiments, they found no visible difference in
protein picture for the mutants and wild type.
Question 5:
There are many bacteria use TTSS in
their virulence. Of these species may
include Shigella (bacillary dysentery), Salmonella
(Typhoid fever), Escherichia coli (food poisoning)
Burkholderia (glanders), Yersinia (plague), Chlamydia
(Chlamydia) and Pseudomonas. The high prevalence
of TTSS of pathogens may be due to TTSS
cassette can be transferred horizontally between species.
As an example I will use the bacterium Shigella.
The most common cause of shigellainfektion is
contamination of food with faecal matter. After you have
eating infected food or water will bacterium
invade its host through epithelial cells of the colon
through its TTSS. This injects the IPAD
protein into epithelial cells, which triggers it to
incorporate the bacterium. This vakuole breaking down
of the divisional IpaB and IPAC proteins. Here may
now divide, and furthermore it may by
ICSA protein "take over" epithelial actin
polymeriseringsapparat which it uses to
be shot around the cell, and into other
neighboring cells, which then becomes infected. Total causes
Shigella approx. 165 million annual cases of
dysentery, and approx. A million deaths - almost
exclusively in developing countries
