lysis gene E counter-selection cassette in S. marcescens
A counter-selection marker would be more attractive if it has a
universal application. In the interest of generality, application of
lysis gene E counter-selection cassette in other bacteria should
be tested.
S. marcescens , a rod-shaped gram-negative bacterium, have the
ability to produce a variety of valuable metabolites, such as
prodigiosin, chitinase and serratiopeptidase(Emruzi et al., 2018; Pan et
al., 2019; Velez-Gomez, Melchor-Moncada, Veloza, & Sepulveda-Arias,
2019; Yip et al., 2019). We select S. marcescens to test the
counter-selection effect of lysis gene E cassette. Unfortunately,
high-temperature condition like 42 °C is not suitable for the growth ofS. marcescens (W. Chen et al., 2021), We resorted to the
AraC/PBAD regulatory system which function well in this
species. At first, E-GmR fragment with short
homologous arms was amplified and transformed into competent
MG1655[pKD46] to substitute the CDS of Red recombinases in plasmid
pKD46 to construct the goal plasmid pKD-EG. Therefore, expression of
lysis gene E is thoroughly controlled by AraC/PBAD inducible
system (Fig. 3A). After identification (Fig. 3B), plasmid pKD-EG was
transformed into S. marcescens GY1 for functional testing.
GY1[pKD-EG] grew well in LB
broth, but it cannot survive in LB broth supplemented with 0.4%
arabinose, and the LB broth is very clear even 18h after inoculation
(Fig. 3C). This result indicates that expression of lysis gene Ethrough AraC/PBAD regulatory system could effectively
kill its host.
In genomic seamless modification, counter-selection marker usually works
in single copy. Therefore, the counter-selection effect of lysis geneE inserted in the chromosome must be evaluated. The 2,303 bpPBAD-E-GmR (including AraC/PBAD
regulatory system, CDS of lysis gene E and gentamycin resistance geneGm R ) double selection cassette was therefore
inserted into pigA CDS of S. marcescens. The successfully
constructed strain was named as GY4 (Fig. 3D). Obvious differences can
be seen when GY4 grown in LB broth without or with the addition of 0.4%
arabinose (Fig. 3E). This result indicates that single copy of
counter-selection marker gene E is also efficient. It is further
verified by the spot dilution experiment: GY4 grew well on LB plate,
while it cannot survive on arabinose added plate and only a faintly
layer of dead cells was observed (Fig. 3F). It should be noted thatpigA is an indispensably gene for the synthesis of prodigiosin, a
kind of red pigment, therefor GY1 shows red phenotype while pigAgene mutated GY4 is white.
Having verified the lethality of chromosomal expressing lysis geneE in S. marcescens , reverse mutation experiment was
finally conducted to test its counter-selection effect in genomic
modification. Using ssDNA mediated Red recombination, we tried to repair
the insertion inactivation of pigA gene in GY4 (Fig. S1).
Numerous red colonies dotted with a few white colonies were observed
after transported GY4 with ssDNA, while only sparse white colonies
appear in the control group without adding ssDNA (Fig. 4A). Colony in
red indicates that the ability of synthesizing prodigiosin was repaired
due to successful reverse mutation, which is verified by colony PCR
identification (Fig. 4B).
dsDNA mediated homologous recombination was also tested. Partial
fragment of T7 RNA polymerase (~ 1,000 bp in length) was
amplified and transported into GY4 to substitute thePBAD-E-GmR cassette. 4 out of
10 of randomly selected colonies were true recombinants (Fig. 4C).
All these results suggest that lysis gene E counter-selection
cassette can be used in S. marcescens for genomic seamless
modification.
Improving selection
stringency frequency through combining E and kil
In genomic seamless modification, selection stringency frequency is a
decisive factor that influences counter-selection and therefor an
indicator for evaluating an excellent counter-selection marker(Khetrapal
et al., 2015). The higher the selection stringency frequency is, the
lower the background it causes. Low background is very conducive to the
selection of recombinants during seamless modification. The selection
stringency frequency of lysis E under the control of promoterpL at ack locus in MG1655 is about
2.7×10−7 (Fig. 5A), performing better than thekil counter-selection cassette constructed in our previous work
(Wei Chen et al., 2019). However, it is still much lower than the best
counter-selection system, inducible toxins system(Khetrapal et al.,
2015).
We tried to increase the selection stringency by combing the two
counter-selection marker genes. At first, CDS of lysis gene Ewith a consensus RBS sequence (AAGGAGATATACAT) and gentamycin resistance
gene GmR were inserted immediately behind the
stop codon of kil gene at ack locus of E. coliMG-10. In this constructed strain CWE-3, kil and E were
expressed as a bi-cistron under the control of promoter pL and repressor
cI857 (Fig. S2). We named this combining counter-selection cassette askil-sd-E . Then the selection stringency of these
counter-selection systems was compared. At the ack locus,
selection stringency frequency of E is
2.7×10−7, several fold lower than that of kil(8.7×10−7). While selection stringency frequency ofkil-sd-E was significantly decreased to
4.9×10−8 (Fig. 5A), which is very close to the best
reported inducible toxins system.
In consideration that insertion sites may influence stringency, another
non-essential gene locus araB were selected for the further
analysis. Selection stringency frequency of E(2.9×10−6) and kil (2.1×10−6)
are almost at the same level. But selection stringency frequency ofkil-sd-E dropped sharply to 3.2×10−8 at this
locus, about 65- to 90- fold lower than the above two counter-selection
system (Fig. 5B). This result hinds that
co-expression of kil andE in the form of bi-cistron can significantly increase their
selection stringency in E. coli to the degree to the best
reported inducible toxins system.
To explore whether it also works in other bacteria, S. marcescenswas used for the subsequent research. At first,E-GmR and kil-sd-E-GmRwere amplified from plasmid pBBR-E and strain CWE-3 separately and then
were used to substitute the CDS of araB to construct the goal
strain MG-4A and MG-4B (Fig. S3). Sole expression of E or
co-expression of kil and E were both under the control of
AraC/PBAD in the two strains. After adding 0.4%
arabinose into LB broth, both MG-4A and MG-4B can’t grow normally. In
particular, the LB broth was very clear in the MG-4B cultivating tube
18h after inoculation, while slightly turbid cultivated bacteria can be
seen in the MG-4A cultivating tube (Fig. 5C). These results indicate
that E and kil-sd-E counter-selection marker under the
control of AraC/PBAD function well in E. coli . It
also hints that co-expression of kil and E is of better
lethal effect than the sole expression of E .
AraC/PBAD-kil-sd-E-GmR cassette
was then introduced into S. marcescens to insert into the CDS ofpigA . The goal strain was named as GY5. Selection stringency
frequency of GY5 (1.4×10−7) is about 10- fold lower
than that of GY4 (1.9×10−6) (Fig. 5D). The result
hints that counter-selection system in GY5 should perform better than
GY4. This was then verified by the ssDNA mediated mutation repair:
compared with GY4 (Fig. 4A), no matter in the control group or in the
ssDNA added group, fewer white colonies were shown on GY5 (Fig. 5E).
When 1,000 bp T7 RNA polymerase gene fragment was used to substitute the
AraC/PBAD-kil-sd-E-GmR in GY5,
6 out of 10 randomly selected colonies were correct (Fig. 5G). The ratio
of correct recombinants is also higher than that in GY4 (Fig. 4C).
All these results show that co-expression of kil and E can
elevate the selection stringency by orders of magnitude in multiple
species and decrease the number of escaping colonies during seamless
modification.