Background
Foreigner DNA fragments are usually needed to be introduced into
bacteria. They can be constructed into replicative plasmids, plasmids
are inherently unstable, limiting the application of this strategy in
researches with high requirements for the stability of results. As a
better alternative, exogenous DNA fragments should be irreversibly
incorporated into chromosome or other stable DNA molecule inside the
cell (Heap et al., 2012). For this sake, DNA fragments of interest could
be combined with positive selection markers, typically using an
antibiotic resistance gene, allowing desired recombinant cells to be
selected and isolated after a single-crossover or double-crossover
recombination. Positive selection markers can also be removed through
specific procedure, such as FLP- or CRE-mediated recombination (Meyers,
Lewandoski, & Martin, 1998), but frt or loxp site will
leave behind inevitably (Lee et al., 2001). Generally speaking, for the
sake of biosecurity or eliminating unanticipated effects, residual of
resistance genes or frt and loxp scars on the genome is
not expected during functional genome analysis and metabolic engineering
research.
This issue can be overcome using a two-step selection method by
combining positive-negative selection cassette. In particular, a
positive-negative selection cassette could be introduced and
recombinants are selected according to positive selection markers, then
exogenous DNA fragments are transformed into cells to replace the
above-mentioned cassette and the final desired recombinants are selected
against the counter-selection gene.
Many positive selection markers are highly efficient so recombinants
could be easily acquired in the first recombination event. In contrast,
counter-selection markers are not so satisfactory. The main problem of
counter-selection markers is that high background causes great
difficulties for counter-selection, especially in the case of low
recombination efficiency (Imam et al., 2000; Muyrers et al., 2000; Y.
Zhang, F. Buchholz, J. P. Muyrers, & A. F. Stewart, 1998). Several
research groups are being dedicated to developing and optimizing better
counter-selection markers(DeVito, 2008; Ma et al., 2015; Wang et al.,
2014; Wong et al., 2005) and some novel counter-selection marker genes
have been reported. Among them tetA -sacB and inducible
toxins system are the most outstanding (Khetrapal et al., 2015; Li,
Thomason, Sawitzke, & Costantino, 2013). It is worth mentioning that
inducible toxins system possesses the highest selection stringency
(1.1×10-8 to 3.31×10-8) which even
comes up to that of positive antibiotic markers in some strains
(Khetrapal et al., 2015). Despite these achievements, excellent
counter-selection markers are still scanty. In view of the situation, we
are motivated to develop robust counter-selection marker and provide new
strategy for optimizing counter-selection efficiency.
Lysis gene E of bacteriophage PhiX174 was discovered to have a
role in lysing Escherichia coli in 1966 for the first time
(Hutchison & Sinsheimer, 1966), and the subsequent researches shows
that expression of this gene is sufficient to cause lysis of E.
coli (Henrich, Lubitz, & Plapp, 1982; Young & Young, 1982). Lysis
gene E codes for a 91-aa membrane protein with hydrophobic
moieties at its N-terminal end that oligomerizes into a transmembrane
tunnel structure(Blasi, Linke, & Lubitz, 1989; Witte & Lubitz, 1989).
This specific tunnel structure is associated with the fusion of the
inner and outer membranes of Gram-negative bacteria, leaving behind
empty envelopes devoid of all cytoplastic content (Hajam, Dar, Won, &
Lee, 2017; Witte, Wanner, Sulzner, & Lubitz, 1992). Non-living
Gram-negative bacterial empty envelopes caused by E protein are knows as
Bacterial Ghosts (BG), representing a potential platform both for potent
candidate vaccines and for technical applications in white biotechnology
(Hajam et al., 2017; Langemann et al., 2010; Won, Hajam, & Lee, 2017).
BGs of many Gram-negative strains have been prepared successfully,
including Salmonella typhimurium , Salmonella enteritidis ,Bordetella bronchiseptica , Vibrio cholerde ,Mannbeimia haemolytica , Pseudomonas aeruginosa , etc
(Langemann et al., 2010). In view of the universal and potent killing
effect mediated by lysis protein E in Gram-negative strains, we focus
our effort on developing lysis E gene as a universal marker gene
to try to expand the existing counter-election tool chest, which is
especially useful for host strains lack of facile genetic tools.
This study reports on the introduction of lysis gene E of PhiX174
to create “seamless” genetic manipulations in Gram-negative strains.
Under the control of the lambda pL promoter and
AraC/PBAD inducible system, lysis gene E can
effectively promote the death of E. coli and Serratia
marcescens , and the selection stringency of lysis gene E is
similar to the next best reported tetA-sacB double-negative
selection system. By co-expressing lysis gene E and kilnegative gene (kil-sd-E counter-selection cassette) through a
classic RBS (Ribosome Binding Site) sequence, the selection stringency
frequency is even comparable with the best counter-selection system,
inducible toxins system. By introducing araC gene harboring
plasmid, the selection stringency frequency of kil-sd-E arrives
at the level of 10−9 both in E. coli and inS. marcescens , This is the lowest selection stringency frequency
for counter-selection reported so far, exceeding the best counter
selection system, inducible toxins system, several fold.
Due to the universal lethal of
expression of lysis protein E, counter selection cassette based on lysisE or kil-sd-E could be applicable to many, if not all,
Gram-negative strains. And the strategy reported in this article of
combining two short counter-selection genes could be used to increase
the counter-selection efficiency of other negative markers, which can
provide more powerful tools to the genome editing toolbox.