Control of bacterial lipopolysaccharide modifications and rare
metal nanoparticle production
Akinori KATO

Kindai University

Research aims
The PmrA/PmrB two-component regulatory system confers cationic peptide
polymyxin B and ferric iron resistance to Salmonella enterica by modifying phosphoryl
groups in lipopolysaccharide (LPS). LPS is the major component of the outer membraneof Gram-negative bacteria (Fig. 1) 1, 2). Recently, we have shown that reciprocal controlof the PmrA/PmrB system and LPS modifications, in combination with Ugd/PbgPEand PmrC-mediated modifications of phosphoryl groups affecting the lipid A moiety of
LPS and the PmrR dependent inhibition of pyrophosphorylated lipid A formation,(catalyzed by LpxT), all act to decrease the net negative charge of the LPS. This in turn
hinders the accessibility of Fe3+ to the cell surface, as well as to the Fe3+ sensor PmrB
(Fig. 1) 3, 4).
Shewanella oneidensis is a free-living Gram-negative bacterium that can produce
nanoparticles (NPs) from several metal ions, including iron, gold, platinum, silver, andpalladium. S. oneidensis can therefore be used for the recycling of precious and rare
metals, although it is important to understand and improve the physiology and functionof the organism to fully realize its potential. In this study, the LPS-modifying geneclusters of S. enterica were utilized to alter the binding affinity and specificity of S.
oneidensi, in turn limiting
the types of metal NPs
produced from a mixtureof several metal ion
species, improving themetal recovery technology.
Fig.1.Regulatory network of the LPS modification genes that confers
resistance to iron and polymyxin in Salmonella enterica

Methods(1) Prior to theintroduction of the S.
enterica LPS modification
gene clusters into S.
oneidensis, several
constructs were created.
FRT sites were used todefine a chromosomal target
DNA fragment that encoded
the LPS modification
enzymes. These would thenbe used for in vivo specificcloning (Fig. 2).

Fig. 2. Model illustrating processes of the P22 phage- andFlp-mediated in vivo cloning.

Two consecutive lambda Red-mediated recombination events were induced using
two copies of the same antibiotic marker. Following this, P22 transduction was utilized
to prepare and transfer insert DNA from the donor strain harboring the target segment toa recipient strain containing the vector plasmid (Fig. 2). Some phage particles injected
into Salmonella chromosomal DNAs would include the LPS modification gene cluster,
KmR, and the FRT sites. This provided the targets for chance recombination andintegration into FRT sites in the pBAC108L-FRT plasmid in the recipient strain (Fig.
2).
(2) In order to use the periplasmic space of S. oneidensis as a platform for specific metal
NP production, genes encoding the major c-type cytochrome were disrupted using theallelic exchange vector pDMS197. Upstream and downstream DNA fragments (500 bp)
of the target gene cluster site were cloned into pDMS197, creating thepDMS197-up-FRT-down construct. An FRT site, which could also be used as a site ofintegration for the LPS modifying gene cluster segmented by the two copies of FRT,
was inserted between the upstream and downstream DNA fragments.
(3) To construct defective mutants of the two-component regulatory system in S.
oneidensis, a generalized procedure was developed. The pDMS197 plasmid wasemployed as a vector to clone a portion of each target site in the response regulator(RR)/histidine kinase (HK) operons (except for the sacB gene).
(4) The types of metal species in NPs produced by the S. oneidensis mutant strain
containing the LPS modification gene clusters were analyzed by a scanning electron
microscope using Energy Dispersive X-ray (SEM/EDX).
The LPS modification gene clusters on the chromosome were dissected by FRT site andantibiotic marker-FRT sequence (i.e., Km-FRT) in a Salmonella strain. P22 phagelysate was prepared from this donor strain. The recipient strain possessed anotherantibiotic marker (e.g. Sp) at a site corresponding to the original antibiotic marker-FRT
insertion site in the donor strain. This was used to counter-select and prevent
RecA-dependent homologous recombination, and harbored the pCP20 andpBAC108L-FRT plasmids respectively. A portion of the P22 phage particles package
and transfer the target DNA, together with the marker (i.e. Km) and FRT sites, from thedonor chromosome into the recipient strain. This results in the site-specific integrationof the target segment into the pBAC108L-FRT plasmid.
Results
Accurate in vivo cloning5) employing the P22 phage and FLP recombinase was used
to introduce the relatively large DNA segments containing LPS modification geneclusters from S. enterica into S. oneidensis. Approximately 70% of clones contained acorrect insert of up to at least 20 kb in length. The LPS gene cluster that had been
fragmented by FRTs was cloned into the FRT site of the pBAC108L-FRT vector.
Importantly, the insert from the clone can be transferred into other plasmids possessing
an FRT via FLP-mediated exchange. The plasmid pDMS197-up-FRT-down wasdesigned with a targeted FRT site to delete the major outer membrane c-type
cytochrome in S. oneidensis. After the exchange reaction between the two plasmids, theLPS gene cluster was introduced into the S. oneidensis chromosome using conjugationtransfer and homologous recombination. To construct defective mutants of thetwo-component regulatory system of S. oneidensis, part of the coding region for the firstgene in each RR/HK operon was cloned into a pDMS197 vector. After conjugativetransfer into S. oneidensis, and a single crossover event, target genes were disrupted byintegration of the pDMS197 plasmid. This will likely influence downstream HK and RR
genes. Analysis of the metal NPs produced by the S. oneidensis mutant strainsconstructed in this study is ongoing.
Conclusions
In S. oneidensis, genetic tools that allow wide-scale gene disruptions and introductionof large gene clusters are relatively limited. Therefore, it is important to develop
improved strategies for the cloning and transfer target DNA fragments, even in a donorbacterium. We have developed a cloning method that enables the insertion of large DNA
fragments in vivo, such as the LPS gene clusters. Moreover, the FRT sites derived fromthe construction steps of the in vivo cloning method were successfully reused fortargeting DNA delivery into the S. oneidensis chromosome. To fully realize the potentialof S. oneidensis mutant strains containing the LPS modifying genes, it is necessary tooptimize expression of genes derived from Salmonella, a distinct genus of Shewanella,
to increase the metal selectivity. Continuous breeding of the strains constructed in thisstudy and repeat analyses of metal NPs profiles by SEM/EDX will be the nextchallenge.
References
1) Kato A, Groisman EA. (2004) Connecting two-component regulatory systems by a proteinthat protects a response regulator from dephosphorylation by its cognate sensor.
Genes Dev. 18: 2302-2313.
2) Kato A, Latifi T, Groisman EA. (2003) Closing the loop: the PmrA/PmrB two-component
system negatively controls expression of its posttranscriptional activator PmrD.
Proc. Natl. Acad. Sci. U S A. 100: 4706-4711.
3) Kato A, Chen HD, Latify T, Groisman EA. (2012) Reciprocal Control Between a Bacterium'sRegulatory System and the Modification Status of its Lipopolysaccharide. Mol Cell. 47:897-908.
4) Kato A, Higashino N, and Utsumi R. (2016) Fe3+-dependent epistasis between theCpxR-activated loci and the PmrA-activated LPS modification loci in Salmonella enterica. J.
Gen. Appl. Microbiol. in press.
5) Kato A. (2016) In vivo cloning of large chromosomal segments into a BAC derivative bygeneralized transduction and recombineering in Salmonella enterica. J. Gen. Appl. Microbiol.
doi 10.2323/jgam.2016.04.003.