56NISR Research GRANT
2007 Young Investigator Research Grant

Characterization of the diversity of lipoprotein-targetingmechanisms in proteobacteria
Shin-ichiro NARITA
The Institute of Molecular and Cellular Biosciences, The University of Tokyo

Research aims

Results

Bacterial lipoproteins constitute a subset of membraneproteins with their N-terminal cysteine residues modified
with a lipid moiety. Lipoproteins are synthesized in the cytoplasm as precursors, translocated across the inner membrane and modified with lipids at periplasmic surface of theinner membrane1). In Gram-negative bacteria, lipoproteinsdestined for the outer membrane are escorted to their finaldestination by the function of the Lol system that is composed of a lipoprotein-specific ABC transporter, a periplasmic carrier protein and an outer membrane receptor protein,
whereas inner membrane lipoproteins are not transportedby the Lol system and remain in the inner membrane2).
Molecular mechanisms involved in outer-membrane targeting of lipoproteins have been studied using Eschericiha coli
as a model organism. Other Gram-negative bacteria also
posses homologous genes to lol genes of E. coli, and therefore it is assumed that Lol system-mediated transport is thecommon mechanism for outer-membrane localization oflipoproteins in Gram-negative bacteria. However, whileb ,d ,g -proteobacteria have complete set of genes for Lolproteins, a -proteobacteria lack homologous gene for theouter membrane receptor protein, LolB. To investigate therole of Lol proteins in a -proteobacteria, genes for Lol proteins were cloned from the nitrogen-fixing rhizobia
Mesorhizobium loti.

The chromosome of M. loti contains two orthologues oflolA (mll4242 and mll8064, which are referred here as
lolA1 and lolA2, respectively), lolC and lolD but not lolB
nor lolE 4). In E. coli, the lipoprotein-specific ABC transporter is composed of two molecules of nucleotide-binding
protein LolD and one molecule each of integral membraneprotein LolC and LolE, which share 26% sequence identity
each other5). In contrast, the lipoprotein-specific ABCtransporter of M. loti may consist of two molecules each of
LolC and LolD.
LolA1 and LolA2 were synthesized in E. coli as fusion
proteins with signal peptides of E. coli OmpF. Periplasmic
fractions were prepared from each culture and mature
LolA1 and LolA2 were purified by utilizing hexahistidine
tag attached at the C-terminus of each protein. Althoughboth proteins could be purified, LolA2 became insoluble
immediately after purification and therefore was not further
characterized. Genes encoding LolC and hexahistidinetagged LolD were cloned as an operon into a single plasmid
and these proteins were synthesized in E. coli. Membrane
fraction was prepared and proteins were solubilized by ndodecyl-b -D-maltopyranoside, and subjected to an immobilized metal affinity chromatography. LolC was co-purified
with hexahistidine-tagged LolD with a stoichiometry of 1 :1, supporting the assumption that the lipoprotein-specific
ABC transporter of M. loti is composed of two molecules
each of LolC and LolD.
Lipoprotein-releasing activity of LolA and LolCDE canbe monitored by using E. coli spheroplasts6). E. coli cells
expressing both LolC and LolD were converted into spheroplasts, and release of an endogenous outer membrane
lipoprotein Lpp was monitored. Upon addition of LolA1,
Lpp appeared in the supernatant fraction of spheroplasts,
indicating that Lpp was released from the inner membraneby forming a soluble complex with LolA1. On the contrary,
Lpp was not detected in the supernatant fraction even in thepresence of LolA1 when spheroplasts were prepared from
E. coli cells that was transformed with a vector. This results
indicates that the LolCD complex is responsible for LolA1dependent release of Lpp from spheroplasts.

Methods
Two orthologous genes for lolA (mll4242 and mll8064)
were amplified by PCR from the chromosomal DNA of M.
loti MAFF 303099, and cloned into plasmids so that eachDNA sequence for the mature region of LolA is inserted
between those for the signal peptide of E. coli OmpF andC-terminal hexahistidine tag. The chromosomal region containing two orthologous genes for lolC and lolD was also
amplified from the M. loti genome and cloned into a plasmid with a sequence that encodes hexahistidine tag attached at the C-terminus of LolD. Lol proteins were expressed in E. coli C43 and purified with immobilized metal
affinity chromatography using TALON Metal Affinity
Resin (Clontech). Lipoprotein release assay from E. coli
spheroplasts was performed according to the method described by Matsuyama et al 3).

Conclusion
Lol system is characterized by formation of soluble intermediate of lipoprotein and LolA in the periplasmic space.

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In this study, lipoprotein-releasing reaction mediated byLolA1 and LolCD of M. loti was reconstructed in E. coli.
From these results, it was suggested that Lol system is responsible for outer membrane localization of lipoproteins ina -proteobacteria, even though they lack orthologous genefor lolB. Although it has become clear that lipoproteins are
released as soluble complexes with LolA1 from the inner
membrane into the periplasmic space, how they are incorporated into the outer membrane remain unknown. Further
analysis on the interaction of lipoprotein-LolA1 complex
and the outer membrane of M. loti is needed to understandthe evolution of lipoprotein-targeting pathway in Gramnegative bacteria.

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References
1)

Pugsley AP: The complete general secretory pathway in gram-negative bacteria. Microbiol. Rev. 1993; 57: 50–108.

Narita S, Matsuyama S, Tokuda H: Lipoprotein trafficking in Escherichia coli. Arch. Microbiol. 2004; 182: 1–6.
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Watanabe A, Idesawa K, Ishikawa A, Kawashima K, Kimura T,Kishida Y, Kiyokawa C, Kohara M, Matsumoto M, Matsuno A,Mochizuki Y, Nakayama S, Nakazaki N, Shimpo S, Sugimoto M,Takeuchi C, Yamada M, Tabata S: Complete genome structure of thenitrogen-fixing symbiotic bacterium Mesorhizobium loti. DNA Res.
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Narita S, Tanaka K, Matsuyama S, Tokuda H: Disruption of lolCDE,encoding an ATP-binding cassette transporter, is lethal for Escherichia coli and prevents release of lipoproteins from the innermembrane. J. Bacteriol. 2002; 184: 1417–1422.