129NISR RESEARCH GRANT
2003 Young Investigator Research Grant

Molecular Dissection of the Selenomonas ruminantium Cell
Envelope and Lysine Decarboxylase Involved in the Biosynthesis of a Polyamine Covalently Linked to the Cell Wall Peptidoglycan Layer
Yumiko TAKATSUKA
Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science,
Tohoku University
The wild type of Selenomonas ruminantium subsp. lactilytica, which is a strictly anaerobic, Gram-negative bacterium isolated from sheep rumen, requires one of the normal saturated volatile fatty acids with 3 to 10 carbon atoms
for its growth in a glucose medium; however, no such obligate requirement of fatty acid is observed when the cells
are grown in a lactate medium. This bacterium is characterized by a unique structure of the cell envelope and a novel
lysine decarboxylase and its regulatory protein.
S. ruminantium possesses neither free nor bound formsof Braun lipoprotein which plays an important role of themaintenance of the structural integrity of the cell surface ingeneral Gram-negative bacteria. However, S. ruminantium
has cadaverine which links covalently to the peptidoglycan
as a pivotal constituent for the cell division.
In this study, we did the molecular cloning of the genes
encoding a major outer membrane protein of 45 kDa(45 kDa protein, S. ruminanitum lysine decarboxylase(LDC), and its regulatory protein of 22-kDa (22-kDa protein; P22) which has similar characteristics to that of antizyme of ornithine decarboxylase in eukaryotic cells, andthe molecular dissection of these proteins for understanding
the regulation of cadaverine biosynthesis.
(1) Cloning of the gene encoding of a major outermembrane protein of 45 kDa (45 kDa protein)
The nucleotide sequence of the gene encoding a major
outer membrane protein of 45 kDa in Selenomonas ruminantium was determined. The structure gene of 44 kDa protein gene consisted of 1296 base pairs. An open reading
frame that could encode a 44,990 dalton polypeptide consisting of 418 amino acid residues was assigned. Inspectionof the amino acid sequence deduced from nucleotide sequence of 45 kDa protein gene and that from native 45 kDa
protein clarified that pre-matured 45 kDa protein contains a
typical signal sequence at the N-terminus. The amino acidsequence of the predicted matured 45 kDa protein correlated exactly with the known N-terminal 23 amino acid sequence of the native protein. The nucleotide sequence ofthe 5-flanking region showed the presence of the consensus sequence of ribosome binidng site, Pribnow box and theRNA polymerase recognition site in Escherichia coli. E.
coli Bla21(DE3) with the plasmid pOM44, which contains
the 45 kDa protein gene expressed the gene to produce
45 kDa protein.
(2)

Regulation of S. ruminantium LDC production

In mammalian cells and tissues, the turnover of ornithine
decarboxylase (ODC) (EC 4.1.1.17) is very rapid andhighly regulated. The degradation of mammalian ODC catalyzed by the 26S proteasome is accelerated by ODC antizyme, a trigger protein involved in the specific degradation of eukaryotic ODC. In prokaryote, S20, L26 and L34
ribosomal proteins were identified as antizymes in Escherichia coli whose overproduction decrease the level ofODC protein and its mRNA. However, the direct evidence
on the existence of antizyme in E. coli has not been obtained. Previously, we found cadaverine covalently linked tothe peptidoglycan in Selenomonas ruminantium, a strictly
anaerobic bacterium, which is an essential constituent of
the peptidoglycan to maintain the integrity of the cell envelope. We also found that cadaverine is synthesized constitutively from L-lysine catalyzed by LDC (EC 4.1.1.18) andtransferred to the D-glutamic acid residue of the lipid intermediate for the synthesis of the peptidoglycan by lipid intermediate: cadaverine transferase. S. ruminantium LDC
was purified and characterized and its gene(ldc) was cloned,
and the following findings became evident. I) S. ruminantium LDC consists of two identical monomeric subunits
with 45 kDa and decarboxylates both L-lysine and L-ornithine with similar Km and Vmax values and its decarboxylating activities toward both substrates were completely prevented by DFMO which had been known to be a
specific inhibitor of eukaryotic ODC. II) The amino acidsequence of S. ruminantium LDC is 35% identical and 53
to 60% similar to those of eukaryotic ODCs and that 26
amino acid residues, all of which are implicated either incontributing to PLP- and substrate-binding domains or information of the homodimeric forms of eukaryotic ODCs,
are conserved in S. ruminantium LDC. III) S. ruminantium
LDC has a homologous sequence to that of mouse antizyme
binding region in mouse ODC. IV) S. ruminantium LDC is130classified into fold type III to which eukaryotic ODCs but
not bacterial ODC and LDC are belonging. In addition, S.
ruminantium has no typical ODC with decarboxylase activity exclusively toward L-ornithine and our LDC involves inthe synthesis of putrescine. These finding clarified that S.
ruminantium LDC and eukaryotic ODC are resemble eachother in both biochemical and biophysical characteristics
except for the broad range of the substrate specificity in S.
ruminantium LDC.
The production of LDC in S. ruminantium is highly regulated and is strictly linked to the growth phase of the bacteria, i.e. a drastic decrease in LDC activity occurred on entry
into the stationary phase of cell growth, which was due tothe rapid degradation of LDC. In the proceeding paper, weisolated a new protein of 22 kDa, which is induced in putrescine-grown cells as a regulating factor for the degradation of LDC in S. ruminantium cells by ATP-dependent
protease. Take together with the characteristics of our LDC,
22-kDa protein is a possible direct counterpart of mammalian ODC antizyme. Here, we showed that the aminoacid sequence of the protein is 47 identical and 60–66%
similar to those of reported ribosomal L10 protein of bacteria. The recombinant 22-kDa protein, which was expressedin E. coli, was purified and the following findings were evident. I) The 22-kDa protein itself has no degradation activ-

ity toward LDC but it was required for the degradation of S.
ruminantium LDC by ATP-dependent proteases in cell-freesystem. II) The mouse ODC was degraded in our cell-freesystem containing the 22-kDa protein preparation insteadof mouse antizyme. III) S. ruminantium LDC was degradedin our cell-free system containing mouse antizyme preparation instead of the 22-kDa protein preparation. S. ruminantium LDC was degraded in ODC degradation system containing 22-kDa preparation or mouse antizyme preparation.
IV) The production of 22-kDa protein is induced by putrescine but not cadaverine added in the culture. V) Boththe 22-kDa protein and mouse antizyme preparations themselves inhibit LDC/ODC activities of S. ruminantium LDC
preparation in the cell-free system. VI) Both 22-kDa protein and mouse antizyme bind to S. ruminantium LDC.
However, they did not bind to S. ruminantium LDC mutant
in antizyme binding region, in which three Lys residues
K103, K123, K126 were replaced by Ala, resulting in nulldegradation of the mutant LDC in cell-free system. Thus,
we conclude that the 22-kDa protein is a direct counterpartof mammalian ODC antizyme which is required for thedegradation of S. ruminantium LDC by ATP-dependent
protease. We propose to designate 22-kDa protein and its
gene prokaryote antizyme (ProAZ) and proAZ, respectively.