Jpn. J. Protozool. Vol. 41, No. 2. (2008)169

Short communication
Dictyostelium proteins bearing motifs conserved in penicillin-binding
proteins and beta-lactamases
Yosuke UMEDA, Kosuke NOMOTO, Takeshi YONEMICHI, Kentaro TAMURA
and Hiro YASUKAWA*Graduate School of Science and Engineering, University of Toyama, Toyama
930-8555, Japan.
Key words: Dictyostelium discoideum, LACTB, penicillin-binding protein, beta-lactamase

SUMMARY
The soil-dwelling eukaryotic microorganism
Dictyostelium discoideum encodes a protein bearing SNTK, SNN and KTG sequences(DDB0168572) and a protein bearing SISK andHTG sequences (DDB0233562). These sequences are similar to SXXK, S(Y)XN and K(H)
T(S)G sequences, which are motifs conserved inpenicillin-binding proteins and beta-lactamases.
RT-PCR analysis showed that D. discoideum
expressed genes for DDB0168572 andDDB0233562 in the growth phase and had decreased expression levels of those genes in thedevelopmental phase. A mutant in which the*Corresponding author
Tel:81-76-445-6875
Fax: 81-76-445-6703
E-mail: hiro@eng.u-toyama.ac.jpReceived: 14 March 2008; Accepted: 24 July 2008.

gene for DDB0168572 was disrupted grew anddeveloped as did the parental strain, indicating that
DDB0168572 is dispensable for D. discoideum.
Loss of DDB0233562 is thought to be fatal to D.
discoideum because no gene-disruption mutant
was established.
Horizontal transfer and evolutional changes ofgenes have become recognized as a mechanism of
genome evolution (Iyer et al., 2004; Jain et al.,
2002; Richards et al., 2003). Transferred genes
have evolved and proteins encoded play importantroles in the host. Some of these proteins would
remain as domains conserved in bacterial proteins.
Finding and analyzing eukaryotic genes encoding
such proteins are important to reveal genome evolution. Database searches have shown that eukaryotes encode proteins bearing SXXK and HTG
sequences, which are similar to SXXK, S(Y)XNand K(H)T(S)G motifs conserved in bacterial penicillin-binding proteins (PBPs) and beta-lactamases(Liobikas et al., 2006; Smith et al., 2001). The

170D. discoideum proteins bearing SXXK, S(Y)eukaryotic protein designated LACTB has been
identified in various eukaryotic organisms, suggesting that it plays an important role in eukaryotes. PBPs and beta-lactamases function in synthesis of peptidoglycan and inactivation of betalactam antibiotics, respectively. These functions
are required for bacteria but not for eukaryotes;
therefore, the function of LACTB is not known.
We previously reported that the cellular slime
mold Dictyostelium discoideum encodes a protein
similar to PBP (Yasukawa et al., 2003). This protein, PscA (same as DDB0168572), bears SNTK,
SNN and KTG sequences. We carried out further
screening in the online informatics resource for D.
discoideum (http://dictybase.org/) and found an
additional protein bearing the motifs. The accession number of the protein is DDB0233562.
Amino acid sequences around the SXXK, S(Y)XNand K(H)T(S)G motifs in LACTBs, Escherichia
coli PBPs, E. coli beta-lactamases, DDB0168572
and DDB0233562 are presented in Fig. 1.

Expression levels of the genes for
DDB0168572 and DDB0233562 in an axenic
strain of D. discoideum, Ax2, were analyzed byRT-PCR. RNA samples prepared from growing
cells and developing cells were used as templates.
Reaction products were electrophoresed and photographed (Fig. 2). The results show that D. discoideum expressed the genes in the growth phaseand had reduced the expression levels of the genes
in the developmental phase. We obtained almost
the same results from two independent experiments.
We conducted experiments to investigate theimportance of DDB0168572 and DDB0233562 in
D. discoideum by transforming Ax2 with knockout
constructs of the genes that were disrupted by agene cassette conferring blasticidin S (BS) resistance on D. discoideum (a bsr cassette; 1.35 kb insize) (Fig. 3A and B).
Ax2 was transformed by a construct to disrupt
the gene for DDB0168572, cultured with BS for

Fig. 1. Sequence alignment of the conserved motifs in LACTBs, E. coli PBPs, E. coli beta-lactamases, DDB0168572
and DDB0233562. The conserved amino acids in the motifs are typed in white on a black background. The accession
numbers of the LACTBs are P83111 (Homo sapiens), P83095 (Bos taurus), AAG37911 (Mus musculus), XP_217181(Rattus norvegicus), NP_001025717 (Gallus gallus) and O62481 (Caenorhabditis elegans). The accession numbers ofthe E. coli proteins are P02918 (PBP1a), P02919 (PBP1b), P76577 (PBP1c), P0AD65 (PBP2), P0AD68 (PBP3),
P24228 (PBP4), P0AEB2 (PBP5), P08506 (PBP6), P0AFI5 (PBP7), P33013 (DacD), P00811 (AmpC) and P0AD70(AmpH).

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Fig. 2. Developmental expression patterns of DDB0168572 and DDB0233562. Changes in gene expression during thelife cycle of D. discoideum were analyzed by RT-PCR. Total RNA samples extracted from growing amoebae (v) anddeveloping cells (6 – 24 h) were used as templates. Reaction products amplified by 26 cycles (DDB0168572) and 25cycles (DDB0233562) following reverse transcription are shown. Reaction was performed by the use of a Primescript
One Step RT-PCR Kit (Takara Bio, Shiga, Japan). Primers used to amplify mRNA for DDB0168572 are 5’GATGGTAATATTCCAAGTGCTTGGGAATGGG (P1) and 5’-CAACGCCACCATCTTCCAACAATGCTGAG (P2).
The DDB0233562 mRNA was amplified by the primers 5’-CCAATGGAAACTTCAAGTAAATTAAGAGTTGCGAG
TATTTC (P3) and 5’-GCTGATTCAATTACTAAACCTAATAAAGTGTAACCAAAAGTTG (P4). The EtBr staining
pattern of rRNAs and illustrations of the multicellular structures are presented.

Fig. 3. Disruption of the genes for DDB0168572 and DDB0233562.
(A) Construct used in disruption of the gene encoding DDB0168572. The bsr cassette was inserted into the EcoRI site inthe DDB0168572 cDNA. The gene for DDB0168572 is not interrupted by an intron. The restriction site for EcoRI (Ec) isindicated. Arrowheads indicate primers P1 and P2. (B) Construct used in disruption of the gene encoding DDB0233562.
The bsr cassette was inserted into the NcoI site in the DDB0233562 cDNA. The genomic sequence encoding DDB0233562
is interrupted by an intron. Restriction sites for NcoI (Nc) are indicated. Arrowheads indicate primers P3 and P4. (C) PCR
analysis. Disruption of the gene for DDB0168572 was confirmed by PCR. Genomic DNA samples purified from the cells
were used as templates. The enzyme used in the reaction was ExTaq (Takara Bio, Shiga, Japan). Primers used are P1 andP2. Lanes 1 and 2 are Ax2 and the null mutant, respectively. (D) RT-PCR analysis. Disruption of the gene for
DDB0168572 was confirmed by RT-PCR. Total RNA samples purified from the cells were used as templates. The EtBr
staining pattern of rRNAs is presented (bottom). Lanes 1 and 2 are Ax2 and the null mutant, respectively.

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D. discoideum proteins bearing SXXK, S(Y)selection, and subjected to analysis. GenomicDNA extracted from a selected clone was used as atemplate for PCR. Comparison of the sizes of thePCR products indicated that the gene in the BSresistant strain was disrupted by insertion of the1.35-kb fragment (Fig. 3C). Expression of the gene
was examined by RT-PCR using total RNA purified from the cells. The DNA fragments were amplified from the RNA of Ax2 but not from theRNA of the BS-resistant strain (Fig. 3D). Theseresults indicate that the gene for DDB0168572 inthe clone was disrupted by the insertion of the bsrcassette. The gene disruption mutant we established grew and developed normally under laboratory conditions (data not shown).
Ax2 was also transformed by a construct todisrupt the gene for DDB0233562. BS-resistantcells were observed in culture medium 6 days after
transformation but did not survive for more than 8days in the medium. Three sets of the experiments
were conducted, but no stable transformant wasestablished, indicating that disruption of the geneis fatal to D. discoideum.
In summary, the current results indicate that
DDB0168572 is dispensable for D. disocideum,whereas DDB0233562 is essential for this organism.
The genes for DDB0168572 and DDB0233562
might have been horizontally transferred frombacteria, one of them evolving into the gene essential for the host. Further analysis of DDB0168572
and DDB0233562 is needed to reveal evolution of
the genes and genome.
ACKNOWLEDGMENTS We thank members of
the Dictyostelium cDNA project team for providing the cDNA clones for DDB0168572 (SSA619)
and DDB0233562 (VFG665).

REFERENCES
Iyer, L. M., Aravind, L., Coon, S.L., Klein, D.C.
and Koonin, E.V. (2004) Evolution of cell-cellsignaling in animals: did late horizontal genetransfer from bacteria have a role? Trends
Genet., 20, 292-299.
Jain, R., Rivera, M.C., Moore, J.E. and Lake, J.A.
(2002) Horizontal gene transfer in microbial
genome evolution. Theor. Popul. Biol., 61, 489-495.
Liobikas, J., Polianskyte, Z., Speer, O., Thompson,J., Alakoskela, J.M., Peitsaro, N., Franck, M.,
Whitehead, M.A., Kinnunen, P.J. and Eriksson, O. (2006) Expression and purification ofthe mitochondrial serine protease LACTB as
an N-terminal GST fusion protein in Escherichia coli. Protein Expr. Purif., 45, 335342.
Richards, T.A., Hirt, R.P., Williams, B.A. and Embley, T.M. (2003) Horizontal gene transfer andthe evolution of parasitic protozoa. Protist,154, 17-32.
Smith, T.S., Southan, C., Ellington, K., Campbell,D., Tew, D.G. and Debouck, C. (2001) Identification, genomic organization, and mRNA
expression of LACTB, encoding a serine beta-lactamase-like protein with an aminoterminal transmembrane domain. Genomics,78, 12-14.
Yasukawa, H., Kuroita, T., Tamura, K. and Yamaguchi, K. (2003) Identification of the penicillin-sensitive carboxypeptidase in the cellularslime mold Dictyostelium discoideum. Biol.
Pharm. Bull., 26, 1018-1020.