Microbiology Comment

Patatin-like proteins: a new

family of lipolytic enzymes
present in bacteria?
Patatins are a group of plant storage
glycoproteins that show lipid acyl
hydrolase activity (Andrews et al., 1988).
The patatin-associated lipolytic activity
may be a means of defence against plant
parasites (Strickland et al., 1995) and has
been shown to function in plant signal
transduction (Holk et al., 2002). Hirschberg
et al. (2001) found that potato patatin B2
and human cytosolic phospholipase A2
(cPLA2) share conserved domains of
protein homology and therefore predicted
that patatin B2, like cPLA2, contains an
active-site dyad instead of the more
common Ser-His-Asp (or Glu) triad of
lipolytic enzymes (Schrag & Cygler, 1997).
This was confirmed by the crystal structure
of the potato patatin isozyme Pat17 which
together with mutagenesis studies revealed
that the active site consists of a Ser-Asp
dyad (Rydel et al., 2003). Recently, Sato
et al. (2003) and Phillips et al. (2003)
have shown that ExoU, a type III
secreted cytotoxin and virulence factor
of Pseudomonas aeruginosa with lipase as
well as phospholipase A activity, contains
defined regions of protein homology to
potato patatin B2 and cPLA2 with a putative
catalytic Ser-Asp dyad. In animal models
and human infections, the toxicity of
ExoU has been linked to the development
of lung injury, sepsis and bacterial
dissemination (Allewelt et al., 2000;
Finck-Barbancon et al., 1997; Hauser et al.,
1998; Kurahashi et al., 1999). Thus, ExoU
is both an important virulence factor of
P. aeruginosa and the first patatin-like
bacterial hydrolase characterized. However,
it has yet to be determined whether
patatin-like proteins (PLPs) are found in
bacteria other than P. aeruginosa.
To find out whether PLPs exist in bacteria
other than P. aeruginosa, we searched (A) all
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bacterial genomes available for patatin

B2 and P. aeruginosa ExoU homologues
by means of the BLAST algorithm
(http://www.ncbi.nlm.nih.gov/sutils/
genom_table.cgi) (Altschul et al., 1997)
and (B) we applied the key word patatin
to screen the 123 accessible completed
bacterial genomes using the text search tool
of the PEDANT server (http://pedant.gsf.de).
The PEDANT server provides extensive data
from computational analysis of genome
sequences by algorithms such as PROSITE,
PFAM, BLAST and others. For search (A),
the BLAST alignment, we only obtained a
minimal number of hits for PLPs in
bacteria (<20 for patatin B2 or ExoU,
BLAST expect value1). On the other
hand, we found a high number of hits
(>200) when we used the PEDANT server
text search. In 134 proteins (PFAM domain
expect value0?5), text search hits for
patatin originated from the detection of
the patatin domain by the PFAM algorithm
(Bateman et al., 2002), indicating that only
short amino acid sequences of homology
were conserved within related proteins.
This might also explain why the application
of the BLAST algorithm did not lead to
adequate results. Genes encoding PLPs
were detected in 55 of the 123 completed
bacterial genomes including a wide range
of Gram-positive and Gram-negative
bacteria, such as Bacillus sp., Brucella sp.,
Rickettsia sp., Staphylococcus aureus,
Yersinia pestis and many others.
Moreover, we observed that particularly
some animal pathogen and plant
pathogen/symbiont genomes contain
a high number of PLP genes, e.g.
Bradyrhizobium japonicum (n=8),
Leptospira interrogans (n=6),
Mesorhizobium loti (n=6), Mycobacterium
tuberculosis (n=8) and Ralstonia
solanacearum (n=5). We were interested
in whether pathogens/symbionts possess
more PLP genes than non-pathogens.
Thus, we compared the number of PLP
genes present per 1000 open reading
frames (ORFs) in the two groups and
found that the genomes of the pathogens/
symbionts showed a significantly higher
number of PLP genes than the genomes of
non-pathogens, more precisely 0?800?50
and 0?530?24 (P0?01, Students t-test),
respectively. The number of PLP genes
also varied between different species of a
genus. For example, the genome of
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Microbiology Comment

Fig. 1. Alignment of selected bacterial PLPs with conserved patatin domains. Sixty proteins with patatin-like domains (PFAM
domain expect value5?561019) were aligned by the CLUSTAL W method (Thompson et al., 1994) using the program
55
MEGALIGN (DNASTAR). Conserved protein domains of a selection of PLPs (PFAM domain expect values from 6610
to
2?661038) representing different bacterial genera and including proteins with different N- or C-terminal extensions are listed
in the order of increasing PFAM expect values. Residues which were conserved in more than 80 % of the 60 aligned proteins
are marked bold. An arrow designates the members of the catalytic dyad in patatin B2 (Rydel et al., 2003). D, The number of
amino acid residues present before and after the conserved blocks. Conserved regions of patatin B2 (modified from
Hirschberg et al., 2001) and of P. aeruginosa ExoU are shown for comparison.
http://mic.sgmjournals.org

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Microbiology Comment

Mycobacterium leprae included only

one PLP gene, whereas the genome
of M. tuberculosis contained eight
corresponding genes. This finding is in
agreement with a previous report which
stated that M. leprae originated by a
process of reductive evolution resulting in
a minimal gene set sufficient for survival
in its highly specialized niche (Cole et al.,
2001). In contrast, 52 % of the proteome
of M. tuberculosis was derived from gene
duplication (Tekaia et al., 1999), leading to
a higher flexibility in utilizing nutrients.
Furthermore, the genome of pathogenic
Escherichia coli O157 : H7 encoded a higher
number of PLP genes (n=4) than the
genome of non-pathogenic strain E. coli
K-12 (n=2), which again implies a
correlation between the number of PLP
genes and virulence. Hence, we speculate
that a high number of PLP genes might
confer an advantage to the bacterium in
(i) interaction with the host, (ii) adaptation
to various environments and (iii)
competition with other micro-organisms.
To determine whether the bacterial PLPs
share all characteristic domains with their
eukaryotic counterparts, 60 bacterial PLPs
(PFAM domain expect value5?5610219)
were aligned by the CLUSTAL W method
(Thompson et al., 1994). Bacterial PLPs
were found to possess four conserved
domains (block IIV) similar to those in
potato patatin B2 (Fig. 1). Block I consists
of a glycine-rich region with a conserved
arginine or lysine residue which probably
serves as an oxyanion hole. Block II is
located in proximity to block I (about
1020 aa distance) and comprises the
hydrolase motif G-X-S-X-G with the
putative active-site serine (Schrag &
Cygler, 1997). Block III contains a
conserved serine, which may be an
important structural element as a potential
phosphorylation site or due to its capacity
to form hydrogen bonds. The adjacent
highly conserved proline residues (in
blocks III and IV) may be important for
the proper conformation of the protein.
In addition to many structural features
shared both by eukaryotic and bacterial
PLPs, we nevertheless observed that in
block III bacterial and eukaryotic PLPs
show different conserved motifs. While
bacterial PLPs possessed the conserved
sequence A-S-X-X-X-P, eukaryotic patatin
homologues contained the conserved
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amino acids A-A-P, as seen by CLUSTAL W

alignment of 20 eukaryotic PLPs (Fig. 1,
and data not shown). Block IV includes the
putative active-site aspartate which is the
second member of the catalytic dyad in
patatin Pat17 (Rydel et al., 2003). Successive
to the domain with the active-site aspartate,
Hirschberg et al. (2001) have defined an
additional region of protein homology for
potato patatin B2 and human cPLA2
containing a conserved serine. We found
that this domain was present in at least 15
other eukaryotic patatin homologues (data
not shown) but was not conserved in the
respective prokaryotic proteins (Fig. 1).
We therefore conclude that eukaryotic
and bacterial PLPs share many but not all
structural features, suggesting that they
originated from a common ancestor and
were modified to support/expand the
specific lifestyle of an organism.

proteins. Some bacterial proteins with

N-terminal extensions showed a predicted
cyclic nucleotide-binding domain (e.g.
Caulobacter crescentus gi13423668,
L. interrogans gi24214875, M. tuberculosis
gi13883156) and therefore might be
regulated by cAMP or cGMP (McCue
et al., 2000). Several of the proteins with
C-terminal extensions possessed a putative
bacterial-surfaceantigen domain (e.g.
Pseudomonas putida gi26991199, Vibrio
cholerae gi9655035, Vibrio vulnificus
gi27361160), which is also present in
outer-membrane proteins such as D15
from Haemophilus influenzae (Loosmore
et al., 1997). Furthermore, P. aeruginosa
ExoU (gi2429143) contains a C-terminal
extension, which has been described to
be essential for its cytotoxic activity
(Finck-Barbancon & Frank, 2001; Hauser
et al., 1998).

Arpigny & Jaeger (1999) classified bacterial

esterases and lipases into eight groups based
on their amino acid sequence. Bacterial
PLPs do not show any homology to known
groups of bacterial lipases as evaluated by
BLAST (Altschul et al., 1997) or PFAM
domain search (Bateman et al., 2002) (data
not shown), indicating that the amino
acid sequences of PLPs and established
groups of lipolytic enzymes are not closely
related. Second, except for the G-X-S-X-G
lipase motif which is common even in
distinct lipase families, the conserved
domains presented here for bacterial PLPs
do not exhibit the same features as found
for other bacterial lipases (Fig. 1) (Arpigny
& Jaeger, 1999). Finally, the lipases classified
by Arpigny & Jaeger (1999) either possess
a catalytic Ser-Asp-His triad or a Ser-His
dyad which makes the putative Ser-Asp
catalytic dyad of patatin homologues
unique among bacterial lipases (Rydel et al.,
2003). Since the prokaryotic PLPs appear
to be more related to their eukaryotic
counterparts than to any other group of
bacterial lipases, we propose that they
comprise a new group of bacterial
lipolytic enzymes.

We have herein presented evidence that

genes of PLPs are found in many bacterial
genomes. Therefore, it is conceivable that
many bacteria possess as-yet-unidentified
lipolytic enzymes. Since these proteins
show catalytic domains and conserved
regions distinct from all groups of lipolytic
enzymes known so far in bacteria, we
believe that they comprise a new group
of hydrolytic enzymes. With the exception
of P. aeruginosa cytotoxin ExoU, none of
the bacterial PLPs have been characterized
at present. Consequently, the identification
of the patatin domain may be a valuable
clue for characterizing these putative
hydrolases and could help discover new
virulence factors.

Finally, while comparing the size of

bacterial PLPs, we noticed that several of
them possess a N- or C-terminal extension
(300 aa) preceding or successive to the
conserved blocks, respectively (Fig. 1). An
additional domain search revealed that
many PLPs are two- or multi-domain

Acknowledgements
We thank Winlet Sheba Shadrach for
helpful suggestions with regard to the
manuscript.
Sangeeta Banerji and Antje Flieger
Robert Koch-Institut, Nordufer 20,
D-13353 Berlin, Germany
Correspondence: Antje Flieger
(fliegera@rki.de)
Allewelt, M., Coleman, F. T., Grout, M.,
Priebe, G. P. & Pier, G. B. (2000). Acquisition

of expression of the Pseudomonas aeruginosa

ExoU cytotoxin leads to increased bacterial
virulence in a murine model of acute
pneumonia and systemic spread. Infect
Immun 68, 39984004.

Microbiology 150

Microbiology Comment

Altschul, S. F., Madden, T. L., Schaffer, A. A.,

Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J.
(1997). Gapped BLAST and PSI-BLAST: a new

generation of protein database search programs.

Nucleic Acids Res 25, 33893402.
Andrews, D. L., Beames, B., Summers, M. D.
& Park, W. D. (1988). Characterization of the

lipid acyl hydrolase activity of the major potato

(Solanum tuberosum) tuber protein, patatin,
by cloning and abundant expression in a
baculovirus vector. Biochem J 252, 199206.
Arpigny, J. L. & Jaeger, K.-E. (1999). Bacterial
lipolytic enzymes: classification and properties.
Biochem J 343, 177183.
Bateman, A., Birney, E., Cerruti, L. & 7 other
authors (2002). The Pfam protein families

database. Nucleic Acids Res 30, 276280.

Cole, S. T., Eiglmeier, K., Parkhill, J. & 41
other authors (2001). Massive gene decay in

the leprosy bacillus. Nature 409, 10071011.

Finck-Barbancon, V., Goranson, J., Zhu, L.,
Sawa, T., Wiener-Kronish, J. P., Fleiszig, S. M.,
Wu, C., Mende-Mueller, L. & Frank, D. W.
(1997). ExoU expression by Pseudomonas

aeruginosa correlates with acute cytotoxicity

and epithelial injury. Mol Microbiol 25,
547557.
Finck-Barbancon, V. & Frank, D. W. (2001).

in Mycobacterium tuberculosis. Genome Res 10,

204219.
Phillips, R. M., Six, D. A., Dennis, E. A.
& Ghosh, P. (2003). In vivo phospholipase

activity of the Pseudomonas aeruginosa

cytotoxin ExoU and protection of mammalian
cells with phospholipase A2 inhibitors. J Biol
Chem 278, 4132641332.
Rydel, T. J., Williams, J. M., Krieger, E.,
Moshiri, F., Stallings, W. C., Brown, S. M.,
Pershing, J. C., Purcell, J. P. & Alibhai, M. F.
(2003). The crystal structure, mutagenesis,

and activity studies reveal that patatin is a lipid

acyl hydrolase with a Ser-Asp catalytic dyad.
Biochemistry 42, 66966708.
Sato, H., Frank, D. W., Hillard, C. J. & 9 other
authors (2003). The mechanism of action of

the Pseudomonas aeruginosa-encoded type III

cytotoxin, ExoU. EMBO J 22, 29592969.
Schrag, J. D. & Cygler, M. (1997). Lipases and
alpha/beta hydrolase fold. Methods Enzymol
284, 85107.
Strickland, J. A., Orr, G. L. & Walsh, T. A.
(1995). Inhibition of Diabrotica larval growth

by patatin, the lipid acyl hydrolase from potato

tubers. Plant Physiol 109, 667674.
Tekaia, F., Gordon, S. V., Garnier, T.,
Brosch, R., Barrell, B. G. & Cole, S. T.
(1999). Analysis of the proteome of

Multiple domains are required for the toxic

activity of Pseudomonas aeruginosa ExoU.
J Bacteriol 183, 43304344.

Mycobacterium tuberculosis in silico. Tuber

Lung Dis 79, 329342.

Hauser, A. R., Kang, P. J. & Engel, J. N. (1998).

Thompson, J. D., Higgins, D. G. & Gibson, T. J.

(1994). CLUSTAL W: improving the sensitivity

PepA, a secreted protein of Pseudomonas

aeruginosa, is necessary for cytotoxicity and
virulence. Mol Microbiol 27, 807818.
Hirschberg, H. J. H. B., Simons, J.-W. F. A.,
Dekker, N. & Egmond, M. R. (2001). Cloning,

expression, purification and characterization of

patatin, a novel phospholipase A. Eur J Biochem
268, 50375044.

of progressive multiple sequence alignment

through sequence weighting, position-specific
gap penalties and weight matrix choice. Nucleic
Acids Res 22, 46734680.

DOI 10.1099/mic.0.26957-0

Holk, A., Rietz, S., Zahn, M., Quader, H.

& Scherer, G. F. E. (2002). Molecular

identification of cytosolic, patatin-related

phospholipases A from Arabidopsis with
potential functions in plant signal transduction.
Plant Physiol 130, 90101.
Kurahashi, K., Kajikawa, O., Sawa, T.,
Ohara, M., Gropper, M. A., Frank, D. W.,
Martin, T. R. & Wiener-Kronish, J. P. (1999).

Pathogenesis of septic shock in Pseudomonas

aeruginosa pneumonia. J Clin Invest 104,
743750.
Loosmore, S. M., Yang, Y.-P., Coleman,
D. C., Shortreed, J. M., England, D. M.
& Klein, M. H. (1997). Outer membrane

protein D15 is conserved among Haemophilus

influenzae species and may represent a universal
protective antigen against invasive disease. Infect
Immun 65, 37013707.
McCue, L. A., McDonough, K. A. & Lawrence,
C. E. (2000). Functional classification of

cNMP-binding proteins and nucleotide cyclases

with implications for novel regulatory pathways

http://mic.sgmjournals.org

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