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J. Biol. Chem., Vol. 277, Issue 28, 25096-25105, July 12, 2002
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From the
Received for publication, December 21, 2001, and in revised form, April 25, 2002
Eleven bacteria capable of utilizing cyanophycin
(cyanophycin granule polypeptide (CGP)) as a carbon source for growth
were isolated. One isolate was taxonomically affiliated as
Pseudomonas anguilliseptica strain BI, and the
extracellular cyanophycinase (CphE) was studied because utilization of
cyanophycin as a carbon source and extracellular cyanophycinases were
hitherto not described. CphE was detected in supernatants of CGP
cultures and purified from a corresponding culture of strain BI
employing chromatography on the anion exchange matrix Q-Sepharose and
on an arginine-agarose affinity matrix. The mature form of the
inducible enzyme consisted of one type of subunit with
Mr = 43,000 and exhibited high specificity for
CGP, whereas proteins and synthetic polyaspartic acid were not
hydrolyzed or were only marginally hydrolyzed. Degradation products of
the enzyme reaction were identified as aspartic acid-arginine dipeptides ( Cyanophycin (cyanophycin granule polypeptide, CGP)1 is
a naturally occurring poly(amino acid),
which is synthesized in most cyanobacteria as nitrogen, carbon, and
energy storage compound in the early stationary growth phase (1, 2).
The water-insoluble CGP is accumulated intracellularly in the form of
membraneless granules (3) and is degraded by the cells when growth is
resumed. The backbone of this unique biopolymer consists of
Although much information has been obtained concerning the
non-ribosomal biosynthesis of CGP, which is catalyzed by the
cyanophycin synthetase (CphA; see Ref. 7 and cited references therein), only a few reports are available on the intracellular degradation of
CGP. Intracellular CGP degradation was first observed in crude extracts
of soluble proteins prepared from cells of Anabaena
cylindrica (5). The corresponding enzyme, cyanophycinase (CphB),
was purified from a recombinant E. coli harboring the
cphB gene from Synechocystis sp. PCC6803 and
characterized in detail (8). Dipeptides consisting of arginine plus
aspartic acid and free arginine were identified as products of CGP
degradation in addition to small amounts of aspartic acid (8). In
contrast to intracellular degradation, nothing is known about the
extracellular decomposition of this biopolymer by bacteria or other microorganisms.
In this study, we demonstrate for the first time that CGP can be easily
degraded and utilized as the sole carbon source for growth by a variety
of non-cyanobacterial eubacteria isolated from different habitats.
Because it is known that CGP is resistant to a wide range of
commercially available proteases (4, 9), these bacteria must possess an
enzyme specialized for CGP degradation. We report on the isolation of a
strain of the species Pseudomonas anguilliseptica
and describe the substrate utilization capabilities of this bacterium
and the purification of an extracellular cyanophycinase (extracellular
CGPase (CphE)) from culture supernatants of cells grown on CGP.
Furthermore, CphE was biochemically characterized to reveal the
degradation mechanism and to identify the cleavage products. In
addition, the CGPase gene (cphE) of the isolated P. anguilliseptica strain BI was cloned and characterized.
Bacterial Strains and Growth of Bacteria--
CGP-degrading
bacteria isolated in this study are listed in Table I. These strains
were either grown on Standard I complex medium (Merck) or grown
on basic inorganic medium B (10) for CGP degradation and substrate
utilization experiments. The concentrations of CGP and other carbon
sources added to the medium are indicated in the text. All isolates
were grown at 30 °C. The following microorganisms were used as
reference strains in substrate utilization assays on solid CGP medium
(see below) with 0.05% (w/v) glucose as an additional carbon source:
E. coli K12 (wild type), Pseudomonas putida
KT2440 (11), Micrococcus luteus (DSMZ 348), Bacillus subtilis 168+ (DSMZ 402), and Bacillus
megaterium (DSMZ 319). For CGP production, an E. coli
DH1 strain harboring plasmid pMa/c5-914::cphA
expressing cphA from Synechocystis PCC6803 (12)
was employed (see below). E. coli strains were usually grown
at 37 °C in Luria-Bertani (LB) medium or terrific broth (TB) complex
medium (13).
Preparation of Selective Medium for the Detection and Isolation
of CGP-degrading Microorganisms--
Samples from different sources
were spread on solid basic inorganic medium B (10) supplemented with
trace element solution SL 7 (14) and overlaid with 0.5% (w/v) agar
containing 0.2% (w/v) of CGP. For the preparation of the overlay agar,
diethyl ether-sterilized CGP was first dissolved in 0.1 N
HCl and then added to sterile medium under vigorous stirring to avoid
the formation of inhomogeneous CGP precipitates. For adjustment of the
pH value, an equal volume of 0.1 N NaOH was added before
pouring plates.
Isolation, Manipulation, and Analysis of DNA--
For isolation
of plasmid DNA, the lithium preparation method was applied (15). Total
genomic DNA of P. anguilliseptica strain BI was isolated
according to the method of Rao et al. (16). After partial
digestion with the endonuclease PstI, genomic DNA fragments
were ligated to the cosmid vector pHC79 (17), and E. coli
strain S17-1 (18) was used as a recipient for transduction of the
cosmid library. A Gigapack® III XL packaging extract
(Stratagene, La Jolla, CA) was employed for the packaging of DNA and
subsequent infection of strain S17-1 as described by the manufacturer.
E. coli strain XL1-Blue (Stratagene) was
used in combination with pBluescript SK
5'-infrared fluorescence dye 800-labeled synthetic
oligonucleotides (MWG-Biotech, Ebersberg, Germany) were used as
primers, and a Sequi Therm EXCEL TM II long-read cycle sequencing kit
(Epicentre Technologies, Madison, WI) was employed for DNA-sequencing
according to the "primer-hopping strategy" (19). Analysis was done
in 6% (w/v) acrylamide gels using Sequagel XR®
(acrylamide/urea), Complete® (buffer reagent)
solutions (National Diagnostics, Atlanta, GA), and buffer
containing 89 mM Tris, 89 mM boric acid, and 2 mM EDTA in a LI-COR 4000L automatic sequencing apparatus
(MWG-Biotech). Nucleic acid sequence data and deduced amino acid
sequences were analyzed with the sequence analysis software CAP (Contig
Assembly Program; (20)), ClustAlX 1.8 (21), and Genamics Expression 1.1.
The 16-S rRNA gene was amplified from total DNA (see above)
using oligonucleotide primers as described before (22). After purification of the PCR products with a NucleoTrap®CR kit
(Macherey-Nagel, Düren, Germany), their nucleotide sequences were
determined as described above. The 16-S rDNA sequence was aligned with
published sequences from representative Pseudomonas species
from the National Center for Biotechnology Information (NCBI) data base.
Nucleotide Sequence Accession Numbers--
The nucleotide and
amino acid sequence data reported here for cphE have been
submitted to the NCBI data base under accession number AY065671. The
16-S rRNA gene sequence data of P. anguilliseptica strain BI
were deposited in the NCBI data base under accession number
AF439803.
Cyanophycin Production--
For production of native CGP,
Synechocystis sp. strain PCC6308 was cultivated in
full-strength BG11 medium (23) in an 80-liter closed tubular
glass photobioreactor as described before (24). Also, a recombinant
E. coli DH1 harboring plasmid
pMa/c5-914::cphA (see above) with a
temperature-sensitive inducible promoter was employed for the
production of CGP. A 42-liter Biostat UD30 stainless steel
bioreactor (B. Braun Biotech International, Melsungen,
Germany) with TB complex medium was used for production as described
previously (12).
Isolation of Cyanophycin--
After cell harvest, CGP was
isolated according to the method of Simon (25), which was modified by
applying only one washing step at each Triton X-100®
concentration and two additional centrifugation steps of the acidic and
neutralized suspensions, respectively. The purity of CGP was controlled
both by SDS-PAGE with subsequent Coomassie staining (26) and by HPLC
analysis after acid hydrolysis of the polymer and subsequent
derivatization of amino groups with o-phtaldialdehyde
reagent (27).
Taxonomic Determination with Physiological Tests--
Motility
and Gram behavior were determined as described before. Oxidase
(Bactident® Oxidase test strips from Merck) and catalase tests were
performed according to standard protocols. Further determinations were
done by using the API 20NE test kit (BioMérieux, Marcy-l'Etoile, France).
Analytical Methods--
Reversed phase HPLC was used to
determine the products of enzymatic CGP degradation as described by the
method for the quantitative determination of amino acids (27).
Electrospray ionization mass spectrometry (ESI) was applied for
identification of the final degradation product of CGP by mass
determination and structural analysis (28). All measurements were
performed employing a Quattro LCZ system (Micromass, Manchester, UK)
with a nanospray inlet.
Purification of the Extracellular Cyanophycinase from P. anguilliseptica Strain BI--
A cell-free supernatant from a CGP
culture was obtained by sedimentation of the cells in the late
exponential growth phase by centrifugation and subsequent filtration of
the supernatant through a 0.2-µm nitrocellulose membrane. All steps
were carried out at 4 °C and in the presence of 50 mM
sodium phosphate buffer (pH 8.3). Further components added to the
buffer are mentioned below. After concentration in an ultrafiltration
chamber (Amicon, Beverly, MA) using a YM10 membrane, the buffered
solution was applied onto a MonoQ HR5/5 anion exchange column (Amersham
Biosciences). After washing the column with 2 bed volumes of buffer,
CGPase was eluted with a linear NaCl gradient (0-1 M)
employing an increase of NaCl concentration of 17 mM/ml and
a total flow rate of 1 ml/min. Active fractions (1 ml) were detected
after the transfer of 10 µl of the respective eluates onto CGP
overlay plates (see above) by the occurrence of halos after 5-40 min
of incubation at 30 °C. Fractions with high activity were combined,
desalted by ultrafiltration (see above), and applied onto an
arginine-agarose column (5-ml bed volume; Sigma). For selective elution
of the enzyme, an arginine gradient (0-1 M) was applied.
To avoid nonspecific protein binding and to prevent the enzyme from
binding in the presence of high arginine concentrations, the buffer in
addition contained 100 mM NaCl.
SDS-PAGE of active enzyme fractions or CGP samples was performed in
11.5% polyacrylamide gels according to standard protocols (29).
Proteins were visualized by the Coomassie staining method (26). An
"in-gel" renaturation method described for activity staining with
proteases after SDS-PAGE (30) was used to obtain reactivated CGPase
after separation of enzyme subunits according to their apparent
molecular mass under denaturating conditions. The ability of
reactivated CGPase to form degradation halos was tested by the
application of a thin CGP-agar layer (see above) on top of
buffer-pretreated gels. Protein concentrations were determined by the
procedure of Bradford (31).
Characterization of the Purified CGPase--
For determination
of the substrate specificity of the CGPase, the purified recombinant
enzyme was incubated at 30 °C in 1 ml of 50 mM sodium
phosphate buffer (pH 8.3) with various polypeptide substrates. Each
reaction contained 1 mg of the respective substrate and 1.6 µg of
enzyme. The reaction was stopped after 120 min by incubation at
70 °C for 5 min. After centrifugation, 100-µl aliquots of
supernatant were incubated at 95 °C for 5 min in the presence of
1.25% ninhydrin (Merck) in 1 ml of total reaction volume.
Subsequently, they were assayed photometrically at 570 nm for the
presence of released hydrolysis products. Bovine casein (Hammersten
grade) was from Merck, bovine serum albumin (BSA) was from Roth
(Karlsruhe, Germany), and
poly( Enrichment and Isolation of CGP-degrading Bacteria--
To screen
for CGP-degrading bacteria, samples from typical habitats of
CGP-producing cyanobacteria were plated onto solid mineral medium
containing CGP as the sole carbon source. Due to the insolubility of
CGP at neutral pH, the agar was turbid. Colonies of CGP-degrading
microorganisms were recognized because of the formation of degradation
halos, which appeared after 12-18 h of incubation at 30 °C (Fig.
1A). Based on this feature,
axenic cultures of nine bacterial strains were finally isolated
from Baltic sea water, different pond sediments, and sewage sludge
(Table I). In addition to the newly
isolated CGP-degrading strains, other bacteria from our culture
collection were also tested, and two additional strains with CGP
degradation capability were detected (strains BE2 and PAS1, Table
I). However, E. coli K12, P. putida KT2440,
M. luteus, B. subtilis 168+, and
B. megaterium were not able to cause formation of halos on
CGP overlay agar plates, although some of these bacteria
(e.g. B. subtilis) are known to use proteins as
nutrients.
Characterization of CGP-degrading Bacteria through Substrate
Utilization Patterns and Taxonomic Classification--
All isolates
tested (from Baltic sea water, pond sediment, and sewage sludge,
i.e. "A, B, and D series"; Table I) were Gram-negative, oxidase- and catalase-positive rod-shaped bacteria. With the exception of isolate DXIII, all strains showed motility. Applying the API 20NE
test kit, two isolates (DIII and DIV) revealed acceptable identification profiles. Both strains were taxonomically affiliated as
strains of the species Pseudomonas alcaligenes (Table
II). As listed in Table I, most strains
isolated in this study showed growth on the amino acid constituents of
CGP, i.e. on aspartic acid and arginine. For most strains,
growth with arginine was faster than with aspartic acid. Only isolate
BI showed no growth on aspartic acid. Strain PAS 1 from the culture
collection of our institute also did not grow on aspartic acid as the
sole carbon source. None of the isolates was able to grow on synthetic
poly( Taxonomic Classification of Isolate BI by 16-S rRNA Gene (rDNA)
Sequence Analysis--
For several reasons, isolate BI was a good
candidate for a more detailed investigation of CGP degradation.
Therefore, the taxonomic position of the isolate was determined.
Analysis of the 16-S rDNA sequence of isolate BI revealed 98% identity
to the nucleotide sequence of all three P. anguilliseptica
strains available at the NCBI data base including the P. anguilliseptica type strain NCIMB 1949. Maximum sequence identity
to other species of the genus Pseudomonas was only in the
range of 95-96% (Fig. 2). Therefore,
the new isolate was referred to as P. anguilliseptica strain
BI.
Preliminary 16-S rDNA sequence data of strain PAS 1 (about 1000 bp)
revealed that this strain most probably belongs to the genus
Streptomyces. This finding corresponds well with the
streptomycete-like habitus of this strain, e.g. the
formation of exospores in aging colonies.
Growth Kinetics of P. anguilliseptica Strain BI--
The ability
of P. anguilliseptica strain BI to grow on CGP as the sole
carbon source was investigated in more detail. Therefore, growth of
this strain on CGP and on its amino acid constituents as well as on the
non-related substrate citrate was monitored over 24 h (Fig.
3). Living cell counts for the
cyanophycin culture revealed that growth of the cells started at about
4 h of incubation (data not shown) after inoculation from a
citrate culture. The turbidity caused by suspended CGP particles
disappeared visibly during incubation. Strain BI grew best with a
combination of arginine and aspartic acid if these amino acids were
provided at a molar ratio according to their proportional masses in the
CGP molecule (248 Klett units). Growth on CGP led to a maximum optical
density of 202 Klett units, which is in the range of the OD of
the citrate culture. Slightly weaker growth was detected for the
arginine culture (182 Klett units). No increase or change of the OD
occurred in the control (sterile medium containing citrate) or in
mineral salt medium containing aspartic acid as the sole carbon source (Fig. 3). During growth on CGP, 46% (w/w) of the polymer was converted into cellular dry matter by P. anguilliseptica strain
BI.
Purification of the Extracellular CGPase from P. anguilliseptica
Strain BI--
The extracellular CGPase of P. anguilliseptica was purified to electrophoretic homogeneity from
CGP-grown cultures by the application of anion exchange chromatography
on Q-Sepharose followed by L-arginine-agarose affinity
chromatography (Fig. 1C). The latter is usually used for
different purposes, e.g. purification of transfer RNA
molecules (34). The L-arginine-agarose matrix was highly specific for the binding of CGPase under the employed conditions, revealing a high affinity of the enzyme to this matrix. Therefore, an
arginine gradient (0-1 M) in sodium phosphate buffer was
applied for the elution of the CGPase. To further reduce nonspecific
binding of other proteins, the sodium phosphate buffer additionally
contained 100 mM NaCl. In the absence of NaCl, arginine had
to be applied at concentrations higher than 3 M for total
release of the enzyme from the affinity matrix, again reflecting the
high affinity of the CGPase to the arginine residues coupled to the
matrix. During enzyme purification, active fractions were detected by
their ability to cause rapid halo formation on CGP overlay agar plates
(Fig. 1B). These halos occurred mostly within a few minutes
to 2 h, and their diameters indicated the activity of the enzyme
in the sample.
SDS-polyacrylamide gel electrophoresis revealed an apparent molecular
mass of the subunits of the enzyme of 43 kDa (Fig.
1C). By the employment of an in-gel renaturation method,
previously used for the detection of proteases in SDS-polyacrylamide
gels (30), in combination with the subsequent application of a thin CGP-agar layer on top of the "renaturated" gel, it was possible to
detect reactivated CGPase by the formation of a degradation halo at the
position corresponding to a protein of the expected molecular mass of
43 kDa (Fig. 1D). This finding clearly proved that the
43-kDa protein represented the subunit of the CGPase and that the
enzyme consisted of only one type of subunit or of subunits with
identical apparent molecular masses.
Purification of the P. anguilliseptica CGPase from a Recombinant
Strain of E. coli--
The heterologously produced enzyme was purified
in the same way from the soluble fraction of E. coli cells
harboring pBluescript SK Cloning and Analysis of the Extracellular CGPase Gene from P. anguilliseptica BI--
Applying the restriction endonuclease
PstI for partial digestion of genomic DNA from P. anguilliseptica BI, fragments with a broad size range were
obtained and subsequently ligated to the cosmid vector pHC79. After
transduction of the cosmid library into E. coli S17-1,
~3000 tetracycline-resistant clones were obtained and tested for
their ability to degrade CGP by transfer onto LB agar plates overlaid
with a thin layer of CGP-containing medium. After 3 days of
incubation at 37 °C, one colony was detected that was surrounded by
a halo, indicating degradation of the polymer in the CGP layer. The
plasmid containing the CGPase-encoding genomic fragment from P. anguilliseptica BI (26 kbp) was isolated from the respective
clone. Twelve subfragments were obtained after total digestion with
PstI and ligated to the vector pBluescript SK Biochemical Characterization of the CGPase--
P.
anguilliseptica strain BI produced the extracellular CGPase only
when CGP was present in the medium. In complex Standard I medium, no
enzyme activity was detected. As substrate utilization patterns of
strain BI indicated (Table I), no correlation occurred between the
ability to degrade CGP and the utilization of other polyamide
substrates, including BSA, poly(
Using purified recombinant CGPase, degradation of cyanobacterial CGP
(Synechocystis PCC6308) was visualized by SDS-PAGE and subsequent nonspecific protein staining (Fig.
5). Degradation of the high molecular
weight polydisperse biopolymer (about 43-100 kDa; lane 2)
to low molecular weight material (lanes 3-9) is
demonstrated in Fig. 5. The lack of detectable high molecular weight
material after 165 min of incubation (lane 10) corresponded
well with the nearly exclusive detection of the final degradation
product of CGP (see HPLC and ESI analysis below), indicating total
degradation of CGP having occurred within that time. A splitting of the
initial molecule population (lane 2) into two populations of
molecules exhibiting two different molecular weight ranges was observed during the incubation (Fig. 5, lanes 3-9).
The composition of the amino acid constituents of CGPs varies depending
on the source of CGP. Cyanobacterial CGP isolated from
Synechocystis PCC6308 contains only aspartic acid plus
arginine, whereas in CGP isolated from cells of a recombinant E. coli expressing the PCC6803 cyanophycin synthetase gene, some
arginine residues were replaced by lysine residues (6). The final
degradation products of the enzyme reaction on both CGPs were
determined. As shown in Fig. 6,
degradation of the cyanobacterial CGP led to the formation of only one
detectable degradation product after separation in HPLC, whereas
recombinant polymer produced by E. coli led to the
formation of two main products. Therefore, it seemed likely that
dipeptides of
To elucidate the mechanism of CGP degradation, C-terminal
L-[U-14C]arginyl-labeled CGP was synthesized
using the cyanophycin synthetase from Synechocystis PCC6308.
Incubation of labeled CGP with recombinant CGPase resulted in an
immediate release of radioactivity that continued for ~7 min (Fig.
8). Together with the observation that after addition of CGPase, the release of
Inhibitor studies showed that the extracellular CGPase of P. anguilliseptica BI is strongly inhibited by the serine protease inhibitors phenylmethylsulfonyl fluoride and
Pefabloc® (63 and 92%,
respectively; Table III). The inhibition
of the enzyme by Pefabloc® was additionally confirmed by
the inhibition of halo formation (Table III). CGPase activity was only
slightly (and inhibitor concentration independently) decreased by the
thiol protease inhibitor leupeptin (13%). The observed decrease in
activity in this case is most probably due to interference with the
"o-phtaldialdehyde-derivatization" method necessary for
HPLC analysis. The application of the metalloprotease inhibitor EDTA
led to a strong reduction of the release of detectable Molecular Characterization of the Extracellular CGPase Gene (CphE)
from P. anguilliseptica--
The coding gene for CphE from P. anguilliseptica strain BI was identified by its heterologous
active expression from a gene library of total DNA in E. coli S17-1 (see above). One E. coli clone harboring a
26-kbp fragment of P. anguilliseptica genomic DNA exhibited
the ability to cause the formation of degradation halos after 3 days of
incubation on CGP overlay agar plates. After subcloning of smaller DNA
fragments in pBluescript SK
Alignment of the deduced amino acid sequence of cphE
with proteins exhibiting an acceptable sequence similarity (27-32% in conserved regions) revealed a 3-amino acid motif most probably representing the catalytical triad also present in other serine type
proteases (Fig. 9). The differences between CphE, CphB enzymes, and the
most closely related protein, a hypothetical protein of Caulobacter crescentus (Fig.
11), reveal the relatively isolated position for CphE among all known enzymes involved in CGP
metabolism.
Employing a newly developed CGP mineral medium, CGP-degrading
bacteria were isolated from different habitats where cyanobacteria and
concomitantly CGP were expected to be present. Every sample applied to
the CGP medium, regardless of the origin of the sample, led to the
identification of Gram-negative bacteria capable of utilizing CGP as
the sole carbon source for growth. This finding revealed the abundance
of CGP-degrading eubacteria, especially of the genus
Pseudomonas, to which three isolates (P. anguilliseptica BI, P. alcaligenes DIII and DIV) were
taxonomically assigned in this study. In addition, strain PAS1, which
most probably belongs to the genus Streptomyces, exhibited
good growth on CGP, indicating that the capability to degrade CGP
occurs also among Gram-positive bacteria. Occurrence of so many
CGP-degrading bacteria is not surprising because cyanobacteria
represent a large and metabolically highly diverse group of bacteria.
Representatives of cyanobacteria occur in almost any aquatic and
terrestrial environment exposed to light, and in some of these
environments, cyanobacteria are the predominant microorganisms.
Furthermore, most cyanobacteria are able to synthesize CGP. Moreover,
the finding that CGP-like polymers are also synthesized by eubacteria
not belonging to the group of cyanobacteria (35) suggests that such
degradation mechanisms are even present in obligate heterotrophic
microbial communities. In conclusion, CGP is probably an abundant
biopolymer in natural environments. However, studies on the
extracellular degradation of CGP were limited in the past due to the
difficulties of producing sufficient amounts of this biopolymer by
cultivation of cyanobacteria.
Because this enzyme, after CphA and CphB, is the third bacterial enzyme
that is involved in CGP metabolism, and because it is localized
extracellularly, the cyanophycinase of P. anguilliseptica enzyme was referred to as CphE. As demonstrated in this study, CphE of
P. anguilliseptica BI was only synthesized if CGP was available as substrate for growth, indicating a specific induction of
the enzyme by CGP or its degradation products. Moreover (Table I), it
also became obvious that there was no correlation between the abilities
to degrade CGP and to hydrolyze other polyamide substrates. Most of the
other polyamide substrates were only poorly utilized by the employed
strains. For example, the naturally occurring poly( Studies on the substrate specificity of CphE of P. anguilliseptica BI showed that the enzyme is highly specific for
CGP. Therefore, consistent with the findings mentioned above, CGP seems
to be exclusively hydrolyzed by the employment of specialized CGPases. The lack of release of significant amounts of ninhydrin-positive degradation products from bovine casein, BSA, or synthetic
poly( Purification of CphE from culture supernatants to electrophoretic
homogeneity became very efficient by utilizing the high affinity of
this CGPase toward immobilized arginine residues, thus allowing
purification of CphE by the application of only two chromatography
steps and with high activity yield. The strong binding of the
extracellular CGPase to immobilized arginine residues was not only
observed during protein purification employing an arginine-agarose
column. Also, the binding of CphE to the natural substrate CGP during
cultivation occurred with such high affinity that soluble enzyme
activity was detected in culture supernatants of CGP-degrading bacteria
in liquid medium only after all CGP particles in the medium had visibly
disappeared, and the enzyme was thereby released from the substrate.
After cloning of the cphE gene from P. anguilliseptica BI and its heterologous functional expression in
E. coli, sufficient amounts of the enzyme were obtained for
further biochemical characterization. HPLC and ESI analysis identified
dipeptides as degradation products of the enzyme reaction. In the case
of cyanobacterial CGP, Molecular characterization of cphE revealed a DNA sequence
that encodes a protein with a similarity of only 27-28% to
intracellular CGPases (CphB) from cyanobacteria in conserved regions.
In contrast to the intracellular CGPase from Synechocystis
sp. PCC6803, which has an apparent molecular mass of 27 kDa (8), the
molecular mass of extracellular CphE from P. anguilliseptica BI was significantly higher (43 kDa). The
dendrogram shown in Fig. 11 demonstrates the isolated position of
CphE.
The amino acids Ser169, Glu185, and
His222 of CphE may be the catalytic active residues
responsible for the hydrolytic cleavage of the CphE was active when expressed heterologously in E. coli but
was not secreted by recombinant cells. The finding of an N-terminal leader peptide in the amino acid sequence deduced from cphE
(Fig. 9) suggested an export mechanism for CphE with specific
recognition of the signaling peptide and cleavage of the leader
sequence during export in P. anguilliseptica. Similarities
of this leader peptide to that of a potential chemotaxis transducer
identified in the Pseudomonas aeruginosa PAO1 genome (49),
which is according to its function most probably located in the
cytoplasmatic membrane, support the assumption that CphE is also
membrane-directed. However, the amino acid sequence of the N terminus
of CphE produced in E. coli did not deviate from that of
native mature (extracellular) CphE, indicating that the suggested
leader peptide sequence might be cleaved off in the cytoplasm of
E. coli cells.
CGPases are most probably commonly employed enzymes for degradation of
a widespread, and therefore, in cases of biomass degradation, often
released biopolymer. This is indicated by its high specificity and
affinity toward CGP-like material. The enzyme makes the dipeptide building blocks quickly available to cells that possess appropriate proteins for the uptake or further cleavage of We thank Dr. Tran Hai for the provision of
the strains PAS I and Synechocystis PCC6308 (SK19-1) and
Francis Hezayen for the provision of poly( *
This work was supported by Bayer (Leverkusen).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The amino acid sequence(s) of this protein can be accessed
through NCBI Protein Database under NCBI accession numbers AF439803 and
AY065671.
¶
To whom correspondence should be addressed. Tel.:
49-251-833-9821; Fax: 49-251-833-8388; E-mail:
steinbu@uni-muenster.de.
Published, JBC Papers in Press, May 1, 2002, DOI 10.1074/jbc.M112267200
The abbreviations used are:
CGP, cyanophycin granule polypeptide (cyanophycin);
CphA, cyanophycin
synthetase;
CphB, cyanophycinase;
CphE, extracellular cyanophycinase;
BSA, bovine serum albumin;
MS, mass spectrometry;
ESI, electrospray
ionization mass spectrometry;
HPLC, high performance liquid
chromatography.
Isolation of Cyanophycin-degrading Bacteria, Cloning and
Characterization of an Extracellular Cyanophycinase Gene
(cphE) from Pseudomonas anguilliseptica Strain
BI
THE cphE GENE FROM P. ANGUILLISEPTICA BI
ENCODES A CYANOPHYCIN-HYDROLYZING ENZYME*
,,¶
Institut für Mikrobiologie,
Westfälische Wilhelms-Universität Münster,
Corrensstrasse 3, D-48149 Münster, Germany and the
§ Institut für Organische Chemie, Westfälische
Wilhelms-Universität Münster, Corrensstrasse 40, D-48149 Münster, Germany
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Asp-Arg) by high performance liquid chromatography and
electrospray ionization mass spectrometry. The corresponding gene
(cphE, 1254 base pairs) was identified in subclones of a cosmid gene library of strain BI by heterologous active expression in
Escherichia coli, and its nucleotide sequence was
determined. The enzyme exhibited only 27-28% amino acid sequence
identity to intracellular cyanophycinases occurring in cyanobacteria.
Analysis of the amino acid sequence of cphE revealed a
putative catalytic triad consisting of the motif
GXSXG plus a histidine and most probably
a glutamate residue. In addition, the strong inhibition of the enzyme
by Pefabloc® and phenylmethylsulfonyl fluoride indicated
that the catalytic mechanism of CphE is related to that of serine type
proteases. Quantitative analysis on the release of -Asp-Arg
dipeptides from C-terminal labeled CGP gave evidence for an
exo-degradation mechanism.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-amino--carboxyl-linked L-aspartic acid monomers.
Most of the -carboxylic groups are covalently bound to the -amino
groups of L-arginine residues (4, 5); in recombinant
Escherichia coli expressing cyanobacterial CGP-synthesizing
enzymes (see below), a significant fraction of arginine is replaced by
lysine (6).
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
(Stratagene) for
cloning of a PstI restriction subfragment (2600 bp),
sequence analysis of cphE, and heterologous production of the enzyme.
,-D/L-aspartic acid)
(Mr = 11,000) was obtained from Bayer
(Leverkusen, Germany). Labeling experiments were performed by
enzymatic elongation of the C terminus of a CGP primer (32). L-[U-14C]arginine was incorporated into the
polymer chain using purified cyanophycin synthetase from
Synechocystis sp. strain PCC6308 heterologously produced in
E. coli (27). Labeled CGP was incubated at 30 °C with 1.6 µg of CGPase in 50 mM sodium phosphate buffer (pH 8.3) under vigorous shaking. For heat inactivation of the enzyme, 50-µl samples were transferred to test tubes containing 500 µl of water preincubated at 70 °C. After 5 min of inactivation, the samples were
transferred onto ice for 5 min to allow for CGP reprecipitation. After
subsequent centrifugation, 50 µl of supernatant were mixed with 500 µl of Hydroluma® Scintillation mixture (J. T. Baker, Inc.). Radioactivity was measured with a model LS 6500 scintillation counter (Beckman Instruments (27)).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Detection of the extracellular cyanophycinase
of P. anguilliseptica strain BI. A,
halo formation during isolation of bacteria on CGP-mineral medium.
B, routine test for enzyme activity in chromatography
fractions. C, nonspecific protein staining (Coomassie; (26))
of samples from fast protein liquid chromatography purification after
separation in SDS-polyacrylamide gels. Lane 1,
molecular mass standard proteins; lane 2, supernatant of CGP
culture broth; lane 3, protein pattern after anion exchange
chromatography; lane 4, after arginine-agarose affinity
chromatography; 10 µg of protein of each sample was applied to the
gel. D, activity staining (black arrow) after
SDS-PAGE, in-gel renaturation, and the subsequent application of a
CGP-agar layer (lane 1). Lane 2, molecular mass
standard proteins.
Growth of CGP-degrading bacteria on compounds related to cyanophycin or
other polyamide substrates
,-D/L-aspartic acid) (data not
shown). With poly(
-D-glutamic acid) as the sole carbon
source, only isolate DXIII and to some extent also P. alcaligenes strain BE2 (33) showed growth (Table I). With bovine
serum albumin as the sole carbon source, only strain PAS I and P. alcaligenes strain BE2 exhibited good or poor growth,
respectively. Citrulline and ornithine, two putative degradation
products of arginine, were not utilized as carbon sources for growth by
any of the bacteria investigated in this study. The only exceptions
were isolate AVN, which was able to grow on
ornithine, and B. subtilis 168+, which
utilized citrulline (data not shown).
Substrate utilization of CGP-degrading bacteria in the API 20NE
physiological determination assay
-glucosidase
test; -galactosidase test; anaerobic utilization of glucose
(fermentation); aerobic substrate utilization tests: glucose,
arabinose, mannose, manitol, N-acetyl-glucosamine, maltose,
gluconate, adipate, phenyl-acetate.
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Fig. 2.
Taxonomic classification of the isolated
P. anguilliseptica strain BI. The affiliation of
strain BI is based on 16-S rRNA gene sequence homologies to closely
related bacterial strains, i.e. strains of the genus
Pseudomonas. The new isolate BI groups with the three
P. anguilliseptica strains from the NCBI data base (98%
sequence identity), including the type strain NCIMB 1949. Many other
strains of the genus Pseudomonas, of which some examples are
depicted above, showed sequence identities of 95-96% to strain BI.
The bar represents the number of expected changes per
sequence position of those positions changing at the median rate.
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Fig. 3.
Growth of P. anguilliseptica
BI in liquid medium B. Cells of strain BI were grown in
30-ml cultures in 300-ml Klett flasks on 0.5% (w/v) cyanophycin
(Mr = 25,000-100,000, 
); arginine (
);
aspartic acid (
); arginine + aspartic acid (
); or citrate (
).
In the case of using arginine and aspartic acid as cosubstrates
(together, 0.5% (w/v)), these amino acids were added according to
their proportional masses in the cyanophycin molecule (i.e.
57% (w/w) Arg to 43% (w/w) Asp). The sterile control (
) contained
0.5% (w/v) of citrate. The cultures were inoculated from a 10-ml
overnight citrate preculture using 1 ml as inoculum. The incubation of
the cultures was done at 30 °C on a rotary shaker (120 rpm). Growth
was recorded in a Klett-Summerson photometer.
::cphE (for
construction, see below) grown in TB medium. The enzyme was not
excreted from the cells when it was produced by E. coli. In
culture supernatants, enzyme activity was not detectable. In addition,
halo formation on CGP overlay LB agar plates occurred only after 3 days
of incubation, thus indicating that the release of the enzyme
presumably occurred due to partial cell lysis during aging of the
cells. By this purification method, the activity of the heterologously
expressed enzyme was enriched 15-fold, and 49% of the total activity
was recovered as confirmed by the calculation of halo forming units in
a plate diffusion assay using purified enzyme as a standard (data not shown).
.
Subsequent transformation of E. coli XL1-Blue with the
resulting mixture of pBluescript SK construction products
led to the identification of a bacterial colony capable of forming
halos on a CGP overlay plate.
-D-glutamic acid), or
synthetic poly(,-D/L-aspartic acid) (data
not shown). This indicated that the CGPase is not employed by the
bacterium for nonspecific hydrolysis of polyamide substrates. This was
also confirmed by studies on the substrate specificity of the purified enzyme. Using the enzyme purified from the recombinant strain of
E. coli (see above), the release of degradation products
from the polyamide substrates CGP, BSA, bovine casein, and
poly(,-D/L-aspartic acid) was
investigated by employing ninhydrin reagent for detection of
released amino groups (Fig. 4). After
2 h of incubation in the presence of purified enzyme, only CGP
samples showed a significant release of ninhydrin-positive degradation
product, whereas BSA and bovine casein samples revealed only a very
weak release of reactive products amounting to 3.9 or 6.4% of that
obtained with CGP, respectively (Fig. 4). From synthetic
poly(,-D/L-aspartic acid), no release of
ninhydrin-positive material was detected (Fig. 4).
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Fig. 4.
Substrate specificity of the CGPase
(CphE). Recombinant CphE (1.6 µg of purified enzyme) was
incubated for 120 min with 1 mg of the indicated polypeptides. The
formation of hydrolysis products was detected by employing the
ninhydrin method and measuring the increase of ninhydrin activity at
570 nm. poly(Asp),
poly( ,-D/L-aspartic acid).
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Fig. 5.
Decrease of the molecular mass of CGP during
incubation of the polymer in the presence of purified recombinant
CphE. CGPase was inactivated before electrophoresis (SDS-PAGE) by
incubation at 70 °C for 5 min. Lanes 1 and 11, molecular
mass standard proteins; lane 2, 25 µg of cyanobacterial
CGP in 50 mM sodium phosphate buffer (pH 8.3); lanes
3-10, 5, 11, 17, 25, 37, 51, 69, and 165 min after the addition
of 16 µg of purified recombinant CphE. Cyanophycin was visualized by
Coomassie staining (26).
-Asp-Arg or of -Asp-Arg plus -Asp-Lys,
respectively, were formed during the degradation process. A degradation
mechanism producing oligomers would have led to the formation of more
than two different products in the case of the recombinant CGP,
resulting most probably in the appearance of more than two peaks in the
HPLC chromatogram. Final proof for the presence of the dipeptide was
obtained by the application of ESI on the isolated degradation
product of cyanobacterial CGP (see above). The degradation products
gave a strong signal in the positive ion ESI/MS spectrum at
m/z 290 (Fig.
7A, [M + H]+), corresponding to a molecular mass of 289 Da, as it is calculated for the -Asp-Arg dipeptide. Furthermore,
analysis of the m/z 290 molecule by ESI/MS/MS
revealed a fragmentation pattern characteristic for the structure of
the -Asp-Arg dipeptide (Fig. 7B). The peaks at
m/z 175 and m/z 116 represent the molecules occurring after fragmentation of the dipeptide
at the -amide bond. This is indicated by the 
115 arrow
pointing at m/z 175 ([M + H HO2C-CHNH2-CH=C=O]+) and the 174
arrow pointing at m/z 116 ([HO2C-CHNH2-CH=C=O + H]+),
respectively (Fig. 7B).
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Fig. 6.
Detection of degradation products of the
CGPase reaction by HPLC analysis. Analysis of products of the
degradation of cyanobacterial CGP isolated from
Synechocystis PCC6308 (A) and of recombinant CGP
isolated from E. coli DH1
(pMa/c5-914::cphA) containing lysine residues
randomly incorporated as arginine substitutes (B).
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Fig. 7.
Positive ion ESI/MS analysis of the CGP
degradation product (A) and ESI/MS/MS analysis
(B) of the m/z 290 peak
from panel A. The ESI/MS peak
m/z 290 corresponds to the mass of the protonated
-Asp-Arg dipeptide [M + H]+ (A). The
fragmentation pattern of the [M + H]+ ion from
panel A is in accordance with the structure of the
-Asp-Arg dipeptide (B). The loss of 115 and 174 atomic
mass units indicates fragmentation at the -amide bond.
m/z 273 and m/z 158 correspond to a loss of an ammonia moiety from the [M + H]+ and the [M + H 115]+,
respectively.
-Asp-Arg dipeptides was
detectable by HPLC analysis within 30 s (data not shown), an
exo-degradation mechanism proceeding (at least partially) from the C
terminus of CGP seems to be most likely for the enzyme reaction.
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Fig. 8.
Degradation of
L-[U-14C]-arginyl-labeled CGP.
The release of labeled hydrolysis products is shown as an increase in
scintillation counts.
-Asp-Arg
dipeptides in the enzyme reaction (Table III). This was most probably
due to the formation of precipitates occurring during derivatization.
The ability of the enzyme to form degradation halos in CGP overlay agar
in the presence of 30-500 mM EDTA was, however, not
affected. Only the tryptophan oxidant N-bromosuccinimide totally prevented the release of -Asp-Arg and the formation of degradation halos on CGP overlay plates. The latter occurred after 30 min of incubation together with CGPase, even if
N-bromosuccinimide was applied at concentrations 
1
mM. Therefore, a tryptophan residue may be involved in the
process of CGP degradation (compare Fig. 9).
Inhibition of the CGPase by group-specific protease inhibitors
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Fig. 9.
Alignment of deduced amino acid sequence of
CGPase CphE from P. anguilliseptica strain BI with
protein sequences of highest similarity (NCBI data base). A
potential signaling peptide preceding the N terminus of CphE is
underlined. The N terminus (shaded in light
gray) was determined by peptide sequencing of purified native
CGPase. The proposed residues of the catalytic triad are
shaded in gray and are indicated by the symbols
,
, and 
for serine, aspartic acid (or glutamic acid), and
histidine. A potential alternative catalytic aspartic acid residue of
CphE is shaded in light gray. The conserved
Gly-Xaa-Ser-Xaa-Gly motif of serine type proteases is boxed.
Three tryptophan residues whose oxidation could have lead to enzyme
inactivation (compare Table III) are underlined and in
bold letters. For sequence alignment, the ClustAlX 1.8 program was employed (21). PCC6308, CphB from
Synechocystis sp. PCC6308; PCC6803, CphB from
Synechocystis sp. PCC6803; A. variabilis, CphB
from Anabaena variabilis; C. crescentus,
hypothetical protein from C. crescentus; PepE,
-aspartyl dipeptidase from S. typhimurium LT2.
, one transformant of E. coli XL1-Blue was identified that was also able to form
degradation halos on CGP plates. This clone harbored a 2.6-kbp fragment
of P. anguilliseptica DNA. By DNA sequence analysis of the
cloned fragment, the N terminus of CphE, which was determined by
N-terminal amino acid sequence analysis (compare Fig. 9), was
rediscovered in antilinear orientation toward the lacZ
promoter of the vector; the gene can therefore be assumed to be under
the control of its own promoter. Upstream of the N terminus of the
mature enzyme, which was determined by N-terminal sequencing, a
probable leader peptide of 21 amino acids was found in the deduced
amino acid sequence of the gene (Figs. 9 and
10). 9-14 base pairs upstream of the
methionine codon (ATG) of the leader peptide sequence, a purine-rich
sequence GGAGAA was detected, indicating a potential ribosome binding
site (Shine-Dalgarno-sequence) in the complementary mRNA transcript
of the gene. An open reading frame of 1,254 bp with TAA as stop codon
was identified corresponding to a theoretical protein mass of 42.4 kDa
for the mature CphE protein if the mass of the leader peptide (2.4 kDa)
is not considered. This corresponds well with the apparent molecular
mass of the enzyme (subunit) that was detected by SDS-PAGE (43 kDa,
compare Fig. 1). The pI of the mature enzyme (i.e. the
extracellular form of CphE) was calculated to be 5.92.
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Fig. 10.
Similarity of the N-terminal leader peptide
of CphE (deduced amino acid sequence) from P. anguilliseptica strain BI to N-terminal amino acid sequences
of other proteins. Amino acids present in two or in all sequences
depicted are shaded in gray. At the amino acid
residue positions 1 and 3, short chained amino acids are present
(potential recognition site for peptide cleavage). A hydrophilic
arginine residue and serine residues preceding a hydrophobic
leucine-rich domain were found in CphE and in the potential chemotaxis
transducer of P. aeruginosa PAO1 (49). In the sequence of
the Kex1 protein (precursor of a subtilisin type serine protease from
Kluyveromyces lactis (50, 51)), two serine residues followed
by a leucine and isoleucine-rich hydrophobic domain were detected as
well (compare boxed hydrophobic sequences).
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Fig. 11.
Amino acid sequence similarity of CphE to
intracellular CGPases (CphB) and other proteins. The
cyanobacterial CGPases form a phylogenetically related group.
The strongest similarity of CphE was found to a hypothetical protein of
C. crescentus (32% identity over 325 amino acids, NCBI data
base search). The isolated position of CphE among CGPases is revealed
by the distance between CphE and any other related protein, including
the -aspartyl dipeptidase (PepE) from S. typhimurium,
previously described as a related protein of intracellular CGPases
(8).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-glutamic acid), which is an extracellular
polymer of various Gram-positive bacteria (36-39), was only accepted
by isolate DXIII and by P. alcaligenes strain BE2, which was
previously isolated on poly(-D-glutamic acid)-containing
medium (33). On the other side, by cultivation of non CGP-degrading
reference strains including typical protease producing bacteria
(i.e. B. subtilis and B. megaterium) on CGP overlay agar plates, the resistance of the polymer against typical bacterial proteases, of which B. subtilis produces a
great variety (40-45), became clearly evident. This is consistent with the finding of the total resistance of CGP to a variety of commercially available proteases (4, 9).
,-D/L-aspartic acid), which is used
as a biodegradable substitute for non-degradable polyacrylates in
detergents (46), during incubation in the presence of purified enzyme
(Fig. 4) clearly demonstrated the high specificity of CphE for CGP. A
similar high specificity toward CGP was also described for the
intracellular enzyme (CphB) of Synechocystis sp. PCC6803
(8).
-Asp-Arg dipeptides occurred (Figs. 6 and 7)
as described for the intracellular cleavage of CGP in the
cyanobacterium Synechocystis sp. PCC6803 (8). Degradation experiments employing enzymatically C-terminal
L-[U-14C]arginyl-labeled CGP revealed a
continuous and immediate release of radioactive degradation product
after addition of CGPase (Fig. 8). Because -Asp-Arg dipeptides were
detectable immediately after addition of the enzyme, an exo-degradation
mechanism proceeding from the C terminus of the CGP molecule and
release of the dipeptides by successive cleavage of the -amide bonds
of the polymer backbone seems to be most likely for CphE.
-amide bonds of the
polymer backbone (Fig. 9). Accordingly, the catalytic mechanism is
suggested to be that of a serine type protease. This finding is in good
agreement with the detected sensitivity of CphE toward serine protease
inhibitors (compare Table III). In contrast to most serine type
proteases, the characteristic aspartic acid residue of the catalytic
triad is replaced by glutamic acid. The same amino acid replacement was
observed for the intracellular CGPase of Synechocystis sp.
PCC6803 or other cyanobacteria (see Ref. 8 and compare Fig. 5). In the
predicted sequence of a hypothetical protein from C. crescentus, which showed the highest similarity to CphE in a NCBI
data base search (32% identity over 325 amino acids) and exhibited a
similar molecular mass, an aspartic acid residue typical for the
catalytic triad of most serine proteases was present (Fig. 5).
Therefore, Asp188 of CphE, which is according to the
alignment close to the position of the proposed catalytic glutamic acid
residues of CphB proteins (8), must be considered as another potential
residue that is involved in catalysis, instead of Glu185.
The proposed catalytic aspartic acid residue of the PepE protein, which
represents an aspartyl-dipeptidase from Salmonella
typhimurium (47, 48), has been suggested to be in a corresponding
position as compared with the conserved glutamic acid residues of CphB proteins (8); therefore, it seems on the other hand more likely that
Glu185 is catalytically active.
-linked amino acid
dimers. It should be emphasized that CphE is an extracellular enzyme.
It is therefore not involved in the mobilization of intracellular storage polymer CGP; for this, CGP-accumulating bacteria possess intracellular CGPases referred to as CphB (8).
ACKNOWLEDGEMENTS
-D-glutamic
acid). We gratefully acknowledge Martin Krehenbrink for the performance
of the CGP labeling experiments.
FOOTNOTES
ABBREVIATIONS
REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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