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System. Appl. Microbiol. 25, 183–188 (2002)
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Accumulation of Poly(3-hydroxybutyrate) from Octanoate
in Different Pseudomonas Belonging to the rRNA Homology
Group I
STÉPHANE DIARD1, JEAN-PHILIPPE CARLIER2, ELISABETH AGERON1, PATRICK A.D. GRIMONT1, VALÉRIE LANGLOIS3,
PHILIPPE GUÉRIN3, and ODILE M.M. BOUVET1
1
Unité de Biodiversité des Bactéries Pathogènes Emergentes, Institut National de la Santé et de la Recherche Médicale U389, Institut
Pasteur, Paris, France
2
Centre National de Référence des Bactéries Anaérobies, Institut Pasteur, Paris, France
3
Laboratoire de Recherche sur les Polymères, UMR CNRS C7581, Université Paris XII, Thiais, France
Received: April 9, 2002
Summary
It is admitted that one of the characteristics of pseudomonads is their inability to accumulate poly(3-hydroxybutyrate). In this paper, we show that poly(3-hydroxyoctanoate) synthesis is restricted to Pseudomonas rRNA homology group I, which includes both fluorescent and nonfluorescent species. However, within the genus Pseudomonas, the P. aeruginosa complex can be subdivided into two groups : the
“P. aeruginosa group”, which includes P. aeruginosa, P. alcaligenes, P. citronellolis, P. mendocina, produce poly(3-hydroxyoctanoate) from octanoate and the “P. oleovorans group” which includes the type
strain of P. oleovorans, P. pseudoalcaligenes and two Pseudomonas sp., produce poly(3-hydroxybutyrate) during cultivation on octanoate.
Strain GPo1 (ATCC 29347) formely identified as P. oleovorans and known to produce various mediumside-chain PHAs such as poly(3-hydroxyoctanoate) has been reclassified in the P. putida complex.
Key words: Pseudomonas – octanoate – poly(3-hydroxyalcanoates) – polyester
Introduction
Many bacteria are able to accumulate poly(3-hydroxyalcanoates) (PHA) as a carbon and energy reserve. PHAs
are very promising bacterial polyesters with chemical and
physical properties that make them suitable for industrial
and medical applications (ANDERSON and DAWES, 1990;
BRAUNEGG et al., 1998; MADISON and HUISMAN, 1999;
WILLIAMS et al., 1999). Poly(3-hydroxybutyrate) (P-3HB)
is the most common type of short-side-chain PHA and
the ability of bacteria to accumulate P-3HB is often used
as a taxonomic characteristic. Such accumulation occurs
in a wide variety of Gram-positive and Gram-negative
bacteria which accumulate P-3HB from media containing
acetate, butyrate, glucose, gluconate or n-alkanoates.
Pseudomonas putida GPo1 (CIP 105816 = ATCC
29347) commonly know as P. oleovorans GPo1 (VANBEILEN et al., 2001) and a number of Pseudomonas
species accumulate various medium-side-chain PHAs
(msc-PHA) such as poly(3-hydroxyoctanoate) (P-3HO)
when n-alcohols, n-alkanoates or gluconate are provided
as carbon sources. The biosynthetic pathways involve either β-oxidation or fatty acid biosynthesis intermediates
for msc-PHA production (MADISON and HUISMAN, 1999).
Furthermore, P. putida GPo1 is also able to oxidize medium-chain-length n-alkanes to their corresponding fatty
acids because it contains the catabolic OCT plamid,
which encodes an alkane hydroxylase complex (DE SMET
et al. 1983). Therefore, although the P-3HO is structurally related to P-3HB, different synthesis routes are used.
Abbreviations:
PHA – poly(3-hydroxyalcanoate)
P-3HB – poly(3-hydroxybutyrate)
P-3HO – poly(3-hydroxyoctanoate)
3-HB – 3-hydroxybutyrate
3-HHx – 3-hydroxyhexanoate
3-HO – 3-hydroxyoctanoate
3-HD – 3- hydroxydecanoate
0723-2020/02/25/02-183 $ 15.00/0
184
S. DIARD et al.
The genus Pseudomonas comprises a taxon of
metabolically versatile organisms capable of utilizing a
wide range of simple and complex organic compounds
(STANIER et al., 1966). The genus Pseudomonas sensu
stricto should be restricted to Pseudomonas rRNA group
I of PALLERONI (PALLERONI et al., 1972; 1973; DE VOS
and DE LEY, 1983) which includes both fluorescent and
nonfluorescent species. Recently, YAMAMOTO et al.
(2000) on the basis of combined gyrB and rpoD nucleotide sequences showed that pseudomonads diverged
into two major clusters. Most species of the cluster I
occur in the clinical environment whereas many of the
constituents of the cluster II are saprophytic or
pathogenic for plants and fungi.
It is admitted that one of the characteristics of pseudomonads is their inability to accumulate P-3HB. In fact,
we show that different Pseudomonas species (P. oleovorans type strain CIP 59.11T, P. pseudoalcaligenes and two
Pseudomonas sp.) belonging to the P. aeruginosa complex synthesize P-3HB when grown on octanoate. The
taxonomic position of P. putida GPo1 has been determined by 16S rRNA sequencing and DNA relatedness.
Methods
Bacterial strains and growth conditions
A total of 49 strains [24 type strains and 25 strains used in ribotyping and Biotype-100 studies (BROSCH et al., 1996; GRIMONT et al., 1996)] belonging to the genus Pseudomonas were
studied. All bacteria were cultivated at 30 °C in 500 ml Erlenmeyer flasks containing 50 ml of mineral salt medium and agitated to 200 rpm. Composition of the mineral salt medium was
as follow: CaCl2, 2H2O: 15 mg; MgSO4, 7H2O: 123 mg;
KH2PO4: 680 mg; K2HPO4, 3H2O: 2610 mg; NaCl: 7000 mg;
(NH4)2SO4: 525 mg for 1 liter of distilled water. The medium
was complemented with 10 ml of microelements (1 liter of microelements stock solution contains: H3PO4: 1.15 ml; FeSO4,
7H2O: 56 mg; ZnSO4, 7H2O: 29 mg; MnSO4, 4H2O: 22 mg;
CuSO4, 5H2O: 2.5 mg; Co(NO3)2, 6H2O: 3 mg; H3BO3: 6 mg)
(VERON, 1975). Octanoate was provided as concentration of 20
mM and octane was supplied to a final concentration of 20 %
(v/v) when needed.
Determination of PHA
Cells were harvested, lyophilized and methanolysed using
method described by BRAUNEGG et al. (1978), BRANDL et al.
(1988) and modified by JAN et al. (1995). Cells were treated
with 10 % sulfuric acid in methanol-chloroform at 60 °C for
16 hours to convert fatty acids to their corresponding methyl esters. The methyl esters formed were then analyzed by gas chromatography (Shimadzu GC-14B equiped with a Carbowax 20M
25 m × 0.25 mm, df = 0.25 µm). The cell density and the peak
area ratios of the monomers and the internal standard (o-toluic
acid) allow determination of the amount of polymer accumulated by the cells and its composition.
Genetics methods
The procedure used for the DNA hybridization experiments
(the S1 nuclease-trichloracetic acid method) and the thermal stability of duplexes have been described previously (CROSA et al.,
1973; GRIMONT et al., 1980). The difference between the melting point of a heteroduplex and the melting point of a homoduplex is the ∆Tm value (BRENNER, 1978; GRIMONT, 1988).
Phenotypic characterization
Carbon source utilization tests of P. putida GPo1, P. putida
biotype A and P. monteilii were performed using Biotype-100
strips (BioMérieux, La Balme Les Grottes, France). Galleries
were incubated at 30 °C for 4 days with record at 2 and 4 day.
Results and Discussion
The classification of the genus Pseudomonas has considerably benefited from the nutritional studies of
STANIER et al. (1966), nucleic acid homology experiments
(PALLERONI et al., 1972; 1973; DE VOS and DE LEY, 1983)
and 16S rRNA sequence comparison (MOORE et al.,
1996; WOESE, 1987).
The genus Pseudomonas is now restricted to rRNA
group I (KERSTERS et al., 1996). The majority of the
rRNA group II pseudomonads have been transferred to
the genera Burkholderia with the type species Burkholderia cepacia and Ralstonia with the type species Ralstonia
picketti. Pseudomonas lemoignei, one of first known P3HB producing prokaryotes (LEMOIGNE, 1926) belongs to
the Burkholderia-Ralstonia rRNA sublineages.
Pseudomonads of rRNA group III are now classified in
the family Comamonadaceae with the genera Comamonas, Delftia, Acidovorax, and Hydrogenophaga, all
containing previously named Pseudomonas species.
rRNA group IV contains the genus Brevundimonas and
the rRNA group V pseudomonads (P. maltophilia) have
been assigned to the genus Stenotrophomonas.
Independently, the composition of polyesters of different pseudomonads grown on octanoate have been analyzed (HUISMAN et al., 1989; TIMM and STEINBUECHEL,
1990). When we confronted their results to the new classification, it appears that species described as producing
P-3HB are not related to the genus Pseudomonas sensu
stricto but belong to new genera. Effectively, P. cepacia,
P. gladioli, P. glathei and P. caryophylli are now classified
in the genus Burkholderia, P. solanacearum in the genus
Ralstonia, P. testosteroni in the genus Comamonas, P. delafieldii in the genus Acidovorax, P. palleronii, P. pseudoflava, P. taeniospiralis in the genus Hydrogenophaga
and P. mixta in the genus Telluria, which constitutes a
separate rRNA sublineage in the β-subclass of the Proteobacteria (KERSTERS et al., 1996).
Occurrence of PHA in the genus Pseudomonas
In this study, we investigated the PHA composition of
different pseudomonads belonging to the rRNA homology group I. A total of 48 validly described Pseudomonas
species were cultivated in mineral medium supplemented
with octanoate and analyzed for PHA (Table 1).
We observed that in the intragenic cluster I defined by
YAMAMOTO et al. (2000), the Pseudomonas aeruginosa
complex is heterogenous. P. aeruginosa, P. citronellolis
and P. mendocina produced P-3HO from octanoate. The
PHAs were always composed of approximately 8 to 20
mol% 3-hydroxyhexanoate (3-HHx), 77 to 86 mol% 3hydroxyoctanoate (3-HO) and 2 to 9 mol% 3-hydroxy-
Accumulation of P-3HB in different Pseudomonas
185
Table 1. Accumulation and composition of PHA in different Pseudomonas grown on octanoate belonging to the rRNA homology
group I (classification according to YAMAMOTO et al., 2000).
Strain
Content
(% of cell
dry wt)
3HB
3HHx
3HO
3HD
14.4
3.3
16.2
6.3
3.7
34.1
55.2
49.3
47.4
40.0
32.6
Tr
Tr
0
0
0
0
0
99.4
99.7
100
99.6
99.5
99.4
–
–
8.0
13.9
20.3
12.4
11.8
0
0
0
0
0
0
–
–
86.0
77.4
77.2
79.8
79.2
0
0.1
0
0
0.2
0
–
–
6.0
8.8
2.5
7.8
9.0
0.6
0.2
0
0.4
0.3
0.6
–
–
0
0
0
0
0
0
0
0
0
0
0
–
–
0
0
0
0
0
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Intragenic cluster II
Pseudomonas putida complex
P. putida bv A CIP 52.191T
P. putida bv A LMG 5834
Pseudomonas sp. GPo1 CIP 105816
P. monteilii CIP 104883T
P. plecoglossicida CIP 106493T
14.1
12.3
9.6
4.6
28.0
0
0
0
0
0
10.3
6.0
15.8
9.6
16.3
87.7
91.6
81.6
85.4
82.7
2.0
2.3
2.5
5.0
1.0
0
0
0
0
0
Pseudomonas syringae complex
P. cichorii CIP 106704T
P. ficuserectae LMG 5694T
P. syringae CIP 106698T
P. viridiflava CIP 106699T
P. viridiflava LMG 12647
P. caricapapayae CIP 106736T
P. savastanoi pv savastanoi CIP 103721T
8.5
1.7
0
0
NG
NG
NG
0
0
–
–
NG
NG
NG
21.5
53.2
–
–
NG
NG
NG
75
46.8
–
–
NG
NG
NG
3.5
0
–
–
NG
NG
NG
0
0
–
–
NG
NG
NG
Pseudomonas fluorescens complex
Pseudomonas fluorescens lineage
P. fluorescens bt A LMG 1794T
P. fluorescens bt A LMG 14565
P. fluorescens bt C LMG 14576
P. synxantha CIP 99.22T
P. marginalis pv alfalfae LMG 2214T
P. marginalis pv marginalis LMG 2210
P. marginalis pv pastinacae LMG 2238
P. mucidolens CIP 103298T
P. tolaasii CIP 106735T
0
4.5
0
Tr
1.8
1.4
5.6
NG
NG
0
0
–
–
0
0
0
NG
NG
0
26.1
–
–
28.9
30.5
30.7
NG
NG
0
69.1
–
–
59.2
51.0
64.9
NG
NG
0
4.8
–
–
11.9
18.6
4.4
NG
NG
0
0
–
–
0
0
0
NG
NG
Pseudomonas chlororaphis lineage
P. agarici CIP 106703T
P. chlororaphis CIP 63.22T
P. aureofasciens CIP 103295T
P. aspleni LMG 2137
P. corrugata CIP 105514T
P. fluorescens bt G LMG 5940
P. fluorescens bt G LMG 14675
P. fluorescens bt G LMG 14676
P. fluorescens bt F LMG 5939
P. putida bv B LMG 14683
P. putida bv B LMG 1246
0
17.8
16.9
3.8
8.2
4.7
1.7
7.3
13.8
26.4
26.4
–
0
0
0
0
0
0
0
0
0
0
–
20.2
20.1
20.9
20.9
31.1
32.2
28.4
21.5
27.8
26.7
–
77.6
77.9
72.3
74.4
63.9
51.7
68.7
76.3
71.0
72.1
–
2.2
2.0
6.8
4.7
5.0
16.1
2.9
2.1
1.2
1.1
–
0
0
0
0
0
0
0
0
0
0
Intragenic cluster I
Pseudomonas aeruginosa complex
P. aeruginosa CIP 100720T
P. aeruginosa LMG 5031
P. citronellolis CIP 104381T
P. mendocina CIP 75.21T
P. mendocina LMG 6396
P. oleovorans CIP 59.11T
Pseudomonas sp. LMG 13970
Pseudomonas sp. LMG13971
P. pseudoalcaligenes CIP 66.14T
P. pseudoalcaligenes CIP 61.21
P. pseudoalcaligenes LMG 6036
P. alcaligenes CIP 101034T
P. alcaligenes CIP 104974
Pseudomonas stutzeri complex
P. stutzeri CIP 103022T
P. stutzeri CIP 63.21
P. stutzeri LMG 2332
P. stutzeri LMG 6397
P. balearica CIP 105297T
Composition (mol %)
3HDD
Abbreviation: 3HB – 3-hydroxybutyrate; 3HHx – 3-hydroxhexanoate; 3HO – 3-hydoxyoctanoate; 3HD – 3-hydroxydecanoate;
3HDD – 3 hydroxydodecanoate; CIP – Collection de l'Institut Pasteur; T – Type strain; LMG – Laboratorium voor Microbiologie
universiteit Gent; Tr – trace amounts; bv – biovar; NG – no growth; pv – pathovar; bt – biotype.
186
S. DIARD et al.
decanoate (3-HD). In contrast, P. oleovorans type strain,
P. pseudoalcaligenes strains and two Pseudomonas sp.
formed polyester of P-3HB when grown on octanoate.
The polymer amounted to 33 to 55% of the cellular dry
matter. Only traces of polymer (less than 1 mol% of cellular dry weight) were detected in P. alcaligenes strains
grown on octanoate.
Strains belonging to the P. stutzeri complex were unable to accumulate PHA at all. These results are consistent with published studies (TIMM and STEINBUECHEL,
1990).
The second major cluster is more homogeneous than
the cluster I. The majority of species belonging to ‘P. putida complex’ and P. chlororaphis lineage in the ‘P. fluorescens complex’ synthesized P-3HO consisting of approximately 6 to 32 mol% 3-HHx, 52 to 92 mol% 3-HO
and 1 to 16 mol% 3-HD. The polymer amounted to 2 to
28% of the cellular dry matter. P-3HB was never found.
It was interesting to observe that in the P. fluorescens lineage, the three pathovars of P. marginalis and P. fluorescens biotype A LMG 14565 were the sole strains to
produce P-3HO. Strains belonging to P. syringae complex
seemed unable to synthesize PHA except P. cichorii and P.
ficuserectae.
Taxonomic position of P. putida (oleovorans) GPo1
Recently, VAN BEILEN et al. (2001) have shown that the
16S rRNA gene sequence of P. putida GPo1 was closely
related to P. putida biotype A. Some articles report that
16S rRNA sequence analysis are not useful in defining
species (STACKEBRANDT and GOEBEL, 1994). In order to
determine its taxonomic position, DNA-DNA hybridization experiments were performed with Pseudomonas
species selected by 16S rRNA gene sequence analysis. The
results of analysis of the 16S rRNA gene sequence
showed that species which had the highest similarity
(99%) with P. putida GPo1 were P. putida biovar A CIP
52.191T, P. monteilii CIP 101883T (ELOMARI et al., 1997)
and P. plecoglossicida CIP 106493T (NISHIMORI et al.,
2000). DNA-DNA hybridization were performed with
strains of selected species. The levels of hybridization between the strain GPo1 and P. putida biovar A CIP
52.191T and P. monteilii CIP 101883T were 58 to 59%.
The ∆Tm values were 6.2 °C and 4.9 °C respectively, indicating that these organisms are closely related although
not in the same species. The level of reassociation with
P. plecoglossicida CIP 106493T was low (25%). The levels of reassociation between P. putida GPo1 and other
species were low (< 25%). The phylogenetic definition of
a species generally includes strains with approximately
70% or greater DNA-DNA relatedness and a ∆Tm value
of 5 °C or less (both values must be considered) (WAYNE
et al., 1987; GRIMONT, 1988). The DNA relatedness data
suggest that GPo1 represents a new species although percent relatedness and ∆Tm values are close to the accepted
thresholds for the species. Because of recent recommendations not to name a new species based on a single strain
(CHRISTENSEN et al., 2001) refrained us from naming a
new species.
Table 2. Differenciation of Pseudomonas sp. GPo1 from P.
putida biovar A and P. monteilii.
substrate
D-Mannose
D-Ribose
L-Arabinose
L-Tartrate
meso-Tartrate
Tricarballylate
D-Gluconate
D-Galacturonate
Itaconate
Pseudomonas sp.
GPo1
P. putida bv A
P. monteilii
CIP 52.191T
CIP 104883T
+
+
–
+
+
+
–
–
–
–
–
–
+
+
–
+
+
+
–
+
+
–
–
+
+
+
–
+, growth in 1–2 day; –, no growth
Phenotypically, Pseudomonas sp. GPo1 can be differentiated from P. putida biotype A and P. monteilii by assimilation of D-mannose, D-ribose, L-arabinose, L-tartrate, meso-tartrate, tricarballylate, D-gluconate, Dgalacturonate and itaconate (Table 2). Pseudomonas sp.
GPo1 use the following compounds as sole carbon
sources: D-glucose, D-fructose, glycerol, D-saccharate,
mucate, D-malate, L-malate, cis-aconitate, trans-aconitate, citrate, 2-ketogluconate, D-gluconate, phenylacetate, protocatechuate, 4-hydroxybenzoate, quinate, benzoate, trigonelline, betaine, putrescine, 4-aminobutyrate,
histamine, DL-lactate, caprate, caprylate, L-histidine,
succinate, fumarate, glutarate, DL-glycerate, 5-aminovalerate, ethanolamine, 3-hydroxybutyrate, L-aspartate,
L-glutamate, L-proline, D-alanine, L-alanine, L-serine,
malonate, propionate, L-tyrosine, 2-ketoglutarate.
The following compounds are not use as sole carbon
sources: D-galactose, D-trehalose, L-sorbose, D-melibiose, sucrose, D-raffinose, maltotriose, maltose, lactose,
lactulose, 1-O-methyl-β-galactoside, 1-O-methyl-α-galactoside, D-cellobiose, gentiobiose, 1-O-methyl-D-glucoside,
esculin, D-xylose, palatinose, L-rhamnose, L-fucose, Dmelezitose, D-arabitol, L-arabitol, xylitol, dulcitol, Dtagatose, myo-inositol, D-mannitol, maltitol, D-turanose,
D-sorbitol, adonitol, hydroxyquinoline-β-glucuronide, Dlyxose, i-erythritol, 1-O-methyl-D-glucoside, 3-O-methylD-glucose, D-tartrate, 5-ketogluconate, tryptophan, Nacetyl-D-glucosamine, gentisate, 3-hydroxybenzoate, 3phenylpropionate, m-coumarate, tryptamine, D-glucosamine.
The oxidation of n-alkanes to carboxylic acids has
been extensively studied (CHAKRABARTY et al., 1973; VAN
BEILEN et al., 2001). The plasmid OCT enables Pseudomonas sp. GPo1 to use C6-C12 alkanes as a sole
source of energy and carbon (BAPTIST et al., 1963). The
ability of Pseudomonas species to grow on octane were
studied. It appears that, except Pseudomonas sp. GPo1
and P. citronellolis CIP 104 381, none could use octane as
carbon source.
A large variety of bacteria, plants and animal tissues
produce P-3HB whereas P-3HO synthesis is restricted to
Accumulation of P-3HB in different Pseudomonas
Pseudomonas rRNA group I. However, within the genus
Pseudomonas, the P. aeruginosa complex can be subdivided into two groups: the ‘P. aeruginosa group’, which
included P. aeruginosa, P. alcaligenes, P. citronellolis,
P. mendocina, produce P-3HO from octanoate and the
‘P. oleovorans group’ which included the type strain of
P. oleovorans, P. pseudoalcaligenes and two Pseudomonas sp., produce P-3HB during cultivation on octanoate.
Acknowledgements
S. DIARD is the recipient of a doctoral fellowship from the
Ministère de l’Enseignement Supérieur et de la Recherche.
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Corresponding author:
ODILE M. M. BOUVET, Unité des Pathogènes et Fonctions des
Cellules Epithéliales Polarisées, Institut National de la Santé et
de la Recherche Médicale U 510, Faculté de Pharmacie, Université Paris XI, F-92296 Chatenay-Malabry, France
Tel.: ++33-1-46 83 58 43; Fax: ++33-1-46 83 58 44;
e-mail: [email protected]