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Critical Reviews in Microbiology, 31:55–67, 2005
c Taylor & Francis Inc.
Copyright ISSN: 1040-841X print / 1549-7828 online
DOI: 10.1080/10408410590899228
Ecological and Agricultural Significance of Bacterial
Polyhydroxyalkanoates
Daniel Kadouri, Edouard Jurkevitch, and Yaacov Okon
Department of Plant Pathology and Microbiology, and The Otto Warburg Minerva Center for
Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences,
The Hebrew University of Jerusalem, Rehovot, Israel
Susana Castro-Sowinski
Department of Plant Pathology and Microbiology, and The Otto Warburg Minerva Center for
Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences,
The Hebrew University of Jerusalem, Rehovot, Israel and Departamento de Bioquı́mica, Instituto
Clemente Estable (IIBCE), Unidad Asociada de Bioquı́mica, Instituto de Biologı́a, Facultad de Ciencias,
Universidad de la República. Av. Italia, 3318, 11600, Montevideo, Uruguay
noates (PHAs), and are homopolymers or copolymers containing different alkyl groups at the β position (Anderson & Dawes
1990). PHAs are structurally simple macromolecules accumulated as discrete granules to levels as high as 90% of the cell dry
weight and are generally believed to play a role as a sink for carbon and reducing equivalents when other nutrient supplies are
limited, and when the population is not growing exponentially in
batch culture (Senior & Dawes 1973; Williams & Peoples 1996;
Madison & Huisman 1999). Alternatively, PHAs are accumulated when growing in a continuous culture at constant dilution
rate under nitrogen limitation (Dawes 1986). These molecules
exhibit material properties that are similar to those of some common plastics such as polypropylene (Byrom 1987). The many
different PHAs that have been identified to date are primarily linear; head-to-tail polyesters composed of 3-hydroxy fatty
acid monomers. In these polymers, the carboxyl group of one
monomer forms an ester bond with the hydroxyl group of the
neighboring monomer. The chemical diversity of PHAs is large,
and includes short, medium, and long chain polymers, homoand heteropolymers, and many types of substituted groups. For
a detailed review see Kim and Lenz (2001). These variations are
the basis for the diversity of the PHA polymer family and for their
vast array of potential applications (Byrom 1987; Williams &
Peoples 1996; Madison & Huisman 1999). The industrial interest in PHAs as plastics products has been extensively reviewed
and is not in the scope of this review (van der Walle et al. 2001).
We will mainly focus on the ecological and agricultural significance of PHAs.
The biosynthesis and degradation of PHA is a cyclic mechanism that has been earlier proposed in Azotobacter beijerinckii
and Hydrogenomonas eutropha (Senior & Dawes 1973; Oeding
& Schlegel 1993).
Polyhydroxyalkanoates (PHAs) are a group of carbon and energy storage compounds that are accumulated during suboptimal
growth by many bacteria, and intracellularly deposited in the form
of inclusion bodies. Accumulation of PHAs is thought to be used by
bacteria to increase survival and stress tolerance in changing environments, and in competitive settings where carbon and energy
sources may be limited, such as those encountered in the soil and
the rhizosphere. Understanding the role that PHAs play as internal storage polymers is of fundamental importance in microbial
ecology, and holds great potential for the improvement of bacterial inoculants for plants and soils. This review summarizes the
current knowledge on the ecological function of PHAs, and their
strategic role as survival factors in microorganisms under varying
environmental stress is emphasized. It also explores the phylogeny
of the PHA cycle enzymes, PHA synthase, and PHA depolymerase,
suggesting that PHA accumulation was earlier acquired and maintained during evolution, thus contributing to microbial survival in
the environment.
Keywords
Polyhydroxyalkanoates (PHA); Poly-β-Hydroxybutyrate
(PHB); Bacterial Survival; PHA Metabolism; PHA Synthase; PHA Depolymerase; Phylogeny
1.
INTRODUCTION
A wide variety of taxonomically different groups of microorganisms are known to produce intracellular energy and carbon
storage compounds, generally described as polyhydroxyalka-
Received 29 September 2004; accepted 13 October 2004.
Address correspondence to Yaacov Okon, Department of Plant
Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem,
P.O. Box 12, Rehovot 76100, Israel. E-mail: [email protected]
55
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D. KADOURI ET AL.
1996; Jendrossek & Handrick 2002). In contrast, intracellular
PHA depolymerases degrade intracellular PHA granules in the
accumulating strain, in order to mobilize PHA in the absence of
a suitable exogenous carbon source or energy source (Saegusa
et al. 2001). Therefore, the former can be seen as “scavenging”
enzymes, not necessarily found in PHA-accumulating organisms (e.g., fungi), while the latter are truly engaged in cellular
PHA cycling.
2.
FIG. 1. Key enzyme reactions of the anabolism and catabolism of PHA in
Azospirillum brasilense (produces only PHB).
The synthesis and utilization of PHAs are mediated by PHA
synthases (PhaC), intracellular PHA depolymerases (PhaZ),
phasins (PhaP), and regulators (PhaR). The PhaR regulator/PhaP
phasin system plays an important role in promoting PHA production (York et al. 2001, 2002). PHA accumulation is a widely
distributed prokaryotic phenotype. Since the identification and
characterization of the first enzyme involved in PHA synthesis
(For Azospirillum brasilense see Figure 1) more than 60 genes
from a wide range of bacteria have been cloned or identified as
putative PHA biosynthetic genes (see section on phylogeny).
Within these enzymes, PhaC is the only one exclusively involved in PHA biosynthesis and it is responsible for the polymerization of β-hydroxyalkanoyl-CoA monomers into poly-βhydroxyalkanoate. Bacterial PHA synthases can be classified
in three classes depending on the number of carbon atoms in
the monomers (Rehm & Steinbüchel 1999): (1) Class I PhaCs,
are also called short-chain-length (SCL) PHA synthases, are
composed of one type of subunit, and preferentially use three
to five carbon monomers as substrates; (2) Class II PhaCs,
called medium-chain-length (MCL) PHA synthases, are also
composed of one type of subunits and use monomers with 6 to
14 carbons as substrates; and, (3) Class III PhaCs that preferentially use three to five carbon-long substrates and are composed
of two different subunits (Steinbüchel et al. 1992).
The PHA degradation pathway as described in most bacteria, begins with the depolymerization of PHA to D-β-hydroxybutyrate monomers by PHA depolymerase (encoded by phaZ)
(For Azospirillum brasilense see Figure 1). PHA degradation can
whether proceed intracellularly or extracellularly (Jendrossek &
Handrick 2002). Extracellular PHA depolymerases are secreted
by many bacteria from very varied environments under aerobic and anaerobic conditions, for the utilization of PHAs left
in the environment after the lysis of cells in which the compounds were accumulated (Tanio et al. 1982; Jensrossek et al.
WITH A LITTLE HELP FROM MY PHA DURING
ENVIRONMENTAL STRESS
The function of PHAs as intracellular carbon storage compounds that can be mobilized and used when carbon acts as a
limiting resource has been the subject of most of the research
in assigning a role for these compounds (Macrae & Wilkinson
1958; Sierra & Gibbons 1962; Hippe 1967; Hippe & Schlegel
1967). Under certain circumstances, free-living bacterial cells
with a higher content of PHA may survive longer than those
with a lower PHA content, either because they are not subjected
to some additional adverse factors, or because they can utilize
their reserve material longer and more efficiently (Dawes &
Senior 1973; Matin et al. 1979).
It has been shown that PHA accumulation plays a major role
in some bacteria that live in close association with eukaryotes.
PHA accumulation was involved in promoting long-term survival under starvation conditions of the intracellular parasite of
amoebae, Legionella pneumophila (James et al. 1999). Interestingly, PHA accumulation was promoted during bacterial growth
under iron-limitation into the amoeba host which therefore may
induce a PHA-rich phenotype, rendering the bacteria more fit for
extracellular survival in low-nutrient environments. In rhizobia,
bacteria that are characterized by a free-living and a symbiotic
stage with a eukaryotic host, the impact of the bacteria-host
relationship on PHA accumulation is diverse (see below).
Soil is a heterogeneous, discontinuous, and structured environment with a high diversity of microhabitats in which conditions can change rapidly (Postma et al. 1989). Bacterial cells in
soil face different stresses, such as low nutrient availability and
detrimental physical, chemical, or biological factors, all fluctuating in time and space. To cope with this changing, often
oligotrophic environment, soil bacteria have developed various
survival strategies (van Elsas & van Overbeek 1992). It has been
proposed that the accumulation and degradation of PHA is one
such strategy by which bacteria can improve establishment, proliferation, and survival in competitive settings such as soil and
rhizosphere (Okon & Itzigsohn 1992). Additionally, the conditions for bacterial PHA production can be met in soil when a
high C:N ratio prevails, as is mostly the case in the rhizosphere,
where it is estimated to be at about 20. Nitrogen availability
may become a limiting factor for bacterial growth, especially
in some nitrogen-poor sites. These conditions of suboptimal
growth are conducive to the production of PHAs (Madison &
Huisman 1999).
SIGNIFICANCE OF BACTERIAL POLYHYDROXYALKANOATES
Supporting data for PHA production in telluric environments
was provided by Wang and Bakken (1998) who screened 63 soil
bacteria samples for PHA production and assessed that PHA
accumulation is an aid in the survival of starving soil bacteria.
They found that soil bacteria within the pseudomonads, coryneform and bacilli produce PHA. In addition, Azospirillum and
Azotobacter, as well as other rhizosphere bacteria where shown
to produce PHA in culture under a C:N ratio of 20 (Itzigsohn
et al. 1995). The free-living Gram-negative nitrogen-fixing rhizobacteria belonging to the genus Azospirillum are extensively
used as plant growth promoting rhizobacteria (PGPR) (Okon
& Vanderleyden 1997). These bacteria, which are of agronomical importance, are also models for understanding the physiology and ecology of inoculants, and for deciphering traits important for survival, colonization, and effectiveness. One such
trait appears to be the secretion of plant-like growth promoting
substances such as auxins, gibberellins, and cytokinins by the
bacteria, leading to an increase in root surface area, and to an
enhancement of water and mineral uptake (Okon & Kapulnik
1986; Fallik et al. 1994; Okon & Vanderleyden 1997; Burdman
et al. 2000; Steenhoudt & Vanderleyden 2000). Production and
accumulation of PHA in A. brasilense, which was examined
by Tal and Okon (1985) and Tal et al. (1990a), may also be one
such trait. Under appropriate conditions, A. brasilense cells may
accumulate above 75% of their dry weight exclusively as polyβ-hydroxybutyrate (PHB) (Tal & Okon 1985; Tal et al. 1990a;
Itzigsohn et al. 1995). It was shown that the C:N ratio in the
medium, and the oxygen partial pressure are controlling factors
for PHA production in Azospirillum (Nur et al. 1981; Paul et al.
1990; Tal et al. 1990b).
Increased survival and respiration under starvation conditions
were detected in A. brasilense containing higher amount of PHA
(Tal & Okon 1985). In addition, A. brasilense mutants defective
in their capability to produce PHA (phaC− ) (Kadouri et al. 2002)
or to degrade PHA (phaZ − ) (Kadouri et al. 2003a) survived
shorter than the wild-type strain. As with Ralstonia eutropha
(Handrick et al. 2000), accumulated PHA in A. brasilense can
also support cell multiplication in the absence of an exogenous
carbon source (Kadouri et al. 2002). The term “survival” sensus stricto may therefore not precisely cover the impact of PHA
because it may imply the arrest of multiplication. “Stress alleviation” may be more appropriate. In addition, PHA as a sole
energy source was shown to support nitrogenase activity and
aerotaxis, two physiological features that are extremely energy
consuming (Tal & Okon 1985).
The production of cell types, such as spore and cyst production is yet another survival strategy but some evidence has
been provided that spore formation and germination may be
linked with PHA biosynthesis and utilization. In Bacillus cereus
cells that had accumulated PHA, the polymer disappeared
after sporulation, while the degradation products were incorporated into the spore. In that case PHA may serve as a
carbon and energy reserve for sporulation (Kominek &
Halvorson 1965; Nakata 1965). Moreover, López et al. (1995)
57
observed that in a PHA negative mutant of Bacillus megaterium,
sporulation occurred immediately after exposure to river water,
while survival of vegetative cells was clearly decreased as compared to wild type, pointing that in an oligotrophic environment,
cells depleted of intracellular carbon source may be committed to earlier sporulation than normal cells. The mutant spores
needed a heat shock for germination, suggesting that PHA or its
degradation products are involved in this process (López et al.
1995).
Similar results were found in Azotobacter vinelandii in which
PHA was utilized as a carbon and energy source during encystment (Lin & Sadoff 1968; Segura et al. 2003). However, mutations in the phaB and phaC genes in A. vinelandii had no impact
on encystment or cyst viability under laboratory conditions, but
the possibility that under natural conditions, PHA metabolism
does have such an impact cannot be ruled out (Segura et al.
2003).
Several other works, in which different PHA-producing bacteria were incubated under starvation condition in natural oligotrophic environments, or under conditions mimicking natural
settings, showed that wild-type strains containing PHAs survived starvation better than either PHA-polymerase or PHAdepolymerase mutants (López et al. 1995; Ruiz et al. 1999).
Under such conditions, morphological changes associated with
starvation were largely delayed in the wild-type strains of Pseudomonas oleovorans (Ruiz et al. 2001). Changes from rod-like
to coccus-like shapes are often part of the starvation response,
a major regulator of which is the rpoS gene (Lange & HengerAronis 1991; Gentry et al. 1993).
In summary, PHA production is a widespread trait, lending
support to the hypothesis that this is a central feature of survival
physiology when cells are faced with starvation and in response
to sporulation and encysment. However, the ability to produce
PHA is apparently not absolute for improved survival ability
during this type of stress: Non-PHA producing bacterial strains
isolated from soil survived starvation periods equally as well as
strains capable of PHA accumulation (Wang & Bakken 1998),
suggesting that PHA enhances the survival of some, but not all,
of the bacteria, which must then rely on alternative strategies.
In PHA-producing bacteria, PHA is a major determinant for
overcoming periods of carbon and energy starvation, and may
represent a basic feature for so-called “environmental bacteria.”
This point will be further developed in the section dealing with
phylogeny.
Besides its defined role in starvation alleviation, PHA compounds appear to endow the producing cells with increased endurance to other types of environmental stresses, as was shown
when P. oleovorans and a PHA depolymerization-minus mutant
strain were used to assess the effect of PHA on survival and resistance to various stress agents. When exposed to 20% ethanol,
the wild-type strain survived better than the PHA depolymeraseminus strain, with 23% and 9% of the cells surviving the challenge, for the wild-type and mutant strains, respectively. Also,
a 47◦ C heat shock was less damaging to the wild type than to
58
D. KADOURI ET AL.
the mutant strain (Ruiz et al. 2001). Similar observations were
made for psychrotrophic strains of Rhizobium under cold stress
(Sardesai et al. 2001).
An extensive analysis of the role of PHA in the protection
of A. brasilense cells exposed to physical and chemical stresses
was performed, with the aim of understanding how central parameters such as quality, longevity, reliability, and efficacy of
commercial bacterial inoculants for agricultural uses can be improved. After exposure of A. brasilense to stress and adverse conditions such as UV-irradiation, desiccation, and osmotic pressure, the survival of PHA-rich bacteria was higher than that
of PHA-poor ones (Tal & Okon 1985). Similar results were
obtained working with phaC (Kadouri et al. 2002) and phaZ
mutants of A. brasilense (Kadouri et al. 2003a; Kadouri et al.
2003b). The ability of these mutants to tolerate various stresses,
such as ultraviolet irradiation, heat, osmotic pressure, osmotic
shock, desiccation, and to grow in the presence of hydrogen
peroxide, was significantly affected. Thus, as the two PHA mutants, one blocked in the anabolic (phaC mutant), the second
in the catabolic route (phaZ mutant), are similarly altered, increased resistance to the described stresses can be traced to a
normal functioning of the PHA cycle, and not exclusively to the
presence of the polymer.
The mechanisms by which the PHA cycle favors stress alleviation are not yet fully understood. Early work on R. eutropha suggested an association of PHA utilization with respiration and oxidative phosphorylation (Hippe & Schlegel 1967).
It was found that a rise in ATP and guanosine tetraphosphate
(ppGpp) levels was concomitant with PHA degradation. This
phenomenon was only observed in wild type P. oleovorans and
not in a PHA depolymerase-deficient strain unable to degrade
the polymer (Ruiz et al. 2001). The effector ppGpp was shown to
increase mRNA translation of the central stationary phase regulator rpoS (Brown et al. 2002), which upregulates resistance
to environmental insults such as ethanol, H2 O2 , high temperature, or high salt concentration (Lange & Henger-Aronis 1991;
Samiguet et al. 1995; Ramos-González and Molin 1998; Ruiz
et al. 2001). The sigma subunit RpoS activates the expression
of genes involved in cell survival in cessation of growth and
provides cross-protection to various stresses. Recently, it was
found that the enhanced cross-tolerance to different stress agents
during PHA-depolymerization in P. oleovorans is related to an
increase in the intracellular concentration of RpoS (Ruiz et al.
2004). Ruiz et al. (2004) suggested that there is an association
between PHA depolymerization and the stress tolerance phenotype, controlled by RpoS. In addition, Peralta-Gil et al. (2002)
showed that one of the promoters that control PHA synthesis in
A. vinelandii is regulated by RpoS. It seems that to respond properly to diverse stresses, PHA-producing bacteria require the rpoS
gene product, this product induces expression of many genes,
including the ones responsible for PHA depolymerization, and
allows the organism to mediate changes in cellular physiology and structure and to adapt, resist, and survive under stress
conditions.
The data gathered in these various studies suggest a complex
role for PHA in stress alleviation. However, the evidence clearly
shows a relationship between PHA metabolism and cell alleviation under stress, mainly environmental stress. The granules
may offer physical protection in stresses such as UV radiation,
protecting DNA from damage, and increase resistance to desiccation, but normally functioning PHA anabolic and catabolic
pathways seem to be essential to provide increased stress protection. While stress response players such as rpoS and ppGpp
(or their functional homologues in different organisms) are central in determining the type and the strength of the response
by redirecting cell resources to the synthesis of the appropriate effectors, the PHA cycle could be responsible for providing
the fuel necessary for this response, and therefore could also
determine its intensity.
3.
ENERGY FLOW AND PHA METABOLISM
PHA is generally overproduced and accumulated when more
reducing power is being generated than consumed, due to the
limitation for other syntheses (Dawes 1986; Babel et al. 2001).
On the other side of the cycle, PHAs can serve as sources of
NADH and ultimately, ATP. This energy-generation capacity
can be used to fuel various energy-consuming pathways which
in the absence of PHA would be slowed down or blocked. For example, Rothermich et al. (2000) reported the PHA accumulation
during the night and degradation during the day of PHA from a
variety of photosynthetic benthic microbial mats obtained from
soil, compost, and sewage sludge. The authors suggest that the
accumulation of PHA at night appears to be related to routine
dark energy metabolism and is not influenced by the availability
of organic nutrients.
Among others, energy taxis are involved in chemotaxis to a
carbon source and it appears to be important in plant-microbe interaction. It has been shown that A. brasilense exhibited stronger
chemotaxis toward attractants such as fructose, malate, or sweet
corn seeds exudates in a semi-solid agar test, than the phaC mutant (Kadouri et al. 2003b). Nevertheless, the mutant exhibited
a three-time increase in swimming velocity over the wild-type
strain (Kadouri et al. 2002; Kadouri et al. 2003b). Under the
tested conditions (carbon-free medium), this increased motility
did not provide the mutant with an elevated chemotactic response, probably because of the absence of PHA as a storage
compound that could be utilized during the starvation period. Increase motility was not observed in a phaZ mutant that accumulated high PHA levels but was incapable of degrading it (Kadouri
et al. 2003a). The reduced power produced during PHA degradation likely energized the chemotactic process in the environment,
where sources of reducing power are low. In A. brasilense, PHA
oxidation involves a specific NADH-dependent dehydrogenase,
which competes for TCA-cycle intermediates in the electron
transport system (Tal et al. 1990a, 1990b). When PHA accumulation is disrupted, more resources are accessible to the TCA
cycle, resulting in an increased motility in the phaC mutant as
SIGNIFICANCE OF BACTERIAL POLYHYDROXYALKANOATES
compared to the wild type. Therefore, a similar motility of the
phaZ mutant and of the wild-type strains is probably due to the
inability of the mutant to generate excess reducing power, as was
likely the case in the phaC mutant when PHA polymerization
was disrupted. Several lines of evidence suggest that taxis in
plant-associated bacteria is metabolism dependent (Grishanin
et al. 1991), which explains that chemotaxis is dependent to
PHA metabolism. The redox state of the rhizosphere is one of
the most important parameters for maintaining this ecological
system. Thus the energy taxis, driven by PHA catabolism, toward metabolizable substrates in plant root exudates may play
a major role in plant-microbe interactions.
Another feature of phaC mutants in A. brasilense, is a considerable increase in excreted exopolysaccharide (EPS), when
grown under a high C:N ratio over the wild-type strain. In such
mutants, EPS production may act as a sink for carbon and reducing equivalents which are diverted from the blocked PHAsynthesis pathway. Azospirilla are known for their capacity to
aggregate and flocculate under diverse stress conditions, and
some studies have suggested that EPS are involved in cell aggregation and in root adhesion (Burdman et al. 2000). Recently,
Bahat-Samet et al. (2004) showed that the arabinose content of
A. brasilense EPS plays a role in cell aggregation. The phaC mutant was more aggregative, and exhibited an increased (five to six
times) ability to adhere to roots relative to the wild type (Kadouri
et al. 2002; Kadouri et al. 2003b). In contrast, EPS production
in the wild-type strain, as well as its aggregation capability was
higher than that of the phaZ mutant under the same conditions
(Kadouri et al. 2003a). Burdman et al. (2000) suggested that
cell aggregation could increase survival of Azospirillum cells
under diverse stress conditions. The attachment of the bacteria to plant roots, within bacterial aggregates and flocs, is a requirement for the establishment of the bacterial-root association.
Thus, both PHA accumulation and cell aggregation could constitute a protected model of growth that allows survival in a hostile environment. This phenomenon of cell aggregation—PHA
metabolism—may also be important during root colonization
where cell aggregation is commonly observed.
In Rhizobium spp.,PHA formation decreases the amount of
reducing equivalents that could otherwise be used for nitrogen
fixation during the symbiosis of the bacteria with their host
plants, as observed when a PHA− strain of Rhizobium etli exhibited a higher and prolonged nitrogenase activity in nodules
(Cevallos et al. 1996). As a consequence, plants inoculated with
the PHA− mutants had higher nitrogen content. Along similar
lines, it can be mentioned that mutations in the nitrogenaseencoding genes that resulted in the absence of nitrogen fixation
by Bradyrhizobium japonicum, brought about an increase in
PHA synthesis. It therefore seems that PHA synthesis can serve
as an alternative pathway for the regeneration of reducing equivalents in the nodule (Hahn et al. 1984). Like R. etli, Azorhizobium
caulinodans also accumulates PHA in both symbiotic and freeliving state, but A. caulinodans phaC mutant was totally devoid
of nitrogenase activity ex plant and induced nodules devoid of
59
bacteria (Mandon et al. 1998). Interestingly, nitrogenase activity
of the mutant was partially restored by constitutive expression
of the nifA gene. Mandon et al. (1998) suggested that PHA is
required for maintaining the reducing power of the cell and that
nifA expression adapts nitrogen fixation to the carbon and the
reducing equivalents available in the nodule. Also Vassileva and
Ignatov (2002) studied the relationship between PHA formation
and nitrogenase activity in Galega orientalis—Rhizobium galegae symbiosis. They reported high acetylene reduction activity
parallel to PHA degradation when applying low concentrations
of plant growth promoters and polyamine modulators. In contrast to R. etli, A. caulinodans, and R. galegae that forms PHA in
both the free-living and bacteroid states, S. meliloti bacteroids
typically do not deposit PHA. S. meliloti strains defective in PHA
formation were not affected in their ability to induce nodule formation. Nodules developed at the same timing and had the same
appearance on alfalfa plants (Medicago sativa) inoculated with
the wild type or the mutant strains. Additionally, the acetylenereducing activity, a measure of the nitrogenase activity, was not
affected in these PHA defective mutants when compared to the
wild-type strain. It was concluded that an effective symbiosis between S. meliloti and alfalfa is not affected by PHA formation,
and that a reduced competition for reducing equivalent may be
a reason for the efficiency of nitrogen fixation in alfalfa nodules
(Hahn et al. 1984).
The data herein suggest that a sensitive regulatory mechanism
that controls the synthesis or degradation of PHA in bacteroids is
present and point its importance to maintain an effective symbiosis. A well-regulated PHA cycle therefore appears to be central
both for a balanced use of the available energy (as seen with
the swimming velocity in A. brasilense and nitrogen fixation
in Rhizobium spp., R. galegae, and A. caulinodans) and for a
balanced distribution of the carbon resources (as seen with EPS
production and aggregation), at least when a high C:N ratio
prevails.
4.
PHA: AN ECOLOGICAL EDGE?
Although single strain inoculation experiments done with S.
meliloti PHA− mutants and wild type strains suggested that competent symbiosis between S. meliloti and alfalfa is not affected by
PHA formation (Povolo et al. 1994), results from co-inoculation
experiments suggested that the wild-type bacteria outnumbered
PHA mutant bacteria by more than 200 to 1 (Willis & Walker
1998). This result indicates that the phaC mutant strains were
less competitive, and that PHA production may provide an advantage to the strain during nodule initiation or invasion. Although in the case of S. meliloti, the mutation was not disadvantageous after the establishment of symbiosis, pleiotropic effects
may have reduced competitiveness of the free-living bacteria
in the rhizosphere. On the other hand, rhizobia are positively
chemotactic toward a variety of amino acids, dicarboxylic acids,
and sugars, toward the nodulation gene-inducing flavonoids secreted by the roots of its host. Mutants defective in motility or
chemotaxis are impaired in their ability to compete for sites of
60
D. KADOURI ET AL.
nodule initiation on the host root (Caetano-Anollés et al. 1988).
If like A. brasilense, S. meliloti PHA catabolism is involved
in energy supply to the chemotaxis, it is reasonable that phaC
mutant strains are less competitive than the wild type.
No differences were detected in a range of carbon sources
utilized by a wild type and a phaC mutant of A. brasilense, but
the generation time of the former was always shorter than that
of the later (Kadouri et al. 2003b). Similar findings were reported with A. brasilense phaZ (Kadouri et al. 2003a) and R. etli
phaC mutants (Cevallos et al. 1996). It was observed that a mutation in the phaC gene severely impaired the ability of R. etli to
grow in minimal media supplemented with glucose or pyruvate.
Such consequences may further hinder the competitiveness in
the environment of mutants defective in PHA accumulation or
utilization (Okon & Itzigsohn 1992).
The possession of relatively high growth rates seems to be
an obviously important factor for rhizosphere competence. Although it was suggested that carbon is not a limiting factor
for microorganisms in the rhizosphere (Cheng et al. 1996), the
ability of r-type soil inhabitants to grow quickly, that is, to attain short growth rates on the available compounds in the rhizosphere, probably affect their capacity for root colonization
(Simons et al. 1996; Jjemba & Alexander 1999). However, the
impact of a functional PHA cycle on root colonization is not
always evident as shown in a study in which plant root colonization by wild type and phaC mutant strains of A. brasilense
were evaluated under sterile and non-sterile conditions in soil.
Both root colonization and plant growth promoting effects were
similar under each of the tested conditions (Kadouri et al. 2002).
While the lack for influence of the mutation on these parameters
may stem from the optimal plant growth conditions as well as the
high inoculum level used in the study (which can reduce the plant
growth promoting effect and blur differences in colonization efficiency, respectively), it is left to be seen if under inoculation
and growth conditions found in the field, the impaired stress
resistance and physiological changes observed in cells with a
disrupted PHA cycle are translated in reduced colonization and
growth promotion.
Studies carried out with the potential bioremediation and biocontrol agent, B. megaterium, on sterile and non-sterile microcosms soil showed that in non-sterile soil the total cell number
(vegetative cells plus spores) of a PHA accumulating strain was
higher than that of the PHA-negative mutant (López et al. 1998).
Similar result was obtained in homogeneous aquatic microcosms
(López et al. 1995). These results suggest that PHA accumulation contributed to the survival capabilities and spore quality in
homogeneous and heterogeneous environments enriched with
organic matter such as crop field soil; thus contributing in the
ecological edge of B. megaterium in its natural environment.
5.
INOCULANT CARRIERS—AN ULTIMATE MODEL
FOR STRESS SURVIVAL
Inoculant preparations for agricultural uses can constitute
particularly stressful environments. Bacterial cells may have to
be stored for long periods, and should also survive desiccation
and possibly hot conditions. A major feature inoculants should
possess is a high capacity to maintain high survival rates of the
bacterial cells within the carrier itself. Most of the research done
in this field is based on improving carrier properties by adding
elements that can prolong survival, such as nutrients, or other
synthetic products (López et al. 1998). The vast amount of information gathered on Azospirilla throughout the years suggests
that for an inoculant to be successful—to provide efficient root
colonization—the type of carrier material is not the only important parameter, but the metabolic state of the cells and their
capability to use intercellular storage material can be of significance for survival within the carrier itself. This knowledge originated from studies showing that while the carriers may vary,
plant growth promotion effects were more consistent with A.
brasilense inoculants containing high amount of PHA (Fallik
& Okon 1996). Confirmation of these results was obtained in
field experiments carried out in Mexico with maize and wheat,
where better consistency in increasing crop yield was obtained
using peat inoculants prepared with PHA-rich Azospirillum cells
(Dobbelaere et al. 2001). In addition, studies carried out with the
A. brasilense phaC mutant showed that among different inoculant carriers (peat, sianic sand, perlite), peat sustained the highest
populations of inoculated bacteria and perlite the lowest. In all
cases, the wild-type strain survived better than the mutant but the
variations between the carriers were very large. It was thus concluded that the production of PHA is of critical importance for
improving the shelf life, efficiency, and reliability of commercial
inoculants (Kadouri et al. 2003b).
6.
A PHYLOGENETIC OVERVIEW OF PHA SYNTHESIS
AND DEGRADATION
A BLASTP search enabled us to identified more than 170
and 60 PhaC and PhaZ homologous proteins, respectively. For
further phylogenetic studies, a subset of 67 PhaC (61 from Proteobacteria, 3 from Gram positive bacteria and 3 sequences
from Cyanobacteria) and 46 PhaZ (all proteobacterial in origin) representative proteins annotated as known or putative were
chosen. Environmental sequences and sequences with E-values
worse than 10−3 were not included in this study. In addition, 16S
rDNA gene sequences were used to infer phylogenetic trees for
the species and for comparative (congruence) purposes. Multiple alignments of protein sequences were performed using the
CLUSTAL W program (Thompson et al. 1994) and edited for
graphic presentation with GeneDoc (Nicholas et al. 1997). Phylogenetic trees were inferred with maximum-parsimony (heuristic search factor of 2) and neighbor-joining (p-distance matrix)
analyses, using MEGA2 (Kumar et al. 2001).
In almost all cases, the PhaC protein trees were congruent with the 16S rDNA data, clustering as phylogenetic taxa
(Figures 2 and 3), suggesting the existence of “genotypic” clusters that corresponded to traditional species designations. The
PhaC tree showed a topology in agreement to that reported by
SIGNIFICANCE OF BACTERIAL POLYHYDROXYALKANOATES
61
FIG. 2. Phylogenetic tree of PhaC generated using the Neighbor-Joining (NJ) distance method. Similar topology was generated using Maximum Parsimony
(MP) method (data not shown). Accession Numbers are given between parentheses. Numbers in brackets identify clusters. An asterisk labels sequences showing
discrepancies with the 16S rDNA phylogeny of the cluster.
62
D. KADOURI ET AL.
FIG. 3. Phylogenetic relationship based on the analysis of the 16S rRNA gene among the bacterial groups used in this analysis generated using the NeighborJoining (NJ) distance method.
SIGNIFICANCE OF BACTERIAL POLYHYDROXYALKANOATES
Steinbüchel and Hein (2001) and Rehm (2003). However, the
tree presented in our work includes 67 instead of 36 and 58 sequences, respectively. Six clusters were evident (Figure 2). The
first monophyletic cluster exclusively contained members of the
Pseudomonadaceae, with Class II synthases (Cluster 1) and was
constituted of two divergent groups. The other γ -Proteobacteria
belonged to a different, separated cluster (Cluster 3). Within
this cluster, Aeromonas caviae that synthesizes copolyesters
of MCL-PHAs was found (Shimamura et al. 1994; Doi et al.
1995). Although the PhaC belonging to α-Proteobacteria, was
found in two, well separated and defined clusters (4 and 5),
their relative positions remain uncertain due to low bootstrap
values. The larger cluster (Cluster 4) included a PhaC from A.
brasilense that exclusively synthesizes homopolymers of polyhydroxyburyrate (Itzigsohn et al. 1995). Within the smaller cluster 5 was found Rhodobacter sphaeroides that only produces
SCL-PHA (Clemente et al. 2000). A group of PhaCs originating
from β-Proteobacteria appeared to form cluster 2. This cluster
included R. eutropha, Azotobacter spp., and Chromobacterium
violaceum. R. eutropha’s PhaC is a representative member of
the Class I PhaCs and the most intensively studied synthase.
R. eutropha (Chua et al. 1998) and C. violaceum (Kolibachuk
et al. 1999) synthesize copolyesters of 3-hydroxybutyrate-co-3hydroxyvalerate (3HB-3HV), but it has also been reported that R.
eutropha produces 3-hydroxybutyrate-co-3-mercaptopropionic
acid or co-3,3’-thiodipropionic acid (Lutke-Eversloh and
Steinbüchel 2003). In addition, Azotobacter PhaC has also been
identified as a Class I PhaC (Pettinari et al. 2001). Cluster
2 and the two Pseudomonadaceae branches appeared to derive from a common ancestor, with a high degree of confidence. The clustering of Azotobacter with the β-Proteobacteria
is unambiguous. Cluster 6 was used as an outgroup and included PhaCs from cyanobacteria, Bacillus species as well as
from two γ -Proteobacteria, Allochromatium vinosum (formally
called Chromatium), and Thiocystis violacea, all member of
Class III PhaC (Hein et al. 1998; McCool & Cannon 2001; Yuan
et al. 2001). To summarize, while clusters 1 and 6 PhaCs were
class I and class III PHAs, respectively, the PhaCs from the other
clusters were involved in the synthesis of widely different types
of PHAs.
Although the PhaC and 16S rDNA data were congruent for
individual clusters, the branching orders were not necessarily
identical. The clear separation of the Pseudomonaceae’s PhaCs
from those of the other γ -Proteobacteria, and the large distance between the clusters formed by organisms belonging to
this group—which also reflects their phylogenetic grouping, as
well as the presence of Azotobacter within cluster 2, support a
very ancient split, and most probably a duplication event within
cluster 1. The few incongruence found between the PhaC and
the 16S rDNA phylogenies could be explained by very large
distances separating the organisms and low bootstrap values
(R. prowazekii), or possibly, horizontal gene transfer (HGT) (P.
putida in β-Proteobacteria). In the case of R. prowazekii, the GC
content of the gene matches the average of the genome, so HGT
63
does not appear to have taken place, or it was a very ancient event.
Interestingly, this is the only occurrence of a phaC-like gene in
a pathogen, all the others originating from “environmental bacteria,” such as soil or waterborne bacteria. Moreover, no such
function seems to be present in Rickettsia conorii, a closely related pathogen. Whether R. prowazekii indeed produces PHAs is
unknown. To survive and grow under fluctuating environmental
conditions, including periods of unbalanced nutrient and energy
supply, microorganisms may use several strategies, including
PHA accumulation, which can provide the energy and carbon
necessary to maintain metabolic activities and protein synthesis
under detrimental situations. The PhaC protein appears to be
highly conserved, it is widely distributed within the Bacteria
domain (we can suppose that it is present in the many known
PHA producing bacterial species), even though to the best of
our knowledge, no PHA production by δ- or ε-Proteobacteria
has been reported. We also have presented a large body of data
supporting a role for PHA in alleviating environmental stresses.
We believe these data lend strong support to the hypothesis that
PHA metabolism is an essential feature for survival of many
bacteria in the environment, or in other words, endows the cells
with an ecological advantage.
The mechanism and regulation of PHA mobilization is poorly
understood, but a clear distinction exists between intracellular
and extracellular PHA degradation by way of the PhaZ enzymes
(Jendrossek & Handrick 2002). Herein, we focus on the intracellular PhaZ (i-PhaZ). The i-PhaZ of P. oleovorans, R. rubrum,
R. eutropha, and Paracoccus denitrificans are the best studied
(Huisman et al. 1991; Merrick et al. 1999; Gao et al. 2001;
Saegusa et al. 2001; Ueda et al. 2002; York et al. 2003). Most of
the clusters of the phylogenetic tree for the PhaZ enzyme presented here clearly show well-supported internal structures, but
their orders are often undefined (Figure 4). Moreover, taking
exception from the Pseudomonadaceae’s PhaZs, which along
with those of two β-Proteobacteria were only very distantly related to the other PhaZs, and from two other clusters containing
few sequences, none of the clusters were homogeneous, that is,
they all contained one or more sequences not congruent at the
class level with the 16S rDNA data. It has been reported that R.
eutropha possesses three different PhaZs (York et al. 2003). In
addition, we detected two copies of PhaZ in the genome of Raltonia metallidurans and Burkholderia fungorum. R. eutropha
PhaZ2 and PhaZ3 clustered independently, but were associated
with the PhaZ of R. metallidurans. R. eutropha PhaZ1 and PhaZ3
clustered with PhaZs of other β-Proteobacteria, but the PhaZ3
sequence diverged from the PhaZ1 one. This multiplication of
depolymerases in one organism may reflect the diversity of the
PHAs it is able to produce. The incongruence within the mentioned cluster and within the rest of the tree maybe signs of HGT,
duplication events, or of parallel evolution of proteins originally
performing various functions. In other words, while the synthetic process appeared to be stringently conserved, a number of
options for using the polymer as a substrate may have developed
in parallel or were laterally acquired.
64
D. KADOURI ET AL.
FIG. 4. Phylogenetic tree of PhaZ generated using the Neighbor-Joining (NJ) distance method. Similar topology was generated using Maximum Parsimony
(MP) method (data not shown). Accession Numbers are given between parentheses.
SIGNIFICANCE OF BACTERIAL POLYHYDROXYALKANOATES
TABLE 1
PHA in bacterial environmental fitness and agriculture
Features
Cell survival under starvation
in batch and continuous
culture (days)
Cell survival under nutrient
limitation in water, soil,
rhizosphere and
phyllosphere
Cell survival in inoculant
carriers
Establishment of inoculum in
soil and plant surfaces
Energy source and flow for
cell motility, chemotaxis,
aerotaxis and biological
nitrogen fixation
Sporulation, cyst formation
and germination
Control of exopolysaccharide
production
Endurance under
environmental stress: heat,
UV-irradiation, desiccation,
osmotic stress, osmotic
shock, ethanol and H2 O2
Balanced use of available
energy and distribution of
carbon resources
7.
Selected references
Tal and Okon 1985; Anderson
and Dawes 1990;
James et al. 1999.
Okon and Itzigsohn 1992;
López et al. 1995;
Ruiz et al. 1999.
Fallik and Okon 1996;
Dobbelaere et al. 2001;
Kadouri et al. 2003b.
Kadouri et al. 2002;
Kadouri et al. 2003b.
Tal and Okon 1985; Cevallos
et al. 1996; Kadouri et al.
2002.
Kominek and Halvorson
1965; Segura et al. 2003.
Kadouri et al. 2002.
Tal and Okon 1985; Kadouri
et al. 2003a; Kadouri et al.
2003b.
Dawes 1986; Rothermich
et al. 2000; Babel et al.
2001.
CONCLUDING REMARKS
PHA-related features influencing bacterial fitness are summarized in Table 1. In order to survive in nutrient-poor ecosystems, bacteria use different strategies, among them, the use of
PHAs as intracellular carbon storage compounds. Many works
have revealed that cells with higher content of PHA can survive
longer than those with lower amounts, and that PHA degraded
elements can be used rapidly for numerous metabolic needs.
Accumulation of PHA can provide the cell with the ability to
endure a variety of harmful physical and chemical stresses, either directly linked to the presence of the polyester itself (PHA
granules) or through a cascade of events concomitant with PHA
degradation and the expression of genes involved in protection
against damaging agents. The first type of events may be hypothesized to occur when PHAs offer physical protection to damage
and may include the use of PHAs as carbon source under nutrient deficiency. In the second type of event, PHAs function as
a source of power (reducing equivalents) that can be mobilized
to enact reaction to the stress (maybe such as ATP-dependent
repair mechanisms, osmoregulation). In both cases, a normally
65
functioning PHA cycle may enable the maintenance of optimal
metabolic capabilities (including nitrogen fixation and chemotaxis) under sub-optimal conditions, permitting not only survival
but also cell proliferation. The capability of PHA to provide the
cell with an endogenous carbon reservoir to aid in stress endurance, and to support quick growth on the nutrients accessible,
is an important factor for soil competence, which can probably
affect their capacity to colonize plants. The growth rate (replication times, generation times) of bacteria in the soil, rhizosphere,
phyllosphere, and inoculants is apparently much slower than in
batch culture under laboratory conditions. While in the latter
PHAs are utilized and depleted after several days under starvation, in nature the PHAs could be aiding bacterial division and
survival for much longer periods. In addition, the phylogenetic
analysis of PHA metabolism proteins evidences that PHA accumulation is an essential feature for survival of many bacteria
in the environment, or in other words, endows the cells with an
ecological advantage.
One of the disciplines in which the information gathered can
be of benefit is the field of bacteria inoculants used for plant protection and plant growth promotion. When implementing bacteria to plants and soils, the capability of the microorganism to
establish itself, survive and proliferate and, is of utmost importance. Understanding and manipulating this feature may be of
great agro-industrial interest.
ACKNOWLEDGMENTS
We thank Y. Davidov for discussions. This review was supported by “The Israel Science Foundation” founded by “The
Academy of Sciences and Humanities,” and by the European
Union-5th Framework contract QLK3-CT-2000-31759-ECOSAFE. The work of S. Castro-Sowinski at The Hebrew University of Jerusalem was supported by Lady Davis Trust Fellowship.
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