ISPUB.

COM The Internet Journal of Microbiology

Volume 9 Number 1

Phosphate Solubilization: Their Mechanism Genetics And

Application
N Ahmed, S Shahab

Citation
N Ahmed, S Shahab. Phosphate Solubilization: Their Mechanism Genetics And Application. The Internet Journal of
Microbiology. 2009 Volume 9 Number 1.

Abstract
The global necessity to increase agricultural production from a steadily decreasing and degrading land resource base has
placed considerable strain on agro ecosystems (Tilak, 2005). Current strategy is to maintain and improve agricultural
productivity exclusively via the use of chemical fertilizers. Although the use of chemical fertilizers is credited with nearly fifty
percent increase in agricultural production but they are closely associated with environmental pollution and health hazards
(Gaur and Gaind, 1999). Many synthetic fertilizers contain acids, such as sulfuric acid and hydrochloric acid, which tend to
increase the acidity of the soil, reduce the soil's beneficial organism population and interfere with plant growth. Generally,
healthy soil contains enough nitrogen-fixing bacteria to fix sufficient atmospheric nitrogen to supply the needs of growing plants.
However, continued use of chemical fertilizers may destroy these nitrogen-fixing bacteria. Furthermore, chemical fertilizers may
affect plant health. For example, citrus trees tend to yield fruits that are lower in vitamin C when treated with synthetic fertilizer.
Lack of trace elements in soil regularly dosed with chemical fertilizers is not uncommon. This lack of vital micronutrients can
generally be attributed to the use of chemical fertilizers. On the other hand Biofertilizer adds nutrients to soil.Environmentally
friendly biotechnological approaches offer alternatives to chemical fertilizers (Dobbelaere et al., 2003). Given the negative
environmental impacts of chemical fertilizers and their increasing costs, the use of PGPB is thus being considered as an
alternative or a supplemental way of reducing the use of chemicals in agriculture (De Weger et al., 1995; Gerhardson, 2002,
Postma, et al., 2003; Welbaum, 2004)

INTRODUCTION dosed with chemical fertilizers is not uncommon. This lack

The global necessity to increase agricultural production from
a steadily decreasing and degrading land resource base has
placed considerable strain on agro ecosystems (Tilak, 2005).
Current strategy is to maintain and improve agricultural
productivity exclusively via the use of chemical fertilizers.
Although the use of chemical fertilizers is credited with
nearly fifty percent increase in agricultural production but
they are closely associated with environmental pollution and
health hazards (Gaur and Gaind, 1999). Many synthetic
fertilizers contain acids, such as sulfuric acid and
hydrochloric acid, which tend to increase the acidity of the
soil, reduce the soil's beneficial organism population and
interfere with plant growth. Generally, healthy soil contains
enough nitrogen-fixing bacteria to fix sufficient atmospheric
nitrogen to supply the needs of growing plants. However,
continued use of chemical fertilizers may destroy these
nitrogen-fixing bacteria. Furthermore, chemical fertilizers
may affect plant health. For example, citrus trees tend to
yield fruits that are lower in vitamin C when treated with
synthetic fertilizer. Lack of trace elements in soil regularly
of vital micronutrients can generally be attributed to the use
of chemical fertilizers. On the other hand Biofertilizer adds
nutrients to soil.
Environmentally friendly biotechnological approaches offer
alternatives to chemical fertilizers (Dobbelaere et al., 2003).
Given the negative environmental impacts of chemical
fertilizers and their increasing costs, the use of PGPB is thus
being considered as an alternative or a supplemental way of
reducing the use of chemicals in agriculture (De Weger et
al., 1995; Gerhardson, 2002, Postma, et al., 2003; Welbaum,
2004)
It has been estimated that in some soil up to 75% of applied
phosphate fertilizer may become unavailable to the plant
because of mineral phase reprecipitation (Goldstein, 1986;
Sundara et al., 2002). Phosphate-solubilizing bacteria (PSB)
are able to convert insoluble phosphates into soluble forms
(Illmer and Schinner, 1995; Hilda et al., 2000ab; Peix et al.,
2001 ab; Viverk and Singh, 2001; Sudhakara et al., 2002)
and have therefore been used to enhance the solubilization of

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Phosphate Solubilization: Their Mechanism Genetics And Application

reprecipitated soil P for crop improvement (Shekhar et al., ORGANIC PHOSPHATE

2000; Young et al., 1986; Young, 1990). A second major component of soil P is organic matter.
Organic forms of P may constitute 30–50% of the total
PHOSHATE AVAILABILITY IN SOIL
phosphorus in most soils, although it may range from as low
Phosphorus (P) is one of the major essential macronutrients
as 5% to as high as 95% (Paul and Clark, 1988). Organic P
for biological growth and development (Ehrlich, 1990). It is
in soil is largely in the form of inositol phosphate (soil
present at levels of 400–1200 mg/kg of soil (Fernandez,
phytate). It is synthesized by microorganisms and plants and
1988). The concentration of soluble P in soil is usually very
is the most stable of the organic forms of phosphorus in soil,
low, normally at levels of 1 ppm or less then 1ppm
accounting for up to 50% of the total organic P (Dalal, 1977;
(Goldstein, 1994). The cell might take up several P forms
Anderson 1980; Harley and Smith, 1983). Other organic P
but the greatest part is absorbed in the forms of Phosphate
compounds in soil are in the form of phosphomonoesters,
(Beever and Burns, 1980).
phosphodiesters including phospholipids and nucleic acids,
Figure 1 and phosphotriesters.Of the total organic phosphorus in soil,
Figure 1: Phosphate cycle ( only approximately 1% can be identified as nucleic acids or
their derivatives (Paul and Clark, 1988). Various studies
have shown that only approximately 1–5 ppm of
phospholipids phosphorus occurs in soil, although values as
high as 34 ppm have been detected (Paul and Clark, 1988).
Large quantities of xenobiotic phosphonates, which are used
as pesticides, detergent additives, antibiotics, and flame
retardants, are released into the environment. These C-P
compounds are generally resistant to chemical hydrolysis
and biodegradation, but several reports have documented
Mineral forms of phosphorus are represented in soil by microbial P release from these sources (Ohtake, 1996;
primary minerals, such as apatite, hydroxyapatite, and McGrath, 1998).
oxyapatite. They are found as part of the stratum rock and
Figure 2
their principal characteristic is their insolubility. In spite of
Figure 2: Figure showing complexity of average soil
that, they constitute the biggest reservoirs of this element in
soil because, under appropriate conditions, they can be
solubilized and become available for plants and
microorganisms. Mineral phosphate can be also found
associated with the surface of hydrated oxides of Fe, Al, and
Mn, which are poorly soluble and assimilable. This is
characteristic of ferralitic soils, in which hydration and
accumulation of hydrated oxides and hydroxides of Fe takes
place, producing an increase of phosphorus fixation capacity
(Fernandez, 1988).

There are two components of P in soil, organic and inorganic

phosphates. A large proportion is present in insoluble forms,
and therefore, not available for plant nutrition. Inorganic P
occurs in soil, mostly in insoluble mineral complexes, some
of these appearing after the application of chemical
fertilizers. These precipitated forms cannot be absorbed by
plants. Organic matter, on the other hand, is an important
reservoir of immobilized P that accounts for 20–80% of soil
P (Richardson, 1994).
(http:/ www.physicalgeography.net/fundamentals/10t.html)
ORGANIC PHOSPHATE SOLUBILIZATION
Organic phosphate solubilization is also called
mineralization of organic phosphorus, and it occurs in soil at
the expense of plant and animal remains, which contain a
large amount of organic phosphorus compounds. The
decomposition of organic matter in soil is carried out by the
action of numerous saprophytes, which produce the release

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Phosphate Solubilization: Their Mechanism Genetics And Application
of radical orthophosphate from the carbon structure of the
molecule. The organophosphonates can equally suffer a
process of mineralization when they are victims of
biodegradation (McGrath, 1995). The microbial
mineralization of organic phosphorus is strongly influenced
by environmental parameters; in fact, moderate alkalinity
favors the mineralization of organic phosphorus (Paul and
Clark, 1988) The degradability of organic phosphorous
compounds depend mainly on the physicochemical and
biochemical properties of their molecules, e.g. nucleic acids,
phospholipids, and sugar phosphates are easily broken down,
but phytic acid, polyphosphates, and phosphonates are
decomposed more slowly (Ohtake, 1996; McGrath, 1995;
McGrath 1998).
Phosphorus can be released from organic compounds in soil
by three groups of enzymes:
Nonspecific phosphatases, which perform dephosphorylation
of phospho-ester or phosphoanhydride bonds in organic
matter
Phytases, which specifically cause P release from phytic acid
Phosphonatases and C–P Lyases, enzymes that perform C–P
cleavage in organophosphonates
The main activity apparently corresponds to the work of acid
phosphatases and phytases because of the predominant
presence of their substrates in soil.
Figure 3
Figure 3: Mineralization of organic compounds within soil
(Source:http://grunwald.ifas.ufl.edu/Nat_resources/organic_
matter/som.gif)
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INORGANIC PHOSPHATE MINERALIZATION
Several reports have suggested the ability of different
bacterial species to solubilize insoluble inorganic phosphate
compounds, such as tricalcium phosphate, dicalcium
phosphate, hydroxyapatite, and rock phosphate (Goldstein,
1986). In two thirds of all arable soils, the pH is above 7.0,
so that most mineral P is in the form of poorly soluble
calcium phosphates (CaPs). Microorganisms must assimilate
P via membrane transport, so dissolution of CaPs to Pi
(H2PO4) is considered essential to the global P cycle.
Evaluation of samples from soils throughout the world has
shown that, in general, the direct oxidation pathway provides
the biochemical basis for highly efficacious phosphate
solubilization in Gram-negative bacteria via diffusion of the
strong organic acids produced in the periplasm into the
adjacent environment. Therefore, the quinoprotein glucose
dehydrogenase (PQQGDH) may play a key role in the
nutritional ecophysiology of soil bacteria. MPS bacteria may
be used for industrial bioprocessing of rock phosphate ore (a
substituted fluroapatite) or even for direct inoculation of
soils as a ‘biofertilizer’ analogous to nitrogen-fixing
bacteria. Both the agronomic and ecological aspects of the
direct oxidation mediated MPS trait. (Gold stein et al., 2003)
Among the bacterial genera with this capacity are
Pseudomonas, Bacillus, Rhizobium, Burkholderia,
Achromobacter, Agrobacterium, Microccocus, Aereobacter,
Flavobacterium and Erwinia (Babu-khan et al 1995;
Goldstein, 1987; Sperber 1958; Rodríguez and Fraga, 1999).
MECHANISM OF PHOSPHATE
SOLUBILIZATION
A number of theories have been proposed to explain the
mechanism of phosphate solubilization. Important among
them are:
Acid production theory
Proton and enzyme theory
ACID PRODUCTION THEORY
According to this theory, the process of phosphate
solubilization by PSM is due to the production of organic
acids which is accompanied by the acidification of the
medium (Puente et al., 2004). A decrease in the pH of the
filtrate from the initial value of 7.0 to a final value of 2.0
was recorded by many workers (Gaur and Sachar 1980;
Gaind and Gaur 1990, 1991; Illmer and Schinner, 1992). The
analysis of culture filtrates of PSMs has shown the presence
Phosphate Solubilization: Their Mechanism Genetics And Application
of number of organic acids such as malic, glyoxalic,
succinic, fumaric, tartaric, alpha keto butyric, oxalic, citric,
2-ketogluconic and gluconic acid (Lapeyrie et al., 1991;
Cuningham and kuaick, 1992; Ilmer and schiner, 1995;
Fasim et al., 2002, Gadd 1997; Kim et al., 1997)
The amount and type of the organic acid produced varied
with the microorganism. The organic acids released in the
culture filtrates react with the insoluble phosphate. The
amount of soluble phosphate released depends on the
strength and type of acid. Aliphatic acids are found to be
more effective in P solubilization then phenolic acids and
citric acids. Fumaric acid has highest P solubilizing ability.
Tribasic and dibasic acids are also more effective than
monobasic acids. In the presence of tribasic acids and
dibasic acids, a secondary effect appears due to ability of
these acids to form unionized association compounds with
calcium thereby removing calcium from the solution and
increasing soluble phosphate concentration (Gaur and
Gaind,1999).
Organic acids contribute to the lowering of solution pH as
they dissociate in a pH dependent equilibrium, into their
respective anion(s) and proton(s). Organic acids buffer
solution pH and will continue to dissociate as protons are
consumed by the dissolution reaction (Welch et al., 2002).
Similarly, microorganisms often export organic acids as
anions (Duro and Serrano 1981, Konings 1985, Netik et al.,
1997).
Besides organic acids, inorganic acids such as nitric and
sulphuric acids are also produced by the nitrifying bacteria
and thiobacillus during the oxidation of nitrogenous or
inorganic compounds of sulphur which react with calcium
phosphate and convert them into soluble forms (Gaur and
Gaind, 1999).
The most efficient mineral phosphate solubilization (MPS)
phenotype in Gram negative bacteria results from
extracellular oxidation of glucose via the quinoprotein
glucose dehydrogenase to gluconic acid (Kpomblekou and
Tabatabai, 1994; Hilda and Fraga, 1999; Hilda et al., 2000).
The resulting pH change and reduction potential are thought
to be responsible for the dissolution of phosphate in the
culture medium.
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Figure 4
Figure 4: Production of gluconic acid via the alternative
extracellular oxidation pathway of glucose metabolism.
(Source: http://www.ucc.ie/biomerit/simon%20image.gif)
Gluconic acid biosynthesis is carried out by the glucose
dehydrogenase (GDH) enzyme and the co-factor,
pyrroloquinoline quinone (PQQ). Goldstein and Liu (1987)
cloned a gene from Erwinia herbicola that is involved in
mineral phosphate solubilization. The expression of this
gene allowed production of gluconic acid and mineral
phosphate solubilization activity in E.coli HB101.
Gluconic acid is the principal organic acid produced by
Pseudomonas sp. (Illmer and Schinner, 1992), Erwinia
herbicola (Liu et al., 1992) Pseudomonas cepacia (Goldstein
et al., 1994) and Burkholderia cepacia (Rodríguez and Fraga
1999) Rhizobium leguminosarum (Halder et al., 1990)
Rhizobium meliloti (Halder and Chakrabartty, 1993) and
Bacillus firmus (Banik and Dey, 1982) produce noticeable
amounts of 2-ketogluconic acid. Fasim et al., (2002) have
reported bacterial solubilization of insoluble zinc oxide and
zinc phosphate, mediated by the production of gluconic and
2-ketogluconic acid. Other organic acids, such as lactic,
isovaleric, isobutyric, acetic, glycolic, oxalic, malonic and
succinic acids are also generated by different phosphate
solubilizing bacteria (Rodríguez and Fraga 1999).
Goebel and Krieg (1984) showed that gluconic acid was not
formed during growth of either A. brasilense or A. lipoferum
on fructose (a common carbon source for both), and was
detected only during growth of glucose. Rodríguez (2004)
reported that A. brasilense can produce gluconic acid in vitro
when grown on fructose and amended with glucose as an
inducer for gluconic acid production and have in vitro
phosphate solubilizing capability.
Glucose is the precursor for synthesis of gluconic acid
(Rodrigues etal 2004). This has suggested that Phosphate
Phosphate Solubilization: Their Mechanism Genetics And Application

solubilization in these strains is mediated by glucose or
gluconic acid metabolism. As solubilization of phosphate
preceded detection of gluconic acid in the medium, perhaps
even low levels of the acid (below the detection level of
HPLC) started to dissolve the sparingly soluble phosphate.
Alternatively, consumption of gluconic acid by growing
cells could also take place. In A. brasilense, reduction in the
quantity of soluble phosphate after incubation for 48 h can
be explained as auto consumption of soluble phosphate by
the growing bacterial population (Rodriguez et al., 2000).
process of nitrate accumulation in soils might play an
important role for the weathering of rock in general.
The nature and type of acid production is mainly dependent
on the carbon source (Reyes et al., 1999). In general, oxalic,
citric, and gluconic acid, are strong solubilizing agents of
feldspar, biotite, and phyllosilicates, (Torre et al., 1993)
Figure 5
Table 1: Gluconic acid production by various bacterial
strains

The latter may result from production of gluconic acid and

NH4 + uptake, which may release protons to the medium. In
the faster growing A. brasilense strains, perhaps the cells
used more NO-3 at the end of the incubation time, thereby
releasing OH-, which may account for the higher pH after 48
h. The metabolic mechanism by which gluconic acid was
produced was not explored (Rodríguez et al., 2004)

The P-solubilizing capability of gluconic acid was much

higher as compared to 2-keto-gluconic acid in the filtrate
from strain CC-Al74 culture. The process of acidification
and chelation by gluconic acid and 2-keto gluconic acid
dissolved tri calcium phosphate (TCP) in cultural medium.
The chelation property of gluconic acid enables it to form
insoluble complex. Insoluble metal forms may be solubilized
by protons, with Ca++ liberating phosphates (Kpomblekou
and Tabatabai, 1994; Reyes et al., 1999; Shekhar et al.,
2000).
Protons can be pumped into the external medium by various
membrane associated pumps which set up ionic gradients for
the acquisition of nutrients (Jones and Gadd, 1990; Sigler,
and Hofer, 1991; Gadd, 1993). In addition, protons arise
from produced organic acids which also possess an organic
acid anion which is usually capable of forming a complex
with metal cation (Burgstaller,. and Schinner, 1993; Hughes
and Poole, 1991).
The production of citric or gluconic acid and the extrusion of
+
H result from membrane transport mechanisms was
described as possible mechanism for dissolving rock-
phosphate from hydroxy apatite, iron phosphate, and
aluminum phosphate by Penicillium rugulosum (Reyes et al.,
1999). These processes are influenced by the sources of the
nitrogen, phosphate, and carbon. Citric acid production and
the resulting amount of phosphate dissolution are increased
if nitrate is the only nitrogen source. Because citric acid is
involved not only in the dissolution of phosphate but also in
dissolution of iron and other metals from minerals, the
PROTON AND ENZYME THEORY
Esterase type enzymes are known to be involved in
liberating phosphorus from organic phosphatic compounds.
PSMs (phosphate solubilizing microorganisms) are also
known to produce phosphatase enzyme along with acids
which cause the solubilization of P in aquatic environment
(Alghazali et al., 1986). Illmer and Schinner (1995) reported
that out of the four efficient phosphates solubilizing
microbes, Penicillium aurathiogriseum, Penicilllium
simplicissimum, Aspergillus niger and Pseudomonas sp only
A .niger could produce orgainic acid. Two most probable
explanations for this are:
Solubilization without acid production is due to the release
of protons accompanying respiration or ammonium
assimilation (Taha et al., 1969; Kucey 1983; Dighton and
Boddy 1989; Parks et al., 1990)
More solubilization occurs with ammonium salts than with
nitrate salts as the nitrogen source in the media (Gaur and
Gaind, 1999).
Besides these two mechanisms the production of chelating
substances (Luo et al., 1993) H2S, CO2 (Kapoor et al., 1989)
mineral acids, siderophores (Bossier et al., 1988)
biologically active substances like indole acetic acids,

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Phosphate Solubilization: Their Mechanism Genetics And Application

gibberllines and cytokinins (Kucey et al., 1988) are also
correlated with Phosphate solubilization. Chelation involves
the formation of two or more coordinate bonds between an
anionic or polar molecule and a cation, resulting in a ring
structure complex (Whitelaw, 2000). Organic acid anions,
with oxygen containing hydroxyl and carboxyl groups, have
the ability to form stable complexes with cations such as
Ca2+, Fe2+, Fe3+, and Al3+, that are often bound with phosphate
in poorly forms (Jones ,1998 ; Kucey 1988)
Dissolution of phosphate in soil is a very important process
for plant growth. Several studies have shown that the
phosphate uptake by plants can be markedly increased by
either mycorrhizal fungi (Azcon-Aguilar et al., 1986) or
inoculation of soil with species capable of solubilizing free
phosphate, such as P. Bilaii (Cunningham and Kuiack 1992;
Uzair et al., 2006).
PHOSPHATE –PLANT INTERACTION
Phosphorus is one of one of the major plant nutrient limiting
plant growth. It plays a key role in nutrition of plants as it
promotes development of deeper roots. The average soil is
rich in phosphorus as it contains about 0.05% (w/w)
phosphorus (Barber, 1984) but only one tenth of this is
available to plants approximately 95–99% is present in the
form of insoluble phosphates and hence cannot be utilized
by the plants and due to its poor solubility and chemical
fixation in the soil (Gaurand Gaind, 1999) causing a low
efficiency of soluble P fertilizers.
To increase the availability of phosphorus for plants, large
amounts of fertilizer is used on a regular basis. But after
application, a large proportion of fertilizer phosphorus is
quickly transferred to the insoluble forms. Therefore, very
little percentage of the applied phosphorus is used, making
continuous application necessary. (Abd Alla, 1994).
Soils microorganisms are involved in a range of processes
that affect Phosphate transformation and thus influence the
subsequent availability of phosphate to plant roots
(Richardson, 2001). Free living phosphate solubilizing
microorganisms (PSM) are always present in soils. The
populations of inorganic Phosphate solubilizing
microorganisms are sometimes very low, less than 102 CFU
-1
g of soil as observed in a soil in Northern Spain (Peix et al.,
2001). In four Quebec soils the number of root free PSM
-1
ranged from 2.5-to 3 x 106 CFU g of soil and they
represented from 26- 46% of the total soil microflora
(Chabot et al., 1993). As observed with other soil microbes
the number of PSM is more important in the rhizosphere
than in non rhizosphere soil (Kucey et al., 1989), and the
number of phosphate solubilizing bacteria is more important
than that of fungi (Kucey, 1983). However, inoculation
studies aimed to improving P nutrition in plants involved
bacteria and fungi, and is commercially available in Western
Canada as the phosphate inoculant Jumpstart (Philom Bios,
Saskatoon, Sask.). They are sold for wheat, canola, mustard
and other legumes and contain an bacterial strain of
Penicillium Bilaii. (http://www.philombios.ca/).
PLANT GROWTH PROMOTING BACTERIA
Although plant growth promoting bacteria occur in soil,
usually their numbers are not high enough to compete with
other bacteria commonly established in the rhizosphere.
Therefore, for agronomic utility, inoculation of plants by
target microorganisms at a much higher concentration than
those normally found in soil is necessary to take advantage
of their beneficial properties for plant yield enhancement.
(Igual, 2001)

Figure 6
Figure 5: Soil and root interactions

(http://www.sare.org/publications/bsbc/fig3_3.jpg)

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Phosphate Solubilization: Their Mechanism Genetics And Application
Figure 7
Figure 6: Microbial activities in the soil for plant growth
promotion.
(Source: http://www.treepower.org/soils/soil-benefits.jpg)
In recent years, interest in soil microorganisms that can
promote plant growth has been increased considerably. The
use of PGPRs to control soil borne pathogens is a practice
with a promising future, because the Montreal Protocol (an
international treaty to protect the earth from the detrimental
effects) proposes the elimination of toxic chemicals. This
has forced the plant scientists to look for new alternatives to
replace fertilizers. A number of different bacteria have been
reported to promote plant growth, including Azotobacter sp.,
Azospirillum sp., Pseudomonas sp., Acetobacter sp.,
Burkholderia sp. and Bacillus sp. (Rodrigues and Fraga
1999)
MECHANISM OF PLANT GROWTH PROMOTION
PGPR use one or more of direct or indirect mechanisms of
action to improve plant growth and health. These
mechanisms can probably be active simultaneously or
sequentially at different stages of plant growth. They include
1. Phosphate solubilization,
2. Biological nitrogen fixation
3. Biological control of plant pathogens
4. Improvement of other plant nutrients uptake
5. Phytohormone production like indole-3-acetic acid
and indole butyric acid
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The Phosphate solubilization effect seems to be the most
important mechanism of plant growth promotion in
moderately to fertile soils (Chabot et al., 1998). Strains from
the genera Pseudomonas, Bacillus and Rhizobium are among
the most powerful P solubilizers (Rodriguez and Fraga,
1999)
Rhizobia have well known beneficial symbiotic atmospheric
nitrogen fixing symbiosis with legumes, have an excellent
potential to be used as PGPR with non legumes (Antoun et
al., 1998).
Biological control of plant pathogens and deleterious
microbes, through the production of antibiotics, lytic
enzymes, hydrogen cyanide, and siderophores. Induction of
the systemic resistance against many pathogens, insect and
nematodes is also a recent indirect mechanism of action of
PGPR (Ramamoorthy et al., 2001; Zehnder et al., 2001)
They assist competition for nutrients and space which
significantly improve plant health and promote growth as
evidenced by increases in seedling emergence, vigor and
yield (Antoun and Kloepper, 2001).
Some PGPR have the enzyme 1-aminocyclopropane-1-
carboxylate (ACC) deaminase, which hydrolyses ACC, the
immediate precursor of ethylene in plants (Glick et al.,
1995). By lowering ethylene concentration in seedlings and
thus its inhibitory effect, these PGPR stimulate seedlings
root length (Glick et al., 1998).
PGPR can promote mycorrhizal functioning. Recently for
example, Villegas and Fortin (2001) showed an interesting
specific synergistic interaction between the Phosphate
solubilizing bacterium Pseudomonas aeruginosa and the AM
fungus Glomus intraradices. No synergistic effect was
observed with the other two Phosphate solubilizing bacteria
tested (Pseudomonas putida and Serratia plymuthica)
showing the specificity of this interaction.
All these traits that can be present in PGPR, illustrate how it
is complex and difficult to associate the promotion of plant
growth with Phosphate solubilization, and they explain in
part the reason of obtaining better responses from plant
inoculated with a mixture of PGPR. (Hodge, 2000) This has
also forced plant scientist to look for PGPRs having more
than one above-mentioned PGPRs trait in it.
The amelioration of phosphate deficiency by the application
of costly and environmentally hazardous phosphate
fertilizers is not an ideal solution and has generated serious
Phosphate Solubilization: Their Mechanism Genetics And Application
issues about the continued viability of current agriculture
practice. This has led to a search for more environmentally
friendly and economically feasible strategies to improve
crop production in low phosphorus soils. In an ideal manner,
such strategies should enable the efficient use of phosphate
solubilizing microorganisms. Several scientists have
reported the ability of different bacterial species to solubilize
insoluble inorganic phosphate compounds, such as
tricalcium phosphate and dicalcium phosphate (Gaur and
Gaind, 1999).
ROLE OF PHOSPHATE SOLUBILIZING
BACTERIA AS BIOFERTILIZER
Biofertilizers are organisms that enrich the nutrient quality
of soil. The main sources of biofertilizers are bacteria, fungi,
and cyanobacteria (blue-green algae). Plants have a number
of relationships with fungi, bacteria, and algae. After the
introduction of chemical fertilizers during the last century,
farmers were happy of getting increased yield in agriculture.
But slowly chemical fertilizers started displaying their ill-
effects such as leaching out, polluting water basins,
destroying flora and fauna including friendly organisms,
making the crop more susceptible to the attack of diseases,
reducing the soil fertility and thus causing irreparable
damage to the eco system (Rodrigues and Fraga 1999).
The principle behind this strategy is that microbes have
various abilities which could be exploited for better farming
practices. Some of them help in combat diseases while some
have the ability to degrade soil complex compounds into
simpler forms which are utilized by plants for their growth.
They are extremely beneficial in enriching the soil by
producing organic nutrients for the soil. To convert insoluble
phosphates to a form accessible to the plants, like
orthophosphate, is an important trait for a PGPB for
increasing plant yields (Hilda et al., 2006). Microbes having
the ability to dissolve appreciable amount of phosphates is
not rare. Some of them are already used as commercial
biofertilizers for agricultural improvements (Rodrigues and
Fraga 1999).The use of microbial products has certain
advantages over conventional chemicals: they are considered
safer than many of the chemicals now in use; they do not
accumulate in the food chain; the target organisms seldom
develop resistance as is the case when chemical agents are
used; and biofertilizing agents are not considered harmful to
ecological processes or the environment (Rodrigues and
Frega 1999).
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Figure 8
Table 2: Use of plant growth promoting bacteria as
biofertilizers
According to Statistics, the worldwide transaction amount of
fertilizer is roughly US$40 billion. Out of this, 135 million
metric tons of chemical fertilizer is applied each year, with
sales volume of about US$30 billion. Although there are no
clear application statistics for biofertilizer, however, its sales
volume is estimated to be as much as US$3 billion.
Commercial biofertilizers claiming to undergo phosphate
solubilization using mixed bacterial cultures have been
developed. Examples of these are: ‘Phylazonit-M’
(permission at No. 9961, 1992, by the Ministry of
Agriculture of Hungary), a product containing Bacillus
Megaterium; Azotobacter Chroococcum, which allows an
increase in N and P supply to the plants; and the product
known as ‘KYUSEI EM’ (EM Technologies, Inc.) (
Rodrigues and Fraga 1999).
PSBS AS PLANT GROWTH PROMOTERS
The substantial number of bacterial species, mostly those
associated with the plant rhizosphere, may exert a beneficial
effect upon plant growth. This group of bacteria has been
termed “plant growth promoting bacteria” or PGPB, and
among them, some phosphatesolubilizing bacteria (PSB) are
already used as commercial biofertilizers for agricultural
improvements. (SubbaRao1993; Rodríguez and Fraga 1999)
Among PGPR, phosphate solubilizing bacteria (PSB) are
considered as promising biofertilizers since they can supply
plants with phosphorus (P) from sources otherwise poorly
available. Beneficial effects of the inoculation with PSB to
many crop plants have been described by numerous authors
(Antoun et al., 1998; Chabot et al., 1998; Pal, 1998; Peix et
Phosphate Solubilization: Their Mechanism Genetics And Application

al., 2001a, b; Sarawgi et al., 1999; Tomar et al., 1996). PHYTOHORMONES

Moreover, synergistic interactions on plant growth have
been observed by co-inoculation of PSB with other bacteria,
such as Azospirillum (Alagawadi and Gaur, 1992; Belimov
et al., 1995) and Azotobacter (Kundu and Gaur, 1984), or
with vesicular arbuscular mycorrhizae (Toro et al., 1997,
1998).In addition many Pseudomonas strains, promote plant
growth by mechanisms such as the production of plant
growth regulators and vitamins, enhancement of plant
nutrient uptake and suppression of pathogenic or deleterious
organisms (Davison, 1998; Glick, 1995; O’Sullivanand
O’Gara, 1992).
Plant hormones also known as plant growth substances are
naturally occurring chemicals that control plant growth and
development. They regulate the rate at which individual part
of a plant growth, integrate growth of those parts to form the
whole organism and control reproduction. Since 1937,
gibberllin, ethylene, cytokinin and abscisic acid have joined
auxin as phytohormones and regarded as”classical
five”(Ranjan2003).
Figure 11
Figure 8: Effect of Auxin at molecular level

PHOSPHATE SUPPLY AND DEMAND

The expected annual growth rate in world demand for
phosphate fertilizers is about 2.8 percent until 2012 (Figure
2), for an increase of 5 million tonnes P2O5compared with
2006.

About 58 percent of this growth will take place in Asia;

consumption growth in South Asia is projected to surpass
growth in East Asia at almost 5 percent/year. Rapid growth
will also occur in East Europe and Central Asia (from a low
base) and in Latin America. Phosphate fertilizer The primary auxin in plants is indole- 3- acetic acid.
consumption will continue to decline marginally in West Although other compounds with auxin activity such as
Europe and Central Europe. indole butyric acid, phenyl acetic acid are also present in
plants but little is known about their physiological role
Figure 9
(Normanly et al., 1995).

At the molecular level, auxins influence cell division, cell

elongation and cell differentiation (Davies, 1995).At the
macroscopic level, auxins direct vascular development,
promotes apical dominance and lateral root formation, and
mediate gravitropism and phototropism (Davies, 1995).
Indole -3-acetic acid (IAA) and indole-3-butyric acid (IBA)
are two endogenous auxins that can be interconverted
(Ludwig-Miuller, 2000; Baartel et al., 1997).

World phosphoric acid capacity is expected to increase by

18 percent (8.1 million tonnes) by 2012. The largest increase
is expected towards the end of the projection period in West
Asia, when significant additional supply capacity should
become operational in Saudi Arabia. Other significant
expansions are scheduled in Africa (Morocco), China, and
Latin America. In all other regions, phosphoric acid capacity
is expected to remain almost constant or increase marginally
during the period.(www.cfo)

PRODUCTION OF GROWTH STIMULATING

9 of 19
Phosphate Solubilization: Their Mechanism Genetics And Application
Figure 12
Figure 9: Chemical structure of PQQ (4, 5-dihydro-4, 5-
dioxo-1H-pyrrolo- [2, 3-f] quinoline-2, 7, 9-tricarboxylic
acid)
(Source:http://www.dlarborist.com/treetrends/2005/05/27/au
xin_action_s.jpg)
IAA is the main auxin in plants, controlling many important
physiological processes including cell enlargement and
division, tissue differentiation, and responses to light and
gravity (Taiz and Zeiger, 1998). Bacterial IAA producers
(BIPs) have the potential to interfere with any of these
processes by input of IAA into the plant’s auxin pool. The
consequence for the plant is usually a function of (i) the
amount of IAA that is produced and (ii) the sensitivity of the
plant tissue to changes in IAA concentration. A root, for
instance, is one of the plant’s organs that is most sensitive to
fluctuations in IAA, and its response to increasing amounts
of exogenous IAA extends elongation of the primary root,
formation of lateral and adventitious roots, (Davies, 1995).
IBA was found to increase root development in the
propagation of stem cuttings (Ranjan, et al., 2003). They
also produced by various bacteria which live in association
with plants. The ability of auxins to stimulate adventitious
root formation is well documented (Weisman et al., 1989).
IAA is the main auxin in plants, controlling many important
physiological processes including cell enlargement and
division, tissue differentiation, and responses to light and
gravity. Plant growth promoting effects exerted by some
plant beneficial bacteria are due to the bacterial production
10 of 19
of plant hormones such as IAA, cytokinins, and gibberellins.
These plant growth hormones increase growth rates and
improve yields of the host plants. It has been shown that
continuous supply of Auxin via cotton wick stimulates
growth of two dwarf mutants of pea (Yang et al.,
1996).Indole butyric acid was found to increase root
developments (Ranjan, et al., 2003). IAA and IBA are two
endogenous auxins that can be inter converted (Ludwig-
Miuller, 2000; Baartel et al., 1996). Karcaz and Burdach,
2002 reported that IAA is the principal regulator of plant
elongation in maize coleoptile. Induction of lateral roots
with increased no of root hairs and root lateral is a growth
response attributed to IAA production by other bacteria,
which improves their nutrient uptake efficiency (Okon
1985).
GENETIC BASIS OF PSBS (PHOSPHATE
SOLUBILIZING BACTERIA)
The genetic basis of phosphate solubilization is not well
understood. The production of organic acids is considered to
be the principal mechanism for mineral phosphate
solubilization. It could be assumed that any gene involved in
organic acid synthesis might have an effect on this character.
Several acid phosphatase genes from Gram negative bacteria
have been isolated and characterized (Rossolini et al., 1998).
For example, the acpA gene isolated from Francisella
tularensis expresses an acid phosphatase with optimum
action at pH 6, with a wide range of substrate specificity
(Reilly et al., 1996). Also, genes encoding nonspecific acid
phosphatases class A (PhoC) and class B (NapA) isolated
from Morganella morganii are very promising .It showed the
highest extracellular phytase activity, and diluted culture
filtrates of these strains stimulated growth of maize seedlings
under limited phosphate in the presence of phytate. (Idris et
al., 2002).
For bacteria whose genes express in E. coli, it is possible to
use plasmid cloning systems to ‘trap’ PQQGDH genes from
highly efficacious MPS bacteria via functional
complementation. E. coli K12 and derivatives such as DH5α
constitutively synthesize apoGDH but do not synthesize
PQQ. Therefore, screening genomic libraries of mineral
phosphate solubilization (MPS) bacteria for gluconic acid
production traps PQQ biosynthesis genes (Liu et al., 1992).
Conversely, screening in E. coli AG121, a Tn5 knockout of
gcd in HB101 with exogenous PQQ can trap apoGDH genes.
In addition, he identified several novel DNA fragments that
induce gluconic acid production in DH5α or HB101, but not
Phosphate Solubilization: Their Mechanism Genetics And Application

in AG121 (Babu khan et al., 1995: krishnaraj, 2001). These
clones have no sequence homology to known PQQ genes but
at least two have some homology to trans-membrane or
periplasmic proteins that could mediate an environmental
signal. Goldstein develops a strategy to open the funnel,
thereby increasing the rate of catalysis with a concomitant
increase in gluconic acid production and CaP solubilization.
(Goldstein, 2003)
GENETIC BASIS OF INORGANIC PHOSPHATE
SOLUBILIZATION
The genetic basis of mineral phosphate solubilization i.e. the
MPS phenotype is not well understood (Goldstein and Liu,
1987). Because the production of organic acids is considered
to be the principal mechanism for mineral phosphate
solubilization, it could be assumed that any gene involved in
organic acid synthesis might have an effect on this character.
dehydrogenase. PQQ belongs to the family of quinone
cofactors that has been recognized as the third class of redox
cofactors following pyridine nucleotide- and flavin-
dependent cofactors.
PQQ is a prosthetic group required by several bacterial
dehydrogenases, including methanol dehydrogenase (MDH)
of Gram negative methylotrophs and some glucose
dehydrogenases. PQQ is derived from two amino acids,
tyrosine and glutamic acid (Houck, 1991,Van Kleef, 1988),
but the pathway for its biosynthesis is unknown.
Figure 13
Figure 10: Alignment of pqq gene cluster. Equivalent genes
have the same pattern

Very little is known regarding the genetic regulation

governing the mineral phosphate solubilization trait. In fact,
the information about the genetic or biochemical
mechanisms involved in the synthesis of the GDH-PQQ halo
enzyme is scant, and variations between constitutive and
inducible phenotypes are observed among several bacterial
species (Goldstein, 1994). Glucose, gluconate, manitol, and
glycerol are among the possible inducers of the halo enzyme
activity (Vanschie, 1987)

Goldstein and Liu (1987) cloned a gene from Erwinia

herbicola that is involved in mineral phosphate solubilization
by screening the antibiotic resistant recombinants from a
genomic library in a medium containing hydroxyapatite as
the source of P. The expression of this gene allowed
production of gluconic acid and mineral phosphate
solubilization activity in E.coli HB101. Sequence analysis of
this gene (Liu, 1992) suggested its probable involvement in
the synthesis of the enzyme pyrroloquinoline quinone (PQQ)
synthase, which directs the synthesis of PQQ, a co-factor
necessary for the formation of the holoenzyme glucose
dehydrogenase (GDH)-PQQ. This enzyme catalyzes the
formation of gluconic acid from glucose by the direct
oxidation pathway. (Rodrigues and Fraga, 1999;
Goldstein1995; Goldstein 2003)
PYRROLOQUINOLINE QUINONE (PQQ)
PQQ (4, 5-dihydro-4, 5-dioxo-1H-pyrrolo- [2, 3- ]
quinoline-2, 7, 9-tricarboxylic acid) PQQ is an aromatic,
tricyclic ortho-quinone that serves as the redox cofactor for
several bacterial dehydrogenases. (Fig. 9) Among the best-
known examples are methanol dehydrogenase and glucose
(Source:
http://www.chris-anthony.co.uk/reseach%20pics/pqq.jpg)
All carbon and nitrogen atoms of PQQ are derived from
conserved tyrosine and glutamate residues of the PQQA
peptide. R1 and R3 represent the N- and C-terminal portions
of PQQA, respectively. R2 represent a three-amino-acid
linker between Glu and Tyr.
PQQ is an important cofactor of bacterial dehydrogenases,
linking the oxidation of many different compounds to the
respiratory chain. PQQ was the first of the class of quinone
cofactors that have been discovered in the last 18 years and
make up the prosthetic group of quinoproteins (Duine, 1991;
Klinman &Mu, 1994, Klinman, 1996). Although plants and
animals do not produce PQQ themselves, PQQ has invoked
considerable interest because of its presence in human milk
and its remarkable antioxidant properties. Recently, the first
potential eukaryotic PQQ dependent enzyme [(aminoadipic
6-semialdehyde-dehydrogenase (AASDH; U26)] has been

11 of 19
Phosphate Solubilization: Their Mechanism Genetics And Application
identified, indicating that PQQ may function as a vitamin in
mammals as well (Duine1999).
PRESENT STATUS, DISTRIBUTION AND
SIGNIFICANCE OF PQQ
PQQ was discovered in 1979 from a bacterium, and
afterward it was reported to be in common foods. Because
PQQ-deprived mice showed several abnormalities, such as
poor development and breakable skin, PQQ has been
considered as a candidate for vitamin. It was a mystery, that
until 2003 it was not identified as vitamin. Since the first
vitamin (now called vitamin B1) was discovered in 1910 by
Dr. U. Suzuki, thirteen substances have been recognized as
vitamins; the latest one was vitamin B12 found in 1948.So it
takes 55 years to discover “PQQ” a previously identified
substance as new vitamin ( Choi 2008; Kashara and
kato2003).
After it had been established that PQQ occurs in several
bacterial enzymes, a logical next question was whether it
also occurs in higher organisms. Perhaps stimulated by the
reports (Paz, 1988) that PQQ occurs at high levels in certain
body fluids and tissues of mammalian milk, and in citrus
fruits, several reports followed in which beneficial effects
were ascribed to its administration, e.g. that a diet
supplemented with PQQ improved the “health” of mice
substantially or prevented the outbreak of certain diseases
(Duine, 1999).
The quinoprotein glucose dehydrogenase (GCD) has been
demonstrated in a number of microorganisms including the
enteric bacteria Klebsiella pneumoniae, E. coli and
Salmonella typhimurium, and in Acinetobacter,
Pseudomonas, Agrobacterium and Gluconobacter species. In
most cases, GCD is a membrane bound enzyme which
oxidizes glucose to gluconate in the periplasmic space
(Duine, 1991). The formed gluconate is subsequently taken
up in a number of organisms via a specific transport system
and further metabolized. The reducing equivalents from
glucose are donated via PQQ to the respiratory chain.
Surprisingly, in some of the organisms mentioned above
only apo-GCD is found. Thus, K. pneumoniae synthesizes a
holo-GCD (Neijssel et al., 1983) but the closely related E.
coli and S. typhimurium can synthesize only apo-GCD but
not PQQ (Hommes et al., 1984, 1986). However, apo-GCDs
in these and other organisms can be converted into active
holo-GCDs by the addition of extracellular PQQ (Ameyama
et al., 1986; Hommes et al., 1986, 1984; Van Schie et al.,
1987).
12 of 19
It has been concluded that organisms like E. coli and S.
typhimurium lack the genes to synthesize PQQ and it
remains unclear what the role of such apo-GCDs could be. It
has been suggested that organisms that are unable to
synthesize PQQ could scavenge it from their surroundings
and activate their apo-quinoproteins (Matsushita & Adachi,
1993).
BIOSYNTHETIC ROUTE OF PQQ
The biosynthetic route of PQQ has not been elucidated yet,
but it has been proposed that glutamate and tyrosine are
precursors of PQQ (Houck et al., 1988, 1991; Van Kleef &
Duine, 1988). In all cases studied, the pqq operon contains a
small gene; in the case of K. pneumoniae, this is pqqA
which can encode a small peptide of 23-29 a containing a
glutamate and tyrosine residue at conserved positions.
Synthesis of the small peptide has been demonstrated
(Velterop, 1995) and it is thought to be the precursor of PQQ
(Goosen et al., 1989, 1992; Meulenberg et al., 1992). The K.
pneumoniae pqqF gene product shows similarities to
proteases like E. coli protease III (or pitrilysin) and
insulinases and it has been proposed that the PqqF enzyme is
involved in cleavage of the PqqA precursor (Meulenberg et
al., 1992).
Expression of the six K. pneumoniae pqq genes in E. coli
results in PQQ synthesis and an active GCD (Meulenberg et
al., 1990, 1992). Most likely PQQ biosynthesis takes place
in the cytoplasm and PQQ can be released into the medium.
Since the PQQ binding site of GCD faces the periplasm
(Yamada et al., 1993), it is clear that both complementation
of E. coli with the K. pneumoniae pqq genes as well as
addition of PQQ can result in an active GCD (Matsushita,
1997). From growth studies with E. coli ptsHI mutants
complemented with the complete set of K. pneumoniae pqq
genes or with plasmids lacking one of the six pqq genes and
from measurement of PQQ biosynthesis in the growth
medium, it was concluded that each of the six genes is
required for PQQ biosynthesis (Velterop et al., 1995).
In Methylobacterium extorquens AM1, the genes for PQQ
synthesis are found in two clusters, pqqAB (C/D) E and
pqqFG (Toyama et al., 1997). These gene designations
standardize the nomenclature with that of K. pneumoniae.
These genes in Methylobacterium strains were formerly
called pqqDGCBA (Morris et al., 1994) and pqqEF
(Springer et al., 1996), respectively. In M. extorquens AM1,
pqqC and pqqD are not separate genes. Instead, they are
fused into a single gene, pqqCD (Toyama et al., 1997).
Phosphate Solubilization: Their Mechanism Genetics And Application
Transcriptional analysis of pqqA in M. extorquens AM1
showed that a short mRNA containing only pqqA was
produced at a much higher level than the mRNA for pqqAB
(Ramamoorthi & Mary, 1995). Similar results were obtained
in the case of K. pneumoniae by using pqqTn5::lacZ operon
fusions on the chromosome (Velterop et al., 1995).These
results are also consistent with the hypothesis that pqqA is a
PQQ precursor, because stoichiometric amounts rather than
catalytic amounts are required if the peptide is the substrate
for PQQ synthesis (Velterop et al .,1995)..
GENETICS OF PQQ
Genes involved in PQQ synthesis have been cloned from
Acinetobacter calcoaceticus (Goosen et al., 1989), K.
pneumoniae (Meulenberg et al., 1992), Pseudomonas
fluorescens CHA0 (Schnider et al., 1995), Methylobacterium
organophilum DSM 760 (Biville et al., 1989) and M.
extorquens AM1 (Morris et al., 1994). In A. calcoaceticus,
five pqq genes were identified and sequenced, designated
IV, V, I, II and III (Goosen et al., 1989). In K. pneumoniae,
genes analogous to those were identified and designated
pqqABCDE, and in addition, a sixth gene was found
immediately downstream of pqqE, designated pqqF
(Meulenberg et al., 1992) In Methylobacterium strains, a
five gene cluster (designated pqqDGCBA) was identified by
complementation analysis (Biville et al., 1989; Morris et al.,
1994), and sequence data from M. extorquens AM1 showed
that the first three of these genes (pqqDGC) were analogous
to pqqABC of K. pneumoniae (Morris et al., 1994). In P.
fluorescens, genes analogous to pqqFAB of K. pneumoniae
have also been sequenced (Schnider et al., 1995).
Figure 14
Table 3: Percentage similarities between pqqBCDE genes of
different bacteria
(Source: Felder et al., 2000)
In all four sequences, a small gene is present that encodes a
13 of 19
peptide of 22-29 amino acids, which contains conserved
tyrosine and glutamate residues. Since tyrosine and
glutamate are the probable precursors for PQQ synthesis
(Van Kleef and Duine, 1988; Houck et al., 1991), it has been
proposed that this peptide is the precursor from which PQQ
is synthesized (Goosen et al., 1992). However, the
biochemical steps of PQQ synthesis are still unknown.
Velterop et al., (1995) examined PQQ synthesis in vitro.
A series of experiments was carried out in which cell
extracts of Escherichia coli containing all but one of the Pqq
proteins were combined with those containing the missing
Pqq protein. PQQ was produced in only one of these sets,
that involving PqqC. E. coli cells containing a clone
encoding all but the PqqC protein apparently produced an
intermediate of PQQ, found both in the culture medium and
in the cells. However, the mount of the intermediate was low
and it was unstable (Velterop1995).
{image:14}
(Source: Felder et al., 2000)
Genes involved in PQQ biosynthesis have been cloned from
several organisms. Five A. calcoaceticus pqq genes, pqqIV,
V, I, II, and III (Goosen et al., 1989, Goosen et al., 1987),
and six K. pneumoniae pqq genes, pqqA, B, C, D, E, and F
(Mellenberg 1990, Mellenberg et al., 1992), were cloned and
sequenced. Comparison of the deduced amino acid
sequences showed that the proteins encoded by the first five
genes of the K. pneumoniae pqq operon (pqqABCDE) show
similarity to the proteins encoded by the corresponding A.
calcoaceticus genes (49 to 64% identical amino acid
residues). The K. pneumoniae pqqF gene encodes a protein
that shows similarity to E. coli protease III and other
proteases (Mellenberg et al., 1992), but its equivalent has not
yet been found in A. calcoaceticus. Recently, three M.
extorquens AM1 pqq genes, pqqD, G, and C, have been
cloned and sequenced (Morris et al., 1994); pqqC was only
partly sequenced. The encoded proteins showed similarity to
the K. pneumoniae PqqA, B, and C proteins and the A.
calcoaceticus PqqIV, V, and I proteins, respectively. Four
additional pqq genes have been detected in M. extorquens by
isolation of mutants and complementation studies. From
similar studies, six (possibly seven) pqq genes have been
postulated in M. organophilum DSM760 (Biville, 1989).
Finally, a DNA fragment cloned from Erwinia herbicola
contained a gene encoding a protein similar to K.
pneumoniae PqqE and A. calcoaceticus PqqIII (Liu, et al.,
Phosphate Solubilization: Their Mechanism Genetics And Application
1992). Except for the K. pneumoniae PqqF protein, none of
the Pqq proteins shows similarity to other proteins in the
database. One of the pqq genes is small and may encode a
polypeptide of 24 amino acids (PqqIV, A. calcoaceticus), 23
amino acids (PqqA, K. pneumoniae), or 29 amino acids
(PqqD, M. extorquens AM1). Interestingly, these putative
polypeptides contain conserved glutamate and tyrosine
residues (positions 15 and 19, respectively, in K.
pneumoniae and the equivalents in A. calcoaceticus and M.
extorquens). Those residues have been suggested previously
as precursors in PQQ biosynthesis. Replacement of Glu-16
by Asp and Tyr-20 by Phe in A. calcoaceticus PqqIV
abolished PQQ biosynthesis. A frame shift in K.
pneumoniae pqqA had the same result (Melulenberg et al.,
1992). It was suggested that the PqqA/PqqIV polypeptide
might act as a precursor in PQQ biosynthesis (Goosen 1989;
Goosen 1992; Melulenberg et al., 1992).
In what way could a 24-amino-acid polypeptide be involved
in PQQ synthesis? Its small size makes a direct enzymatic
function in the conversion of glutamate and tyrosine to PQQ
unlikely. A regulatory role of the gene IV product in the
expression of the other pqq genes is also improbable, since
previous experiments already showed that gene IV is also
essential for PQQ synthesis in an E. coli strain. In these
experiments, the expression of the pqq genes from A.
calcoaceticus was under control of the E. coli lac promoter,
which excludes a transcriptional control of these genes by
the gene IV peptide. Therefore, it is likely that the small
polypeptide has a more direct role in synthesis of the
coenzyme (Velterop1995).
Seven genes, called pqq genes, are required for PQQ
biosynthesis in M. extorquens AM1, but their functions are
unknown (Moris et al., 1994; Nunn and Lidstrom 1986a,
Nunn and Lidstrom 1986b). The pqqD gene encodes a small
polypeptide of 29 amino acids containing conserved tyrosine
and glutamate residues separated by three amino acids
(Moris et al., 1994). Tyrosine and glutamate have been
shown to be the precursors of PQQ biosynthesis, and it has
been proposed that the peptide might serve as the substrate
for PQQ biosynthesis (Goosen, 1992; Houck, 1991;
Meulenberg1992).
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Phosphate Solubilization: Their Mechanism Genetics And Application

Author Information
Nuzhat Ahmed
Centre for Molecular Genetics, University of Karachi

Sadaf Shahab
Centre for Molecular Genetics, University of Karachi

19 of 19