Pseudomonas putida

In Pseudomonas putida, a Tn5 insertion within the ttg2A gene, encoding an MKL
family ABC, renders the cells sensitive to toluene (Vermeij et al., 1999).

From: ABC Proteins, 2003

Related terms:

Plasmid, Pseudomonas, Nested Gene, Bacterium, Biofilm, Degradation, Microor-

ganism, Escherichia coli

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Branched-Chain Amino Acids, Part B

Kunapuli T. Madhusudhan, John R. Sokatch, in Methods in Enzymology, 2000

Introduction
Branched-chain keto acid dehydrogenase (Bkd) is an important member of the --
keto acid dehydrogenase family of enzymes. This multienzyme complex is induced in
Pseudomonas putida1 and Pseudomonas aeruginosa2 by growth in media containing
branched-chain amino acids or branched-chain keto acids. The enzyme has been
characterized from several sources, including P. putida,3 P. aeruginosa,2 bovine kid-
ney,4 rabbit liver,5 rat kidney,6 and Bacillus subtilis.7 The purified branchedchain keto
acid dehydrogenase from P. putida and P. aeruginosa contains three components: E1
( 2 2, the dehydrogenase–decarboxylase), E2 (the transacylase), and E3 (lipoamide
dehydrogenase) or Lpd-Val.2,8 The genes of the bkd operon are bkdA1 and bkdA2,
encoding E1 and E1 , respectively; bkdB, encoding the E2 component; and lpdV,
encoding Lpd-Val.9−11 The mRNA of the bkd operon of P. putida encoding subunits
of the multienzyme complex is polycistronic and all four genes are tightly linked.11

BkdR is encoded in P. putida by bkdR, which is divergently transcribed from the

bkd operon. Chromosomal inactivations of bkdR in P. putida result in loss of
branched-chain keto acid dehydrogenase activity and are complemented by supply-
ing BkdR in trans. Therefore, expression of the bkd operon is positively regulated by
BkdR.12,13 In addition, mutations in bkdR have no effect on either branched-chain
amino acid transport or transamination in P. putida.12 BkdR shares 37.5% amino
acid identity with Lrp, the leucine-responsive protein of Escherichia coli,12,14 and is the
second member of this family to be characterized. Lrp is a global transcriptional reg-
ulator that regulates the expression of several operons either in a leucinedependent
or leucine-independent manner.15 Lrp complements bkdR mutations in P. putida.12
Anti-Lrp antibodies recognize BkdR but anti-BkdR antibody does not recognize Lrp
on Western blots.

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Basics of Biotechnology
David P. Clark, Nanette J. Pazdernik, in Biotechnology (Second Edition), 2016

Other Bacteria in Biotechnology

Other bacteria besides E. coli are used to produce biotechnology products. Bacillus
subtilis is a Gram-positive bacterium that is used as a research organism to study
the biology and genetics of Gram-positive organisms. Bacillus can form hard spores
that can survive almost indefinitely. It is also used in biotechnology. For industrial
production, secreting proteins through the single membrane of Gram-positive
bacteria is much easier than secreting them through the double membrane of
Gram-negative bacteria; therefore, Bacillus strains are used to make extracellular
enzymes such as proteases and amylases on a large scale.

Pseudomonas putida is a bacterium that normally lives in water. It is a Gram-neg-

ative bacterium like E. coli but is commonly used in environmental studies because
it is able to degrade many aromatic compounds. Streptomyces coelicolor is a soil
bacterium that is Gram positive. This organism degrades cellulose and chitin, and
also produces a large number of different antibiotics. Another example of a common
industrial microorganism is Corynebacterium glutamicum, which is used to produce
L-glutamic acid and L-lysine for the biotechnology industry.

Many different bacteria are used for biotechnology research because of their unique
qualities.

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Immobilized Cells
J.V. Sinisterra, H. Dalton, in Progress in Biotechnology, 1996
Microorganism and growth medium
Pseudomonas putida UV4, was provided by ICI Biological Products, Billingham U.K.
The cell culture conditions have been described in the literature(8). The bacteria
for immobilization were harvested after 24 h of incubation time. The concentration
of cells in the culture medium was measured by absorption at 600πm. The dry
cell weight (dew) was experimentally measured by drying at 120 °C to constant
weight, one pellet obtained by centrifugation of 40 ml of cell culture. The obtained
equivalence between bacteria concentration ( 106 cells/ml cell culture) and the dry
weight (dcw) was: 106 cells/ml cell culture = 3.4 10- 5 g. dew.

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Pseudomonas putida–based cell facto-

ries
Justyna Mozejko-Ciesielska, in Microbial Cell Factories Engineering for Production
of Biomolecules, 2021

Abstract
Pseudomonas putida is a metabolically versatile bacterium grown in various habitats
that have been intensively studied with regard to its potential to produce various
products of industrial relevance. Due to its special features like robust metabolism,
a broad tolerance to toxic compounds and oxidative stress, and easiness of genetic
modification, this bacterium is becoming microbial efficient cell factories for
many natural products with diverse biological functions. This chapter provides an
overview of P. putida as a host microorganism for the recombinant synthesis of
applicable bioproducts such as polyhydroxyalkanoates, surfactants, terpenoids, and
prodigiosin. This overview of bioproducts that could be produced by heterologous
gene expression and strain engineering confirms P. putida potential to be a work-
horse for industrial applications being a good provider of the molecular machinery.

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SEQUENCE DETERMINATION OF
THE PSEUDOMONAS PUTIDA CY-
TOCHROME P-450: ISOLATION AND
PARTIAL SEQUENCES OF THE CYS-
TEINE PEPTIDES1
Mitsuru Haniu, ... Irwin C. Gunsalus, in Microsomes, Drug Oxidations and Chemical
Carcinogenesis, Volume 1, 1980

I INTRODUCTION
Pseudomonas putida contains a camphor hydroxylating system which requires
NADH-putidaredoxin reductase, putidaredoxin and cytochrome P–450 (Gunsalus
et al, 1978). For investigating the structure-function relationships of the individual
components as well as a multienzyme complex, for a comparison of the chemical
structures of the individual components from different living species, for the com-
plete structural determination of the protein components by crystal X-ray diffraction
studies as well as for a variety of other important reasons, the amino acid sequences
of the protein components are essential. Thus far, our laboratory has sequenced
the putidaredoxin, an iron sulfur protein (Tanaka et al, 1974). Currently, our group
is sequencing the cytochrome P–450, the mixed function oxidase of the hydrox-
ylating system. Besides our major goal of completely sequencing the cytochrome
P-450cam, the role of the individual residues in the protein from a structure–function
standpoint is of interest. Probably, the amino acid side chains which play very vital
roles and can be studied by a variety of physicochemical procedures are the cysteine
residues. However, before we could carry out chemical modification experiments
of the cysteine residues, it was essential that a peptide fractionation procedure be
developed to isolate the individual cysteine containing peptides and a knowledge of
their sequences. In this present report, a procedure for the isolation of the cysteine
containing tryptic peptides and the partial sequences around the cysteine residues
are presented.

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The versatility of Pseudomonas putida

in the rhizosphere environment
Lázaro Molina, ... Juan-Luis Ramos, in Advances in Applied Microbiology, 2020

6 Plant growth promoting properties

Pseudomonas putida establishes a commensal relationship with plants. As described
above, plants feed P. putida in the rhizosphere through root exudates and in turn the
microbe helps the plants to grow by producing plant hormone precursors, favoring
the mobilization of nutrients and producing antibiotics that prevent the growth of
pathogens. Fig. 1 shows some of the positive effects of P. putida on stimulation of
plant growth. For instance, BIRD1 stimulates the formation of secondary roots which
help to increase the plant's root surface and concomitantly nutrient assimilation. The
increase in nutrient uptake results in a significantly larger sized plant in early stages
of growth and also because Pseudomonas prevents the growth of phytopathogens the
general state of the plants is better and the quality and quantity of the fruit is also
improved. One of the key molecules behind these positive effects is the overpro-
duction of indole acetic acid (IAA), a well-known phytohormone that P. putida BIRD1
produces through two parallel pathways (Roca et al., 2013). P. putida also produces
organic acids and phosphatases that help to mobilize insoluble inorganic phosphate
and release phosphate from organophosphorous compounds, respectively, making
phosphate available for plant uptake. In fact, having P. putida associated with some
crops can save as much as 50% on phosphorous rich fertilizers which in turn has an
enormous environmental benefit (Roca et al., 2013). Furthermore, Molina, Ramos,
and Espinosa-Urgel (2006) demonstrated the importance of capturing iron from soil
for the colonization of roots by P. putida. In fact, a feature that facilitates proliferation
of P. putida in the root is that they produce specific siderophores to solubilize
iron, and that in addition to their specific siderophore transport systems, they are
endowed with external membrane receptors able to capture iron chelated by foreign
siderophores (xenosiderophores) that are produce by other bacterial species (Roca et
al., 2013; Molina et al., 2006). This set of iron capturing systems makes P. putida an
authentic “thief ” of iron in soil. This “skill” not only helps them to acquire iron to
satisfy their own needs, but prevents the use of iron by other microbes which in turn
inhibits the growth of phytopathogens, a property that makes P. putida an indirect
biocontrol agent.

Pseudomonas putida can also play a role as a direct biocontrol agent; the presence of
this microorganism in the rhizosphere induces the plant systemic response, protect-
ing the plant host against pathogen infection and proliferation (Matilla et al., 2010),
a process that is characterized by the production of high levels of reactive oxygen
species (ROS) (Baxter, Mittler, & Suzuki, 2014). For this reason, proliferation in this
environment requires a high capacity of ROS removal. P. putida is endowed with a
large number of oxidative stress response genes encoding superoxide dismutases,
catalases, betaine (aldehyde) dehydrogenase and other enzymes, which play a role in
the detoxification of abundant free radicals in the rhizospheric environment (Matilla
et al., 2010).

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Aerobic Respiration—Chemoorgan-
otrophic Bacteria
H.W. Doelle, in Bacterial Metabolism (Second Edition), 1975

Lysine Metabolism
Pseudomonas putida utilizes lysine via two distinct pathways (59, 321, 599). l-Lysine
appears to be the substrate for the acyclic pathway and d-lysine for the cyclic pathway
leading via pipecolate (Fig. 7.19).

Fig. 7.19. Lysine metabolism by Pseudomonas putida(reprinted with permission of the

authors and the American Society of Biological Chemists) (267b).
Each of these pathways is selectively induced by l- or d-lysine and the appropri-
ate intermediates. The enzyme that catalyzes the conversion of d-lysine to Δ'--
piperideine-2-carboxylate is not known as yet. A Δ'-piperideine-2-carboxylate reduc-
tase (l-pipecolate: NADP 2-oxidoreductase; 59a), however, was found responsible for
the formation of l-pipecolate. The further metabolism of l-pipecolate is identical to
that described in Chapter 8.

Apart from the racemase (EC 5.1.1.5) (155a, 354), which can inter-convert d- and
l-lysine, an l-lysine 2-monooxygenase [l-lysine: oxygen 2-oxidoreductase (decar-
boxylating), EC 1.13.12.2] (120a, 131, 371, 371a) appears to be responsible for the
conversion to -aminovaleramide. The release of the amide group is obtained by
the action of -aminovaleramide amidase (321), which results in the formation of
aminovalerate. The next amino group is transferred to -ketoglutarate by a -amino-
valerate transaminase (155b) producing glutamic acid and glutaric semialdehyde.
The last intermediate, glutaric acid, is formed with the aid of an NAD+-dependent
glutaric semialdehyde dehydrogenase (glutarate-semialdehyde: NADP oxidoreduc-
tase, EC 1.2.1.20) (155c). Glutaric acid is further metabolized as described in Chapter
8.

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An introduction to microbial cell facto-

ries for production of biomolecules
Nisarg Gohil, ... Vijai Singh, in Microbial Cell Factories Engineering for Production
of Biomolecules, 2021

2.5 Pseudomonas putida as a cell factory

Pseudomonas putida is a versatile, ubiquitous, amenable, genetically accessible
alternative potent strain having the capacity to be used in industries for large-scale
production of diverse compounds due to its oxidative stress resistance, low nutri-
tional requirement, and high tolerance to toxins, high temperature, extreme pH, and
solvents (Kim and Park, 2014; Lemire et al., 2017; Hernandez-Arranz et al., 2019). It
is a promising alternative to E. coli for isoprenoid production (Hernandez-Arranz et
al., 2019). Besides this, it is also known for its ability to degrade aromatic compounds
significantly (Nogales et al., 2017). To date, it has been used to produce isoprenoids
such as limonene (Loeschcke and Thies, 2015), geranic acid (Mi et al., 2014), zeax-
anthin (Beuttler et al., 2011), and -carotene (Loeschcke et al., 2013; Sánchez-Pas-
cuala et al., 2019); aromatic compounds such as trans-cinnamate, l-phenylalanine,
2-phenylethanol, p-coumarate, p-hydroxybenzoate, and phenol (Molina-Santiago et
 al., 2016; Calero et al., 2018); amino acids such as N-methylglutamate (Mindt et al.,
2018); polyhydroxyalkanoates (Wang et al., 2011a); d-glucosaminic acid (Wu et al.,
2011); and amino acid-derived compounds such as phenol (Wierckx et al., 2005),
p-coumarate (Nijkamp et al., 2007), catechol (Prakash et al., 2010), and aliphatic
alcohols (Bosetti et al., 1992) (refer Table 5).

Table 5. Important biomolecules produced by Pseudomonas putida cell factory.

Limonene Loeschcke and Thies (2015)

Geranic acid Mi et al. (2014)
Zeaxanthin Beuttler et al. (2011)
-Carotene Loeschcke et al. (2013) and Sánchez-Pas-
cuala et al. (2019)

Trans-Cinnamate, l-phenylalanine, Molina-Santiago et al. (2016) and Calero

2-phenylethanol, p-coumarate, p-hydroxyben- et al. (2018)
zoate
N-Methylglutamate Mindt et al. (2018)
Polyhydroxyalkanoates Wang et al. (2011a)

d-Glucosaminic acid Wu et al. (2011)

Phenol Wierckx et al. (2005)

p-Coumarate Nijkamp et al. (2007)

Catechol Prakash et al. (2010)
Aliphatic alcohols Bosetti et al. (1992)

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