A novel pathway for the biodegradation of 3-nitrotoluene in Pseudomonas putida

Abstract: 3-Nitrotoluene was degraded when incubated with the resting cells of Pseudomonas putida OU83. Most of the 3-nitrotoluene (70%) was metabolized via reduction of the nitro group to form 3-aminotoluene (3-AT). A minor portion (30%) was degraded through a novel pathway involving oxidation of 3-NT to form 3-nitrophenol through a series of intermediary metabolites: 3-nitrobenzyl alcohol, 3-nitrobenzaldehyde and 3-nitrobenzoic acid. Degradation of 3-nitrophenol occurred with the formation of a transient intermediary metabolite, hydroxynitroquinone, which was further degraded with the near stoichiometric release of nitrite into the medium. 3-Nitrotoluene-induced cells showed increased oxygen consumption with 3-nitrotoluene, 3-nitrobenzaldehyde, 3-nitrobenzoate, and 3-nitrophenol as substrates in comparison to uninduced cells. Cell extracts prepared from strain OU83 contained benzylalcohol dehydrogenase and benzaldehyde dehydrogenase activities. The experimental evidence suggests a novel pathway for the degradation of 3-NT in which C-1 elimination is catalyzed by a cofactor-independent deformylase, rather than a decarboxylase or dioxygenase.     

Broad spectrum and narrow spectrum mercury resistance encoded by different plasmids of a Pseudomonas putida strain

Abstract Pseudomonas putida strain H harbours two plasmids of different sizes. It was demonstrated that the large plasmid pPGH1 confers a broad spectrum resistance to mercurials, whereas the small plasmid pPGH2 confers a narrow spectrum one. Under the influence of the small plasmid the resistance of cells against poisoning with 2,4-di-chlorophenol or o -cresol increases in comparison to cells without this plasmid. Both plasmids proved to be not self-transmissible, but pPGH1 is transferable by mobilisation by means of the IncP-1 vectors R68.45 or RP1.     

Prime plasmids derived from the IncP-10 plasmid R91-5 in Pseudomonas putida

Abstract Plasmid primes carrying various fragments of Pseudomonas putida chromosome have been derived from pMO22, a derivative of R91-5 loaded with Tn 501 . These prime plasmids transfer efficiently to P. aeruginosa where they effectively complement various auxotrophic markers. Proof of prime plasmid formation has been provided by the high-frequency transfer of plasmid and chromosomal markers, the unselected cotransfer of either plasmid or chromosomal markers into P. aeruginosa and by transformation of both plasmid and chromosomal markers using prime plasmid DNA. Such prime plasmids have been used to map the location of new markers on the P. putida chromosome.     

Production of 3-hydroxy-n-phenylalkanoic acids by a genetically engineered strain of Pseudomonas putida

Overexpression of the gene encoding the poly-3-hydroxy-n-phenylalkanoate (PHPhA) depolymerase (phaZ) in Pseudomonas putida U avoids the accumulation of these polymers as storage granules. In this recombinant strain, the 3-OH-acyl-CoA derivatives released from the different aliphatic or aromatic poly-3-hydroxyalkanoates (PHAs) are catabolized through the -oxidation pathway and transformed into general metabolites (acetyl-CoA, succinyl-CoA, phenylacetyl-CoA) or into non-metabolizable end-products (cinnamoyl-CoA). Taking into account the biochemical, pharmaceutical and industrial interest of some PHA catabolites (i.e., 3-OH-PhAs), we designed a genetically engineered strain of P. putida U (P. putida U fadBA-phaZ) that efficiently bioconverts (more than 80%) different n-phenylalkanoic acids into their 3-hydroxyderivatives and excretes these compounds into the culture broth.     

Transfer and expression of a hydrocarbon-degrading plasmid pHCL from Pseudomonas putida to marine bacteria

Transfer of a catabolic plasmid from Pseudomonas putida to indigenous marine bacteria and obligate halophilic bacteria was carried out under both in vitro and in situ conditions. The marine recipients, which could not otherwise grow on hydrocarbon substrates, were able to degrade them after the horizontal transfer of the catabolic plasmid from P. putida. Mating conducted on nutrient plates yielded comparatively more transconjugants than in broth mating under laboratory conditions (10⁶ c.f.u./ml). The transconjugants stably maintained the plasmid when they were maintained in seawater amended with selective pressure (antibiotics/Hg (25 g/l) even after 30 days, whereas under non-selective conditions they progressively lost the plasmid after 24 days. The expression of the plasmid in the marine recipients was investigated by gas chromatographic analysis. The overall objective of this study is to evolve a novel strategy for bioremediation of oil spills and the results of the present study suggest that the present approach would offer a better solution for the removal of harmful substances from the environment by avoiding serious interference with the microbial flora of the ecosystem.     

Frequent conjugative transfer accelerates adaptation of a broad-host-range plasmid to an unfavorable Pseudomonas putida host

IncP-1 plasmids are known to be promiscuous, but it is not understood if they are equally well adapted to various species within their host range. Moreover, little is known about their fate in bacterial communities. We determined if the IncP-1beta plasmid pB10 was unstable in some Proteobacteria, and whether plasmid stability was enhanced after long-term carriage in a single host and when regularly switched between isogenic hosts. Plasmid pB10 was found to be very unstable in Pseudomonas putida H2, and conferred a high cost (c. 20% decrease in fitness relative to the plasmid-free host). H2(pB10) was then evolved under conditions that selected for plasmid maintenance, with or without regular plasmid transfer (host-switching). When tested in the ancestral host, the evolved plasmids were more stable and their cost was significantly reduced (9% and 16% for plasmids from host-switched and nonswitched lineages, respectively). Our findings suggest that IncP-1 plasmids can rapidly adapt to an unfavorable host by improving their overall stability, and that regular conjugative transfer accelerates this process.     

Biotransformation of corn bran derived ferulic acid to vanillic acid using engineered Pseudomonas putida KT2440

Abstract

Ferulic acid is a fraction of the phenolics present in cereals such as rice and corn as a component of the bran. Substantial amounts of waste bran are generated by the grain processing industry and this can be valorized via extraction, purification and conversion of phenolics to value added chemical products. Alkaline alcohol based extracted and purified ferulic acid from corn bran was converted to vanillic acid using engineered Pseudomonas putida KT2440. The strain was engineered by rendering the vanAB gene nonfunctional and obtaining the mutant defective in vanillic acid metabolism. Biotransformation of ferulic acid using resting Pseudomonas putida KT2440 mutant cells resulted in more than 95?±?1.4% molar yield from standard ferulic acid; while the corn bran derived ferulic acid gave 87?±?0.38% molar yield. With fermentation time of less than 24?h the mutant becomes a promising candidate for the stable biosynthesis of vanillic acid at industrial scale.     

Short Communication: Carbon source-starvation-induced precipitation of copper by Pseudomonas putida strain S4

Pseudomonas putida strain S4, when starved of carbon source, precipitated Cu2+ in the medium. The precipitate, apart from containing copper, consisted of phosphate and hydroxide residues. While high acid phosphatase activity provided the necessary phosphates for Cu2+-precipitation, hydroxyl residues generated by metal efflux pathway may be used for metal hydroxide precipitation. This phenomenon could be exploited in the biorecovery of copper from different sources.     

Comparisons of the transferability of plasmids pCAR1, pB10, R388, and NAH7 among Pseudomonas putida at different cell densities

The transferability of plasmids pCAR1, pB10, R388, and NAH7 was compared using the same donor-recipient system at different cell density combinations in liquid or on a solid surface. pCAR1 was efficiently transferred in liquid, whereas the other plasmids were preferentially transferred on a solid surface. Difference of liquid or solid affected the transfer frequency especially at lower cell densities.     

Survival of genetically engineered Pseudomonas fluorescens in soil in competition with the parent strain

Abstract The population dynamics of two genetically engineered Pseudomonas fluorescens strains, D5 and C5t, introduced into a loamy sand soil, in competition with a spontaneous antibiotic-resistant mutant of the corresponding wildtype strain was studied. Strain D5 contained an insertion of transposon Tn5 in its genome, whereas strain C5t was obtained by insertion of Tn 5 :: tox , a Tn 5 -derivative containing a Bacillus thuringiensis var. morrisoni δ-endotoxin gene, into the chromosome using a suicide vector system. Southern hybridization analysis demonstrated the absence of vector sequences, and the presence of single copies of either Tn 5 or Tn 5 :: tox in the respective strains. Western blotting and a bio-assay on larvae of Anopheles stephensi suggested the tox gene was functional in clone C5t. Both D5 and C5t were prototrophic and their generation times in minimal medium were slightly below that of the corresponding wild-type strain. Tn 5 and Tn 5 :: tox were stable in both clones during growth in minimal medium for 16 generations. During growth in competition with the wild-type strain, D5 competed well, however C5t was outcompeted from 50 to below 3% of the population in 40 generations. During growth in competition in the sterile loamy sand, both strains were outcompeted by the parent strain; strain C5t was less competitive than D5. In non-sterile loamy sand, the introduced mixed populations showed a slow decline; both C5t and D5 were outcompeted by the parent strain. The decreased fitness of both modified strains, although significant, was considered to be small in ecological terms. Further, the addition of 10% bentonite clay to the loamy sand resulted in a significant enhancement of survival of the mixed populations, and a stabilization of the proportions between the modified strains and the parent. Finally, there was a trend towards a decrease in the proportion modified strain/parent strain in both mixes in the rhizosphere of wheat.     

Physiological role of phosphatidylcholine in the Pseudomonas putida A ATCC 12633 response to tetradecyltrimethylammonium bromide and aluminium

Aims:  To evaluate the effect of tetradecyltrimethylammonium bromide (TTAB) and aluminium stresses on the phospholipid (PL) composition of Pseudomonas putida A ATCC 12633.
Methods and Results:  Pseudomonas putida were grown with TTAB in the presence or absence of AlCl₃, and the PL composition was analysed. The presence of TTAB resulted in an increase in phosphatidylglycerol and phosphatidic acid levels (6- and 20-fold, respectively) with respect to the levels in cells grown without the surfactant. With AlCl₃, phosphatidylcholine (PC) increased (threefold) and cell-free extracts contained approximately threefold more phosphatidylcholine synthase activities than extracts without AlCl₃, indicating that the PC level is dependent upon activation of this enzyme.
Conclusions:  The negative charges of the headgroups of PL are the primary membrane-associated factors for the response to TTAB. PC are involved in cellular responses to binding Al³⁺ and should be viewed as a temporary reservoir of available Al³⁺ to allow a more efficient utilization of TTAB by Ps. putida .
Significance and Impact of the Study:  The changes in the PL of Ps. putida in the presence of TTAB and AlCl₃ indicate that different responses are utilized by bacteria to maintain optimal PL composition in the presence of such environmental pollutants.     

Production and characterization of a new bioemulsifier from Pseudomonas putida ML2

AIMS: To characterize the bioemulsifier produced by a nonfluorescent strain of Pseudomonas putida isolated from a polluted sediment and to determine the influence of pH, temperature, media composition, and carbon and nitrogen source on growth and emulsifying activity. METHODS AND RESULTS: Different indexes were employed to determine the emulsifying properties of culture supernatants of P. putida ML2 in defined and complex media. Surface tension of cell-free supernatants was measured. Purification and chemical analysis of the emulsifier was performed. Confirmed results indicate that a polysaccharide with hexasaccharide repeating units is responsible for the emulsifying activity in a mineral medium with glucose as sole carbon source. Moreover, an emulsifier is produced when growing on naphthalene. CONCLUSIONS: Culture media composition influences the amount and the properties of the emulsifier produced by this P. putida strain. Under nitrogen limiting conditions, a polysaccharide is responsible for the emulsifying activity in defined mineral media. In complex nitrogen rich medium, a different kind of emulsifier is produced. The exopolymer may contribute to hydrocarbons solubilization. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first exopolysaccharide with emulsifying properties produced by a Pseudomonas strain reported to the present. Also chemical composition is significantly different from previous reports. This strain has potential use in bioremediation and the purified polysaccharide may be used in food and cosmetic industry. Moreover, the production of the exopolymer may play a role on biofilm formation.     

FTIR Spectroscopic Study of Biogenic Mn-Oxide Formation by Pseudomonas putida GB-1

Biomineralization in heterogeneous aqueous systems results from a complex association between pre-existing surfaces, bacterial cells, extracellular biomacromolecules, and neoformed precipitates. Fourier transform infrared (FTIR) spectroscopy was used in several complementary sample introduction modes (attenuated total reflectance [ATR], diffuse reflectance [DRIFT], and transmission) to investigate the processes of cell adhesion, biofilm growth, and biological Mn-oxidation by Pseudomonas putida strain GB-1. Distinct differences in the adhesive properties of GB-1 were observed upon Mn oxidation. No adhesion to the ZnSe crystal surface was observed for planktonic GB-1 cells coated with biogenic MnO _x , whereas cell adhesion was extensive and a GB-1 biofilm was readily grown on ZnSe, CdTe, and Ge crystals prior to Mn-oxidation. IR peak intensity ratios reveal changes in biomolecular (carbohydrate, phosphate, and protein) composition during biologically catalyzed Mn-oxidation. In situ monitoring via ATR-FTIR of an active GB-1 biofilm and DRIFT data revealed an increase in extracellular protein (amide I and II) during Mn(II) oxidation, whereas transmission mode measurements suggest an overall increase in carbohydrate and phosphate moieties. The FTIR spectrum of biogenic Mn oxide comprises Mn-O stretching vibrations characteristic of various known Mn oxides (e.g., “acid” birnessite, romanechite, todorokite), but it is not identical to known synthetic solids, possibly because of solid-phase incorporation of biomolecular constituents. The results suggest that, when biogenic MnO _x accumulates on the surfaces of planktonic cells, adhesion of the bacteria to other negatively charged surfaces is hindered via blocking of surficial proteins.     

Stability and conjugal transfer kinetics of a TOL plasmid in Pseudomonas aeruginosa PAO 1162

Abstract Batch mating experiments were employed to study the kinetics of the conjugal transfer of a TOL plasmid, using the transconjugant strain Pseudomonas aeruginosa PAO 1162 (TOL) as the plasmid donor and Pseudomonas putida PB 2442 and Pseudomonas aeruginosa PAO 1162N as the plasmid recipients. Transfer rates from PAO 1162 (TOL) to PAO 1162N and PB 2442 measured for exponentially grown PAO 1162 (TOL) were 1.81 × 10⁻¹⁴ (standard error (S.E.) 1.25 × 10⁻¹⁵) ml·cell⁻¹min⁻¹ and 3.32 × 10⁻¹³ (S.E. 4.42 × 10⁻¹⁴) ml·cell⁻¹min⁻¹, respectively. The instability of the TOL plasmid in PAO 1162 (TOL) was evaluated under conditions that were non-selective for maintenance of the TOL catabolic functions. The measured rates of instability were 6.7 10⁻⁶ to 8.3 10⁻⁶ min⁻¹, and the loss of the catabolic functions was mainly caused by structural instability of the plasmid.     

Characteristics of a newly created bioluminescent Pseudomonas putida harboring TOL plasmid for use in analysis of a bioaugmentation system

Insertion of a bacterial lux operon into the chromosome of Pseudomonas putida mt-2 holding TOL plasmid, yielded a new bioluminescent strain of P. putida BLU. Both in the cultures containing toluene and m-toluic acid as the sole carbon sources, P. putida BLU showed the same specific growth rate and cell yield as those of the wild strain. The bioluminescence output in the cell growth phases correlated with the cell concentration, indicating that the bioluminescent P. putida BLU can be monitored and quantified in a mixed culture in real time by the luminescence detection.     

Sequence analysis of the major outer membrane protein gene of an ovine abortion strain of Chlamydia psittaci

The major outer membrane protein (MOMP) gene from an ovine abortion strain of Chlamydia psittaci (S26/3) has been cloned and sequenced. The gene shows the features of other chlamydial MOMPs but comparison with the previously reported sequence for the ovine abortion isolate A22/M has revealed substantial sequence divergence which is clustered into the same four intramolecular regions as the sequence variation found between C. trachomatis serovars. Subsequent restriction enzyme analysis of A22/M DNA has shown that it has an avian-type genomic profile and thus the comparison is between types rather than between strains.