Isolation of a Pseudomonas stutzeri strain that degrades o-xylene

A Pseudomonas stutzeri strain capable of growing on o-xylene was isolated from enrichment cultures. The organism grew on 2,3- and 3,4-dimethylphenol but not on 2-methylbenzyl alcohol, o-tolualdehyde, or o-toluate. P. stutzeri was not able to utilize m-xylene, p-xylene, or 1,2,4-trimethylbenzene, but growth was observed in the presence of the corresponding alcohols and acids. From the Pseudomonas cultures supplied with o-xylene, 2,3-dimethylphenol was isolated and identified. When resting P. stutzeri cells were incubated with 2,3-dimethylphenol, the reaction mixture turned greenish yellow and showed spectral properties identical to those of the 3,4-dimethylcatechol meta ring fission product. Catechol 2,3-oxygenase was induced by growth on o-xylene or on 2,3- or 3,4-dimethylphenol. The suggested hypothesis is that the first metabolic steps of growth on o-xylene involve the direct oxygenation of the aromatic nucleus, followed by meta pathway reactions.     

Isolation of a Pseudomonas stutzeri strain that degrades o-xylene.

A Pseudomonas stutzeri strain capable of growing on o-xylene was isolated from enrichment cultures. The organism grew on 2,3- and 3,4-dimethylphenol but not on 2-methylbenzyl alcohol, o-tolualdehyde, or o-toluate. P. stutzeri was not able to utilize m-xylene, p-xylene, or 1,2,4-trimethylbenzene, but growth was observed in the presence of the corresponding alcohols and acids. From the Pseudomonas cultures supplied with o-xylene, 2,3-dimethylphenol was isolated and identified. When resting P. stutzeri cells were incubated with 2,3-dimethylphenol, the reaction mixture turned greenish yellow and showed spectral properties identical to those of the 3,4-dimethylcatechol meta ring fission product. Catechol 2,3-oxygenase was induced by growth on o-xylene or on 2,3- or 3,4-dimethylphenol. The suggested hypothesis is that the first metabolic steps of growth on o-xylene involve the direct oxygenation of the aromatic nucleus, followed by meta pathway reactions.     

Characterization of Esterase A, a Pseudomonas stutzeri A15 Autotransporter

Autotransporters are a widespread family of proteins, generally known as virulence factors produced by Gram-negative bacteria. In this study, the esterase A (EstA) autotransporter of the rice root-colonizing beneficial bacterium Pseudomonas stutzeri A15 was characterized. A multiple sequence alignment identified EstA as belonging to clade II of the GDSL esterase family. Autologous overexpression allowed the investigation of several features of both autotransporter proteins and GDSL esterases. First, the correctly folded autotransporter was shown to be present in the membrane fraction. Unexpectedly, after separation of the membrane fraction, EstA was detected in the N-laurylsarcosine soluble fraction. However, evidence is presented for the surface exposure of EstA based on fluorescent labeling with EstA specific antibodies. Another remarkable feature is the occurrence of a C-terminal leucine residue instead of the canonical phenylalanine or tryptophan residue. Replacement of this residue with a phenylalanine residue reduced the stability of the β-barrel. Regarding the esterase passenger domain, we show the importance of the catalytic triad residues, with the serine and histidine residues being more critical than the aspartate residue. Furthermore, the growth of an estA-negative mutant was not impaired and cell mobility was not disabled compared to the wild type. No specific phenotype was detected for an estA-negative mutant. Overall, P. stutzeri A15 EstA is a new candidate for the surface display of proteins in environmentally relevant biotechnological applications.     

Genetic characterization and evolutionary implications of a chromosomally encoded naphthalene-degradation upper pathway from Pseudomonas stutzeri AN10.

Pseudomonas stutzeri strain AN10 is a naphthalene-degrading strain whose dissimilatory genes are chromosomally encoded. We sequenced a total of 11514bp including the entire naphthalene-degradation upper pathway (nah) of P. stutzeri AN10. Nine open reading frames, nahAaAbAcAdBFCED, encoding the enzymes for the degradation of naphthalene to salicylate, were identified. The nah genes of P. stutzeri AN10 have been compared with genes encoding isofunctional proteins from other Pseudomonas naphthalene-degradation upper pathways. The implications of the sequence homologies to the evolution of aromatic catabolic pathways are discussed. Our findings indicate that this entire catabolic module of P. stutzeri AN10 was recruited from other microorganisms and a short period of time has elapsed after its incorporation within the P. stutzeri AN10 genome. Comparisons also suggest the coexistence of two entire nah upper pathways in a host strain, and further recombination between them. These events could accelerate the evolution of modern catabolic pathways.     

Expression of expansin genes is correlated with growth in deepwater rice.

Expansins are a family of proteins that catalyze long-term extension of isolated cell walls. Previously, two expansin proteins have been isolated from internodes of deepwater rice, and three rice expansin genes, Os-EXP1, Os-EXP2, and Os-EXP3, have been identified. We report here on the identification of a fourth rice expansin gene, Os-EXP4, and on the expression pattern of the rice expansin gene family in deepwater rice. Rice expansin genes show organ-specific differential expression in the coleoptile, root, leaf, and internode. In these organs, there is increased expression of Os-EXP1, Os-EXP3, and Os-EXP4 in developmental regions where elongation occurs. This pattern of gene expression is also correlated with acid-induced in vitro cell wall extensibility. Submergence and treatment with gibberellin, both of which promote rapid internodal elongation, induced accumulation of Os-EXP4 mRNA before the rate of growth started to increase. Our results indicate that the expression of expansin genes in deepwater rice is differentially regulated by developmental, hormonal, and environmental signals and is correlated with cell elongation.     

Microbiological degradation of quinoline by Pseudomonas stutzeri: the coumarin pathway of quinoline catabolism

A Gram-negative, oxidase positive, polar flagellated rod, characterised as Pseudomonas stutzeri, has been isolated from sewage by enrichment culture on quinoline. The organism utilizes quinoline as the sole source of carbon, nitrogen and energy, and liberates UV absorbing and phenolic metabolites during its growth on quinoline. 2-Hydroxyquinoline, 2,8-dihydroxyquinoline, 8-hydroxycoumarin and 2,3-dihydroxyphenylpropionic acid have been isolated as the transformation products of quinoline by this bacterium. Quinoline, 2-hydroxyquinoline, and 8-hydroxycoumarin were rapidly oxidised by quinoline-adapted cells; 2,3-dihydroxyphenylpropionic acid oxidation was also demonstrated by Warburg respirometry but 2,8-dihydroxyquinoline was not oxidised. A pathway for quinoline catabolism by P. stutzeri and the probable mechanisms for formation of 8-hydroxycoumarin are suggested.     

Isolation and identification of N2-fixing microorganisms from the rhizosphere of Capparis spinosa (L.)

Four bacterial strains, Pseudomonas stutzeri var. mendocina, Comamonas sp., Agrobacterium tumefaciens biovar. 2 and Sphingobacterium sp., isolated from the rhizosphere of wild-grown caper (Capparis spinosa L.) plants were able to fix N₂ as shown by their growth in nitrogen-free medium and by the acetylene reduction test. P. stutzeri var. mendocina and Comamonas sp. contained DNA homologous to the Klebsiella pneumoniae M5a1 nifHDK genes. No hybridization was found with total DNA from either A. tumefaciens biovar. 2 or Sphingobacterium sp. using nifHDK probes from either K. pneumoniae or Rhizobium meliloti.     

Genetic diversity and horizontal transfer of genes involved in oxidation of reduced phosphorus compounds by Alcaligenes faecalis WM2072

Enrichment was performed to isolate organisms that could utilize reduced phosphorus compounds as their sole phosphorus sources. One isolate that grew well with either hypophosphite or phosphite was identified by 16S rRNA gene analysis as a strain of Alcaligenes faecalis. The genes required for oxidation of hypophosphite and phosphite by this organism were identified by using transposon mutagenesis and include homologs of the ptxD and htxA genes of Pseudomonas stutzeri WM88, which encode an NAD-dependent phosphite dehydrogenase (PtxD) and 2-oxoglutarate-dependent hypophosphite dioxygenase (HtxA). This organism also has the htxB, htxC, and htxD genes that comprise an ABC-type transporter, presumably for hypophosphite and phosphite transport. The role of these genes in reduced phosphorus metabolism was confirmed by analyzing the growth of mutants in which these genes were deleted. Sequencing data showed that htxA, htxB, htxC, and htxD are virtually identical to their homologs in P. stutzeri at the DNA level, indicating that horizontal gene transfer occurred. However, A. faecalis ptxD is very different from its P. stutzeri homolog and represents a new ptxD lineage. Therefore, this gene has ancient evolutionary roots in bacteria. These data suggest that there is strong evolutionary selection for the ability of microorganisms to oxidize hypophosphite and phosphite.     

Type IV pilus genes pilA and pilC of Pseudomonas stutzeri are required for natural genetic transformation, and pilA can be replaced by corresponding genes from nontransformable species

Pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a P. stutzeri JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of Pseudomonas aeruginosa, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The pilC mutant had no pili and was defective in twitching motility. Further sequencing revealed that pilC is clustered in an operon with genes homologous to pilB and pilD of P. aeruginosa, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a pilA gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of pilA abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of (3)H-labeled P. stutzeri DNA that were bound to competent parental cells and taken up were strongly reduced in the pilC and pilA mutants. Remarkably, the cloned pilA genes from nontransformable organisms like Dichelobacter nodosus and the PAK and PAO strains of P. aeruginosa fully restored pilus formation and transformability of the P. stutzeri pilA mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium P. stutzeri function in DNA uptake for transformation and that their role in this process is not confined to the species-specific pilin.     

The rice inoculant strain Alcaligenes faecalis A15 is a nitrogen-fixing Pseudomonas stutzeri.

The taxonomic position of the nitrogen-fixing rice isolate A15, previously classified as Alcaligenes faecalis, was reinvestigated. On the basis of its small subunit ribosomal RNA (16S rRNA) sequence this strain identifies as Pseudomonas stutzeri. Phenotyping and fatty acid profiling confirm this result. DNA:DNA hybridisations, using the optical renaturation rate method, between strain A15 and Pseudomonas stutzeri LMG 11199T revealed a mean DNA-binding of 77%. The identification was further corroborated by comparative sequence analysis of the oprF gene, which encodes the major outer membrane protein of rRNA homology group I pseudomonads. Furthermore we determined the nifH sequence of this strain and of two putative diazotrophic Pseudomonas spp. and made a comparative analysis with sequences of other diazotrophs. These Pseudomonas NifH sequences cluster with NifH sequences isolated from the rice rhizosphere by PCR and of proteobacteria from the beta and gamma subclasses.     

An airlift biofilm reactor for the biodegradation of phenol by Pseudomonas stutzeri OX1

Phenol bioconversion by Pseudomonas stutzeri OX1 using either free or immobilized cells was investigated with the aim of searching for optimal operating conditions of a continuous bioconversion process. The study was developed by analyzing: (a) free-cell growth and products of phenol bioconversion by batch cultures of P. stutzeri; (b) growth of P. stutzeri cells immobilized on carrier particles; (c) bioconversion of phenol-bearing liquid streams and the establishment and growth of an active bacterial biofilm during continuous operation of an internal-loop airlift bioreactor. We have confirmed that free Pseudomonas cultures are able to transform phenol through the classical meta pathway for the degradation of aromatic molecules. Data indicate that bacterial growth is substrate-inhibited, with a limiting phenol concentration of about 600 mg/L. Immobilization tests revealed that a stable bacterial biofilm can be formed on various types of solid carriers (silica sand, tuff, and activated carbon), but not on alumina. Entrapment in alginate beads also proved to be effective for P. stutzeri immobilization. Continuous bioconversion of phenol-bearing liquid streams was successfully obtained in a biofilm reactor operated in the internal-circulation airlift mode. Phenol conversion exceeded 95%. Biofilm formation and growth during continuous operation of the airlift bioreactor were quantitatively and qualitatively assessed.     

Isolation and characterization of a benzoylformate decarboxylase and a NAD+/NADP+-dependent benzaldehyde dehydrogenase involved in D-phenylglycine metabolism in Pseudomonas stutzeri ST-201

Following induction with D-phenylglycine both d-phenylglycine aminotransferase activity and benzoylformate decarboxylase activity were observed in cultures of Pseudomonas stutzeri ST-201. Induction with benzoylformate, on the other hand, induced only benzoylformate decarboxylase activity. Purification of the benzoylformate decarboxylase, followed by N-terminal sequencing, enabled the design of probes for hybridization with P. stutzeri ST-201 genomic DNA libraries. Sequencing of two overlapping genomic DNA restriction fragments revealed two open reading frames which were denoted dpgB and dpgC. Sequence alignments suggested that the genes encoded a thiamin-diphosphate-dependent decarboxylase and an aldehyde dehydrogenase, respectively. Both genes were isolated and expressed in Escherichia coli. The dpgB gene product was confirmed as a benzoylformate decarboxylase while the dpgC gene product was characterized as a NAD+/NADP+-dependent benzaldehyde dehydrogenase. In keeping with their high sequence identities (both greater than 85%) the kinetic properties of the two enzymes were similar to those of the homologous enzymes in the mandelate pathway of Pseudomonas putida ATCC 12633. However, Pseudomonas stutzeri ST-201 was unable to grow on either isomer of mandelate, and sequencing indicated that the dpgB gene did not form part of an operon. Thus it appears that the two enzymes form part of a d-phenylglycine, rather than mandelate, degrading pathway.     

Biology of Pseudomonas stutzeri.

Pseudomonas stutzeri is a nonfluorescent denitrifying bacterium widely distributed in the environment, and it has also been isolated as an opportunistic pathogen from humans. Over the past 15 years, much progress has been made in elucidating the taxonomy of this diverse taxonomical group, demonstrating the clonality of its populations. The species has received much attention because of its particular metabolic properties: it has been proposed as a model organism for denitrification studies; many strains have natural transformation properties, making it relevant for study of the transfer of genes in the environment; several strains are able to fix dinitrogen; and others participate in the degradation of pollutants or interact with toxic metals. This review considers the history of the discovery, nomenclatural changes, and early studies, together with the relevant biological and ecological properties, of P. stutzeri.