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Metabolic flux analysis revealed that in Pseudomonas putida KT2440 about 50% of glucose taken up by the cells is channeled through the 2-ketogluconate peripheral pathway. This pathway is characterized by being compartmentalized in the cells. In fact, initial metabolism of glucose to 2-ketogluconate takes place in the periplasm through a set of reactions catalyzed by glucose dehydrogenase and gluconate dehydrogenase to yield 2-ketogluconate. This metabolite is subsequently transported to the cytoplasm, where two reactions are carried out, giving rise to 6-phosphogluconate, which enters the Entner-Doudoroff pathway. The genes for the periplasmic and cytoplasmic set of reactions are clustered in the host chromosome and grouped within two independent operons that are under the control of the PtxS regulator, which also modulates its own synthesis. Here, we show that although the two catabolic operons are induced in vivo by glucose, ketogluconate, and 2-ketogluconate, in vitro we found that only 2-ketogluconate binds to the regulator with an apparent KD (equilibrium dissociation constant) of 15 [micro]M, as determined using isothermal titration calorimetry assays. PtxS is made of two domains, a helix-turn-helix DNA-binding domain located at the N terminus and a C-terminal domain that binds the effector. Differential scanning calorimetry assays revealed that PtxS unfolds via two events characterized by melting points of 48.1[degrees]C and 57.6[degrees]C and that, in the presence of 2-ketogluconate, the unfolding of the effector binding domain occurs at a higher temperature, providing further evidence for 2-ketogluconate--PtxS interactions. Purified PtxS is a dimer that binds to the target promoters with affinities in the range of 1 to 3 [micro]M. Footprint analysis revealed that PtxS binds to an almost perfect palindrome that is present within the three promoters and whose consensus sequence is 5'-TGAAACCGGTTTCA-3'. This palindrome overlaps with the RNA polymerase binding site. doi: 10.1128/JB.00520-10

FinR is required for the induction of fpr (ferredoxin-[NADP.sup.+] reductase) under superoxide stress conditions in Pseudomonas putida. Many proteobacteria harbour FinR homologues in their genome as a putative LysR-type protein. Three cysteine residues (at positions 150, 239 and 289 in P. putida FinR) are conserved in all FinR homologues. When these conserved cysteines, along with two other cysteine residues present in FinR, were individually mutated to serines, the FinR remained active, unlike SoxR and OxyR in Escherichia coli. The results of our in vitro DNA-binding assay with cellular extracts showed that FinR binds directly to the fpr promoter region. In order to identify the FinR functional domain for sensing superoxide stress, we employed random and site-directed mutagenesis of FinR. Among 18 single amino acid mutants, three mutants (T39A, R194A and E225A) abolished fpr induction without any alteration of their DNA-binding ability, whereas other mutants also abrogated their DNA-binding abilities. Interestingly, two mutants (L215P and D51A) appeared to be constitutively active, regardless of superoxide stress conditions. Ferrous iron depletion, ferric iron addition and fdxA (ferredoxin) gene deletion also participate in the regulation of fpr. These data indicate that FinR has unusual residues for redox sensing and that the redox-sensing mechanism of FinR differs from the well-known mechanisms of OxyR and SoxR. DOI 10.1099/mic.0.034181-0

Pseudomonas putida DOT-T1E was used as a model to develop a 'phenomics' platform to investigate the ability of P. putida to grow using different carbon, nitrogen, and sulfur sources and in the presence of stress molecules. Results for growth of wild-type DOT-T1E on 90 different carbon sources revealed the existence of a number of previously uncharted catabolic pathways for compounds such as salicylate, quinate, phenylethanol, gallate, and hexanoate, among others. Subsequent screening on the subset of compounds on which wild-type DOT-TIE could grow with four knockout strains in the global regulatory genes [DELTA]crc, [DELTA]crp, [DELTA]cyoB, and [DELTA]ptsN allowed analysis of the global response to nutrient supply and stress. The data revealed that most global regulator mutants could grow in a wide variety of substrates, indicating that metabolic fluxes are physiologically balanced. It was found that the Crc mutant did not differ much from the wild-type regarding the use of carbon sources. However, certain pathways are under the preferential control of one global regulator, i.e., metabolism of succinate and D-fructose is influenced by CyoB, and L-arginine is influenced by PtsN. Other pathways can be influenced by more than one global regulator; i.e., L-valine catabolism can be influenced by CyoB and Crp (cyclic AMP receptor protein) while phenylethylamine is affected by Crp, CyoB, and PtsN. These results emphasize the cross talk required in order to ensure proper growth and survival. With respect to N sources, DOT-T1E can use a wide variety of inorganic and organic nitrogen sources. As with the carbon sources, more than one global regulator affected growth with some nitrogen sources; for instance, growth with nucleotides, dipeptides, D-amino acids, and ethanolamine is influenced by Crp, CyoB, and PtsN. A surprising finding was that the Crp mutant was unable to flourish on ammonium. Results for assayed sulfur sources revealed that CyoB controls multiple points in methionine/cysteine catabolism while PtsN and Crc are needed for N-acetyl-L-cysteamine utilization. Growth of global regulator mutants was also influenced by stressors of different types (antibiotics, oxidative agents, and metals). Overall and in combination with results for growth in the presence of various stressors, these phenomics assays provide multifaceted insights into the complex decision-making process involved in nutrient supply, optimization, and survival. doi: 10.1128/JB.01129-09

A fatty acyl coenzyme A synthetase (FadD) from Pseudomonas putida CA-3 is capable of activating a wide range of phenylaikanoic and alkanoic acids. It exhibits the highest rates of reaction and catalytic efficiency with long-chain aromatic and aliphatic substrates. FadD exhibits higher [k.sub.cat] and [K.sub.m] values for aromatic substrates than for the aliphatic equivalents (e.g., 15-phenylpentadecanoic acid versus pentadecanoic acid). FadD is inhibited noncompetitively by both acrylic acid and 2-bromooctanoic acid. The deletion of thefadD gene from P. putida CA-3 resulted in no detectable growth or polyhydroxyalkanoate (PHA) accumulation with 10-phenyldecanoic acid, decanoic acid, and longer-chain substrates. The results suggest that FadD is solely responsible for the activation of long-chain phenylalkanoic and alkanoic acids. While the CA-3[DELTA]fadD mutant could grow on medium-chain substrates, a decrease in growth yield and PHA accumulation was observed. The PHA accumulated by CA-3AfadD contained a greater proportion of short-chain monomers than did wild-type PHA. Growth of CA-3[DELTA]fadD was unaffected, but PHA accumulation decreased modestly with shorter-chain substrates. The complemented mutant regained 70% to 90% of the growth and PHA-accumulating ability of the wild-type strain depending on the substrate. The expression of an extra copy of fadD in P. putida CA-3 resulted in increased levels of PHA accumulation (up to 1.6-fold) and an increase in the incorporation of longer-monomer units into the PHA polymer. doi: 10.1128/JB.01016-09

In the presence of copper, Pseudomonasputida activates transcription of cinAQ via the two-component system CinS-CinR. The CinS-CinR TCS was responsive to 0.5 [micro]M copper and was specifically activated only by copper and silver. Modeling studies of CinS identified a potential copper binding site containing H37 and H147. CinS mutants with H37R and H147R mutations had an almost 10-fold reduced copper-dependent induction of cinAQ compared to the wild type.

Pseudomonas putida CBB5 was isolated from soil by enrichment on caffeine. This strain used not only caffeine, theobromine, paraxanthine, and 7-methylxanthine as sole carbon and nitrogen sources but also theophylline and 3-methylxanthine. Analyses of metabolites in spent media and resting cell suspensions confirmed that CBB5 initially N demethylated theophylline via a hitherto unreported pathway to 1- and 3-methylxanthines. NAD(P)H-dependent conversion of theophylline to 1- and 3-methylxanthines was also detected in the crude cell extracts of theophylline-grown CBB5. 1-Methylxanthine and 3-methylxanthine were subsequently N demethylated to xanthine. CBB5 also oxidized theophylline and 1- and 3-methylxanthines to 1,3-dimethyluric acid and 1- and 3-methyluric acids, respectively. However, these methyluric acids were not metabolized further. A broad-substrate-range xanthine-oxidizing enzyme was responsible for the formation of these methyluric acids. In contrast, CBB5 metabolized caffeine to theobromine (major metabolite) and paraxanthine (minor metabolite). These dimethylxanthines were further N demethylated to xanthine via 7-methylxanthine. Theobromine-, paraxanthine-, and 7-methylxanthine-grown cells also metabolized all of the methylxanthines mentioned above via the same pathway. Thus, the theophylline and caffeine N-demethylation pathways converged at xanthine via different methylxanthine intermediates. Xanthine was eventually oxidized to uric acid. Enzymes involved in theophylline and caffeine degradation were coexpressed when CBB5 was grown on theophylline or on caffeine or its metabolites. However, 3-methyixanthine-grown CBB5 cells did not metabolize caffeine, whereas theophylline was metabolized at much reduced levels to only methyluric acids. To our knowledge, this is the first report of theophylline N demethylation and coexpression of distinct pathways for caffeine and theophylline degradation in bacteria.

We developed a high-throughput quantitative capillary assay and demonstrated that Pseudomonas putida strains F1 and PRS2000 were attracted to cytosine, but not thymine or uracil. In contrast, Pseudomonas aeruginosa PAO1 was not chemotactic to any pyrimidines. Chemotaxis assays with a mutant strain of F1 in which the putative methyl-accepting chemotaxis protein-encoding gene Pput_0623 was deleted revealed that this gene (designated mcpC) encodes a chemoreceptor for positive chemotaxis to cytosine. P. putida F1 also responded weakly to cytidine, uridine, and thymidine, but these responses were not mediated by mcpC. Complementation of the F1 [DELTA]mcpC mutant XLF004 with the wild-type gene restored chemotaxis to cytosine. In addition, introduction of this gene into P. aeruginosa PAO1 conferred the ability to respond to cytosine. To our knowledge, this is the first report of a chemoreceptor for cytosine.

Northern blot analysis and a GFP-based reporter assay showed that zwf-1, which encodes glucose-6-phosphate dehydrogenase, was highly induced when Pseudomonas putida KT2440 was cultured in minimal medium containing glucose or gluconate. However, zwf-1 expression was not detected in the presence of pyruvate or succinate. The use of a knockout mutant of HexR, a putative transcription regulator, resulted in constitutively high expression of zwf-1, regardless of the carbon source. An electrophoretic mobility shift assay showed that HexR protein binds to the zwf-1 promoter region and that HexR binding is inhibited by 2-keto-3-deoxy-6-phosphogluconate (KDPG). Despite the presence of gluconate, the edd mutant (non-KDPG producer) was not able to induce the zwf-1 gene. The eda mutant (KDPG overproducer) featured a constitutively high level of zwf-1 expression. Interestingly, zwf-1 was also highly expressed in the presence of oxidative stress-inducing reagents. The level of zwf-1 induction in wild-type cells undergoing oxidative stress did not differ significantly from that observed with the hexR mutant under normal conditions. Interestingly, the hexR mutant was more tolerant of oxidative stress than the wild-type. Expression of zwf-1 was induced by oxidative stress in the edd mutant. Thus, KDPG, a real inducer of zwf-1 gene expression, was not necessary for oxidative-stress induction. In vitro binding of HexR to its cognate promoter region was diminished by menadione and cumene hydroperoxide. The data suggested that HexR might be a dual-sensing regulator of zwf-1 induction that is able to respond to both KDPG and oxidative stress.

A two-component system formed by a sensor histidine kinase and a response regulator has been identified as an element participating in cell density signal transduction in Pseudomonas putida KT2440. It is a homolog of the Pseudomonas aeruginosa RoxS/RoxR system, which in turn belongs to the RegA/RegB family, described in photosynthetic bacteria as a key regulatory element. In KT2440, the two components are encoded by PP_0887 (roxS) and PP_0888 (roxR), which are transcribed in a single unit. Characterization of this two-component system has revealed its implication in redox signaling and cytochrome oxidase activity, as well as in expression of the cell density-dependent gene ddcA, involved in bacterial colonization of plant surfaces. Whole-genome transcriptional analysis has been performed to define the P. putida RoxS/RoxR regulon. It includes genes involved in sugar and amino acid metabolism and the sulfur starvation response and elements of the respiratory chain (a cbb3 cytochrome oxidase, Fe-S clusters, and cytochrome c-related proteins) or genes participating in the maintenance of the redox balance. A putative RoxR recognition element containing a conserved hexamer (TGCCAG) has also been identified in promoters of genes regulated by this two-component system.

Pseudomonas putida G7 carries a naphthalene-catabolic and self-transmissible plasmid, NAH7, which belongs to the IncP-9 incompatibility group. Adjacent to the putative origin of conjugative transfer (oriT) of NAH7 are three genes, traD, traE, and traF, whose functions and roles in conjugation were previously unclear. These three genes were transcribed monocistronically and thus were designated the traD operon. Mutation of the three genes in the traD operon resulted in 10- to [10.sup.5]-fold decreases in the transfer frequencies of the plasmids from Pseudomonas to Pseudomonas and Escherichia coli and from E. coli to E. coli. On the other hand, the traD operon was essential for the transfer of NAH7 from E. coli to Pseudomonas strains. These results indicated that the traD operon is a host-range modifier in the conjugative transfer of NAH7. The TraD, TraE, and TraF proteins were localized in the cytoplasm, periplasm, and membrane, respectively, in strain G7 cells. Our use of a bacterial two-hybrid assay system showed that TraE interacted in vivo with other essential components for conjugative transfer, including TraB (coupling protein), TraC (relaxase), and MpfH (a channel subunit in the mating pair formation system).

The aerobic catabolism of nicotinic acid (NA) is considered a model system for degradation of N-heterocyclic aromatic compounds, some of which are major environmental pollutants; however, the complete set of genes as well as the structural-functional relationships of most of the enzymes involved in this process are still unknown. We have characterized a gene cluster (nic genes) from Pseudomonas putida KT2440 responsible for the aerobic NA degradation in this bacterium and when expressed in heterologous hosts. The biochemistry of the NA degradation through the formation of 2,5-dihydroxypyridine and maleamic acid has been revisited, and some gene products become the prototype of new types of enzymes with unprecedented molecular architectures. Thus, the initial hydroxylation of NA is catalyzed by a two-component hydroxylase (NicAB) that constitutes the first member of the xanthine dehydrogenase family whose electron transport chain to molecular oxygen includes a cytochrome c domain. The [Fe.sup.2+]-dependent dioxygenase (NicX) converts 2,5-dihydroxypyridine into N-formylmaleamic acid, and it becomes the founding member of a new family of extradiol ring-cleavage dioxygenases. Further conversion of N-formylmaleamic acid to formic and maleamic acid is catalyzed by the NicD protein, the only deformylase described so far whose catalytic triad is similar to that of some members of the [alpha]/ [beta]-hydrolase fold superfamily. This work allows exploration of the existence of orthologous gene clusters in saprophytic bacteria and some pathogens, where they might stimulate studies on their role in virulence, and it provides a framework to develop new biotechnological processes for detoxification/ biotransformation of N-heterocyclic aromatic compounds. ring-cleavage dioxygenase | nicotinic acid hydroxylase | heterocyclic compounds

The genome of Pseudomonas putida KT2440 encodes only five recognizable proteins belonging to the phosphoenolpyruvate (PEP)-carbohydrate phosphotransferase system (PTS). Two of these PTS constituents (FruA and FruB) form a complete system for fructose intake. The other three products, encoded byptsP ([EI.sup.Ntr]),ptsO (NPr), and ptsN ([EIIA.sup.Ntr]), comprise a branch of the system unrelated to sugar traffic but thought to have an influence on coordination of N and C metabolism. We used a genetic approach to clarify the course of high-energy phosphate through this reduced set of PTS proteins. To this end, we monitored the phosphorylation state in vivo of the [EIIA.sup.Ntr] enzyme in various genetic backgrounds and growth conditions. Our results show that the source of phosphate available to the system is PEP and that the primary flow of phosphate through the N/C-sensing PTS proceeds from PEP to [EI.sup.Ntr] to NPr to [EIIA.sup.Ntr]. We also found that in the presence of fructose, unlike in the presence of succinate, [EIIA.sup.Ntr] can be phosphorylated in aptsP strain but not in aptsP fruB double mutant. This result revealed that the fructose transport system has the ability to cross talk in vivo with the N-related PTS branch. The data reported here thus document an unexpected connection in vivo between the sugar-dependent and sugar-independent PTSs.

Pseudomonas putida KT2440 channels glucose to the central Entner-Doudoroff intermediate 6-phosphogluconate through three convergent pathways. The genes for these convergent pathways are clustered in three independent regions on the host chromosome. A number of monocistronic units and operons coexist within each of these clusters, favoring coexpression of catabolic enzymes and transport systems. Expression of the three pathways is mediated by three transcriptional repressors, HexR, GnuR, and PtxS, and by a positive transcriptional regulator, GItR-2. In this study, we generated mutants in each of the regulators and carried out transcriptional assays using microarrays and transcriptional fusions. These studies revealed that HexR controls the genes that encode glucokinase/glucose 6-phosphate dehydrogenase that yield 6-phosphogluconate; the genes for the Entner-Doudoroff enzymes that yield glyceraldehyde-3-phosphate and pyruvate; and gap-1, which encodes glyceraldehyde-3-phosphate dehydrogenase. GItR-2 is the transcriptional regulator that controls specific porins for the entry of glucose into the periplasmic space, as well as the gtsABCD operon for glucose transport through the inner membrane. GnuR is the repressor of gluconate transport and gluconokinase responsible for the conversion of gluconate into 6-phosphogluconate. PtxS, however, controls the enzymes for oxidation of gluconate to 2-ketogluconate, its transport and metabolism, and a set of genes unrelated to glucose metabolism.

According to in silico analysis, the genome of Pseudomonas putida KT2440 encodes at least four Zn/Cd/Pb efltux transporters--two P-type ATPases (CadA1 and CadA2) and two czc chemiosmotic transporters (Czc-CBA1 and CzcCBA2). In this study we showed that all these transporters are functional, but under laboratory conditions only two of them were involved in the mediation of heavy metal resistance in P. putida KT2440. CadA2 conferred [Cd.sup.2+] and [Pb.sup.2+] resistance, whereas CzcCBA1 was involved in export of [Zn.sup.2+], [Cd.sup.2+], and possibly [Pb.sup.2+]. CadA1, although nonfunctional in P. putida, improved [Zn.sup.2+] resistance and slightly improved [Cd.sup.2+] resistance when it was expressed in Escherichia coli. CzcCBA2 contributed to Zn resistance of a czcA1-defective P. putida strain or when the CzcA2 subunit was overexpressed in a transporter-deficient strain. It seemed that CzcA2 could complex with CzcCl and CzcB1 subunits and therefore complement the loss of CzcA1. The CzcCBA2 transporter itself, however, did not function. Expression of cadA1, cadA2, and czcCBA1 was induced by heavy metals, and the expression levels were dependent on the growth medium and growth phase. Expression of cadA2 and czcCBA1 was nonspecific; both genes were induced by [Zn.sup.2+], [Cd.sup.2+], [Pb.sup.2+], [Ni.sup.2+], [Co.sup.2+], and [Hg.sup.2+]. On the other hand, remarkably, expression of cadA1 was induced only by [Zn.sup.2+]. Possible roles of distinct but simultaneously functioning transporters are discussed.

We describe an ATP-binding cassette (ABC) transporter in Pseudomonas putida KT2440 that mediates the uptake of glutamate and aspartate. The system (AatJMQP, for acidic amino acid transport) is encoded by an operon involving genes PP1071-PP1068. A deletion mutant with inactivated solute-binding protein (KTaatJ) failed to grow on Glu and Gin as sole sources of carbon and nitrogen, while a mutant lacking a functional nucleotide-binding domain (KTaatP) was able to adapt to growth on Glu after an extended lag phase. Uptake of Glu and Asp by either mutant was greatly impaired at both low and high amino acid concentrations. The purified solute-binding protein AatJ exhibited high affinity towards Glu and Asp ([K.sub.d]=0.4 and 1.3 [micro]M, respectively), while Gin and Asn as well as dicarboxylates (succinate and fumarate) were bound with much lower affinity. We further show that the expression of AatJMQP is controlled by the [sigma].sup.54]-dependent two-component system AauRS. Binding of the response regulator AauR to the aat promoter was examined by gel mobility shift assays and DNase I footprinting. By in silico screening, the AauR-binding motif (the inverted repeat TTCGGNNNNCCGAA) was detected in further P. putida KT2440 genes with established or putative functions in acidic amino acid utilization, and also occurred in other pseudomonads. The products of these AauR-responsive genes include the [H.sup.+]/Glu symporter GltP, a periplasmic glutaminase/asparaginase, AnsB, and phosphoenolpyruvate synthase (PpsA), a key enzyme of gluconeogenesis in Gram-negative bacteria. Based on these findings, we propose that AauR is a central regulator of acidic amino acid uptake and metabolism in pseudomonads.

This work describes a regulatory network of Pseudomonas putida controlled in response to nitrogen availability. We define NtrC as the master nitrogen regulator and suggest that it not only activates pathways for the assimilation of alternative nitrogen sources but also represses carbon catabolism under nitrogen-limited conditions, possibly to prevent excessive carbon and energy flow in the cell.

Biofilms exist in a variety of habitats that are routinely or periodically not saturated with water, and residents must integrate cues on water abundance (matric stress) or osmolarity (solute stress) into lifestyle strategies. Here we examine this hypothesis by assessing the extent to which alginate production by Pseudomonas putida strain rot-2 and by other fluorescent pseudomonads occurs in response to water limitations and how the presence of alginate in turn influences biofilm development and stress tolerance. Total exopolysaccharide (EPS) and alginate production increased with increasing matric, but not solute, stress severity, and alginate was a significant component, but not the major component, of EPS. Alginate influenced biofilm architecture, resulting in biofilms that were taller, covered less surface area, and had a thicker EPS layer at the air interface than those formed by an mr-2 algD mutant under water-limiting conditions, properties that could contribute to less evaporative water loss. We examined this possibility and show that alginate reduces the extent of water loss from biofilm residents by using a biosensor to quantify the water potential of individual cells and by measuring the extent of dehydration-mediated changes in fatty acid composition following a matric or solute stress shock. Alginate deficiency decreased survival of desiccation not only by P. putida but also by Pseudomonas aeruginosa PAO1 and Pseudomonas syringae pv. syringae B728a. Our findings suggest that in response to water-limiting conditions, pseudomonads produce alginate, which influences biofilm development and EPS physiochemieal properties. Collectively these responses may facilitate the maintenance of a hydrated microenvironment, protecting residents from desiccation stress and increasing survival.

Pseudomonas putida KT2440(pWW0) can use toluene via the TOL plasmid-encoded catabolic pathways and can use glucose via a series of three peripheral chromosome-encoded routes that convert glucose into 6-phosphogluconate (6PG), namely, the glucokinase pathway, in which glucose is transformed to 6PG through the action of glucokinase and glucose-6-phosphate dehydrogenase. Alternatively, glucose can be oxidized to gluconate, which can be phosphorylated by gluconokinase to 6PG or oxidized to 2-ketogluconate, which, in turn, is converted into 6PG. Our results show that KT2440 metabolizes glucose and toluene simultaneously, as revealed by net flux analysis of [sup.13]C]glucose. Determination of glucokinase and gluconokinase activities in glucose metabolism, gene expression assays using a fusion of the promoter of the Pu TOL upper pathway to 'lacZ, and global transcriptomic assays revealed simultaneous catabolite repression in the use of these two carbon sources. The effect of toluene on glucose metabolism was directed to the glucokinase branch and did not affect gluconate metabolism. Catabolite repression of the glucokinase pathway and the TOL pathway was triggered by two different catabolite repression systems. Expression from Pu was repressed mainly via PtsN in response to high levels of 2-dehydro-3-deoxygluconate6-phosphate, whereas repression of the glucokinase pathway was channeled through Crc.

The isolation of a Pseudomonas putida DTB bacterium capable of utilizing N,N-diethyl-m-toluamide (DEET) as sole carbon and energy source and hydrolyzing DEET into 3-methylbenzoate and diethylamine is reported.

Hybrid-hollow-fiber membrane bioreactors were developed for the enhanced cometabolic biotransformation of phenol and 4-chlorophenol (4-cp) by Pseudomonas putida ATCC49451. Bioreactor performance was investigated, compared, and analyzed under batch and continuous operating modes. The spinning solutions contained polysulfone (PS), N-methyl-2-pyrrolidone, and various weight ratios of granular activated carbon (GAC) (GAC: PS of 0, 1:4, and 1:2). The bioreactor fabricated with 1:2 GAC hybrid-hollow-fiber membranes demonstrated the best performance for the removal of phenol and 4-cp, both under batch and continuous operations. Under batch operation, 500 mg [L.sup.-1] phenol and 4-cp were completely removed within 23 h in the bioreactor, compared with 26 and 30 h for the 1:4 GAC and GAC free bioreactors. Sorption, biotransformation, desorption, and bioregeneration were identified as the four steps for substrate removal during batch operation. The 1:2 GAC hollow-fiber membrane bioreactor also manifested superiority over the other two during continuous operation for start up and the transient phase after shock loadings of the feed. 300 mg [L.sup.-1] phenol and 4-cp were completely removed in the 1:2 GAC hybrid-hollow-fiber membrane bioreactor whereas 4-cp was not completely removed in the other two bioreactors at a feed rate of 30 mLh [h.sup.-1]. From the experimental results, it was inferred that at steady state, biotransformation was achieved through the dynamic equilibrium among sorption, desorption, and biotransformation rates established within the bioreactors. DOI: 10.1061/(ASCE)0733-9372(2007)133:4(404) CE Database subject headings: Hybrid method; Reactors; Transformations: Biological treatment; Phenol.

As reported, the two-component system ColRS is involved in two completely different processes. It facilitates the root colonization ability of Pseudomonas fluorescens and is necessary for the Tn4652 transposition-dependent accumulation of phenol-utilizing mutants in Pseudomonas putida. To determine the role of the ColRS system in P. putida, we searched for target genes of response regulator ColR by use of a promoter library. Promoter screening was performed on phenol plates to mimic the conditions under which the effect of ColR on transposition was detected. The library screen revealed the porin-encoding gene oprQ and the alginate biosynthesis gene algD occurring under negative control of ColR. Binding of ColR to the promoter regions of oprQ and algD in vitro confirmed its direct involvement in regulation of these genes. Additionally, the porin-encoding gene omp[A.sub.PP0773] and the type I pilus gene csuB were also identified in the promoter screen. However, it turned out that omp[A.sub.PP0773] and csuB were actually affected by phenol and that the influence of ColR on these promoters was indirect. Namely, our results show that ColR is involved in phenol tolerance of P. putida. Phenol MIC measurement demonstrated that a colR mutant strain did not tolerate elevated phenol concentrations. Our data suggest that increased phenol susceptibility is also the reason for inhibition of transposition of Tn4652 in phenol-starving colR mutant bacteria. Thus, the current study revealed the role of the ColRS two-component system in regulation of membrane functionality, particularly in phenol tolerance of P. putida.

The Pseudomonas putida KT2440 TOL upper pathway is repressed under nonlimiting conditions in cells growing in chemostat with succinate as a carbon source. A demonstration that the ptsN gene product [IIA.sup.Ntr] participates in this repression is presented.

Hydrogenophaga intermedia strain S1 and Agrobacterium radiobacter strain $2 form a mixed bacterial culture which degrades sulfanilate (4-aminobenzenesulfonate) by a novel variation of the [beta]-ketoadipate pathway via 4-sulfocatechol and 3-sulfomuconate. It was previously proposed that the further metabolism of 3-sulfomuconate is catalysed by modified 3-carboxy-cis, cis-muconate-lactonizing enzymes (CMLEs) and that these 'type 2' enzymes were different from the conventional CMLEs ('type 1') from the protocatechuate pathway in their ability to convert 3-sulfomuconate in addition to 3-carboxy-cis,cis-muconate. In the present study the genes for two CMLEs (pcaB2S1 and pcaB2S2) were cloned from H. intermedia S1 and A. radiobacter S2, respectively. In both strains, these genes were located close to the previously identified genes encoding the 4-sulfocatechol-converting enzymes. The gene products of pcaB2Sl and pcaB2S2 were therefore tentatively identified as type 2 enzymes involved in the metabolism of 3-sulfomuconate. The genes were functionally expressed and the gene products were shown to convert 3-carboxy-cis,cis-muconate and 3-sulfomuconate. 4-Carboxymethylene-4-sulfo-but-2en-olide (4-sulfomuconolactone) was identified by HPLC-MS as the product, which was enzymically formed from 3-sulfomuconate. His-tagged variants of both CMLEs were purified and compared with the CMLE from the protocatechuate pathway of Pseudomonas putida PRS2000 for the conversion of 3-carboxy-cis,cis-muconate and 3-sulfomuconate. The CMLEs from the 4-sulfocatechol pathway converted 3-sulfomuconate with considerably higher activities than 3-carboxy-cis,cis-muconate. Also the CMLE from P. putida converted 3-sulfomuconate, but this enzyme demonstrated a clear preference for 3-carboxy-cis, cis-muconate as substrate. Thus it was demonstrated that in the 4-sulfocatechol pathway, distinct CMLEs are formed, which are specifically adapted for the preferred conversion of sulfonated substrates.

A [sigma.sup.54] dependent two-component system, the AauR-AauS that controls the uptake and metabolism of acidic amino acids in Pseudomonas putida was identified. The results confirmed that the aau system is a major regulator of acidic amino acid uptake and utilization.

The metabolically versatile soil bacterium Pseudomonas putida has to cope with numerous abiotic stresses in its habitats. The stress responses of P. putida KT2440 to 4[degrees]C, pH 4.5, 0.8 M urea, and 45 mM sodium benzoate were analyzed by determining the global mRNA expression profiles and screening for stress-intolerant non-auxotrophic Tn5 transposon mutants. In 392 regulated genes or operons, 36 gene regions were differentially expressed by more than 2.5-fold, and 32 genes in 23 operons were found to be indispensable for growth during exposure to one of the abiotic stresses. The transcriptomes of the responses to urea, benzoate, and 4[degrees]C correlated positively with each other but negatively with the transcriptome of the mineral acid response. The CbrAB sensor kinase, the cysteine synthase CysM, PcnB and VacB, which control mRNA stability, and BipA, which exerts transcript-specific translational control, were essential to cope with cold stress. The cyo operon was required to cope with acid stress. A functional PhoP, PtsP, RelA/SpoT modulon, and adhesion protein LapA were necessary for growth in the presence of urea, and the outer membrane proteins OmlA and FepA and the phosphate transporter PstBACS were indispensable for growth in the presence of benzoate. A lipid A acyltransferase (PP0063) was a mandatory component of the stress responses to cold, mineral acid, and benzoate. Adaptation of the membrane barrier, uptake of phosphate, maintenance of the intracellular pH and redox status, and translational control of metabolism are key mechanisms of the response of P. putida to abiotic stresses.

Esterase (EST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl DL-[beta]-acetylthioisobutyrate (DL-MATI) to produce D-[beta]-acetylthioisobutyric acid (DAT), serving as a key intermediate for the synthesis of angiotensin-converting enzyme inhibitors. The EST gene was cloned and expressed in Escherichia coli; the recombinant protein is a non-disulfide-linked homotrimer with a monomer molecular weight of 33,000 in both solution and crystalline states, indicating that these ESTs function as trimers. EST hydrolyzed DL-MATI to produce DAT with a degree of conversion of 49.5% and an enantiomeric excess value of 97.2% at an optimum pH of about 8 to 10 and an optimum temperature of about 57 to 67[degrees]C. The crystal structure of EST has been determined by X-ray diffraction to a resolution of 1.6 [Angstrom], confirming that EST is a member of the [alpha]/[beta] hydrolase fold superfamily of enzymes and includes a catalytic triad of Ser97, Asp227, and His256. The active site is located approximately in the middle of the molecule at the end of a pocket ~12 [Angstrom] deep. EST can hydrolyze the methyl ester group without affecting the acetylthiol ester moiety in DL-MATI. The examination of substrate specificity of EST toward other linear esters revealed that the enzyme showed specific activity toward methyl esters and that it recognized the configuration at C-2.

The substrate specificity of polyhydroxyalkanoate (PHA) synthase 1 (PhaC[1.sub.Pp], class II) from Pseudomonas putida Gpo1 (formerly known as Pseudomonas oleovorans Gpo1) was successfully altered by localized semirandom mutagenesis. The enzyme evolution system introduces multiple point mutations, designed on the basis of the conserved regions of the PHA synthase family, by using PCR-based gene fragmentation with degenerate primers and a reassembly PCR. According to the opaqueness of the colony, indicating the accumulation of large amounts of PHA granules in the cells, 13 PHA-accumulating candidates were screened from a mutant library, with Pseudomonas putida GPp104 PH[A.sup.-] as the host. The in vivo substrate specificity of five candidates, L1-6, D7-47, PS-A2, PS-C2, and PS-E1, was evaluated by the heterologous expression in Ralstonia eutropha PHB-4 supplemented with octanoate. Notably, the amount of 3-hydroxybutyrate (short-chain-length [SCL] 3-hydroxyalkanoate [3-HA] unit) was drastically increased in recombinants that expressed evolved mutant enzymes L1-6, PS-A2, PS-C2, and PS-E1 (up to 60, 36, 50, and 49 mol%, respectively), relative to the amount in the wild type (12 mol%). Evolved enzyme PS-E1, in which 14 amino acids had been changed and which was heterologously expressed in R. eutropha PH[B.sup.-]4, not only exhibited broad substrate specificity (49 mol% SCL 3-HA and 51 mol% medium-chain-length [MCL] 3-HA) but also conferred the highest PHA production (45% dry weight) among the candidates. The 3-HA and MCL 3-HA units of the PHA produced by R. eutropha PH[B.sup.-]4/pPS-E1 were randomly copolymerized in a single polymer chain, as analytically confirmed by acetone fractionation and the [sup.13]C nuclear magnetic resonance spectrum.

Pseudomonas putida KT2440 is a soil microorganism that attaches to seeds and efficiently colonizes the plant's rhizosphere. Lysine is one of the major compounds in root exudates, and P. putida KT2440 uses this amino acid as a source of carbon, nitrogen, and energy. Lysine is channeled to [delta]-aminovaleric acid and then further degraded to glutaric acid via the action of the davDT gene products. We show that the davDT genes form an operon transcribed from a single [[sigma].sup.70]-dependent promoter. The relatively high level of basal expression from the davD promoter increased about fourfold in response to the addition of exogenous lysine to the culture medium. However, the true inducer of this operon seems to be [delta]-aminovaleric acid because in a mutant unable to metabolize lysine to [delta]-aminovaleric acid, this compound, but not lysine, acted as an elector. Effective induction of the P. putida [P.sub.davD] promoter by exogenously added lysine requires efficient uptake of this amino acid, which seems to proceed by at least two uptake systems for basic amino acids that belong to the superfamily of ABC transporters. Mutants in these ABC uptake systems retained basal expression from the davD promoter but exhibited lower induction levels in response to exogenous lysine than the wild-type strain.

Acinetobacter baumannii causes severe infections in compromised patients, survives on abiotic surfaces in hospital environments and colonizes different medical devices. In this study the analysis of the processes involved in surface attachment and biofilm formation by the prototype strain 19606 was initiated. This strain attaches to and forms biofilm structures on plastic and glass surfaces, particularly at the liquid-air interface of cultures incubated stagnantly. The cell aggregates, which contain cell stacks separated by water channels, formed under different culture conditions and were significantly enhanced under iron limitation. Electron and fluorescence microscopy showed that pili and exopolysaccharides are part of the cell aggregates formed by this strain. Electron microscopy of two insertion derivatives deficient in attachment and biofilm formation revealed the disappearance of pili-like structures and DNA sequencing analysis showed that the transposon insertions interrupted genes with the highest similarity to hypothetical genes found in Pseudomonas aeruginosa, Pseudomonas putida and Vibrio parahaemolyticus. Although the products of these genes, which have been named csuC and csuE, have no known functions, they are located within a polycistronic operon that includes four other genes, two of which encode proteins related to chaperones and ushers involved in pili assembly in other bacteria. Introduction of a copy of the csuE parental gene restored the adherence phenotype and the presence of pili on the cell surface of the csuE mutant, but not that of the csuC derivative. These results demonstrate that the expression of a chaperone-usher secretion system, some of whose components appear to be acquired from unrelated sources, is required for pili formation and the concomitant attachment to plastic surfaces and the ensuing formation of biofilms by A. baumannii cells.

The cellular levels of the alternative sigma factor [[sigma].sup.54] of Pseudomonas putida have been examined in a variety of growth stages and culture conditions with a single-chain Fv antibody tailored for detection of scarce proteins. The levels of [[sigma].sup.54] were also monitored in P. putida strains with knockout mutations in ptsO or ptsN, known to be required for the C-source control of the [[sigma].sup.54]-dependent Pu promoter of the TOL plasmid. Our results show that ~80 [+ or -] 26 molecules of [[sigma].sup.54] exist per cell. Unlike that in relatives of Pseudomonas (e.g., Caulobacter), where fluctuations of [[sigma].sup.54] determine adaptation and differentiation when cells face starvation, [[sigma].sup.54] in P. putida remains unexpectedly constant at different growth stages, in nitrogen starvation and C-source repression conditions, and in the ptsO and ptsN mutant strains analyzed. The number of [[sigma].sup.54] molecules per cell in P. putida is barely above the predicted number of [[sigma].sup.54]-dependent promoters. These figures impose a framework on the mechanism by which Pu (and other [[sigma].sup.54]-dependent systems) may become amenable to physiological control.

Research has been conducted on DEH transposon from Pseudomonas putida strain PP3. The function, the structure of this transposon and the full sequence of DEH element, including ISPpu12, have been investigated, and the results from the experiments studying its transposition and associated effects are presented.

Research has been conducted on Pseudomonas putida mt-2 archetypal tolyene-xylene catabolic plasmid pWW0. The identification of the insertion sequence ISPpu12 on this plasmid has been carried out, and the details are reported.

Research has been conducted on Pseudomonas putida (S)-Mandelate dehydrogenase. The catalytic role of arginine residues Arg 165 and Arg 277 present in these enzymes and the importance of Arg165/Arg277 pair have been investigated and the results are reported.

Results indicate that depending on the carbon source used, supplied interms of aromatic hydrocarbons, bacteria exhibit different carbon and hydrogen stable isotope fractionation during substrate degradation as shown by Pseudomonas putida, strain mt-2, strain F1, and strain NCIB9816 and Ralstonia picketti strain PK01.

Results show that 2-aminophenoxazin-3-one-7-carboxylate is formed via oxidative dimerization of 4-amino-3-hydroxybenzoate, which undergoes replacement of the 4-amino group by a hydroxyl to form protocatechuate. Data indicate that 4-hydroxylaminobenzoate lyase converts 4-hydroxylaminobenzoate to 3,4-dihydroxybenzoate (protocatechuate).

Research has been conducted on hydroxyectoine. Results suggest that for Pseudomonas putida hydroxyectoine is superior to trehalose as a desiccation protectant and can be a drying excipient for the live microorganisms which are recalcitrant to trehalose.

Pseudomonas putida toluate dioxygenase activity with the effector XylS is influenced by both meta- and ortho-substituted benzoates, reflecting vestigial activity of the effector in regulating the TOL meta operon. Data suggest that the specificity of XylS reflects a dual regulatory role.

Research has been conducted on Pseudomonas putida benzoate 1,2-dioxygenase system. The use of the two-component Rieske dioxygenase system in demonstrating the ability of its oxygenase component to leave both metal centers in the oxidized states is described.

A mutant strain of Pseudomonas putida (LU2201) expressing a novel ethylphenylacetate (EPA) esterase was isolated after mutagenesis of P. putida LU6456. The DNA fragment conferring the EPA+ phenotype was cloned and characterized. It included the structural genes of the 2,3-butanediol catabolic pathway, including acoC, which catalyzes the acetyl transfer step (i.e., dihydrolipoamide acetyltransferase). The acoC gene product from LU2201 was expressed in E. coli and shown to be responsible for the novel EPA-esterase activity. Biochemical characterization revealed that the enzyme still retained the natural dihydrolipoamide acetyltransferase activity. Sequence comparison with the corresponding wild-type gene indicated a single-base pair change leading to a I238V replacement in the respective protein. Biochemical characterization of wild-type AcoC enzyme showed that it too was able to catalyze EPA hydrolysis despite the apparent absence of this activity in cells grown in the presence of EPA. Unlike the wild type, EPA serves as an inducer in the mutant strain. Inverse PCR was used to clone the putative regulator acoR from the aco operon in both wild type and mutant strains. Sequence analyses indicated an S14R exchange in the AcoR from the P. putida mutant. This mutation was located in the N-terminal region responsible for regulatory functions, presumably by interaction with signal molecules. This may account for the ability of EPA to induce expression of acoC only in the mutant strain. Key words: Pseudomonas putida, ethylphenylacetate esterase, acetoin cleaving system, acoC gene, dihydrolipoamide acetyl-transferase, E2 component, acoR gene.

Research has been conducted on the ketosteroid isomerase from Pseudomonas putida biotype B. The structural and functional roles of the tyrosine residues' phenyl rings in the active site of P. putida ketosteroid isomerase have been investigated and discussed.

Research has been conducted on the physiological changes of the plant saprophyte Pseudomonas putida. The effect of the 6 h of attachment to a silicon surface on the physiological changes of R. putida has been investigated via the protein profile analysis and mRNA expression patterns and the results are reported.

Research has been conducted on the recombinant phage antibody Phab. The strategy for the selection of this antibody which recognizes both XylR and other members of XylR class of regulators has been developed and the results demonstrate that this strategy helped to visualize the fluctuations in the intracellular XylR levels of Pseudomonas putida.

Research has been conducted on the adrenodoxin-type Escherichia coli ferredoxin. The crystal structure of this ferredoxin has been determined via the multiple-wavelength anomalous dispersion method and the results indicate that its structure is similar to the bovine adrenodoxin and Pseudomonas putida putidaredoxin structures.

Research points out that in the putidaredoxin-P450cam redox couple the complex fomation occurs electrostatically whereas in case putidaredoxin-NADH-putidaredoxin reductase, the association is driven by nonelectrostatic interactions as shown by laser flash photolysis technique.

Research has been conducted on the Pseudomonas putida transposon Tn4652. Results indicate that the Tn4652 transposase promoter's transcription is controlled by the stationary-phase-specific sigma factor sigma (super)S.

Research has been conducted on the isolated toluene-tolerant strain Pseudomonas putida DOT-T1E. Results indicate that it can grow on high p-hydroxybenzoate concentrations.

Research has been conducted on the alkyl-isocyanides capable of binding to both ferric and ferrous heme iron in cytochrome P450. The crystallographic and spectrophotometric properties of the isocyanide complexes of Pseudomonas putida cytochrome P450cam and Fusarium oxysporum cytochrome P450nor have been investigated.