Isolation of Environmental Bacteria Able to Degrade Sterols and/or Bile Acids: Determination of Cholesterol Oxidase and Several Hydroxysteroid Dehydrogenase Activities in Rhodococcus, Gordonia, and Pseudomonas putida.

Interest about the isolation and characterization of steroid-catabolizing bacteria has increased over time due to the massive release of these recalcitrant compounds and their deleterious effects or their biotransformation derivatives as endocrine disruptors for wildlife, as well as their potential use in biotechnological approaches for the synthesis of pharmacological compounds. Thus, in this chapter, an isolation protocol to select environmental bacteria able to degrade sterols, bile acids, and androgens is shown. Moreover, procedures for the determination of cholesterol oxidase or different hydroxysteroid dehydrogenase activities in Pseudomonas putida DOC21, Rhodococcus sp. HE24.12, Gordonia sp. HE24.4J and Gordonia sp. HE24.3 are also detailed.

Identification and Characterization of Some Genes, Enzymes, and Metabolic Intermediates Belonging to the Bile Acid Aerobic Catabolic Pathway from Pseudomonas.

The study of the catabolic potential of microbial species isolated from different habitats has allowed the identification and characterization of bacteria able to assimilate bile acids and/or other steroids (e.g., testosterone and 4-androsten-3,17-dione) under aerobic conditions through the 9,10-seco pathway. From soil samples, we have isolated several strains belonging to genus Pseudomonas that grow efficiently in chemically defined media containing some cyclopentane-perhydrophenanthrene derivatives as carbon sources. Genetic and biochemical studies performed with one of these bacteria (P. putida DOC21) allowed the identification of the genes and enzymes belonging to the route involved in bile acids and androgens, the 9,10-seco pathway in this bacterium. In this manuscript, we describe the most relevant methods used in our lab for the identification of the chromosomal location and nucleotide sequence of the catabolic genes (or gene clusters) encoding the enzymes of this pathway, and the tools useful to establish the role of some of the enzymes that participate in this route.

Xerotolerance: A New Property in Exiguobacterium Genus.

The highly xerotolerant bacterium classified as Exiguobacterium sp. Helios isolated from a solar panel in Spain showed a close relationship to Exiguobacterium sibiricum 255-15 isolated from Siberian permafrost. Xerotolerance has not been previously described as a characteristic of the extremely diverse Exiguobacterium genus, but both strains Helios and 255-15 showed higher xerotolerance than that described in the reference xerotolerant model strain Deinococcus radiodurans. Significant changes observed in the cell morphology after their desiccation suggests that the structure of cellular surface plays an important role in xerotolerance. Apart from its remarkable resistance to desiccation, Exiguobacterium sp. Helios strain shows several polyextremophilic characteristics that make it a promising chassis for biotechnological applications. Exiguobacterium sp. Helios cells produce nanoparticles of selenium in the presence of selenite linked to its resistance mechanism. Using the Lactobacillus plasmid pRCR12 that harbors a cherry marker, we have developed a transformation protocol for Exiguobacterium sp. Helios strain, being the first time that a bacterium of Exiguobacterium genus has been genetically modified. The comparison of Exiguobacterium sp. Helios and E. sibiricum 255-15 genomes revealed several interesting similarities and differences. Both strains contain a complete set of competence-related DNA transformation genes, suggesting that they might have natural competence, and an incomplete set of genes involved in sporulation; moreover, these strains not produce spores, suggesting that these genes might be involved in xerotolerance.

Catabolism of biogenic amines in Pseudomonas species.

Biogenic amines (BAs; 2-phenylethylamine, tyramine, dopamine, epinephrine, norepinephrine, octopamine, histamine, tryptamine, serotonin, agmatine, cadaverine, putrescine, spermidine, spermine and certain aliphatic amines) are widely distributed organic molecules that play basic physiological functions in animals, plants and microorganisms. Pseudomonas species can grow in media containing different BAs as carbon and energy sources, a reason why these bacteria are excellent models for studying such catabolic pathways. In this review, we analyse most of the routes used by different species of Pseudomonas (P. putida, P. aeruginosa, P. entomophila and P. fluorescens) to degrade BAs. Analysis of these pathways has led to the identification of a huge number of genes, catabolic enzymes, transport systems and regulators, as well as to understanding of their hierarchy and functional evolution. Knowledge of these pathways has allowed the design and collection of genetically manipulated microbes useful for eliminating BAs from different sources, highlighting the biotechnological applications of these studies.

Steroids as Environmental Compounds Recalcitrant to Degradation: Genetic Mechanisms of Bacterial Biodegradation Pathways.

Steroids are perhydro-1,2-cyclopentanophenanthrene derivatives that are almost exclusively synthesised by eukaryotic organisms. Since the start of the Anthropocene, the presence of these molecules, as well as related synthetic compounds (ethinylestradiol, dexamethasone, and others), has increased in different habitats due to farm and municipal effluents and discharge from the pharmaceutical industry. In addition, the highly hydrophobic nature of these molecules, as well as the absence of functional groups, makes them highly resistant to biodegradation. However, some environmental bacteria are able to modify or mineralise these compounds. Although steroid-metabolising bacteria have been isolated since the beginning of the 20th century, the genetics and catabolic pathways used have only been characterised in model organisms in the last few decades. Here, the metabolic alternatives used by different bacteria to metabolise steroids (e.g., cholesterol, bile acids, testosterone, and other steroid hormones), as well as the organisation and conservation of the genes involved, are reviewed.

Identification and expression of the 11ß-steroid hydroxylase from Cochliobolus lunatus in Corynebacterium glutamicum.

Hydroxylation of steroids has acquired special relevance for the pharmaceutical industries. Particularly, the 11ß-hydroxylation of steroids is a reaction of biotechnological importance currently carried out at industrial scale by the fungus Cochliobolus lunatus. In this work, we have identified the genes encoding the cytochrome CYP103168 and the reductase CPR64795 of C. lunatus responsible for the 11ß-hydroxylase activity in this fungus, which is the key step for the preparative synthesis of cortisol in industry. A recombinant Corynebacterium glutamicum strain harbouring a plasmid expressing both genes forming a synthetic bacterial operon was able to 11ß-hydroxylate several steroids as substrates. This is a new example to show that the industrial strain C. glutamicum can be used as a suitable chassis to perform steroid biotransformation expressing eukaryotic cytochromes.

Histamine catabolism in Pseudomonas putida U: identification of the genes, catabolic enzymes and regulators.

In this study, the catabolic pathway required for the degradation of the biogenic amine histamine (Hin) was genetically and biochemically characterized in Pseudomonas putida U. The 11 proteins (HinABCDGHFLIJK) that participate in this pathway are encoded by genes belonging to three loci hin1, hin2 and hin3 and by the gene hinK. The enzymes HinABCD catalyze the transport and oxidative deamination of histamine to 4-imidazoleacetic acid (ImAA). This reaction is coupled to those of other well-known enzymatic systems (DadXAR and CoxBA-C) that ensure both the recovery of the pyruvate required for Hin deamination and the genesis of the energy needed for Hin uptake. The proteins HinGHFLKIJ catalyze the sequential transformation of ImAA to fumaric acid via N2 -formylisoasparagine, formylaspartic acid and aspartic acid. The identified Hin pathway encompasses all the genes and proteins (transporters, energizing systems, catabolic enzymes and regulators) needed for the biological degradation of Hin. Our work was facilitated by the design and isolation of genetically engineered strains that degrade Hin or ImAA and of mutants that accumulate Ala, Asp and Hin catabolites. The implications of this research with respect to potential biotechnological applications are discussed.

Identification and Characterization of the Genes and Enzymes Belonging to the Bile Acid Catabolic Pathway in Pseudomonas.

The study of the catabolic potential of microbial species isolated from different habitats has allowed the identification and characterization of bacteria able to assimilate bile acids and other steroids (e.g., testosterone and 4-androsten-3,17-dione). From soil samples, we have isolated several strains belonging to genus Pseudomonas that grow efficiently in chemical defined media containing some cyclopentane-perhydro-phenantrene derivatives as carbon sources. Genetic and biochemical studies performed with one of these bacteria (P. putida DOC21) allowed the identification of the genes and enzymes belonging to the 9,10-seco pathway, the route involved in the aerobic assimilation of steroids. In this manuscript, we describe the most relevant methods required for (1) isolation and characterization of these species; (2) determining the chromosomal location, nucleotide sequence, and functional analysis of the catabolic genes (or gene clusters) encoding the enzymes from this pathway; and (3) the tools employed to establish the role of some of the proteins that participate in this route.

Steroid 11-Alpha-Hydroxylation by the Fungi Aspergillus nidulans and Aspergillus ochraceus.

Steroids are a group of natural compounds derived from the cyclopentane-perhydro-phenantrene nucleus that have a great interest for the pharmaceutical industries as a consequence of their physiological effects. Among their functions are anti-inflammatory, immunosuppressive, or contraceptive activities. Nowadays, microbial transformation of steroid precursors is winning relevance opposite to the chemical synthesis, since it allows for decreasing time, expenses, and environmental pollution. Pharmaceutical industry tends to use cholesterol and phytosterols as starting materials due to their low cost. Aspergillus ochraceus and Aspergillus nidulans, a fungus whose biochemistry and genetics are well known, have been chosen because of their capacity of 11-α-hydroxylation over some steroids which confers on them their anti-inflammatory properties. We have cloned the genes encoding the 11-α-hydroxylase enzymatic activities with the aim to introduce them in other microorganisms, such as Mycobacterium smegmatis, used in the industry to split the side chain of phytosterols, and thus creating recombinant microorganisms able to generate useful steroids from cheap precursors in just one-step fermentation.

The phasin PhaF controls bacterial shape and size in a network-forming strain of Pseudomonas putida.

Pseudomonas putida N, a poly-3-hydroxyalkonate (PHA)-producing bacterium, showing ampicillin resistance, is an unusual strain. In the presence of this antibiotic, it grows as giant cells (25-50µm) forming complex networks inter-connected by micro-tubular structures. The transformation of this bacterium with a plasmid containing the gene phaF, which encodes a phasin involved in the molecular architecture of the PHA-granules, (i) restores the wild-type phenotype by reducing both bacterial size and length (coco-bacilli ranging between 0.5 and 3µm), and (ii) increases ampicillin resistance by more than 100-fold.

The loss of function of PhaC1 is a survival mechanism that counteracts the stress caused by the overproduction of poly-3-hydroxyalkanoates in Pseudomonas putidaΔfadBA.

The poly-3-hydroxylkanoate (PHA)-overproducing mutant Pseudomonas putida U ΔfadBA (PpΔfadBA) lacks the genes encoding the main ß-oxidation pathway (FadBA). This strain accumulates enormous amounts of bioplastics when cultured in chemically defined media containing PHA precursors (different n-alkanoic or n-aryl-alkanoic acids) and an additional carbon source. In medium containing glucose or 4-hydroxy-phenylacetate, the mutant does not accumulate PHAs and grows just as the wild type (P. putida U). However, when the carbon source is octanoate, growth is severely impaired, suggesting that in PpΔfadBA, the metabolic imbalance resulting from a lower rate of ß-oxidation, together with the accumulation of bioplastics, causes severe physiological stress. Here, we show that PpΔfadBA efficiently counteracts this latter effect via a survival mechanism involving the introduction of spontaneous mutations that block PHA accumulation. Surprisingly, genetic analyses of the whole pha cluster revealed that these mutations occurred only in the gene encoding one of the polymerases (phaC1) and that the loss of PhaC1 function was enough to prevent PHA synthesis. The influence of these mutations on the structure of PhaC1 and the existence of a protein-protein (PhaC1-PhaC2) interaction that explains the functionality of the polymerization system are discussed herein.

Functional analyses of three acyl-CoA synthetases involved in bile acid degradation in Pseudomonas putida†DOC21.

Pseudomonas putida DOC21, a soil-dwelling proteobacterium, catabolizes a variety of steroids and bile acids. Transposon mutagenesis and bioinformatics analyses identified four clusters of steroid degradation (std) genes encoding a single catabolic pathway. The latter includes three predicted acyl-CoA synthetases encoded by stdA1, stdA2 and stdA3 respectively. The ΔstdA1 and ΔstdA2 deletion mutants were unable to assimilate cholate or other bile acids but grew well on testosterone or 4-androstene-3,17-dione (AD). In contrast, a ΔstdA3 mutant grew poorly in media containing either testosterone or AD. When cells were grown with succinate in the presence of cholate, ΔstdA1 accumulated Δ(1/4) -3-ketocholate and Δ(1,4) -3-ketocholate, whereas ΔstdA2 only accumulated 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC). When incubated with testosterone or bile acids, ΔstdA3 accumulated 3aα-H-4α(3'propanoate)-7aß-methylhexahydro-1,5-indanedione (HIP) or the corresponding hydroxylated derivative. Biochemical analyses revealed that StdA1 converted cholate, 3-ketocholate, Δ(1/4) -3-ketocholate, and Δ(1,4) -3-ketocholate to their CoA thioesters, while StdA2 transformed DHOPDC to DHOPDC-CoA. In contrast, purified StdA3 catalysed the CoA thioesterification of HIP and its hydroxylated derivatives. Overall, StdA1, StdA2 and StdA3 are acyl-CoA synthetases required for the complete degradation of bile acids: StdA1 and StdA2 are involved in degrading the C-17 acyl chain, whereas StdA3 initiates degradation of the last two steroid rings. The study highlights differences in steroid catabolism between Proteobacteria and Actinobacteria.

The 3,4-dihydroxyphenylacetic acid catabolon, a catabolic unit for degradation of biogenic amines tyramine and dopamine in Pseudomonas putida U.

Degradation of tyramine and dopamine by Pseudomonas putida U involves the participation of twenty one proteins organized in two coupled catabolic pathways, Tyn (tynABFEC tynG tynR tynD, 12 338 bp) and Hpa (hpaR hpaBC hpaHI hpaX hpaG1G2EDF hpaA hpaY, 12 722 bp). The Tyn pathway catalyses the conversion of tyramine and dopamine into 4-hydroxyphenylacetic acid (4HPA) and 3,4-dihydroxyphenylacetic acid (3,4HPA) respectively. Together, the Tyn and Hpa pathways constitute a complex catabolic unit (the 3,4HPA catabolon) in which 3,4HPA is the central intermediate. The genes encoding Tyn proteins are organized in four consecutive transcriptional units (tynABFEC, tynG, tynR and tynD), whereas those encoding Hpa proteins constitute consecutive operons (hpaBC, hpaG1G2EDF, hpaX, hpaHI) and three independent units (hpaA, hpaR and hpaY). Genetic engineering approaches were used to clone tyn and hpa genes and then express them, either individually or in tandem, in plasmids and/or bacterial chromosomes, resulting in recombinant bacterial strains able to eliminate tyramine and dopamine from different media. These results enlarge our biochemical and genetic knowledge of the microbial catabolic routes involved in the degradation of aromatic bioamines. Furthermore, they provide potent biotechnological tools to be used in food processing and fermentation as well as new strategies that could be used for pharmacological and gene therapeutic applications in the near future.

Genetic analyses and molecular characterization of the pathways involved in the conversion of 2-phenylethylamine and 2-phenylethanol into phenylacetic acid in Pseudomonas putida U.

In Pseudomonas putida U two different pathways (Pea, Ped) are required for the conversion of 2-phenylethylamine and 2-phenylethanol into phenylacetic acid. The 2-phenylethylamine pathway (PeaABCDEFGHR) catalyses the transport of this amine, its deamination to phenylacetaldehyde by a quinohaemoprotein amine dehydrogenase and the oxidation of this compound through a reaction catalysed by a phenylacetaldehyde dehydrogenase. Another catabolic route (PedS(1)R(1)ABCS(2)R(2)DEFGHI) is needed for the uptake of 2-phenylethanol and for its oxidation to phenylacetic acid via phenylacetaldehyde. This implies the participation of two different two-component signal-transducing systems, two quinoprotein alcohol dehydrogenases, a cytochrome c, a periplasmic binding protein, an aldehyde dehydrogenase, a pentapeptide repeat protein and an ABC efflux system. Additionally, two accessory sets of elements (PqqABCDEF and CcmABCDEFGHI) are necessary for the operation of the main pathways (Pea and Ped). PqqABCDEF is required for the biosynthesis of pyrroloquinoline quinone (PQQ), a prosthetic group of certain alcohol dehydrogenases that transfers electrons to an independent cytochrome c; whereas CcmABCDEFGHI is required for cytochrome c maturation. Our data show that the degradation of phenylethylamine and phenylethanol in P. putida U is quite different from that reported in Escherichia coli, and they demonstrate that PeaABCDEFGHR and PedS(1)R(1)ABCS(2)R(2)DEFGHI are two upper routes belonging to the phenylacetyl-CoA catabolon.

Poly-3-hydroxyalkanoate synthases from Pseudomonas putida U: substrate specificity and ultrastructural studies.

The substrate specificity of the two polymerases (PhaC1 and PhaC2) involved in the biosynthesis of medium-chain-length poly-hydroxyalkanoates (mcl PHAs) in Pseudomonas putida U has been studied in vivo. For these kind of experiments, two recombinant strains derived from a genetically engineered mutant in which the whole pha locus had been deleted (P. putida U Δpha) were employed. These bacteria, which expresses only phaC1 (P. putida U Δpha pMC-phaC1) or only phaC2 (P. putida U Δpha pMC-phaC2), accumulated different PHAs in function of the precursor supplemented to the culture broth. Thus, the P. putida U Δpha pMC-phaC1 strain was able to synthesize several aliphatic and aromatic PHAs when hexanoic, heptanoic, octanoic decanoic, 5-phenylvaleric, 6-phenylhexanoic, 7-phenylheptanoic, 8-phenyloctanoic or 9-phenylnonanoic acid were used as precursors; the highest accumulation of polymers was observed when the precursor used were decanoic acid (aliphatic PHAs) or 6-phenylhexanoic acid (aromatic PHAs). However, although it synthesizes similar aliphatic PHAs (the highest accumulation was observed when hexanoic acid was the precursor) the other recombinant strain (P. putida U Δpha pMC-phaC2) only accumulated aromatic PHAs when the monomer to be polymerized was 3-hydroxy-5-phenylvaleryl-CoA. The possible influence of the putative three-dimensional structures on the different catalytic behaviour of PhaC1 and PhaC2 is discussed.

Genetic and ultrastructural analysis of different mutants of Pseudomonas putida affected in the poly-3-hydroxy-n-alkanoate gene cluster.

Functional analyses of the different proteins involved in the synthesis and accumulation of polyhydroxyalkanoates (PHAs) in P. putida U were performed using a mutant in which the pha locus had been deleted (PpUDeltapha). These studies showed that: (i) Pha enzymes cannot be replaced by other proteins in this bacterium, (ii) the transformation of PpDeltapha with a plasmid containing the locus pha fully restores the synthesis of bioplastics, (iii) the transformation of PpDeltapha with a plasmid harbouring the gene encoding the polymerase PhaC1 (pMCphaC1) permits the synthesis of polyesters (even in absence of phaC2ZDFI); however, in this strain (PpUDeltapha-pMCphaC1) the number of PHAs granules was higher than in the wild type, (iv) the expression of phaF in PpUDeltapha-pMCphaC1 restores the original phenotype, showing that PhaF is involved in the coalescence of the PHAs granules. Furthermore, the deletion of the phaDFI genes in P. putida U considerably decreases (> 70%) the biosynthesis of PHAs consisting of hydroxyalkanoates with aliphatic constituents, and completely prevents the synthesis of those ones containing aromatic monomers. Additional experiments revealed that the deletion of phaD in P. putida U strongly reduces the synthesis of PHA, this effect being restored by PhaF. Moreover, the overexpression of phaF in P. putida U, or in its DeltafadBA mutant, led to the collection of PHA over-producer strains.

Biochemical evidence that phaZ gene encodes a specific intracellular medium chain length polyhydroxyalkanoate depolymerase in Pseudomonas putida KT2442: characterization of a paradigmatic enzyme.

Polyhydroxyalkanoates (PHAs) can be catabolized by many microorganisms using intra- or extracellular PHA depolymerases. Most of our current knowledge of these intracellular enzyme-coding genes comes from the analysis of short chain length PHA depolymerases, whereas medium chain length PHA (mcl-PHA) intracellular depolymerization systems still remained to be characterized. The phaZ gene of some Pseudomonas putida strains has been identified only by mutagenesis and complementation techniques as putative intracellular mcl-PHA depolymerase. However, none of their corresponding encoded PhaZ enzymes have been characterized in depth. In this study the PhaZ depolymerase from P. putida KT2442 has been purified and biochemically characterized after its overexpression in Escherichia coli. To facilitate these studies we have developed a new and very sensitive radioactive method for detecting PHA hydrolysis in vitro. We have demonstrated that PhaZ is an intracellular depolymerase that is located in PHA granules and that hydrolyzes specifically mcl-PHAs containing aliphatic and aromatic monomers. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. We have modeled the three-dimensional structure of PhaZ complexed with a 3-hydroxyoctanoate dimer. Using this model, we found that the enzyme appears to be built up from a corealpha/beta hydrolase-type domain capped with a lid structure with an active site containing a catalytic triad buried near the connection between domains. All these data constitute the first biochemical characterization of PhaZ and allow us to propose this enzyme as the paradigmatic representative of intracellular endo/exo-mcl-PHA depolymerases.

Acetyl-CoA synthetase from Pseudomonas putida U is the only acyl-CoA activating enzyme induced by acetate in this bacterium.

The gene (acs) encoding the acetyl-CoA synthetase (Acs) in Pseudomonas putida U has been cloned, sequenced and expressed in different microbes. The protein has been purified and characterized from a biochemical, structural and evolutionary point of view. Disruption or deletion of acs handicapped the bacterium for growth in a chemically defined medium containing acetate; this ability was regained when P. putida U was transformed with a plasmid carrying this gene. By contrast, all the acs knock-out mutants could assimilate n-alkanoic acids having a carbon length greater than C2, suggesting that other acyl-CoA activating enzymes (different from Acs) are involved in the catabolism of these compounds. However, these enzymes that can replace the function played by Acs in vivo are not induced by acetate.

A genetically engineered strain of Pseudomonas putida as a useful tool for identifying new therapeutic herbicides.

A genetically engineered strain of Pseudomonas putida U designed for the identification of new therapeutic herbicides has been obtained. In this bacterium, deletion of the homogentisate gene cluster (hmgRABC) confers upon this mutant huge biotechnological possibilities since it can be used: (i) as a target for testing new specific herbicides (p-hydroxy-phenylpyruvate dioxygenase inhibitors); (ii) to identify new therapeutic drugs-effective in the treatment of alkaptonuria and other related tyrosinemia - and (iii) as a source of homogentisic acid in a plant-bacterium association.

A two-component hydroxylase involved in the assimilation of 3-hydroxyphenyl acetate in Pseudomonas putida.

The complete catabolic pathway involved in the assimilation of 3-hydroxyphenylacetic acid (3-OH-PhAc) in Pseudomonas putida U has been established. This pathway is integrated by the following: (i) a specific route (upper pathway), which catalyzes the conversion of 3-OH-PhAc into 2,5-dihydroxyphenylacetic acid (2,5-diOH-PhAc) (homogentisic acid, Hmg), and (ii) a central route (convergent route), which catalyzes the transformation of the Hmg generated from 3-OH-PhAc, l-Phe, and l-Tyr into fumarate and acetoacetate (HmgABC). Thus, in a first step the degradation of 3-OH-PhAc requires the uptake of 3-OH-PhAc by means of an active transport system that involves the participation of a permease (MhaC) together with phosphoenolpyruvate as the energy source. Once incorporated, 3-OH-PhAc is hydroxylated to 2,5-diOH-PhAc through an enzymatic reaction catalyzed by a novel two-component flavoprotein aromatic hydroxylase (MhaAB). The large component (MhaA, 62,719 Da) is a flavoprotein, and the small component (MhaB, 6,348 Da) is a coupling protein that is essential for the hydroxylation of 3-OH-PhAc to 2,5-diOH-PhAc. Sequence analyses and molecular biology studies revealed that homogentisic acid synthase (MhaAB) is different from the aromatic hydroxylases reported to date, accounting for its specific involvement in the catabolism of 3-OH-PhAc. Additionally, an ABC transport system (HmgDEFGHI) involved in the uptake of homogentisic acid and two regulatory elements (mhaSR and hmgR) have been identified. Furthermore, the cloning and the expression of some of the catabolic genes in different microbes presented them with the ability to synthesize Hmg (mhaAB) or allowed them to grow in chemically defined media containing 3-OH-PhAc as the sole carbon source (mhaAB and hmgABC).