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

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

Catabolism of phenylacetic acid in Escherichia coli. Characterization of a new aerobic hybrid pathway

13 pages, 8 figures, 4 tables.-- PMID: 9748275 [PubMed].The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X97452.The paa cluster of Escherichia coli W involved in the aerobic catabolism of phenylacetic acid (PA) has been cloned and sequenced. It was shown to map at min 31.0 of the chromosome at the right end of the mao region responsible for the transformation of 2-phenylethylamine into PA. The 14 paa genes are organized in three transcription units: paaZ and paaABCDEFGHIJK, encoding catabolic genes; and paaXY, containing the paaX regulatory gene. The paaK gene codes for a phenylacetyl-CoA ligase that catalyzes the activation of PA to phenylacetyl-CoA (PA-CoA). The paaABCDE gene products, which may constitute a multicomponent oxygenase, are involved in PA-CoA hydroxylation. The PaaZ protein appears to catalyze the third enzymatic step, with the paaFGHIJ gene products, which show significant similarity to fatty acid beta-oxidation enzymes, likely involved in further mineralization to Krebs cycle intermediates. Three promoters, Pz, Pa, and Px, driven the expression of genes paaZ, paaABCDEFGHIJK, and paaX, respectively, have been identified. The Pa promoter is negatively controlled by the paaX gene product. As PA-CoA is the true inducer, PaaX becomes the first regulator of an aromatic catabolic pathway that responds to a CoA derivative. The aerobic catabolism of PA in E. coli represents a novel hybrid pathway that could be a widespread way of PA catabolism in bacteria.This work was supported by Grants AMB94-1038-C02-02 and AMB97-063-C02-02 from Comisión Interministerial de Ciencia y Tecnología, Spain.Peer reviewe

Steroid catabolism in bacteria: Genetic and functional analyses of stdH and stdJ in Pseudomonas putida DOC21

Pseudomonas putida DOC21 assimilates a large variety of steroids, including bile acids, via a single 9, 10-seco pathway. Two specific mutants knocked down in stdH and stdJ were obtained by deletion (strains P. putida DOC21ΔstdH and P. putida DOC21ΔstdJ). Analysis of these mutants revealed that both had lost the ability to fully degrade bile acids and that the genes stdH and stdJ are involved in oxidation of the A and B rings of the polycyclic steroid structure. Moreover, whereas P. putida DOC21ΔstdH and P. putida DOC21ΔstdJ were unable to degrade testosterone or 4-androstene-3,17-dione (AD), P. putida DOC21ΔstdJ was also unable to assimilate androsta-1,4-diene-3,17-dione (ADD). When cultured in medium containing lithocholate and succinate, P. putida DOC21ΔstdH and P. putida DOC21ΔstdJ accumulated AD and ADD, respectively. Genetic and bioinformatics analyses revealed that: (i) stdH encodes a 3-ketosteroid-Δ1-dehydrogenase; (ii) StdJ is the reductase component of a 3-ketosteroid 9α-hydroxylase; (iii) the trans-expression of stdH and stdJ in the corresponding mutant restored the lost catabolic function(s), and (iv) full steroid metabolism by P. putida DOC21ΔstdH was restored by its expression of kstD2, but not kstD1 or kstD3, of Rhodococcus ruber Chol-4. Our results shed light on the systems used by bacteria to oxidize the A and B rings of steroid compounds. In addition, as the mutants described herein were able to synthesize two pharmaceutically important synthons, AD and ADD, they may be of value in industrial applications

A new class of glutamate dehydrogenases (GDH). Biochemical and genetic characterization of the first member, the AMP-requiring NAD-specific GDH of Streptomyces clavuligerus

14 pages, 10 figures, 6 tables.-- PMID: 10924516 [PubMed].The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AF218569.A new class of glutamate dehydrogenase (GDH) is reported. The GDH of Streptomyces clavuligerus was purified to homogeneity and characterized. It has a native molecular mass of 1,100 kDa and exists as an alpha(6) oligomeric structure composed of 183-kDa subunits. GDH, which requires AMP as an essential activator, shows a maximal rate of catalysis in 100 mm phosphate buffer, pH 7.0, at 30 degrees C. Under these conditions, GDH displayed hyperbolic behavior toward ammonia (K(m), 33 mm) and sigmoidal responses to changes in alpha-ketoglutarate (S(0.5) 1.3 mm; n(H) 1.50) and NADH (S(0.5) 20 microm; n(H) 1.52) concentrations. Aspartate and asparagine were found to be allosteric activators. This enzyme is inhibited by an excess of NADH or NH(4)(+), by some tricarboxylic acid cycle intermediates and by ATP. This GDH seems to be a catabolic enzyme as indicated by the following: (i) it is NAD-specific; (ii) it shows a high value of K(m) for ammonia; and (iii) when S. clavuligerus was cultured in minimal medium containing glutamate as the sole source of carbon and nitrogen, a 5-fold increase in specific activity of GDH was detected compared with cultures provided with glycerol and ammonia. GDH has 1,651 amino acids, and it is encoded by a DNA fragment of 4,953 base pairs (gdh gene). It shows strong sequence similarity to proteins encoded by unidentified open reading frames present in the genomes of species belonging to the genera Mycobacterium, Rickettsia, Pseudomonas, Vibrio, Shewanella, and Caulobacter, suggesting that it has a broad distribution. The GDH of S. clavuligerus is the first member of a class of GDHs included in a subfamily of GDHs (large GDHs) whose catalytic requirements and evolutionary implications are described and discussed.This investigation was supported by Comisión Interministerial de Ciencia y Tecnología, Madrid, Spain, Grant AMB97-0603-C02-01, Fondo Europeo de Desarrollo Regional Grant 1FD97-0245, and Junta de Castilla y León Grant LE 42/96.Peer reviewe

Plasmids containing the same origin of replication are useful tools to perform biotechnological studies in Pseudomonas putida U and in E. coli DH10B

Plasmids containing the same origin of replication (pBBR1MCS-2 KmR and pBBR1MCS-3 TcR) have been used to express simultaneous and independently different proteins in P. putida U and in E. coli. Thus, when P. putida was transformed with different genetic constructions made in the same plasmid (pBBR1MCS-3 TcR), or with plasmids containing the same replication origin but with different antibiotic resistant genes (KmR and TcR), they co-existed inside the same microbe. Furthermore, when E. coli DH10B was transformed with the plasmids recovered from the recombinant P. putida U, we noticed that all the bacteria isolated from single colonies are resistant to Km and Tc, suggesting that these plasmids were also present in E. coli. This observation facilitates the genetic manipulation of these strains (i.e. avoiding the use of different plasmids in double or multiple complementation experiments), and could be an interesting tool to approach many metabolic and biotechnological studies

Phenylacetyl-Coenzyme A Is the True Inducer of the Phenylacetic Acid Catabolism Pathway in Pseudomonas putida U

Aerobic degradation of phenylacetic acid in Pseudomonas putida U is carried out by a central catabolism pathway (phenylacetyl-coenzyme A [CoA] catabolon core). Induction of this route was analyzed by using different mutants specifically designed for this objective. Our results revealed that the true inducer molecule is phenylacetyl-CoA and not other structurally or catabolically related aromatic compounds

Specific Gene Expression in <i>Pseudomonas Putida</i> U Shows New Alternatives for Cadaverine and Putrescine Catabolism

Pseudomonas putida strain U can be grown using, as sole carbon sources, the biogenic amines putrescine or cadaverine, as well as their catabolic intermediates, ɣ-aminobutyrate or δ-aminovalerate, respectively. Several paralogs for the genes that encode some of the activities involved in the catabolism of these compounds, such as a putrescine-pyruvate aminotransferase (spuC1 and spuC2 genes) and a ɣ-aminobutyrate aminotransferase (gabT1 and gabT2 genes) have been identified in this bacterium. When the expression pattern of these genes is analyzed by qPCR, it is drastically conditioned by supplying the carbon sources. Thus, spuC1 is upregulated by putrescine, whereas spuC2 seems to be exclusively induced by cadaverine. However, gabT1 increases its expression in response to different polyamines or aminated catabolic derivatives from them (i.e., ɣ-aminobutyrate or δ-aminovalerate), although gabT2 does not change its expression level concerning no-amine unrelated carbon sources (citrate). These results reveal differences between the mechanisms proposed for polyamine catabolism in P. aeruginosa and Escherichia coli concerning P. putida strain U, as well as allow a deeper understanding of the enzymatic systems used by this last strain during polyamine metabolism

Strategy for Cloning Large Gene Assemblages as Illustrated Using the Phenylacetate and Polyhydroxyalkanoate Gene Clusters

We report an easy procedure for isolating chromosome-clustered genes. By following this methodology, the entire set of genes belonging to the phenylacetic acid (PhAc; 18-kb) pathway as well as those required for the synthesis and mobilization of different polyhydroxyalkanoates (PHAs; 6.4 kb) in Pseudomonas putida U were recovered directly from the bacterial chromosome and cloned into a plasmid for the first time. The transformation of different bacteria with these genetic constructions conferred on them the ability to either degrade PhAc or synthesize bioplastics (PHAs)

The Homogentisate Pathway: a Central Catabolic Pathway Involved in the Degradation of l-Phenylalanine, l-Tyrosine, and 3-Hydroxyphenylacetate in Pseudomonas putida

Pseudomonas putida metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate. Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate. Whereas the phh, tyr, and hpd genes are not linked in the P. putida genome, the hmgABC genes appear to form a single transcriptional unit. Gel retardation assays and lacZ translational fusion experiments have shown that hmgR encodes a specific repressor that controls the inducible expression of the divergently transcribed hmgABC catabolic genes, and homogentisate is the inducer molecule. Footprinting analysis revealed that HmgR protects a region in the Phmg promoter that spans a 17-bp palindromic motif and an external direct repetition from position −16 to position 29 with respect to the transcription start site. The HmgR protein is thus the first IclR-type regulator that acts as a repressor of an aromatic catabolic pathway. We engineered a broad-host-range mobilizable catabolic cassette harboring the hmgABC, hpd, and tyrB genes that allows heterologous bacteria to use Tyr as a unique carbon and energy source. Remarkably, we show here that the catabolism of 3-hydroxyphenylacetate in P. putida U funnels also into the homogentisate central pathway, revealing that the hmg cluster is a key catabolic trait for biodegradation of a small number of aromatic compounds