Julián Perera

Histone H4 from the fruit fly Ceratitis capitata. Circular dichroism studies

Conformational studies on histone H4 from Ceratitis capitata have been carried out. The circular dichroism of histone solutions has been measured at different conditions of pH and ionic strength. Similar experiments have been also carried out with the homologous histone from chicken erythrocytes and both results are compared. Histones H4 from either Ceratitis capitata or higher animals show very close conformational stability.

Molecular characterization of three 3-ketosteroid-Delta(1)-dehydrogenase isoenzynnes of Rhodococcus ruber strain Chol-4

Rhodococcus ruber strain Chol-4 isolated from a sewage sludge sample is able to grow on minimal medium supplemented with steroids, showing a broad catabolic capacity. This paper reports the characterization of three different 3-ketosteroid-Δ(1)-dehydrogenases (KstDs) in the genome of R. ruber strain Chol-4. The genome of this strain does not contain any homologues of a 3-keto-5α-steroid-Δ(4)-dehydrogenase (Kst4d or TesI) that appears in the genomes of Rhodococcus erythropolis SQ1 or Comamonas testosteroni. Growth experiments with kstD2 mutants, either a kstD2 single mutant, kstD2 double mutants in combination with kstD1 or kstD3, or the triple kstD1,2,3 mutant, proved that KstD2 is involved in the transformation of 4-androstene-3,17-dione (AD) to 1,4-androstadiene-3,17-dione (ADD) and in the conversion of 9α-hydroxy-4-androstene-3,17-dione (9OHAD) to 9α-hydroxy-1,4-androstadiene-3,17-dione (9OHADD). kstD2,3 and kstD1,2,3 R. ruber mutants (both lacking KstD2 and KstD3) did not grow in minimal medium with cholesterol as the only carbon source, thus demonstrating the involvement of KstD2 and KstD3 in cholesterol degradation. In contrast, mutation of kstD1 does not alter the bacterial growth on the steroids tested in this study and therefore, the role of this protein still remains unclear. The absence of a functional KstD2 in R. ruber mutants provoked in all cases an accumulation of 9OHAD, as a branch product probably formed by the action of a 3-ketosteroid-9α-hydroxylase (KshAB) on the AD molecule. Therefore, KstD2 is a key enzyme in the AD catabolism pathway of R. ruber strain Chol-4 while KstD3 is involved in cholesterol catabolism.

Cholesterol Degradation by Gordonia cholesterolivorans

ABSTRACT This paper reports physiological and genetic data about the type strain Gordonia cholesterolivorans, a strain that is able to degrade steroid compounds containing a long carbon side chain such as cholesterol (C27), cholestenone (C27), ergosterol (C28), and stigmasterol (C29). The length of the carbon side chain appears to be of great importance for this bacterium, as the strain is unable to grow using steroids with a shorter or nonaliphatic carbon side chain such as cholic acid (C24), progesterone (C21), testosterone, androsterone, 4-androstene-3,17-dione (all C19), and further steroids. This study also demonstrates that the degradation of cholesterol is a quite common feature of the genus Gordonia by comparing Gordonia cholesterolivorans with some other species of this genus (e.g., G. sihwensis, G. hydrophobica, G. australis, and G. neofelifaecis). Pyrosequencing of the genome of G. cholesterolivorans led to the identification of two conventional cholesterol oxidase genes on an 8-kb and a 12.8-kb genomic fragment with genetic organizations that are quite unique as compared to the genomes of other cholesterol-degrading bacteria sequenced so far. The identified two putative cholesterol oxidases of G. cholesterolivorans are both intracellularly acting enzymes of the class I type. Whereas one of these two cholesterol oxidases (ChoOx-1) shows high identity with an oxidoreductase of the opportunistic pathogen G. bronchialis and is not transcribed during growth with cholesterol, the other one (ChoOx-2) appears phylogenetically closer to cholesterol oxidases from members of the genus Rhodococcus and is transcribed constitutively. By using targeted gene disruption, a G. cholesterolivorans ChoOx-2 gene mutant strain that was unable to grow with steroids was obtained.

ChoG is the main inducible extracellular cholesterol oxidase of Rhodococcus sp. strain CECT3014

Cholesterol catabolism has been reported in different bacteria and particularly in several Rhodococcus species, but the genetic of this complex pathway is not yet very well defined. In this work we report the isolation and sequencing of a 9.8 kb DNA fragment of Rhodococcus sp. strain CECT3014, a bacterial strain that we here identify as a Rhodococcus erythropolis strain. In this DNA fragment we found several ORF that are probably involved in steroid catabolism, and choG, a gene encoding a putative cholesterol oxidase whose functional characterization we here report. ChoG protein is a class II cholesterol oxidase with all the structural features of the enzymes of this group. The disruption of the choG gene does not alter the ability of strain CECT3014 cells to grow on cholesterol, but it abolishes the production of extracellular cholesterol oxidase. This later effect is reverted when the mutant cells are transformed with a plasmid expressing choG. We conclude that choG is the gene responsible for the inducible extracellular cholesterol oxidase activity of strain CECT3014. This activity distributes between the cellular membrane and the culture supernatant in a way that suggests it is produced by the same ChoG protein that occurs in two different locations. RT-PCR transcript analysis showed a dual scheme of choG expression: a low constitutive independent transcription, plus a cholesterol induced transcription of choG into a polycistronic kstD-hsd4B-choG mRNA.

Gordonia cholesterolivorans sp. nov., a cholesterol- degrading actinomycete isolated from sewage sludge

The taxonomic position of the cholesterol-degrading strain Chol-3(T), isolated from a sewage sludge sample, was clarified using a polyphasic taxonomic approach. Phylogenetic analysis of its 16S rRNA gene sequence, whole-cell fatty acid profile and mycolic acid composition revealed that this isolate is a member of the genus Gordonia with the species Gordonia sihwensis, G. hydrophobica and G. shandongensis being the nearest phylogenetic neighbours. The results of DNA-DNA hybridization against its phylogenetically closest neighbours as well as the results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain Chol-3(T) from the other Gordonia species with validly published names. Strain Chol-3(T) therefore merits recognition as a member of a novel species within the genus Gordonia, for which the name Gordonia cholesterolivorans sp. nov. is proposed. The type strain is Chol-3(T) (=CECT 7408(T) =DSM 45229(T)).

Genetic characterization of the styrene lower catabolic pathway of Pseudomonas sp. strain Y2.

Pseudomonas sp. strain Y2 is a styrene-degrading bacterium, which initiates the catabolism of this compound via its transformation into phenylacetate by the sequential oxidation of the vinyl side chain. The styrene upper catabolic gene cluster (sty genes) had been localized in a 9.2-kb chromosomal region. This report describes the isolation, sequencing and analysis of an adjacent 20.5-kb chromosomal region that contains the genes of the styrene lower degradative pathway (paa genes), which are involved in the transformation of phenylacetate into aliphatic compounds that can enter the Krebs cycle. Hence, Pseudomonas sp. strain Y2 becomes the first microorganism whose entire styrene catabolic cluster has been completely characterized. Analysis of the paa gene cluster has revealed the presence of 17 open reading frames as well as gene duplications and gene reorganizations that are absent in other phenylacetate catabolic clusters described so far. The functionality of these genes has been proved by means of both complementation experiments on Pseudomonas putida mutants and in vitro enzymatic assays. Moreover, a DNA cassette encoding the whole styrene lower pathway has been constructed and has been used to expand the ability of Pseudomonas strains to degrade phenylacetic acid. For the first time, two functional phenylacetate-CoA ligases have been identified in an aerobic phenylacetic acid degradation pathway. Although the upper and lower styrene catabolic clusters are adjacent in the Pseudomonas sp. strain Y2 chromosome, their particular base composition and codon usage suggest a distinct evolutionary history.

Coregulation by Phenylacetyl-Coenzyme A-Responsive PaaX Integrates Control of the Upper and Lower Pathways for Catabolism of Styrene by Pseudomonas sp. Strain Y2

The P(styA) promoter of Pseudomonas sp. strain Y2 controls expression of the styABCD genes, which are required for the conversion of styrene to phenylacetate, which is further catabolized by the products of two paa gene clusters. Two PaaX repressor proteins (PaaX1 and PaaX2) regulate transcription of the paa gene clusters of this strain. In silico analysis of the P(styA) promoter region revealed a sequence located just within styA that is similar to the reported PaaX binding sites of Escherichia coli and the proposed PaaX binding sites of the paa genes of Pseudomonas species. Here we show that protein extracts from some Pseudomonas strains that have paaX genes, but not from a paaX mutant strain, can bind and retard the migration of a P(styA) specific probe. Purified maltose-binding protein (MBP)-PaaX1 fusion protein specifically binds the P(styA) promoter proximal PaaX site, and this binding is eliminated by the addition of phenylacetyl-coenzyme A. The sequence protected by MBP-PaaX1 binding was defined by DNase I footprinting. Moreover, MBP-PaaX1 represses transcription from the P(styA) promoter in a phenylacetyl-coenzyme A-dependent manner in vitro. Finally, the inactivation of both paaX gene copies of Pseudomonas sp. strain Y2 leads to a higher level of transcription from the P(styA) promoter, while heterologous expression of the PaaX1 in E. coli greatly decreases transcription from the P(styA) promoter. These findings reveal a control mechanism that integrates regulation of styrene catabolism by coordinating the expression of the styrene upper catabolic operon to that of the paa-encoded central pathway and support a role for PaaX as a major regulatory protein in the phenylacetyl-coenzyme A catabolon through its response to the levels of this central metabolite.

Construction of a bacterial biosensor for styrene

A new bacterial biosensor for styrene has been developed and characterized. A translational fusion of the lacZ gene to the sty promoter of Pseudomonas sp. strain Y2 has been inserted into miniTn5. Transposition of the recombinant transposon to the chromosome of Pseudomonas sp. strain Y2 resulted in a whole-cell biosensor able to detect and degrade styrene. In this biosensor, the endogenous StyS/StyR system detects the presence of styrene and turns on the expression of the exogenous reporter gene from the transferred construction. Other compounds such as toluene, epoxystyrene, phenylacetaldehyde and 2-phenylethanol also induced expression of beta-galactosidase although quantitative differences in their effect are clearly detected. Non-inducing compounds affect differently the sensitivity to inducing compounds when present in a mixture.

The mer operon of the acidophilic bacterium Thiobacillus T3.2 diverges from its Thiobacillus ferrooxidans counterpart

Abstract The chromosomal mercury resistance (mer) region of the acidophilic bacterium Thiobacillus T3.2 was cloned, characterized, and compared to reported homologous sequences. The Thiobacillus T3.2 mer resistance system is organized as an operon that transcribes into a polycistronic mRNA encoding the Hg2+ ion transport T and Mer P proteins and the mercuric reductase MerA. In contrast to the Thiobacillus ferrooxidans mer determinant, no merC gene was detected. Transcription of structural genes is regulated by the product of the regulatory merR gene. On the basis of sequence data and expression experiments in E. coli, both merTPA and merR transcription units could be located close to each other and in different strands, with their promoters (PTPA and PR, respectively) overlapping the putative MerR binding site in the intergenic operator/promoter (O/P) region. Amino acid sequences of mer gene products were compared to their homologs. Some sequence features, such as the number and position of cysteine residues, are unique for the Mer proteins of this bacterium. Similarities (−10 and −35 boxes are 19 bp apart in both PR and PTPA promoters) and differences (inverted repeats in the Thiobacillus T3.2 MerR-binding site are 2 bp shorter than in Thiobacillus ferrooxidans) exist between the O/P intergenic regions of both Thiobacilli. In vivo experiments showed inducible expression of mercury resistance in E. coli cells transformed with the entire Thiobacillus T3.2 mer genetic determinant (structural plus regulatory genes), and little or no expression in clones containing only the structural merT, merP, and merA genes.

Design of catabolic cassettes for styrene biodegradation

A broad-host range metabolic cassette has been designed that, under the control of the Ptac promoter, expresses the styABCD catabolic genes from Pseudomonas sp. Y2, which are responsible for the transformation of styrene into phenylacetic acid (styrene upper pathway). This novel cassette confers to phenylacetic acid-degrading bacteria the ability to grow efficiently on styrene as the sole carbon and energy source. By combining both the sty cassette and the archetypal pWW0 TOL plasmid into the well-known Pseudomonas putida F1 aromatic biodegrader, we have constructed a novel derivative strain that shows one of the largest degradative potentials so far described for aromatic hydrocarbons, because it is able to use BTEX compounds (benzene, toluene, ethylbenzene and xylenes) and styrene as a source of carbon and energy. Furthermore, the sty cassette was engineered within a mini-transposon and endowed with a gene containment system, based on the toxic effect of the colicin E3 RNase, to reduce its lateral spread to other hosts. This contained cassette lacks defined transcriptional regulatory signals and, thus, it becomes an alternative strategy to select recombinant strains that efficiently express the desired phenotype from housekeeping regulatory elements.