Understanding the metabolism of the tetralin degrader Sphingopyxis granuli strain TFA through genome-scale metabolic modelling.

Sphingopyxis granuli strain TFA is an α-proteobacterium that belongs to the sphingomonads, a group of bacteria well-known for its degradative capabilities and oligotrophic metabolism. Strain TFA is the only bacterium in which the mineralisation of the aromatic pollutant tetralin has been completely characterized at biochemical, genetic, and regulatory levels and the first Sphingopyxis characterised as facultative anaerobe. Here we report additional metabolic features of this α-proteobacterium using metabolic modelling and the functional integration of genomic and transcriptomic data. The genome-scale metabolic model (GEM) of strain TFA, which has been manually curated, includes information on 743 genes, 1114 metabolites and 1397 reactions. This represents the largest metabolic model for a member of the Sphingomonadales order thus far. The predictive potential of this model was validated against experimentally calculated growth rates on different carbon sources and under different growth conditions, including both aerobic and anaerobic metabolisms. Moreover, new carbon and nitrogen sources were predicted and experimentally validated. The constructed metabolic model was used as a platform for the incorporation of transcriptomic data, generating a more robust and accurate model. In silico flux analysis under different metabolic scenarios highlighted the key role of the glyoxylate cycle in the central metabolism of strain TFA.

Study of the microbiological quality, prevalence of foodborne pathogens and product shelf-life of Gilthead sea bream (Sparus aurata) and Sea bass (Dicentrarchus labrax) from aquaculture in estuarine ecosystems of Andalusia (Spain).

This study was aimed at characterizing microbiologically Gilthead sea bream (Sparus aurata) and Sea bass (Dicentrarchus labrax) produced in two estuarine ecosystems in Andalusia (Spain): the estuary of the river Guadalquivir (La Puebla del Río, Sevilla) (A), and the estuary of the river Guadiana (Ayamonte, Huelva) (B). The collected fish individuals and water were analysed for hygiene indicator microorganisms and pathogens. The statistical analysis of results revealed that microbial counts for the different microbiological parameters were not statistically different for fish type. On the contrary, considering anatomic part, viscera showed significantly higher concentrations for Enterobacteriaceae, total coliforms and for Staphylococcus spp. coagulase +. Furthermore, location A showed in water and fish higher levels for lactic acid bacteria, aerobic mesophilic bacteria, Enterobacteriaceae, total coliforms and Staphylococcus spp. coagulase +. Neither Listeria monocytogenes, nor Salmonella spp. were detected, though Vibrio parahaemolyticus was identified, molecularly, in estuarine water in location B. The predictive analysis demonstrated that the initial microbiological quality could have an impact on product shelf-life, being longer for location B, with better microbiological quality. Results stress the relevance of preventing the microbiological contamination of water in estuary production systems in order to assure the quality and safety of Gilthead sea bream and Sea bass.

Biodegradation of Tetralin: Genomics, Gene Function and Regulation.

Tetralin (1,2,3,4-tetrahydonaphthalene) is a recalcitrant compound that consists of an aromatic and an alicyclic ring. It is found in crude oils, produced industrially from naphthalene or anthracene, and widely used as an organic solvent. Its toxicity is due to the alteration of biological membranes by its hydrophobic character and to the formation of toxic hydroperoxides. Two unrelated bacteria, Sphingopyxis granuli strain TFA and Rhodococcus sp. strain TFB were isolated from the same niche as able to grow on tetralin as the sole source of carbon and energy. In this review, we provide an overview of current knowledge on tetralin catabolism at biochemical, genetic and regulatory levels in both strains. Although they share the same biodegradation strategy and enzymatic activities, no evidences of horizontal gene transfer between both bacteria have been found. Moreover, the regulatory elements that control the expression of the gene clusters are completely different in each strain. A special consideration is given to the complex regulation discovered in TFA since three regulatory systems, one of them involving an unprecedented communication between the catabolic pathway and the regulatory elements, act together at transcriptional and posttranscriptional levels to optimize tetralin biodegradation gene expression to the environmental conditions.

SuhB, a small non-coding RNA involved in catabolite repression of tetralin degradation genes in Sphingopyxis granuli strain TFA.

Global dRNA-seq analysis of transcription start sites combined with in silico annotation using Infernal software revealed the expression of 91 putative non-coding sRNA in Sphingopyxis granuli TFA cells grown on different carbon sources. Excluding housekeeping sRNAs, only one additional sRNA, which belongs to the Rfam SuhB family (RF00519), was detected by Infernal but with an incorrect size according to the experimental results. SuhB is highly conserved across the Sphingopyxis genus. Expression data revealed that SuhB is present in rapidly growing TFA cells. A suhB deletion mutant exhibited de-repression of tetralin degradation (thn) gene expression and higher amounts of their LysR-type activator, ThnR, under conditions of carbon catabolite repression (CCR). Interaction between SuhB and the 5'UTR of thnR mRNA was demonstrated in vitro. Moreover, co-immunoprecipitation experiments, combined with fluorescence measurements of gfp fusions to the 5'UTR of thnR mRNA and the phenotype of an hfq deletion mutant, suggest the involvement of Hfq in this interaction. Taken together, these data support an Hfq-mediated repressive role for SuhB, on ThnR mRNA translation that prevents thn gene induction. SuhB, which is a highly conserved sRNA in the Sphingopyxis genus, is the first identified element directly involved in CCR of thn gene expression in S. granuli strain TFA.

Genetic dissection of independent and cooperative transcriptional activation by the LysR-type activator ThnR at close divergent promoters.

Regulation of tetralin biodegradation operons is one of the examples of unconventional LysR-type mediated transcriptional regulation. ThnR activates transcription from two divergent and closely located promoters PB and PC. Although ThnR activates each promoter independently, transcription from each one increases when both promoters are together. Mutational analysis of the intergenic region shows that cooperative transcription is achieved through formation of a ThnR complex when bound to its respective sites at each promoter, via formation of a DNA loop. Mutations also defined ThnR contact sites that are important for independent transcriptional activation at each promoter. A mutation at the PB promoter region, which abolishes its independent transcription, does not affect at all PB transcription in the presence of the divergent promoter PC, thus indicating that the complex formed via DNA loop can compensate for the deficiencies in the correct protein-DNA interaction at one of the promoters. Combination of mutations in both promoters identifies a region at PC that is not important for its independent transcription but it is essential for cooperative transcription from both promoters. This work provides new insights into the diversity and complexity of activation mechanisms used by the most abundant type of bacterial transcriptional regulators.

Harnessing the power of microbial metabolism.

Microorganisms are rich repositories of genetic material encoding many activities of potential interest. Recent advances make identifying and exploiting the metabolic treasures of uncultured microbes an easier proposition. Improved expression vectors and metagenomic screening techniques make it easier to identify activities of interest. Synthetic biology and efficient genome editing techniques allow microbial genomes to be modified almost without restriction. Computational approaches based on organism-wide analysis of transcription, protein synthesis and metabolic fluxes make it possible to accurately predict the outcome of the metabolic processes and modifications required for optimization. Together these advances represent a major breakthrough in microbial biotechnology that is expected to yield new generations of tailor-made biocatalysts suitable for multiple biotechnological applications.

Genomic analysis of the nitrate-respiring Sphingopyxis granuli (formerly Sphingomonas macrogoltabida) strain TFA.

BACKGROUND: Sphingomonads are Alphaproteobacteria that belong to the Sphingomonas, Novosphingobium, Sphingopyxis or Sphingobium genera, They are physiologically diverse and broadly distributed in nature, playing important roles in oligotrophic environments and in the degradation of recalcitrant polyaromatic compounds, Sphingopyxis is a poorly studied genus of which only one representative (S. alaskensis RB2256) has been deeply characterized. In this paper we analyze the genomic features of S. granuli strain TFA (formerly Sphingomonas macrogoltabida) in comparison with the available Sphingopyxis sequenced genomes, to describe common characteristics of this genus and to highlight unique characteristics of strain TFA. RESULTS: The TFA genome has been assembled in a single circular chromosome of 4.7 Mb. Genomic sequence analysis and proteome comparison re-assigned the TFA strain to the Sphingopyxis genus and the S. granuli species. Some regions of the TFA genome show high similarity (ca. 100%) to other bacteria and several genomic islands have been detected. Pathways for aromatic compound degradation have been predicted but no growth of TFA has been detected using these as carbon or nitrogen sources. Genes for nitrate respiration have been identified as TFA exclusive. Experimental data on anaerobic growth of TFA using nitrate as a terminal electron acceptor are also provided. CONCLUSIONS: Sphingopyxis representatives form a compact phylogenetic group (with the exception of S. baekryungensis DSM 16222) that share several characteristics, such as being naturally resistant to streptomycin, having only one ribosomal operon, a low number of prophages and CRISPR sequences, absence of selenoproteins and presence of ectoin and other biosynthesis pathways for secondary metabolites. Moreover, the TFA genome organization shows evidence of the presence of putative integrative and conjugative elements (ICE) responsible for the acquisition of several characteristics by horizontal transfer mechanisms. Sphingopyxis representatives have been described as strict aerobes but anaerobic growth using nitrate as a terminal electron acceptor might confer an environmental advantage to the first S. granuli strain characterized at genomic level.

Combination of degradation pathways for naphthalene utilization in Rhodococcus sp. strain TFB.

Rhodococcus sp. strain TFB is a metabolic versatile bacterium able to grow on naphthalene as the only carbon and energy source. Applying proteomic, genetic and biochemical approaches, we propose in this paper that, at least, three coordinated but independently regulated set of genes are combined to degrade naphthalene in TFB. First, proteins involved in tetralin degradation are also induced by naphthalene and may carry out its conversion to salicylaldehyde. This is the only part of the naphthalene degradation pathway showing glucose catabolite repression. Second, a salicylaldehyde dehydrogenase activity that converts salicylaldehyde to salicylate is detected in naphthalene-grown cells but not in tetralin- or salicylate-grown cells. Finally, we describe the chromosomally located nag genes, encoding the gentisate pathway for salicylate conversion into fumarate and pyruvate, which are only induced by salicylate and not by naphthalene. This work shows how biodegradation pathways in Rhodococcus sp. strain TFB could be assembled using elements from different pathways mainly because of the laxity of the regulatory systems and the broad specificity of the catabolic enzymes.

Involvement of a putative cyclic amp receptor protein (CRP)-like binding sequence and a CRP-like protein in glucose-mediated catabolite repression of thn genes in Rhodococcus sp. strain TFB.

Glucose catabolite repression of tetralin catabolic genes in Rhodococcus sp. strain TFB was shown to be exerted by a protein homologous to transcriptional regulators of the cyclic AMP receptor (CRP)-FNR family. The protein was detected bound to putative CRP-like boxes localized at the promoters of the thnA1 and thnS genes.

ThnY is a ferredoxin reductase-like iron-sulfur flavoprotein that has evolved to function as a regulator of tetralin biodegradation gene expression.

Previous genetic studies in Sphingomonas macrogolitabida strain TFA have established that expression of genes involved in tetralin biodegradation (thn genes) requires the function of the LysR type activator ThnR and also ThnY. Sequence comparison indicated that ThnY is homologous to bacterial oxygenase-coupled NAD(P)H-dependent ferredoxin reductases. However, ThnY showed substitutions in highly conserved positions of the pyridine nucleotide binding domain of these ferredoxin reductases. ThnY expression is co-regulated with all other genes required for tetralin biodegradation, and presumably thnY is part of the thnCA3A4RY operon. ThnY has been purified, and its biochemical and functional properties were characterized. ThnY was found to be a monomeric orange-brown iron-sulfur flavoprotein (estimated mass of 37,000 Da) containing one non-covalently attached flavin adenine dinucleotide and one plant type ferredoxin 2Fe-2S cluster. It can be efficiently reduced by dithionite, but reduction by pyridine nucleotides was very poor. Consistently, ThnY-dependent reduction of cytochrome c, ferricyanide, or 2,6-dichlorophenolindophenol using NAD(P)H as the electron donor was undetectable or very weak. The addition of ThnY to electrophoretic mobility shift assays containing ThnR and a probe bearing two thn divergent promoters resulted in a 3-fold increase in protein-DNA complex formation affinity, which indicates that ThnY directly promotes thn transcription activation by ThnR.

Involvement of poly(3-hydroxybutyrate) synthesis in catabolite repression of tetralin biodegradation genes in Sphingomonas macrogolitabida strain TFA.

The tetralin biodegradation genes of Sphingomonas macrogolitabida strain TFA are repressed by catabolite. Insertion mutants in which thn genes are transcribed in the presence of a preferential carbon source and tetralin, bear the insertion in phaC, encoding a poly(3-hydroxybutyrate) (PHB) synthase, a key enzyme in PHB synthesis. Mutant complementation with phaC genes from either Ralstonia euthropha or TFA restored PHB accumulation and the wild-type regulatory pattern of thn genes, thus indicating that this accumulation is a signal for carbon sufficient conditions that prevents expression of thn catabolic genes in this α-proteobacteria.

Tetralin-induced and ThnR-regulated aldehyde dehydrogenase and beta-oxidation genes in Sphingomonas macrogolitabida strain TFA.

A new cluster of genes has been found downstream of the previously identified thnA2 gene. The gene products are similar to nonacylating aldehyde dehydrogenases (ThnG) and to proteins representing a complete beta-oxidation pathway (ThnH to ThnP). ThnG has a nonacylating NAD-dependent pimelic semialdehyde dehydrogenase activity that renders pimelic acid a seven-carbon dicarboxylic acid. For further metabolism via beta-oxidation, pimelic acid could be acylated by a constitutive acyl coenzyme A (acyl-CoA) ligase found in Sphingomonas macrogolitabida strain TFA or by ThnH, which would transfer CoA from a previously acylated molecule. The first round of beta-oxidation is expected to render glutaryl-CoA and acetyl-CoA. Glutaryl-CoA dehydrogenase (ThnN) would catalyze the oxidation and decarboxylation of glutaryl-CoA and yield crotonyl-CoA, which enters the central metabolism via acetyl-CoA. Mutagenesis studies have shown that these genes are not essential for growth on tetralin or fatty acids, although a thnG disruption mutant showed threefold less pimelic semialdehyde dehydrogenase activity. Transcriptional analysis indicated that these genes are induced by tetralin, subjected to catabolite repression, and regulated by the same regulatory factors previously identified to regulate other thn structural genes. In the present study, transcription initiation upstream of thnH and thnM has been detected by primer extension analysis, and putative promoters were identified by sequence analysis. In addition, binding of the activator ThnR to its putative binding sites at the PH and PM promoter regions has been characterized. These results provide a complete characterization of the biodegradation pathway of tetralin to central metabolites and describe the transcriptional organization of the thn operons in S. macrogolitabida strain TFA.

Co-ordinated regulation of two divergent promoters through higher-order complex formation by the LysR-type regulator ThnR.

The genes required for tetralin biodegradation by Sphingomonas macrogolitabida strain TFA are clustered in two divergent and closely linked operons. ThnR, a LysR-type regulator, activates transcription from each operon in response to tetralin. The regulatory thnR gene is co-transcribed with the catabolic genes thnC, thnA3 and thnA4, resulting in positive autoregulation. ThnR binds with different affinity to two primary binding sites, designated B and C, in the intervening region between the two operons and makes additional contact with secondary sites that extend towards the promoters. In addition, ThnR may interact with itself when bound to each site via the formation of a DNA loop, as evidenced by the distortion of the DNA between the primary binding sites and the elimination of the higher-order complexes following the introduction of a half-turn of the DNA helix between the primary binding sites. Transcription from each promoter is not fully independent since mutations in each binding site affected transcription from both promoters. Based on these results, we propose a model of transcription activation that involves the formation of a complex structure by interactions between ThnR molecules bound to distant binding sites and favours transcription from one promoter to the detriment of the other.

Molecular and biochemical characterization of the tetralin degradation pathway in Rhodococcus sp. strain TFB.

The tetralin biodegradation pathway in Rhodococcus sp. strain TFB, a Gram-positive bacterium resistant to genetic manipulation, was characterized using a proteomic approach. Relative protein expression in cell free extracts from tetralin- and glucose-grown cells was compared using the 2D-DIGE technique. Identification of proteins specifically expressed in tetralin-grown cells was used to characterize a complete set of genes involved in tetralin degradation by reverse genetics. We propose a tetralin degradation pathway analogous to that described for Sphingomonas macrogolitabida strain TFA. TFB thn genes are organized into three operons; two contain all of the structural genes and are transcribed in the same direction, while the third operon, thnST, is transcribed in the opposite direction and encodes a two-component regulatory system, whose transcription is higher in tetralin-grown cells. In addition to tetralin induction, TFB thn structural genes are subject to glucose repression. Primer extension assays and translational thnA1::gfp and thnS::gfp fusions were used to characterize putative promoter regions. A mutational analysis of the thnA1 promoter region allowed us to define nucleotides within the cis regulatory elements that are important for the control of thn gene expression.

Integrated response to inducers by communication between a catabolic pathway and its regulatory system.

Efficient gene regulation of metabolic pathways implies that the profile of molecules inducing the pathway matches that of the molecules that are metabolized. Gratuitous induction, a well-known phenomenon in catabolic pathways, is the consequence of differences in the substrate and inducer profiles. This phenomenon is particularly evident in pathways for biodegradation of organic contaminants that can be induced by a variety of molecules similar to the real substrates. Analysis of the regulation of tetralin biodegradation genes in mutant strains with mutations that affect each component of the initial dioxygenase enzymatic complex indicated that the response of the regulatory system to potential inducers is altered differently depending on the mutated component. Based on the expression phenotypes of a number of single or double mutants, we propose a model that represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent efficient induction by a molecule that is not a real substrate. This communication allows a better fit of the substrate and inducer profiles, thus minimizing gratuitous induction, without a requirement for optimal coevolution to match the specificity of catabolic enzymes and their regulatory systems. Modulation of the regulatory system in this way not only provides a more appropriate response to potential inducers recognized by the regulatory system but also may properly adjust the levels of gene expression to the substrate availability.

Molecular analysis of the 21-kb bacteriocin-encoding plasmid pEF1 from Enterococcus faecium 6T1a.

The complete 21,344-bp DNA sequence of the bacteriocin-encoding plasmid pEF1 from Enterococcus faecium 6T1a was determined. Thirty-four putative open reading frames which could code for proteins longer than 42 amino acids were found. Those included the structural genes encoding for the previously described bacteriocins enterocin I and J (also named as enterocins L50A and L50B). After comparison to sequences in public databases, analysis of the gene organization of pEF1 suggests a modular structure with three different functional domains: the replication region, the bacteriocin region and the mobilization plus UV-resistance region. This genetic mosaic structure most probably evolved through recombination events promoted by transposable elements. The hypothesis that the bacteriocin cluster on pEF1 could act as a functional plasmid stabilization module in E. faecium 6T1a is discussed.

Proteomic and transcriptional characterization of aromatic degradation pathways in Rhodoccocus sp. strain TFB.

Rhodococcus sp. strain TFB is a versatile gram-positive bacterium able to grow on a wide variety of aromatic compounds as carbon and energy sources. Since the strain is refractory to genetic analysis, a proteomic approach was used to study the metabolic pathways involved in the catabolism of such compounds by analyzing differentially induced proteins. The most marked difference was observed when the proteome profiles of phthalate-grown cells were compared with those cultured in the presence of tetralin- or naphthalene, suggesting that different metabolic pathways are involved in the degradation of mono- and polyaromatic compounds. Comparison with the proteome of glucose-grown cells indicated that each pathway was specifically induced by the corresponding aromatic compound. A combination of proteomics and molecular biology led to the identification of 14 proteins (65-80% identical to known Pht proteins) that describe a complete pathway for the catabolism of phthalate to central metabolites via intradiol cleavage of protochatechuic acid. Chaperonins were also induced in phthalate-grown cells, indicating that growth on this compound induces a stress response. Absence of catabolite repression by glucose was observed by both transcriptional and proteome analysis, suggesting that Rhodococcus sp. strain TFB may have advantages over other tightly regulated strains in bioremediation.

The locus responsible for production of plantaricin S, a class IIb bacteriocin produced by Lactobacillus plantarum LPCO10, is widely distributed among wild-type Lact. plantarum strains isolated from olive fermentations.

The genes plsA and plsB encoding for production of plantaricin S (Pls), a two-peptide bacteriocin produced by Lactobacillus plantarum LPCO10, are commonly distributed among wild-type Lact. plantarum strains isolated from olive fermentations. Among 68 independent isolates from different olive processing plants in South Spain, 15 of them were shown to produce bacteriocins that were active against other lactic acid bacteria, as well as spoilage and pathogenic bacteria. On the basis of PCR amplification and hybridization with specific probes, the Pls operon was detected in all the bacteriocin producer strains but not in the non-producer ones. Purification and subsequent amino acid sequencing of the bacteriocin produced by some of the 15 isolates yielded both the alpha and beta peptides from Pls. These results suggest that bacteriocin production contributes an ecological advantage for the wild-type Lact. plantanum strains in the colonization of the spontaneous, traditional olive fermentation process.