Belén Floriano

Bacteriocin production and competitiveness of Lactobacillus plantarum LPCO10 in olive juice broth, a culture medium obtained from olives

A culture medium, named olive juice broth, which resembles the natural environment of Lactobacillus plantarum in the traditional Spanish-style green olive fermentation was obtained from green olives. In this medium, the bacteriocin-producing L. plantarum LPCO10 strain was able to produce bacteriocin throughout the incubation time (15 days). Bacteriocin purification from olive juice broth was achieved by a protocol including ammonium sulphate precipitation of cell-free, L. plantarum LPCO10 culture supernatants, and cation-exchange, hydrophobic-interaction and reversed-phase chromatographies. In a series of mixed cultures in olive juice broth, L. plantarum LPCO10 was able to dominate the bacteriocin-sensitive L. plantarum 128/2 strain, whereas the non-bacteriocin-producing, LPCO10 strain derivative, L. plantarum 55-1 strain did not show such capability. These results indicated that olive juice broth may be a valuable experimental substitute for olive fermentation brine in gaining more knowledge about the role of the bacteriocin-producing L. plantarum strains in the control of the Spanish-style green olive fermentation.

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.

Regulation of Tetralin Biodegradation and Identification of Genes Essential for Expression of thn Operons

The tetralin biodegradation genes of Sphingomonas macrogolitabida strain TFA are clustered in two closely linked and divergent operons. To analyze expression of both operons under different growth conditions, transcriptional and translational gene fusions of the first genes of each operon to lacZ have been constructed in plasmids unable to replicate in Sphingomonas and integrated by recombination into the genome of strain TFA. Expression analysis indicated that the transcription of both genes is induced in similar ways by the presence of tetralin. Gene expression in both operons is also subjected to overimposed catabolic repression. Two additional genes named thnR and thnY have been identified downstream of thnCA3A4 genes. ThnR is similar to LysR-type regulators, and mutational analysis indicated that ThnR is strictly required for expression of the thn operons. Unlike other LysR-type regulators, ThnR does not repress its own synthesis. In fact, ThnR activates its own expression, since thnR is cotranscribed with the thnCA3A4 genes. ThnY is similar to the ferredoxin reductase components of dioxygenase systems and shows the fer2 domain, binding a Cys4[2Fe-2S] iron sulfur center, and the FAD-binding domain, common to those reductases. However, it lacks the NAD-binding domain. Intriguingly, ThnY has a regulatory role, since it is also strictly required for expression of the thn operons. Given the similarity of ThnY to reductases and the possibility of its being present in the two redox states, it is tempting to speculate that ThnY is a regulatory component connecting expression of the thn operons to the physiological status of the cell.

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.

Tetralin-Induced and ThnR-Regulated Aldehyde Dehydrogenase and β-Oxidation Genes in Sphingomonas macrogolitabida Strain TFA

ABSTRACT 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 β-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 β-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 β-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.

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.

Identification of a hydratase and a class II aldolase involved in biodegradation of the organic solvent tetralin.

ABSTRACT Two new genes whose products are involved in biodegradation of the organic solvent tetralin were identified. These genes, designated thnE and thnF, are located downstream of the previously identified thnD gene and code for a hydratase and an aldolase, respectively. A sequence comparison of enzymes similar to ThnE showed the significant similarity of hydratases involved in biodegradation pathways to 4-oxalocrotonate decarboxylases and established four separate groups of related enzymes. Consistent with the sequence information, characterization of the reaction catalyzed by ThnE showed that it hydrated a 10-carbon dicarboxylic acid. The only reaction product detected was the enol tautomer, 2,4-dihydroxydec-2-ene-1,10-dioic acid. The aldolase ThnF showed significant similarity to aldolases involved in different catabolic pathways whose substrates are dihydroxylated dicarboxylic acids and which yield pyruvate and a semialdehyde. The reaction products of the aldol cleavage reaction catalyzed by ThnF were identified as pyruvate and the seven-carbon acid pimelic semialdehyde. ThnF and similar aldolases showed conservation of the active site residues identified by the crystal structure of 2-dehydro-3-deoxy-galactarate aldolase, a class II aldolase with a novel reaction mechanism, suggesting that these similar enzymes are class II aldolases. In contrast, ThnF did not show similarity to 4-hydroxy-2-oxovalerate aldolases of other biodegradation pathways, which are significantly larger and apparently are class I aldolases.

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.

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.