A new and promiscuous α/ß hydrolase from Acinetobacter tandoii DSM 14970 T inactivates the mycotoxin ochratoxin A.

The presence of ochratoxin A (OTA) in food and feed represents a serious concern since it raises severe health implications. Bacterial strains of the Acinetobacter genus hydrolyse the amide bond of OTA yielding non-toxic OTα and L-ß-phenylalanine; in particular, the carboxypeptidase PJ15_1540 from Acinetobacter sp. neg1 has been identified as an OTA-degrading enzyme. Here, we describe the ability to transform OTA of cell-free protein extracts from Acinetobacter tandoii DSM 14970 T, a strain isolated from sludge plants, and also report on the finding of a new and promiscuous α/ß hydrolase (ABH), with close homologs highly distributed within the Acinetobacter genus. ABH from A. tandoii (AtABH) exhibited amidase activity against OTA and OTB mycotoxins, as well as against several carboxypeptidase substrates. The predicted structure of AtABH reveals an α/ß hydrolase core composed of a parallel, six-stranded ß-sheet, with a large cap domain similar to the marine esterase EprEst. Further biochemical analyses of AtABH reveal that it is an efficient esterase with a similar specificity profile as EprEst. Molecular docking studies rendered a consistent OTA-binding mode. We proposed a potential procedure for preparing new OTA-degrading enzymes starting from promiscuous α/ß hydrolases based on our results. KEY POINTS: • AtABH is a promiscuous αß hydrolase with both esterase and amidohydrolase activities • AtABH hydrolyses the amide bond of ochratoxin A rendering nontoxic OTα • Promiscuous αß hydrolases are a possible source of new OTA-degrading enzymes.

Non-redundant functionality of Lactiplantibacillus plantarum phospho-ß-glucosidases revealed by carbohydrate utilization signatures associated to pbg2 and pbg4 gene mutants.

AIM: To increase our knowledge on the functionality of 6-phospho-ß-glucosidases linked to phosphoenolpyruvate-dependent phosphotransferase systems (PTS) that are encountered in high redundancy in the Lactiplantibacillus plantarum WCFS1 genome. METHODS AND RESULTS: Two L. plantarum WCFS1 gene mutants that lacked one of the 6-phospho-ß-glucosidases, ∆pbg2 (or ∆lp_0906) or ∆pbg4 (or ∆lp_2777) were constructed and the metabolic impact of these mutations assessed by high-throughput phenotyping (Omnilog). The ∆pbg2 mutant displayed a reduced metabolic performance, having lost the capacity to utilize 20 out of 57 carbon (C)-sources used by the wild-type strain. Conversely, the ∆pbg4 mutant conserved the capacity to metabolize most of the C-sources preferred by the wild type strain. This mutant utilized 56 C-sources albeit the range of substrates used and hence its metabolic profiling differed from that of the WCFS1 strain. The ∆pbg2 mutant notably reduced or abolished the capacity to metabolize substrates related to pentose and glucoronate interconversions and was unable to assimilate fatty acids or nucleosides as sole C-sources for growth. The ∆pbg4 mutant acquired the capacity to utilize efficiently glycogen, indicating an efficient supply of glucose from this source. CONCLUSION: Lactiplantibacillus plantarum gene mutants that lack individual 6-phospho-ß-glucosidases display very different carbohydrate utilization signatures showing that these enzymes can be crucial to determine the capacity of L. plantarum to consume different C-sources and hence for the nutrition and physiology of this microorganism.

Production and Digestibility Studies of ß-Galactosyl Xylitol Derivatives Using Heterogeneous Catalysts of LacA ß-Galactosidase from Lactobacillus Plantarum WCFS1.

The synthesis of ß-galactosyl xylitol derivatives using immobilized LacA ß-galactosidase from Lactobacillus plantarum WCFS1 is presented. These compounds have the potential to replace traditional sugars by their properties as sweetener and taking the advantages of a low digestibility. The enzyme was immobilized on different supports, obtaining immobilized preparations with different activity and stability. The immobilization on agarose-IDA-Zn-CHO in the presence of galactose allowed for the conserving of 78% of the offered activity. This preparation was 3.8 times more stable than soluble. Since the enzyme has polyhistidine tags, this support allowed the immobilization, purification and stabilization in one step. The immobilized preparation was used in synthesis obtaining two main products and a total of around 68 g/L of ß-galactosyl xylitol derivatives and improving the synthesis/hydrolysis ratio by around 30% compared to that of the soluble enzyme. The catalyst was recycled 10 times, preserving an activity higher than 50%. The in vitro intestinal digestibility of the main ß-galactosyl xylitol derivatives was lower than that of lactose, being around 6 and 15% for the galacto-xylitol derivatives compared to 55% of lactose after 120 min of digestion. The optimal amount immobilized constitutes a very useful tool to synthetize ß-galactosyl xylitol derivatives since it can be used as a catalyst with high yield and being recycled for at least 10 more cycles.

The use of Lactobacillus plantarum esterase genes: a biotechnological strategy to increase the bioavailability of dietary phenolic compounds in lactic acid bacteria.

In Lactobacillus plantarum the metabolism of hydroxybenzoic and hydroxycinnamic acid derivatives follows a similar two-step pathway, an esterase action followed by a decarboxylation. The L. plantarum esterase genes involved in these reactions have been cloned into pNZ8048 or pT1NX plasmids and transformed into technologically relevant lactic acid bacteria. None of the strains assayed can hydrolyse methyl gallate, a hydroxybenzoic ester. The presence of the L. plantarum tannase encoding genes (tanALp or tanBLp) on these bacteria conferred their detectable esterase (tannase) activity. Similarly, on hydroxycinnamic compounds, esterase activity for the hydrolysis of ferulic acid was acquired by lactic acid bacteria when L. plantarum esterase (JDM1_1092) was present. This study showed that the heterologous expression of L. plantarum esterase genes involved in the metabolism of phenolic acids allowed the production of healthy compounds which increase the bioavailability of these dietary compounds in food relevant lactic acid bacteria.

Geranyl Functionalized Materials for Site-Specific Co-Immobilization of Proteins.

Different materials containing carboxylic groups have been functionalized with geranyl-amine molecules by using an EDC/NHS strategy. Chemical modification of the support was confirmed by XRD, UV-spectrophotometer, and FT-IR. This geranyl-functionalized material was successfully applied for four different strategies of site-selective immobilization of proteins at room temperature and aqueous media. A reversible hydrophobic immobilization of proteins (lipases, phosphoglucosidases, or tyrosinase) was performed in neutral pH in yields from 40 to >99%. An increase of the activity in the case of lipases was observed from a range of 2 to 4 times with respect to the initial activity in solution. When chemically or genetically functionalized cysteine enzymes were used, the covalent immobilization, via a selective thiol-alkene reaction, was observed in the presence of geranyl support at pH 8 in lipases in the presence of detergent (to avoid the previous hydrophobic interactions). Covalent attachment was confirmed with no release of protein after immobilization by incubation with hydrophobic molecules. In the case of a selenium-containing enzyme produced by the selenomethionine pathway, the selective immobilization was successfully yielded at acidic pH (pH 5) (89%) much better than at pH 8. In addition, when an azido-enzyme was produced by the azide-homoalanine pathway, the selective immobilization was successful at pH 6 and in the presence of CuI for the click chemistry reaction.

High-resolution mapping of tuberculosis transmission: Whole genome sequencing and phylogenetic modelling of a cohort from Valencia Region, Spain.

BACKGROUND: Whole genome sequencing provides better delineation of transmission clusters in Mycobacterium tuberculosis than traditional methods. However, its ability to reveal individual transmission links within clusters is limited. Here, we used a 2-step approach based on Bayesian transmission reconstruction to (1) identify likely index and missing cases, (2) determine risk factors associated with transmitters, and (3) estimate when transmission happened. METHODS AND FINDINGS: We developed our transmission reconstruction method using genomic and epidemiological data from a population-based study from Valencia Region, Spain. Tuberculosis (TB) incidence during the study period was 8.4 cases per 100,000 people. While the study is ongoing, the sampling frame for this work includes notified TB cases between 1 January 2014 and 31 December 2016. We identified a total of 21 transmission clusters that fulfilled the criteria for analysis. These contained a total of 117 individuals diagnosed with active TB (109 with epidemiological data). Demographic characteristics of the study population were as follows: 80/109 (73%) individuals were Spanish-born, 76/109 (70%) individuals were men, and the mean age was 42.51 years (SD 18.46). We found that 66/109 (61%) TB patients were sputum positive at diagnosis, and 10/109 (9%) were HIV positive. We used the data to reveal individual transmission links, and to identify index cases, missing cases, likely transmitters, and associated transmission risk factors. Our Bayesian inference approach suggests that at least 60% of index cases are likely misidentified by local public health. Our data also suggest that factors associated with likely transmitters are different to those of simply being in a transmission cluster, highlighting the importance of differentiating between these 2 phenomena. Our data suggest that type 2 diabetes mellitus is a risk factor associated with being a transmitter (odds ratio 0.19 [95% CI 0.02-1.10], p < 0.003). Finally, we used the most likely timing for transmission events to study when TB transmission occurred; we identified that 5/14 (35.7%) cases likely transmitted TB well before symptom onset, and these were largely sputum negative at diagnosis. Limited within-cluster diversity does not allow us to extrapolate our findings to the whole TB population in Valencia Region. CONCLUSIONS: In this study, we found that index cases are often misidentified, with downstream consequences for epidemiological investigations because likely transmitters can be missed. Our findings regarding inferred transmission timing suggest that TB transmission can occur before patient symptom onset, suggesting also that TB transmits during sub-clinical disease. This result has direct implications for diagnosing TB and reducing transmission. Overall, we show that a transition to individual-based genomic epidemiology will likely close some of the knowledge gaps in TB transmission and may redirect efforts towards cost-effective contact investigations for improved TB control.

Unravelling the diversity of glycoside hydrolase family 13 α-amylases from Lactobacillus plantarum WCFS1.

BACKGROUND: α-Amylases specifically catalyse the hydrolysis of the internal α-1, 4-glucosidic linkages of starch. Glycoside hydrolase (GH) family 13 is the main α-amylase family in the carbohydrate-active database. Lactobacillus plantarum WCFS1 possesses eleven proteins included in GH13 family. Among these, proteins annotated as maltose-forming α-amylase (Lp_0179) and maltogenic α-amylase (Lp_2757) were included. RESULTS: In this study, Lp_0179 and Lp_2757 L. plantarum α-amylases were structurally and biochemically characterized. Lp_2757 displayed structural features typical of GH13_20 subfamily which were absent in Lp_0179. Genes encoding Lp_0179 (Amy2) and Lp_2757 were cloned and overexpressed in Escherichia coli BL21(DE3). Purified proteins showed high hydrolytic activity on pNP-α-D-maltopyranoside, being the catalytic efficiency of Lp_0179 remarkably higher. In relation to the hydrolysis of starch-related carbohydrates, Lp_0179 only hydrolysed maltopentaose and dextrin, demonstrating that is an exotype glucan hydrolase. However, Lp_2757 was also able to hydrolyze cyclodextrins and other non-cyclic oligo- and polysaccharides, revealing a great preference towards α-1,4-linkages typical of maltogenic amylases. CONCLUSIONS: The substrate range as well as the biochemical properties exhibited by Lp_2757 maltogenic α-amylase suggest that this enzyme could be a very promising enzyme for the hydrolysis of α-1,4 glycosidic linkages present in a broad number of starch-carbohydrates, as well as for the investigation of an hypothetical transglucosylation activity under appropriate reaction conditions.

Bacterial tannases: classification and biochemical properties.

Tannin acyl hydrolases, also known as tannases, are a group of enzymes critical for the transformation of tannins. The study of these enzymes, which initially evolved in different organisms to detoxify and/or use these plant metabolites, has nowadays become relevant in microbial enzymology research due to their relevant role in food tannin transformation. Microorganisms, particularly bacteria, are major sources of tannase. Cloning and heterologous expression of bacterial tannase genes and structural studies have been performed in the last few years. However, a systematic compilation of the information related to all recombinant tannases, their classification, and characteristics is missing. In this review, we explore the diversity of heterologously produced bacterial tannases, describing their substrate specificity and biochemical characterization. Moreover, a new classification based on sequence similarity analysis is proposed. Finally, putative tannases have been identified in silico for each group of tannases taking advantage of the use of the "tannase" distinctive features previously proposed.

Unravelling the Reduction Pathway as an Alternative Metabolic Route to Hydroxycinnamate Decarboxylation in Lactobacillus plantarum.

Lactobacillus plantarum is the lactic acid bacterial species most frequently found in plant-food fermentations where hydroxycinnamic acids are abundant. L. plantarum efficiently decarboxylates these compounds and also reduces them, yielding substituted phenylpropionic acids. Although the reduction step is known to be induced by a hydroxycinnamic acid, the enzymatic machinery responsible for this reduction pathway has not been yet identified and characterized. A previous study on the transcriptomic response of L. plantarum to p-coumaric acid revealed a marked induction of two contiguous genes, lp_1424 and lp_1425, encoding putative reductases. In this work, the disruption of these genes abolished the hydroxycinnamate reductase activity of L. plantarum, supporting their involvement in such chemical activity. Functional in vitro studies revealed that Lp_1425 (HcrB) exhibits hydroxycinnamate reductase activity but was unstable in solution. In contrast, Lp_1424 (HcrA) was inactive but showed high stability. When the hcrAB genes were co-overexpressed, the formation of an active heterodimer (HcrAB) was observed. Since L. plantarum reductase activity was only observed on hydroxycinnamic acids (o-coumaric, m-coumaric, p-coumaric, caffeic, ferulic, and sinapic acids), the presence of a hydroxyl group substituent on the benzene ring appears to be required for activity. In addition, hydroxycinnamate reductase activity was not widely present among lactic acid bacteria, and it was associated with the presence of hcrAB genes. This study revealed that L. plantarum hydroxycinnamate reductase is a heterodimeric NADH-dependent coumarate reductase acting on a carbon-carbon double bond.IMPORTANCELactobacillus plantarum is a bacterial species frequently found in the fermentation of vegetables where hydroxycinnamic acids are present. The bacterial metabolism on these compounds during fermentation plays a fundamental role in the biological activity of hydroxycinnamates. L. plantarum strains exhibit an as yet unknown reducing activity, transforming hydroxycinnamates to substituted phenylpropionic acids, which possess higher antioxidant activity than their precursors. The protein machinery involved in hydroxycinnamate reduction, HcrAB, was genetically identified and characterized. The heterodimeric NADH-dependent coumarate reductase HcrAB described in this work provides new insights on the L. plantarum metabolic response to counteract the stressful conditions generated by food phenolics.

Ethylphenol Formation by Lactobacillus plantarum: Identification of the Enzyme Involved in the Reduction of Vinylphenols.

Ethylphenols are strong odorants produced by microbial activity that are described as off flavors in several foods. Lactobacillus plantarum is a lactic acid bacterial species able to produce ethylphenols by the reduction of vinylphenols during the metabolism of hydroxycinnamic acids. However, the reductase involved has not been yet uncovered. In this study, the involvement in vinylphenol reduction of a gene encoding a putative reductase (lp_3125) was confirmed by the absence of reduction activity in the Δlp_3125 knockout mutant. The protein encoded by lp_3125, VprA, was recombinantly produced in Escherichia coli VprA was assayed against vinylphenols (4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol), and all were reduced to their corresponding ethylphenols (4-ethylphenol, 4-ethylcatechol, and 4-ethylguaiacol). PCR and high-performance liquid chromatography (HPLC) detection methods revealed that the VprA reductase is not widely distributed among the lactic acid bacteria studied and that only the bacteria possessing the vprA gene were able to produce ethylphenol from vinylphenol. However, all the species belonging to the L. plantarum group were ethylphenol producers. The identification of the L. plantarum VprA protein involved in hydroxycinnamate degradation completes the route of degradation of these compounds in lactic acid bacteria.IMPORTANCE The presence of volatile phenols is considered a major organoleptic defect of several fermented alcoholic beverages. The biosynthesis of these compounds has been mainly associated with Brettanomyces/Dekkera yeasts. However, the potential importance of lactic acid bacteria in volatile phenol spoilage is emphasized by reports describing a faster ethylphenol production by these bacteria than by yeasts. The genetic identification of the bacterial vinylphenol reductase involved in volatile phenol production provides new insights into the role of lactic acid bacteria in the production of these off flavors. The development of a molecular method for the detection of ethylphenol-producing bacteria could be helpful to design strategies to reduce the bacterial production of vinylphenols in fermented foods.

A Diverse Range of Human Gut Bacteria Have the Potential To Metabolize the Dietary Component Gallic Acid.

The human gut microbiota contains a broad variety of bacteria that possess functional genes, with resultant metabolites that affect human physiology and therefore health. Dietary gallates are phenolic components that are present in many foods and beverages and are regarded as having health-promoting attributes. However, the potential for metabolism of these phenolic compounds by the human microbiota remains largely unknown. The emergence of high-throughput sequencing (HTS) technologies allows this issue to be addressed. In this study, HTS was used to assess the incidence of gallate-decarboxylating bacteria within the gut microbiota of healthy individuals for whom bacterial diversity was previously determined to be high. This process was facilitated by the design and application of degenerate PCR primers to amplify a region encoding the catalytic C subunit of gallate decarboxylase (LpdC) from total metagenomic DNA extracted from human fecal samples. HTS resulted in the generation of a total of 3,261,967 sequence reads and revealed that the primary gallate-decarboxylating microbial phyla in the intestinal microbiota were Firmicutes (74.6%), Proteobacteria (17.6%), and Actinobacteria (7.8%). These reads corresponded to 53 genera, i.e., 47% of the bacterial genera detected previously in these samples. Among these genera, Anaerostipes and Klebsiella accounted for the majority of reads (40%). The usefulness of the HTS-lpdC method was demonstrated by the production of pyrogallol from gallic acid, as expected for functional gallate decarboxylases, among representative strains belonging to species identified in the human gut microbiota by this method.IMPORTANCE Despite the increasing wealth of sequencing data, the health contributions of many bacteria found in the human gut microbiota have yet to be elucidated. This study applies a novel experimental approach to predict the ability of gut microbes to carry out a specific metabolic activity, i.e., gallate metabolism. The study showed that, while gallate-decarboxylating bacteria represented 47% of the bacterial genera detected previously in the same human fecal samples, no gallate decarboxylase homologs were identified from representatives of Bacteroidetes The presence of functional gallate decarboxylases was demonstrated in representative Proteobacteria and Firmicutes strains from the human microbiota, an observation that could be of considerable relevance to the in vivo production of pyrogallol, a physiologically important bioactive compound.

Identification of a highly active tannase enzyme from the oral pathogen Fusobacterium nucleatum subsp. polymorphum.

BACKGROUND: Tannases are tannin-degrading enzymes that have been described in fungi and bacteria as an adaptative mechanism to overcome the stress conditions associated with the presence of these phenolic compounds. RESULTS: We have identified and expressed in E. coli a tannase from the oral microbiota member Fusobacterium nucleatum subs. polymorphum (TanBFnp). TanBFnp is the first tannase identified in an oral pathogen. Sequence analyses revealed that it is closely related to other bacterial tannases. The enzyme exhibits biochemical properties that make it an interesting target for industrial use. TanBFnp has one of the highest specific activities of all bacterial tannases described to date and shows optimal biochemical properties such as a high thermal stability: the enzyme keeps 100% of its activity after prolonged incubations at different temperatures up to 45 °C. TanBFnp also shows a wide temperature range of activity, maintaining above 80% of its maximum activity between 22 and 55 °C. The use of a panel of 27 esters of phenolic acids demonstrated activity of TanBFnp only against esters of gallic and protocatechuic acid, including tannic acid, gallocatechin gallate and epigallocatechin gallate. Overall, TanBFnp possesses biochemical properties that make the enzyme potentially useful in biotechnological applications. CONCLUSIONS: We have identified and characterized a metabolic enzyme from the oral pathogen Fusobacterium nucleatum subsp. polymorphum. The biochemical properties of TanBFnp suggest that it has a major role in the breakdown of complex food tannins during oral processing. Our results also provide some clues regarding its possible participation on bacterial survival in the oral cavity. Furthermore, the characteristics of this enzyme make it of potential interest for industrial use.

Role of APOBEC3H in the Viral Control of HIV Elite Controller Patients.

Background APOBEC3H (A3H) gene presents variation at 2 positions (rs139297 and rs79323350) leading to a non-functional protein. So far, there is no information on the role played by A3H in spontaneous control of HIV. The aim of this study was to evaluate the A3H polymorphisms distribution in a well-characterized group of Elite Controller (EC) subjects. Methods We analyzed the genotype distribution of two different SNPs (rs139297 and rs79323350) of A3H in 30 EC patients and compared with 11 non-controller (NC) HIV patients. Genotyping was performed by PCR, cloning and Sanger sequencing. Both polymorphisms were analyzed jointly in order to adequately attribute the active or inactive status of A3H protein. Results EC subjects included in this study were able to maintain a long-term sustained spontaneous HIV-viral control and optimal CD4-T-cell counts; however, haplotypes leading to an active protein were very poorly represented in these patients. We found that the majority of EC subjects (23/30; 77%) presented allelic combinations leading to an inactive A3H protein, a frequency slightly lower than that observed for NC studied patients (10/11; 91%). Conclusions The high prevalence of non-functional protein coding-genotypes in EC subjects seems to indicate that other innate restriction factors different from APOBEC3H could be implicated in the replication control exhibited by these subjects.

Differential Gene Expression by Lactobacillus plantarum WCFS1 in Response to Phenolic Compounds Reveals New Genes Involved in Tannin Degradation.

Lactobacillus plantarum is a lactic acid bacterium that can degrade food tannins by the successive action of tannase and gallate decarboxylase enzymes. In the L. plantarum genome, the gene encoding the catalytic subunit of gallate decarboxylase (lpdC, or lp_2945) is only 6.5 kb distant from the gene encoding inducible tannase (L. plantarumtanB [tanBLp ], or lp_2956). This genomic context suggests concomitant activity and regulation of both enzymatic activities. Reverse transcription analysis revealed that subunits B (lpdB, or lp_0271) and D (lpdD, or lp_0272) of the gallate decarboxylase are cotranscribed, whereas subunit C (lpdC, or lp_2945) is cotranscribed with a gene encoding a transport protein (gacP, or lp_2943). In contrast, the tannase gene is transcribed as a monocistronic mRNA. Investigation of knockout mutations of genes located in this chromosomal region indicated that only mutants of the gallate decarboxylase (subunits B and C), tannase, GacP transport protein, and TanR transcriptional regulator (lp_2942) genes exhibited altered tannin metabolism. The expression profile of genes involved in tannin metabolism was also analyzed in these mutants in the presence of methyl gallate and gallic acid. It is noteworthy that inactivation of tanR suppresses the induction of all genes overexpressed in the presence of methyl gallate and gallic acid. This transcriptional regulator was also induced in the presence of other phenolic compounds, such as kaempferol and myricetin. This study complements the catalog of L. plantarum expression profiles responsive to phenolic compounds, which enable this bacterium to adapt to a plant food environment.IMPORTANCELactobacillus plantarum is a bacterial species frequently found in the fermentation of vegetables when tannins are present. L. plantarum strains degrade tannins to the less-toxic pyrogallol by the successive action of tannase and gallate decarboxylase enzymes. The genes encoding these enzymes are located close to each other in the chromosome, suggesting concomitant regulation. Proteins involved in tannin metabolism and regulation, such GacP (gallic acid permease) and TanR (tannin transcriptional regulator), were identified by differential gene expression in knockout mutants with mutations in genes from this region. This study provides insights into the highly coordinated mechanisms that enable L. plantarum to adapt to plant food fermentations.

Structural basis of the substrate specificity and instability in solution of a glycosidase from Lactobacillus plantarum.

Statistics from structural genomics initiatives reveal that around 50-55% of the expressed, non-membrane proteins cannot be purified and therefore structurally characterized due to solubility problems, which emphasized protein solubility as one of the most serious concerns in structural biology projects. Lactobacillus plantarum CECT 748T produces an aggregation-prone glycosidase (LpBgl) that we crystallized previously. However, this result could not be reproduced due to protein instability and therefore further high-resolution structural analyses of LpBgl were impeded. The obtained crystals of LpBgl diffracted up to 2.48Å resolution and permitted to solve the structure of the enzyme. Analysis of the active site revealed a pocket for phosphate-binding with an uncommon architecture, where a phosphate molecule is tightly bound suggesting the recognition of 6-phosphoryl sugars. In agreement with this observation, we showed that LpBgl exhibited 6-phospho-ß-glucosidase activity. Combination of structural and mass spectrometry results revealed the formation of dimethyl arsenic adducts on the solvent exposed cysteine residues Cys211 and Cys292. Remarkably, the double mutant Cys211Ser/Cys292Ser resulted stable in solution at high concentrations indicating that the marginal solubility of LpBgl can be ascribed specifically to these two cysteine residues. The 2.30Å crystal structure of this double mutant showed no disorder around the newly incorporated serine residues and also loop rearrangements within the phosphate-binding site. Notably, LpBgl could be prepared at high yield by proteolytic digestion of the fusion protein LSLt-LpBgl, which raises important questions about potential hysteretic processes upon its initial production as an enzyme fused to a solubility enhancer.

Synthesis and structural characterization of raffinosyl-oligofructosides upon transfructosylation by Lactobacillus gasseri DSM 20604 inulosucrase.

A new process based on enzymatic synthesis of a series of raffinose-derived oligosaccharides or raffinosyl-oligofructosides (RFOS) with degree of polymerization (DP) from 4 to 8 was developed in the presence of raffinose. This process involves a transfructosylation reaction catalyzed by an inulosucrase from Lactobacillus gasseri DSM 20604 (IS). The main synthesized RFOS were structurally characterized by nuclear magnetic resonance (NMR). According to the elucidated structures, RFOS consist of ß-2,1-linked fructose unit(s) to raffinose: α-D-galactopyranosyl-(1 → 6)-α-D-glucopyranosyl-(1↔2)-ß-D-fructofuranosyl-((1 ← 2)-ß-D-fructofuranoside)n (where n refers to the number of transferred fructose moieties). The maximum yield of RFOS was 33.4 % (in weight respect to the initial amount of raffinose) and was obtained at the time interval of 8-24 h of transfructosylation reaction initiated with 50 % (w/v) of raffinose. Results revealed the high acceptor and donor affinity of IS towards raffinose, being fairly comparable with that of sucrose for the production of fructooligosaccharides (FOS), including when both carbohydrates coexisted (sucrose/raffinose mixture, 250 g L(-1) each). The production of RFOS was also attempted in the presence of sucrose/melibiose mixtures; in this case, the predominant acceptor-product formed was raffinose followed by a minor production of a series of oligosaccharides with varying DP. The easiness of RFOS synthesis and the structural similarities with both raffinose and fructan series of oligosaccharides warrant the further study of the potential bioactive properties of these unexplored oligosaccharides.

An outbreak of monophasic and biphasic Salmonella Typhimurium, and Salmonella Derby associated with the consumption of dried pork sausage in Castellon (Spain).

An outbreak of S.Typhimurium occurred in several towns and cities in the province of Castellon (Spain) between 23 February and 27 May 2011. On April 5, the microbiology laboratory of a hospital in Castellon alerted the health authorities to the increase in S.Typhimurium isolated in fecal culture of children with gastroenteritis. The serotype and phage-type of 83 positive cases of S.Typhimurium isolated in these period included 49 monophasic/biphasic S.Typhimurium phage type 138, phage type 193, S.Derby, and 34 other S.Typhimurium phage-types. The median of age of patients was 4 years with a range of 0.6-80 years, and the 18% of patients were hospitalised. Two incident matched case-control studies were carried out; the first with S.Typhimurium phage type 138, 193, and S.Derby cases and the second with the other cases. The two studies found that the consumption of brand X dried pork sausage, purchased in a supermarket chain A, was associated with the disease (matched Odds Ratio [mOR]=13.74 95% Confidence Interval [CI] 4.84-39.06 and mOR=8.20 95% CI 2.32-28.89), respectively). S.Typhimurium phage type 193 and S.Derby were isolated in the food taken from the household of two patients and from the supermarket chain's A central warehouse. The pulsed-field gel electrophoresis study confirmed the similarity of the strains from the patients and the food. On May 25 2011, a national food alert led to the withdrawal of the food from the chain A and the outbreak ended.

Enantioselective oxidation of galactitol 1-phosphate by galactitol-1-phosphate 5-dehydrogenase from Escherichia coli.

Galactitol-1-phosphate 5-dehydrogenase (GPDH) is a polyol dehydrogenase that belongs to the medium-chain dehydrogenase/reductase (MDR) superfamily. It catalyses the Zn(2+)- and NAD(+)-dependent stereoselective dehydrogenation of L-galactitol 1-phosphate to D-tagatose 6-phosphate. Here, three crystal structures of GPDH from Escherichia coli are reported: that of the open state of GPDH with Zn(2+) in the catalytic site and those of the closed state in complex with the polyols Tris and glycerol, respectively. The closed state of GPDH reveals no bound cofactor, which is at variance with the conformational transition of the prototypical mammalian liver alcohol dehydrogenase. The main intersubunit-contacting interface within the GPDH homodimer presents a large internal cavity that probably facilitates the relative movement between the subunits. The substrate analogue glycerol bound within the active site partially mimics the catalytically relevant backbone of galactitol 1-phosphate. The glycerol binding mode reveals, for the first time in the polyol dehydrogenases, a pentacoordinated zinc ion in complex with a polyol and also a strong hydrogen bond between the primary hydroxyl group and the conserved Glu144, an interaction originally proposed more than thirty years ago that supports a catalytic role for this acidic residue.

A Lactobacillus plantarum esterase active on a broad range of phenolic esters.

Lactobacillus plantarum is the lactic acid bacterial species most frequently found in the fermentation of food products of plant origin on which phenolic compounds are abundant. L. plantarum strains showed great flexibility in their ability to adapt to different environments and growth substrates. Of 28 L. plantarum strains analyzed, only cultures from 7 strains were able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate. As revealed by PCR, only these seven strains possessed the est_1092 gene. When the est_1092 gene was introduced into L. plantarum WCFS1 or L. lactis MG1363, their cultures acquired the ability to degrade hydroxycinnamic esters. These results support the suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on the L. plantarum strains analyzed. The Est_1092 protein was recombinantly produced and biochemically characterized. Surprisingly, Est_1092 was able to hydrolyze not only hydroxycinnamic esters, since all the phenolic esters assayed were hydrolyzed. Quantitative PCR experiments revealed that the expression of est_1092 was induced in the presence of methyl ferulate, an hydroxycinnamic ester, but was inhibited on methyl gallate, an hydroxybenzoic ester. As Est_1092 is an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase and tannase activities, its presence on some L. plantarum strains provides them with additional advantages to survive and grow on plant environments.

Molecular adaptation of Lactobacillus plantarum WCFS1 to gallic acid revealed by genome-scale transcriptomic signature and physiological analysis.

BACKGROUND: Gallic acid (GA) is a model hydroxybenzoic acid that occurs esterified in the lignocellulosic biomass of higher plants. GA displays relevant biological activities including anticancer properties. Owing to its antimicrobial and cellulase-inhibiting activities, GA also imposes constraints to the fermentability of lignocellulosic hydrolysates. In depth-knowledge of the mechanisms used by tolerant microorganisms to adapt to hydroxybenzoic acids would be a step forward to improve the bioavailability of GA or select/engineer production hosts with improved metabolic traits for the bioconversion of pretreated lignocellulosic biomass. RESULTS: Whole genome transcriptional profiling using DNA microarrays was used to characterize the molecular response of Lactobacillus plantarum WCFS1 to GA. Expression levels of 14 and 40 genes were differentially regulated at 1.5 and 15 mM GA, respectively. The transcriptomic analysis identified a marked induction of genes with confirmed or related roles to gastrointestinal survival, the repression of genes coding for certain ABC-type transporters and modulation of genes involved in the control of intracellular ammonia levels, among other responses. Most notably, a core set of genes dedicated to produce GA from polyphenols (tanB Lp ), decarboxylate GA to pyrogallol (lpdB, lpdC and lpdD) and transport functions (lp_2943) was highly overexpressed at both GA concentrations. Correspondingly, resting cells of strain WCFS1 induced by GA, but not their non-induced controls, produced pyrogallol. Gene expression and organization of genes involved in GA metabolism suggested a chemiosmotic mechanism of energy generation. Resting cells of L. plantarum induced by GA generated a membrane potential and a pH gradient across the membrane immediately upon addition of GA. Altogether, transcriptome profiling correlated with physiological observations indicating that a proton motive force could be generated during GA metabolism as a result of electrogenic GA uptake coupled with proton consumption by the intracellular gallate decarboxylase. CONCLUSIONS: The combination of transcriptome and physiological analyses revealed versatile molecular mechanisms involved in the adaptation of L. plantarum to GA. These data provide a platform to improve the survival of Lactobacillus in the gut. Our data may also guide the selection/engineering of microorganisms that better tolerate phenolic inhibitors present in pretreated lignocellulosic feedstocks.