Dps Gene Research Articles

Involvement of OxyR and Dps in the repression of replication initiation by DsrA small RNA in Escherichia coli

Regulation of the cell cycle process is an effective measure to ensure the stability and fidelity of genetic material during the reproduction of bacteria under different stresses. The small RNA DsrA helps bacteria adapt to environments by binding to multiple targets, but its association with the cell cycle remains unclear. Detection by flow cytometry, we first found that the knockout of dsrA promoted replication initiation, and corresponding overexpression of DsrA inhibited replication initiation in Escherichia coli. The absence of the chaperone protein Hfq, the DNA replication negative regulator protein Dps, or the transcription factor OxyR, was found to cause DsrA to no longer inhibit replication initiation. Excess DsrA promotes expression of the oxyR and dps gene, whereas β-galactosidase activity assay showed that deleting oxyR limited the enhancement of dps promoter transcriptional activity by DsrA. OxyR is a known positive regulator of Dps. Our data suggests that the effect of DsrA on replication initiation requires Hfq and that the upregulation of Dps expression by OxyR in response to DsrA levels may be a potential regulatory pathway for the negative regulation of DNA replication initiation.

Complementary Roles of Two DNA Protection Proteins from Deinococcus geothermalis.

The roles of two interrelated DNA protection protein in starved cells (Dps)-putative Dps Dgeo_0257 and Dgeo_0281-as orthologous proteins to DrDps1 for DNA binding, protection, and metal ion sensing were characterised in a Deinococcus geothermalis strain. Dgeo_0257 exhibited high DNA-binding affinity and formed a multimeric structure but lacked the conserved amino acid sequence for ferroxidase activity. In contrast, the Dgeo_0281 (DgDps1) protein was abundant in the early exponential phase, had a lower DNA-binding activity than Dgeo_0257, and was mainly observed in its monomeric or dimeric forms. Electrophoretic mobility shift assays demonstrated that both purified proteins bound nonspecifically to DNA, and their binding ability was affected by certain metal ions. For example, in the presence of ferrous and ferric ions, neither Dgeo_0257 nor Dgeo_0281 could readily bind to DNA. In contrast, both proteins exhibited more stable DNA binding in the presence of zinc and manganese ions. Mutants in which the dps gene was disrupted exhibited higher sensitivity to oxidative stress than the wild-type strain. Furthermore, the expression levels of each gene showed an opposite correlation under H2O2 treatment conditions. Collectively, these findings indicate that the putative Dps Dgeo_0257 and DgDps1 from D. geothermalis are involved in DNA binding and protection in complementary interplay ways compared to known Dps.

Sequence, structure, and function of the Dps DNA-binding protein from Deinococcus wulumuqiensis R12

Deinococcus wulumuqiensis R12, which was isolated from arid irradiated soil in Xinjiang province of China, belongs to a genus that is well-known for its extreme resistance to ionizing radiation and oxidative stress. The DNA-binding protein Dps has been studied for its great contribution to oxidative resistance. To explore the role of Dps in D. wulumuqiensis R12, the Dps sequence and homology-modeled structure were analyzed. In addition, the dps gene was knocked out and proteomics was used to verify the functions of Dps in D. wulumuqiensis R12. Docking data and DNA binding experiments in vitro showed that the R12 Dps protein has a better DNA binding ability than the Dps1 protein from D. radiodurans R1. When the dps gene was deleted in D. wulumuqiensis R12, its resistance to H2O2 and UV rays was greatly reduced, and the cell envelope was destroyed by H2O2 treatment. Additionally, the qRT-PCR and proteomics data suggested that when the dps gene was deleted, the catalase gene was significantly down-regulated. The proteomics data indicated that the metabolism, transport and oxidation–reduction processes of D. wulumuqiensis R12 were down-regulated after the deletion of the dps gene. Overall, the data conformed that Dps protein plays an important role in D. wulumuqiensis R12.

Generation of polyclonal antibodies against recombinant Agrobacterium tumefaciens decaprenyl diphosphate synthase produced in Escherichia coli

Coenzyme Q (CoQ), an electron carrier of the mitochondrial respiratory chain and a potent antioxidant, consists of a benzoquinone head and an isoprenoid side chain of varying numbers of isoprene units, 10 in humans, animals and some microbes including Agrobacterium tumefaciens (CoQ10), 9 in Arabidopsis and cereals (CoQ9) and 8 in Escherichia coli (CoQ8). The synthesis of CoQ10 side chain is catalyzed by a single side chain length determining enzyme, decaprenyl diphosphate synthase (DPS) which is encoded by a dps gene. We report here, the isolation and sequencing of a dps gene from A. tumefaciens strain LBA4404 and its cloning in pET28a expression vector of E. coli BL21 (DE3) strain to produce and purify recombinant DPS protein for the production of its antibodies in rabbit. The dps gene isolated from A. tumefaciens consists of 1077 bp efficient for encoding a polypeptide of 359 amino acids with a molecular mass of around 39 kDa. DPS protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside in bacterial culture to a final concentration of 1.0 mM. Recombinant protein tagged with histidine at N-terminal was purified by immobilized metal-affinity chromatography. The purified DPS protein was capable of producing polyclonal antibodies in rabbit whose specificity to DPS enzyme was confirmed by the western blot analysis. Expression of dps gene in E. coli extended the side chain length of CoQ8 to 10 isopenoid units and produced CoQ10 in addition to CoQ8. This is the first report on the production of polyclonal antibodies against recombinant DPS protein and demonstration of their utility.

DNA-binding protein from starvation cells traps intracellular free-divalent iron and plays an important role in oxidative stress resistance in Acetobacter pasteurianus NBRC 3283

Acetobacter pasteurianus accumulates reactive oxygen species (ROS). ROS are produced by electron and oxygen coupling in the electron transport chain in the intracellular environment during the stationary and in the acetic acid over-oxidation phases in the presence of ethanol, thereby exposing cell to oxidative stress. In this study, to reveal the resistance mechanism to oxidative stress in A.pasteurianus, we focused on DNA-binding protein from starvation cells (Dps) and analyzed the function of Dps against oxidative stress. When Dps under the copresence of plasmid DNA was exposed to H2O2 and divalent iron, plasmid DNA fragmentation was suppressed under the presence of Dps; however, DNA binding was not observed, revealing a defensive activity for oxidative damage. In addition, this finding revealed that Dps incorporates a divalent iron intracellularly, forming a ferroxidase center. Moreover, levels of hydroxyl radicals produced by Fenton reaction under the presence of H2O2 and divalent iron were decreased by the addition of Dps, resulting in the suppression of the Fenton reaction. Through fluorescence microscopy using a divalent-iron-specific fluorescent probe, we found that, in dps gene disruptants, the accumulation of the divalent iron increased, and the dps gene disruptants showed higher sensitivity to H2O2 than the wild-type. These result strongly suggested that Dps traps intracellular free-divalent iron and plays an important role in the oxidative stress resistance of A.pasteurianus NBRC 3283 after the acetic acid fermentation phase.

The functional identification of Dps in oxidative stress resistance and virulence of Riemerella anatipestifer CH-1 using a new unmarked gene deletion strategy

Excessive iron in the bacterial cytoplasm can potentiate the production of harmful reactive oxygen species (ROS). Riemerella anatipestifer (R. anatipestifer, RA), a gram-negative bacterium, encodes an iron uptake system, but its iron detoxification mechanism is unknown. Here, the dps gene of R. anatipestifer CH-1 (RA-CH-1) was deleted using sacB as a counterselection marker. The dps mutant was more sensitive to H2O2 than the wild type in iron-rich conditions but not in iron-limited conditions, suggesting that Dps prevents H2O2-induced damage through iron binding. However, the dps mutant and wild type were identically sensitive to bactericidal antibiotics, and antibiotic treatment did not enhance RA-CH-1 ROS production. Furthermore, Dps prevents DNA damage by binding DNA. The RA-CH-1 dps transcript level was higher in the stationary phase than in the early and exponential phases and was increased by OxyR in the presence of H2O2. Finally, duckling colonization by the dps mutant was similar to that by the wild type at 48 h postinfection but significantly lower at 60 h postinfection, suggesting that RA-CH-1 Dps is not involved in host invasion but increases resistance to host clearance. Dps thus likely plays an important role in R. anatipestifer physiology and pathogenesis through protecting against oxidative stress.

Dps Protects Enterohemorrhagic Escherichia coli against Acid-Induced Antimicrobial Peptide Killing.

Dps, a DNA-binding protein from starved cells in Escherichia coli, is part of the bacterial defense system that protects DNA against various cellular stresses. Our lab previously demonstrated that a novel antimicrobial peptide, WRWYCR, enhances acid-induced killing of enterohemorrhagic Escherichia coli (EHEC) and ameliorates infection in a Citrobacter rodentium mouse model of EHEC infection. WRWYCR has previously been shown to compromise DNA damage repair and to increase chelatable iron within the cell. These findings, combined with the effects of peptide and acid stress on DNA damage, suggest a key defense role for Dps in peptide-induced killing of EHEC. The goal of this study is to evaluate the role of Dps in peptide-induced killing of EHEC through survival assays and flow cytometric analyses of DNA damage and hydroxyl radical formation. Our results demonstrate that disruption of the dps gene in stationary-phase EHEC O157:H7 cells, but not in exponential-phase cells, enhances acid-, peptide-, and peptide-acid-induced killing relative to that of wild-type (WT) EHEC. Using flow cytometric analysis, we have also demonstrated increased levels of hydroxyl radicals in peptide-treated wild-type EHEC relative to those in the untreated control. Disruption of the dps gene further increases this. These findings indicate that peptide treatment of EHEC enhances the formation of hydroxyl radicals, likely through the Fenton reaction, thereby contributing to the killing action of the peptide, and that dps protects against peptide killing of EHEC. This study provides important insights into peptide WRWYCR-mediated killing of EHEC, which could be exploited in the development of more effective antimicrobials.IMPORTANCE The research presented in this paper explores the role of the DNA-binding protein Dps as a key defense mechanism of enterohemorrhagic Escherichia coli (EHEC) strains in protecting against killing by the novel antimicrobial peptide WRWYCR. Our results demonstrate that Dps protects against peptide-induced killing of EHEC through direct protection against acid stress and hydroxyl radical formation, both of which are mechanisms targeted by the antimicrobial peptide. This study provides important insights into peptide WRWYCR-mediated killing of EHEC, which could be exploited in the development of more effective antimicrobials through specific targeting of Dps in order to allow a more potent response to the antimicrobial WRWYCR.

Global DNA Compaction in Stationary-Phase Bacteria Does Not Affect Transcription

In stationary-phase Escherichia coli, Dps (DNA-binding protein from starved cells) is the most abundant protein component of the nucleoid. Dps compacts DNA into a dense complex and protects it from damage. Dps has also been proposed to act as a global regulator of transcription. Here, we directly examine the impact of Dps-induced compaction of DNA on the activity of RNA polymerase (RNAP). Strikingly, deleting the dps gene decompacted the nucleoid but did not significantly alter the transcriptome and only mildly altered the proteome during stationary phase. Complementary invitro assays demonstrated that Dps blocks restriction endonucleases but not RNAP from binding DNA. Single-molecule assays demonstrated that Dps dynamically condenses DNA around elongating RNAP without impeding its progress. We conclude that Dps forms a dynamic structure that excludes some DNA-binding proteins yetallows RNAP free access to the buried genes, a behavior characteristic of phase-separated organelles.

Knockout of pprM Decreases Resistance to Desiccation and Oxidation in Deinococcus radiodurans

Deinococcus radiodurans has attracted a great interest in the past decades due to its extraordinary resistance to ionizing radiation and highly efficient DNA repair system. Recent studies indicated that pprM is a putative pleiotropic gene in D. radiodurans and plays an important role in radioresistance and antioxidation, but its underlying mechanisms are poorly elucidated. In this study, pprM mutation was generated to investigate resistance to desiccation and oxidative stress. The result showed that the survival of pprM mutant under desiccation was markedly retarded compared to the wild strain from day 7-28. Furthermore, knockout of pprM increases the intercellular accumulation of ROS and the sensibility to H2O2 stress in the bacterial growth inhibition assay. The absorbance spectrum experiment for detecting the carotenoid showed that deinoxanthin, a carotenoid that peculiarly exists in Deinococcus, was reduced in the pprM mutant in the pprM mutant. Quantitative real time PCR showed decreased expression of three genes viz. CrtI (DR0861, 50%),CrtB (DR0862, 40%) and CrtO (DR0093, 50%), which are involved in deinoxanthin synthesis, and of Dps (DNA protection during starving) gene (DRB0092) relevant to ion combining and DNA protection in cells. Our results suggest that pprM may affect antioxidative ability of D. radiodurans by regulating the synthesis of deinoxanthin and the concentration of metal ions. This may provide new clues for the treatment of antioxidants.

Genomic and transcriptomic analyses reveal adaptation mechanisms of an Acidithiobacillus ferrivorans strain YL15 to alpine acid mine drainage.

Acidithiobacillus ferrivorans is an acidophile that often occurs in low temperature acid mine drainage, e.g., that located at high altitude. Being able to inhabit the extreme environment, the bacterium must possess strategies to copy with the survival stress. Nonetheless, information on the strategies is in demand. Here, genomic and transcriptomic assays were performed to illuminate the adaptation mechanisms of an A. ferrivorans strain YL15, to the alpine acid mine drainage environment in Yulong copper mine in southwest China. Genomic analysis revealed that strain has a gene repertoire for metal-resistance, e.g., genes coding for the mer operon and a variety of transporters/efflux proteins, and for low pH adaptation, such as genes for hopanoid-synthesis and the sodium:proton antiporter. Genes for various DNA repair enzymes and synthesis of UV-absorbing mycosporine-like amino acids precursor indicated hypothetical UV radiation—resistance mechanisms in strain YL15. In addition, it has two types of the acquired immune system–type III-B and type I-F CRISPR/Cas modules against invasion of foreign genetic elements. RNA-seq based analysis uncovered that strain YL15 uses a set of mechanisms to adapt to low temperature. Genes involved in protein synthesis, transmembrane transport, energy metabolism and chemotaxis showed increased levels of RNA transcripts. Furthermore, a bacterioferritin Dps gene had higher RNA transcript counts at 6°C, possibly implicated in protecting DNA against oxidative stress at low temperature. The study represents the first to comprehensively unveil the adaptation mechanisms of an acidophilic bacterium to the acid mine drainage in alpine regions.

The DNA–mimic antirestriction proteins ArdA ColIB-P9, Arn T4, and Ocr T7 as activators of H-NS-dependent gene transcription

The antirestriction proteins ArdA ColIb-P9, Arn T4 and Ocr T7 specifically inhibit type I and type IV restriction enzymes and belong to the family of DNA-mimic proteins because their three-dimensional structure is similar to the double-helical B-form DNA. It is proposed that the DNA-mimic proteins are able to bind nucleoid protein H-NS and alleviate H-NS-silencing of the transcription of bacterial genes. Escherichia coli lux biosensors were constructed by inserting H-NS-dependent promoters into a vector, thereby placing each fragment upstream of the promoterless Photorhabdus luminescens luxCDABE operon. It was demonstrated that the DNA-mimic proteins ArdA, Arn and Ocr activate the transcription of H-NS-dependent promoters of the lux operon of marine luminescent bacteria (mesophilic Aliivibrio fischeri and psychrophilic Aliivibrio logei), and the dps gene from E. coli. It was also demonstrated that the ArdA antirestriction protein, the genes of which are located on transmissive plasmids ColIb-P9, R64, PK101, decreases levels of H-NS silencing of the PluxC promoter during conjugation in the recipient bacteria.

The effect of oxidative stress on gene expression of Shiga toxin-producing Escherichia coli (STEC) O157:H7 and non-O157 serotypes

Understanding the survival mechanisms used by Shiga toxin-producing Escherichia coli (STEC), including O157:H7 and non-O157 serotypes, is important for minimizing contamination of fresh produce and occurrence of foodborne outbreaks. Recent outbreaks linked to leafy green vegetables and sprouted seeds have prompted researchers to focus on investigating decontamination strategies. Several studies showed that hydrogen peroxide (H2O2) treatment has been effective in reducing pathogens on fresh produce. As such, the effect of hydrogen peroxide on stress-associated and virulence gene expression in six STEC isolates was investigated in this study. Logarithmic phase cells of E. coli O157:H7 (EDL933) and non-O157 serotypes, including E. coli O26:H11 (EC20070549), O103:H2 (EC19970811), O104:H4 (NML#11-3088), O111:NM (EC20070546) and O145:NM (EC19970355) were exposed to 2.5mM H2O2 for 40min and gene expression was evaluated using quantitative real-time PCR. Different patterns of gene expression were observed in E. coli O157:H7 and non-O157 serotypes. Particularly, Shiga toxin gene stx2 was upregulated in O157:H7, but not in O104:H4. Moreover, stx1 was significantly upregulated in STEC O157:H7, but only slightly upregulated Stx1-positive non-O157 serotypes. However genes related to motility (fliC) and intimin gene (eae) were downregulated in most strains. Stress-associated sodA gene encoding manganese superoxide dismutase was significantly upregulated in all serotypes. The dps gene coding for non-specific DNA binding protein was upregulated in O145:NM, O111:NM, O103:H2 and O26:H11. However genes related to cold shock (cspC) and acid resistance (gadW) were significantly downregulated in all strains tested. The results of this study provide a basic understanding of the oxidative stress impact on survival and virulence of non-O157 serotype STEC strains.

Modes of Escherichia coli Dps Interaction with DNA as Revealed by Atomic Force Microscopy.

Multifunctional protein Dps plays an important role in iron assimilation and a crucial role in bacterial genome packaging. Its monomers form dodecameric spherical particles accumulating ~400 molecules of oxidized iron ions within the protein cavity and applying a flexible N-terminal ends of each subunit for interaction with DNA. Deposition of iron is a well-studied process by which cells remove toxic Fe2+ ions from the genetic material and store them in an easily accessible form. However, the mode of interaction with linear DNA remained mysterious and binary complexes with Dps have not been characterized so far. It is widely believed that Dps binds DNA without any sequence or structural preferences but several lines of evidence have demonstrated its ability to differentiate gene expression, which assumes certain specificity. Here we show that Dps has a different affinity for the two DNA fragments taken from the dps gene regulatory region. We found by atomic force microscopy that Dps predominantly occupies thermodynamically unstable ends of linear double-stranded DNA fragments and has high affinity to the central part of the branched DNA molecule self-assembled from three single-stranded oligonucleotides. It was proposed that Dps prefers binding to those regions in DNA that provide more contact pads for the triad of its DNA-binding bundle associated with one vertex of the protein globule. To our knowledge, this is the first study revealed the nucleoid protein with an affinity to branched DNA typical for genomic regions with direct and inverted repeats. As a ubiquitous feature of bacterial and eukaryotic genomes, such structural elements should be of particular care, but the protein system evolutionarily adapted for this function is not yet known, and we suggest Dps as a putative component of this system.

Механизмы регуляции экспрессии гена dps Escherichia coli в условиях стресса: реконструкция по кинетическим данным

Механизмы регуляции экспрессии гена dps Escherichia coli в условиях стресса: реконструкция по кинетическим данным

A PerR-like protein involved in response to oxidative stress in the extreme bacterium Deinococcus radiodurans

Response and defense systems against reactive oxygen species (ROS) contribute to the remarkable resistance of Deinococcus radiodurans to oxidative stress induced by oxidants or radiation. However, mechanisms involved in ROS response and defense systems of D. radiodurans are not well understood. Fur family proteins are important in ROS response. Only a single Fur homolog is predicted by sequence similarity in the current D. radiodurans genome database. Our bioinformatics analysis demonstrated an additional guanine nucleotide in the genome of D. radiodurans that is not in the database, leading to the discovery of another Fur homolog DrPerR. Gene disruption mutant of DrPerR showed enhanced resistance to hydrogen peroxide (H2O2) and increased catalase activity in cell extracts. Real-time PCR results indicated that DrPerR functions as a repressor of the catalase gene katE. Meanwhile, derepression of dps (DNA-binding proteins from starved cells) gene under H2O2 stress by DrPerR point to its regulatory role in metal ions hemostasis. Thus, DrPerR might function as a Fur homolog protein which is involved in ROS response and defense. These results help clarify the complicated regulatory network that responds to ROS stress in D. radiodurans.

The Campylobacter jejuni Dps Homologue Is Important forIn VitroBiofilm Formation and Cecal Colonization of Poultry and May Serve as a Protective Antigen for Vaccination

In this work, we investigated the Campylobacter jejuni dps (DNA binding protein from starved cells) gene for a role in biofilm formation and cecal colonization in poultry. In vitro biofilm formation assays were conducted with stationary-phase cells in cell culture plates under microaerophilic conditions. These studies demonstrated a significant (>50%) reduction in biofilm formation by the C. jejuni dps mutant compared to that by the wild-type strain. Studies in poultry also demonstrated the importance of the dps gene in host colonization by C. jejuni. Real-time PCR analysis of mRNA extracted from the cecal contents of poultry infected with wild-type C. jejuni indicated that the dps gene is upregulated 20-fold during poultry colonization. Cecal colonization was greater than 5 log CFU lower in chicks infected with the dps mutant than chicks infected with the wild-type C. jejuni strain. Moreover, the dps mutant failed to colonize 75% of the chicks following challenge with 10(5) CFU. Preliminary studies were conducted in chicks by parenteral vaccination with a recombinant Dps protein or through oral vaccination with a recombinant attenuated Salmonella enterica strain synthesizing the C. jejuni Dps protein. No reduction in C. jejuni was noted in chicks vaccinated with the parenteral recombinant protein, whereas, a 2.5-log-unit reduction of C. jejuni was achieved in chicks vaccinated with the attenuated Salmonella vector after homologous challenge. Taken together, this work demonstrated the importance of Dps for biofilm formation and poultry colonization, and the study also provides a basis for continued work using the Dps protein as a vaccine antigen when delivered through a Salmonella vaccine vector.

Role of the Dickeya dadantii Dps protein

During infection, the phytopathogenic enterobacterium Dickeya dadantii has to cope with iron-limiting conditions and the production of reactive oxygen species by plant cells. A tight control of the bacterial intracellular iron content is necessary for full virulence of D. dadantii: previous studies have shown that the ferritin FtnA and the bacterioferrtin Bfr, devoted to iron storage, contribute differentially to the virulence of this species. In this work, we investigated the role of the Dps miniferritin in iron homeostasis in D. dadantii. We constructed a Dps-deficient mutant by reverse genetics. This mutant grew like the wild-type stain under iron starvation and showed no decreased iron content. However, the dps mutant displayed an increased sensitivity to hydrogen peroxide in comparison to the wild-type strain. This hydrogen peroxide susceptibility only occurs when bacteria are in the stationary phase. Unlike the bfr and the ftnA mutants, the dps mutant is not affected in its pathogenicity on host plants. The dps gene expression is induced at the stationary phase of growth. The Sigma S transcriptional factor is necessary for this control. Furthermore, dps expression is positively regulated by the oxidative stress response regulator OxyR during the exponential growth phase, after hydrogen peroxide treatment. These results indicate that the Dps miniferritin from D. dadantii has a minor role in iron homeostasis, but is important in conferring tolerance to hydrogen peroxide and for survival of cells that enter the stationary phase of growth.

Bacterioferritin: Properties, structural and functional organization of the dps gene regulatory region

The paper considers the properties of bacterioferritin Dps, which is involved in the sequestering of iron ions, forms the ferrihydrite core inside the protein cavity, and functions as a major nucleoid protein. Experimental evidence on the effect of microwave irradiation on the dps gene expression is presented. The structural and functional organization of its regulatory region is analyzed, and the technological prospects of bacterioferritin application for designing new materials with desired properties are discussed.

ACampylobacter jejuniDps Homolog Has a Role in Intracellular Survival and in the Development of Campylobacterosis in Neonate Piglets

Iron acquisition is an absolute requirement by most microorganisms for host survival. In this work, we investigated the Campylobacter jejuni iron binding Dps protein for a potential role in virulence. In vitro assays using J774A.1 macrophage-like cells demonstrated a 2.5 log reduction in C. jejuni survival of the Dps mutant and a reduction of four logs in invasion of HEp-2 epithelial cells compared to the wild-type strain. To examine the role of the dps gene in host pathogenesis, the piglet model was used in C. jejuni challenge studies. In vivo inoculation studies of newborn piglets with wild-type C. jejuni demonstrated an 11-fold upregulation of the dps gene and intestinal lesion production typical of campylobacteriosis in humans. In contrast, piglets inoculated with the dps mutant were not colonized and remained normal throughout the study period. Mucosal lesion production was restored in piglets inoculated with the complemented Dps mutant strain. Based on these results, we conclude that the C. jejuni Dps homolog is a virulence factor in the production of campylobacteriosis, and warrants further investigation.

Population dynamics of lactic acid bacteria during spontaneous malolactic fermentation in industrial cider

A deeper knowledge on the complex microbial ecology of malolactic fermentation (MLF) in spontaneous cider production is essential to understand the cider making process, thus, applicable to develop adequate malolactic starters. Trends in food fermentation focussed on the isolation of proper wild-type strains from traditional products to be used as starter cultures, with the aim of conducting industrial production processes without losing their unique flavour and product characteristics. A mixed inoculum consisted of samples taken from the four main areas of the most traditional cider making region in Spain was used to carry out MLF under controlled laboratory conditions. A culture-based approach was used to investigate the diversity of lactic acid bacteria (LAB) by molecular tools. By monitoring MLF, population dynamics involved in the fermentation process were assessed. Interactions of different microorganisms adapted to a common environment, competition and resistance during the process, give rise to dominant species showing selective advantages. The main aim of this work was the identification of dominant species during the process in order to obtain potential starter cultures with a clear industrial utility. The fermentative capacity of a representative strain belonging to each isolated species, after analysis of interspecific polymorphisms in 16S rRNA sequencing of 84 isolates, was compared. Molecular characterization for the presence of gene coding for exopolysaccharide production ( dps gene amplification), acrolein production ( pdu gene amplification) and biogenic amine production ( tyrdc , hdc and odc genes amplification) was carried out to verify the suitability of the selected strains.