Production Of Degrading Enzymes Research Articles

Poly(dl-lactide)-degrading enzyme production by immobilized Actinomadura keratinilytica strain T16-1 in a 5-L fermenter under various fermentation processes

Abstract Background Poly( dl -lactic acid), or PDLLA, is a biodegradable polymer that can be hydrolyzed by various types of enzymes. The protease produced by Actinomadura keratinilytica strain T16-1 was previously reported to have PDLLA depolymerase activity. However, few studies have reported on PDLLA-degrading enzyme production by bacteria. Therefore, the aims of this study were to determine a suitable immobilization material for PDLLA-degrading enzyme production and optimize PDLLA-degrading enzyme production by using immobilized A. keratinilytica strain T16-1 under various fermentation process conditions in a stirrer fermenter. Results Among the tested immobilization materials, a scrub pad was the best immobilizer, giving an enzyme activity of 30.03 U/mL in a shake-flask scale. The maximum enzyme activity was obtained at aeration 0.25 vvm, agitation 170 rpm, 45°C, and 48 h of cultivation time. Under these conditions, a PDLLA-degrading enzyme production of 766.33 U/mL with 15.97 U/mL·h productivity was observed using batch fermentation in a 5-L stirrer fermenter. Increased enzyme activity and productivity were observed in repeated-batch (942.67 U/mL and 19.64 U/mL·h) and continuous fermentation (796.43 U/mL and 16.58 U/mL·h) at a dilution rate of 0.013/h. Scaled-up production of the enzyme in a 10-L stirrer bioreactor using the optimized conditions showed a maximum enzyme activity of 578.67 U/mL and a productivity of 12.06 U/mL·h. Conclusions This research successfully scaled-up the enzyme production to 5 and 10 L in a stirrer fermenter and is helpful for many applications of poly(lactic acid).

Solid state bioconversion of lignocellulosic residues by Inonotus obliquus for production of cellulolytic enzymes and saccharification

White rot fungi have been usually considered for lignin degradation and ligninolytic enzyme production. To understand whether the white rot fungus Inonotus obliquus was able to produce highly efficient cellulase system, the production of cellulolytic enzyme cocktails was optimized under solid state fermentation. The activities of CMCase, FPase, and β-glucosidase reached their maximum of 27.15IU/g, 3.16IU/g and 2.53IU/g using wheat bran at 40% (v/w) inoculum level, initial pH of 6.0 and substrate-moisture ratio of 1:2.5, respectively. The enzyme cocktail exhibited promising properties in terms of high catalytic activity at 40–60°C and at pH 3.0–4.5, indicating that the cellulolytic enzymes represent thermophilic and acidophilic characteristics. Saccharification of raw wheat straw and rice straw by the cellulolytic enzyme cocktail sampled on Day 12 resulted in the release of reducing sugar of 130.24mg/g and 125.36mg/g of substrate after 48h of hydrolysis, respectively.

Enhancement of poly(L-lactide)-degrading enzyme production by Laceyella sacchari LP175 using agricultural crops as substrates and its degradation of poly(L-lactide) polymer

Optimization of the medium for poly(L-lactide) (PLLA)-degrading enzyme production in shake flask experiments was investigated using agricultural products as substrates. Cassava chips and soybean meal were the best carbon and nitrogen sources, respectively, as determined by the one-factor-at-a-time method. Enzyme production was significantly enhanced by the addition of phosphates, but was suppressed by the addition of ammonium salt. The maximum enzyme production, 65.5 U/mL, was obtained from the optimized medium consisting of 4.64 g/L cassava chips, 1.53 g/L soybean meal and 0.31 g/L PLLA powder using central composite design in the basal medium. The optimal physical factors in 3 L airlift fermenter were 50 °C, pH 7.0 and 0.5 vvm aeration rate for 18 h, yielded 94.4 U/mL. The crude enzyme was shown to be able to hydrolyze PLLA powder (91%) at 50 °C in 72 h which showed high efficiency for recycling of PLLA polymer and reducing the global environmental problem.

Synergistic effect of Chryseobacterium gleum sp. SUK with ACC deaminase activity in alleviation of salt stress and plant growth promotion in Triticum aestivum L.

Soil salinity is major abiotic stresses affecting morphological, biochemical and physiological processes of plant growth. Chryseobacterium gleum sp. SUK isolated from salt-stressed soil exhibited ACC (1-aminocyclopropane-1-carboxylate) deaminase activity with IAA (indole acetic acid), siderophore, ammonia, hydrogen cyanide production, 2% salt tolerance and fungal cell wall degrading enzyme production (cellulase, protease). The isolate also showed a poultry feather degrading activity which is the main waste material of poultry industry and opulent source of proteins, amino acids, nitrogen, phosphorous, calcium, potassium, manganese, zinc and copper. Application of feather-degraded lysate with the degrading isolate, C. gleum sp. SUK denotes triple role of bioformulation to surmount salinity stress, management of poultry waste disposal and utilization of feathers degraded products as a biostimulant for better growth of plants as well as strain SUK having multifarious plant growth promoting traits. Wheat crops exposed to salt stressor were inoculated with studied bioformulation. Results of plant analysis showed improvement in root and shoot length, fresh and dry weight, chlorophyll, proteins, amino acids, phenolics, flavonoids content and decreased level of proline. In addition, Na+ uptake was decreased and K+ uptake was increased. Therefore, application of novel bioformulation could increase the yield of crops by ameliorating growth of plants and alleviating the salinity stress.

Effective enhancement of polylactic acid-degrading enzyme production by Amycolatopsis sp. strain SCM_MK2-4 using statistical and one-factor-at-a-time approaches

ABSTRACTThis study aims to find the optimal medium and conditions for polylactic acid (PLA)-degrading enzyme production by Amycolatopsis sp. SCM_MK2-4. Screening of the most effective components in the enzyme production medium by Plackett–Burman design revealed that the silk cocoon and PLA film were the most significant variables enhancing the PLA-degrading enzyme production. After an response surface methodology, a maximum amount of PLA-degrading enzyme activity at 0.74 U mL−1 was predicted and successfully validated at 95% after 0.39% (w/v) silk cocoon and 1.62% (w/v) PLA film were applied to the basal medium. The optimal initial pH value, temperature, and inoculum size were evaluated by a method considering one-factor-at-a-time. The values were recorded at an initial pH in the range of 7.5–9.0, a temperature of 30–32°C, and an inoculum size of 4–10%. The highest activity of approximately 0.95 U mL−1 was achieved after 4 days of cultivation using the optimized medium and under optimized conditions in a shake flask. Upscaling to the use of a 3-L stirred tank fermenter was found to be successful with a PLA-degrading activity of 5.53 U mL−1; which represents a 51-fold increase in the activity compared with that obtained from the nonoptimized medium and conditions in the shake flask.

Medium pH in submerged cultivation modulates differences in the intracellular protein profile of Fusarium oxysporum

ABSTRACTFusarium oxysporum is a filamentous fungus that damages a wide range of plants and thus causes severe crop losses. In fungal pathogens, the genes and proteins involved in virulence are known to be controlled by environmental pH. Here, we report the influence of culture-medium pH (5, 6, 7, and 8) on the production of degradative enzymes involved in the pathogenesis of F. oxysporum URM 7401 and on the 2D-electrophoresis profile of intracellular proteins in this fungus. F. oxysporum URM 7401 was grown in acidic, neutral, and alkaline culture media in a submerged bioprocess. After 96 hr, the crude extract was processed to enzyme activity assays, while the intracellular proteins were obtained from mycelium and analyzed using 2D electrophoresis and mass spectrometry. We note that the diversity of secreted enzymes was changed quantitatively in different culture-medium pH. Also, the highest accumulated biomass and the intracellular protein profile of F. oxysporum URM 7401 indicate an increase in metabolism in neutral–alkaline conditions. The differential profiles of secreted enzymes and intracellular proteins under the evaluated conditions indicate that the global protein content in F. oxysporum URM 7401 is modulated by extracellular pH.

Producción de enzimas ligninolíticas durante la degradación del herbicida paraquat por hongos de la pudrición blanca

Paraquat is a widely used herbicide in agriculture. Its inappropriate use and wide distribution represents a serious pollution problem for soil and water. White rot fungi are capable of degrading pollutants having a similar structure to that of lignin, such as paraquat. This study evaluated the degradation effect of paraquat on the production of ligninolytic enzymes by white rot fungi isolated from the South of Mexico. Six fungal strains showed tolerance to the herbicide in solid culture. Three of the six evaluated strains showed levels of degradation of 32, 26 and 47% (Polyporus tricholoma, Cilindrobasidium laeve and Deconica citrispora, respectively) after twelve days of cultivation in the presence of the xenobiotic. An increase in laccase and manganese peroxidase (MnP) activities was detected in the strains showing the highest percentage of degradation. Experiments were done with enzyme extracts from the extracellular medium with the two strains showing more degradation potential and enzyme production. After 24hours of incubation, a degradation of 49% of the initial paraquat concentration was observed for D. citrispora. These results suggest that paraquat degradation can be attributed to the presence of extracellular enzymes from white rot fungi. In this work the first evidence of the biodegradation potential of D. citrispora and Cilindrobasidium leave is shown.

CCK1, a PMK1-type MAP kinase is required for hyphal growth, pigmentation, conidiation, enzyme activity, osmotic stress response, and pathogenicity in Corynespora cassiicola

Corynespora cassiicola is a fungal pathogen causing Corynespora Leaf Fall disease (CLF) in rubber trees (Hevea brasiliensis). CLF has great economic impact in rubber-producing countries. However, the molecular mechanisms involved in the pathogenicity of C. cassiicola remain poorly understood. Mitogen-activated protein kinase (MAPK) is one of the major signaling pathways responsible for regulation of fungal development and virulence in many plant fungal pathogens. The C. cassiicola cck1 gene encodes a MAPK that is homologous to the rice pathogen Magnaporthe grisea MAPK Pmk1 protein. In this study, targeted cck1 gene disruption and complementation experiments were carried out in C. cassiicola. Compared to the wild-type and CΔcck1 complement, the Δcck1 mutant had more pigmentation and exhibited dramatically reduced aerial mycelial mass and mycelial growth rates, along with the inability to produce conidia or conidiophores. In addition, the Δcck1 hyphae were darker in color and had less branches and septae with visibly condensed protoplasm. The Δcck1 mutant was more resistant to hyperosmotic stress, and was able to produce greater amount of laccase and lesser cellulase in vitro; however, the production of laccase was delayed. Importantly, Δcck1 mycelia and crude toxin were less virulent upon inoculation of rubber tree leaves. These findings indicate that the C. cassiicola CCK1 MAP kinase is a versatile regulator involved in nearly every aspect of plant fungal pathogenicity including mycelial growth, development, differentiation, pigmentation, conidiation, production of extracellular degradation enzymes, osmoregulation, and virulence.

Mixed colonies of Aspergillus niger and Aspergillus oryzae cooperatively degrading wheat bran

In both natural and man-made environments, microorganisms live in mixed populations, while in laboratory conditions monocultures are mainly used. Microbial interactions are often described as antagonistic, but can also be neutral or cooperative, and are generally associated with a metabolic change of each partner and cause a change in the pattern of produced bioactive molecules. A. niger and A. oryzae are two filamentous fungi widely used in industry to produce various enzymes (e.g. pectinases, amylases) and metabolites (e.g. citric acid). The co-cultivation of these two fungi in wheat bran showed an equal distribution of the two strains forming mixed colonies with a broad range of carbohydrate active enzymes produced. This stable mixed microbial system seems suitable for subsequent commercial processes such as enzyme production. XlnR knock-out strains for both aspergilli were used to study the influence of plant cell wall degrading enzyme production on the fitness of the mixed culture. Microscopic observation correlated with quantitative PCR and proteomic data suggest that the XlnR Knock-out strain benefit from the release of sugars by the wild type strain to support its growth.

Optimization of liquid-state fermentation conditions for the glyphosate degradation enzyme production of strain Aspergillus oryzae by ultraviolet mutagenesis

ABSTRACTThis study aimed to obtain strains with high glyphosate-degrading ability and improve the ability of glyphosate degradation enzyme by the optimization of fermentation conditions. Spore from Aspergillus oryzae A-F02 was subjected to ultraviolet mutagenesis. Single-factor experiment and response surface methodology were used to optimize glyphosate degradation enzyme production from mutant strain by liquid-state fermentation. Four mutant strains were obtained and named as FUJX 001, FUJX 002, FUJX 003, and FUJX 004, in which FUJX 001 gave the highest total enzyme activity. Starch concentration at 0.56%, GP concentration at 1,370 mg/l, initial pH at 6.8, and temperature at 30°C were the optimum conditions for the improved glyphosate degradation endoenzyme production of A. oryzae FUJX 001. Under these conditions, the experimental endoenzyme activity was 784.15 U/100 ml fermentation liquor. The result (784.15 U/100 ml fermentation liquor) was approximately 14-fold higher than that of the original strain. The result highlights the potential of glyphosate degradation enzyme to degrade glyphosate.

In vitro growth and cell wall degrading enzyme production by Argentinean isolates of Macrophomina phaseolina, the causative agent of charcoal rot in corn

Macrophomina phaseolina is a polyphagous phytopathogen, causing stalk rot on many commercially important species. Damages caused by this pathogen in soybean and maize crops in Argentina during drought and hot weather have increased due its ability to survive as sclerotia in soil and crop debris under non-till practices. In this work, we explored the in vitro production of plant cell wall-degrading enzymes [pectinases (polygalacturonase and polymethylgalacturonase); cellulases (endoglucanase); hemicellulases (endoxylanase) and the ligninolytic enzyme laccase] by several Argentinean isolates of M. phaseolina, and assessed the pathogenicity of these isolates as a preliminary step to establish the role of these enzymes in M. phaseolina–maize interaction. The isolates were grown in liquid synthetic medium supplemented with glucose, pectin, carboxymethylcellulose or xylan as carbon sources and/or enzyme inducers and glutamic acid as nitrogen source.Pectinases were the first cell wall-degrading enzymes detected and the activities obtained (polygalacturonase activity was between 0.4 and 1.3U/ml and polymethylgalacturonase between 0.15 and 1.3U/ml) were higher than those of cellulases and xylanases, which appeared later and in a lesser magnitude. This sequence would promote initial tissue maceration followed by cell wall degradation. Laccase was detected in all the isolates evaluated (activity was between 36U/l and 63U/l). The aggressiveness of the isolates was tested in maize, sunflower and watermelon seeds, being high on all the plants assayed. This study reports for the first time the potential of different isolates of M. phaseolina to produce plant cell wall-degrading enzymes in submerged fermentation.

Cholesterol crystals activate Syk and PI3 kinase in human macrophages and dendritic cells

Background and aimsCholesterol crystals are a key component of atherosclerotic lesions where they promote pro-inflammatory cytokine production and plaque destabilization. Antagonists of inflammatory mediators and agents that dissolve or prevent the formation of cholesterol crystals are being explored as potential therapeutics for atherothrombosis. We sought to identify signalling molecules activated following exposure of immune cells to cholesterol crystals with the view to identifying novel therapeutic targets. MethodsHuman macrophages and dendritic cells (DC) were exposed to cholesterol crystals and activation of signalling molecules was assessed by immunoblotting. The role of Syk and PI3K in crystal-induced interleukin (IL)-1 production was determined by ELISA using specific kinase inhibitors. Real-time PCR was employed to examine the role of Syk/PI3K in cholesterol crystal-induced expression of S100 proteins and MMPs. ResultsExposure of human macrophages and DC to cholesterol crystals induced robust activation of Syk and PI3K within 2–5 min. Pharmacological inhibition of Syk/PI3K reduced crystal-induced IL-1α/β production by approximately 80%. Activation of the downstream MAP kinases, MEK and ERK, was suppressed following inhibition of Syk and PI3K. Finally, inhibition of both Syk and PI3K significantly reduced cholesterol crystal-induced S100A8 and MMP1 gene expression by >70% while inhibition of PI3K also reduced S100A12 expression. ConclusionCholesterol crystals activate specific cell signalling pathways which drive the production of inflammatory cytokines and degradative enzymes known to contribute to disease initiation and progression. These molecular events are dependent on activation of Syk and PI3K, hence, they represent potential therapeutic targets for the treatment of cholesterol crystal-related pathologies.

Identification and characterization of endophytic bacteria isolated from in vitro cultures of peach and pear rootstocks.

Endophytes are microorganisms which live symbiotically with almost all varieties of plant and in turn helping the plant in a number of ways. Instead of satisfactory surface sterilization approaches, repeatedly occurring bacterial growth on in vitro rootstock cultures of peach and pear was identified and isolated as endophytic bacteria in our present study. Five different isolates from peach rootstocks were molecularly identified by 16S rRNA gene sequencing as Brevundimonas diminuta, Leifsonia shinshuensis, Sphingomonas parapaucimobilis Brevundimonas vesicularis, Agrobacterium tumefaciens while two endophytic isolates of pear were identified as Pseudoxanthomonas mexicana, and Stenotrophomonas rhizophilia. Identified endophytes were also screened for their potential of plant growth promotion according to indoleacetic acid (IAA) production, nitrogen fixation, solubilization of phosphate and production of siderophore. All seven endophytic isolates have shown positive results for IAA, nitrogen fixation and phosphate solubilization tests. However, two out of seven isolates showed positive results for siderophore production. On the basis of these growth promoting competences, isolated endophytes can be presumed to have significant influence on the growth of host plants. Future studies required to determine the antimicrobial susceptibility profile and potential application of these isolates in biological control, microbial biofertilizers and degradative enzyme production.

Transgenic Plant-Produced Hydrolytic Enzymes and the Potential of Insect Gut-Derived Hydrolases for Biofuels.

Various perennial C4 grass species have tremendous potential for use as lignocellulosic biofuel feedstocks. Currently available grasses require costly pre-treatment and exogenous hydrolytic enzyme application to break down complex cell wall polymers into sugars that can then be fermented into ethanol. It has long been hypothesized that engineered feedstock production of cell wall degrading (CWD) enzymes would be an efficient production platform for of exogenous hydrolytic enzymes. Most research has focused on plant overexpression of CWD enzyme-coding genes from free-living bacteria and fungi that naturally break down plant cell walls. Recently, it has been found that insect digestive tracts harbor novel sources of lignocellulolytic biocatalysts that might be exploited for biofuel production. These CWD enzyme genes can be located in the insect genomes or in symbiotic microbes. When CWD genes are transformed into plants, negative pleiotropic effects are possible such as unintended cell wall digestion. The use of codon optimization along with organelle and tissue specific targeting improves CWD enzyme yields. The literature teaches several important lessons on strategic deployment of CWD genes in transgenic plants, which is the focus of this review.

Cordycepin inhibits LPS-induced inflammatory and matrix degradation in the intervertebral disc.

Cordycepin is a component of the extract obtained from Cordyceps militaris and has many biological activities, including anti-cancer, anti-metastatic and anti-inflammatory effects. Intervertebral disc degeneration (IDD) is a degenerative disease that is closely related to the inflammation of nucleus pulposus (NP) cells. The effect of cordycepin on NP cells in relation to inflammation and degeneration has not yet been studied. In our study, we used a rat NP cell culture and an intervertebral disc (IVD) organ culture model to examine the inhibitory effects of cordycepin on lipopolysaccharide (LPS)-induced gene expression and the production of matrix degradation enzymes (MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5) and oxidative stress-associated factors (nitric oxide and PGE2). We found a protective effect of cordycepin on NP cells and IVDs against LPS-induced matrix degradation and macrophage infiltration. In addition, western blot and luciferase assay results demonstrated that pretreatment with cordycepin significantly suppressed the LPS-induced activation of the NF-κB pathway. Taken together, the results of our research suggest that cordycepin could exert anti-inflammatory and anti-degenerative effects on NP cells and IVDs by inhibiting the activation of the NF-κB pathway. Therefore, cordycepin may be a potential treatment for IDD in the future.

Evaluation of plant cell wall degrading enzyme production by Clostridium thermocellum B8 in the presence of raw agricultural wastes

Plant cell wall represents an important source of fermentable sugars for second generation bioethanol production. However, cellulosic biomass hydrolysis still is a bottleneck to bioethanol production in an efficient and low cost process. Thermophilic bacteria have been studied as a source of cellulolytic enzymes for cellulosic biomass deconstruction, as their enzymes present unique features compatible with current industrial process conditions. The present study was carried out to evaluate the use of different agro-industrial wastes as suitable carbon sources for growth and enzyme secretion by a strain of Clostridium thermocellum isolated from goat rumen. C. thermocellum B8 was able to grow on/degrade microcrystalline cellulose, Sugar cane bagasse/Straw and Cotton waste, and produced different sets of cellulases and hemicellulases in their presence. The enzymatic mixtures produced by C. thermocellum (B8) showed activities over a broad range of temperatures (50–70 °C). Highest values were obtained between 60 and 70 °C, at a pH range from 5 to 7, decreasing at alkaline pH values. In addition, enzymes displayed thermostability, with CMCase and xylanase activities maintaining maximum values over 12 days at 50 °C.

Propionibacterium acnes Recovered from Atherosclerotic Human Carotid Arteries Undergoes Biofilm Dispersion and Releases Lipolytic and Proteolytic Enzymes in Response to Norepinephrine Challenge In Vitro.

In the present study, human atherosclerotic carotid arteries were examined following endarterectomy for the presence of the Gram-positive bacterium Propionibacterium acnes and its potential association with biofilm structures within the arterial wall. The P. acnes 16S rRNA gene was detectable in 4 of 15 carotid artery samples, and viable P. acnes was one among 10 different bacterial species recoverable in culture. Fluorescence in situ hybridization analysis of 5 additional atherosclerotic carotid arteries demonstrated biofilm bacteria within all samples, with P. acnes detectable in 4 samples. We also demonstrated that laboratory-grown cultures of P. acnes biofilms were susceptible to induction of a biofilm dispersion response when challenged with physiologically relevant levels of norepinephrine in the presence of iron-bound transferrin or with free iron. The production and release of lipolytic and proteolytic extracellular enzymes by P. acnes were shown to increase in iron-induced dispersed biofilms, and these dispersion-induced P. acnes VP1 biofilms showed increased expression of mRNAs for the triacylglycerol lipases PPA2105 and PPA1796 and the hyaluronate lyase PPA380 compared to that in untreated biofilms. These results demonstrate that P. acnes can infect the carotid arteries of humans with atherosclerosis as a component of multispecies biofilms and that dispersion is inducible for this organism, at least in vitro, with physiologically relevant levels of norepinephrine resulting in the production and release of degradative enzymes.

Characterization of Trichoderma isolates against Sclerotium rolfsii, the collar rot pathogen of Amorphophallus – A polyphasic approach

The aim of the present study was to characterize ten selected isolates of Trichoderma with differential inhibitory potential over the collar rot pathogen of Amorphophallus, Sclerotium rolfsii. Use of a polyphasic approach which combined the morphological, molecular and biochemical attributes revealed inter specific and intra specific diversity among different isolates. Strains with the best in vitro antagonistic capacity were Trichoderma harzianum (Tr9) and Trichoderma asperellum (Tr10). In general, relationship was observed between the anatgonistic capacity of the Trichoderma spp. and their in vitro biocontrol performance, cell wall degrading enzyme production, protein profiles and the RAPD and AFLP finger print. The pot experiment revealed a comparative success of Tr9 in controlling collar rot incidence in Amorphophallus over Tr10. This is the first report of intensive characterization of Trichoderma strains with the integration of the above data sets against S. rolfsii, in an attempt to explore their biocontrol potential in managing collar rot disease.

Statistical Optimization for Simultaneous Production of PLA Degrading and Raw Starch Degrading Enzymes by Thermophilic Filamentous Bacterium, Laceyella sacchari LP175 Using Agricultural Crops as Substrates

Statistical Optimization for Simultaneous Production of PLA Degrading and Raw Starch Degrading Enzymes by Thermophilic Filamentous Bacterium, Laceyella sacchari LP175 Using Agricultural Crops as Substrates

Computational modeling, docking and molecular dynamics of the transcriptional activator ComA bound to a newly-identified functional DNA binding site

Quorum sensing is the mechanism by which bacterial cells communicate to each other in response to changes in cell density. Secreted signaling molecules reach other bacteria and trigger an internal physiological response, like production of degradative enzymes and antibiotics, competence development, sporulation and pathogenesis. In Bacillus subtilis, the transcriptional activator ComA regulates several genes of the quorum sensing response. ComA binds to recognition elements (RE) in bacterial promoters, activating transcription. The DNA binding domain of ComA has four α-helices, which contain a helix-turn-helix (HTH) motif. Several promoters in B. subtilis have known inverted repeat motifs (RE1 and RE2), where a ComA dimer can bind. However, a third and fourth recognition-like element (RE3 and RE4) in a direct repeat (DR) arrangement have recently been identified downstream of the known ComA box. Currently there is no structural information on how a DR would bind to ComA. Here, we present computational results supported by experiments that the DR (RE3 and RE4) form a functional domain for recognition of a ComA dimer. Flexible protein-DNA docking was used to get insight into the putative binding mode of ComA bound to the new direct repeat. The lowest-energy docked conformations of the ComA-DNA complex were tested for dynamic stability with explicit molecular dynamics simulations. Clustering of the sampled conformations was used to select a representative structure of the ComA-DR- DNA complex. In our results, the second α-helix of the HTH contributes most of the DNA recognition, binding to the major grooves of the DR-DNA, and interacting mostly with the DNA bases. We pinpoint specific ComA-DNA interactions that may have a key role for recognition and affinity. Furthermore, the physical interaction between ComA and the new DR was demonstrated in vitro, and its functionality was confirmed in vivo. Our results strongly support the hypothesis that ComA dimers can bind to direct repeats, and provide an atomistic model for its recognition. Additionally, we suggest specific interactions for fine-tuning of transcription which will be tested experimentally.