Conjugational delivery of chromosomal integrative constructs for gene expression in the carbendazim-degrading <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> D-1

Rhodococcus strains not only have been widely used in industries but also have a potential ability of producing new structural natural products. Integration of heterologous genes into chromosomes of Rhodococcus strains for gene expression can facilitate the studies and applications of these strains. A conjugation system was optimized in order to transfer enhanced green fluorescent protein (EGFP) encoding gene as a reporter from Escherichia coli into Rhodococcus erythropolis D-1. The influence of three native ribosome binding sites (RBSs) and two designed RBSs on the target protein production in R. erythropolis D-1 was also characterized. An efficient conjugation system of R. erythropolis D-1 was established to integrate EGFP gene into its chromosome. Among of five RBSs, RBS3 showed the highest translational activity in R. erythropolis D-1.     

Psychometric validation of the EuroQoL 5-Dimension 5-Level (EQ-5D-5L) in Chinese patients with adolescent idiopathic scoliosis

Background

Scoliosis is a common spinal deformity that occurs often during adolescence. Previous studies suggested that adolescent idiopathic scoliosis (AIS) patients can have various aspects of their lives being affected, due to disease presentation and/or treatment received. It is important to define a reliable instrument based on which the affected patients’ health-related quality of life can be assessed. This study aims to assess the validity, reliability and sensitivity of the EuroQoL 5-dimension 5-level (EQ-5D-5L) in Chinese patients with AIS.

Methods

Adolescent idiopathic scoliosis patients of Chinese descent were prospectively recruited to complete both the traditional Chinese versions of the EQ-5D-5L and the refined Scoliosis Research Society-22 (SRS-22r) questionnaires. Patients’ demographic profiles and corresponding clinical parameters including treatment modalities, spinal curve pattern and magnitude, and duration of bracing were recorded. Telephone interviews were then conducted at least two weeks later for the assessment of test-retest reliability. Statistical analysis was performed: construct validity of the EQ-5D-5L domains were assessed using Spearman’s correlation test against the SRS-22r; whereas intra-class correlation coefficient (ICC) was used to assess the test-retest reliability, and agreement over the test-retest period was expressed in percentages. Also, the sensitivity of the EQ-5D-5L in differentiating various clinical known groups was determined by effect size, independent t-test and analysis of variance.

Results

A total of 227 AIS patients were recruited. Scores of domains of the EQ-5D-5L correlated significantly (r: 0.57-0.74) with the scores of the SRS-22r domains that were intended to measure similar constructs, supporting construct validity. The EQ-5D-5L domain responses and utility scores showed good test-retest reliability (ICC: 0.777; agreement: 76.4?-98.1 %). Internal consistency was good (Cronbach’s α: 0.78) for the EQ-5D-5L utility score. The EQ-5D-5L utility score was sensitive in detecting differences between subjects who had different treatment modalities and bracing duration, but not for curve pattern and its magnitude.

Conclusions

The EQ-5D-5L is found to be a valid, reliable and sensitive measure to assess the health-related quality of life in Chinese AIS patients. This potentiates the possibility of utilizing the EQ-5D-5L to estimate AIS patients’ health-related quality of life, based on which the outcome of various treatment options can eventually be evaluated.

     

Detection of Carboxymethyl Cellulase Activity in Acetobacter xylinum KU-1

We detected carboxymethyl cellulase activity in a crude extract of Acetobacter xylinum KU-1. The enzyme activity was detected when glycerol, d-fructose, d-mannitol, d-glucose, d-arabitol, d-sorbitol, or carboxymethyl cellulose was used as a carbon source. The optimum pH was found to be 4.0, while the optimum temperature was 50°C. The enzyme activity was inhibited characteristically by the addition of Hg²⁺.     

2-Keto-l-gulonic Acid Fermentation

A number of bacterial strains from type culture collections and natural sources were examined in their metabolic characteristics toward sorbitol and l-sorbose.

Paper chromatographic analyses of sorbitol and l-sorbose metabolites obtained from the cultures of various bacteria revealed that the organisms producing 2-keto-l-gulonic acid from sorbitol were merely found in the genera Acetobacter, Gluconobacter and Pseudomonas, whereas those producing the acid from l-sorbose were distributed in the twelve genera of bacteria: Acetobacter, Alcaligenes, Aerobacter, Azotobacter, Bacillus, Escherichia, Gluconobacter, Klebsiella, Micrococcus, Pseudomonas, Serratia and Xanthomonas.

G. melanogenus, which was characterized by excellent production of 2-keto-l-gulonic acid from sorbitol, also formed several other sugars and sugar acids as the sorbitol metabolites. These compounds were identified to be d-fructose, l-sorbose, d-mannonic acid, L-idonic acid, 2-keto-d-gluconic acid and 5-keto-d-mannonic acid, respectively, by means of two-dimensional paper chromatography.

Bacteria producing 2-keto-l-gulonic acid from sorbitol were usually isolated from fruits but not from soil.     

Prenatal screening of fetal ventriculoarterial connections: benefits of 4D technique in fetal heart imaging

Background

Identification of prenatal ventriculoarterial connections in fetuses with conotruncal anomalies (CTA) remains one of the greatest challenges for sonographers performing screening examinations. Herein, we propose a novel protocol of 4D volume analysis that identifies ventriculoarterial connections and evaluate its clinical utility in routine screenings.

Methods

Twenty-nine cases of transposition of the great arteries (TGA), 22 cases of double-outlet right ventricle (DORV), 36 cases of tetralogy of Fallot (TOF), 14 cases of truncus arteriosus (TCA), and randomly selected 70 normal fetuses were reviewed in this study. All cases were evaluated using 2D data alone (2D method), post-processing volumes with no exact algorithm (4D-1 method), or with the proposed algorithm (4D-2 method), or using the 2D and 4D data together (combined method). Comparisons were made to evaluate the detection rate of ventriculoarterial connections for these different methods.

Results

During 18–28 gestational weeks, the detection rate of 4D-2 modality was satisfactory. The detection rate of the combined method was significantly higher than 2D method in the identification of TGA, TOF, and TCA. The detection rate of 4D-1 method was significantly lower than 4D ?2 modality for CTA fetuses. During late pregnancy, the detection rate for both 4D modalities was very low due to the poor quality of the 4D volumes.

Conclusions

We proposed a detailed protocol, which allowed the examiner to identify fetal ventriculoarterial connections by 4D volumes. Inclusion of blood information into the volumes improved diagnosis. Our findings suggest that the incorporation of 4D STIC into routine screenings could improve the detection for TGA, TOF, and TCA.

     

Molecular identification of yeast,lactic and acetic acid bacteria species during spoilage of tchapalo,a traditional sorghum beer from Côte d’Ivoire

Yeasts, lactic and acetic acid bacteria are responsible of microbial spoilage of alcoholic beverages. However species involved in deterioration of sorghum beer produced in Côte d’Ivoire has not been investigated. This study was carried out to identify species of yeast, LAB and AAB during spoilage of tchapalo in order to define the best strategy for beer preservative. Thus, a total of 210 yeasts, LAB and AAB were isolated from samples of tchapalo stored at ambient temperature and at 4 °C for 3 days. Based on PCR–RFLP of the ITS region and the sequencing of D1/D2 domain, yeast isolates were assigned to seven species (Saccharomyces cerevisiae, Candida tropicalis, Rhodotorula mucilaginosa, Trichosporon asahii, Kluyveromyces marxianus, Meyerozyma guilliermondii and Trichosporon coremiiforme). During the storage at ambient temperature and at 4 °C, S. cerevisiae was the predominant species (>?76%). Excepted R. mucilaginosa, occurrence of non-Saccharomyces species was sporadic. LAB species detected in fresh samples using molecular methods were Pediococcus acidilactici, Lactobacillus paracasei, Lb. curvatus, Lb. fermentum and Weisssella paramesenteroides. P. acidilactici was the dominant species (47.8%) followed by Lb. paracasei (17.5%). W. paramesenteroides and Lb. fermentum were not detected during the spoilage at ambient temperature while at 4 °C W. paramesenteroides and Lb. paracasei have not been detected. For AAB, the species found were Acetobacter pasteurianus sub paradoxus and Acetobacter cerevisiae. These species were common to all samples during spoilage and A. pasteurianus sub paradoxus was the most frequently detected.     

Untersuchungen über die Wirkung einiger 1,2,4-Triazolderivate auf das Wachstum der Keimlinge vonSinapis alb a L.

При рассмотреии физи ологических влияий д веадцати е испытывае мых до сих пор ?леметов — девяти д ериватов 1, 2, 4 — триазола III-XI, поодому из соедиеий 1, 3, 4-оксадиазолового XII и т етразиалового ряда и, акоец, II — замещеого амида икотиовой кисл оты — а всходящие раст еия Sinapis alba L., было устаовлео:

1. 1.
   Ни одно из соединений не проявляет гербици дных действий.     
   
   

Preparation of New Glycoheptofurano[2,1-d]Imidazolidine-2-Thiones., Isomerizations to Acyclic-Sugar C-Nucleoside Analogues of Imidazole

Abstract

1-Methyl- and 1-aryl-(1,2-dideoxy-D-glyofurano)[2,1-d]-imidazolidine-2-thiones having the configurations β-D-glycero-L-gluco (4), β-D-glycero-D-ido (5—8), α-D glycerol-D-galacto (9—10) and β-D-glycero-D-talo (11, 12) are prepared by reaction of 2-amino-2-deoxy-aldoses with methyl and aryl isothiocyanates. 1-Aryl-(1,2-dideoxy–β-D-glycero-L-gluco-heptofurano)[2,1-d]imidazolidine-2-thiones (1—3) have been converted into 1-aryl-4-(D-galacto-pentitol-1-yl)-4-imidazo-line-2-thiones (24—26) by acid catalysed isomerization.     

Polyol Dehydrogenases in the Soluble Fraction of Acetobacter melanogenum

Polyol dehydrogenases of Acetobacter melanogenum were investigated. Three polyol dehydrogenases, i. e. NAD⁺-linked d-mannitol dehydrogenase, NAD⁺-linked sorbitol dehydrogenase and NADP⁺-linked d-mannitol dehydrogenase, in the soluble fraction of the organism were purified 12-fold, 8-fold and 88-fold, respectively, by fractionation with ammonium sulfate and DEAE-cellulose column chromatography. NAD⁺-linked sorbitol dehydrogenase reduced 5-keto-d-fructose (5KF) to l-sorbose in the presence of NADH, whereas NADP⁺-linked d-mannitol dehydrogenase reduced the same substrate to d-fructose in the presence of NADPH. It was also shown that NAD⁺-linked d-mannitol dehydrogenase was specific for the interconversion between d-mannitol and d-fructose and that this enzyme was very unstable in alkaline conditions.     

Toxic polyketides produced by Fusarium sp., an endophytic fungus isolated from Melia azedarach

A new isocoumarin derivative named fusariumin (1), together with two known related resorcylic acid lactones aigialomycin D (2) and pochonin N (3), has been isolated from the cultures of Fusarium sp. LN-10, an endophytic fungus originated from the leaves of Melia azedarach. Their structures were established on the basis of extensive spectroscopic analyzes including 1D- and 2D- NMR (¹H-¹H COSY, HSQC, HMBC, and NOESY) experiments. Compounds 1-3 displayed significant growth inhibitory activity against the brine shrimp (Artemia salina).     

Genomics and Biochemistry of Metabolic Pathways for the C<Subscript>1</Subscript> Compounds Utilization in Colorless Sulfur Bacterium <Emphasis Type="Italic">Beggiatoa leptomitoformis</Emphasis> D-402

The metabolic pathways of one-carbon compounds utilized by colorless sulfur bacterium Beggiatoa leptomitoformis D-402 were revealed based on comprehensive analysis of its genomic organization, together with physiological, biochemical and molecular biological approaches. Strain D-402 was capable of aerobic methylotrophic growth with methanol as a sole source of carbon and energy and was not capable of methanotrophic growth because of the absence of genes of methane monooxygenases. It was established that methanol can be oxidized to CO₂ in three consecutive stages. On the first stage methanol was oxidized to formaldehyde by the two PQQ (pyrroloquinolinequinone)-dependent methanol dehydrogenases (MDH): XoxF and Mdh2. Formaldehyde was further oxidized to formate via the tetrahydromethanopterin (H₄MPT) pathway. And on the third stage formate was converted to CO₂ by NAD⁺-dependent formate dehydrogenase Fdh2. Finally, it was established that endogenous CO₂, formed as a result of methanol oxidation, was subsequently assimilated for anabolism through the Calvin–Benson–Bassham cycle. The similar way of one-carbon compounds utilization also exists in representatives of another freshwater Beggiatoa species—B. alba.     

Anthocyanins ofImpatiens holstii

1. 1.
   Byly chromatografovány antokyany t?í typ?Impatiens holstii. Bylo zji?-těno, ?e typ A (f. nana, sorta Záre, s ?arlatovými květy) obsahuje ve stonku—gluko-sidy malvidinu, v petalech—glukosidy pelargonidinu a peonidinu, v sepalech— glukosid malvidinu a cyanidinu, v pylu—glukosidy a snad i xylosidy malvidinu; typ B (f. nana, sorta Zá?e, s ?ervenofialovými květy) obsahuje ve stonku— malvidin, v petalech—peonidin, v sepalech—malvidin a cyanidin, v pylu— malvidin; typ C (vzp?ímený typ, s ?ervenofialovými květy) obsahuje v petalech—malvidin a peonidin, v sepalech—malvidin a cyanidin, v pylu—malvidin. U typ? ? a ? eukry nebyly sledovány.     
   
   

Surface-<Emphasis Type="Italic">engineered Saccharomyces cerevisiae</Emphasis> displaying α-acetolactate decarboxylase from <Emphasis Type="Italic">Acetobacter aceti</Emphasis> ssp <Emphasis Type="Italic">xylinum</Emphasis>

Objectives

To convert α-acetolactate into acetoin by an α-acetolactate decarboxylase (ALDC) to prevent its conversion into diacetyl that gives beer an unfavourable buttery flavour.

Results

We constructed a whole Saccharomyces cerevisiae cell catalyst with a truncated active ALDC from Acetobacter aceti ssp xylinum attached to the cell wall using the C-terminal anchoring domain of α-agglutinin. ALDC variants in which 43 and 69 N-terminal residues were absent performed equally well and had significantly decreased amounts of diacetyl during fermentation. With these cells, the highest concentrations of diacetyl observed during fermentation were 30 % less than those in wort fermented with control yeasts displaying only the anchoring domain and, unlike the control, virtually no diacetyl was present in wort after 7 days of fermentation.

Conclusions

Since modification of yeasts with ALDC variants did not affect their fermentation performance, the display of α-acetolactate decarboxylase activity is an effective approach to decrease the formation of diacetyl during beer fermentation.

     

Investigation of microorganisms involved in kefir biofilm formation

Kefir is a natural fermentation agent composed of various microorganisms. To address the mechanism of kefir grain formation, we investigated the microbial role in forming kefir biofilms. The results showed that a biofilm could be formed in kefir-fermented milk and the biofilm forming ability reached the maximum at 13 days. The strains Kluyveromyces marxianus, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus kefiri, Lactobacillus sunkii and Acetobacter orientalis were isolated from kefir biofilms by the streak-plate method. These microorganisms were analysed with respect to biofilm forming properties, including their surface characterisation (hydrophobicity and zeta potentials) and the microbial aggregation. The results indicated that Klu. marxianus possessed the strongest biofilm forming properties with the strongest hydrophobicity, lowest zeta potential and greatest auto-aggregation ability. When Klu. marxianus and Ac. orientalis were co-cultured with kefir LAB strains respectively, it was found that mixing Klu. marxianus with Lb. sunkii produced the highest co-aggregation ability. These results elucidated the mechanism of kefir biofilm formation and the microorganisms involved.     

Refining the late Silurian sea-level history of the Prague Syncline—a case study based on the Přídolí GSSP (Czech Republic)

A 50-m-thick section in the Po?áry quarry, Prague Syncline (Czech Republic) spanning the upper Silurian (uppermost Ludlow and P?ídolí) to the lowermost Devonian (Lochkovian) has been studied using sedimentological and physical stratigraphical (gamma-ray spectrometry) methods combined with conodont biostratigraphy. Conodont data demonstrate the presence of local conodont biozones: “Ozarkodina” crispa (uppermost Ludlow)—Zieglerodina zellmeri (base of P?ídolí)—Zieglerodina ivochlupaci—Delotaxis detorta—“Ozarkodina” eosteinhornensis s.s.—Zieglerodina klonkensis—Icriodus hesperius-optima (lowermost Lochkovian). The studied section represents a transgressive–regressive facies succession characterized by a transition from distal calciturbidites deposited in a distally steepened carbonate platform during transgression and sea-level highstand (crispa to lower ivochlupaci zones) to mixed calciturbidites/coarse-grained bioclastic limestones (falling-stage systems tract, upper ivochlupaci to lower klonkensis zones). The upper part of the succession consists of bioclastic limestones corresponding to a subtidal setting between storm-wave and fair-weather wave base (lowstand systems tract in upper klonkensis and hesperius-optima zones). This interpretation corresponds to the late Silurian global sea-level pattern as recorded in other regions. Thus the depositional system is interpreted as mostly driven by eustasy with short-lived periods of influence from local tectonics.     

D-2-Hydroxyglutarate does not mimic all the IDH mutation effects,in particular the reduced etoposide-triggered apoptosis mediated by an alteration in mitochondrial NADH

Somatic mutations in isocitrate dehydrogenase (IDH)-1 and -2 have recently been described in glioma. This mutation leads to a neomorphic enzymatic activity as the conversion of isocitrate to alpha ketoglutarate (αKG) is replaced by the conversion of αKG to D-2-hydroxyglutarate (D-2HG) with NADPH oxidation. It has been suggested that this oncometabolite D-2HG via inhibition of αKG-dioxygenases is involved in multiple functions such as epigenetic modifications or hypoxia responses. The present study is aimed at deciphering how the mutant IDH can affect cancer pathogenesis, in particular with respect to its associated oncometabolite D-2HG. We show that the overexpression of mutant IDH in glioma cells or treatment with D-2HG triggered an increase in cell proliferation. However, although mutant IDH reduced cell sensitivity to the apoptotic inducer etoposide, D-2HG exhibited no effect on apoptosis. Instead, we found that the apoptotic effect was mediated through the mitochondrial NADH pool reduction and could be inhibited by oxamate. These data show that besides D-2HG production, mutant IDH affects other crucial metabolite pools. These observations lead to a better understanding of the biology of IDH mutations in gliomas and their response to therapy.Gliomas are the most common type of human brain tumors and can be classified based on clinical and pathological criteria in four grades. The grade IV glioma, commonly known as glioblastoma multiforme (GBM), is the most invasive form and has a dismal prognosis with <5% patient survival at 5 years. These GBM can develop de novo (primary GBM) or through the progression from low-grade tumors (secondary GBM). Although these two types of GBM are histologically similar, primary and secondary GBM exhibit distinct genetic patterns. A recent integrated genome analysis of human GBM shows that 12% of these tumors have a mutation in the gene encoding isocitrate dehydrogenase 1 (IDH1) and to a lesser extent in IDH2 gene.¹ This mutation is present in >90% secondary GBMs, whereas it is present in <5% primary GBMs.² Mutations in IDH1 and IDH2 have also been identified in acute myeloid leukemia (AML)³ and chondrosarcomas.⁴ The occurrence of IDH mutations predicts a significantly longer survival for patients affected by GBM or grade III gliomas.^{1, 2} Whether this difference is driven by IDH mutations or reflects other fundamental biological differences between primary and secondary GBM is, as yet, unclear. For example, the prognostic significance of IDH mutations may be secondary to their prevalence among younger patients, as age is a well-known prognostic factor in gliomas.⁵ In AML, the prognostic significance of IDH mutations is more ambiguous. Several studies have reported that IDH mutations do not affect the prognosis in AML, whereas other studies have found that IDH mutations are associated with an increased or decreased risk of relapse when compared with IDH wild-type patients.^{6, 7}The human genome has five IDH genes coding for three different IDH isoforms, the activities of which depend on either nicotinamide adenine dinucleotide (NAD+) for IDH3 or nicotinamide adenine dinucleotide phosphate (NADP+) for IDH1 and IDH2. Both IDH2 and IDH3 are located in the mitochondria where they participate in the TCA cycle, whereas IDH1 is mostly cytosolic.⁸ To date, all reported mutations are located in the IDH1 and IDH2 genes and result in an amino-acid substitution at residues located in the enzymatic active site, respectively, R132 for IDH1 and R140 or R172 for IDH2. This mutation disrupts the normal enzymatic function of IDH, that is, the conversion of isocitrate to alpha ketoglutarate (αKG) with the concomitant production of NADPH. Instead, mutant IDH displays a neomorphic activity converting αKG into D-2-hydroxyglutarate (D-2HG), although reducing NADPH.⁹ As a result, mutant IDH may alter the redox state of cells, modulate the activity of metabolic and epigenetic tumor suppressor enzymes that use αKG as a co-substrate.¹⁰ Loss of IDH function may also alter normal mitochondrial function and promote a metabolic switch in cancer cells to glycolysis.^{11, 12}Mutant IDH is widely believed to have the ability to transform cells by modulating αKG-dependent enzymes. D-2HG and αKG are structurally similar suggesting that D-2HG may act as a competitive inhibitor of αKG-dioxygenases including prolyl hydroxylase involved in HIF-1α stability, histone demethylases and the Ten-Eleven Translocation (TET) family of 5-methylcytosine hydroxylases involved in epigenetic modifications of DNA.^{13, 14} In fact, IDH mutations lead to numerous metabolic abnormalities besides D-2HG production. Deciphering the relative importance of either D-2HG production, αKG or NADPH reduction in cancer pathogenesis remains to be determined. In this paper, we show that mutant IDH increases cell proliferation and reduces etoposide (ETO)-induced cell death through different metabolic pathways. Although cell proliferation changes are mediated through D-2HG, alteration in the mitochondrial NADH pool is involved in the response to apoptosis.     

Succession of Selected Strains of Acetobacter pasteurianus and Other Acetic Acid Bacteria in Traditional Balsamic Vinegar

The application of a selected Acetobacter pasteurianus strain for traditional balsamic vinegar production was assessed. Genomic DNA was extracted from biofilms after enrichment cultures on GYC medium (10% glucose, 1.0% yeast extract, 2.0% calcium carbonate) and used for PCR/denaturing gradient gel electrophoresis, 16S rRNA gene sequencing, and enterobacterial repetitive intergenic consensus/PCR sequencing. Results suggested that double-culture fermentation is suitable for traditional balsamic vinegar acetification.The use of selected starter cultures (SSC) in fermented food production is widely applied throughout the food industry, in particular for wine, dairy products, sausages, and a variety of vegetables (3, 11). The advantages of their use are related to the improvement of the process control, hygiene, and quality with respect to fermented foods obtained through indigenous fermentation. Vinegar is one of the fermented beverages produced without SSC inoculation, in both small- and large-scale production, mainly for the following reasons: (i) the majority of vinegars have low commercial value, and often technological innovation is not considered profitable, and (ii) there is limited knowledge of the ecophysiology of acetic acid bacteria (AAB) due to the difficulty in accessing, sampling, isolating, and preserving strains (2, 12, 15, 16, 17). Among vinegars, traditional balsamic vinegar (TBV) is an Italian aged condiment produced by “seed vinegar,” the so-called “mother of vinegar” that is an indigenous starter culture withdrawn from acetifying vinegar through back-slopping procedures. The raw material is a fermented and cooked grape must (here indicated as must) at a soluble solids content ranging from 20 to 60°Bx (10). TBV production is regulated by denomination of protected origin guidelines that specify procedures and final product features. In particular, the raw material characteristics, the production process (e.g., must cooking, alcoholic fermentation, acetic oxidation, and ageing), features of the production area (no environmental condition management is permitted), and analytical and sensorial parameters are stated as follows: acidity (not less than 4.5% [wt/wt], expressed as grams of acetic acid per 100 g of product), density at 20°C (not less than 1.240 g per liter), color, aroma, and taste. The production is performed in wood barrels, and the process is carried out by sequential refilling to acetify the must and replace the volume lost by evaporation. AAB grow on the surface of liquid by biofilm formation. No addition of any substance can be made except for the acetifying must as a starter (7). Microbial studies of TBV reported culture-dependent and -independent approaches to evaluating AAB occurrence in TBV musts (5, 10). These studies highlighted the occurrence of Gluconacetobacter europaeus as a widespread indigenous species, as well as Acetobacter pasteurianus, Acetobacter aceti, and Acetobacter malorum. However, no comprehensive studies of AAB diversity and the correlation between species occurrence and technological steps of TBV production have been published, due mainly to the difficulty of easy access to AAB microflora in vinegar matrix by both culture-dependent and -independent approaches.Regarding production technology, at least one drawback of current production procedures has been acknowledged. It concerns the difficulty of start-up acetification, which affects the minimum acidity value required for the final product. In fact, some studies showed that many variables regulate AAB growth and activity. Above all is the sugar concentration among substrates and the temperature among physical parameters. To efficiently control the acetification start-up, it is necessary to understand the function of AAB responsible for the initial colonization of musts and to investigate the microbial succession suitable to complete the acetification. Our previous researches on TBV showed that AAB strains exhibit different growing abilities. In particular, strains of Acetobacter pasteurianus grow quickly on laboratory synthetic media, wine, and cooked must. In contrast, strains belonging to G. europaeus do not grow or grow very slowly on cooked and fermented must (9, 10).The goal of this study was to implement a laboratory SSC to test it on a factory scale for TBV production purposes. In particular, we focused our attention on the effect of A. pasteurianus strain AB0220 on the acetification and dynamics of species at the end of the process. The SSC effectiveness was assessed by monitoring analytical parameters (acetic acid, ethanol, and pH), species succession, and strain persistence during three stages by the following molecular analyses: PCR/denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene sequencing, and enterobacterial repetitive intergenic consensus (ERIC)/PCR sequencing using genomic DNA extracted from biofilms recovered on GYC (10% glucose, 1.0% yeast extract, 2.0% calcium carbonate) plates.