Brzi postupak identifikacije bakterija octenog vrenja tijekom submerzne industrijske proizvodnje octa, lokalizacijom 16S rRNA pomoću hibridizacije in situ i protočne citometrije

Acetic acid bacteria are involved in many biotechnological processes such as vitamin C, gluconic acid, miglitol or acetic acid production, and others. For a technologist trying to control the industrial process, the ability to follow the microbiological development of the process is thus of importance. During the past few years hybridization in a combination with flow cytometry has often been used for this purpose. Since vinegar is a liquid, it is an ideal matrix for flow cytometry analysis. In this work we have constructed a specific probe for highly acetic acid-resistant species of the acetic acid bacteria and a protocol for in situ hybridization, which in combination with flow cytometry enables direct monitoring of bacteria producing vinegar with >10 % of acetic acid. The approach was successfully applied for monitoring microbiota during industrial vinegar production.Bakterije octenog vrenja koriste se u mnogim biotehnološkim procesima, između ostalog u proizvodnji vitamina C, miglitola (inhibitora α-glucozidaze), glukonske ili octene kiseline. Tijekom industrijske proizvodnje izrazito je važno omogućiti praćenje aktivnosti mikroorganizama radi kontrole postupka, pa se u tu svrhu u novije vrijeme često koristi hibridizacija in situ u kombinaciji s protočnom citometrijom. Ocat je tekućina, pa je idealan medij za protočnu citometriju. Pripremom nove specifične probe za sojeve bakterija octenog vrenja koji mogu rasti pri većim udjelima octene kiseline, te izradom novog protokola hibridizacije in situ, u kombinaciji s protočnom citometrijom omogućeno je izravno praćenje bakterija u proizvodnji octa s udjelom octene kiseline većim od 10 %. Metoda je uspješno upotrijebljena za praćenje mikrobiote tijekom industrijske proizvodnje octa

Molecular profiling and identification of methanogenic archaeal species from rabbit caecum

During a comparison of 16S rDNA PCR-denaturant gradient gel electrophoresis (DGGE) profiles of methanogenic archea from rumen fluid, rabbit caecum and pig feces, a unique band common to all rabbit caecum samples was observed. DGGE profiling also showed that the methanogen community from the New Zealand White adult rabbits is different and less complex than the methanogen communities from the rumen and pig feces. Small subunit ribosomal gene sequences of methanogenic archaea were subsequently retrieved from the constructed rabbit caecum 16S rDNA gene library. Results of the phylogenetic analysis indicated that rabbit caecum is inhibited by members of the genus Methanobrevibacter and is possibly one-species dominanted, because all the retrived sequences exhibited similarity values of 99% or higher. This species may well be a novel species of the genus Methanorevibacter. It belongs to a distinct phylogenetic group containing methanobrevibacter woesei, Methanobrevibacter thaueri and Methanobrevibacter gottschalkii strains isolated from animal faeces, and Methanobrevibactersmithii from the predominanting methanogen population of the human large bowel

Prebavna mikrobiota kot dejavnik pri razvoju debelosti

Porast debelosti v moderni družbi je povezan z večjo pojavnostjo z debelostjo povezanih bolezni in predstavlja veliko finančno breme za javno zdravstvo. Pomembno odkritje na področju mikrobiologije prebavnega trakta sesalcev je povezano z vlogo prebavne mikrobiote pri razvoju debelosti. Z novimi molekularnimi metodami in poskusi z gnotobiotskimi živalmi so do neke mere pojasnili udeležbo prebavne mikrobiote pri uravnavanju telesne mase in energijskega ravnovesja gostitelja. Prebavna mikrobiota vpliva na vnos hranil in porabo energije iz hrane in pospešuje shranjevanje le-te v maščobna tkiva s procesi fermentacije, olajšane absorpcije in tudi z vplivom na izražanje gostiteljevih genov (protein Fiaf) ter na aktivnost gostiteljevih encimov (proteinska kinaza AMPK). Pri debelih miših in ljudeh je prebavna mikrobiota dokazano bolj učinkovita pri izkoriščanju energije iz hrane kot pri suhih osebkih. Obstajajo značilne razlike v sestavi mikrobne združbe glede na debel oz. suh fenotip. V prebavilih debelih živali in ljudi se dosledno kae povišan dele predstavnikov bakterij iz debla Firmicutes na račun zmanjšanja predstavnikov debla Bacteroidetes, obe prevladujoči debli pa v prebavilih sesalcev skupaj predstavljata do 90 % vseh bakterij. Izkazalo se je, da je prebavna mikrobiota udeležena tudi pri patofiziologiji debelosti preko dejavnikov, kot je mikrobni LPS. Rezultati raziskav kažejo, da lahko spremembe deleža maščob v hrani vplivajo na sestavo mikrobne združbe ter da te spremembe vplivajo na pojavnost metabolnih bolezni. Odpira se novo področje manipulacije prebavne mikrobiote za zdravljenje debelosti in z njo povezanih bolezni.The increased prevalence of obesity in modern society is associated with incidence of obesity related diseases and represents a financial burden on public health. Important discovery in the field of microbial ecology of the gut was the possible involvement of the gut microbiota in obesity development. Using new molecular techniques and gnotobiotic animal models has revealed the relation between the regulation of body mass and energy balance of the host with the microbial community of the gut. Gut microbiota affects nutrient intake, facilitate the extraction of energy from food and promote storage of the calories in host adipose tissue through processes of fermentation, absorption and through the effect on the expression of host genes (e.g. Fiaf) and the activity of host enzymes (e.g. AMPK). In obese mice and humans the gut microbiota is clearly able to obtain energy from food more effectively as in the lean subjects. There are significant differences in the composition of microbial communities in relation to fat vs. lean phenotype. In the gut of obese animals and humans the increased proportion of the Firmicutes at the expense on Bacteroidetes was consistently detected. Both are the dominant bacterial groups in mammalian gastrointestinal tract, accounting together for 90% of all bacteria. It has been shown that gut microbiota is involved also in patophysiology of obesitz through factors such as microbial LPS. Existing results show that high fat diet can affect the composition of microbial community in the gut and that these changes can further affect the incidence of metabolic disease. This evidence potentially opens a new field of manipulation of the gut microbiota as a new strategy to treat obesity and related diseases

Optimization of the DGGE band identification method

Denaturant gradient gel electrophoresis (DGGE) enables insight into the diversity of the studied microbial communities on the basis of separation of PCR amplification products according to their nucelotide sequence composition. However, the success of the method is accompanied by the inherent appearance of various sequence artifacts that bias the impression of community structure by generating additional bands representing no virtual microbes. PCR-DGGE artifacts require optimization of the method when aiming at the phylogenetic identification of the selected DGGE bands. The aim of our study was to develop a procedure which will increase the reliability of the identification. Samples of rumen fluid were used for the optimization since they contain a complex microbial community that supports the generation of artifactual bands. An optimized procedure following band excision and elution of microbial DNA is proposed including nuclease treatment, selection of DNA polymerase with proofreading activity, and cloning prior to sequencing and identification analysis

Inability of Prevotella bryantii to form a functional Shine-Dalgarno interaction reflects unique evolution of ribosome binding sites in Bacteroidetes

The Shine-Dalarno (SD) sequence is a key element directing the translation to initiate at the authentic start codons and also enabling translation initiation to proceed in 5\u27 untranslated mRNA regions (5\u27-UTRs) containing moderately strong secondary structures. Bioinformatic analysis of almost forty genomes from the major bacterial phylum Bacteroidetes revealed, however, a general absence of SD sequence, drop in GC content and consequently reduced tendency to form secondary structures in 5\u27-UTRs. The experimentsusing the Prevotella bryantii TC-11 expression system were in agreement with this findings: neither addition nor omission of SD sequence in the unstructured 5\u27-UTR affected the level of the reporter protein, non-specific nuclease NucB. Further, NucB level in P. bryantii TC1-1, contrary to hMGFP level in Eschericihia coli, was five times lower when SD sequence formed part of the secondary structure with a folding energy -5,2 kcal/mol. Also, the extended SDsequences did not affect protein levels as in E. coli. It seems therefore that a functional SD interaction does not take place during the translation initiation in P. bryantii TC1-1 and possibly other members of phylum Bacteroidetes although the anti SD sequence is present in 16S rnRNA genes of their genomes. We thus propose that in the absence of the SD sequence interaction, the selection of genuine start codons in Bacteroidetes is accomplished by binding of ribosomal protein S1 to unstructured 5\u27UTR as opposed to coding region which is inaccessible due to mRNA secondary structure. Additionally, we found that sequence logos of region preceding the start codons may be used as taxonomical markers. Depending on whether complete sequence logo or only part of it, such as information content and base proportion at specific positions, is used, bacterial genera or families and insome cases even bacterial phyla can be distinguised

Iskanje konjugativnega transpozona v vampni bakteriji Prevotella bryantii B[down]14

Only few plasmids and bacteriophages have been described to date in ruminal prevotella strains, therefore it appears plausible that the genetic exchange in these organisms must exploit other routes. Large conjugative transposons make possible the gene exchange process in bacteria from the genus Bacteroides, the phylogenetic relatives of ruminal prevotellas. The access to fully or partially finished genome sequences of Bacteroides and Prevotella representatives made possible the search for conserved regions within putative conjugative transposons. Multiple sequence alignment of known and putative conjugative transposon gene sequences of Bacteroides thetaiotaomicron, Prevotella intermedia, Bacteroides fragilis and Tanerella sp. was used to locate partially conserved regions within most preserved conjugative transposition genes, traG, and to construct appropriate degenerated oligonucleotide primers. These were used to amplify genome fragments from ruminal prevotella strains. Sequence analysis of the subcloned PCR products revealed the presence of a hypothetical gene in the genome of Prevotella bryantii B14, similar to the ORF BF2880 from B. fragilis YCH46, which is a part of a large conjugative transposon. Inverse PCRs were designed and performed to confirm the initial findings. A partial map of P. bryantii B14 putative conjugative transposon region was constructed, indicating an intergeneric horizontal gene transfer.Ker sevi rodu Prevotella iz vampa le izjemoma posedujejo plazmide in je opisanih le nekaj bakteriofagov, je zelo verjetno, da izmenjava genov pri teh organizmih vključuje druge poti. Veliki konjugativni transpozoni omogočajo prenos genov pri rodu Bacteroides, filogenetskih sorodnikih vampnih prevotel. Dostop do delno ali v celoti sekvenciranih genomov predstavnikov Bacteroides in Prevotella je omogočil iskanje ohranjenih regij znotraj domnevnih konjugativnih transpozonov. S poravnavo več sekvenc znanih ali domnevnih genov konjugativnih transpozonov iz vrst Bacteroides thetaiotaomicron, Prevotella intermedia, Bacteroides fragilis in rodu Tanerella smo določili delno ohranjene regije v najbolj ohranjenem genu konjugativne transpozicije, traG, in jih uporabili za izdelavo primernih začetnih oligonukleotidov za pomnoevanje dela gena pri vampnih sevih iz rodu Prevotella. Analiza sekvenc subkloniranih pomnokov je pri P. bryantii B14 razkrila prisotnost hipotetičnega gena, podobnega odprtemu čitalnemu okvirju BF2880 seva B. fragilis YCH46, ki je del velikega konjugativnega transpozona. Z inverzno verižno reakcijo s polimerazo smo potrdili prvotne ugotovitve. Izdelali smo delno mapo regije domnevnega konjugativnega transpozona pri P. bryantii B14, ki nakazuje, da je prišlo do med rodovnega horizontalnega prenosa genov

Distinct approaches for the detection and removal of chimeric 16S rRNA sequences can significantly affect the outcome of between-site comparisons

Comparative analyses of 16S rRNA clone libraries represent a standard tool in microbial ecology. Chimeric sequences are generally removed prior to such compariosns. A literature survey revealed a general pattern: (1) most commonly a single chimera identification approach (CIA) has been used(2) putative chimeras have routinely been discarded without manual examination(3) chimera filtered datasets have been submitted to repositories. To explore the effects of various CIAs on the study of microbial ß-diversity relationships using complete primary data, 4 bacterial and 4 archaeal clone libraries were generated from a submarine spring and analyzed together with 3 bacterial and 3 archaeal published primary datasets. The primary datasets were compared with their 8 different CIA filtered datasets using Chimera_check,CCODE, Pintail, Chimera Slayer and Ballerophon, the last with 4 different settings. When CIA filtered datasets were pooled accoring to the CIA used, no significant differnces between them could be observed, although there was not complete congruency between the different CIAs. When CIA filtered datasets of the same clone library were compared, generally no significant differences could be observed. In contrast, when CIA filtered datasets of different clone libraries were compared, the statistical significance of the relationships shifted from significant to insignificant orvice-versa in many cases depending on the CIA used. This precludes a correct identification of ß-diversity. To solve this problem, we treated all CIA filtered datasets and primary data of a single clone library as CIA replicated in non-parametric MANOVA. This enabled unambiguous delineation of environmental samples by taking into account all CIA introduced data modifications

Identification of bacterial contaminants from calcium carbonate filler production lines and an evaluation of biocide based decontamination procedures

<p>The aim of this study was to analyze the bacterial community in the production line of a calcium carbonate filler production company and to investigate possible causes for bacterial presence. Throughout 2012, 24 carbonate slurry and six groundwater samples were analyzed. <i>Pseudomonas</i> and <i>Microbacterium</i> were the most frequent contaminants in the slurry, whereas <i>Pseudomonas</i> and <i>Brevundimonas</i> dominated the groundwater samples. Of the 43 different bacterial strains isolated, only five were found both in the slurry and the groundwater, indicating that the latter was not a major source of contamination. The efficacy of 54 commercial biocidal formulations was tested against an artificial bacterial consortium composed of selected slurry isolates. A formulation containing 7.5–15% (v v^–1) bronopol and 1.0–2.5% (v v^–1) [chloroisothiazolinone (CIT) + methylisothiazolinone (MIT)] exhibited the highest efficacy. Of the possible causes for bacterial presence, sporogenesis and biocide adsorption to carbonate particles were found to be less probable compared to bacterial adsorption to particles, and the acquisition of resistance to biocides.</p