Suchergebnisse

[Background]: The chaperone ClpB, a bacterial protein, is a conformational antigen-mimetic of α-melanocyte-stimulating hormone (α-MSH) implicated in body weight regulation in mice. We here investigated the potential associations of gut bacterial ClpB-like gene function with obesity status and gut microbiota in humans. ; [Results]: Gut microbiota ClpB KEGG function was negatively associated with body mass index, waist circumference, and total fat mass (DEXA). The relative abundance (RA) of several phyla and families directly associated with ClpB was decreased in subjects with obesity. Specifically, the RA of Rikenellaceae, Clostridiaceae and not assigned Firmicutes were lower in subjects with obesity and positively associated with gut bacterial ClpB-like gene function (not assigned Firmicutes (r = 0.405, FDR = 2.93 × 10−2), Rikenellaceae (r = 0.217, FDR = 0.031), and Clostridiaceae (r = 0.239, FDR = 0.017)). The gut bacterial ClpB-like gene function was also linked to specific plasma metabolites (hippuric acid and 3-indolepropionic acid) and fecal lupeol. The α-MSH-like epitope similar to that of Escherichia coli ClpB was also identified in some sequences of those bacterial families. After fecal transplantation from humans to mice, the families that more contributed to ClpB-like gene function in humans were also associated with ClpB-like gene function in mice after adjusting for the donor's body mass index (not assigned Firmicutes (r = 0.621, p = 0.003), Prevotellaceae (r = 0.725, p = 4.1 × 10−7), Rikenellaceae (r = 0.702, p = 3.9 × 10−4), and Ruminococcaceae (r = 0.526, p = 0.014)). Clostridiaceae (r = − 0.445, p = 0.038) and Prevotellaceae RA (r = − 0.479, p = 0.024) and were also negatively associated with weight gain in mice. The absolute abundance (AA) of Prevotellaceae in mice was also positively associated with the gut bacterial ClpB-like gene function in mice. DESeq2 identified species of Prevotellaceae, both negatively associated with mice' weight gain and positively with gut bacterial ClpB-like gene function. ; [Conclusions]: In summary, gut bacterial ClpB-like gene function is associated with obesity status, a specific gut microbiota composition and a plasma metabolomics profile in humans that could be partially transplanted to mice. ; This work was partially supported by research grants FIS (PI15/01934) from the Instituto de Salud Carlos III from Spain, SAF2015-65878-R from Ministry of Economy and Competitiveness, Prometeo/2018/A/133 from Generalitat Valenciana, Spain and also by Fondo Europeo de Desarrollo Regional (FEDER) funds, European Commission (FP7, NeuroPain #2013-602891), the Catalan Government (AGAUR, #SGR2017-669, ICREA Academia Award 2015), the Instituto de Salud Carlos III (RTA, #RD16/0017/0020), and the European Regional Development Fund (No. 01.2.2-LMT-K-718-02-0014). María Arnoriaga-Rodríguez is funded by Instituto de Salud Carlos III, Río Hortega (CP19/00190). Jordi Mayneris-Perxachs is funded by Instituto de Salud Carlos III, Miguel Servet (CP18/00009). The project has also been 65% cofinanced by the European Regional Development Fund (ERDF) through the Interreg V-A Spain-France-Andorra programme (POCTEFA 2014-2020). POCTEFA aims to reinforce the economic and social integration of the French–Spanish–Andorran border. Its support is focused on developing economic, social and environmental cross-border activities through joint strategies favouring sustainable territorial development. ; Peer reviewed

Background The chaperone ClpB, a bacterial protein, is a conformational antigen-mimetic of α-melanocyte-stimulating hormone (α-MSH) implicated in body weight regulation in mice. We here investigated the potential associations of gut bacterial ClpB-like gene function with obesity status and gut microbiota in humans. Results Gut microbiota ClpB KEGG function was negatively associated with body mass index, waist circumference, and total fat mass (DEXA). The relative abundance (RA) of several phyla and families directly associated with ClpB was decreased in subjects with obesity. Specifically, the RA of Rikenellaceae, Clostridiaceae and not assigned Firmicutes were lower in subjects with obesity and positively associated with gut bacterial ClpB-like gene function (not assigned Firmicutes (r = 0.405, FDR = 2.93 × 10−2), Rikenellaceae (r = 0.217, FDR = 0.031), and Clostridiaceae (r = 0.239, FDR = 0.017)). The gut bacterial ClpB-like gene function was also linked to specific plasma metabolites (hippuric acid and 3-indolepropionic acid) and fecal lupeol. The α-MSH-like epitope similar to that of Escherichia coli ClpB was also identified in some sequences of those bacterial families. After fecal transplantation from humans to mice, the families that more contributed to ClpB-like gene function in humans were also associated with ClpB-like gene function in mice after adjusting for the donor's body mass index (not assigned Firmicutes (r = 0.621, p = 0.003), Prevotellaceae (r = 0.725, p = 4.1 × 10−7), Rikenellaceae (r = 0.702, p = 3.9 × 10−4), and Ruminococcaceae (r = 0.526, p = 0.014)). Clostridiaceae (r = − 0.445, p = 0.038) and Prevotellaceae RA (r = − 0.479, p = 0.024) and were also negatively associated with weight gain in mice. The absolute abundance (AA) of Prevotellaceae in mice was also positively associated with the gut bacterial ClpB-like gene function in mice. DESeq2 identified species of Prevotellaceae, both negatively associated with mice' weight gain and positively with gut bacterial ClpB-like gene function. Conclusions In summary, gut bacterial ClpB-like gene function is associated with obesity status, a specific gut microbiota composition and a plasma metabolomics profile in humans that could be partially transplanted to mice. ; This work was partially supported by research grants FIS (PI15/01934) from the Instituto de Salud Carlos III from Spain, SAF2015-65878-R from Ministry of Economy and Competitiveness, Prometeo/2018/A/133 from Generalitat Valenciana, Spain and also by Fondo Europeo de Desarrollo Regional (FEDER) funds, European Commission (FP7, NeuroPain #2013-602891), the Catalan Government (AGAUR, #SGR2017-669, ICREA Academia Award 2015), the Instituto de Salud Carlos III (RTA, #RD16/0017/0020), and the European Regional Development Fund (No. 01.2.2-LMT-K-718-02-0014). María Arnoriaga-Rodríguez is funded by Instituto de Salud Carlos III, Río Hortega (CP19/00190). Jordi Mayneris-Perxachs is funded by Instituto de Salud Carlos III, Miguel Servet (CP18/00009). The project has also been 65% cofinanced by the European Regional Development Fund (ERDF) through the Interreg V-A Spain-France-Andorra programme (POCTEFA 2014-2020). POCTEFA aims to reinforce the economic and social integration of the French–Spanish–Andorran border. Its support is focused on developing economic, social and environmental cross-border activities through joint strategies favouring sustainable territorial development.

Background: The chaperone ClpB, a bacterial protein, is a conformational antigen-mimetic of α-melanocyte-stimulating hormone (α-MSH) implicated in body weight regulation in mice. We here investigated the potential associations of gut bacterial ClpB-like gene function with obesity status and gut microbiota in humans. Results: Gut microbiota ClpB KEGG function was negatively associated with body mass index, waist circumference, and total fat mass (DEXA). The relative abundance (RA) of several phyla and families directly associated with ClpB was decreased in subjects with obesity. Specifically, the RA of Rikenellaceae, Clostridiaceae and not assigned Firmicutes were lower in subjects with obesity and positively associated with gut bacterial ClpB-like gene function (not assigned Firmicutes (r = 0.405, FDR = 2.93 × 10-2), Rikenellaceae (r = 0.217, FDR = 0.031), and Clostridiaceae (r = 0.239, FDR = 0.017)). The gut bacterial ClpB-like gene function was also linked to specific plasma metabolites (hippuric acid and 3-indolepropionic acid) and fecal lupeol. The α-MSH-like epitope similar to that of Escherichia coli ClpB was also identified in some sequences of those bacterial families. After fecal transplantation from humans to mice, the families that more contributed to ClpB-like gene function in humans were also associated with ClpB-like gene function in mice after adjusting for the donor's body mass index (not assigned Firmicutes (r = 0.621, p = 0.003), Prevotellaceae (r = 0.725, p = 4.1 × 10-7), Rikenellaceae (r = 0.702, p = 3.9 × 10-4), and Ruminococcaceae (r = 0.526, p = 0.014)). Clostridiaceae (r = - 0.445, p = 0.038) and Prevotellaceae RA (r = - 0.479, p = 0.024) and were also negatively associated with weight gain in mice. The absolute abundance (AA) of Prevotellaceae in mice was also positively associated with the gut bacterial ClpB-like gene function in mice. DESeq2 identified species of Prevotellaceae, both negatively associated with mice' weight gain and positively with gut bacterial ClpB-like gene function. Conclusions: In summary, gut bacterial ClpB-like gene function is associated with obesity status, a specific gut microbiota composition and a plasma metabolomics profile in humans that could be partially transplanted to mice. Video Abstract. ; This work was partially supported by research grants FIS (PI15/01934) from the Instituto de Salud Carlos III from Spain, SAF2015-65878-R from Ministry of Economy and Competitiveness, Prometeo/2018/A/133 from Generalitat Valenciana, Spain and also by Fondo Europeo de Desarrollo Regional (FEDER) funds, European Commission (FP7, NeuroPain #2013-602891), the Catalan Government (AGAUR, #SGR2017-669, ICREA Academia Award 2015), the Instituto de Salud Carlos III (RTA, #RD16/0017/0020), and the European Regional Development Fund (No. 01.2.2-LMT-K-718-02-0014). María Arnoriaga-Rodríguez is funded by Instituto de Salud Carlos III, Río Hortega (CP19/00190). Jordi Mayneris-Perxachs is funded by Instituto de Salud Carlos III, Miguel Servet (CP18/00009). The project has also been 65% cofinanced by the European Regional Development Fund (ERDF) through the Interreg V-A Spain-France-Andorra programme (POCTEFA 2014-2020). POCTEFA aims to reinforce the economic and social integration of the French–Spanish–Andorran border. Its support is focused on developing economic, social and environmental cross-border activities through joint strategies favouring sustainable territorial development.

One: Introduction -- 1 The Bacterial Cell -- Two: Gene Expression -- 2 RNA and Protein Production -- 3 Mutation -- Three: Gene Transfer -- 4 Plasmids -- 5 Bacteriophages -- 6 Reactions of DNA -- 7 Investigation of Gene Structure and Function -- Four: Gene Regulation -- 8 Operon Control -- 9 Control of Bacterial Gene Expression -- 10 Control of Extrachromosomal Genetic Elements.

Bacterial genes are typically grouped into operons defined as clusters of adjacent genes encoding for proteins that fill related roles and are transcribed into a single polycistronic mRNA molecule. This simple organization provides an efficient mechanism to coordinate the expression of neighboring genes and is at the basis of gene regulation in bacteria. Here, we report the existence of a higher level of organization in operon structure that we named noncontiguous operon and consists in an operon containing a gene(s) that is transcribed in the opposite direction to the rest of the operon. This transcriptional architecture is exemplified by the genes menE-menC-MW1733-ytkD-MW1731 involved in menaquinone synthesis in the major human pathogen Staphylococcus aureus. We show that menE-menC-ytkD-MW1731 genes are transcribed as a single transcription unit, whereas the MW1733 gene, located between menC and ytkD, is transcribed in the opposite direction. This genomic organization generates overlapping transcripts whose expression is mutually regulated by transcriptional interference and RNase III processing at the overlapping region. In light of our results, the canonical view of operon structure should be revisited by including this operon arrangement in which cotranscription and overlapping transcription are combined to coordinate functionally related gene expression. ; This work was supported by the Spanish Ministry of Economy and Competitiveness Grants BIO2014-53530-R and BIO2017-83035-R (Agencia Española de Investigacion/Fondo Europeo de Desarrollo Regional, European Union). A.T.-A. is supported by the European Research Council under the European Union's Horizon 2020 research and innovation programme Grant Agreement 646869.

Bacterial infections have been on the rise world-wide in recent years and have a considerable impact on human well-being in terms of attributable deaths and disability-adjusted life years. Yet many mechanisms underlying bacterial pathogenesis are still poorly understood. Here, we introduce the BacFITBase database for the systematic characterization of bacterial proteins relevant for host infection aimed to enable the identification of new antibiotic targets. BacFITBase is manually curated and contains more than 90 000 entries with information on the contribution of individual genes to bacterial fitness under in vivo infection conditions in a range of host species. The data were collected from 15 different studies in which transposon mutagenesis was performed, including top-priority pathogens such as Acinetobacter baumannii and Campylobacter jejuni, for both of which increasing antibiotic resistance has been reported. Overall, BacFITBase includes information on 15 pathogenic bacteria and 5 host vertebrates across 10 different tissues. It is freely available at www.tartaglialab.com/bacfitbase. ; This study was supported by the Spanish Ministerio de Ciencia, Innovación y Universidades [SAF2015-72518-EXP, SAF2017-82158-R, RYC-2012-09999]; European Research Council [RIBOMYLOME_309545]; European Union's Horizon 2020 Research and Innovation Programme [727658, IASIS]; Spanish Ministry of Economy and Competitiveness [BFU2014-55054-P, BFU2017-86970-P]; Spanish Ministry of Economy and Competitiveness; 'Centro de Excelencia Severo Ochoa 2013–2017'; CERCA Programme of the Generalitat de Catalunya; European Union's Horizon 2020 Research and Innovation Programme, Marie Sklodowska-Curie Individual Fellowship [793135, 'DeepRNA' to B.L.]; European Society of Clinical Microbiology and Infectious Diseases, Research Grant 2016 (ESCMID). Funding for open access charge: Spanish Ministerio de Ciencia, Innovación y Universidades [SAF2017-82158-R]

International audience ; The rise of antibiotic resistance is not only a challenge for human and animal health treatments, but is also posing the risk of spreading among bacterial populations in foodstuffs. Farmed fish-related foodstuffs, the food of animal origin most consumed worldwide, are suspected to be a reservoir of antibiotic resistance genes and resistant bacterial hazards. However, scant research has been devoted to the possible sources of diversity in fresh fillet bacterial ecosystems (farm environment including rivers and practices, and factory environment). In this study bacterial communities and the antibiotic resistance genes of fresh rainbow trout fillet were described using amplicon sequencing of the V3-V4 region of the 16S rRNA gene and high-throughput qPCR assay. The antibiotic residues were quantified using liquid chromatography/mass spectrometry methods. A total of 56 fillets (composed of muscle and skin tissue) from fish raised on two farms on the same river were collected and processed under either factory or laboratory sterile filleting conditions. We observed a core-bacterial community profile on the fresh rainbow trout fillets, but the processing conditions of the fillets has a great influence on their mean bacterial load (3.38 ± 1.01 log CFU/g vs 2.29 ± 0.72 log CFU/g) and on the inter-individual diversity of the bacterial community. The bacterial communities were dominated by Gamma- and Alpha-proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. The most prevalent genera were Pseudomonas, Escherichia-Shigella, Chryseobacterium, and Carnobacterium. Of the 73 antibiotic residues searched, only oxytetracycline residues were detected in 13/56 fillets, all below the European Union maximum residue limit (6.40-40.20 μg/kg). Of the 248 antibiotic resistance genes searched, 11 were found to be present in at least 20% of the fish population (tetracycline resistance genes tetM and tetV, β-lactam resistance genes bla (DHA) and bla (ACC), macrolide resistance gene mphA, vancomycin resistance genes vanTG and vanWG and multidrug-resistance genes mdtE, mexF, vgaB and msrA) at relatively low abundances calculated proportionally to the 16S rRNA gene.

International audience ; The rise of antibiotic resistance is not only a challenge for human and animal health treatments, but is also posing the risk of spreading among bacterial populations in foodstuffs. Farmed fish-related foodstuffs, the food of animal origin most consumed worldwide, are suspected to be a reservoir of antibiotic resistance genes and resistant bacterial hazards. However, scant research has been devoted to the possible sources of diversity in fresh fillet bacterial ecosystems (farm environment including rivers and practices, and factory environment). In this study bacterial communities and the antibiotic resistance genes of fresh rainbow trout fillet were described using amplicon sequencing of the V3-V4 region of the 16S rRNA gene and high-throughput qPCR assay. The antibiotic residues were quantified using liquid chromatography/mass spectrometry methods. A total of 56 fillets (composed of muscle and skin tissue) from fish raised on two farms on the same river were collected and processed under either factory or laboratory sterile filleting conditions. We observed a core-bacterial community profile on the fresh rainbow trout fillets, but the processing conditions of the fillets has a great influence on their mean bacterial load (3.38 ± 1.01 log CFU/g vs 2.29 ± 0.72 log CFU/g) and on the inter-individual diversity of the bacterial community. The bacterial communities were dominated by Gamma- and Alpha-proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. The most prevalent genera were Pseudomonas, Escherichia-Shigella, Chryseobacterium, and Carnobacterium. Of the 73 antibiotic residues searched, only oxytetracycline residues were detected in 13/56 fillets, all below the European Union maximum residue limit (6.40-40.20 μg/kg). Of the 248 antibiotic resistance genes searched, 11 were found to be present in at least 20% of the fish population (tetracycline resistance genes tetM and tetV, β-lactam resistance genes bla (DHA) and bla (ACC), macrolide resistance gene mphA, vancomycin ...

PMCID: PMC4482672 ; [Background]: Precise temporal and spatial expression of the clustered Hox genes is essential for patterning the developing embryo. Temporal activation of Hox genes was shown to be cluster-autonomous. However, gene clustering appears dispensable for spatial colinear expression. Generally, a set of Hox genes expressed in a group of cells instructs these cells about their fate such that the differential expression of Hox genes results in morphological diversity. The spatial colinearity is considered to rely both on local and long-range cis regulation. [Results]: Here, we report on the global deregulation of HoxA and HoxD expression patterns upon inactivation of a subset of HOXA and HOXD proteins. [Conclusions]: Our data suggest the existence of a >self-regulation> mechanism, a process by which HOX proteins establish and/or maintain the spatial domains of the Hox gene family and we propose that the functionally dominant HOX proteins could contribute to generating the spatial parameters of Hox expression in a given tissue, i.e., HOX controlling the establishment of the ultimate HOX code. ; Grant sponsor: the Spanish Government; Grant number: BFU2011-24972; Grant sponsor: the Canadian Institutes for Health Research; Grant number: MOP-82880; Grant number: 126110. This work was supported by the Spanish Government to M.R. and by the Canadian Institutes for Health Research as well as the Canada Research Chair program to M.K. R.S was supported by a Formación Profesorado Universitario fellowship from the Spanish Ministry of Science and Innovation and currently supported by the Angelo Pizzagalli postdoctoral fellowship. ; Peer Reviewed

The rise of antibiotic resistance is not only a challenge for human and animal health treatments, but is also posing the risk of spreading among bacterial populations in foodstuffs. Farmed fish-related foodstuffs, the food of animal origin most consumed worldwide, are suspected to be a reservoir of antibiotic resistance genes and resistant bacterial hazards. However, scant research has been devoted to the possible sources of diversity in fresh fillet bacterial ecosystems (farm environment including rivers and practices, and factory environment). In this study bacterial communities and the antibiotic resistance genes of fresh rainbow trout fillet were described using amplicon sequencing of the V3-V4 region of the 16S rRNA gene and high-throughput qPCR assay. The antibiotic residues were quantified using liquid chromatography/mass spectrometry methods. A total of 56 fillets (composed of muscle and skin tissue) from fish raised on two farms on the same river were collected and processed under either factory or laboratory sterile filleting conditions. We observed a core-bacterial community profile on the fresh rainbow trout fillets, but the processing conditions of the fillets has a great influence on their mean bacterial load (3.38 ± 1.01 log CFU/g vs 2.29 ± 0.72 log CFU/g) and on the inter-individual diversity of the bacterial community. The bacterial communities were dominated by Gamma- and Alpha-proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. The most prevalent genera were Pseudomonas, Escherichia-Shigella, Chryseobacterium, and Carnobacterium. Of the 73 antibiotic residues searched, only oxytetracycline residues were detected in 13/56 fillets, all below the European Union maximum residue limit (6.40–40.20 μg/kg). Of the 248 antibiotic resistance genes searched, 11 were found to be present in at least 20% of the fish population (tetracycline resistance genes tetM and tetV, β-lactam resistance genes bla(DHA) and bla(ACC), macrolide resistance gene mphA, vancomycin resistance genes vanTG and vanWG ...

Ever since World War II, antibiotics have been medicine's number one asset in fighting microbial infection, one of the leading causes of death worldwide. Misuse of antibiotics has, however, led to rapid spread of antibiotic resistance among bacteria and ensuing development of multiple resistant pathogens. Therefore, antibiotics are rapidly losing their antimicrobial value, which can be seen a failure of society to protect one of its valuable resources. The use of antibiotics in food production animals is strictly controlled by the European Union. Veterinary use is regulated to prevent spreading of resistance due to unwarranted use and to prevent antibiotic residues in food products. EU legislation establishes maximum residue limits (MRLs) of veterinary medicinal products in foodstuffs of animal origin, and enforces countries to establish and execute a national monitoring plan of animal products to implement food control measures. Among samples selected for monitoring, suspect noncompliant samples are screened for and then subjected to confirmatory analysis to establish the identity and concentration of the contaminant. Screening methods for antibiotic residues are typically based on microbiological growth inhibition, whereas physico-chemical methods are used for confirmatory analysis. In this study, antibiotic whole-cell biosensor assays were examined as a novel screening method. Utilizing a tetracycline-specific bioluminescent whole-cell biosensor, a screening method for tetracycline residues in poultry meat was developed. Assay sensitization to meet the EU MRLs was achieved by improving tetracycline accumulation into the biosensor cells with a combination of membrane-permeabilizing agent polymyxin B and chelating agent EDTA. The result was a rapid, simple and cost-effective high-throughput screening method that could detect all four veterinary relevant tetracyclines and their 4-epimer metabolites in poultry meat with sensitivity below the MRLs. The study also provided proof of antimicrobial activity of tetracycline 4-epimer metabolites, a quality previously thought absent from 4-epidoxycycline. Nisin is a lantibiotic, a peptide antibiotic produced by lactococci. The industrial use of nisin as a food preservative (E234) and maximum allowed levels set by the EU warrant developing methods for nisin quantification in foods. In this study, a bioluminescent whole-cell biosensor for nisin was constructed and utilized in determining nisin concentrations in milk. The developed assay was rapid and simple to perform, and required no sample pretreatment except dilution. Sensitivity of the assay was in the sub-picogram per ml level, exceeding the performance of all previously published methods. The assay was also used in determining nisin-production efficiency by quantifying nisin in growth medium of a nisin-producing Lactococcus strain. Simultaneously, nisin producers could be distinguished from non-producers. This idea was expanded in a follow-up study, which utilized the nisin biosensor in screening for nisin producers in raw milk. Screening was based on simple overlay of raw milk cultures and identification of nisin producers by a bioluminescent zone surrounding the nisinogenic colony. The seven identified nisinogenic colonies were divided in three groups by genetic fingerprinting,and characterized as nisin variant Z producing Lactococcus lactis subsp. lactis. In addition, four nisin A producers were identified in a panel of 91 dairy lactococcal strains. Specificity studies showed that only nisin and not other bacteriocin peptides induced bioluminescence in the sensor strain. Also, all nisin-gene harboring colonies induced bioluminescence, with the exception of one lactococcal strain shown to carry a nonfunctional nisin gene. The development of novel inducible whole-cell biosensors for different groups of antimicrobials can be limited by the lack of regulatory elements specifically responsive for these substances. In this study, we characterized DNA and ligand binding of the macrolide antibiotic-responsive repressor protein, MphR(E). The protein was modified by rational design of mutations to improve DNA affinity and dimerization. DNA and ligand binding as well as macrolide-induced dissociation from DNA were studied by fluorescence anisotropy and mass spectrometry. Mutants with improved DNA affinity and retained ligand binding and dissociation characteristics were identified. One mutant surprisingly formed a covalent dimer through disulfide bridge formation. This was shown to improve DNA affinity, but ligand binding and induction was impaired. Ligand binding spectrum of MphR(E) was shown to cover macrolides with a 14-membered lactone ring structure, but macrolides with a 16-membered ring or lincosamides showed no binding. MphR(E) and its mutants showed interesting novel characteristics that could benefit biosensor design. In conclusion, this study shows the applicability of whole-cell biosensors in developing simple, robust and cost-effective screening methods for antimicrobials in food products. These methods show high sensitivity and specificity towards the target analyte, and can be used in semi-quantitative to quantative analysis. In addition to residue monitoring, whole-cell biosensors can be used for producer identification. The identified nisin producers can find use as protective starter cultures in fermented food production. The modified repressor MphR(E) shows promise as an improved regulator of reporter gene production in whole-cell biosensor applications, and is an example of purposeful effort to develop regulatory elements for novel biosensor designs. ; Antibiootit ovat olleet tärkein bakteeri-infektioiden hoitokeino aina toisesta maailmansodasta lähtien. Bakteeri-infektiot ovat maailmanlaajuisten kuolemansyytilastojen kärkisijoilla. Antibioottien huolimaton käyttö on kuitenkin johtanut antibioottiresistenssin nopeaan leviämiseen bakteerien keskuudessa sekä useille antibiooteille resistenttien patogeenien kehittymiseen. Tämän vuoksi antibiootit menettävät nopeasti antimikrobiaalista voimakkuuttaan, mitä voidaan pitää yhteiskunnan kyvyttömyytenä suojella arvokasta pääomaansa. Euroopan Unioni valvoo antibioottien käyttöä eläimissä, joita hyödynnetään elintarvikkeiden tuotannossa. Eläinlääketieteellistä käyttöä säädellään resistenssin leviämisen ja ruoassa esiintyvien antibioottijäämien estämiseksi. EU:n lainsäädäntö osoittaa enimmäisjäämärajat eläinlääkinnällisille aineille eläinperäisissä elintarvikkeissa ja velvoittaa jäsenvaltiot laatimaan ja toteuttamaan eläimistä saatavien elintarvikkeiden kansallisen vierasainevalvontaohjelman. Valvontaan valittujen näytteiden joukosta seulotaan näytteet, joiden epäillään sisältävän jäämiä, joiden luonne ja pitoisuus varmistetaan lisäanalyysillä. Seulonnassa käytetään tavallisesti mikrobiologiseen kasvuinhibitioon perustuvia menetelmiä, ja varmistukseen fysikaalis-kemiallisia analyysejä. Tässä tutkimuksessa tarkasteltiin kokosolubioantureilla tehtävien antibioottimääritysten soveltuvuutta antibioottijäämien seulontamenetelmäksi. Tetrasykliinispesifistä bioluminoivaa kokosolubiosensoria käyttäen kehitettiin seulontamenetelmä tetrasykliinijäämien tunnistamiseksi kananlihasta. Määritys saatiin herkistettyä EU:n enimmäisjäämärajojen tasolle helpottamalla tetrasykliinien pääsyä bioanturisoluun solukalvon läpäisykykyä lisäävällä polymyksiini B:llä sekä kahdenarvoisia kationeja kelatoivalla EDTA:lla. Tuloksena oli nopea, yksinkertainen ja kustannustehokas seulontamenetelmä, jolla oli korkea suoritusteho. Menetelmä kykeni havaitsemaan kaikki neljä eläinlääketieteessä käytettävää tetrasykliiniä sekä niiden 4-epimeeri aineenvaihduntatuotteet kananlihassa enimmäisjäämärajat alittavissa pitoisuuksissa. Tutkimus myös tuotti todisteita tetrasykliinien 4-epimeerien antimikrobiaalisesta aktiivisuudesta, joka aiemmin arveltiin puuttuvan 4-epidoksisykliiniltä. Nisiini on laktokokkien tuottama lantibiootti eli peptidiantibiootti. Nisiinin käyttö elintarviketeollisuudessa säilöntäaineena (E234) sekä EU:n nisiinille asettama sallittu enimmäismäärä elintarvikkeissa luovat tarpeen nisiinin määritysmenetelmille. Tässä tutkimuksessa rakennettiin bioluminoiva nisiinispesifinen kokosolubioanturi, jota käytettiin määrittämään nisiinipitoisuuksia maidossa. Kehitetty määritys oli nopea ja yksinkertainen, eikä vaatinut laimennusta monimutkaisempaa näytteen esikäsittelyä. Määrityksen herkkyys oli alle pg/ml mittaluokassa, ja se oli herkin koskaan julkaistu nisiinimääritys. Määritystä käytettiin myös nisiinintuotannon tehokkuuden arvioinnissa mittaamalla nisiinipitoisuus sitä tuottavan Lactococcus lactis -kannan kasvumediumista. Samanaikaisesti nisiinintuottaja voitiin erottaa nisiiniä tuottamattomista kannoista. Tätä ajatusta tarkasteltiin laajemmin jatkotutkimuksessa,jossa nisiinibioanturia käytettiin seulomaan nisiinintuottajakantoja raakamaidosta. Yksinkertainen seulontamenetelmä perustui raakamaitobakteerien viljelmien peittämiseen ohuella bioanturikerroksella, jolloin nisiinintuottajapesäkkeiden ympärille muodostui biolumine-senssivyöhyke. Seitsemän tunnistettua nisiinintuottajapesäkettä jakautuivat geneettisen sormen-jäljen perusteella kolmeen ryhmään, ja ne olivat kaikki Lactococcus lactis subsp. lactis -alalajiin kuuluvia nisiini Z -variantin tuottajia. Lisäksi 91 laktokokkikannan paneelista tunnistettin kolme nisiini A -variantin tuottajaa. Spesifisyystutkimukset osoittivat, että vain nisiini indusoi bioluminesenssin bioanturibakteerissa eivätkä muut bakteriosiinipeptidit. Lisäksi kaikki nisiinigeeniä kantavat pesäkkeet indusoivat bioluminesenssin. Poikkeuksena oli yksi laktokokkikanta, jonka todettiin kantavan toimimatonta nisiinigeeniä. Tietyille antibioottiryhmille spesifisen vasteen antavien säätelyelementtien puute saattaa vaikeuttaa kokosolubioanturien kehittämistä. Tässä tutkimuksessa karakterisoitiin makrolidispesifisen repressoriproteiini MphR(E):n DNA- ja ligandinsitomisominaisuuksia. Proteiinia muokattiin rationaalisen mutaatiosuunnittelun keinoin tarkoituksena tuottaa DNA- ja dimerisoitumisominaisuuksiltaan parempia mutantteja. DNA- ja ligandisitoutumista sekä makrolidiligandien indusoimaa dissosiaatiota DNA:sta tutkittiin fluoresenssianisotropialla ja massaspektrometrialla. Tutkimuksessa löydettiin mutantteja, joilla oli villityypin proteiinia parempi DNA-affiniteetti sekä ennallaan säilynyt kyky sitoa ligandeja ja indusoitua sitomisen vaikutuksesta. Yksi mutanteista muodosti rikkisillan avulla kovalenttisen dimeerin vastoin odotuksia. Kovalentti dimerisaatio paransi DNA-affiniteettia, mutta haittasi ligandin sitomista sekä induktiota. MphR(E):n ligandikirjo kattoi 14-jäsenisen laktonirenkaan makrolidit, mutta ei 16-jäsenisen renkaan makrolideja eikä linkosamideja. MphR(E) ja sen mutantit osoittivat mielenkiintoisia uusia ominaisuuksia, jotka voivat hyödyttää bioantureiden suunnittelua. Johtopäätöksenä voidaan sanoa, että tämä tutkimus osoittaa kokosolubioanturien soveltuvan yksinkertaisten, luotettavien ja kustannustehokkaiden seulontamenetelmien kehittämiseen antibioottijäämien osoittamiseen elintarvikkeista. Nämä menetelmät osoittavat suurta herkkyyttä ja spesifisyyttä analyyttimolekyyliä kohtaan, ja niitä voidaan käyttää semikvantitatiiviseen sekä kvantitatiiviseen analysiin. Jäämien havainnoinnin lisäksi bioantureita voidaan käyttää antimikrobiaalisten aineiden tuottajien tunnistamiseen. MphR(E)-mutantit ovat lupaavia paranneltua säätelykykyä osoittavia reportterigeenin tuoton repressoreita käytettäväksi bioanturisovelluksissa. Ne ovat myös esimerkki rationaalisesta säätelyelementtien parantelusta uusien bioanturisovellusten kehittämiseksi.