Azithromycin, a macrolide antibiotic, specifically binds to the 50S ribosomal subunit of bacterial ribosomes and inhibits translation. Azithromycin also prevents 50S ribosomal subunit assembly by binding to a 50S ribosomal subunit precursor particle. When exposed to azithromycin, several ribonucleases in wild-type Escherichia coli cells degrade antibiotic-bound 50S precursor particles. Presumably, cells expressing one or more mutated ribonucleases will degrade the antibiotic-bound precursor less efficiently, resulting in increased sensitivity to the antibiotic. To test this, eight ribonucleaseûdeficient strains of Escherichia coli were grown in the presence or absence of azithromycin. Cell viability, growth rates, and protein synthesis rates were measured. Degradation of 23S rRNA was examined by hybridization with a 23S specific probe. Ribonuclease II and polynucleotide phosphorylase mutants demonstrated hypersensitivity to the antibiotic and showed a greater extent of 23S rRNA accumulation, suggesting that these two ribonucleases are important for 23S rRNA turnover in azithromycin-treated Escherichia coli.

The 16S, 23S, and 5S ribosomal RNA (rRNA) genes are highly conserved sequences in bacteria. For this reason, rRNA genes are often used for phylogenetic classification. On the other hand, the regions between the structural sequences, known as intergenic spacer regions (ITS), are under less evolutionary pressure to be conserved. Because they are not as highly conserved, they can be used to differentiate strains of the same bacterial specie. The purpose of this study was to evaluate the 16S-23S ITS of Flavobacterium columnare, an important pathogen of cultured fish, by comparing the 16S-23S ITS sequences from 70 isolates. We developed two PCR assays that amplify overlapping regions of one large previously identified ITS. The primers targeted the 16S sequence and isoleucine tRNA encoding sequences and the 23S sequence and alanine tRNA encoding sequences. The PCR products were cloned and sequenced. We also targeted I-CeuI restriction fragments from the ATCC type strain that were separated by pulse field gel electrophoresis and analyzed the 16S-23S ITS regions. We found that the genome of this species harbors at least 6 intergenic spacer regions that are very similar and contain the same tRNA encoding sequences. This suggests that earlier studies that used the ITS for distinguishing between strains of Flavobacterium columnare may be comparing sequences from different structural RNA operons and thus have misleading data.

Molts bacteris del grup fluorescent del gènere Pseudomonas són capaços de controlar malalties de les plantes causades per fongs i bacteris fitopatògens (ACBs) o mostren activitat com a bacteris promotors del creixement de les plantes (BPCPs). S'han descrit diversos metabòlits que intervenen de manera important en la seva activitat com a ACBs i BPCPs entre els quals en destaquen el 2,4-diacetilfloroglucinol (Phl), àcid fenazin-1-carboxílic (PCA), Pirrolnitrina (Prn), àcid cianhídric (HCN), àcid 3-indolacètic (IAA), sideròfors i quitinases.L'objectiu principal del nostre treball ha estat la comparació de les característiques d'un grup de Pseudomonas del grup fluorescent utilitzant una aproximació polifàsica amb la finalitat d'establir possibles relacions entre algunes de les característiques i la capacitat d'actuar com a ACB o BPCP.Atesa la importància en el biocontrol de la producció de metabòlits com Phl, PCA i Prn, l'objectiu preliminar ha estat la recerca i obtenció de soques productores d'aquests metabòlits. Per assolir aquest objectiu s'ha emprat una aproximació molecular basada en la detecció dels gens biosintètics implicats en la seva producció en lloc de la detecció directa dels metabòlits per evitar els efectes que poden tenir les condicions de cultiu en la inducció o repressió de la seva síntesi. S'han realitzat diferents protocols basats (i) en la cerca assistida de productors mitjançant l'ús de marcadors fenotípics i posterior confirmació per PCR i, (ii) en l'ús de la PCR per a la detecció dels gens directament dels extractes bacterians, d'enriquiments d'aquests extractes i la realització de la hibridació en colònies per al posterior aïllament. La cerca assistida de productors de Phl mitjançant marcadors fenotípics i posteriorment la utilització de tècniques moleculars (amplificació per PCR del gen phlD), ha estat el millor mètode en el tipus de mostres processades en el nostre treball, on la proporció de productors és relativament baixa. En total s'han aïllat a partir de diversos ambients 4 soques portadores dels gens de la síntesi de PCA, 15 de Phl i 1 de Prn.S'ha constituït una col·lecció de 72 soques de Pseudomonas del grup fluorescent que inclou 18 aïllats propis portadors dels gens biosintètics necessaris per la producció de Phl PCA i Prn; 6 soques de referència procedents de col·leccions de cultius tipus, 14 soques productores dels diferents antibiòtics cedides per altres investigadors i una selecció de 34 soques procedents d'un treball previ realitzat en el nostre grup de recerca. A la col·lecció s'hi troben soques candidates a ACB i BPCP de diverses malalties i plantes.Les 72 soques s'han caracteritzat fenotípica i genotípicament. La caracterització fenotípica s'ha portat a terme mitjançant la identificació a nivell d'espècie amb galeries API 20NE i proves bioquímiques específiques; la producció de metabòlits com PCA, Phl, Prn, IAA, HCN, quitinases i sideròfors mitjançant l'ús de diferents tècniques; antagonisme in vitro en diversos medis enfront dos fongs (Stemphylium vesicarium i Penicillium expansum) i tres bacteris fitopatògens (Erwinia amylovora, Pseudomonas syringae pv. syringae i Xanthomonas arboricola pv. juglandis); l'eficàcia de la inhibició de la infecció en bioassaigs in vivo sobre material vegetal enfront els fongs P. expansum en poma i S. vesicarium en fulles de perera i enfront el bacteri E. amylovora en fruits immadurs de perera i, finalment, en assaigs de promoció de creixement en dos portaempelts comercials de Prunus. Cal destacar que P. expansum causa la podridura blava en pomes i peres en postcollita, S. vesicarium la taca bruna de la perera i E. amylovora el foc bacterià de les rosàcies.El nombre de soques de Pseudomonas, sobre el total de les 72 estudiades, productores d'IAA (4) i quitinases (6) és baix, mentre que és elevat en el cas del HCN (32), que a més està associat a la producció de Phl. Els resultats obtinguts en l'antagonisme in vitro han mostrat en el cas dels bacteris que és dependent del patogen indicador i del medi de cultiu. La presència o absència de ferro no sembla ser un factor que potencií l'antagonisme. En el cas dels fongs no s'ha observat però, influència del medi de cultiu emprat. En el total de 72 soques s'ha observat un percentatge baix de soques que manifesten antagonisme en tots els medis assajats vers 3 o 4 dels patògens (7). Solament 2 d'aquestes 7 soques han mostrat ser també efectives en bioassaigs d'inhibició de les infeccions causades per 2 dels 3 patògens assajats. Algunes de les soques efectives en els bioassaigs no són antagonistes in vitro en cap dels medis assajats enfront el mateix patogen. En el cas de la promoció del creixement, s'han observat més soques promotores del creixement del portaempelts de prunera Marianna 2624 que no en l'híbrid de presseguer-ametller GF677 i les eficàcies assolides són també majors en el cas de Marianna 2624, detectant una elevada especificitat soca/portaempeltsLa caracterització genotípica s'ha realitzat mitjançant l'anàlisi dels polimorfismes en la longitud dels fragments de restricció de DNA ribosomal (RFLP-rDNA) i l'anàlisi dels polimorfismes en la longitud dels fragments de macrorestricció genòmica de DNA cromosòmic separats per electroforesi en camp polsant (MRFLP-PFGE). Ambdues anàlisis van mostrar una gran heterogeneïtat genètica entre les soques caracteritzades i no s'ha pogut relacionar les agrupacions obtingudes amb les característiques fenotípiques o capacitat d'actuar com a ACB o BPCP. Els patrons de macrorestricció genòmica (MRFLP-PFGE) del bacteri model P. fluorescens EPS288 són estables en el temps i independents de les condicions de cultiu assajades al laboratori o en mostres naturals, mostrant ser una tècnica eficaç en la identificació de reaïllats de mostres naturals inoculades prèviament amb el bacteri.Una selecció de soques que comparteixen el fet de produir floroglucinol s'han caracteritzat mitjançant RFLP i seqüenciació del gen phlD. S'ha establert una relació entre les agrupacions obtingudes en les anàlisis RFLP-rDNA, RFLP-phlD i les seqüències del gen. En l'anàlisi filogenètica de les seqüències del gen phlD s'ha observat un elevat grau de polimorfisme obtenint-se 3 agrupacions principals. Les agrupacions semblen relacionar-se amb els patrons de producció de metabòlits (Phl, HCN i Prn en una primera agrupació; Phl i HCN en la segona i solament Phl en la tercera), però aquestes no s'han pogut relacionar amb l'origen geogràfic de les soques o la seva activitat com a ACBs i/o BPCP.Amb les dades obtingudes de la caracterització fenotípica i genotípica s'ha realitzat una anàlisi multivariant (correspondències, correlacions d'Spearman i de freqüències amb variables categòriques). S'ha demostrat la importància de disposar d'una tècnica que permeti depurar una col·lecció de soques descartant les soques genèticament idèntiques, ja que influeixen en els resultats de les anàlisis. Pels tres patògens assajats com a indicadors i els dos portaempelts emprats, no s'ha observat cap correlació entre la inhibició de la infecció o la promoció del creixement amb les característiques fenotípiques i genotípiques de les soques que fos significatiu i consistent en les tres tècniques emprades. / Many bacteria pertaining to the fluorescent Pseudomonas group are able to control plant diseases caused by bacterial and fungal plant pathogens (BCAs). Also, some of them exhibit activity as plant growth promoting bacteria (PGPBs). Several metabolites like 2,4-diacetylphloroglucinol (PHL), phenazin-1-carboxilic acid (PCA), pyrrolnitrin (Prn), hydrogen cyanide (HCN), indol-3-acetic acid, siderophores and chitinases has been described accounting for their ability of being BCAs or PBPBs.The aim of the present work was to compare the phenotypic and genotypic characteristics of a selected group of fluorescent Pseudomonas by means of a polyphasic approach in order to establish relationships with the ability of being BCA and PGPB.Due to the importance of production of metabolites like Phl, PCA and Prn in biocontrol, the preliminary objective of this work was to search and isolate of metabolite producing strains. To achieve this objective a molecular approach was used based on the detection of biosynthetic genes, which are implicated in the metabolite production, instead of direct detection of the metabolites. The procedure was performed to avoid the effect of culture conditions on induction or repression of metabolite synthesis. Two procedures were used (i) assisted search of metabolite producing strains by means of phenotypic markers and subsequent confirmation by PCR analysis, (2) direct use of PCR for gene detection in direct extracts or enrichments from samples and subsequent colony-hybridization for assistance in strain isolation. The best method for isolation of Phl producers in the type of samples processed in the present work, where the frequency of Phl producers is very low, was the assisted search by means of phenotypic markers followed of a confirmation by PCR amplification of phlD gene. Four strains harboring the PCA genes, 15 strains with Phl genes, and one with Prn genes were isolated.A collection of 72 strains of Pseudomonas pertaining to the fluorescent group was made including 18 isolates derived from the present work which harbor the biosynthetic genes for PhL, PCA and Prn production; 6 strains from reference collections; 14 strains producing several antibiotics which were supplied by other colleagues; and a selection of 34 strains from a previous work performed by our research group. Within the collection there are many strains with promising potential as BCAs and PGPBs of several diseases and plant hosts.The collection was characterized phenotypically and genotypically. The phenotypic characterization included identification at the species level with the aid of API20NE kit and several specific biochemical tests; the production of metabolites such as PCA, Phl, Prn, IAA, HCN, chitinases and siderophores; in vitro antagonism in several culture media against two fungi (Stemphylium vesicarium and Penicillium expansum) and three bacterial pathogens (Erwinia amylovora, Pseudomonas syringae pv. syringae and Xanthomonas arboricola pv. juglandis); efficacy in biossays of inhibition of infection of plant material by P. expansum on apple fruit tissue and S. vesicarium on pear leaves, E. amylovora on immature pear fruit, and of growth promotion in two commercial Prunus rootstocks. P. expansum causes blue mold rot of apple and pear in postharvest, S. vesicarium cause brown spot of pear and E. amylovora is the causal agent of fire blight of rosaceous plants.The number of strains, over a total of 72 studied, producing IAA and chitinases was very low (4 producing IAA and 6 producing chitinases), whereas a high number of HCN producing strains (32) was detected which was associated to Phl production. In vitro antagonism against bacteria was highly dependent on indicator pathogen and growth medium, but the presence or absence of iron did not seem to affect. However, in the case of antagonism against fungi, no influence of the medium composition was observed. Among the collection a low frequency of strains exhibiting antagonism against 3 or 4 pathogens a total of 7 strains was observed in all tested media. Only two out of the seven strains were effective in infection inhibition bioassays caused by two of the three pathogens tested. Some of the effective strains in the in vivo assays were not antagonist for the indicator pathogen in any of the media tested. In the case of plant growth promoting strains, the growth in rootstock Marianna 2624 was more stimulated than in GF677, and there was a strain-host specificity. Genotypic characterization of strains was performed by means of restriction fragment length polymorphism analysis of the ribosomal DNA (RFLP-rDNA) and macrorestriction fragment length polymorphism analysis of chromosomal DNA separated by pulsed field gel electrophoresis (MRFLP-PFGE). Both techniques showed a high level of genotypic diversity within the collection of strains, and no relationships were observed between clusters and phenotypic characteristics or ability to be BCA or PGPB. Patterns of MRFLP-PFGE for the model bacterium P. fluorescens EPS288 were found stable within time and independent of cultivation conditions in the laboratory or under natural conditions. Therefore the method is highly suitable for identification of strains reisolated from natural samples previously inoculated with the bacterium.A further selection of strains which produce phloroglucinol were characterized by RFLP analysis and phlD sequencing. The groups observed were consistent among the RFLP-rDNA, RFLP-phlD and gene sequence data. Phylogenetic analysis obtained using phlD sequences showed a high level of polymorphism revealing three main clusters. These clusters appear to be related with metabolite production: a first cluster producing Phl, HCN and Prn, a second producing Phl and HCN, and a third producing only Phl. However, this groups were related neither to the geographic origin of strains nor to the activity as BCA or PGPB.A multivariate analysis (correspondence, pairwise Spearman rank correlation, and frequency analysis)was performed using data of phenotypic and genotypic characterization of strains. The results emphasize the importance of discarding from the database those strains being genetically identical because skew the final results. The three types of analysis did not revealed a significant and consistent relationship between the activity of infection inhibition or growth promotion of the strains and their characteristics.

Abstract The aim of this work was to assess and improve the enrichment cultivation of food-contaminating bacteria prior to detection by means of RNA-based sandwich hybridization assay (SHA). The examples of beer-spoiling lactic acid bacteria (LAB) and food-borne Salmonella Typhimurium were selected based on their relevance in Finnish food industry. Also universal challenges affecting on the selection of the enrichment cultivation procedure are discussed, including some potential possibilities for improved enrichment cultivation. The results of this study may therefore be used for the assessment of the efficiency of bacterial cultivation in other applications. The evaluation of the enrichment cultivation procedures prior to SHA lead to following conclusions: i) the enrichment cultivation procedure is necessary prior to rRNA-based SHA, and it directly influences the accuracy of SHA; ii) the improvement of the enrichment cultivation may allow faster recovery and growth of bacteria; iii) the improved recovery of bacteria can be achieved by reducing environmental stress factors in the enrichment culture; and iv) the growth of bacteria may be accelerated by assuring the selectivity of medium and allowing accessibility to growth factors. Several growth factors were studied by means of full factorial design and response surface modeling. Measured cell densities, as well as predicted lag-times and maximum growth rates in the bacterial cultures were used as responses. The results show that small shifts in the cultivation conditions extend the lag-time and decrease the growth rate of both LAB and Salmonella. Besides adjusting the temperature and pH, the growth of LAB was facilitated by reducing osmotic and oxidative stresses in the enrichment medium. In this study, a novel enzyme controlled glucose delivery system was used for the first time in the enrichment cultivation of food-contaminating bacteria. The glucose delivery system improved the growth of LAB in single strain cultures and in actual brewing process samples. The recovery of injured Salmonella was also enhanced by using the glucose delivery system together with selective siderophore ferrioxamine E, both in terms of reduced lag-times and increased growth rates. Based on the SHA, the adjusted BPW broth enhanced the molecular detection of heat-injured Salmonella in meat.

Flera olika arter av mjölksyrabakterier som ingår i släktena Lactobacillus och Bifidobacterium har hittats hos bin och i deras honung. Idag finns ingen effektiv metod för identifiering av bakterierna. Syftet med detta projekt är att utveckla en metod för snabb identifiering genom att hitta lämpliga primers till olika mjölksyrabakterier och därmed få fram en Polymeraskedjereaktion (PCR) metod. Ribosomal ribonukleinsyra (rRNA) generna eller 16S-23S rRNA intergenic spacer region (ISR) används ofta vid design av primers, som därefter används i PCR för att identifiera olika bakterier. Deoxiribonukleinsyra (DNA) visualiseras i agarosgelen med hjälp av SYBRgreen I som fluorescens på ultraviolett (UV)-ljusbord. I detta projekt har 16S rRNA och 16S-23S rRNA ISR amplifierats i enkel PCR och multiplex PCR och visualiserats i agarosgel i försök att identifiera mjölksyrabakterierna. 16S rRNA har visat sig ha mycket liten variation mellan bakterierna och ansågs därför inte lämplig att använda för identifiering av närbesläktade arter. 16S-23S rRNA ISR visade större variation, fram för allt mellan lactobacillerna och bifidobakterierna. Gruppering av bakterierna med hjälp av multiplex PCR gjordes med viss framgång, med undantag av några bakterier som inte hamnade i den förväntade gruppen. Dock behövs fler försök för att stödja dessa resultat. / Several different lactic acid bacterium (LAB) species from the genera Lactobacillus and Bifidobacterium was discovered in bees and in their honey. Today there is no rapid and reliable method to identify these LAB. Therefore a rapid polymerase chain reaction (PCR) method to identify the LAB is needed. The aim of this project is to find primers suitable for the different LAB. Ribosomal ribonucleic acid (rRNA) genes or 16S-23S rRNA intergenic spacer region (ISR) are often used to designing of primers followed by PCR assays, for identification of different bacteria. To visualize deoxyribonucleic acid (DNA) in agarose gels, SYBRgreen I was used as fluorescence and then viewed under ultraviolet (UV) light. In this project the 16S rRNA and 16S-23S rRNA ISR was used as a target in a PCR and a multiplex PCR amplification. The PCR product was analyzed in agarose gel in an attempt to identify the LAB. 16S rRNA sequence have to little variation and is not suitable to identify closely related species. 16S-23S rRNA ISR sequence exhibits greater variations, especially between Lactobacillus and Bifidobacterium. Differentiation of the bacteria into groups by multiplex PCR was done with good result, except for some of the bacteria that did not end up in the expected group. More studys is needed to support these results.

Hansson, L. Detektion av makrolidresistens hos Mycoplasma genitalium med Panther Fusion. Examensarbete i biomedicinsk laboratorievetenskal 15 högskolepoäng. Malmö universitet: Fakulteten för hälsa och samhälle, institutionen för Biomedicinsk Vetenskap, 2023.   Mycoplasma genitalium är en sexuellt överförbar mikroorganism som infekterar både män och kvinnor, som behandlas oftast med azitromycin med ett ökande problem av antibiotikaresistens. För M. genitalium är makrolidresistens det främsta hotet mot behandling, och har kopplats till fyra punktmutationer i region V i 23S rRNA-genen: A2071G, A2072G, A2072C samt A2071T (M. genitalium G-37, GenBank NR_077054.1). Projektet har undersökt möjligheten att ersätta nuvarande in house realtids-PCR metod för makrolidresistensbestämning med ett integrerat nukleinsyra-reningssteg och realtids-PCR med Panther Fusion (Hologic) hos Klinisk mikrobiologi i Lund. Under projektet analyserades 55 patientprover som samlades under perioden januari-februari 2023 i Region Skåne, som blivit positiva vid M. genitalium testning. Dessa prover har därefter analyserats av personal med nuvarande ABI-metod för resistensbestämning och sedan analyserats på Panther Fusion. Nuvarande ABI-metod resulterade i positiv signal för 91% (50/55) av patientprover positiva vid M. genitalium analys och makrolidresistensmutation hos 25 % (14/55), medan Panther Fusion metoden resulterade i positiv signal för 81 % (45/55) av positiva M. genitalium prover och påvisade resistensmutation hos 20 % (11/55) av proverna.

No description available.

No description available.

CSIR / Translation initiation is a rate limiting step during protein biosynthesis. Initiation occurs by formation of an initiation complex comprising 30S subunit of ribosome, mRNA, initiator tRNA, and initiation factors. The initiator tRNA has a specialized function of binding to ribosomal P site whereas all the other tRNAs are selected in the ribosomal A site. The presence of a highly conserved 3 consecutive G-C base pairs in the anticodon stem of the initiator tRNA has been shown to be responsible for its P-site targeting. The exact molecular mechanism involved in the P-site targeting of the initiator tRNA is still unclear and focus of our study. Using genetic methods, we obtained mutant E. coli strains where initiator tRNA mutants lacking the characteristic 3-GC base pairs can also initiate translation. One such mutant strain, A30, was selected for this study. Using standard molecular genetic tools, the mutation was mapped and identified to be a mutation in a transcription remodeling factor, RapA (A511V). RapA is a transcription recycling factor and it displaces S1 when it performs its transcription recycling activity. We found this mutation to cause an increase in the S1-depleted ribosomes leading to decreased fidelity of translation initiation as the mutant RapA inefficiently displaces S1 from RNA polymerase complex. The mutation in the RapA was also found to cause changes in the transcriptome which leads to downregulation of major genes important for methionine and purine metabolism. Using mass spectrometric analysis, we identified deficiencies of methionine and adenine in the strain carrying mutant RapA. Our lab had previously reported that methionine and S-adenosyl methionine deficiency cause deficiency of methylations in ribosome which in turn decreases the fidelity of protein synthesis initiation. We used strains deleted for two newly identified methyltransferases, namely RlmH and RlmI, for our study and these strains also showed decreased fidelity of initiation. RlmH and RlmI methylate 1915 and 1962 positions of 23S rRNA respectively. We found that deletion of these methyltransferases also caused defects in ribosome biogenesis and compromised activity of ribosome recycling factor. We constructed phylogenetic trees of the initiator tRNA from 158 species which distinctly assembled into three domains of life. We also constructed trees using the minihelix or the whole sequence of species specific tRNAs, and iterated our analysis on 50 eubacterial species. We identified tRNAPro, tRNAGlu, or tRNAThr (but surprisingly not elongator tRNAMet) as probable ancestors of tRNAi. We then determined the factors imposing selection of methionine as the initiating amino acid. Overall frequency of occurrence of methionine, whose metabolic cost of synthesis is the highest among all amino acids, remains almost unchanged across the three domains of life. Our results indicate that methionine selection, as the initiating amino acid was possibly a consequence of the evolution of one-carbon metabolism, which plays an important role in regulating translation initiation. In conclusion, the current study reveals the importance of methylations in ribosome biogenesis and fidelity of translation initiation. It also strongly suggests a co-evolution of the metabolism and translation apparatus giving adaptive advantage to the cells where presence of methionine in the environment can be a signal to initiate translation with methionine initiator tRNA.

Das Pflanzenwachstum fördernde Bakterien (PGPB) kommen ubiquitär sowohl an der Wurzel als auch am Spross der Pflanzen vor und sie können über direkte oder indirekte Mechanismen einen bedeutenden Beitrag zur Stickstoffernährung der Pflanzen leisten. Die vorliegende Arbeit umfasst a) die Isolierung von PGPB, welche das Wachstum verschiedener Pflanzenarten fördern und durch Fusarien verursachte Pflanzenkrankheiten bekämpfen, b) die Analyse der Möglichkeiten Probleme der Pflanzenernährung durch den Einsatz von PGPB zu lösen, c) die Entwicklung neuer molekularbiologischer Methoden zur Messung der Diversität und Aktivität der PGPB. Im Rahmen dieser Arbeit wurden Methoden zur Beschreibung der Diversität von rhizosphären PGPB entwickelt und verbessert um Verbindungen zwischen applizierten PGPB und deren Aktivitäten zu prüfen. Die sensitive quantitative real-time-PCR Methode wurde zur Quantifizierung bzw. zum Nachweis der inokulierten PGPB und zum Nachweis des nitrogenase-reduktase-Gens (nifH), des Markergens für potentiell diazotrophe Bakterien. Bakterienartspezifische Primer wurden aus dem Sequenzvergleich der 16S-23S ISR ausgewählter Bakterienstämme selektiert und Protokolle zur Quantifizierung dieser Bakterienarten erarbeitet. Die nifH Gen Quantifizierung an Pflanzen eröffnet die Möglichkeit Schlüsselorganismen in der assoziativen biologischen Luftstickstoffbindung zu identifizieren und kurzfristige Reaktionen der Bakteriengesellschaften auf Umweltveränderungen und Regulationsmechanismen in situ zu analysieren. / Plant growth promoting bacteria (PGPB) are ubiquitous in both plant root and shoot, and are important contributors to the nitrogen-input of plants exerting their positive effects on plant growth directly or indirectly through different mechanisms. The present work focuses on a) the isolation of PGPB, which promotes the growth of different plant cultures and controls plant diseases caused by Fusarium species, b) the prospects of PGPB to solve plant nutritional problems, c) developing new molecular methods for the assessment of their diversity and activity. In the frame of this thesis, the methods for the description of the diversity of root colonizing PGPB have been developed and improved to provide links between introduced PGPB abundance and activities. The approach used was based on the sensitive real – time PCR detection/quantification of introduced PGBP and the nitrogenase reductase gene (nifH), which served as a marker gene for potential diazotrophs. The amplified 16S-23S ISR sequences of studied bacteria were subjected to strain – specific primer design and a highly specific bacteria quantification protocol were developed. The bacteria quantification protocol was based on real – time PCR using strain specific primers in order to evaluate the colonization ability of studied bacteria, which were inoculated to plant roots. The results presented in this thesis have shown that monitoring of nifH amount in plant root is a suitable and promising approach to link inoculated diazotrophic bacteria abundance and its potential activity. The study of nifH gene abundance in plant offers the opportunity to identify key players in asymbiotic nitrogen fixation, to study short-term community responses in changing environments, or to analyze the effect of regulation in situ.