Identification and validation of reference genes to study the gene expression in Gluconacetobacter diazotrophicus grown in different carbon sources using RT-qPCR

Species of the family Pasteurellaceae play an important role as primary or opportunistic animal pathogens. In veterinary diagnostic laboratories identification of this group of bacteria is mainly done by phenotypic assays while genetic identification based on housekeeping genes is mostly used for research and particularly important diagnostic samples. MALDI-TOF MS seems to represent a promising alternative to the currently practiced cumbersome, phenotypic diagnostics carried out in many veterinary diagnostic laboratories. We therefore assessed its application for animal associated members of the family Pasteurellaceae. The Bruker Biotyper 3.0 database was complemented with reference spectra of clinically relevant as well as commensal animal Pasteurellaceae species using generally five strains per species or subspecies and tested for its diagnostic potential with additional, well characterized field isolates. About 250 strains comprising 15 genera and more than 40 species and subspecies were included in the study, covering most representatives of the family. A high discrimination at the genus and species level was observed. Problematic discrimination was only observed with some closely related species and subspecies. MALDI-TOF MS was shown to represent a highly potent method for the diagnosis of this group of animal pathogens, combining speed, precision and low running costs.     

Emergency contraception: can we benefit from lessons learned?

The aim of the paper was to evaluate current emergency contraception (EC) methods and policies in order to implement lessons learned and maximize potential population impact while introducing dedicated EC pills in Croatia. Literature search for potential reasons for EC failing to show positive population impact and detecting actionable points to be implemented in national guidelines. Six potential reasons for ECs failure to show population impact were evaluated and four actionable points were detected: low use of EC compared to the numbers of risk events, low awareness on EC in general population, differences in efficacy of EC methods and EC vailability. In order to ensure EC's population impact in Croatia it is of a critical relevance to establish continuous education programs for population of women at risk. When recommending an EC method, superior efficacy must be a key decision-making criteria therefore cooper IUD and ulipristal acetate should be our primary options. Counseling is a critical step to ensure maximal efficacy of the EC method, but also to encourage future use of regular contraceptives. Finally, national ECP dispension protocol is needed to close the loop from effective women screening, prompt yet appropriate ECP administration/dispensing towards structured follow up after EC pills intake.     

Azospirillum amazonense inoculation: effects on growth, yield and N2 fixation of rice (Oryza sativa L.)

Bacteria of the genus Azospirillum are considered to be plant-growth promoting bacteria (PGPR) and stimulate plant growth directly either by synthesising phyto-hormones or by promoting nutrition by the process of biological nitrogen fixation (BNF). Although this genus extensively studied, the effects of inoculation and the possible BNF contribution of the Azospirillum amazonense specie are very scarce. The aim of this study was to isolate, characterise and evaluate auxin production and nitrogenase activity of this species and to select, by inoculation of rice plants, promising isolates based on their ability to improve plant growth, yield and the BNF contribution. One hundred and ten isolates obtained from rice were characterised and grouped according to colony features. Forty-two isolates, confirmed as A. amazonense by the fluorescent in situ hybridization (FISH) technique, were tested for auxin production and nitrogenase activity in vitro. Subsequently plant growth promotion related to plant nutrition effect was evaluated, in vitro and in greenhouse experiments. The BNF contribution to plant growth was evaluated using the ¹⁵N isotope dilution technique. All A. amazonense strains tested produced indoles, but only 10% of them showed high production, above 1.33 μM mg protein⁻¹. The nitrogenase activity also was variable and only 9% of isolates showed high nitrogenase activity and the majority (54%) exhibited a low potential. The inoculation of selected strains in rice under gnotobiotic conditions reduced the growth of root and aerial part when compared to the control, showing the negative effects of excess of phytohormone accumulation in the medium. However, in the greenhouse experiment, inoculation of strains of A. amazonense increased grain dry matter accumulation (7 to 11.6%), the number of panicles (3 to 18.6%) and nitrogen accumulation at grain maturation (3.5 to 18.5%). BNF contributions up to 27% were observed for A. amazonense Y2 (wild type strain). The data presented here is the first report that the PGPR effect of A. amazonense for rice plants grown under greenhouse conditions is mainly due the BNF contribution as measured by ¹⁵N isotope dilution technique, in contrast to the hormonal effect observed with other Azospirillum species studied.     

Characterization of amplified polymerase chain reaction glnB and nifH gene fragments of nitrogen-fixing Burkholderia species

AIMS: To clone and sequence polymerase chain reaction (PCR)-amplified glnB and nifH genes of the nitrogen-fixing bacteria Burkholderia brasilensis strain M130, B. tropicalis strain PPe8 and B. kururiensis strain KP23. METHODS AND RESULTS: The glnB and nifH gene fragments were amplified by PCR using universal degenerated primers. A very high percentage of similarity for the nifH (100%) and glnB (96%) genes was observed between strains M130 and KP23. A similarity of 100% for the nifH gene was also observed between strains M130 and PPe8. However, the identity for the glnB gene was 98% and the similarity 88%. The phylogenetic tree of the nifH gene showed a very high degree of similarity to the 16S rDNA gene. CONCLUSIONS: The nitrogen-fixing bacteria of the Burkholderia genus formed a cluster separated from the other species of the genus mainly when the nifH rather than the glnB gene was used to construct the phylogenetic tree. Significance and Impact of the Study: Knowledge of the nifH and glnB gene sequences of B. brasilensis, B. tropicalis and B. kururiensis will support new studies on the diversity of these diazotrophs in natural environments.     

Characterisation and action mode of Gluconacin,a bacteriocin with antimicrobial activity against Xanthomonas albilineans

Gluconacin, a bacteriocin produced by Gluconacetobacter diazotrophicus PAL5, has previously shown a large spectrum of inhibitory activity against beneficial and phytopathogenic bacteria. The present study proposes a three-dimensional (3D) structure for Gluconacin based on modelling and describes some physicochemical characteristics and the effect of the peptide on cells of the phytopathogenic bacterium Xanthomonas albilineans ICMP196. The Gluconacin 3D structural model results demonstrated the 7 α-helices and 11 β-sheets. Growth inhibition of the indicator microorganism, X. albilineans, occurred in the first moments of contact with Gluconacin (0.25 μg μl^?1). Treatment of bacterial cells caused significant loss of inorganic phosphate and UV-absorbing materials. Scanning electron microscopy images showed that Gluconacin-treated X. albilineans cells were fully lysed. These results suggest that the mode of action of this peptide involves altered membrane integrity and increased permeability, resulting in complete cell lysis. Physicochemical characterisation demonstrated stability of biological activity under high temperatures, including autoclaving conditions and low pH. The activity was reduced after treatment with proteases and some surfactants and abolished with ethylenediaminetetraacetic acid (EDTA). The 3D conformation model indicates the possibility of a hydrophobic region, which could possibly interact directly with the cell membrane of target bacteria. In conclusion, the ability of Gluconacin to inhibit growth of phytopathogens of agricultural importance opens up new opportunities for biotechnological applications and, consequently, a reduction in the use of pesticides.     

Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant

Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium, which was originally isolated from the interior of sugarcane plants. The genome of strain PAL5 of G. diazotrophicus has been completely sequenced and a next step is the functional characterization of its genes. The aim of this study was to establish an efficient mutagenesis method, using the commercial Tn5 transposon EZ::Tn5™<KAN-2>Tnp Transposome™ (Epicentre). Up to 1 × 10⁶ mutants per microgram of transposome were generated in a single electroporation experiment. Insertion-site flanking sequences were amplified by inverse PCR and sequenced for 31 mutants. For ten of these mutants, both insertion flanks could be identified, confirming the 9 bp duplication that is typical for Tn5 transposition. Insertions occurred in a random fashion and were genetically stable for at least 50 generations. One mutant had an insertion in a homolog of the flagellar gene flgA, and was therefore predicted to be affected in flagella-dependent traits and used to validate the applied mutagenesis methodology. This mutant lacked flagella and was non-motile on soft agar. Interestingly, it was also strongly affected in the ability to form biofilm on glass wool. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Biological Dinitrogen Fixation in Gramineae and Palm Trees

Biological nitrogen fixation (BNF) in the Gramineae family has been well documented, but a complete understanding of this issue is needed to turn the research into a practical approach. The literature has a long and diverse list of diazotrophic bacteria found colonizing several plant tissues, such as roots, stems, leaves, and trash as well as the rhizosphere. However, only a limited amount of research has focussed on existing associations of N₂-fixing microorganisms with grasses or cereal, especially for BNF inputs and ecological studies under field conditions. The recent discovery of the endophytic diazotroph bacteria such as Acetobacter diazotrophicus, Herbaspirillum spp. and Azoarcus spp. colonizing the interior of sugarcane, rice, Kallar grass (Leptochloa fusca (L.) Kunth), respectively, and other species of grasses as well as cereals has led to a considerable interest in exploring these novel associations. There is a general consensus that plant genotype is a key factor to higher contributions of BNF together with the selection of more efficient bacterial strains. This review summarizes the present data on this field and introduces the discovery of a new group of diazotrophic bacteria colonizing palm trees and therefore opening a future perspective for using these plants, especially African oil palm, to replace diesel as a fuel.