A ClpB Chaperone Knockout Mutant of Mesorhizobium ciceri Shows a Delay in the Root Nodulation of Chickpea Plants

Several molecular chaperones are known to be involved in bacteria stress response. To investigate the role of chaperone ClpB in rhizobia stress tolerance as well as in the rhizobiaplant symbiosis process, the clpB gene from a chickpea microsymbiont, strain Mesorhizobium ciceri LMS-1, was identified and a knockout mutant was obtained. The ClpB knockout mutant was tested to several abiotic stresses, showing that it was unable to grow after a heat shock and it was more sensitive to acid shock than the wild-type strain. A plant-growth assay performed to evaluate the symbiotic performance of the clpB mutant showed a higher proportion of ineffective root nodules obtained with the mutant than with the wild-type strain. Nodulation kinetics analysis showed a 6- to 8-day delay in nodule appearance in plants inoculated with the Delta clpB mutant. Analysis of nodC gene expression showed lower levels of transcript in the Delta clpB mutant strain. Analysis of histological sections of nodules formed by the clpB mutant showed that most of the nodules presented a low number of bacteroids. No differences in the root infection abilities of green fluorescent protein tagged clpB mutant and wild-type strains were detected. To our knowledge, this is the first study that presents evidence of the involvement of the chaperone ClpB from rhizobia in the symbiotic nodulation process

The Symbiotic Performance of Chickpea Rhizobia Can Be Improved by Additional Copies of the clpB Chaperone Gene

The ClpB chaperone is known to be involved in bacterial stress response. Moreover, recent studies suggest that this protein has also a role in the chickpea-rhizobia symbiosis. In order to improve both stress tolerance and symbiotic performance of a chickpea microsymbiont, the Mesorhizobium mediterraneum UPM-Ca36T strain was genetically transformed with pPHU231 containing an extra-copy of the clpB gene. To investigate if the clpB-transformed strain displays an improved stress tolerance, bacterial growth was evaluated under heat and acid stress conditions. In addition, the effect of the extra-copies of the clpB gene in the symbiotic performance was evaluated using plant growth assays (hydroponic and pot trials). The clpB-transformed strain is more tolerant to heat shock than the strain transformed with pPHU231, supporting the involvement of ClpB in rhizobia heat shock tolerance. Both plant growth assays showed that ClpB has an important role in chickpea-rhizobia symbiosis. The nodulation kinetics analysis showed a higher rate of nodule appearance with the clpB-transformed strain. This strain also induced a greater number of nodules and, more notably, its symbiotic effectiveness increased ~60%at pH5 and 83% at pH7, compared to the wild-type strain. Furthermore, a higher frequency of root hair curling was also observed in plants inoculated with the clpB-transformed strain, compared to the wild-type strain. The superior root hair curling induction, nodulation ability and symbiotic effectiveness of the clpB-transformed strain may be explained by an increased expression of symbiosis genes. Indeed, higher transcript levels of the nodulation genes nodA and nodC (~3 folds) were detected in the clpB-transformed strain. The improvement of rhizobia by addition of extracopies of the clpB gene may be a promising strategy to obtain strains with enhanced stress tolerance and symbiotic effectiveness, thus contributing to their success as crop inoculants, particularly under environmental stresses. This is the first report on the successful improvement of a rhizobium with a chaperone gene

Unlocking rhizospheric bacteria secondary metabolism: genome analysis for the discovery of novel antimicrobial compounds

The emergence of antimicrobial resistance in pathogenic agents has raised awareness among society and nowadays is a recognized threat to public health. This problem is aggravated due to the misuse of current antibiotics and the lack of novel antimicrobial compounds1. Soil microorganisms are a potential source of new antibiotics and, thanks to the study of their genomes, we can guide the search for undescribed antimicrobial compounds2,3. We have isolated two bacterial strains from a rhizospheric soil, belonging to the genera Brevibacillus and Streptomyces, which were revealed as antimicrobial agents, inhibiting the growth of bacteria and fungi with different profiles of antimicrobial resistance. We sequenced the genome of these strains using the Illumina MiSeq platform. The gene calling and genome annotation were done through the RAST tool (v2.0)4. antiSMASH (v5.1)5 was used to annotate in depth those genes related to the secondary metabolism of both strains. Genome analyses showed diverse antimicrobial potential encoded within these 2 genomes. In sum, 61 biosynthetic gene clusters (BGCs) related with the secondary metabolism were annotated, of which 16 correspond to the Brevibacillus strain and 45 to the Streptomyces strain. The most abundant BGCs were non-ribosomal peptide synthetase (NRPS), terpenes and siderophores. Interestingly, some of these BGCs showed no similarity to any of the already described ones involved in the production of antimicrobial compounds. Therefore, the genetic machinery encoded in both genomes might provide us the basis for the discovery of novel antibiotics against multidrug resistance pathogens

Role of QseG membrane protein in beneficial enterobacterial interactions with plants and Mesorhizobia

Homologs of qseG gene (coding for the membrane protein QseG), along with the qseEF genes, are present in many Enterobacteriaceae; however, its role in non-pathogenic strains is still unknown. To fill this knowledge gap, we investigated the role of QseG protein of a plant-associated enterobacterium in the interactions with its legume host and in the benefits induced by this enterobacterium in the Mesorhizobium–chickpea symbiosis. Here, we showed that QseG of Kosakonia sp. MH5 is involved in the following processes: (i) the evasion of the plant immune system and (ii) the efficient colonization of chickpea root cells. Furthermore, these features are essential for the beneficial effects of this strain on the Mesorhizobium–chickpea symbiosis. This study demonstrates that the role of QseG is transversal to pathogenic and non-pathogenic enterobacteria and is a step forward to better understanding the molecular bases of plant–bacteria interactions established between legume and beneficial endophytic enterobacteria.ME

Evolution of Microbiota in a Pharmacy Classroom Pre and Post the First Wave of the COVID-19 Pandemic

Microbiota varies over time, therefore during 2019-20 academic year, coinciding with the SARS-COV-2 pandemic, the evolution of it was evaluated in a classroom of the Faculty of Pharmacy of the University of Salamanca with mass spectrometry Matrix-assisted laser desorption ionization time-of-flight. In addition, this study was compared with others, as well as with guidelines of the European Community Commission on indoor air quality./nAfter the study, it was concluded that said classroom had very low contamination degree and that most of microbiota was saprophytic. Furthermore, it can be concluded that quantitatively fungi and quantitatively and qualitatively the bacteria did not present a health risk./nOn the other hand, the most important variables that were found were the influx of people, the frequency of cleaning and ventilation. Finally, this study shows off the lack of regulation that exists on air quality in non-industrial and non-hospital interiors.La microbiota varía con el tiempo, por ello durante el curso académico 2019-20, coincidiendo con la pandemia SARS-COV-2, se evaluó la evolución de la misma en un aula de la Facultad de Farmacia de la Universidad de Salamanca con la espectrometría de masas Matrix-assisted laser desorption ionization time-of-flight. Además, se comparó con otros estudios, así como con las directrices de la Comisión de la Comunidad Europea de calidad del aire de interiores./nTras el estudio se concluyó que dicha aula presentaba un grado de contaminación muy bajo, y que la mayoría de la microbiota era saprofítica. Asimismo, de forma general se puede concluir que de forma cuantitativa los hongos y de forma cuali-cuantitativa las bacterias no presentaban un riesgo para la salud./nPor otro lado, entre las variables más importantes encontramos la afluencia de personas, la frecuencia de limpieza y la ventilación. Finalmente, este estudio recoge la poca normativa que existe sobre la calidad del aire en interiores no industriales y no hospitalarios

The N-fixing legume Periandra mediterranea constrains the invasion of an exotic grass (Melinis minutiflora P. Beauv) by altering soil N cycling

Melinis minutiflora is an invasive species that threatens the biodiversity of the endemic vegetation of the campo rupestre biome in Brazil, displacing the native vegetation and favouring fire spread. As M. minutiflora invasion has been associated with a high nitrogen (N) demand, we assessed changes in N cycle under four treatments: two treatments with contrasting invasion levels (above and below 50%) and two un-invaded control treatments with native vegetation, in the presence or absence of the leguminous species Periandra mediterranea. This latter species was considered to be the main N source in this site due to its ability to fix N2 in association with Bradyrhizobia species. Soil proteolytic activity was high in treatments with P. mediterranea and in those severely invaded, but not in the first steps of invasion. While ammonium was the N-chemical species dominant in plots with native species, including P.mediterranea, soil nitrate prevailed only in fully invaded plots due to the stimulation of the nitrifying bacterial (AOB) and archaeal (AOA) populations carrying the amoA gene. However, in the presence of P. mediterranea, either in the beginning of the invasion or in uninvaded plots, we observed an inhibition of the nitrifying microbial populations and nitrate formation, suggesting that this is a biotic resistance strategy elicited by P. mediterranea to compete with M. minutiflora. Therefore, the inhibition of proteolytic activity and the nitrification process were the strategies elicited by P.mediterranea to constrain M.munitiflora invasion

MALDI-TOF Mass Spectrometry Is a Fast and Reliable Platform for Identification and Ecological Studies of Species from Family Rhizobiaceae

Family Rhizobiaceae includes fast growing bacteria currently arranged into three genera, Rhizobium, Ensifer and Shinella, that contain pathogenic, symbiotic and saprophytic species. The identification of these species is not possible on the basis of physiological or biochemical traits and should be based on sequencing of several genes. Therefore alternative methods are necessary for rapid and reliable identification of members from family Rhizobiaceae. In this work we evaluated the suitability of Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for this purpose. Firstly, we evaluated the capability of this methodology to differentiate among species of family Rhizobiaceae including those closely related and then we extended the database of MALDI Biotyper 2.0 including the type strains of 56 species from genera Rhizobium, Ensifer and Shinella. Secondly, we evaluated the identification potential of this methodology by using several strains isolated from different sources previously identified on the basis of their rrs, recA and atpD gene sequences. The 100% of these strains were correctly identified showing that MALDI-TOF MS is an excellent tool for identification of fast growing rhizobia applicable to large populations of isolates in ecological and taxonomic studies

Plants Probiotics as a Tool to Produce Highly Functional Fruits: The Case of Phyllobacterium and Vitamin C in Strawberries

10 páginas, 1 tabla, 1 figuraThe increasing interest in the preservation of the environment and the health of consumers is changing production methods and food consumption habits. Functional foods are increasingly demanded by consumers because they contain bioactive compounds involved in health protection. In this sense biofertilization using plant probiotics is a reliable alternative to the use of chemical fertilizers, but there are few studies about the effects of plant probiotics on the yield of functional fruits and, especially, on the content of bioactive compounds. In the present work we reported that a strain of genus Phyllobacterium able to produce biofilms and to colonize strawberry roots is able to increase the yield and quality of strawberry plants. In addition, the fruits from plants inoculated with this strain have significantly higher content in vitamin C, one of the most interesting bioactive compounds in strawberries. Therefore the use of selected plant probiotics benefits the environment and human health without agronomical losses, allowing the production of highly functional foods.This work was granted by “Junta de Castilla y León” (Regional Government, Grant SA183A11-2) and MINECO (Central Government, Grant AGL2011-29227). Paula García-Fraile is recipient of a postdoctoral researcher contract from Academy of Sciences of the Czech Republic. José David Flores-Félix was supported by a fellowship of Salamanca University. Marta Marcos García was supported by a fellowship of Fundación Miguel Casado San José. Luís R. Silva is grateful to the financial support from the European Union (FEDER funds through COMPETE) and National Funds (FCT, Fundação para a Ciência e Tecnologia) through project Pest-C/EQB/LA0006/2013 and from the European Union (FEDER funds) under the framework of QREN through Project NORTE-07-0124-FEDER- 000069.Peer reviewe

Control of Brain State Transitions with a Photoswitchable Muscarinic Agonist

Reproducció del document publicat a:https://doi.org/10.1002/advs.202005027The ability to control neural activity is essential for research not only in basic neuroscience, as spatiotemporal control of activity is a fundamental experimental tool, but also in clinical neurology for therapeutic brain interventions. Transcranial-magnetic, ultrasound, and alternating/direct current (AC/DC) stimulation are some available means of spatiotemporal controlled neuromodulation. There is also light-mediated control, such as optogenetics, which has revolutionized neuroscience research, yet its clinical translation is hampered by the need for gene manipulation. As a drug-based light-mediated control, the effect of a photoswitchable muscarinic agonist (Phthalimide-Azo-Iper (PAI)) on a brain network is evaluated in this study. First, the conditions to manipulate M2 muscarinic receptors with light in the experimental setup are determined. Next, physiological synchronous emergent cortical activity consisting of slow oscillations—as in slow wave sleep—is transformed into a higher frequency pattern in the cerebral cortex, both in vitro and in vivo, as a consequence of PAI activation with light. These results open the way to study cholinergic neuromodulation and to control spatiotemporal patterns of activity in different brain states, their transitions, and their links to cognition and behavior. The approach can be applied to different organisms and does not require genetic manipulation, which would make it translational to humans