The Irr and RirA proteins participate in a complex regulatory circuit and act in concert to modulate bacterioferritin expression in Ensifer meliloti 1021

In this work we found that the bfr gene of the rhizobial species Ensifer meliloti, encoding a bacterioferritin iron storage protein, is involved in iron homeostasis and the oxidative stress response. This gene is located downstream of and overlapping the smc03787 open reading frame (ORF). No well-predicted RirA or Irr boxes were found in the region immediately upstream of the bfr gene although two presumptive RirA boxes and one presumptive Irr box were present in the putative promoter of smc03787. We demonstrate that bfr gene expression is enhanced under iron-sufficient conditions and that Irr and RirA modulate this expression. The pattern of bfr gene expression as well as the response to Irr and RirA is inversely correlated to that of smc03787. Moreover, our results suggest that the small RNA SmelC759 participates in RirA- and Irr-mediated regulation of bfr expression and that additional unknown factors are involved in iron-dependent regulation.Fil: Costa, Daniela. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Amarelle, Vanesa. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: O`Brian, Mark R.. State University of New York; Estados UnidosFil: Fabiano, Elena. Instituto de Investigaciones Biológicas "Clemente Estable"; Urugua

Construction and screening of a functional metagenomic library to identify novel enzymes produced by Antarctic bacteria

A metagenomic fosmid library of approximately 52 000 clones was constructed to identify functional genes encoding cold-adapted enzymes. Metagenomic DNA was extracted from a sample of glacial meltwater, collected on the Antarctic Peninsula during the ANTARKOS XXIX Expedition during the austral summer of 2012–2013. Each clone contained an insert of about 35–40 kb, so the library represented almost 2 Gb of genetic information from metagenomic DNA. Activity-driven screening was used to detect the cold-adapted functions expressed by the library. Fifty lipase/esterase and two cellulase-producing clones were isolated, and two clones able to grow on Avicel® as the sole carbon source. Interestingly, three clones formed a brown precipitate in the presence of manganese (II). Accumulation of manganese oxides was determined with a leucoberbelin blue assay, indicating that these three clones had manganese-oxidizing activity. To the best of our knowledge, this is the first report of a manganese oxidase activity detected with a functional metagenomic strategy

Identification of Antarctic culturable bacteria able to produce diverse enzymes of potential biotechnological interest

It is estimated that more than three quarters of the Earth’s biosphere is in perennially cold environments. Despite the extreme environmental conditions of desiccation and freezing, microbes can colonize these habitats through the adaptation of metabolic functions and the synthesis of structurally adapted enzymes. Enzymes within psychrophilic microbes exhibit high specific activity at low and moderate temperature, with low thermostability. In this study we used a classic microbiological approach to isolate Antarctic bacteria with cellulolytic, lipolytic, and ligninolytic activities. From 15 different environmental samples, we generated a collection of approximately 800 bacterial isolates that could grow on R2A or Marine medium at 4°C. This collection was then screened for the presence of the three types of activity at 4°C. We found that 47.7% of the isolates displayed lipolytic activity, 10.2% had cellulase/xylanase activity, and 7.7% showed guaiacol oxidase activity. Of these, 10% displayed two different types of activity, while 0.25% displayed all three types of activity. Our results indicate that cold environments represent outstanding resources for bioprospecting and the study of enzymatic adaptation

Informe final del proyecto: Diseño racional de vectores para la implementación de Pseudomonas y Arthrobacter psicrófilas en la construcción de bibliotecas metagenómicas funcionales especialmente diseñadas para la identificación de enzimas psicrófilas

La metagenómica funcional es una técnica que permiten acceder a la información genética de una comunidad sin la necesidad de cultivar en el laboratorio cada una de las bacterias que la componen. Se basa en la obtención de la información genética de la comunidad (ADN metagenómico), el cual se introduce en una bacteria de laboratorio (hospedero). El hospedero por excelencia es Escherichia coli, quien debe reconocer e interpretar correctamente el fragmento de ADN metagenómico introducido, y así adquirir nuevas capacidades (ej., actividades enzimáticas). Uno de los grandes sesgos de la técnica es que E. coli no siempre es capaz de reconocer e interpretar el ADN introducido, y por lo tanto no es capaz de producir nuevas enzimas de interés. Para sortear este problema se está avanzando en el uso de hospederos distintos a E. coli. Para poder introducir fragmentos de ADN en una bacteria, debemos tener las herramientas moleculares necesarias que nos permitan trabajar con esa bacteria. En este proyecto nos propusimos diseñar algunas de estas herramientas para poder usar como hospederos alternativos a Pseudomonas y Arthrobacter aisladas de la Antártida. Estas bacterias tienen la particularidad de crecer a bajas temperaturas (bacterias psicrófilas). ¿Por qué bacterias psicrófilas? Porque nos interesa buscar actividades enzimáticas que puedan funcionar a bajas temperaturas (enzimas psicrófilas). Las enzimas psicrófilas pueden trabajar a temperatura ambiente, siendo muy codiciadas en algunos procesos biotecnológicos. ¿Por qué? Porque realizar un proceso a temperatura ambiente es más económico porque no se precisa energía para calentar los reactores. Nuestra hipótesis de trabajo es que al usar hospederos psicrófilos en una aproximación de metagenómica funcional aumentaremos la probabilidad de identificar enzimas psicrófilas de interés biotecnológico.Agencia Nacional de Investigación e Innovació

HmuS and HmuQ of Ensifer/Sinorhizobium meliloti degrade heme in vitro and participate in heme metabolism in vivo

Ensifer meliloti is a nitrogen-fixing symbiont of the alfalfa legume able to use heme as an iron source. The transport mechanism involved in heme acquisition in E. meliloti has been identified and characterized, but the fate of heme once inside the cell is not known. In silico analysis of E. meliloti 1021 genome revealed no canonical heme oxygenases although two genes encoding putative heme degrading enzymes, smc01518 and hmuS, were identified. SMc01518 is similar to HmuQ of Bradyrhizobium japonicum, which is weakly homologous to the Staphylococcus aureus IsdG heme-degrading monooxygenase, whereas HmuS is homolog to Pseudomonas aeruginosa PhuS, a protein reported as a heme chaperone and as a heme degrading enzyme. Recombinant HmuQ and HmuS were able to bind hemin with a 1:1 stoichiometry and displayed a Kd value of 5 and 4 lM, respectively. HmuS degrades heme in vitro to the biliverdin isomers IX-b and IX-d in an equimolar ratio. The HmuQ recombinant protein degrades heme to biliverdin IX-d only. Additionally, in this work we demonstrate that humS and hmuQ gene expression is regulated by iron and heme in a RirA dependent manner and that both proteins are involved in heme metabolism in E. meliloti in vivo.Agencia Nacional de Investigación e Innovació

A new small regulatory protein, HmuP, modulates haemin acquisition in Sinorhizobium meliloti

Sinorhizobium meliloti has multiple systems for iron acquisition, including the use of haem as an iron source. Haem internalization involves the ShmR haem outer membrane receptor and the hmuTUV locus, which participates in haem transport across the cytoplasmic membrane. Previous studies have demonstrated that expression of the shmR gene is negatively regulated by iron through RirA. Here, we identify hmuP in a genetic screen for mutants that displayed aberrant control of shmR. The normal induction of shmR in response to iron limitation was lost in the hmuP mutant, showing that this gene positively affects shmR expression. Moreover, the HmuP protein is not part of the haemin transporter system. Analysis of gene expression and siderophore production indicates that disruption of hmuP does not affect other genes related to the iron-restriction response. Our results strongly indicate that the main function of HmuP is the transcriptional regulation of shmR. Sequence alignment of HmuP homologues and comparison with the NMR structure of Rhodopseudomonas palustris CGA009 HmuP protein revealed that certain amino acids localized within predicted β-sheets are well conserved. Our data indicate that at least one of the β-sheets is important for HmuP activity

Elucidación de los sistemas implicados en la captación y utilización de hemina como fuente de hierro nutricional en Sinorhizobium meliloti 1021

Tribunal : Dr. Jorge Monza, Dra. Beatriz Garat, Dra. Silvia Batista

The Comic as an Educational Resource to Teach Microbiology in the Classroom

New educational resources are being implemented as an initiative to foster learning. In order to contribute to the tool-kit of innovative educational resources, we developed a microbiology comic book. The aim of this comic is to provide educators with a fun, accessible and rigorous way to generate awareness of the invisible world that surrounds us and that inhabit us. Bacteria have a record of being harmful and disgusting entities. Mass media, with advertisements of disinfectants, soaps, and house cleaning products, are sending a distorted message about microbes. We must debunk these misconceptions and emphasize the importance of microorganisms, and particularly bacteria, in the environment and our lives. Education is the means to this end, and therefore this comic is intended to help educators to teach microbiology in an attractive, accurate and straightforward way.  Here, we present this educational tool and give some tips of the different themes that can be addressed in the classroom with this resource

Informe final del proyecto: Diseño racional de vectores para la implementación de Pseudomonas y Arthrobacter psicrófilas en la construcción de bibliotecas metagenómicas funcionales especialmente diseñadas para la identificación de enzimas psicrófilas

La metagenómica funcional es una técnica que permiten acceder a la información genética de una comunidad sin la necesidad de cultivar en el laboratorio cada una de las bacterias que la componen. Se basa en la obtención de la información genética de la comunidad (ADN metagenómico), el cual se introduce en una bacteria de laboratorio (hospedero). El hospedero por excelencia es Escherichia coli, quien debe reconocer e interpretar correctamente el fragmento de ADN metagenómico introducido, y así adquirir nuevas capacidades (ej., actividades enzimáticas). Uno de los grandes sesgos de la técnica es que E. coli no siempre es capaz de reconocer e interpretar el ADN introducido, y por lo tanto no es capaz de producir nuevas enzimas de interés. Para sortear este problema se está avanzando en el uso de hospederos distintos a E. coli. Para poder introducir fragmentos de ADN en una bacteria, debemos tener las herramientas moleculares necesarias que nos permitan trabajar con esa bacteria. En este proyecto nos propusimos diseñar algunas de estas herramientas para poder usar como hospederos alternativos a Pseudomonas y Arthrobacter aisladas de la Antártida. Estas bacterias tienen la particularidad de crecer a bajas temperaturas (bacterias psicrófilas). ¿Por qué bacterias psicrófilas? Porque nos interesa buscar actividades enzimáticas que puedan funcionar a bajas temperaturas (enzimas psicrófilas). Las enzimas psicrófilas pueden trabajar a temperatura ambiente, siendo muy codiciadas en algunos procesos biotecnológicos. ¿Por qué? Porque realizar un proceso a temperatura ambiente es más económico porque no se precisa energía para calentar los reactores. Nuestra hipótesis de trabajo es que al usar hospederos psicrófilos en una aproximación de metagenómica funcional aumentaremos la probabilidad de identificar enzimas psicrófilas de interés biotecnológico.Agencia Nacional de Investigación e Innovació