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Marine-derived bacteria are known for producing a wide range of specialized metabolites with antifungal activity against phytopathogens. Assessing the marine bacteria's ability to establish themselves in the rhizosphere and inhibit soil-borne phytopathogens under in vivo conditions could provide evidence that supports their use for biological control, widening biological control agent’s isolation sources. The current study evaluated the antifungal activity of a marine-derived bacterial collection against the tomato phytopathogen Fusarium oxysporum f. sp. lycopersici (FOL). Out of 152 isolates, 28 Firmicutes strains showed antifungal activity against ten isolates of F. oxysporum in in vitro testing. From them, Paenibacillus sp. PNM200 was identified as a potential PGPR and as a biocontrol agent against Fusarium vascular wilt. PNM200 colonized the tomato plant root system, increased plant growth, and reduced vascular wilt severity on greenhouse assays. Interestingly, plants treated with the spent supernatants of PNM200 cultures did not show a decrease in vascular wilt severity. This result indicates that the ability of PNM200 to act as a biocontrol agent against FOL does not depend on the diffusible antifungal metabolites produced during the fermentation process. Other Paenibacillus sp. PNM200 traits like chemotactic response towards tomato root exudates, biofilm, IAA, enzyme activity, and phosphate solubilization may contribute to PNM200 rhizospheric competence and biological control ability. Overall, the obtained results suggest that marine-derived bacteria could be a new source of biological control agents for integrated pest management of soil-borne diseases like Fusarium vascular wilt.

Bacterial Panicle Blight caused by Burkholderia glumae is a major disease of rice, which has dramatically affected rice production around the world in the last years. In this study we describe the assessment of three Streptomyces isolates as biocontrol agents for B. glumae. Additionally, the presence of other plant-growth promoting abilities and their possible beneficial effects upon their inoculation on rice plants was evaluated as an ecological analysis for their future inoculation in rice crops. Two isolates (A20 and 5.1) inhibited growth of virulent B. glumae strains, as well as a wide range of bacterial and fungal species, while a third strain (7.1) showed only antifungal activity. In vitro tests demonstrated the ability of these strains to produce siderophores, Indoleacetic acid (IAA), extracellular enzymes and solubilizing phosphate. Greenhouse experiments with two rice cultivars indicated that Streptomyces A20 is able to colonize rice plants and promote plant growth in both cultivars. Furthermore, an egfp tagged mutant was generated and colonization experiments were performed, indicating that Streptomyces A20 –GFP was strongly associated with root hairs, which may be related to the plant growth promotion observed in the gnotobiotic experiments. In order to characterize the antimicrobial compounds produced by strain A20 bacteria, mass spectrometry analyses were performed. This technique indicated that A20 produced several antimicrobial compounds with sizes below 3 kDa and three of these molecules were identified as Streptotricins D, E and F. These findings indicate the potential of Streptomyces A20 as a biocontrol inoculant to protect rice plants against bacterial diseases.

25 páginas Marine bacteria are considered as promising sources for the discovery of novel biologically active compounds. In this study, samples of sediment, invertebrate and algae were collected from the Providencia and Santa Catalina coral reef (Colombian Caribbean Sea) with the aim of isolating Actinobateria-like strain able to produce antimicrobial and quorum quenching compounds against pathogens. Several approaches were used to select actinobacterial isolates, obtaining 203 strains from all samples. According to their 16S rRNA gene sequencing, a total of 24 strains was classified within Actinobacteria represented by three genera: Streptomyces, Micromonospora, and Gordonia. In order to assess their metabolic profiles, the actinobacterial strains were grown in liquid cultures, and LC-MS-based analyses from ethyl acetate fractions were performed. Based on taxonomical classification, screening information of activity against phytopathogenic strains and quorum quenching activity, as well as metabolic profiling, six out of the 24 isolates were selected for follow-up with chemical isolation and structure identification analyses of putative metabolites involved in antimicrobial activities.

The genus Burkholderia comprises more than 60 species isolated from a wide range of niches. Although they have been shown to be diverse and ubiquitously distributed, most studies have thus far focused on the pathogenic species due to their clinical importance. However, the increasing number of recently described Burkholderia species associated with plants or with the environment has highlighted the division of the genus into two main clusters, as suggested by phylogenetical analyses. The first cluster includes human, animal, and plant pathogens, such as Burkholderia glumae, Burkholderia pseudomallei, and Burkholderia mallei, as well as the 17 defined species of the Burkholderia cepacia complex, while the other, more recently established cluster comprises more than 30 non-pathogenic species, which in most cases have been found to be associated with plants, and thus might be considered to be potentially beneficial. Several species from the latter group share characteristics that are of use when associating with plants, such as a quorum sensing system, the presence of nitrogen fixation and/or nodulation genes, and the ability to degrade aromatic compounds. This review examines the commonalities in this growing subgroup of Burkholderia species and discusses their prospective biotechnological applications.

Pathogenic bacteria interact not only with the host organism but most probably also with the resident microbial flora. In the knot disease of the olive tree (Olea europaea), the causative agent is the bacterium Pseudomonas savastanoi pv. savastanoi (Psv). Two bacterial species, namely Pantoea agglomerans and Erwinia toletana, which are not pathogenic and are olive plant epiphytes and endophytes, have been found very often to be associated with the olive knot. We identified the chemical signals that are produced by strains of the three species isolated from olive knot and found that they belong to the N-acyl-homoserine lactone family of QS signals. The luxI/R family genes responsible for the production and response to these signals in all three bacterial species have been identified and characterized. Genomic knockout mutagenesis and in planta experiments showed that virulence of Psv critically depends on QS; however, the lack of signal production can be complemented by wild-type E. toletana or P. agglomerans. It is also apparent that the disease caused by Psv is aggravated by the presence of the two other bacterial species. In this paper we discuss the potential role of QS in establishing a stable consortia leading to a poly-bacterial disease.

The genus Burkholderia includes over 60 species isolated from a wide range of environmental niches and can be tentatively divided into two major species clusters. The first cluster includes pathogens such as Burkholderia glumae , B. pseudomallei , and B. mallei and 17 well-studied species of the Burkholderia cepacia complex. The other recently established cluster comprises at least 29 nonpathogenic species, which in most cases have been found to be associated with plants. It was previously established that Burkholderia kururiensis , a member of the latter cluster, possesses an N -acyl homoserine lactone (AHL) quorum-sensing (QS) system designated “BraI/R,” which is found in all species of the plant-associated cluster. In the present study, two other BraI/R-like systems were characterized in B. xenovorans and B. unamae and were designated the BraI/R XEN and BraI/R UNA systems, respectively. Several phenotypes were analyzed, and it was determined that exopolysaccharide was positively regulated by the BraIR-like system in the species B. kururiensis , B. unamae , and B. xenovorans , highlighting commonality in targets. However, the three BraIR-like systems also revealed differences in targets since biofilm formation and plant colonization were differentially regulated. In addition, a second AHL QS system designated XenI2/R2 and an unpaired LuxR solo protein designated BxeR solo were also identified and characterized in B. xenovorans LB400 T . The two AHL QS systems of B. xenovorans are not transcriptionally regulating each other, whereas BxeR solo negatively regulated xenI2 . The XenI2/R2 and BxeR solo proteins are not widespread in the Burkholderia species cluster. In conclusion, the present study represents an extensive analysis of AHL QS in the Burkholderia plant-associated cluster demonstrating both commonalities and differences, probably reflecting environmental adaptations of the various species.

This work was aimed at identifying gene sequences in recombinant clones obtained from a genomic library from the Colombian Clostridium sp. IBUN 22A native strain. Nine clones greater than 500 bp were sequenced and analysed using BLAST 2.2.8 to search for complete inserts or their translated open reading frames (ORFs) in GenBank 141.0 and Uniprot 6.6. Seven genes having high similarity to housekeeping genes from different Clostridium species were identified. An 851 bp sequence was located in the pBsIBUN22A-1 clone, having 99.74% identity with the gene encoding the Clostridium butyricum enzyme glycerol dehydratase (DhaBl) which is involved in 1,3-propanediol (1,3-PD) production. Identifying genes in the native IBUN 22A strain formed the starting point for basic research and metabolic engineering aimed at making 1,3-PD production more profitable and increasing knowledge of genes present in the native strain. Key words: glycerol dehydratase, 1,3-propanediol, genomic library, Clostridium, sequence analysis.

LuxR solo transcriptional regulators contain both an autoinducer binding domain (ABD; N-terminal) and a DNA binding Helix-Turn-Helix domain (HTH; C-terminal), but are not associated with a cognate N-acyl homoserine lactone (AHL) synthase coding gene in the same genome. Although a few LuxR solos have been characterized, their distributions as well as their role in bacterial signal perception and other processes are poorly understood. In this study we have carried out a systematic survey of distribution of all ABD containing LuxR transcriptional regulators (QS domain LuxRs) available in the InterPro database (IPR005143), and identified those lacking a cognate AHL synthase. These LuxR solos were then analyzed regarding their taxonomical distribution, predicted functions of neighboring genes and the presence of complete AHL-QS systems in the genomes that carry them. Our analyses reveal the presence of one or multiple predicted LuxR solos in many proteobacterial genomes carrying QS domain LuxRs, some of them harboring genes for one or more AHL-QS circuits. The presence of LuxR solos in bacteria occupying diverse environments suggests potential ecological functions for these proteins beyond AHL and interkingdom signaling. Based on gene context and the conservation levels of invariant amino acids of ABD, we have classified LuxR solos into functionally meaningful groups or putative orthologs. Surprisingly, putative LuxR solos were also found in a few non-proteobacterial genomes which are not known to carry AHL-QS systems. Multiple predicted LuxR solos in the same genome appeared to have different levels of conservation of invariant amino acid residues of ABD questioning their binding to AHLs. In summary, this study provides a detailed overview of distribution of LuxR solos and their probable roles in bacteria with genome sequence information.

A future challenge will be understanding the extensive communication that most likely takes place in bacterial interspecies and interkingdom signaling between plants and bacteria. A major bacterial inter-cellular signaling system in Gram-negative bacteria is LuxI/R quorum sensing (QS) based on the production (via the LuxI-family proteins) and detection (via the LuxR-family proteins) of N-acyl homoserine lactones (AHLs) signaling molecules. LuxR proteins which have the same modular structure of QS LuxRs but are devoid of a cognate LuxI AHL synthase are called solos. LuxR solos have been shown to be responsible to respond to exogenous AHLs produced by neighboring cells as well endogenously produced AHLs. It is now also evident that some LuxR proteins have evolved from the ability to binding AHLs and respond to other molecules/signals. For example, recent research has shown that a sub-family of LuxR solos responds to small molecules produced by plants. This indicates the presence of a uni-directional interkingdom signaling system occurring from plants to bacteria. In addition LuxR solos have now been also implicated to respond to endogenously produced signals which are not AHLs. In this Mini Review article we will discuss current trends and implications of the role of LuxR solos in bacterial responses to other signals using proteins related to AHL quorum sensing systems.

Objective: to analyze the dhaT gene, one of the genes responsible for the 1,3-propanediol (1,3-PD) production, in two native Clostridium strains. Materials and methods: The dhaT gene was amplified by Polimerase Chain Reaction with specific primers designed from Clostridium butyricum VPI1718 operon. Bioinformatics tools like BLASTN, ORF finder, BLASTP and ClustalW were used to determine the identity of the sequence and to assign a function. Results: DNA amplification products were obtained from Colombian Clostridium sp. native strains (IBUN 13A and IBUN 158B) and the Clostridium butyricum DSM 2478 strain, which were sequenced. According to the bioinformatics analysis of the above sequences, a high degree of similarity was found with the dhaT gene of different bacterial species. The highest percentage of identity was obtained with the Clostridium butyricum VPI 1718 strain. Conclusion: knowledge of the physical structure of the 1,3-PD operon in native strains opens the way for developing genetic and metabolic engineering strategies for improving processes productivity. Objetivo: Analizar el gen dhaT, uno de los responsables de la producción de 1,3-propanodiol (1,3-PD), en dos cepas nativas de Clostridium. Materiales y métodos: El gen dhaT fue amplificado por Reacción en Cadena de la Polimerasa, por medio de cebadores específicos diseñados a partir del operon de Clostridium butyricum VPI1718. Herramientas bioinformáticas como BLASTN, ORF finder, BLASTP y ClustalW se usaron para determinar la identidad de la secuencia y asignarle una función. Resultados: Se obtuvieron productos de amplificación de DNA a partir de cepas nativas Colombianas de Clostridium sp. (IBUN 13A y IBUN 158B) y de la cepa Clostridium butyricum DSM 2478, que posteriormente fueron secuenciados. De acuerdo a los análisis bioinformáticos de las secuencias mencionadas, se encontró un alto grado de similitud con el gen dhaT de diferentes especies bacterianas. El más alto porcentaje de identidad se obtuvo con la cepa Clostridium butyricum VPI 1718. Conclusión: El conocimiento de la estructura física del operón 1,3-PD en cepas nativas, abre las puertas al desarrollo de estrategias genéticas y de ingeniería metabólica para mejorar la productividad del proceso. Objetivo: Analisar o gene dhaT, um dos responsáveis pela produção de 1,3-propanodiol (1,3-PD) em duas cepas nativas de Clostridium. Materiais e métodos: O gene dhaT foi amplificado por Reação em Cadeia da Polimerase, usando cevadores específicos sintetizados a partir do operon de Clostridium butyricum VPI1718. Ferramentas de bioinformática, tais como BLASTN, ORF finder, BLASTP e ClustalW foram utilizadas para determinar a identidade da seqüência e atribuir-lhe uma função. Resultados: Obtiveram-se produtos de amplificação do ADN a partir das cepas nativas colombianas de Clostridium sp. (IBUN 13A e IBUN 158B) e da cepa Clostridium butyricum DSM 2478, que foram posteriormente seqüenciados. Segundo a análise bioinformática das seqüências acima mencionadas, encontrou-se um elevado grau de similaridade com o gene dhaT de diferentes espécies bacterianas. A maior porcentagem de identidade foi obtida com a cepa Clostridium butyricum VPI 1718. Conclusão: O conhecimento da estrutura física do operon 1,3-PD em cepas nativas, abre as portas ao desenvolvimento de estratégias de genética e engenharia metabólica para melhorar a produtividade do processo.

76 cepas de las familias Enterobacteriaceae y Pseudomonadaceae fueron conservadas por el método semistock en papel filtro y 68 de las mismas por el método stock de criopreservación, en el banco de cepas y genes del Instituto de Biotecnología de la Universidad Nacional de Colombia (IBUN). La eficacia de los métodos de preservación se realizó mediante la evaluación de la viabilidad a las 24 horas de conservación y fue calculado el porcentaje de recuperación. En conclusión, la preservación en papel filtro y criopreservación son adecuados métodos semistock y stock, respectivamente; ya que el 73.7% de los microorganismos de la familia Enterobacteriaceae y el 82.3% de los microorganismos de la familia Pseudomonadaceae, presentaron un porcentaje de recuperación mayor al 50%. Por otra parte, se implementó una base de datos elaborada en el software Access ®, la cual se implementó con éxito en el IBUN y en la página Web de la Universidad Nacional de Colombia en la dirección electrónica www.ibun.unal.edu.co/instituto/cepario , que está disponible al público en general.