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RESUMEN Las bacterias sostienen la vida de nuestro planeta y aunque solo se conoce alrededor del 1% de su diversidad, muchas de ellas pueden resultar benéficas para ser utilizadas con fines agrícolas, biomédicos y de biorremediación, entre otros. En la actualidad se tiene especial interés en descubrir tanto a nuevas especies bacterianas, así como en estudiar la función que desempeñan los genes que conforman a algunas bacterias de interés; mediante estrategias de mutagénesis y secuenciación. En ambos casos es necesario contar con métodos de preservación de bacterias adecuados que permitan estudios futuros. En el presente trabajo se discuten varias metodologías para preservar a las bacterias de forma correcta, tomando en consideración que se deben mantener sus características fenotípicas, genotípicas y una alta viabilidad. Estas características también asegurarán el mantenimiento del gran potencial biotecnológico que las bacterias resguardan en sus genomas., {"references":["Acinas SG, Marcelino LA, Klepac-Ceraj V & Polz MF (2004) Divergence and redundancy of 16S rRNA sequences in genomes with multiple rrn operons. J. Bacteriol. 186: 2629-2635.","Aithal HN, Kalra VK and Brodie AF (1975) Alteration of Mycobacterium phlei membrane structure by freezing and thawing: reversal by heating. Arch. Biochem. Biophys. 168: 122-132.","Amann RI, Ludwig W & Schleifer K-H (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59: 143-169.","Arias-Barrau E, Olivera ER, Luengo JM, Fernández C, Galan B, García JL, Díaz E & Miñambres B (2004) The homogentisate pathway: a central catabolic pathway involved in the degradation of L-phenylalanine, Ltyrosine, and 3-hydroxyphenylacetate in Pseudomonas putida. J. 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Pseudomonas putida KT2440 encodes 23 alternative sigma factors. The fliA gene, which encodes σ28, is in a cluster with other genes involved in flagella biosynthesis and chemotaxis. Reverse transcriptase-PCR revealed that this cluster is comprised of four independent transcriptional units: flhAF, fleNfliA, cheYZA and cheBmotAB. We generated a nonpolar fliA mutant by homologous recombination and tested its motility, adhesion to biotic and abiotic surfaces, and responses to various stress conditions. The mutant strain was nonmotile and exhibited decreased capacity to bind to corn seeds, although its ability to colonize the rhizosphere of plants was unaffected. The mutant was also affected in binding to abiotic surfaces and its ability to form biofilms decreased by almost threefold. In the fliA mutant background expression of 25 genes was affected: two genes were upregulated and 23 genes were downregulated. In addition to a number of motility and chemotaxis genes, the fliA gene product is also necessary for the expression of some genes potentially involved in amino acid utilization or stress responses; however, we were unable to assign specific phenotypes linked to these genes since the fliA mutant used the same range of amino acids as the parental strain, and was as tolerant as the wild type to stress imposed by heat, antibiotics, NaCl, sodium dodecyl sulfate, H2O2 and benzoate. Based on the sequence alignment of promoters recognized by FliA and genome in silico analysis, we propose that P. putidaσ28 recognizes a TCAAG-t-N12-GCCGATA consensus sequence located between −34 and −8 and that this sequence is preferentially associated with an AT-rich upstream region.

Sistema de autolisis celular para el procesado de la biomasa bacteriana en la producción de polihidroxialcanoatos en Pseudomonas putida KT2440. La presente invención se refiere a una cepa bacteriana de Pseudomonas putida KT2440, que comprende un sistema genético heterólogo lítico donde dicho sistema a su vez comprende la secuencia nucleotídica que codifica la enzima lítica de pared celular endolisina Ejl, la secuencia nucleotídica que codifica la holina Ejh, y una secuencia nucleotídica que codifica un sistema de regulación génica, que a su vez comprende una secuencia nucleotídica promotora de la expresión génica, y una secuencia nucleotídica que codifica y expresa una proteína reguladora de dicha expresión génica. Preferiblemente la cepa es el microorganismo con número de depósito CECT 7659. Además, la presente invención también se refiere a dicha cepa donde el microorganismo Pseudomonas putida KT2440 es mutante en uno o más de los genes to/palo Preferiblemente dicha cepa es el microorganismo con número de depósito CECT 7658. Además, la presente invención se refiere al uso de cualquiera de las cepas descritas para la extracción de polihidroxialcanoato (PHA) sintetizado por dicha bacteria así como a un método para la extracción de dicho compuesto., Consejo Superior de Investigaciones Científicas (España), A1 Solicitud de patente con informe sobre el estado de la técnica

[EN] The present invention relates to a bacterial strain of Pseudomonas putida KT2440, which includes a lytic heterologous genetic system in which said system in tum includes the nucleotide sequence that encodes the lytic enzyme with endolysin cell wall Ejl, the nucleotide sequence that encodes holin Ejh, and a nucleotide sequence that encodes a gene regulation system, which in turn includes a nucleotide sequence promoting the gene expression, and a nucleotide sequence that encodes and expresses a protein regulating said gene expression. The strain is preferably the microorganism with deposit number CECT 7659. The present invention further relates to said strain in which the Pseudo monas putida KT2440 microorganism is mutant in one or more of the tol-pal genes. Said strain is preferably the microorganism with deposit number CECT 7658. The present invention further relates to the use of any ofthe described strains for extracting polyhydroxyalkanoate (PHA) synthesised by said bacterium as well as to a method for extracting said compound., [ES] La presente invención se refiere a una cepa bacteriana de Pseudomonas putida KT2440, que comprende un sistema genético heterólogo lítico donde dicho sistema a su vez comprende la secuencia nucleotídica que codifica la enzima lítica de pared celular endolisina Ejl, la secuencia nucleotídica que codifica la holina Ejh, y una secuencia nucleotídica que codifica un sistema de regulación génica, que a su vez comprende una secuencia nucleotídica promotora de la expresión génica, y una secuencia nucleotídica que codifica y expresa una proteína reguladora de dicha expresión génica. Preferiblemente la cepa es el microorganismo con número de depósito CECT 7659. Además, la presente invención también se refiere a dicha cepa donde el microorganismo Pseudomonas putida KT2440 es mutante en uno o más de los genes tol-pal. Preferiblemente dicha cepa es el microorganismo con número de depósito CECT 7658. Además, la presente invención se refiere al uso de cualquiera de las cepas descritas para la extracción de polihidroxialcanoato (PHA) sintetizado por dicha bacteria así como a un método para la extracción de dicho compuesto., Consejo Superior de Investigaciones Científicas (España), A1 Solicitud de patente con informe sobre el estado de la técnica

Frameshift mutations in a poly(G) track at the flhB gene of Pseudomonas putida DOT-T1E are responsible for the diminished swimming of this strain on semisolid medium, which contrasts with the high swimming ability of P. putida KT2440, which does not exhibit a poly(G) track at the flhB gene. We previously showed that a mutant lacking FlhB was more sensitive to solvents than the wild-type strain (Segura et al., J. Bacteriol., 183:4127-4133, 2001). In this study, we show that swimming ability correlates with solvent tolerance in P. putida DOT-T1E, so that growth conditions favoring a functional flhB gene (growth on semisolid medium) resulted in increased innate tolerance to a sudden toluene shock.