Saturday, May 28, 2011

The stb Operon Balances the Requirements for Vegetative Stability and Conjugative Transfer of Plasmid R388

Catherine Guynet, Ana Cuevas, Gabriel Moncalián*, Fernando de la Cruz*
IBBTEC, Instituto de Biomedicina y Biotecnologia de Cantabria (CSIC-UC-SODERCAN), Facultad de Medicina, Universidad de Cantabria, Santander, Spain

Abstract

The conjugative plasmid R388 and a number of other plasmids carry an operon, stbABC, adjacent to the origin of conjugative transfer. We investigated the role of the stbA, stbB, and stbC genes. Deletion of stbA affected both conjugation and stability. It led to a 50-fold increase in R388 transfer frequency, as well as to high plasmid loss. In contrast, deletion of stbB abolished conjugation but provoked no change in plasmid stability. Deletion of stbC showed no effect, neither in conjugation nor in stability. Deletion of the entire stb operon had no effect on conjugation, which remained as in the wild-type plasmid, but led to a plasmid loss phenotype similar to that of the R388ΔstbA mutant. We concluded that StbA is required for plasmid stability and that StbA and StbB control conjugation. We next observed the intracellular positioning of R388 DNA molecules and showed that they localize as discrete foci evenly distributed in live Escherichia coli cells. Plasmid instability of the R388ΔΔstbA mutant correlated with aberrant localization of the plasmid DNA molecules as clusters, either at one cell pole, at both poles, or at the cell center. In contrast, plasmid molecules in the R388ΔΔstbB mutant were mostly excluded from the cell poles. Thus, results indicate that defects in both plasmid maintenance and transfer are a consequence of variations in the intracellular positioning of plasmid DNA. We propose that StbA and StbB constitute an atypical plasmid stabilization system that reconciles two modes of plasmid R388 physiology: a maintenance mode (replication and segregation) and a propagation mode (conjugation). The consequences of this novel concept in plasmid physiology will be discussed.

Author Summary

The ability of bacteria to evolve and adapt to new environments most often results from the acquisition of new genes by horizontal transfer. Plasmids have a preponderant role in gene exchanges through their ability to transfer DNA by conjugation, a process that transports DNA between bacteria. Besides, plasmids are autonomous DNA molecules that are faithfully transmitted to cell progeny during vegetative cell multiplication. In this study, we report a system composed of two proteins, StbA and StbB, which act to balance plasmid R388 physiology between two modes: a maintenance mode (vertical transmission) and a propagation mode (horizontal transmission). We demonstrate that StbA is essential to ensure faithful assortment of plasmid copies to daughter cells. In turn, StbB is required for plasmid R388 adequate localization for conjugation. This is the first report of a system which reconciles plasmid segregation and conjugation. Furthermore, R388 belongs to the IncW family of conjugative plasmids, which are of particular interest due to their exceptionally broad host range. We show that the StbAB system is conserved among a wide variety of conjugative plasmids, mainly broad host range plasmids. Thus, the Stb system could constitute an interesting therapeutic target to prevent the spread of adaptive genes.

Autoinhibitory regulation of TrwK, an essential VirB4 ATPase in type IV secretion systems

Alejandro Peña, Jorge Ripoll-Rozada, Sandra Zunzunegui, Elena Cabezón, Fernando de la Cruz & Ignacio Arechaga.
Intergenomics Group. Universidad de Cantabria and Instituto de Biomedicina y Biotecnologia de Cantabria (IBBTEC), Spain

Abstract

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. In a previous work we demonstrated that TrwK is able to hydrolyze ATP. Here, based on the structural homology of VirB4 proteins with the DNA-pumping ATPase TrwB coupling protein, we generated a series of variants of TrwK where fragments of the C-terminal domain were sequentially truncated. Surprisingly, the in vitro ATPase activity of these TrwK variants was much higher than that of the wild type enzyme. Moreover, addition of a synthetic peptide containing the amino acid residues comprising this C-terminal region resulted in the specific inhibition of the TrwK variants lacking such domain. These results indicate that the C-terminal end of TrwK plays an important regulatory role in the functioning of the T4SS. 

Friday, May 27, 2011

Association of Composite IS26-sul3 Elements with Highly Transmissible IncI1 Plasmids in Extended-Spectrum-β-Lactamase-Producing Escherichia coli Clones from Humans

Curiao T, Canton R., Garcillán-Barcia M.P., de la Cruz F., Baquero F., Coque T.M.

1Servicio de Microbiología, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain, Unidad de Resistencia a Antibióticos y Virulencia Bacteriana Asociada al Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain, 3CIBER Epidemiología y Salud Pública (CIBERESP), Madrid, Spain, 4Departamento de Biología Molecular e Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria-CSIC-IDICAN, C. Herrera Oria s/n, 39011 Santander, Spain

The association of an IS440-sul3 platform with Tn21 class 1 integrons carried by IncI1 plasmids encoding extended-spectrum β-lactamases (ESBLs; mainly SHV-12 and CTX-M-14) among worldwide Escherichia coli clones of phylogroups A (ST10, ST23, and ST46), B1 (ST155, ST351, and ST359), and D/B2 (ST131) is reported. An in silico comparative analysis of sul3 elements available in the GenBank database shows the evolution of sul3 platforms by hosting different transposable elements facilitating the potential genesis of IS26 composite transposons and further insertion element-mediated promoted arrangements.

Thursday, May 5, 2011

Mariano Barbacid - Doctor Honoris Causa por la Universidad de Cantabria

El próximo día 19 de Mayo a las 19:00h, en el Paraninfo de la Universidad de Cantabria, tendrá lugar  el acto de investidura de Mariano Barbacid como Doctor Honoris Causa por la Universidad de Cantabria, a propuesta del Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC). El día 20 de Mayo impartirá una Conferencia Plenaria en la Facultad de Medicina.
Mariano Barbacid Última actualización Mayo/2010
Mariano Barbacid was born in Madrid in 1949. He was awarded his PhD degree from the Universidad Complutense de Madrid in 1974. From 1974 – 1978 he trained as a Postdoctoral Fellow at the National Cancer Institute (NCI), Bethesda, Maryland, USA.
Mariano BarbacidIn 1978 he set up his own group to work on the molecular biology of human tumours. His work led to the isolation of the first human oncogene, H-Ras, in 1982. Other contributions of special relevance include the identification of Ras oncogenes as targets of chemical carcinogens (1984), the discovery of the Trk family of tyrosine kinase receptors as the signalling receptors for the NGF family of neurotrophic factors (1991) and the physiological role of the cell cycle Cdks (2003 – 2007).
He moved to the NCI campus in Frederick, Maryland (USA), as Head of the Developmental Oncology Section in 1984. In 1988, he joined the Bristol Myers-Squibb Pharmaceutical Research Institute in Princeton, New Jersey where he became Vice President of Oncology Drug Discovery. In 1998 he returned to Madrid to create and direct the CNIO (see Commentary in Cell, 129: 641-644, 2007).
Mariano Barbacid has authored 245 publications including 176 original articles and 24 invited reviews in refereed journals as well as 45 book chapters. The average impact factor of the 200 publications published in peer-reviewed journals is >12. His current Hirsch “h” factor is 86.
The relevance of his work has been recognised through several awards including the Young Investigator Award of the AACR (1986), the Steiner Prize (1988), the Ipsen Prize (1994), The Jimenez Díaz Award (2002), the Brupbacher Cancer Research Prize (2005), and the Medal of Honour of the International Agency for Research on Cancer (WHO) in 2007. He has been an EMBO Member since 1996.

http://www.cnio.es/es/grupos/plantillas/curriculum.asp?pag=14