André M. Grilo

The Second RNA Chaperone, Hfq2, Is Also Required for Survival under Stress and Full Virulence of Burkholderia cenocepacia J2315

Burkholderia cenocepacia J2315 is a highly virulent and epidemic clinical isolate of the B. cepacia complex (Bcc), a group of bacteria that have emerged as important pathogens to cystic fibrosis patients. This bacterium, together with all Bcc strains and a few other prokaryotes, is unusual for encoding in its genome two distinct and functional Hfq-like proteins. In this work, we show results indicating that the 188-amino-acid Hfq2 protein is required for the full virulence and stress resistance of B. cenocepacia J2315, despite the presence on its genome of the functional 79-amino-acid Hfq protein encoded by the hfq gene. Similar to other Hfq proteins, Hfq2 is able to bind RNA. However, Hfq2 is unique in its ability to apparently form trimers in vitro. Maximal transcription of hfq was observed in B. cenocepacia J2315 cells in the early exponential phase of growth. In contrast, hfq2 transcription reached maximal levels in cells in the stationary phase, depending on the CepR quorum-sensing regulator. These results suggest that tight regulation of the expression of these two RNA chaperones is required to maximize the fitness and virulence of this bacterium. In addition, the ability of Hfq2 to bind DNA, not observed for Hfq, suggests that Hfq2 might play additional roles besides acting as an RNA chaperone.

The Burkholderia cenocepacia K56-2 pleiotropic regulator Pbr, is required for stress resistance and virulence.

Burkholderia cenocepacia is one of the most virulent species of the Burkholderia cepacia complex, a group of bacteria that emerged as important pathogens, especially to cystic fibrosis (CF) patients. In this study, we report the identification and characterization of a mutant strain derived form the CF isolate Burkholderia cenocepacia K56-2, carrying a plasposon insertion in a gene, located in a 3516 bp chromosomal region with an atypical G+C content, encoding a 80 amino acid putative regulatory protein named Pbr. Besides its inability to produce phenazines, the B. cenocepacia K56-2 pbr mutant exhibited a pleiotropic phenotype, including impaired survival to oxidative and osmotic stress, aromatic amino acid and prolonged nutrient starvation periods. In addition, the pbr mutant exhibited decreased virulence the nematode Caenorhabditis elegans. Altogether, our results demonstrate the involvement of Pbr on the regulation of phenazine biosynthesis, and an important role for this regulatory protein on several cellular processes related to stress resistance and virulence.

Experimental identification of small non-coding regulatory RNAs in the opportunistic human pathogen Burkholderia cenocepacia J2315.

Small non-coding regulatory RNAs (sRNAs) play important roles in regulating gene expression at the post-transcriptional level and often require the RNA chaperone Hfq. The human opportunistic pathogen Burkholderia cenocepacia J2315 encodes two distinct RNA chaperones, Hfq and Hfq2. The present work describes the experimental identification and validation of 24 sRNAs from B. cenocepacia J2315, based on the co-purification of sRNAs with the bacterium Hfq protein, followed by conversion into cDNA, cloning, computational analysis of sequences and validation by Northern blot analysis. The sRNAs here reported escaped identification by previous studies based on transcriptomics or bioinformatic analyses. Results presented indicate that 3 sRNAs are exclusive to bacteria of the Burkholderia cepacia complex and have no homologues in other bacteria, while the other 21 share homology, at different extents, to sRNAs of other bacterial species.

Hfq: a multifaceted RNA chaperone involved in virulence

Hfq has emerged in recent years as a master regulator of gene expression in bacteria, mainly due to its ability to mediate the interaction of small noncoding RNAs with their mRNA targets, including those related to virulence in Gram-negative bacteria. In this work, we review current knowledge on the involvement of Hfq in the regulation of virulence traits related to secretion systems, alternative sigma factors, outer membrane proteins, polysaccharides and iron metabolism. Recent data from transcriptomics and proteomics studies performed for major pathogens are included. We also summarize and correlate current knowledge on how Hfq protein impacts pathogenicity of bacterial pathogens.

A new methodology combining PCR, cloning, and sequencing of clones discriminated by RFLP for the study of microbial populations: application to an UASB reactor sample.

This work describes a methodology combining DNA extraction, polymerase chain reaction amplification with primers targeting 16S ribosomal RNA genes, cloning, and sequencing of clones previously analyzed by restriction fragment length polymorphism (RFLP), which can be applied to study the microbial diversity in a given habitat. The methodology allows the minimization of the sequencing effort, which is particularly relevant when analyzing large numbers of clones. The methodology does not require particularly skilled personnel and can easily be adaptable to the molecular characterization of virtually any particular microbial population, provided that both adequate primers and suitable restriction enzymes for RFLP analysis of the clone library have been chosen. An example of application is presented, in which a sample taken from a continuously operating upflow anaerobic sludge blanket reactor was analyzed. RFLP analysis of the initial 162 clones with HaeIII allowed the identification of only 28 distinct profiles. As expected, identical RFLP profiles corresponded to identical nucleotide sequences.

Regulation of Hfq mRNA and protein levels in Escherichia coli and Pseudomonas aeruginosa by the Burkholderia cenocepacia MtvR sRNA.

Small non-coding RNAs (sRNAs) are important players of gene expression regulation in bacterial pathogens. MtvR is a 136-nucleotide long sRNA previously identified in the human pathogen Burkholderia cenocepacia J2315 and with homologues restricted to bacteria of the Burkholderia cepacia complex. In this work we have investigated the effects of expressing MtvR in Escherichia coli and Pseudomonas aeruginosa. Results are presented showing that MtvR negatively regulates the hfq mRNA levels in both bacterial species. In the case of E. coli, this negative regulation is shown to involve binding of MtvR to the 5′-UTR region of the hfq Ec mRNA. Results presented also show that expression of MtvR in E. coli and P. aeruginosa originates multiple phenotypes, including reduced resistance to selected stresses, biofilm formation ability, and increased susceptibility to various antibiotics.

Retraction for Ramos et al., The Second RNA Chaperone, Hfq2, Is Also Required for Survival under Stress and Full Virulence of Burkholderia cenocepacia J2315

Volume 193, no. 7, p.1515–1526, 2011. Problems related to images published in this paper have been brought to our attention. Figure 8 contains duplicated images as well as images previously published in articles in Microbiology and Microbial Pathogenesis, i.e., the following: S. A. Sousa, C. G. Ramos, L. M. Moreira, and J. H. Leitão, Microbiology 156:896–908, 2010. http://dx.doi.org/10.1099/mic.0.035139-0. C. G. Ramos, S. A. Sousa, A. M. Grilo, L. Eberl, and J. H. Leitão, Microb. Pathog. 48:168–177, 2010. http://dx.doi.org/10.1016 /j.micpath.2010.02.006. Therefore, we retract the paper.Wedeeply regret this situation and apologize for any inconvenience to the editors and readers of Journal of Bacteriology, Microbial Pathogenesis, and Microbiology.

Retraction for Ramos et al., MtvR Is a Global Small Noncoding Regulatory RNA in Burkholderia cenocepacia

Volume 195, no. 16, p. 3514–3523, 2013. A number of problems related to images published in this paper have been brought to our attention. Figure 1D contains duplicated images in lanes S and LE, and Fig. 4D and 6B contain images previously published in articles in this journal and in Microbiology and Microbial Pathogenesis, i.e., the following: C. G. Ramos, S. A. Sousa, A. M. Grilo, J. R. Feliciano, and J. H. Leitão, J. Bacteriol. 193:1515–1526, 2011. doi:10.1128/JB.01374-11. S. A. Sousa, C. G. Ramos, L. M. Moreira, and J. H. Leitão, Microbiology 156:896–908, 2010. doi:10.1099/mic.0.035139-0. C. G. Ramos, S. A. Sousa, A. M. Grilo, L. Eberl, and J. H. Leitão, Microb. Pathog. 48:168–177, 2010. doi: 10.1016/j.micpath.2010.02.006. Therefore, we retract the paper. We deeply regret this situation and apologize for any inconvenience to the editors and readers of Journal of Bacteriology, Microbial Pathogenesis, and Microbiology.

A RNomics-based strategy identifies regulatory small RNAs in Burkholderia cepacia complex

Regulatory small non-coding RNAs (sRNAs) are know to be involved in bacterial virulence, most often exerting its function in a Hfq-dependent fashion. Burkholderia cepacia complex (Bcc) bacteria are important human opportunistic pathogens, that possess exceptionally large and complex genomes (∼7000 genes) comprising roughly 8 Mb. Within their genomes, it has been predicted in silico 213 sRNAs. Bcc are unique among human pathogens by encoding two distinct and functional Hfq-like proteins. In this work, we describe a strategy based on co-immunoprecipitation of sRNAs with Hfq-like proteins, the cloning and identification of the sRNAs, showing the wide array of possibilities that this RNomics approach endows the researcher for sRNA discovery.

112 Unveiling the roles of small non-coding RNAs and RNA chaperones on the biology of opportunistic pathogens of the Burkholderia cepacia complex

112 Unveiling the roles of small non-coding RNAs and RNA chaperones on the biology of opportunistic pathogens of the Burkholderia cepacia complex C.G. Ramos1, A.M. Grilo1, P.J. da Costa1, J.R. Feliciano1, J.H. Leitao2. 1IBB − Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Tecnico, Lisboa, Portugal; 2IBB-Institute for Biotechnology and Bioengineering, Centre for Chemical and Biological Engineering, Department of Bioengineering, Instituto Superior Tecnico, Lisboa, Portugal