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Vol7SpecialIssueforweb

EuroPneumo Special Issue / pneumonia 2015 Oct 21;7:I–72

P1.41

Systematic nomenclature for the bacterial two-component regulatory

systems, based on genomic, structural and functional analysis of the

pan-genome of Streptococcus pneumoniae

Gustavo Gamez 1, 2 , Diego Sanchez 1, 2 , Frank Mona 1, 2 , Yully Betancur 1, 2 , Andres Castro 1, 2 , Alejandro

Gomez 1, 3 , Jose Mediavilla 4 , Mauricio Corredor 2 , Sven Hammerschmidt 3

1

Basic and Applied Microbiology (MICROBA) Research Group, School of Microbiology, Universidad de Antioquia, Medellin, Colombia; 2 Genetics,

Regeneration and Cancer (GRC) Research Group, University Research Center (SIU), Universidad de Antioquia, Medellin, Colombia; 3 Department

Genetics of Microorganisms, Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany; 4 Public

Health Research Institute, International Center for Public Health, Rutgers University, Newark, USA

Two-component systems (TCSs) represent the single largest paralogous gene family encoding signalling proteins in

Archaea and Eukarya. However, despite the importance of these systems for the regulation of different cellular and

physiological processes, a clear and uniform nomenclature system allowing for their systematic study does not exist.

Here, a thorough bioinformatics search and analysis was performed to identify conserved and distinctive features at the

genomic, structural and functional level, with the aim of devising a systematic and expandable model of nomenclature

for the operons, genes and proteins comprising the TCSs of the pan-genome of Streptococcus pneumoniae. The DNA

and protein sequences of 25 pneumococcal strains, whose genomes are completely sequenced and annotated, were

analysed employing different bioinformatics tools and databases. Twenty-nine different TCS-proteins (14-HKs, 15-RRs)

were identified, indicating a total pan-genomic complement of 15-TCSs, to which this species has access at a population

level. However, the modal complement was 13-TCSs and One-Orphan-RR. The TCS-variome estimation (genetic and

protein variability) confirmed the high-level of conservation among these pneumococcal regulators. Additionally,

through the structural/functional analysis of the TCS-proteins, it was possible to establish the presence of conserved and

distinctive features in terms of presence, absence, number, type, organisation and localisation of the different functional

domains. Moreover, the common names and diversity of functions under the control of the pneumococcal TCSs was

defined by a systematic research of the specialised literature. The identification of both conserved and distinctive

features at the genomic, structural and functional level allowed for the establishment of a systematic and uniform model

of nomenclature for the operons and proteins comprising the TCSs of the pneumococcal pan-genome. This model has

been already extrapolated to other bacterial species and it has the potential to serve as a reference standard to improve

research and understanding of these regulatory systems in prokaryotes.

P1.42

Optimisation of the expression of fluorescent proteins in Streptococcus

pneumoniae

Maria Catalão 1 , Joana Figueiredo 1 , Mafalda Henriques 1 , João Gomes 2 , Sergio Filipe 1

1

Laboratory of Bacterial Cell Surfaces and Pathogenesis, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal;

2

National Institute of Health, Department of Infectious Diseases, Lisbon, Portugal

Streptococcus pneumoniae is a Gram-positive bacterium usually found in association with a range of different types of

infections. The understanding of how these bacteria divide or perform specific tasks important for their survival is a

requirement for the design of efficient strategies to fight bacterial infections. This implies a detailed knowledge not only

of the function of proteins required for the infection process, but also of their localisation and role in complex molecular

machineries. In order to determine the correct subcellular localisation of fluorescent proteins in S. pneumoniae, we

have previously described tools to express derivatives of 4 fluorescent proteins—mCherry, Citrine, CFP and GFP—to

levels that allow visualisation by fluorescence microscopy, by fusing the first 10 amino acids of the S. pneumoniae

protein Wze (the i-tag), upstream of the fluorescent protein. We had proposed that this i-tag extension might facilitate

ribosome accessibility to the ribosome-binding site, thus enhancing protein translation. These tools, which we have now

confirmed that can also be used in other Gram-positive bacteria, namely Lactococcus lactis, Staphylococcus aureus, and

Bacillus subtilis, have been optimised by changing the nucleotide sequence of the i-tag and testing the effect of the first

10 amino acids of other pneumococcal proteins in the increased expression of the fluorescent protein Citrine. We found

that manipulating the structure and stability of the 5’ end of the mRNA molecule, which may influence the accessibility

of the ribosome, is determinant to ensure the expression of a strong fluorescent signal. We therefore propose that the

pneumonia 2015 Volume 7

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