Natural genetic transformation: A novel tool for efficient genetic engineering of the dairy bacterium Streptococcus thermophilus

Streptococcus thermophilus is widely used for the manufacture of yoghurt and Swiss or Italian-type cheeses. These products have a market value of approximately 40 billion dollars per year, making S. thermophilus a species that has major economic importance. Even though the fermentation properties of this bacterium have been gradually improved by classical methods, there is great potential for further improvement through genetic engineering. Due to the recent publication of three complete genome sequences, it is now possible to use a rational approach for designing S. thermophilus starter strains with improved properties. Progress in this field, however, is hampered by a lack of genetic tools. Therefore, we developed a system, based on natural transformation, which makes genetic manipulations in S. thermophilus easy, rapid, and highly efficient. The efficiency of this novel tool should make it possible to construct food-grade mutants of S. thermophilus, opening up exciting new possibilities that should benefit consumers as well as the dairy industry.     

BOX elements modulate gene expression in Streptococcus pneumoniae: impact on the fine-tuning of competence development

More than 100 BOX elements are randomly distributed in intergenic regions of the pneumococcal genome. Here we demonstrate that these elements can affect expression of neighboring genes and present evidence that they are mobile. Together, our findings show that BOX elements enhance genetic diversity and genomic plasticity in Streptococcus pneumoniae.     

Properties and biological role of streptococcal fratricins

Competence for natural genetic transformation is widespread in the genus Streptococcus. The current view is that all streptococcal species possess this property. In addition to the proteins required for DNA uptake and recombination, competent streptococci secrete muralytic enzymes termed fratricins. Since the synthesis and secretion of these cell wall-degrading enzymes are always coupled to competence development in streptococci, fratricins are believed to carry out an important function associated with natural transformation. This minireview summarizes what is known about the properties of fratricins and discusses their possible biological roles in streptococcal transformation.     

Identification of pneumococcal proteins that are functionally linked to penicillin‐binding protein 2b (PBP2b)

The oval shape of pneumococci results from a combination of septal and lateral peptidoglycan synthesis. The septal cross‐wall is synthesized by the divisome, while the elongasome drives cell elongation by inserting new peptidoglycan into the lateral cell wall. Each of these molecular machines contains penicillin‐binding proteins (PBPs), which catalyze the final stages of peptidoglycan synthesis, plus a number of accessory proteins. Much effort has been made to identify these accessory proteins and determine their function. In the present paper we have used a novel approach to identify members of the pneumococcal elongasome that are functionally closely linked to PBP2b. We discovered that cells depleted in PBP2b, a key component of the elongasome, display several distinct phenotypic traits. We searched for proteins that, when depleted or deleted, display the same phenotypic changes. Four proteins, RodA, MreD, DivIVA and Spr0777, were identified by this approach. Together with PBP2b these proteins are essential for the normal function of the elongasome. Furthermore, our findings suggest that DivIVA, which was previously assigned as a divisomal protein, is required to correctly localize the elongasome at the negatively curved membrane region between the septal and lateral cell wall.     

The Colposcopic Atlas of Schistosomiasis in the Lower Female Genital Tract Based on Studies in Malawi,Zimbabwe, Madagascar and South Africa

Background

Schistosoma (S.) haematobium is a neglected tropical disease which may affect any part of the genital tract in women. Female genital schistosomiasis (FGS) may cause abnormal vaginal discharge, contact bleeding, genital tumours, ectopic pregnancies and increased susceptibility to HIV. Symptoms may mimic those typical of sexually transmitted infections (STIs) and women with genital schistosomiasis may be incorrectly diagnosed. An expert consensus meeting suggested that the following findings by visual inspection should serve as proxy indicators for the diagnosis of schistosomiasis of the lower genital tract in women from S. haematobium endemic areas: sandy patches appearing as (1) single or clustered grains or (2) sandy patches appearing as homogenous, yellow areas, or (3) rubbery papules. In this atlas we aim to provide an overview of the genital mucosal manifestations of schistosomiasis in women.

Methodology/Principal findings

Photocolposcopic images were captured from women, between 1994 and 2012 in four different study sites endemic for S. haematobium in Malawi, Zimbabwe, South Africa and Madagascar. Images and specimens were sampled from sexually active women between 15 and 49 years of age. Colposcopic images of other diseases are included for differential diagnostic purposes.

Significance

This is the first atlas to present the clinical manifestations of schistosomiasis in the lower female genital tract. It will be freely available for online use, downloadable as a presentation and for print. It could be used for training purposes, further research, and in clinical practice.     

CellLineMiner: a knowledge portal for human cell lines

Experimental models of human tissues and disease phenotypes frequently rely upon immortalized cell lines, which are easily accessible and simple to use due to their infinite capability of cell division. For decades, cell lines have been used to investigate cellular mechanisms of disease and the efficacy of drugs, most prominently for human cancers. However, the large body of knowledge with respect to human cell lines exists primarily in an unstructured fashion, that is, as free text in the scientific literature. Here we present CellLineMiner, a novel text mining-based web database that provides a comprehensive view of human cell line knowledge. The application offers a simple search in all indexed cell lines, accompanied by a rapid display of all identified literature associations. The CellLineMiner is intended to serve as a knowledge resource companion to the cellular model systems used in biomedical research.

Availability

CellLineMiner is accessible at http://dev.pubgene.com/cellmine     

Fratricide is essential for efficient gene transfer between pneumococci in biofilms

Streptococcus pneumoniae and a number of commensal streptococcal species are competent for natural genetic transformation. The natural habitat of these bacteria is multispecies biofilms in the human oral cavity and nasopharynx. Studies investigating lateral transfer of virulence and antibiotic resistance determinants among streptococci have shown that interspecies as well as intraspecies gene exchange takes place in these environments. We have previously shown that the action of a competence-specific murein hydrolase termed CbpD strongly increases the rate of gene transfer between pneumococci grown in liquid cultures. CbpD is the key component of a bacteriolytic mechanism termed the fratricide mechanism. It is secreted by competent pneumococci and mediates the release of donor DNA from sensitive streptococci present in the same environment. However, in nature, gene exchange between streptococci takes place in biofilms and not in liquid cultures. In the present study, we therefore investigated whether CbpD affects the rate of gene transfer in laboratory-grown biofilms. Our results show that the fratricide mechanism has a strong positive impact on intrabiofilm gene exchange, indicating that it is important for active acquisition of homologous donor DNA under natural conditions. Furthermore, we found that competent biofilm cells of S. pneumoniae acquire a Nov(r) marker much more efficiently from neighboring cells than from the growth medium. Efficient lysis of target cells requires that CbpD act in conjunction with the murein hydrolase LytC. In contrast, the major autolysin LytA does not seem to be important for fratricide-mediated gene exchange in a biofilm environment.     

A hydrophobic patch in the competence-stimulating Peptide, a pneumococcal competence pheromone, is essential for specificity and biological activity

Induction of competence for natural genetic transformation in Streptococcus pneumoniae depends on pheromone-mediated cell-cell communication and a signaling pathway consisting of the competence-stimulating peptide (CSP), its membrane-embedded histidine kinase receptor ComD, and the cognate response regulator ComE. Extensive screening of pneumococcal isolates has revealed that two major CSP variants, CSP1 and CSP2, are found in members of this species. Even though the primary structures of CSP1 and CSP2 are about 50% identical, they are highly specific for their respective receptors, ComD1 and ComD2. In the present work, we have investigated the structural basis of this specificity by determining the three-dimensional structure of CSP1 from nuclear magnetic resonance data and comparing the agonist activity of a number of CSP1/CSP2 hybrid peptides toward the ComD1 and ComD2 receptors. Our results show that upon exposure to membrane-mimicking environments, the 17-amino-acid CSP1 pheromone adopts an amphiphilic alpha-helical configuration stretching from residue 6 to residue 12. Furthermore, the pattern of agonist activity displayed by the various hybrid peptides revealed that hydrophobic amino acids, some of which are situated on the nonpolar side of the alpha-helix, strongly contribute to CSP specificity. Together, these data indicate that the identified alpha-helix is an important structural feature of CSP1 which is essential for effective receptor recognition under natural conditions.