Variability of the 16S-23S rRNA gene internal transcribed spacer in Pseudomonas avellanae strains

The 16S-23S rRNA gene internal transcribed spacer region (ITS1) from 34 strains of Pseudomonas avellanae and some strains of Pseudomonas syringae pathovars was amplified and assessed by restriction fragment length polymorphism (RFLP) using 10 restriction enzymes. In addition, the ITS1 region of four representative P. avellanae strains was sequenced and compared by the neighbour-joining algorithm with that of P. syringae pathovars. Two main groups of P. avellanae strains were observed that did not correlate with their origin. The ITS1 region sequencing revealed a high similarity with the P. syringae complex. One group of P. avellanae strains showed high similarity to P. s. pv. actinidiae and P. s. pv. tomato; another group showed similarity with P. s. pv. tabaci and P. s. pv. glycinea. Two strains clustered with P. s. pv. pisi. The difficulties to unambiguously classify the strains associated with hazelnut decline in Greece and Italy are discussed.     

Repetitive DNA, molecular cytogenetics and genome organization in the King scallop (Pecten maximus)

We studied the structure, organization and relationship of repetitive DNA sequences in the genome of the scallop, Pecten maximus, a bivalve that is important both commercially and in marine ecology. Recombinant DNA libraries were constructed after partial digestion of genomic DNA from scallop with PstI and ApaI restriction enzymes. Clones containing repetitive DNA were selected by hybridisation to labelled DNA from scallop, oyster and mussel; colonies showing strong hybridisation only to scallop were selected for analysis and sequencing. Six non-homologous tandemly repeated sequences were identified in the sequences, and Southern hybridisation with all repeat families to genomic DNA digests showed characteristic ladders of hybridised bands. Three families had monomer lengths around 40 bp while three had repeats characteristic of the length wrapping around one (170 bp), or two (326 bp) nucleosomes. In situ hybridisation to interphase nuclei showed each family had characteristic numbers of clusters indicating contrasting arrangements. Two of the repeats had unusual repetitions of bases within their sequence, which may relate to the nature of microsatellites reported in bivalves. The study of these rapidly evolving sequences is valuable to understand an important source of genomic diversity, has the potential to provide useful markers for population studies and gives a route to identify mechanisms of DNA sequence evolution.     

The sarcomeric myosin heavy chain gene family in the dog: analysis of isoform diversity and comparison with other mammalian species

Sarcomeric myosin heavy chains (MyHC) are the major contractile proteins of cardiac and skeletal muscles and belong to class II MyHC. In this study the sequences of nine sarcomeric MyHC isoforms were obtained by combining assembled contigs of the dog genome draft available in the NCBI database. With this information available the dog becomes the second species, after human, for which the sequences of all members of the sarcomeric MyHC gene family are identified. The newly determined sequences of canine MyHC isoforms were aligned with their orthologs in mammals, forming a set of 38 isoforms, to search for the molecular features that determine the structural and functional specificity of each type of isoform. In this way the structural motifs that allow identification of each isoform and are likely determinants of functional properties were identified in six specific regions (surface loop 1, loop 2, loop 3, converter, MLC binding region, and S2 proximal segment).     

High sensitivity of human centrin 2 toward radiolytical oxidation: C-terminal tyrosinyl residue as the main target

Centrins are calcium-binding proteins that play a significant role in the maintenance of the centrosomal organization, mainly in the continuity between centrosome and microtubular network. Recent data showed that centrosome duplication abnormalities, like overduplication for example, could be due to hydrogen peroxide, suggesting an important impact of oxidative stress. To challenge this hypothesis, we performed one-electron oxidation experiments with human centrin 2, starting from azide radicals. Our results first revealed several intermolecular cross-links generating dimers, tetramers, hexamers, and higher molecular mass species. Dimers result from covalent bond linking the C-terminal tyrosines of each monomer. Second, the methionyl residue at position 19 was oxidized on the monomeric centrin. Further, electron microscopy experiments on centrin 2 showed a preexisting hexameric organization that was stabilized by covalent bonds as a result of irradiation. Overall, these results show that centrin 2 is highly sensitive to ionizing radiation, which could have important consequences on its biological functions.     

BibA: a novel immunogenic bacterial adhesin contributing to group B Streptococcus survival in human blood

By the analysis of the recently sequenced genomes of Group B Streptococcus (GBS) we have identified a novel immunogenic adhesin with anti-phagocytic activity, named BibA. The bibA gene is present in 100% of the 24 GBS strains analysed. BibA-specific IgG were found in human sera from normal healthy donors. The putative protein product is a polypeptide of 630 amino acids containing a helix-rich N-terminal domain, a proline-rich region and a canonical LPXTG cell wall-anchoring domain. BibA is expressed on the surface of several GBS strains, but is also recovered in GBS culture supernatants. BibA specifically binds to human C4-binding protein, a regulator of the classic complement pathway. Deletion of the bibA gene severely reduced the capacity of GBS to survive in human blood and to resist opsonophagocytic killing by human neutrophils. In addition, BibA expression increased the virulence of GBS in a mouse infection model. The role of BibA in GBS adhesion was demonstrated by the impaired ability of a bibA knockout mutant strain to adhere to both human cervical and lung epithelial cells. Furthermore, we calculated that recombinant BibA bound to human epithelial cells of distinct origin with an affinity constant of approximately 10(-8) M for cervical epithelial cells. Hence BibA is a novel multifunctional protein involved in both resistance to phagocytic killing and adhesion to host cells. The identification of this potential new virulence factor represents an important step in the development of strategies to combat GBS-associated infections.     

CcpA mutants with differential activities in Bacillus subtilis

CcpA is the master regulator for carbon catabolite regulation in Bacillus subtilis and regulates more than 300 genes by repression or activation. To revealthe effects of different functional domains of CcpA on various regulatory modes, we compared the activities of CcpA point mutants in activation (alsS, ackA) and repression (xynP, gntR). CcpA variants mutated at residues in the HPrSerP-binding region without allosteric functions are inactive. On the other hand, CcpA variants mutated at residues that change their conformation upon HPrSerP or CrhP binding regulate only ackA. Another set of mutants with alterations in the corepressor-binding region show glucose-independent regulation of xynP. The data presented here demonstrate the involvement of HPrSerP and/or CrhP in activation of ackA and alsS by CcpA. Furthermore, these data also indicate that activation and repression mediated by CcpA may utilize different conformational changes of the protein.     

Quantile regression for longitudinal data using the asymmetric Laplace distribution

In longitudinal studies, measurements of the same individuals are taken repeatedly through time. Often, the primary goal is to characterize the change in response over time and the factors that influence change. Factors can affect not only the location but also more generally the shape of the distribution of the response over time. To make inference about the shape of a population distribution, the widely popular mixed-effects regression, for example, would be inadequate, if the distribution is not approximately Gaussian. We propose a novel linear model for quantile regression (QR) that includes random effects in order to account for the dependence between serial observations on the same subject. The notion of QR is synonymous with robust analysis of the conditional distribution of the response variable. We present a likelihood-based approach to the estimation of the regression quantiles that uses the asymmetric Laplace density. In a simulation study, the proposed method had an advantage in terms of mean squared error of the QR estimator, when compared with the approach that considers penalized fixed effects. Following our strategy, a nearly optimal degree of shrinkage of the individual effects is automatically selected by the data and their likelihood. Also, our model appears to be a robust alternative to the mean regression with random effects when the location parameter of the conditional distribution of the response is of interest. We apply our model to a real data set which consists of self-reported amount of labor pain measurements taken on women repeatedly over time, whose distribution is characterized by skewness, and the significance of the parameters is evaluated by the likelihood ratio statistic.     

Complexity, connectivity, and duplicability as barriers to lateral gene transfer

Background

Lateral gene transfer is a major force in microbial evolution and a great source of genetic innovation in prokaryotes. Protein complexity has been claimed to be a barrier for gene transfer, due to either the inability of a new gene's encoded protein to become a subunit of an existing complex (lack of positive selection), or from a harmful effect exerted by the newcomer on native protein assemblages (negative selection).

Results

We tested these scenarios using data from the model prokaryote Escherichia coli. Surprisingly, the data did not support an inverse link between membership in protein complexes and gene transfer. As the complexity hypothesis, in its strictest sense, seemed valid only to essential complexes, we broadened its scope to include connectivity in general. Transferred genes are found to be less involved in protein-protein interactions, outside stable complexes, and this is especially true for genes recently transferred to the E. coli genome. Thus, subsequent to transfer, new genes probably integrate slowly into existing protein-interaction networks. We show that a low duplicability of a gene is linked to a lower chance of being horizontally transferred. Notably, many essential genes in E. coli are conserved as singletons across multiple related genomes, have high connectivity and a highly vertical phylogenetic signal.

Conclusion

High complexity and connectivity generally do not impede gene transfer. However, essential genes that exhibit low duplicability and high connectivity do exhibit mostly vertical descent.