Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.     

Integration of a transposable DNA sequence which mediates ampicillin resistance into Clo DF13 plasmid DNA: determination of the site and orientation of TnA insertions.

The isolation and characterization of Clo DF13 plasmids containing a transposable DNA sequence (TnA) that specifies for ampicillin resistance is described. The particular transposon is derived from the R plasmid pRI30, and is designated Tn901. In order to determine the site and orientation of Tn901 insertions into the Clo DF13 genome, we made use of restriction endonucleases and heteroduplex mapping. For this purpose, Clo DF13 plasmid DNA and DNA of Clo DF13::Tn901 plasmids were digested with endonucleases HincII, PstI, BamH-I, SalI, and HpaI or with a combination of two of these enzymes. By analysis of the resulting fragmentation patterns, the physical maps of Clo DF13 DNA and Tn901 DNA could be derived. Furthermore, the site and orientation of Tn901 insertions into the Clo DF13 genome could be determined by this approach. The data obtained were verified by heteroduplex mapping. Analysis of 33 independently isolated Clo DF13 recombinant plasmids showed that insertion of Tn901 had occurred at 31 different sites. No preference with respect to the orientation of Tn901 was observed. Insertion of Tn901 into a segment of about 20% of the Clo DF13 genome resulted in the loss of cloacin production, indicating that the structural gene coding for cloacin is located in this area. The sites of Tn901 insertions within Clo DF13 were more or less scattered; however, no Tn901 insertion sites were found in two distinct areas comprising 11 and 17%, respectively, of the Clo DF13 genome. Transposition of Tn901 DNA to the copy mutant Clo DF13-rep3 showed that the β-lactamase activity and the minimal inhibitory concentration of ampicillin were correlated to the number of plasmid copies per cell.     

Algal ecotypes: what they are and what they are not

A salt tolerant ecotype of Scenedesmus armatus from the Baltic Sea is reported. Salinity treatments also reveal variable plasticity of several morphological features of this alga The problem of drawing a sharp distinction between stable and plastic attributes is discussed and it is concluded that the terms ecotype and ecophene, as originally defined, are no longer applicable to algae. A redefinition of the ecotype is proposed.     

Influence of Protein and Ribonucleic Acid Synthesis on the Replication of the Bacteriocinogenic Factor Clo DF13 in Escherichia coli Cells and Minicells

The influence of ribonucleic acid (RNA) and protein synthesis on the replication of the cloacinogenic factor Clo DF13 was studied in Escherichia coli cells and minicells. In chromosomeless minicells harboring the Clo DF13 factor, Clo DF13 deoxyribonucleic acid (DNA) synthesis is slightly stimulated after inhibition of protein synthesis by chloramphenicol or puromycin and continues for more than 8 h. When minicells were treated with rifampin, a specific inhibitor of DNA-dependent RNA polymerase, Clo DF13 RNA and DNA synthesis appeared to stop abruptly. In cells, the Clo DF13 factor continues to replicate during treatment with chloramphenicol long after chromosomal DNA synthesis ceases. When rifampin was included during chloramphenicol treatment of cells, synthesis of Clo DF13 plasmid DNA was blocked completely. Isolated, supercoiled Clo DF13 DNA, synthesized in cells or minicells in the presence of chloramphenicol, appeared to be sensitive to ribonuclease and alkali treatment. These treatments convert a relatively large portion of the covalently closed Clo DF13 DNA to the open circular form, whereas supercoiled Clo DF13 DNA, isolated from non-chloramphenicol-treated cells or minicells, is not significantly affected by these treatments. These results indicate that RNA synthesis and specifically Clo DF13 RNA synthesis are involved in Clo DF13 DNA replication and that the covalently closed Clo DF13 DNA, synthesized in the presence of chloramphenicol, contains one or more RNA sequences. De novo synthesis of chromosomal and Clo DF13-specific proteins is not required for the replication of the Clo DF13 factor. Supercoiled Clo DF13 DNA, isolated from a polA107 (Clo DF13) strain which lacks the 5' --> 3' exonucleolytic activity of DNA polymerase I, is insensitive to ribonuclease or alkali treatment, indicating that in this mutant the RNA sequences are still removed from the RNA-DNA hybrid.     

Epibionts of Asellus aquaticus(L.) (Crustacea, Isopoda): an SEM study

1. This study presents a qualitative and quantitative survey of epibionts infesting two populations of the freshwater isopod Asellus aquaticus (L.). Using scanning electron microscopy, the prevalence, mean intensity, abundance, spatial preferences and distribution of organisms on various external surfaces were ascertained.
2. A diverse community, consisting primarily of protozoa and rotifers, utilised A. aquaticus as a substrate organism. Every individual A. aquaticus supported some form of epizoic life. Peritrich species comprised 89.2% of all epibionts. Carchesium polypinum, Pseudocarchesium aselli, Pseudocarchesium asellicola, Pseudocarchesium simulans and Opercularia hebes dominated the epifauna.
3. Highest epibiont burdens occurred on the mouthparts, the first few ventral segments and the gills and largely comprised the above peritrich species. Most epibionts exhibited some degree of site preference on the host. A distinct gill epifauna existed. Apart from Acineta tuberosa and Vorticella rotunda , which had equal prevalences on dorsal and ventral surfaces in one culture, most species largely avoided the dorsal surface.
4. Epibionts exhibited varying degrees of adaptation to life on a living substrate. They ranged from facultative species such as A. tuberosa , which are capable of colonising abiotic substrata, to Gymnodinoides aselli whose life cycle relies on interaction with A. aquaticus . Highly specialised species tended to exhibit the most pronounced and consistent site restriction.
5. Asellus aquaticus offers a highly tractable system for further study of substrate species–epibiont relationships.     

Algorithm of OMA for large-scale orthology inference

Background  

OMA is a project that aims to identify orthologs within publicly available, complete genomes. With 657 genomes analyzed to date, OMA is one of the largest projects of its kind.