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CLCAs - a family of metalloproteases of intriguing phylogenetic distribution and with cases of substituted catalytic sites.
Get PDFThe zinc-dependent metalloproteases with His-Glu-x-x-His (HExxH) active site motif, zincins, are a broad group of proteins involved in many metabolic and regulatory functions, and found in all forms of life. Human genome contains more than 100 genes encoding proteins with known zincin-like domains. A survey of all proteins containing the HExxH motif shows that approximately 52% of HExxH occurrences fall within known protein structural domains (as defined in the Pfam database). Domain families with majority of members possessing a conserved HExxH motif include, not surprisingly, many known and putative metalloproteases. Furthermore, several HExxH-containing protein domains thus identified can be confidently predicted to be putative peptidases of zincin fold. Thus, we predict zincin-like fold for eight uncharacterised Pfam families. Besides the domains with the HExxH motif strictly conserved, and those with sporadic occurrences, intermediate families are identified that contain some members with a conserved HExxH motif, but also many homologues with substitutions at the conserved positions. Such substitutions can be evolutionarily conserved and non-random, yet functional roles of these inactive zincins are not known. The CLCAs are a novel zincin-like protease family with many cases of substituted active sites. We show that this allegedly metazoan family has a number of bacterial and archaeal members. An extremely patchy phylogenetic distribution of CLCAs in prokaryotes and their conserved protein domain composition strongly suggests an evolutionary scenario of horizontal gene transfer (HGT) from multicellular eukaryotes to bacteria, providing an example of eukaryote-derived xenologues in bacterial genomes. Additionally, in a protein family identified here as closely homologous to CLCA, the CLCA_X (CLCA-like) family, a number of proteins is found in phages and plasmids, supporting the HGT scenario
Sequence-specific Ni(II)-dependent peptide bond hydrolysis for protein engineering: active sequence optimization.
Get PDFIn previous studies we showed that Ni(II) ions can hydrolytically cleave a peptide bond preceding Ser/Thr in peptides of a general sequence RN-(Ser/Thr)-Xaa-His-Zaa-RC, where RN and RC are any peptide sequences. A peptide library screening, assisted by accurate measurements of reaction kinetics for selected peptides, demonstrated the preference for bulky and aromatic residues at variable positions Xaa and Zaa [A. Krężel, E. Kopera, A.M. Protas, A. Wysłouch-Cieszyńska, J. Poznański, W. Bal, J. Am. Chem. Soc., 132 (2010) 3355-3366]. In this work we used a similar strategy to find out whether the next residue downstream to Zaa may influence the reaction rate. Using an Ac-Gly-Ala-Ser-Arg-His-Zaa-Baa-Arg-Leu-NH2 library, with Zaa and Baa positions containing all common amino acids except of Cys, we found a very strong preference for aromatic residues in both variable positions. This finding significantly limits the range of useful Xaa, Zaa and Baa substitutions, thus facilitating the search for optimal sequences for protein engineering applications [E. Kopera, A. Belczyk-Ciesielska, W. Bal, PLoS One 7 (2012) e36350]
Transport of NaYF4: Er3+, Yb3+ upconverting nanoparticles into HeLa cells
Get PDFAn effective, simple and practically useful method to incorporate fluorescent nanoparticles inside live biological cells was developed. The internalization time and concentration dependence of a frequently used liposomal transfection factor Lipofectamine 2000 was studied. An users friendly, one-step technique to obtain water and organic solvents soluble Er3+and Yb3+ doped NaYF4 nanoparticles coated with polyvinylpyrrolidone (PVP) was obtained. The nanoparticles structural analysis confirmed the formation of nanocrystals of desired sizes and spectral properties. Internalization of NaYF4 nanoparticles in HeLa cervical cancer cells were determined at different nanoparticles concentrations and for incubation periods from 3 to 24 hours. The images revealed a redistribution of nanoparticles inside the cell, which increases with incubation time, concentration levels and depends on the presence of the transfection factor. The study identifies, for the first time, factors responsible for an effective endocytosis of the up-converting nanoparticles to HeLa cells. Thus the method could be applied to investigate a wide range of future “smart” theranostic agents. The nanoparticles incorporated into the liposomes appear to be very promising fluorescent probes for imaging real-time cellular dynamics
Structural and functional genomics of plasmid pSinA of Sinorhizobium sp. M14 encoding genes for the arsenite oxidation and arsenic resistance.
Get PDFPlasmid pSinA of Sinorhizobium sp. M14 (Alphaproteobacteria) is the first described, natural, self-transferable plasmid harboring a complete set of genes for oxidation of arsenite. Removal of this plasmid from cells of the host strain caused the loss of resistance to arsenic and heavy metals (Cd, Co, Zn and Hg) and abolished the ability to grow on minimal salt medium supplemented with sodium arsenite as the sole energy source. Plasmid pSinA was introduced into other representatives of Alphaproteobacteria which resulted in acquisition of new abilities concerning arsenic resistance and oxidation, as well as heavy metals resistance. Microcosm experiments revealed that plasmid pSinA can also be transferred via conjugation into other indigenous bacteria from microbial community of As-contaminated soils, including representatives of Alpha- and Gammaproteobacteria. Analysis of "natural" transconjugants showed that pSinA is functional (expresses arsenite oxidase) and is stably maintained in their cells after approximately 60 generations of growth under nonselective conditions. This work clearly demonstrates that pSinA is a self-transferable, broad-host-range plasmid, which plays an important role in horizontal transfer of arsenic metabolism genes
Regulation of toxin-antitoxin systems by proteolysis.
Get PDFToxin-antitoxin systems are widely distributed among many bacterial species, including human pathogens. Regulation of type II TA system functions, where both components are proteins, relies on proteolysis. In this paper, we discuss the significant features of the antitoxin proteins important for proteolysis. Examples of the best described processes of antitoxin degradation are presented as well as the particular case of the ω-ε-ζ TA system
Tandem Multiplication of the IS26-Flanked Amplicon with the blaSHV-5 Gene within Plasmid p1658/97.
Get PDFThe IncF plasmid p1658/97 (~125kb) from Escherichia coli isolates recovered during a clonal outbreak in a Warsaw, Poland, hospital in 1997 contains the extended-spectrum β-lactamase (ESBL) gene blaSHV-5, originated from the Klebsiella pneumoniae chromosome. A region containing the blaSHV-5 gene is flanked by two IS26 copies and its copy number multiplies spontaneously within p1658/97 and RecA-deficient E. coli strains. Here we demonstrate that the amplified IS26-blaSHV-5 units were arranged in tandems, containing up to more than 10 units, which could raise ceftazidime MICs for host strains from 4μg/ml to more than 128μg/ml. Successive deletions within p1658/97, located outside the amplifiable module and encompassing even as little as ~15% of the plasmid, blocked the amplification. Moreover, the complementing re-introduction of the deleted fragments in trans did not restore the process. Similarly, insertions of a 1-kb DNA fragment into the amplicon inhibited its self-multiplication ability. The module was able to transmit into another IS26-containing plasmid by recombination. The results prompted us to speculate that local DNA structure, especially favourable in p1658/97, might have been responsible for the IS26-blaSHV-5 multiplication ability
The distribution of ciliates on Ecology Glacier (King George Island, Antarctica): relationships between species assemblages and environmental parameters
Get PDFCiliates are important consumers of pico- and
nano-sized producers, are nutrient regenerators, and are an
important food source for metazoans. To date, ecological
research on ciliates has focused on marine ecosystems
rather than on glacier habitats. This paper presents the first
major study on ciliates from the Ecology Glacier (South
Shetland Islands, Antarctica). The objective of the study
was to investigate the structure and spatial distribution of
ciliate communities and to identify the environmental
factors determining the structure of the assemblages.
Microbial communities were collected from three habitats:
surface snow, cryoconite holes, and glacier streams. Sampling
was carried out every 3–4 days from January 17 to
February 24, 2012. A total of 18 ciliate taxa were identified.
The species richness, abundance, and biomass of
protozoa differed significantly between the stations studied
with the lowest numbers in streams on the glacier surface
and the highest numbers in cryoconite holes. The RDA
performed to specify the direct relationships between the
abundance of ciliate taxa and environmental variables
showed obvious differences between studied habitats. The
analysis showed that all variables together explained
62.4 % of total variance. However, variables thatsignificantly explained the variance in ciliate communities
in cryoconite holes, snow, and surface streams were temperature,
conductivity, and total nitrogen. Further research
is required to explain the impact of biotic factors influencing
the presence of ciliates, including the abundance of
bacteria, microalgae, and small Metazoa
Genetic diversity in a moulting colony of southern elephant seals in comparison with breeding colonies
Get PDFABSTRACT: Southern elephant seals Mirounga leonina migrate seasonally between pelagic foraging
areas in the Southern Ocean and breeding and moulting sites on subantarctic islands. Here
we characterized genetic diversity of the elephant seal moulting colony from King George Island
(KGI), South Shetlands Archipelago, in comparison with breeding colonies described in earlier
studies. Although KGI serves as a breeding site, the numbers of elephant seals are up to 3 times
higher during the moulting season, suggesting post-breeding immigration from other sites, or that
large numbers of individuals skip the breeding season every year. High haplotype diversity and a
high percentage of shared haplotypes is consistent with the hypothesis of immigration from other
South Atlantic colonies, which is also supported by satellite tracking data from earlier studies.
Estimates of effective population size at both mtDNA and microsatellite loci were unexpectedly
high compared with the census size of the KGI colony, suggesting that they were elevated due to
the presence of immigrants. However, we detected few immigrants and no genetic structure in the
KGI colony, which could result from the genetic similarity between KGI and other breeding
colonies from the South Atlantic, but could also be an effect of an insufficient sampling scheme.
These results show the need for genetic monitoring of southern elephant seal colonies throughout
their annual cycle to better understand the range of their seasonal movements and patterns of
gene flow. The southern elephant seal serves as a model to study links between spatio-temporal
environmental variability, population dynamics, and individual movements, physiology and
reproductive behavior. Understanding the patterns of dispersal and gene flow in this species is
essential to adequately address these general questions
Tobacco LSU-like protein couples sulphur-deficiency response with ethylene signalling pathway
Get PDFMost genes from the plant-specific family encoding Response to Low Sulphur (LSU)-like proteins are strongly induced in sulphur (S)-deficient conditions. The exact role of these proteins remains unclear; however, some data suggest their importance for plants’ adjustment to nutrient deficiency and other environmental stresses. This work established that the regulation of ethylene signalling is a part of plants’ response to S deficiency and showed the interaction between UP9C, a tobacco LSU family member, and one of the tobacco isoforms of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO2A). Increase in ethylene level induced by S deficiency does not take place in tobacco plants with UP9C expressed in an antisense orientation. Based on transcriptomics data, this work also demonstrated that the majority of tobacco’s response to S deficiency is misregulated in plants expressing UP9C-antisense. A link between response to S deficiency, ethylene sensing, and LSU-like proteins was emphasized by changes in expression of the genes encoding ethylene receptors and F-box proteins specific for the ethylene pathway