The acylation state of surface lipoproteins of mollicute Acholeplasma laidlawii

Acylation of the N-terminal Cys residue is an essential, ubiquitous, and uniquely bacterial posttranslational modification that allows anchoring of proteins to the lipid membrane. In gram-negative bacteria, acylation proceeds through three sequential steps requiring lipoprotein diacylglyceryltransferase, lipoprotein signal peptidase, and finally lipoprotein N-acyltransferase. The apparent lack of genes coding for recognizable homologs of lipoprotein N-acyltransferase in gram-positive bacteria and Mollicutes suggests that the final step of the protein acylation process may be absent in these organisms. In this work, we monitored the acylation state of eight major lipoproteins of the mollicute Acholeplasma laidlawii using a combination of standard two-dimensional gel electrophoresis protein separation, blotting to nitrocellulose membranes, and MALDI-MS identification of modified N-terminal tryptic peptides. We show that for each A. laidlawii lipoprotein studied a third fatty acid in an amide linkage on the N-terminal Cys residue is present, whereas diacylated species were not detected. The result thus proves that A. laidlawii encodes a lipoprotein N-acyltransferase activity. We hypothesize that N-acyltransferases encoded by genes non-homologous to N-acyltransferases of gram-negative bacteria are also present in other mollicutes and gram-positive bacteria.     

Molecular analysis of Gaucher disease: distribution of eight mutations and the complete gene deletion in 27 patients from Germany

Gaucher disease is the most common lysosomal storage disease with a high prevalence in the Ashkenazi Jewish population but it is also present in other populations. The presence of eight mutations (1226G, 1448C, IVS2+1, 84GG, 1504T, 1604T, 1342C and 1297T) and the complete deletion of the β-glucocerebrosidase gene was investigated in 25 unrelated non-Jewish patients with Gaucher’s disease in Germany. In the Jewish population, three of these mutations account for more than 90% of all mutated alleles. In addition, relatives of two patients were included in our study. Restriction fragment length polymorphism analysis and sequencing of PCR products obtained from DNA of peripheral blood leukocytes was performed for mutation analysis. Gene deletion was detected by comparison of radioactively labelled PCR fragments of both the functional β-glucocerebrosidase gene and the pseudogene. Among the unrelated patients, 50 alleles were investigated and the mutations identified in 35 alleles (70%), whereas 15 alleles (30%) remained unidentified. The most prevalent mutation in our group of patients was the 1226G (370^Asn→Ser) mutation, accounting for 18 alleles (36%), followed by the 1448C (444^Leu→Pro) mutation, that was found in 12 alleles (24%). A complete gene deletion was present in two alleles (4%). The IVS1+2 (splicing mutation), the 1504T (463^Arg→Cys) as well as the 1342C (409^Asp→His) mutations were each present in one allele (2%). None of the alleles carried the 84GG (frameshift), 1604A (496^Arg→His) or the 1297T (394^Val→Leu) mutation. This distribution is different from the Ashkenazi Jewish population but is similar to other Caucasian groups like the Spanish and Portuguese populations. Our results confirm the variability of mutation patterns in Gaucher patients of different ethnic origin. All patients were divided into nine groups according to their genotype and their clinical status was related to the individual genotype. Genotype/phenotype characteristics of the 1226G, 1448C, and 1342C mutations of previous studies were confirmed by our results. Received: 19 November 1996 / Revised: 29 January 1997     

Proteins of the Rpf (resuscitation promoting factor) family are peptidoglycan hydrolases

The secreted Micrococcus luteus protein, Rpf, is required for successful resuscitation of dormant "non-culturable" M. luteus cells and for growth stimulation in poor media. The biochemical mechanism of Rpf action remained unknown. Theoretical predictions of Rpf domain architecture and organization, together with a recent NMR analysis of the protein structure, indicate that the conserved Rpf domain has a lysozyme-like fold. In the present study, we found that both the secreted native protein and the recombinant protein lyse crude preparations of M. luteus cell walls. They also hydrolyze 4-methylumbelliferyl-beta-D-N,N',N'-triacetylchitotrioside, a synthetic substrate for peptidoglycan muramidases, with optimum activity at pH 6. The Rpf protein also has weak proteolytic activity against N-CBZ-Gly-Gly-Arg-beta-naphthylamide, a substrate for trypsin-like enzymes. Rpf activity towards 4-methylumbelliferyl-beta-D-N,N',N'-triacetylchitotrioside was reduced when the glutamate residue at position 54, invariant for all Rpf family proteins and presumably involved in catalysis, was altered. The same amino acid substitution resulted in impaired resuscitation activity of Rpf. The data indicate that Rpf is a peptidoglycan-hydrolyzing enzyme, and strongly suggest that this specific activity is responsible for its growth promotion and resuscitation activity. A possible mechanism of Rpf-mediated resuscitation is discussed.     

Effect of synthetic cyclopentane β,β′-triketones on amino acid metabolism in roots of buckwheat (<Emphasis Type="Italic">Fagopyrum esculentum</Emphasis> Moench.) seedlings

Germination of buckwheat seeds in solutions of synthetic mono- and tricyclic cyclopentane β,β′-triketones of various concentrations was accompanied by inhibition of seedling root growth and changes in the contents of glutamate, γ-aminobutyric acid, proline, glutamine, and alanine. The monocyclic triketone also affected the amount of isoleucine. It is likely that the increase in proline content is a nonspecific response significant for enhancing stress tolerance in seedlings.     

Rpf proteins are the factors of reactivation of the dormant forms of actinobacteria

As the response to unfavorable growth conditions, nonsporulating mycobacteria transform into the dormant state with the concomitant formation of the specialized dormant forms characterized by low metabolic activity and resistance to antibiotics. Such dormant cells can be reactivated under the influence of several factors including proteins of Rpf (Resuscitation promoting factor) family, which possess peptidoglycan hydrolase activity and were considered to belong to the group of the autocrine growth factors of the bacteria. Remarkable interest toward Rpf family is determined by its par-ticipation in resuscitation of the dormant forms of Mycobacterium tuberculosis, what in turn is the key element in resuscitation of the latent tuberculosis – an infectious disease that affects one third of the World’s population. Experiments with Rpf mutant forms and with strains deleted in these proteins revealed a relationship between the enzymatic activity of this protein and its ability to resuscitate mycobacteria both in vitro and in vivo. This review discusses possible mechanisms of Rpf action including those related to possible participation of the products of mycobacterial Rpf-mediated cell wall hydrolysis (muropeptides) as signaling molecules. The unique ability of Rpf proteins to resuscitate the dormant forms of mycobacteria and to stimulate their proliferation would allow these proteins to occupy their niche in medicine–in diagnostics and in creation of antituberculosis subunit vaccines.     

Scale- and taxon-dependent patterns of plant diversity in steppes of Khakassia,South Siberia (Russia)

The drivers of plant richness at fine spatial scales in steppe ecosystems are still not sufficiently understood. Our main research questions were: (i) How rich in plant species are the natural steppes of Southern Siberia compared to natural and semi-natural grasslands in other regions of the Palaearctic? (ii) What are the main environmental drivers of the diversity patterns in these steppes? (iii) What are the diversity–environment relationships and do they vary between spatial scales and among different taxonomic groups? We sampled the steppe vegetation (vascular plants, bryophytes and lichens) in Khakassia (Russia) with 39 nested-plot series (0.0001–100-m² plot size) and 54 additional 10-m² quadrats across the regional range of steppe types and measured various environmental variables. We measured β-diversity using z-values of power-law species–area relationships. GLM analyses were performed to assess the importance of environmental variables as predictors of species richness and z-value. Khakassian steppes showed both high α- and β-diversity. We found significant scale dependence for the z-values, which had their highest values at small spatial scales and then decreased exponentially. Total species richness was controlled predominantly by heat load index, mean annual precipitation, humus content and soil skeleton content. The positive role of soil pH was evident only for vascular plant species richness. Similar to other studies, we found that the importance of environmental factors strongly differed among taxonomic groups and across spatial scales, thus highlighting the need to study more than one taxon and more than one plot size to get a reliable picture.