The transfer RNA of certain Enterobacteriacae contain 2-methylthiozeatin riboside (ms2io6A) an isopentenyl adenosine derivative

Isopentenyl adenosine derivatives are always located adjacent to the 3' end of the anticodon in transfer RNA and have been implicated in certain biological functions. In the enteric bacterium, E. coli, the derivative is ms2i6A whereas in some plant associated bacteria the derivative is the hydroxylated form, ms2io6A. Anti-i6A immunoadsorbent chromatography has been employed to detect isopentenyl adenosine compounds. In the present study we show that the transfer RNA of three species of enteric bacteria, S. typhimurium, K. pneumoniae, and S. marcescens contains both ms2io6A and ms2i6A. Under the growth conditions utilized the ms2io6A is predominant. The presence of ms2io6A in Enterobacteriacae is particularly noteworthy since in previous work it has been found only in plant-associated species of bacteria.     

Influence of lipopolysaccharide and protein in the cell envelope on recipient capacity in conjugation of Salmonella typhimurium.

In crosses of Salmonella typhimurium FfinP301 lac+ to F- strains of S. typhimurium in broth, recipient strains which were rough mutants affected in the outer core region of the lipopolysaccharide gave an average of 1.4 Lac+ transconjugants per donor cell and over 50% of the donor and recipient cells in mating aggregates, whereas smooth recipient strains gave 0.08 Lac+ transconjugants and few cells in mating aggregates. Strains with mutations affecting the inner core of the lipopolysaccharide were usually poor recipients. When cells were mated on Millipore membrane filters, both smooth and rough strains gave ca. 1.0 Lac+ transconjugants per donor cell. Plasmids in Inc groups FI, FII, M, J, and I beta gave more transconjugants with rough than smooth strains, but there were no difference in crosses with plasmids in Inc groups T, L, P, N, and W. Strains with mutations in the ompA gene (deficient in Omp Ap = 33K = II* = conjugation protein) yielded only 0.02 Lac+ transconjugants per donor cell and few cells in mating aggregates. There was no indication of a deficiency of Omp Ap in smooth strains compared with rough strains. Reduced fertility of smooth recipients may occur because the O side chains of the lipopolysaccharide shield the recipient and reduce the frequency of stabilization of mating aggregates. However, gradient-of-transmission experiments indicated that once these mating aggregates are stabilized, they are equally stable in both smooth and rough recipients. Fertility was high in crosses of S. typhimurium Flac+ to Escherichia coli K-12 F- (0.75 Lac+ transconjugants per donor cell; over 50% of the cells in mating aggregates). In crosses of E. coli K-12 Flac+ to S. typhimurium smooth F-, ca. 10(-5) Lac+ transconjugants per donor cell were obtained; in crosses to rough recipient strains, fertility was increased 14-fold, and when the recipient was defective in the SA and LT host restriction systems, fertility was increased in additional 100-fold. Thus, both the lipopolysaccharide and the protein in the cell envelope of S. typhimurium were shown to be important in the recipient function in F-mediated conjugation.     

Characterization of a thermoactive endoglucanase isolated from a biogas plant metagenome

A metagenomic library from DNA isolated from a biogas plant was constructed and screened for thermoactive endoglucanases to gain insight into the enzymatic diversity involved in plant biomass breakdown at elevated temperatures. Two cellulase-encoding genes were identified and the corresponding proteins showed sequence similarities of 59% for Cel5A to a putative cellulase from Anaerolinea thermolimosa and 99% for Cel5B to a characterized endoglucanase isolated from a biogas plant reactor. The cellulase Cel5A consists of one catalytical domain showing sequence similarities to glycoside hydrolase family 5 and comprises 358 amino acids with a predicted molecular mass of 41.2 kDa. The gene coding for cel5A was successfully cloned and expressed in Escherichia coli C43(DE3). The recombinant protein was purified to homogeneity using affinity chromatography with a specific activity of 182 U/mg, and a yield of 74%. Enzymatic activity was detectable towards cellulose and mannan containing substrates and over a broad temperature range from 40 °C to 70 °C and a pH range from 4.0 to 7.0 with maximal activity at 55 °C and pH 5.0. Cel5A showed high thermostability at 60 °C without loss of activity after 24 h. Due to the enzymatic characteristics, Cel5A is an attractive candidate for the degradation of lignocellulosic material.

     

Versuche zur evolutorischen Entstehung der H?hlentiermerkmale

Ohne ZusammenfassungMit 16 Textabbildungen.     

Characterization of two novel heat-active α-galactosidases from thermophilic bacteria

Two genes (agal1 and agal2) encoding α-galactosidases were identified by sequence-based screening approaches. The gene agal1 was identified from a data set of a sequenced hot spring metagenome, and the deduced amino-acid sequence exhibited 99% identity to an α-galactosidase from the thermophilic bacterium Dictyoglomus thermophilum. The gene agal2 was identified from the whole genome sequence of the thermophile Meiothermus ruber. The amino-acid sequences exhibited structural motifs typical for glycoside hydrolase (GH) family 36 members and were also differentiated into different subgroups of this family. Recombinant production of the heat-active GH36b enzyme Agal1 (87 kDa) and GH36bt enzyme Agal2 (57 kDa) was carried out in E. coli. Agal1 exhibited a specific activity of 1502.3 U/mg at 80 °C, pH 6.5, and Agal2 225.4 U/mg at 60–70 °C, pH 6.5. Half-lives of 14 h (Agal1) and 39 h (Agal2) were obtained at 50 °C, and Agal1 showed half-lives of 4 and 2 h at 70 and 80 °C, respectively. In addition to the natural substrates melibiose, raffinose, and stachyose, 4NP α-d-galactopyranoside was hydrolyzed. Galactose was also liberated from locust bean gum. Both heat-active enzymes are attractive candidates for application in food and feed industry for high-temperature processes for the degradation of raffinose family oligosaccharides.

     

Characierization of monoclonal antibodies specific for isopentenyl adenosine derivatives occurring in transfer RNA

A family of isopentenyl adenosine derivatives are naturally occurring components of transfer RNA and are involved in several different functional roles in the cell. To facilitate the study of the biochemistry of these modified nucleosides we have raised monoclonal antibodies to N⁶-(Δ²-isopentenyl)adenosine and N⁶-(4-hydroxy-3-methyl-but-2-enyl)adenosine. The antibodies show considerable specificity and three characteristic types are distinguishable. The first type have the hydroxylated derivative as the preferred antigen, the second type have isopentenyl adenosine as the preferred antigen and a third type show a specificity for all isopentenyl-containing derivatives.     

Systemic complement activation in age-related macular degeneration

Dysregulation of the alternative pathway (AP) of complement cascade has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the elderly. To further test the hypothesis that defective control of complement activation underlies AMD, parameters of complement activation in blood plasma were determined together with disease-associated genetic markers in AMD patients. Plasma concentrations of activation products C3d, Ba, C3a, C5a, SC5b-9, substrate proteins C3, C4, factor B and regulators factor H and factor D were quantified in patients (n = 112) and controls (n = 67). Subjects were analyzed for single nucleotide polymorphisms in factor H (CFH), factor B-C2 (BF-C2) and complement C3 (C3) genes which were previously found to be associated with AMD. All activation products, especially markers of chronic complement activation Ba and C3d (p<0.001), were significantly elevated in AMD patients compared to controls. Similar alterations were observed in factor D, but not in C3, C4 or factor H. Logistic regression analysis revealed better discriminative accuracy of a model that is based only on complement activation markers Ba, C3d and factor D compared to a model based on genetic markers of the complement system within our study population. In both the controls' and AMD patients' group, the protein markers of complement activation were correlated with CFH haplotypes.This study is the first to show systemic complement activation in AMD patients. This suggests that AMD is a systemic disease with local disease manifestation at the ageing macula. Furthermore, the data provide evidence for an association of systemic activation of the alternative complement pathway with genetic variants of CFH that were previously linked to AMD susceptibility.