Sławomir Dąbrowski

Anaerobic digestion of cattle manure and a mixture of cattle manure with glycerol trioleate (GTO) was studied in lab-scale, continuously stirred tank reactors (CSTR) operated at 37 degrees C. The reactor codigesting manure and lipids exhibited a significantly higher specific methane yield and a higher removal of VS than the reactor treating manure. Microbial population analysis done by cultivation--most probable number (MPN) test and specific methanogenic activity (SMA) measurement, revealed higher MPN and increased SMA of methanogenic populations of biomass from the reactor codigesting manure and lipids. Spatial microbial distribution and activity was studied in digested materials fractionated into size of particles > 200 microm, 50-200 microm and 0.45-50 microm. With manure, the main pool of methanogenic activity from propionate, butyrate and hydrogen was associated with the particles > 200 microm, while the activity of acetotrophic methanogens was uniformly distributed in all fractions. When digesting manure and lipids, an enhanced methanogenesis was detected both for particles > 200 microm and the 50-200 microm fraction. The molecular methods--temperature gradient gel electrophoresis (TGGE), cloning library and sequencing of 16S rDNA--showed presence of a restricted number of archaeal species in both reactors. The vast majority of clones was phylogenetically most closely related to Methanosarcina siciliae.

Phage vB_Tsc2631 infects the extremophilic bacterium Thermus scotoductus MAT2631 and uses the Ts2631 endolysin for the release of its progeny. The Ts2631 endolysin is the first endolysin from thermophilic bacteriophage with an experimentally validated catalytic site. In silico analysis and computational modelling of the Ts2631 endolysin structure revealed a conserved Zn2+ binding site (His30, Tyr58, His131 and Cys139) similar to Zn2+ binding site of eukaryotic peptidoglycan recognition proteins (PGRPs). We have shown that the Ts2631 endolysin lytic activity is dependent on divalent metal ions (Zn2+ and Ca2+). The Ts2631 endolysin substitution variants H30N, Y58F, H131N and C139S dramatically lost their antimicrobial activity, providing evidence for the role of the aforementioned residues in the lytic activity of the enzyme. The enzyme has proven to be not only thermoresistant, retaining 64.8% of its initial activity after 2 h at 95°C, but also highly thermodynamically stable (Tm = 99.82°C, ΔHcal = 4.58 × 10(4) cal mol(-1)). Substitutions of histidine residues (H30N and H131N) and a cysteine residue (C139S) resulted in variants aggregating at temperatures ≥75°C, indicating a significant role of these residues in enzyme thermostability. The substrate spectrum of the Ts2631 endolysin included extremophiles of the genus Thermus but also Gram-negative mesophiles, such as Escherichia coli, Salmonella panama, Pseudomonas fluorescens and Serratia marcescens. The broad substrate spectrum and high thermostability of this endolysin makes it a good candidate for use as an antimicrobial agent to combat Gram-negative pathogens.

Single-stranded-DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination and repair in bacteria, archaea and eukarya. This paper reports the identification and characterization of the SSB-like proteins of the thermophilic bacteria Thermus thermophilus and Thermus aquaticus. These proteins (TthSSB and TaqSSB), in contrast to their known counterparts from mesophilic bacteria, archaea and eukarya, are homodimers, and each monomer contains two ssDNA-binding domains with a conserved OB (oligonucleotide/oligosaccharide-binding) fold, as deduced from the sequence analysis. The N-terminal domain is located in the region from amino acid 1 to 123 and the C-terminal domain is located between amino acids 124 and 264 or 266 in TthSSB and TaqSSB, respectively. Purified TthSSB or TaqSSB binds only to ssDNA and with high affinity. The binding site size for TaqSSB and TthSSB protein corresponds to 30-35 nucleotides. It is concluded that the SSBs of thermophilic and mesophilic bacteria, archaea and eukarya share a common core ssDNA-binding domain. This ssDNA-binding domain was presumably present in the common ancestor to all three major branches of life.

In this study, we present the discovery and characterization of a highly thermostable endolysin from bacteriophage Ph2119 infecting Thermus strain MAT2119 isolated from geothermal areas in Iceland. Nucleotide sequence analysis of the 16S rRNA gene affiliated the strain with the species Thermus scotoductus. Bioinformatics analysis has allowed identification in the genome of phage 2119 of an open reading frame (468 bp in length) coding for a 155-amino-acid basic protein with an Mr of 17,555. Ph2119 endolysin does not resemble any known thermophilic phage lytic enzymes. Instead, it has conserved amino acid residues (His(30), Tyr(58), His(132), and Cys(140)) that form a Zn(2+) binding site characteristic of T3 and T7 lysozymes, as well as eukaryotic peptidoglycan recognition proteins, which directly bind to, but also may destroy, bacterial peptidoglycan. The purified enzyme shows high lytic activity toward thermophiles, i.e., T. scotoductus (100%), Thermus thermophilus (100%), and Thermus flavus (99%), and also, to a lesser extent, toward mesophilic Gram-negative bacteria, i.e., Escherichia coli (34%), Serratia marcescens (28%), Pseudomonas fluorescens (13%), and Salmonella enterica serovar Panama (10%). The enzyme has shown no activity against a number of Gram-positive bacteria analyzed, with the exception of Deinococcus radiodurans (25%) and Bacillus cereus (15%). Ph2119 endolysin was found to be highly thermostable: it retains approximately 87% of its lytic activity after 6 h of incubation at 95°C. The optimum temperature range for the enzyme activity is 50°C to 78°C. The enzyme exhibits lytic activity in the pH range of 6 to 10 (maximum at pH 7.5 to 8.0) and is also active in the presence of up to 500 mM NaCl.