Molecular basis for the maintenance of envelope integrity in Selenomonas ruminantium: cadaverine biosynthesis and covalent modification into the peptidoglycan play a major role.

Polyamine is a small organic polycation composed of a hydrocarbon backbone with multiple amino groups which ubiquitously exists in all living organisms from bacteria to higher animals. The critical step of polyamine biosynthesis generally includes the amino acid-decarboxylating reaction to produce the primary diamines, such as a synthesis of putrescine (NH(3)(+)·(CH(2))(4)·NH(3)(+)) from ornithine, and cadaverine (NH(3)(+)·(CH(2))(5)·NH(3)(+)) from lysine, which are catalyzed by pyridoxal-5'-phosphate (PLP; vitamin B(6))-dependent decarboxylases. Synthesized polyamines are implicated in a wide variety of cytoplasmic reactions such as DNA replication and protein synthesis, and are essential for proper growth of the organisms. However, in Selenomonas ruminantium, a strictly anaerobic Gram-negative bacterium dominant in sheep rumen, cadaverine displays its function in a quite distinctive scheme compared to the general bacteria reported. It serves as an essential constituent of the peptidoglycan for the maintenance of envelope integrity through an interaction with the periplasm-exposed SLH domain of Mep45, the outer membrane protein of this bacterium. Furthermore, cytoplasmic biosynthesis of cadaverine occurs totally in a eukaryotic-like manner rather than in a conventional way of bacteria. Lysine/ornithine decarboxylase (LDC/ODC), a PLP-dependent enzyme responsible for cadaverine synthesis in this bacterium, displays significant homology to the eukaryotic ODC but not to the general bacterial LDC nor ODC, and its activity is tightly regulated by antizyme-mediated proteolysis, a regulatory process generally found in eukaryotes. These findings represent the biological diversity of this bacterium beyond the preexisting knowledge related to the polyamine-physiology, cell envelope-architecture, and the regulatory system for the enzyme. In this review we will describe (i) the cadaverine-containing peptidoglycan of S. ruminantium: its chemical structure, biosynthesis, and biological function, and (ii) cellular biosynthesis of cadaverine by LDC/ODC and its antizyme-mediated regulation. In addition, we will briefly refer to (iii) the phylogenetic position and characteristics of S. ruminantium and its unique cadaverine-physiology.

Cadaverine covalently linked to peptidoglycan is required for interaction between the peptidoglycan and the periplasm-exposed S-layer-homologous domain of major outer membrane protein Mep45 in Selenomonas ruminantium.

The peptidoglycan of Selenomonas ruminantium is covalently bound to cadaverine (PG-cadaverine), which likely plays a significant role in maintaining the integrity of the cell surface structure. The outer membrane of this bacterium contains a 45-kDa major protein (Mep45) that is a putative peptidoglycan-associated protein. In this report, we determined the nucleotide sequence of the mep45 gene and investigated the relationship between PG-cadaverine, Mep45, and the cell surface structure. Amino acid sequence analysis showed that Mep45 is comprised of an N-terminal S-layer-homologous (SLH) domain followed by α-helical coiled-coil region and a C-terminal ß-strand-rich region. The N-terminal SLH domain was found to be protruding into the periplasmic space and was responsible for binding to peptidoglycan. It was determined that Mep45 binds to the peptidoglycan in a manner dependent on the presence of PG-cadaverine. Electron microscopy revealed that defective PG-cadaverine decreased the structural interactions between peptidoglycan and the outer membrane, consistent with the proposed role for PG-cadaverine. The C-terminal ß-strand-rich region of Mep45 was predicted to be a membrane-bound unit of the 14-stranded ß-barrel structure. Here we propose that PG-cadaverine possesses functional importance to facilitate the structural linkage between peptidoglycan and the outer membrane via specific interaction with the SLH domain of Mep45.

Characterization of a major envelope protein from the rumen anaerobe Selenomonas ruminantium OB268.

Cell envelopes from the Gram-negative staining but phylogenetically Gram-positive rumen anaerobe Selenomonas ruminantium OB268 contained a major 42 kDa heat modifiable protein. A similarly sized protein was present in the envelopes of Selenomonas ruminantium D1 and Selenomonas infelix. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of Triton X-100 extracted cell envelopes from S. ruminantium OB268 showed that they consisted primarily of the 42 kDa protein. Polyclonal antisera produced against these envelopes cross-reacted only with the 42 kDa major envelope proteins in both S. ruminantium D1 and S. infelix, indicating a conservation of antigenic structure among each of the major envelope proteins. The N-terminus of the 42 kDa S. ruminantium OB268 envelope protein shared significant homology with the S-layer (surface) protein from Thermus thermophilus, as well as additional envelope proteins containing the cell surface binding region known as a surface layer-like homologous (SLH) domain. Thin section analysis of Triton X-100 extracted envelopes demonstrated the presence of an outer bilayer over-laying the cell wall, and a regularly ordered array was visible following freeze-fracture etching through this bilayer. These findings suggest that the regularly ordered array may be composed of the 42 kDa major envelope protein. The 42 kDa protein has similarities with regularly ordered outer membrane proteins (rOMP) reported in certain Gram-negative and ancient eubacteria.

Localization of phytase in Selenomonas ruminantium and Mitsuokella multiacidus by transmission electron microscopy.

The localization of phytase (myo-inositol-hexaphosphate phosphohydrolase) in the ruminal bacteria, Selenomonas ruminantium JY35 and Mitsuokella multiacidus 46/5(2), was determined with transmission electron microscopy. Phosphate produced from the enzymatic dephosphorylation of the calcium salt of phytic acid is precipitated as calcium phosphate. The calcium is then replaced with lead to produce electron-dense lead phosphate. This deposition of lead phosphate localized phytase in S. ruminantium JY35 and M. multiacidus 46/5(2) to the outer membrane, and confirmed intracellular expression of the enzyme in Escherichia coli pSrP.2, the recombinant clone which possesses the gene (phyA) encoding phytase (phyA) in S. ruminantium.