Isolation of Three New Surface Layer Protein Genes ( slp ) from Lactobacillus brevis ATCC 14869 and Characterization of the Change in Their Expression under Aerated and Anaerobic Conditions

Abstract

Two new surface layer (S-layer) proteins (SlpB and SlpD) were characterized, and three slp genes (slpB, slpC, and slpD) were isolated, sequenced, and studied for their expression in Lactobacillus brevis neotype strain ATCC 14869. Under different growth conditions, L. brevis strain 14869 was found to form two colony types, smooth (S) and rough (R), and to express the S-layer proteins differently. Under aerobic conditions R-colony type cells produced SlpB and SlpD proteins, whereas under anaerobic conditions S-colony type cells synthesized essentially only SlpB. Anaerobic and aerated cultivations of ATCC 14869 cells in rich medium also resulted in S-layer protein patterns similar to those of the S-and R-colony type cells, respectively. Electron microscopy suggested the presence of only a single S-layer with an oblique structure on the cells of both colony forms. The slpB and slpC genes were located adjacent to each other, whereas the slpD gene was not closely linked to the slpB-slpC gene region. Northern analyses confirmed that both slpB and slpD formed a monocistronic transcription unit and were effectively expressed, but slpD expression was induced under aerated conditions. slpC was a silent gene under the growth conditions tested. The amino acid contents of all the L. brevis ATCC 14869 S-layer proteins were typical of S-layer proteins, whereas their sequence similarities with other S-layer proteins were negligible. The interspecies identity of the L. brevis S-layer proteins was mainly restricted to the Nterminal regions of those proteins. Furthermore, Northern analyses, expression of a PepI reporter protein under the control of the slpD promoter, and quantitative real-time PCR analysis of slpD expression under aerated and anaerobic conditions suggested that, in L. brevis ATCC 14869, the variation of S-layer protein content involves activation of transcription by a soluble factor rather than DNA rearrangements that are typical for most of the S-layer phase variation mechanisms known.Surface layers (S layers) are monomolecular crystalline arrays of proteinaceous subunits present in almost all archaea and all major phylogenetic groups of bacteria (26,33). Most of the S layers are composed of subunits of a single protein or glycoprotein species capable of forming symmetrical arrays and covering the cell surface during all stages of growth. Slayer proteins commonly contain high numbers of acidic and hydrophobic amino acids but lack overall amino acid sequence homology with corresponding proteins from unrelated species. Moreover, a low pI is typical for S-layer proteins (31) except for those from different lactobacilli (5, 40) and Methanothermus fervidus (6). Diverse functions have been proposed for S layers, such as acting as molecular sieves, protective coats, molecular and ion traps, cell shape determinants, and promoters for cell adhesion and surface recognition (26). There is also increasing evidence that S-layer-carrying bacteria may use Slayer variation, by expressing alternative S-layer protein genes, for ada...

Effect of Environmental Stresses on S-Layer Production in Lactobacillus acidophilus ATCC 4356

“…The S-layer has been detected in a few species of the genus Lactobacillus (Avall-Jaaskelainen & Palva, 2005). Lactobacilli surface layer proteins (S-layers) are generally monomolecular crystalline arrays exhibiting a morphologically similar, oblique lattice structure and representing 10-15% of the total protein content of the bacterial cell wall (Avall-Jaaskelainen & Palva, 2005;Jakava-Viljanen et al, 2002). Several reports have appeared in which functions of S-layer are described or assumed (Boot et al, 1993;Jakava-Viljanen et al, 2002;Sara & Sleytr, 2000).…”

“…amylovorus, L. gallinarum, L. crispatus, L. brevis, L. gasseri and L. johnsonii (AvallJaaskelainen & Palva, 2005;Jakava-Viljanen et al, 2002;Ventura et al, 2002). There is also increasing evidence that S-layer-carrying bacteria may use S-layer variation, by expressing alternative S-layer protein genes, for adaptation to different stress factors such as the immune response of the host for pathogens and drastic changes in the environmental conditions for nonpathogens Frece et al, 2005;Jakava-Viljanen et al, 2002;Pouwels et al, 1998;Sara & Sleytr, 2000). According to Pouwels' www.intechopen.com study (1998), phase variation or antigenic variation, as a result of inversion of the slp segment, might enable Lactobacillus acidophilus bacteria to better adhere to specific regions of the mucosa.…”

“…Lactobacilli surface layer proteins (S-layers) are generally monomolecular crystalline arrays exhibiting a morphologically similar, oblique lattice structure and representing 10-15% of the total protein content of the bacterial cell wall (Avall-Jaaskelainen & Palva, 2005;Jakava-Viljanen et al, 2002). Several reports have appeared in which functions of S-layer are described or assumed (Boot et al, 1993;Jakava-Viljanen et al, 2002;Sara & Sleytr, 2000). However, no general function has been identified for S-layer proteins, but several lactobacillar S-layers have been identified as putative adhesions with affinity for intestinal epithelial cells, extracellular matrices and/ or to lipoteichoic acid (LTA) of other bacterial species (Avall-Jaaskelainen & Palva, 2005;Buck et al, 2005;Frece et al, 2005;Garrote et al, 2004;Hynonen et al, 2002;Kos et al, 2003;Velez et al, 2007;Vidgren et al, 1992).…”

Cell surface proteins play an important role in probiotic activities of Lactobacillus reuteri

“…Several reports have appeared describing or assuming functions of cell surface proteins which includes its function as a protective sheath against hostile environmental agents, a cell shape determinant, and a sheath to mask properties of the underlying cell wall such as charge and phage receptors [5,6]. There is also increasing evidence that bacteria may employ variation in surface proteins, by expressing alternative cell surface protein genes, for adaptation to different stress factors, such as the immune response of the host for pathogens and drastic changes in the environmental conditions for nonpathogens [6,7]. It has been proposed that cell surface proteins are involved in cell protection and surface recognition and that they could be potential mediators in the initial steps involved in autoaggregation and adhesion [8][9][10].…”

Xylan-mediated aggregation of Lactobacillus brevis and its relationship with the surface properties and mucin-mediated aggregation of the bacteria

“…The cell surface proteins of LAB strains were analyzed as previously described. 18) Harvested cells equivalent to 0.5 mL of broth were suspended in 40 μL of Laemmli sample buffer and incubated for 4 min at 95°C. After centrifugation, the supernatant was analyzed by SDS-PAGE using 10% Mini-PROTEAN TGX Gel (Bio-Rad Laboratories, Hercules, CA) with Tris/glycine/SDS buffer and stained with Coomassie brilliant blue.…”