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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Anaerobe 15 (2009) 74–81 Contents lists available at ScienceDirect Anaerobe journal homepage: www.elsevier.com/locate/anaerobe Molecular biology, genetics and biotechnology Physical and genetic characterization of an outer-membrane protein (OmpM1) containing an N-terminal S-layer-like homology domain from the phylogenetically Gram-positive gut anaerobe Mitsuokella multacida M.L. Kalmokoff a, J.W. Austin b, T.D. Cyr c, M.A. Hefford c, R.M. Teather d, L.B. Selinger e,* a Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada, Kentville, NS, Canada B4N 1J5 b Microbiology Research Division, Bureau of Microbial Hazards, Food Directorate, Health Products and Food Branch, Health Canada, Banting Research Centre, PL#2204A2, Tunney’s Pasture, Ottawa, Canada K1A 0L2 c Centre for Biologics Research, Biologics and Genetic Therapies Directorate, Health Canada Banting Research Centre, PL#2201C, Tunney’s Pasture, Ottawa, Canada K1A 0L2 d Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada T1J 4B1 e Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge, AB, Canada T1K 3M4 article info abstract Article history: Thin sectioning and freeze-fracture-etch of the ovine ruminal isolate Mitsuokella multacida strain 46/5(2) Received 19 February 2008 revealed a Gram-negative envelope ultra-structure consisting of a peptidoglycan wall overlaid by an Received in revised form outer membrane. Sodium-dodecyl-sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of 17 June 2008 whole cells, cell envelopes and Triton X-100 extracted envelopes in combination with thin-section and Accepted 9 January 2009 N-terminal sequence analyses demonstrated that the outer membrane contained two major proteins (45 Available online 20 January 2009 and 43 kDa) sharing identical N-termini (A-A-N-P-F-S-D-V-P-A-D-H-W-A-Y-D). A gene encoding a protein with a predicted N-terminus identical to those of the 43 and 45 kDa outer-membrane proteins Keywords: Outer membrane was cloned. The 1290 bp open reading frame encoded a 430 amino acid polypeptide with a predicted Outer-membrane protein molecular mass of 47,492 Da. Cleavage of a predicted 23 amino acid leader sequence would yield Gram-negative a protein with a molecular mass of 45,232 Da. Mass spectroscopic analysis confirmed that the cloned Porin gene (ompM1) encoded the 45 kDa outer-membrane protein. The N-terminus of the mature OmpM1 Surface-layer homology (SLH) domain protein (residues 24–70) shared homology with surface-layer homology (SLH) domains found in a wide variety of regularly structured surface-layers (S-layers). However, the outer-membrane locale, resistance to denaturation by SDS and high temperatures and the finding that the C-terminal residue was a phenylalanine suggested that ompM1 encoded a porin. Threading analysis in combination with the identification of membrane spanning domains indicated that the C-terminal region of OmpM1 (residues 250–430) likely forms a 16-strand b-barrel and appears to be related to the unusual N-terminal SLH- domain-containing b-barrel-porins previously described in the cyanobacterium Synechococcus PCC6301. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction phytate and to reduce the need to supplement diets with additional phosphates. Mitsuokella multacida is a Gram-negative staining obligatory Although previously classified as Bacteriodes multiacidus [5], anaerobic rod shaped bacterium, having wide distribution in both analysis of 16S rRNA resulted in the re-alignment of this species to the rumen and gastrointestinal tracts of various warm blooded the Group IX of the Clostridium phylum (often referred to as the mammals [1–3]. Recent interest in Mitsuokella spp. results ‘‘Sporomusa branch’’), a group containing a heterogeneous primarily from the discovery of a number of isolates which produce collection of organisms [6,7]. More recently, the species has been phytase activity [3,4]. Phytase producing ruminal bacteria repre- placed into Family VII Acidaminococcaceae within the Firmicutes sent a potential source of this enzyme for inclusion into the diet of phylum in Gram-positive eubacteria [8]. One of the unusual monogastric animals to alleviate excreted phosphate in the form of features of the species within the Family Acidaminococcaceae is that many stain Gram-negative. Indeed electron microscopic examination of thin sections has confirmed the presence of what appears to be an outer membrane in many of these genera * Corresponding author. Tel.: þ1 403 329 2309; fax: þ1 403 329 2082. including the Sporomusa [9], Selenomonas [10], Pectinatus [11], E-mail address: [email protected] (L.B. Selinger). Megasphera [12], Acidaminococcus [13], Anaerovibrio [14], Dialister 1075-9964/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2009.01.001 Author's personal copy M.L. Kalmokoff et al. / Anaerobe 15 (2009) 74–81 75 [15], Dendrosporobacter [16], Veillonella [17,18], and Centipeda [19] resuspended into 5 ml of Tris-buffered-saline (TBS: 150 mM NaCl; to name a few. 10 mM TRIS–HCl; pH 7.5). The resuspended cells were aliquoted Additional evidence also supports that the outer bilayer visible into 1 ml fractions into 10 ml falcon tubes and placed on ice. in thin section represents an outer membrane. For example, Samples were subjected to five 30 s pulses with a micro-tip probe density gradient fractionation of the Selenomonas ruminantium powered by a Branson Sonifier 250 ultrasonicator (Danbury, CT, envelope has demonstrated that the isolated outer-membrane USA.) set to 40% of maximum output. Unbroken cells were removed fraction forms vesicles [20]. Furthermore, in freeze-fracture-etch of by centrifugation (3000 Â g, 5 min). whole cells of both S. ruminantium [21,22] and Pectinatus spp. [11], Cell envelopes were harvested by centrifugation (16,000 Â g, a fracture plane runs through the outer membrane, confirming the 30 min). The envelopes were resuspended into 5 ml of TBS. An bi-laminar structure observed in thin sections. Additional envelope equal volume of TBS containing 2% Triton X-100 (v/v), was added features reported among several genera within the family Acid- and the suspension was mixed for 5 min and harvested as aminococcaceae in common with Gram-negative eubacteria described above. In total, the envelopes were extracted three times include the presence of lipopolysaccharides (LPS) in Selenomonas, with buffer containing Triton X-100 followed by a final wash using Centripeda and Pectinatus [20,23,24] and resistance to ionophoric TBS to remove residual detergent. antibiotics consistent with the presence of an outer membrane For the preparation of cell wall, a portion of the Triton X-100 [25,26]. An additional unusual feature of the Selenomonad cell extracted envelopes was resuspended into a solution containing 1% envelope is the absence of Braun’s lipoprotein [10], which has led SDS and 0.01% b-mercaptoethanol and boiled for a 10 min period. to the suggestion that outer-membrane anchoring may be different The SDS-insoluble portion (cell walls), was recovered by centrifu- than that found in Gram-negative eubacteria [27]. gation (16,000 Â g, 30 min). Cell walls were washed once in water The outer membrane in S. ruminantium contains one or two to remove residual detergent. major proteins [10,22] arranged into a semi-ordered array visible following freeze-fracture-etch through the outer membrane 2.3. Electron microscopy [21,22].InS. ruminantium these proteins share similarities to other regularly ordered outer-membrane proteins (rOMPs) found in Thin sections were prepared using conventional fixation certain Gram-negative eubacteria [28] and similar to porins, exhibit following the method of Austin and co-workers [40]. Thin sections a heat modifiable behaviour in SDS-PAGE [10,22]. A second feature were cut on a Reichert-Jung Ultracut E ultramicrotome (C. Reichert of the major outer-membrane protein in S. ruminantium OB268 was AG, Wien, Austria) and stained with uranyl acetate and lead citrate. that the N-terminus also shared homology with a variety of cell For freeze-fracture etching, early stationary phase cells were wall associated proteins which contain an S-layer homology prepared as previously described [41]. All samples for electron domain (SLH, [29]). SLH domains are involved in the non-covalent microscopy were examined using a Zeiss EM 902 transmission anchoring of outer envelope proteins in bacteria to envelope- electron microscope (Carl Zeiss, Thornwood, N.Y.) operating at associated polysaccharides [29–32]. Proteins which commonly 80 kV with the energy loss spectrometer in place. contain this functional domain include a wide variety of S-layers and other envelope-associated proteins in Gram-positive bacteria [30,33]. 2.4. Cloning ompM1 from M. multacida Here, we report on the ultra-structure of the cellular envelope of M. multacida, the characterization of two major outer-membrane The entire ompM1 gene was cloned in three steps using PCR proteins (42 and 45 kDa), and the cloning of a gene encoding the and genomic DNA extracted from a fresh overnight culture of 45 kDa protein. Furthermore, we provide evidence based on the M. multacida 46/5(2). Genomic DNA was prepared using a modi- predicted secondary structure indicating that the 45 kDa protein fication of a previously described protocol [42].