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Anaerobe 15 (2009) 74–81

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Anaerobe

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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

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[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 g, 5 min). whole cells of both S. ruminantium [21,22] and Pectinatus spp. [11], Cell envelopes were harvested by centrifugation (16,000 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 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]. The 50 end of may represent a porin. ompM1 was discovered serendipitously during the inverse PCR (IPCR) amplification of the 30 end of a phytase gene found 2. Materials and methods upstream of ompM1. In this IPCR a 3 kb fragment was amplified with primers 032IPCR50 R (CTG CAT TGC CGT TGA AGA CA) and 2.1. Cultures and growth medium 032IPCR30 F (AGA TGC CAA TTG ACC CGA AAC) from EcoRI digested and ligated 46/5(2) genomic DNA (Fig. 1). In the second M. multacida 46/5(2) is an ovine ruminal isolate from Scotland step, all of the remaining 30 end of ompM1 gene except 18 nt [Mitsuokella multiacidus 46/5(2); 2] and was maintained frozen as were amplified by IPCR using an intramolecular ligation of EagI glycerol stocks at 20 C [34]. L-10 medium [35] either liquid or digested genomic DNA as a template with IPCR primers solid (1% w/v agar) containing both maltose and glucose (0.1% w/v) 032IPCR50 R and SLIPCR-U1 (CGG CGG CGT TGA TTA CC). The or modified Scott and Dehority medium [36] containing 10% (v/v) remainder of ompM1 was recovered in the final IPCR step using rumen fluid, 0.2% (w/v) glucose, 0.2% (w/v) cellobiose and 0.3% an intramolecular ligation of SacII digested genomic DNA as (v/v) starch [37,38] was used for culturing. Cultures were grown a template with IPCR primers SLAY5055 (TGT AAT CCT CGC TGG under anaerobic and stationary conditions in an atmosphere con- TCA GA) and SLAY4878 (CGT TCA GGG CAT TCG TCT TA). sisting of CO2/H2 (90:10) at a temperature of 37 Corat39 Cin All PCR products were cloned into pGEM-T or pGEM-T Easy 100% CO2 in Hungate tubes containing 5 ml of media. (Promega Corp., Madison, WI). Template pDNA was extracted from Escherichia coli DH5a was purchased from Invitrogen Corpora- overnight cultures of E. coli DH5a with a Qiaprep Spin minipreps tion (Burlington, ON) and used as the cloning host. E. coli cells were DNA purification system (Qiagen Corp., Mississauga, ON) and propagated in liquid or on solid Luria-Bertani (LB) medium [39]. sequenced by automated cycle sequencing at the University of Calgary Core DNA and Protein Services facilities. Primer walking 2.2. Preparation and fractionation of cellular envelopes was used to generate overlapping nucleotide sequence data [39]. Sequence data were analysed and assembled with the aid of Samples of M. multacida for electron microscopy and SDS-PAGE Sequencher version 4.0 (Gene Codes Corp., Ann Arbor, MI) and analyses were prepared from overnight 200 ml cultures. Briefly, MacDNAsis version 3.2 (Hitachi Software Engineering Co., Ltd., San cells were harvested by centrifugation (8000 g, 10 min) and Bruno, CA). Author's personal copy

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Fig. 1. Schematic diagram illustrating the cloning of ompM1 from M. multacida. Three rounds of IPCR (B) were used to clone the entire outer-membrane protein gene as well as part or all of several adjacent coding sequences (A), including part of phytase A (phyA, Selinger, personal communication), an unidentiﬁed ORF and the 50 end of a putative alanine racemase. The relative positions of the primers used in each step are indicated in panel B.

Southern blotting of restriction digested genomic DNA was servers including Porter (http://distill.ucd.ie/porter/), PSIPRED carried out using standard procedures [39]. Genomic DNA was (http://bioinf.cs.ucl.ac.uk/psipred), nnpredict (http://www. restriction digested using PstI, SacI, SacII resolved on a 0.8% agarose cmpharm.ucsf.edu/wnomi/nnpredict.html), Prof (http://www.aber. gel and transferred to Zeta-probeÒ blotting membrane (Bio-Rad ac.uk/wphiwww/prof/) and Jpred (http://www.compbio.dundee. Laboratories Canada Ltd., Mississauga, ON) using the alkaline ac.uk/wwww-jpred/). Predictions of tertiary structure were carried blotting procedure described by the manufacturer. Hybridizations out using 3D-PSSM ([45] http://www.sbg.bio.ic.ac.uk/w3dpssm/). were performed using a probe based on the C-terminal region of Putative membrane spanning domains were identified by using the M. multacida ompM1 DNA sequence. The probe was generated transFold ([46]; http://bioinformatics.bc.edu/clotelab/transFold). by PCR with the following primers CES050EXT (nt 777 – 796; CAG TAA GCA TGG CAA GGA CA) and SLAY4811 (nt 1155 – 1136; GAC ATT 2.7. Mass spectrophotometry TGC CTT GTT GTT GC). Hybridization was carried out overnight at 65 Cin5 SSC, 0.5% (w/v) blocking reagent, 0.1% (w/v) lauryl Protein bands were excised from gels using a scalpel and sulfate and 0.02% (w/v) SDS. The membrane was washed twice for forceps, cut into 1 mm3 pieces and placed in a 96-well plate 5 min at room temperature in 2 SSC, 0.1% (w/v) SDS and twice for (Corning, Corning, NY). Digestion of the separated proteins in the 20 min at 65 Cin0.1 SSC, 0.1% (w/v) SDS. gel pieces with trypsin (Promega Corp., Madison, WI) was accom- plished with a MassPrep station (Micromass, Manchester, UK) 2.5. Protein analysis using the protocol (version 3.5) provided by the robot manufacturer. On completion of the digest and subsequent peptide Sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis extraction, 1 ml of peptide mixture was mixed with 1 mlofa-cyano- (SDS-PAGE) was carried out according to Laemmli [43]. Samples were hydroxycinnamic acid matrix solution (Sigma, Oakville, ON) solubilized by boiling in a solution containing 0.01% b-mercaptoe- together with a lockmass peptide. One microlitre of this mixture thanol for 5 min, then electrophoresed using 12.0% gels. The sepa- was spotted on the target plate and analysed by MALDI-TOF MS rated proteins were visualized by staining with Coomassie blue R-250. (M@LDI, Waters/Micromass, Milford, MA). Both ProteinProbe For N-terminal sequence analysis, samples of Triton X-100 (Waters/Micromass, Milford, MA) software and Mascot Matrix extracted envelopes were solubilized and separated using a 12% Science Inc. software (Tokyo, Japan) were used to search the SDS-PAGE gel. Following completion of the electrophoresis, the peptide mass fingerprints generated against the predicted proteins were electrophoretically transferred onto PVDF membrane sequence of OmpM1. Search parameters allowed for a maximum of [44]. Protein bands were visualized by staining the membrane for 1 missed cleavage and a peptide mass tolerance of þ/0.1 Da. The 2 min in 0.01% methylene blue (w/v), followed by destaining for protein identification was validated by obtaining peptide sequences 5 min in water:acetic acid (95:5 v/v). The first 15 amino acid resi- of individual tryptic peptides by the interpretation of tandem mass dues from each excised protein were determined. Sequencing was spectra obtained with a MicroMass Q-Tof 1 instrument equipped carried out at the University of British Columbia Biotechnology with a nano-electrospray source (Waters Corp., Milford, MA). Laboratory, Vancouver, Canada. The heat modification characteristics of the 43 and 45 kDa 2.8. GenBank accession number outer–membrane proteins were investigated as previously described [22]. Briefly, Triton X-100 extracted envelopes were The GenBank nucleotide sequence accession number for the mixed with an equal volume of solubilization buffer and heated for M. multacida ompM1 gene is DQ641267. a 10 min period at various temperatures ranging from 50 to 100 C. After which, the samples were analysed by SDS-PAGE. 3. Results

2.6. Structure prediction 3.1. Analysis of envelope structure

Secondary structure predictions for the predicted amino acid Electron microscopic examination of thin sections revealed sequence of ompM1 were carried out using a variety of web-based a cell envelope ultra-structure consisting of a murien layer overlaid Author's personal copy

M.L. Kalmokoff et al. / Anaerobe 15 (2009) 74–81 77 by an outer membrane having a ruffled or loose appearance (Fig. 2A N-terminal sequencing of both major proteins yielded an iden- and B). The M. multacida cell envelope appeared to have a Gram- tical sequence (A-A-N-P-F-S-D-V-P-A-D-H-W-A-Y-D), identical to negative ultra-structure; identical to that found in the other genera the previously reported N-terminus of the 45 kDa outer-membrane falling within the Family Acidaminococcaceae [9–19]. protein from S. ruminantium OB268 [22]. Freeze-fracture-etch of whole cells revealed two fracture planes within the cell envelope of whole cells. The first fracture plane, 3.3. Cloning and characterization of ompM1 between the inner and outer leaf of the outer membrane, revealed from M. multacida a grainy surface consisting of densely packed granules of similar size (see Fig. 3A and B). The second fracture plane ran through the The majority (85%) of the M. multacida ompM1 gene was con- cytoplasmic membrane (marked CM) and demonstrated the typical tained on the 30 end of a previously cloned fragment containing irregular grainy appearance (Fig. 3A and 3B) found in fractures a phytase gene (phyA). The remainder of the gene was obtained through the cytoplasmic membrane in other bacteria [41]. through two IPCRs (Fig. 1). An ORF of unknown function and phyA SDS-PAGE analysis of whole cells revealed the presence of two are immediately upstream of ompM1 (Fig. 1). A putative alanine major protein species with estimated masses of 43 and 45 kDa racemase CDS is found adjacent to the 30 end of ompM1. (Fig. 4A, lane 2). Sonic disruption of whole cells and recovery of the The ompM1 CDS is 1290 nt encoding a 430 amino acid poly- cell envelopes indicated that both major proteins remained asso- peptide with a predicted molecular mass of 47,492 Da. Removal of ciated with this insoluble fraction (Fig. 4A, lane 3). Extraction of the a 23 amino acid peptide from the N-terminus yields an cytoplasmic membrane from the crude envelopes using a buffered N-terminus identical with that determined for both the 43 and Triton X-100 solution indicated that both major proteins remained 45 kDa outer-membrane proteins and a predicted mass for the associated with the insoluble washed envelope fraction (Fig. 4A, secreted peptide of 45.2 kDa. The 23 amino acid N-terminal lane 4). Thin sectioning of this material revealed an ultra-structure peptide shared a number of characteristics with other leader consisting of the outer-membrane overlaying the cell wall (Fig. 2C). peptides found in proteins which use the sec-dependent export Further treatment of the Triton X-100 washed envelopes with 1% pathway [49]. This includes its length, a polar N- and C-region SDS and heat, resulted in dissolution of the outer membrane bounding an internal hydrophobic region, and small neutral amino (Fig. 2D) and solubilization of both major proteins (Fig. 4B, lane 4), acid residues at the 1 and 2 positions of the cleavage site. the remaining insoluble material consisting of murien walls only However, the phenylalanine at the 3 position is unusual, (Fig. 2D). Together, these findings indicate an outer-membrane although it does represent a neutral residue. locale for both major proteins. Sequence comparisons with ompM1 and OmpM1 using BlastN and BlastX indicated significant nucleotide (69%) and amino acid 3.2. Characterization of the outer-membrane proteins identity (60%) with only a single previously reported gene (mep45) and its predicted polypeptide (Mep45; BAE86853) from S. rumi- As both major proteins were outer-membrane proteins, their nantium (results not shown). However, a region within the heat denaturation characteristics were further investigated N-terminus of the predicted protein (residues 28–70) shared (Fig. 4B). Treatment of Triton X-100 extracted membranes with SDS significant identity with a corresponding region present on the and heating at various temperatures indicated that both proteins N-terminus of a wide variety of S-layer proteins, various putative did not readily disassociate into monomers until temperatures of cyanobacteria porins, and Ompa from Thermotoga maritima [50], 90 C were reached (Fig. 4B lanes 5 and 6). Porins, the major protein commonly referred to as an S-layer homology (SLH) domain species in the Gram-negative eubacterial outer membrane are (results not shown). SLH domains are involved in the non-covalent similarly very resistant to denaturation by SDS and heating [47,48]. anchoring of proteins to the cell envelope [29].

Fig. 2. Ultra-structure of the M. multacida cellular envelope as determined by thin sectional analysis. (A) Thin section of whole M. multacida cells. Bar equals 200 nm. (B) High magnification of the envelope from thin sections of whole cells. (C) High magnification of thin section of Triton X-100 extracted envelopes. (D) High magnification thin section of SDS extracted Triton X-100 extracted envelopes. CW: cell wall. CM: cytoplasmic membrane. OM: outer membrane. Author's personal copy

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Fig. 3. Freeze-fracture-etch of whole M. multacida cells. Fracture planes are marked OM: outer membrane, CM: cytoplasmic membrane. Direction of shadowing is marked with arrows. Panel A: Freeze-fracture over the outer cell surface with fracture planes running through the outer membrane and cytoplasmic membrane. Panel B: Freeze-fracture-etch through outer membrane and cytoplasmic membrane, note the presence of regularly sized granules in fracture plane through the outer membrane. Bar equals 200 nm (Panel A) and 100 nm (Panel B).

In order to confirm whether ompM1 encoded either major peptides, confirming that although the two proteins shared outer-membrane protein, each protein band was excised from an a conserved N-terminal region, they represent different proteins. SDS-PAGE gel, digested with trypsin, and analysed by MALDI-TOF In order to confirm the presence of an additional gene encoding MS and TOF-MS/MS. Peptide assignments based on the predicted the 43 kDa protein, Southern blot analysis of restricted genomic sequence of OmpM1 for both the 43 and 45 kDa proteins are shown DNA was carried out using a probe encompassing the C-terminal 2/3 in Fig. 5. Overall coverage for the 45 kDa protein was 62% with the portion of the ompM1 gene (Fig. 6). Hybridization to PstI, SalI and identified peptides distributed throughout the entire protein. On SacII restricted DNA yielded multiple hybridization signals, indi- this basis and the similarity in predicted molecular weight, the cating that an additional gene sharing homology with this probe cloned gene was concluded to encode the 45 kDa protein. Coverage was present within the genome of this isolate. We are currently for the 43 kDa protein was limited to peptides falling within the attempting to clone this additional homologous sequence. conserved N-terminal region only. In order to ensure that these peptides were derived from the 43 kDa protein and did not represent contamination with the 45 kDa band, we also carried out 3.4. Secondary and tertiary structure analysis mass spectroscopic analysis of the region from one gel bounded by the 43 and 45 kDa bands. This analysis yielded no detectable Secondary structure predictions for OmpM1 were initially carried out using a variety of web-based servers. Consensus among the various secondary structure predictors indicated the presence of two distinct regions within the protein. The N-terminal SLH- ABcontaining region was predominantly helical (results not shown), 1234 54321 6 whereas significant discordance was found among the various secondary structure predictors regarding the latter 2/3 of the 220.0- 220.0- sequence, where certain algorithms predicted regions being 97.4- predominantly helix and other b sheets for the same stretch of 97.4- residues (results not shown). 66.0- 66.0- While tertiary structure predictors relying on homology to 46.0- 46.0- proteins of known structure were unable to provide a candidate template for modeling, threading analysis using 3D-PSSM fold recognition [45] identified three structural templates all of which were porins, with the best match (E-value ¼ 2.21 102) being the 30.0- 30.0- Rhodobacter blasticus porin 3prn [51]. While threading analysis did provide a template for the C-terminal 230 amino acid residues of OmpM1, there were a significant number of gaps in the alignment 21.5- 21.5- against 3prn (results not shown). Interestingly, the top three matches in the initial PSI-Blast in 3D-PSSM of OmpM1 identified 14.3- 14.3- the Selenomonas major outer-membrane protein (55% sequence identity) as well as two putative cyanobacteria porins (ABA24038 and BAB72791; 18% sequence identity) as being the closest match. Fig. 4. SDS-PAGE analysis of M. multacida. Panel (A) Lane 1: Molecular weight markers While there are a large number of putative porins assigned from (Amersham Biosciences). Lane 2: Whole cells. Lane 3: Sonicated crude cell envelopes. genomic sequencing of various cyanobacteria, only two outer- Lane 4: Triton X-100 extracted cell envelopes. Panel (B) Heat denaturation charac- membrane proteins in Synechococcus PCC6301 (SomA and SomB) teristics of major outer-membrane proteins in Triton X-100 extracted envelopes. have been determined through a functional assay to represent Samples were mixed into solubilization buffer and heated at the following temperatures for 10 min. Lane 1: molecular weight markers. Lane 2: 60 C. Lane 3: 70 C. Lane porins [52]. Consistent with the 3D-PSSM prediction was the fact 4: 80 C. Lane 5: 90 C. Lane 6: 100 C. that the C-terminal residue of OmpM1 was phenylalanine, Author's personal copy

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AANPFSDVPADHWAYDAVSQLAADGVIEGYGDTTFRGNQNITRYEMAQMIAKAMAKTDVSAADKA LIDKLAAEFSDELNNLGVRVSNLERNADNVKWNGKAEYTYYSHRDKDANTKTNDDQLLFRLEPSA EVNSHWHVNARLDASTDLAKDSDDVDSKHGKDSYQDTDVTLKRVWAQGNYGNFQVKLGKFAQI DDDSIFDTSFSGAEVKFGNKVTFTAGAGRQNMNEDSDFTQKFTVGEDTANYQYAGLGYADGKFV GGVDYHHLNSDTFNYAVKGNNKANVEDNANIWLAKAAYRFDKTNALNGFYANNTSADDFDKAWS AQYSYKGAEAENKGTWGAWAAYRYLGQNTDLFSTFDVILAGQKGWEVGANYAPFKNIVATLRYG NGKDLRTDNDIENLFG

Fig. 5. Assignment map of OmpM1 determined through mass spectroscopy. Predicted sequence of OmpM1 is shown. The determined N-terminal sequence from both the 45 and 43 kDa proteins is indicated within the grey box. Peptides from the 45 kDa protein identified by mass spectroscopy are bolded (62% coverage). Peptides identified in the 43 kDa protein are bolded and underlined (8% coverage). a common feature of most outer-membrane channel forming C-terminal region of OmpM1 appeared to form a topology con- proteins [48]. sisting of 16 anti-parallel b-sheets. Consistent with the consensus While crystallographic analysis has demonstrated that porins model for trimeric porins [48,54], the internal loops (T1–T7) were form trans-membrane beta-barrels, the discordance found among short, whereas the external loops (L1–L8) were much longer and the various secondary structure predictors and less than optimal of variable length. The predicted membrane spanning domains, threading of OmpM1 with 3prn, made the identification of putative 10–11 amino acids in length, are consistent with that found in membrane spanning domains within OmpM1 difficult to resolve. In other porins and there are aromatic amino acid residues (Y, W, order to further define a putative secondary structure, we and F, see Fig. 7) located within or in close proximity to the attempted to first identify the membrane spanning domains using interfacial regions defined by several of the putative membrane the wide variety of predictors currently available [46,53], and spanning domains. Loop three (L3) was very long and transFold determine whether the resulting secondary structure model con- predicted that this region to contain several additional membrane formed to the general consensus model for trimeric porins spanning domains (data not shown). Analysis of Mep45 [47,48,54]. (BAE86853) from S. ruminantium yielded an identical secondary With the exception of transFold [46], none of the predictors structural prediction (results not shown). identified any, or at most only a few trans-membrane domains within this region of OmpM1. A putative secondary structure 4. Discussion based on the transFold prediction is shown in Fig. 7. The Despite the phylogenetic placement within the Firmicutes, electron microscopic examination of M. multacida indicated that M 1 2 3 the cell envelope was Gram-negative, identical to that reported in other genera in the Family Acidominococcaceae within the Phylum Firmicutes. Furthermore, freeze-fracture-etch demonstrated the presence of two fracture planes in whole cells, corresponding to the inner cytoplasmic membrane and the outer membrane, consistent with the Gram-negative ultra-structure observed in thin section. The presence of an outer membrane, which would normally be impermeable to the diffusion of hydrophilic solutes, illustrates the requirement for a mechanism to allow for diffusion of small molecules into the cell. Using a combination of detergent fractionation and electron microscopy, it was demonstrated that the outer membrane contained two major proteins. Both were unusual in that they represented the major proteins in whole cells extracts, a finding typically observed with S-layer proteins [33]. However, we found no microscopic evidence to support that either protein represented the monomeric unit of an S-layer, as the only layer external to the peptidoglycan layer was the outer membrane. Furthermore, dissolution of the outer membrane with SDS and heat coincided with solubilization of both proteins, supporting the outer- membrane locale. On these bases, it is likely that the densely packed granules visible in the freeze-fracture-etch through the outer membrane represent these two major proteins. These findings are consistent with previous analyses of the S. ruminantium cell envelope [20,22]. A single ORF (ompM1) was fortuitously discovered downstream of a previously cloned phytase gene. The high degree of identity within the N-terminal region of the predicted protein product of ompM1 with a previously characterized major outer-membrane protein in S. ruminantium [22], led us to initially suspect that this gene encoded an outer-membrane protein. On the basis of the N-terminal identity, the predicted molecular weight and results from mass spectroscopic analysis, ompM1 encoded the 45 kDa Fig. 6. Southern blot of restriction digested genomic DNA hybridized with a digoxigenin labelled probe encompassing the C-terminal region of ompM1. Lane 1: Lamba HindIII major outer-membrane protein (OmpM1). While the 43 kDa ladder. Lane 2: PstI. Lane 3: SacI. Lane 4: SacII. protein (OmpM2) also shared the conserved N-terminal sequence, Author's personal copy

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structures Loop 3 is large and may also fold into the pore [51]. However in OmpM1 Loop 3 is unusually large, containing approx- imately twice as many residues as found in most other porins, although similarly sized Loop 3 regions may also occur in porins from the Vibrio-Photobacterium group [48]. Finally, identical to the putative secondary structure of the unusual porin in the cyano- bacterial species Synechococcus [56], the N-terminal SLH-containing region faces inward, which is consistent with the possibility that the SLH domain may interact with components associated with the peptidoglycan layer.

Acknowledgements

The authors wish to acknowledge the excellent technical assis- tance provide by Sophie D’Aoust, Greg Sanders, Rob Gruninger and Colin Strauss. The authors also thank Jay Yanke for providing the M. multacida isolate. A portion of this work was supported through a grant from the Advanced Foods and Materials Research Network (B. Selinger, J. Austin and M. Kalmokoff) and an NSERC discovery grant (B. Selinger).

References

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