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.

Thin sectioning and freeze-fracture-etch of the ovine ruminal isolate Mitsuokella multacida strain 46/5(2) revealed a Gram-negative envelope ultra-structure consisting of a peptidoglycan wall overlaid by an outer membrane. Sodium-dodecyl-sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of whole cells, cell envelopes and Triton X-100 extracted envelopes in combination with thin-section and N-terminal sequence analyses demonstrated that the outer membrane contained two major proteins (45 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 was cloned. The 1290 bp open reading frame encoded a 430 amino acid polypeptide with a predicted molecular mass of 47,492 Da. Cleavage of a predicted 23 amino acid leader sequence would yield a protein with a molecular mass of 45,232 Da. Mass spectroscopic analysis confirmed that the cloned gene (ompM1) encoded the 45 kDa outer-membrane protein. The N-terminus of the mature OmpM1 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 beta-barrel and appears to be related to the unusual N-terminal SLH-domain-containing beta-barrel-porins previously described in the cyanobacterium Synechococcus PCC6301.

Circular dichroism reveals sensitivity of glucagon solution structure to fluoroalcohols, pH and ionic strength.

Circular dichroism reports that glucagon in solution becomes increasingly helical with the addition of fluoroalcohol (which also decreases solution pH), and with changes in pH and ionic strength. Given the variability of structure observed, these data indicate that care must be taken when comparing results obtained under different solution conditions.

Proteomic and microscopic analysis of biofilms formed by Listeria monocytogenes 568.

Biofilm formation may be important in the colonization of the food-processing environment by the food-borne pathogen Listeria monocytogenes. Listeria monocytogenes 568 formed adherent multicellular layers on a variety of test surfaces following growth at 37 degrees C with multiple transfers of the test surface into fresh medium. Microscopic examination of these adherent layers suggest that the cells were surrounded by extracellular material. The presence of a carbohydrate containing extracellular polymeric matrix was confirmed by labelling hydrated adherent layers with fluorescein-conjugated concanavalin A, indicating that these adherent layers are biofilms. To gain insight into the physiological state of cells in these biofilms, the proteomes from biofilm- and planktonic-grown cells from the same cultures were compared using 2-dimensional polyacrylamide gel electrophoresis. Nineteen proteins, which exhibited higher levels of expression in biofilm-grown cells, were successfully identified from the 2-D gels using a combination of MALDI-TOF and MS/MS. Proteins that were found to be more highly expressed in biofilm-grown cells were involved in stress response, envelope and protein synthesis, biosynthesis, energy generation, and regulatory functions. In biofilm-grown cells, many proteins in the pH range 4-6 ran as multiple spots arranged horizontally across the 2-D gels.

Butyrivibriocin AR10, a new cyclic bacteriocin produced by the ruminal anaerobe Butyrivibrio fibrisolvens AR10: characterization of the gene and peptide.

The gene (bviA) encoding the ruminal bacteriocin butyrivibriocin AR10 was cloned from an EcoRI library by using an oligonucleotide probe based on a partial peptide sequence of the previously isolated peptide. The gene encoded an 80 amino acid prebacteriocin that demonstrated significant identity with the cyclic bacteriocin gassericin A. Negative ion time of flight mass spectroscopic analysis (ESI/MS) indicated a mass of 5981.5 Da for the isolated bacteriocin, a molecular mass that could not be generated by removal of a leader peptide alone. However, an N- to C-terminal cyclization of the predicted mature bacteriocin resulted in a peptide that conformed to the determined mass and charge characteristics. Northern blotting confirmed that expression of bviA mirrored the production of the bacteriocin in both liquid and solid media.

Identification of a new plasmid-encoded sec-dependent bacteriocin produced by Listeria innocua 743.

Listeria innocua 743 produces an inhibitory activity demonstrating broad-spectrum inhibition of Listeria monocytogenes isolates. Gel-electrophoretic analysis of culture supernatants indicated that two inhibitors with different molecular weights were produced by this strain. Insertion of Tn917 into a 2.9 Kb plasmid (pHC743) generated mutants with either an impaired ability or a loss in ability to produce one of the inhibitors. Sequence analysis of the transposon insertion regions revealed the presence of two continuous open reading frames, the first encoding a new pediocin-like bacteriocin (lisA) and the second encoding a protein homologous with genes involved in immunity toward other bacteriocins (lisB). Translation of the bacteriocin gene (lisA) initiates from a noncanonical start codon and encodes a 71-amino-acid prebacteriocin which lacked the double glycine leader peptidase processing site common in other type II bacteriocins. Alignment of the sequence with the processed N termini of related bacteriocins suggests that the mature bacteriocin consists of 43 amino acids, with a predicted molecular mass of 4,484 Da. Mutants containing insertions into lisA were sensitive to the inhibitor, indicating that lisAB forms a single operon and that lisB represents the immunity protein. Cloning of an amplicon containing the lisAB operon into Escherichia coli resulted in expression and export of the bacteriocin. This finding confirms that the phenotype is dependent on the structural and immunity gene only and that export of this bacteriocin is sec dependent. This is the first confirmation of bacteriocin production in a Listeria spp., and it is of interest that this bacteriocin is closely related to the pediocin family of bacteriocins produced by lactic acid bacteria.

In vacuo esterification of carboxyl groups in lyophilized proteins.

A new method is described for the esterification of carboxyl groups in proteins by reaction of the lyophilized protein in vacuo with gaseous alcohol and HCI catalyst. Carboxyl groups are rapidly esterified with no protein degradation. 13C-Methyl or 13C-ethyl esters of the alpha-, gamma- and delta-carboxyl groups could be distinguished by the distinct chemical shifts of their resonances. Within the class of gamma- or delta-esters, the chemical shifts have little variation; however, the chemical shift of a C-terminal esterified alpha-carboxyl group shows a strong dependence on the nature of the C-terminal amino acid and sequence. Iodomethane reacts with deprotonated carboxyl groups in lyophilized proteins to form methyl esters, but unlike the reaction with gaseous methanol/HCI, it does not selectively methylate carboxyl groups. The procedure permits the cost-effective incorporation of isotopic labels and provides a new approach using 13C-NMR spectroscopy for determining the number of different C-termini present in a protein preparation.

Use of the pH memory effect in lyophilized proteins to achieve preferential methylation of alpha-amino groups.

It is demonstrated that the pH memory effect can be used to control the ionization state of amino groups in lyophilized proteins and hence their chemical reactivity toward modifying reagents. When proteins were lyophilized from aqueous solutions at pH values between 6 and 7 and reacted in vacuo with iodomethane, the alpha-amino groups were found to be either preferentially or selectively trimethylated. Reaction with 13C-labeled iodomethane permitted detection and identification of individual trimethylated alpha-amino groups by 13C-NMR spectroscopy as distinct peaks in the spectral region between 52 and 57 ppm. There was adequate sensitivity to detect minor resonances of free alpha-amino groups arising from proteolysis of the major protein or from protein impurities. The resonances of the trimethylated alpha-amino groups in standard amino acids and peptides are sufficiently close to those in the derivatized protein to make a tentative identification of the N-terminal amino acid. It is also demonstrated that advantage can be taken of the pH memory effect to use the preferential 13C-methylation of amino groups to verify whether a protein has a free or blocked amino terminus.

Characterization of MB-1. A dimeric helical protein with a compact core.

MB-1 is a de-novo protein designed to incorporate a large number of the nutritionally important amino acids methionine, lysine, leucine and threonine into a stable four-helix bundle protein. MB-1 has been expressed and purified from Escherichia coli, indicating it was resistant to intracellular proteases [Beauregard, M., Dupont, C., Teather, R.M. & Hefford, M.A. (1995) Bio/Technology 13, 974]. Here we report an analysis of the secondary, tertiary and quaternary structures in MB-1 using circular dichroism, fluorospectroscopy and size-exclusion chromatography. Our data indicate that the MB-1 structure is close to the target structure, an alpha-helical bundle, in many respects and is highly helical in solution. The single tyrosine incorporated into the designed protein as a spectrocopic probe of tertiary structure, is buried in a compact, folded core and becomes accessible on protein denaturation, as per design. Furthermore, MB-1 was found to be native-like in many respects: (a) protein denaturation induced by urea is cooperative and fully reversible; (b) its oligomeric state at moderate concentration is well defined; and (c) MB-1 has very low affinity for 8-anilino-1-naphthalenesulfonic acid (ANSA), leading to enhancement of ANSA fluorescence that resembles that of other native proteins. On the other hand, our analysis revealed two aspects that command further attention. The folding stability of MB-1 as assessed by urea and thermal denaturation is somewhat less than that found for natural globular proteins of similar size. Size-exclusion chromatography experiments and analysis of MB-1 denaturation indicate that MB-1 is dimeric, not monomeric as designed. In light of these results, the utility and the current limitations of our design approach are discussed.

Introduction of potential helix-capping residues into an engineered helical protein.

MB-1 is an engineered protein that was designed to incorporate high percentages of four amino acid residues and to fold into a four-alpha-helix bundle motif. Mutations were made in the putative loop I and III regions of this protein with the aim of increasing the stability of the helix ends. Four variants, MB-3, MB-5, MB-11 and MB-13, have replacements intended to promote formation of an 'N-capping box'. The loop I and III sequences of MB-3 (both GDLST) and MB-11 (GGDST) were designed to cause alphaL C-terminal 'capping' motifs to form in helices I and III. MB-5 has a sequence, GPDST, that places proline in a favourable position for forming beta-turns, whereas MB-13 (GLDST) has the potential to form Schellman C-capping motifs. Size-exclusion chromatography suggested that MB-1, MB-3, MB-5, MB-11 and MB-13 all form dimers, or possibly trimers. Free energies for the unfolding of each of these variants were determined by urea denaturation, with the loss of secondary structure followed by CD spectroscopy. Assuming an equilibrium between folded dimer and unfolded monomer, MB-13 had the highest apparent stability (40.5 kJ/mol, with +/-2.5 kJ/mol 95% confidence limits), followed by MB-11 (39.3+/-5.9 kJ/mol), MB-3 (36.4+/-1.7 kJ/mol), MB-5 (34.7+/-2.1 kJ/mol) and MB-1 (29.3+/-1.3 kJ/mol); the same relative stabilities of the variants were found when a folded trimer to unfolded monomer model was used to calculate stabilities. All of the variants were relatively unstable for dimeric proteins, but were significantly more stable than MB-1. These findings suggest that it might be possible to increase the stability of a protein for which the three-dimensional structure is unknown by placing amino acid residues in positions that have the potential to form helix- and turn-stabilizing motifs.

A consensus residue analysis of loop and helix-capping residues in four-alpha-helical-bundle proteins.

A statistical study was performed on a set of proteins which adopt the four-alpha-helical-bundle tertiary motif in order to determine amino acid occurrences at helix-capping and loop positions. Eight X-ray crystal structures from the Brookhaven Protein Data Bank (PDB) were examined and N", N', Ncap, Ccap, C' and C" residues were assigned. In addition, a set of 55 protein sequences for the analogous proteins from different strains and species was taken from the Protein Information Resource and Swiss-Prot databanks. The residues at the capping and loop positions in this expanded data set were deduced by aligning these sequences with those from the PDB files. Similar trends were observed in the two data sets. In general, polar residues were predominant in the loops, although aromatic residues were also fairly common. Glycine, a highly flexible residue with an excellent 'helix-breaking' ability, was very common at the Ccap, C' and C" residues. Proline, which can force sharp turns in the direction of a peptide backbone, was only common at the N" residue. Residues which can participate in the N-capping box motif were found with high frequency. Capping motifs at the helix C-termini (Schellman and alphaL motifs) were also somewhat common, while another helix N-terminal stabilizing motif, the hydrophobic stable, was not common. The data presented in this study should prove useful for applying the 'consensus residue' approach to the de novo design of loop regions in helical bundle proteins.

Sequence analysis and characterization of pOM1, a small cryptic plasmid from Butyrivibrio fibrisolvens, and its use in construction of a new family of cloning vectors for Butyrivibrios.

As a preliminary step in the development of vector systems, we have isolated and begun to characterize small, cryptic plasmids from several strains of the rumen bacterium Butyrivibrio fibrisolvens. We present here the complete nucleotide sequence of Butyrivibrio plasmid pOM1, which was isolated from B. fibrisolvens Bu49. While it is very similar in size to the previously characterized Butyrivibrio plasmids pRJF1 and pRJF2, pOM1 exhibits a restriction pattern which is quite distinct. Analysis of sequence data reveals that pOM1 contains only two open reading frames of significant length (ORF1 and ORF2), both of which are required for self-replication and maintenance. The protein encoded in ORF1 shows homologies with Pre (plasmid recombination enzyme) proteins encoded in plasmids from gram-positive organisms such as Staphylococcus aureus, Streptococcus agalactiae, Lactobacillus plantarum, and Bacillus thuringiensis. The putative translation product of ORF2, on the other hand, resembles Rep (replication) proteins of a different group of gram-positive plasmids, for which the Staphylococcus plasmid pSN2 is a prototype. Unlike the other characterized-Butyrivibrio plasmids, pOM1 appears to replicate via a rolling-circle mechanism. Experimental evidence showing the presence of a single-stranded replication intermediate consistent with this mechanism is presented. pOM1 has been used in the construction of a new Escherichia coli-B. fibrisolvens shuttle vector, pSMerm1, which has been successfully used to introduce a cloned gene into B. fibrisolvens harboring the pRJF1 plasmid.

Prediction of folding stability and degradability of the de novo designed protein MB-1 in cow rumen.

The authors have recently reported on the design of a protein (MB-1) enriched in methionine, threonine, lysine, and leucine. The protein is intended to be produced by rumen bacteria, in a way that would provide high producing lactating cows with limiting amino acids. In this report, MB-1 stability in the rumen is assessed, i.e., where the protein might be found after cell lysis or after being secreted by rumen bacteria. Current in vitro methods used to predict proteolytic degradability in the rumen were used for MB-1, as well as other natural proteins for comparison. MB-1 was found to be more susceptible to degradation than cytochrome c and ribonuclease A. Data indicate that MB-1 will be rapidly degraded if exposed to the rumen environment without protection. The contribution of folding stability to proteolytic stability was also examined. Rumen liquor components were selected to formulate a solution compatible with constraints of thermal denaturation studies. Denaturation curves show that the natural proteins were folded at rumen temperature. The MB-1 denaturation curves indicated that MB-1 does not unfold in a cooperative transition when heated from 20 to 70 degrees C. This suggests that MB-1 structure may be progressively modified as temperature increases, and that a continuum of conformations are available to MB-1. At 39 degrees C, a significant (50%) portion of MB-1 molecules had their tertiary structure unfolded, contributing to proteolytic degradability. Despite the unusual constraints used in MB-1 design (i.e., a maximized content in selected essential amino acids), results show that MB-1 has structural properties similar to previously reported de novo designed proteins.

Design, expression, and initial characterization of MB1, a de novo protein enriched in essential amino acids.

Using recently emerging protein folding principles we have designed a protein enriched in the essential amino acids methionine, threonine, lysine and leucine. Our preliminary study of consensus residues (based on charge, hydrophobicity and volume) of natural alpha-helical bundle proteins indicated that the residues M, T, K, and L could be inserted in an alpha-helical bundle structure. We therefore attempted to create a stable de novo protein, highly enriched in these essential amino acids, that would adopt the alpha-helical bundle fold. The design process was an iterative one. The consensus residues (based on the properties profile) for bundle helices were found considering the four helices taken together, helices I to IV individually, or only their N- and C-termini. Using these data, the helices in our de novo protein were designed by inserting the residues M, T, K and L as often as possible at positions where their volume, hydrophobicity and charge match the consensus found in natural bundle helices. Short sequences of strong turn formers were used to join the helices and adjust the predicted p1 to 7.7, while a number of local and global factors were used to refine our design. Further, the sequence was checked to eliminate various known protease targets in E. coli. The sequence of our de novo protein, MB1, is: MAT-EDMTDMMTTLFKTMQLLTK-SEPTA-MDEATKTATTMKNHLQNLMQK-TKNKE DMTDMATTYFKTMQLLTK-TEPSA-MDEATKTATTMKNHLQNLMQK-GVA+ ++ , where dashes separate long helices from short, turn forming linkers. A gene coding for this protein was assembled from synthetic oligonucleotides, then fused to the maltose binding protein gene under the control of a tac promoter. The fusion protein was expressed in E. coli, purified and cleaved to yield maltose binding protein and our de novo protein, MB1. MB1 was found to be helical, to have the expected molecular weight (11 kDa) and the expected content (57%) of the essential amino acids M, T, K and L.

Analysis of the sequence of a new cryptic plasmid, pRJF2, from a rumen bacterium of the genus Butyrivibrio: comparison with other Butyrivibrio plasmids and application in the development of cloning vector.

A small cryptic plasmid, pRJF2, from Butyrivibrio fibrisolvens strain OB157 was isolated and sequenced. The plasmid is similar in organisation to the previously sequenced Butyrivibrio plasmid, pRJF1, with two open reading frames, ORF1 and ORF2, flanking a region tentatively identified as the replication origin, and a region of unknown function defined by terminal 79 bp invert repeats. The sequences of ORF1, ORF2, and the presumptive replication origin are highly conserved. The sequence between the 79, bp invert repeats is not, and is therefore presumed to be of lesser functional significance, although the 5' and 3' termini are still highly conserved. The functional importance for plasmid replication of these regions was tested by constructing potential shuttle vectors, each lacking one or more of the regions of interest. When the region between the invert repeats was deleted and replaced by the erythromycin resistance gene from pAM beta 1 together with pUC18, to produce the 7.9 kb chimaeric plasmid pYK4, the construct was successfully transformed into E. coli and B. fibrisolvens by electroporation, and was stably maintained in both hosts. Both ORF1 and ORF2 were required for successful transformation of B. fibrisolvens.

A stable and efficient transformation system for Butyrivibrio fibrisolvens OB156.

A 9.5-kb shuttle vector capable of replication and selection in both Escherichia coli and Butyrivibrio fibrisolvens was constructed. Plasmid pUC118 provided replication functions and ampicillin resistance selection in E. coli. In B. fibrisolvens, replication was controlled by the native plasmid pRJF1 from strain OB156, and selectability was provided by a 3.5-kb fragment of plasmid pAM beta 1 containing the erythromycin resistance gene. Optimum conditions for transformation were 15 kV/cm, 2 h recovery, and plating in an agar overlay on medium containing 10 micrograms erythromycin/ml. Maximum efficiency was 1.1 x 10(5) transformants per micrograms plasmid DNA (average 3 x 10(4)), and restriction mechanisms reduced efficiency by a factor of 2 x 10(2). Nonselective growth for 200 generations gave no measurable loss of plasmid.

The complete nucleotide sequence of a small cryptic plasmid from a rumen bacterium of the genus Butyrivibrio.

The complete nucleotide sequence of a plasmid, designated pRJF1, isolated from a rumen bacterium of the genus Butyrivibrio, has been determined. pRJF1 is a small plasmid (2631 bp) which shows the high AT content (64%) typical of plasmids from gram-positive organisms. Computer analysis of sequence data revealed two major open reading frames encoded on the same strand but in different frames. The smaller, ORF1 (435 bp), is preceded by Shine Dalgarno (SD) and Escherichia coli-10, -35 sequences and is predicted to encode an acidic polypeptide of 145 aa. The putative protein shows little homology to known bacterial proteins except in a small region similar to part of the Pre protein (plasmid recombination enzyme) of Bacillus plasmid pTB913. Pre proteins of the pTB913 family, however, show conservation of several amino acids in the amino terminal region of the protein that may be related to function. This pattern of amino acid conservation is not observed in ORF1. The predicted product of ORF2 (849 bp) is a basic protein of 283 as that shows significant similarity to repA gene products of plasmids from gram-negative (but not gram-positive) bacteria. No typical Shine Dalgarno sequence or -10, -35 sequences precede this coding region. While sequence homologous to nick sites of plasmids from gram-positive organisms that replicate via ssDNA intermediates were also detected in pRJF1, the order of the three consecutive inverted repeats typical of nick sites is not conserved. pRJF1, however, does contain an extended region of directly and inversely repeated sequence motifs reminiscent of theta replication origins.

Bipartite organization of the Bacillus subtilis endo-beta-1,4-glucanase revealed by C-terminal mutations.

The C-terminal boundary of primary sequence of the Bacillus subtilis PAP115 endo-beta-1,4-glucanase (EG) required for stable catalytic activity has been mapped by site-directed mutagenesis using Escherichia coli as host. The 52 kDa cel gene product, EG470 and a 33 kDa mutant (EG300), lacking 170 residues through a nonsense mutation at the leucine-330 codon of the gene, exhibited similar patterns of enzymatic activity and pH optima using cellooligopentaose as substrate. CD spectra indicated that the bulk of the alpha-helical secondary structure in EG470 was contained within EG300. However, relative to EG470, the specific activity of EG300 was 3- to 4-fold lower with amorphous cellulose as substrate and approximately 4- to 5-fold higher with carboxymethylcellulose (soluble cellulose). These results along with data which show that EG470 binding capacity to microcrystalline cellulose is approximately 11 times more than that of EG300, demonstrate the importance of residues 330-499 for non-catalytic binding of cellulose. A construct of the cel gene carrying a deletion of codons 330-499 and an insertion of a nonsense codon at leucine-330, was further used to make mutants EG296 and EG291 with nonsense codon substitutions at arginine-326 and serine-321, respectively. Western analysis using EG-specific antiserum revealed that relative losses in enzymatic activity of EG296 (50%) and EG291 (95%) could be accounted for by the extent of their proteolysis, signifying a marked destabilization of these enzymes by removal of only a few amino acids.

The Glu residue in the conserved Asn-Glu-Pro sequence of two highly divergent endo-beta-1,4-glucanases is essential for enzymatic activity.

We initially aligned 28 different cellulase sequences in pairwise fashion and found half of them have the sequence -Asn-Glu-Pro- located in a region flanked by hydrophobic-rich amino acids. Based on lysozyme as a model, the glutamate residue could be essential for enzyme function. We tested this possibility by site-directed mutagenesis of the genes coding Bacillus polymyxa and Bacillus subtilis endo-beta-1,4-glucanases. The genes and amino acid sequences of these two enzymes show very little similarity. Change of Glu-194 and Glu-169 to the isosteric glutamine form in these respective enzymes resulted in a dramatic loss of CMCase activity which could be restored by reverse mutation. Similar mutations to less-conserved residues, Glu-72 and Glu-147, of the B. subtilis enzyme did not cause any loss of activity.