A genomic perspective on the potential of Actinobacillus succinogenes for industrial succinate production
1 Department of Microbiology and Molecular Genetics, 2215 Biomedical Biophysical Sciences building, Michigan State University, East Lansing, MI 48824, USA
2 Department of Genome Sciences & Medicine, University of Washington, Seattle, WA 98195, USA
3 DOE Joint Genome Institute and Los Alamos National Laboratory, Los Alamos, NM 87545, USA
4 DOE Joint Genome Institute, Walnut Creek, CA 94598, USA
5 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA
6 Department of Microbiology, University of Washington in Seattle, WA 98195, USA
7 Laboratory of Genetics, University of Wisconsin, Madison, WI 53706, USA
8 Department of Zoology, Michigan State University, East Lansing, MI 48824, USA
BMC Genomics 2010, 11:680 doi:10.1186/1471-2164-11-680Published: 30 November 2010
Additional file 1:
Figures S1 to S6. Figure S1: Phylogenetic tree of representative Pasteurellaceae with complete genomes based on 16 S RNA sequences. 16 S rRNA phylogeny was determined using the Michigan State University Ribosomal Database Project tools . Figure S2: Hierarchical clusterings of Pasteurellaceae species according to COGS, PFAM, Enzymes, and TIGRfam classifications. Hierarchical clustering of Pasteurellaceae genomes was done according to COG, Pfam, Enzyme, and TIGRfam functional profiles at the JGI's Integrated Microbial Genomes website . The four functional profile clustering approaches place the two succinogens in a clade separate from other Pasteurellaceae. Figure S3: NUCmer and PROmer alignments of A. succinogenes and M. succiniciproducens, P. multocida, and A. pleuropneumoniae L20. Synteny plots of the whole-genome alignments of A. succinogenes and M. succiniciproducens, A. succinogenes and P. multocida, and A. succinogenes and A. pleuropneumoniae L20 at the nucleotide level (NUCmer) and at the protein level (PROmer). Alignments were performed using the mummer software package . These plots give overviews of the rearrangements that have taken place at the genome level between two bacterial species. Red lines from the bottom left to upper right indicate conservation of nucleotide (NUCmer) or protein (PROmer) sequence, reading in the same direction in both species. Blue lines from upper left to lower right indicate sequence conservation but with sequence inversion between the two species. NUCmer and PROmer comparisons of A. succinogenes with H. influenzae KW20, H. influenzae 028NP, H. somnus, H. ducreyi, and A. pleuropneumoniae JL03 were also performed, but are not shown in this Figure. The NUCmer plots show little to no conservation of genome structure at the nucleotide level between A. succinogenes and any other Pasteurellaceae. PROmer plots reveal that A. succinogenes and M. succiniciproducens are more related to each other than to other Pasteurellaceae. The PROmer plot of A. succinogenes vs. M. succiniciproducens shows that drastic changes in genome structure have occurred as A. succinogenes and M. succiniciproducens evolved divergently from their last common ancestor, indicating that the two succinogens are more distantly related than their functional traits would suggest. Figure S4: Comparison of nucleotide frequencies in Pasteurellaceae uptake signal sequences. Figure S4 shows nucleotide frequencies in the USSs of six representative Pasteurellaceae species containing either USS1 (A. succinogenes, M. succiniciproducens, A. aphrophilus NJ8700, and H. somni 129PT) or USS2 (A. pleuropneumoniae L20 and H. ducreyi 3500HP). USS 9-mer cores were counted and their surrounding sequences reported using a perl script. The output was pasted into a Microsoft Excel spreadsheet to calculate the frequency of each nucleotide occurring at each position, upstream and downstream of the USS core. Nucleotide frequencies in the USSs of sixteen more Pasteurellaceae species containing USS1 (H. influenzae Rd KW20, 028NP, PittEE, PittAA, PittGG, PittHH, PittII, 22.1-21, 22.4-21, 3655, R2846, 2866, and R3021; P. multocida; A. actinomycetemcomitans; and H. somni 2336) and four more Pasteurellaceae species containing USS2 (A. pleuropneumoniae JL03 and 4074, M. haemolytica PHL213, and H. parasuis 29775) were also calculated, but are not shown here. These data are available upon request. Figure S5: A. succinogenes has incomplete pathways for assimilatory sulfate reduction and methionine synthesis. Four-digit numbers are Asuc_ORF (locus tags) numbers and are followed by E.C. numbers. Hyphenated locus tag numbers indicate that the enzyme is encoded by several successive genes. Reaction names: see additional file 2: Table S4. XH, reduced thioredoxin; X+, oxidized thioredoxin. Arrow and number colors: black, product function assumed; blue, probable function assumed; red, possible function assumed. Bold arrows indicate central metabolic pathways. Dotted arrows indicate that A. succinogenes is missing the gene for that function. Figure S6: A. succinogenes has incomplete pathways for biotin, nicotinic acid, pantothenic acid, and pyridoxine synthesis. Four-digit numbers are Asuc_ORF (locus tags) numbers and are followed by E.C. numbers. Hyphenated locus tag numbers indicate that the enzyme is encoded by several successive genes. Reaction names: see additional file 2: Table S4. Arrow and number colors: black, product function assumed; green, putative function assumed; blue, probable function assumed; red, possible function assumed. Bold arrows indicate central metabolic pathways. Gray dotted arrows indicate that A. succinogenes is missing the gene for that function. Metabolites: Alac, 2-acetolactate; AON, 8-amino-7-oxonoanoate; APP, 3-amino-2-oxopropyl phosphate; CoA, coenzyme A; Dbio, dethiobiotin; DCoA, dephospho-CoA; DhP, 2-dehydropantoate; DMB, 2,3-dihydroxy-3-methylbutanoate; dNAD+, deamido-NAD+; DON, 7,8-diaminononanoate; DXP, 1-deoxyxylulose-5-phosphate; Er4P, erythronate-4-phosphate; HPB, 2-oxo-3-hydroxy-4-phosphobutanoate; IAsp, iminoaspartate; MOB, 3-methyl-2-oxobutanoate; NRS, nicotinate ribonucleoside; NRT, nicotinate ribonucleotide; Pan, pantoate; PCA, pimeloyl-CoA; PHT, O-phospho-4-hydroxythreonine; Pim, pimelate; PNP, pyridoxine phosphate; Ppc, 4'-phosphopantothenoyl-cysteine; Ppt, 4'-phosphopantothenate; Ppth, 4'-phosphopantetheine; QNL, quinolinate. Other abbreviations are as in Figure 2.
Format: PDF Size: 2.4MB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 2:
Tables S1 to S5. Table S1: A. succinogenes ORFs encoding sugar transporters and degradation pathways. Table S1 lists all the A. succinogenes transporters, enzymes, and regulatory proteins potentially involved in sugar transport and assimilation, based on our manual annotation of the genome. Annotation criteria are described in the materials and methods section. The ORFs putatively encoding sugar transport and degradation pathways encompass all the sugars A. succinogenes is known to use, except arabitol. The A. succinogenes genome also encodes transporters and degradation pathways for carbon sources A. succinogenes does not metabolize (e.g., pectin). Table S2: A. succinogenes homologs of H. influenzae competency proteins. List of the H. influenzae competency genes and their A. succinogenes homologs, with the likeliness that the A. succinogenes homologs have the same function. A. succinogenes homologs are considered putative if they share 60-75% amino acid identity with the query sequence, probable if they share 40-59% amino acid identity with the query sequence, and possible if they share 25-39% amino acid identity with the query sequence. NA indicates that no suitable homolog was identified in A. succinogenes either due to insufficient alignment length (less than 25% of the query sequence length) or to no hits retrieved from the BLAST search. Table S3: A. succinogenes ORFs encoding central metabolic enzymes. List of A. succinogenes genes encoding enzymes of central metabolism with their locus names and EC numbers. Enzyme names are based on our manual annotation of the genome, using the criteria described in the materials and methods section. Table S4: Partial biosynthetic pathways present in A. succinogenes for amino acids and vitamins required for growth. Cysteine, glutamate, methionine, biotin, nicotinic acid, pantothenate, and pyridoxine are required for A. succinogenes's growth on defined medium. Table S4 lists the components of the cysteine, methionine, biotin, nicotinic acid, pantothenate, and pyridoxine biosynthetic pathways that are present in A. succinogenes. Enzyme names are based on our manual annotation of the genome, using the criteria described in the materials and methods section. This list confirms that A. succinogenes contains an incomplete assimilatory sulfate reduction pathway, but that it is able to synthesize cysteine from sulfide or thiosulfate. It also suggests that A. succinogenes is unable to synthesize Met from L-homocysteine. Table S5: A. succinogenes dicarboxylate transporters. A. succinogenes excretes large amounts of succinate as well as smaller quantities of fumarate, but the succinate and fumarate transporters are unknown. Table S5 lists the twelve possible anaerobic dicarboxylate transporters identified in A. succinogenes. Transporter names are based on our manual annotation of the genome, using the criteria described in the materials and methods section. Percent identity to experimentally characterized transporters is indicated in parentheses. Nine A. succinogenes transporters are similar to the tripartite ATP-independent periplasmic transporter (T.C. 2.A.56) encoded by dctPQM . The other three are related to DcuA, B, and C (T.C. 2.A.13). DcuA, B, and C operate by exchanging an intracellular dicarboxylate (e.g., succinate) for an extracellular dicarboxylate (e.g., fumarate, malate, or aspartate). DcuA and B may also transport Na+ in symport with the dicarboxylates to avoid dissipating the proton motive force . Based on studies performed in E. coli and H. influenzae (see manuscript for references), Asuc_0142, 1999, and 1063 are likely candidates genes for dicarboxylate transport during fumarate respiration and succinate fermentation.
Format: PDF Size: 151KB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 3:
Table S6. Table S6: A. succinogenes and M. succiniciproducens proteins showing similarity to known Pasteurellaceae virulence factors. Table S6 is an extensive list of known Pasteurellaceae virulence factors and their top BLAST hits in the A. succinogenes and M. succiniciproducens genomes associated with the best BLAST hits in A. succinogenes and M. succiniciproducens. Virulence factors are listed by category: cell surface structures, iron acquisition, toxins, and other. Each virulence factor (i.e., query sequence) is identified by its protein name, accession number, PubMed identifier (PMID), source organism, function, and length. Each A. succinogenes or M. succiniciproducens hit is identified by its accession number, locus number (only for A. succinogenes), length, E value, alignment length, percent identity, percent similarity, alignment length in percent of the length of the hit, validity of the hit (i.e., hit and reason columns), and product name in GenBank.
Format: XLSX Size: 128KB Download file
Additional file 4:
Explanation for lack of pathogenicity--extended discussion. The supplementary text contains an extended discussion of alignment of Pasteurellaceae virulence factors against succinogens' genomes. The discussion touches on many of the negative results not reported in the main text.
Format: PDF Size: 91KB Download file
This file can be viewed with: Adobe Acrobat Reader