A genomic perspective on the potential of Actinobacillus succinogenes for industrial succinate production
© McKinlay et al; licensee BioMed Central Ltd. 2010
Received: 4 May 2010
Accepted: 30 November 2010
Published: 30 November 2010
Succinate is produced petrochemically from maleic anhydride to satisfy a small specialty chemical market. If succinate could be produced fermentatively at a price competitive with that of maleic anhydride, though, it could replace maleic anhydride as the precursor of many bulk chemicals, transforming a multi-billion dollar petrochemical market into one based on renewable resources. Actinobacillus succinogenes naturally converts sugars and CO2 into high concentrations of succinic acid as part of a mixed-acid fermentation. Efforts are ongoing to maximize carbon flux to succinate to achieve an industrial process.
Described here is the 2.3 Mb A. succinogenes genome sequence with emphasis on A. succinogenes's potential for genetic engineering, its metabolic attributes and capabilities, and its lack of pathogenicity. The genome sequence contains 1,690 DNA uptake signal sequence repeats and a nearly complete set of natural competence proteins, suggesting that A. succinogenes is capable of natural transformation. A. succinogenes lacks a complete tricarboxylic acid cycle as well as a glyoxylate pathway, and it appears to be able to transport and degrade about twenty different carbohydrates. The genomes of A. succinogenes and its closest known relative, Mannheimia succiniciproducens, were compared for the presence of known Pasteurellaceae virulence factors. Both species appear to lack the virulence traits of toxin production, sialic acid and choline incorporation into lipopolysaccharide, and utilization of hemoglobin and transferrin as iron sources. Perspectives are also given on the conservation of A. succinogenes genomic features in other sequenced Pasteurellaceae.
Both A. succinogenes and M. succiniciproducens genome sequences lack many of the virulence genes used by their pathogenic Pasteurellaceae relatives. The lack of pathogenicity of these two succinogens is an exciting prospect, because comparisons with pathogenic Pasteurellaceae could lead to a better understanding of Pasteurellaceae virulence. The fact that the A. succinogenes genome encodes uptake and degradation pathways for a variety of carbohydrates reflects the variety of carbohydrate substrates available in the rumen, A. succinogenes's natural habitat. It also suggests that many different carbon sources can be used as feedstock for succinate production by A. succinogenes.
Actinobacillus succinogenes is a Gram-negative capnophilic bacterium that was isolated from bovine rumen as part of a search for succinate-producing bacteria . Succinate is an important metabolic intermediate in the rumen, where several bacteria obtain energy by decarboxylating succinate to propionate, which in turn serves as a nutrient for the ruminant [2, 3]. Succinate is used as a specialty chemical in food, agriculture, and pharmaceutical industries, but it has a much greater potential value for augmenting or replacing a multi-billion dollar petrochemical-based bulk chemical market [4, 5]. Succinate production by fermentation of renewable feedstocks is both economically and environmentally attractive. A further environmental benefit is that fermentative succinate production uses CO2, a greenhouse gas, as a substrate.
A. succinogenes is one of the best succinate producers ever described, but it also produces formate and acetate in high concentrations. Flux distribution between succinate and alternative fermentation products is affected by environmental conditions. For example, higher succinate yields can be obtained by increasing the available CO2 and a reductant (e.g., by supplying H2 or by using carbon sources that are more reduced than glucose) . Optimizing the environmental conditions is not sufficient to achieve a homosuccinate fermentation, though. Engineering A. succinogenes's metabolism for homosuccinate production will be most effective if based on an understanding of the enzymes and mechanisms controlling flux distribution. Deciphering the A. succinogenes genome sequence is thus invaluable for defining, understanding, and engineering A. succinogenes metabolic pathways.
There is also much knowledge to be gained by comparing the A. succinogenes genome to its closest relatives. A. succinogenes is a member of the Pasteurellaceae family, which contains thirteen named genera as well as candidates for new taxa . The best known genera are Actinobacillus, Haemophilus, and Pasteurella. At least thirty-two Pasteurellaceae genome sequences (complete and draft) are publicly available, fifteen of which are from different H. influenzae strains. While most Pasteurellaceae are studied for their pathogenic traits, A. succinogenes and its closest relative, Mannheimia succiniciproducens , collectively referred to as "succinogens" in this paper, are studied for their industrially attractive metabolic trait of succinate production. It will be important to confirm lack of pathogenicity in these succinogens before they are recommended for use on an industrial scale. Because A. succinogenes and M. succiniciproducens have never been reported in association with any disease, searching their genome sequences for Pasteurellaceae pathogenicity genes is a logical starting point to assess their potential for non-pathogenicity.
Here we present the first detailed analysis of the A. succinogenes genome sequence with a biotechnological perspective. The A. succinogenes and M. succiniciproducens genome sequences are also examined for known Pasteurellaceae virulence genes.
Chemicals, source strain, growth conditions, and genomic DNA purification
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO). A. succinogenes type strain 130Z (ATCC 55618) was obtained from the American Type Culture Collection (Manassas, VA). To identify the A. succinogenes vitamin auxotrophies, A. succinogenes was grown in the defined medium, AM3 , and then transferred (1:100 dilutions) in parallel into ten tubes containing fresh AM3 medium, each tube lacking a single vitamin. A. succinogenes was considered prototrophic for a vitamin, if growth was maintained for three consecutive transfers in its absence. To confirm the minimal vitamin requirements, A. succinogenes was grown through seven transfers in AM3 containing only the required vitamins. To determine A. succinogenes's ability to grow on various carbon sources, cells were grown anaerobically in Medium B (g/L: NaH2PO4·H2O, 8.5; K2HPO4, 15.5; bactotryptone, 10.0; yeast extract, 5.0; and NaHCO3, 2.1) supplemented with a single carbon source (1 g/L). The initial pH was adjusted to 7.0-7.2. A. succinogenes was considered able to grow on a carbon source when cell yields (absorbance at 660 nm) were higher in medium B supplemented with that carbon source than in non-supplemented medium. Growth data were recorded after each of two serial transfers of three biological replicates. For genomic DNA extraction, A. succinogenes was grown in 100 mL of tryptic soy glucose broth (Becton Dickinson, Sparks, MD) with 25 mM NaHCO3 in a 160-mL anaerobic serum vial at 37°C. The culture was harvested in log phase (~7.7 × 1010 cells) and washed twice in 45 mL of phosphate buffer (g/L: K2HPO4, 15.5; NaH2PO4*H2O, 8.5; NaCl, 1). Genomic DNA was purified using a Qiagen genomic tip protocol with a Qiagen maxiprep column (Valencia, CA) as described in the QIAGEN Genomic DNA Handbook.
Genome sequencing and assembly
Sequencing was performed by the Department of Energy's Joint Genome Institute (JGI). The genome of A. succinogenes was sequenced using a combination of three Sanger genomic libraries: 3 kb pUC18c, 8 kb pMCL200, and 40 kb fosmid libraries. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website . 41,370 Sanger reads were assembled using PGA assembler (Paracel Genome Assembler 2.6.2, Paracel, Pasadena, CA). Possible mis-assemblies were corrected and gaps between contigs were closed by custom primer walks from sub-clones or PCR products. A total of 2,986 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The completed genome of A. succinogenes 130Z contains 43,200 reads. The error rate of the finished genome sequence is less than 1 in 100,000. Together all libraries provided 11× coverage of the genome. The genome sequence of A. succinogenes strain 130Z is available in GenBank under accession number CP000746.
Automated annotation was performed by the Oak Ridge National Laboratory . Open reading frames (ORFs) were identified using three gene caller programs: Critica, Generation, and Glimmer. Translated ORFs were subjected to an automated basic local alignment search tool (BLAST) for proteins  against GenBank's non-redundant database. The translated ORFs were also subjected to searches against KEGG, InterPro (incorporating Pfam, PROSITE, PRINTS, ProDom, SmartHMM, and TIGRFam), and Clusters of Orthologous Groups of proteins (COGs).
The ORFs described in this paper have also been manually annotated. BLAST alignments were examined to assess the correctness of the start codon. DNA sequences upstream of each ORF were examined for a ribosomal binding site (at least 3 nt of the AAGGAGG sequence, 5-10 nt upstream of the start codon) using the web Artemis tool . To assign product names to each ORF, results from BLAST, HMM (i.e., PFAM and TIGRFAM), and domain and motif searches were considered. Most importantly, efforts were made to find a citation of biological function for a homologous gene. If a translated ORF was at least 75% identical to a protein of known function over 75% of the length, or if it belonged to a TIGRFAM equivalog, it was given the associated product name. If a translated ORF was less than 75% identical to a protein of known function, the product name was modified as follows: 60-75% identity over 65% of the length, putative product; 40-64% identity over 40% of the length, probable product; 25-39% identity over 25% of the length, possible product. If a translated ORF was at least 60% identical to a protein of unknown function, it was named a conserved hypothetical protein. If there was no adequate alignment with any protein (less than 25% identity or aligned region is less than 25% of the product length), the translated ORF was named a hypothetical protein.
Other genome analyses
To compare their gene contents, the A. succinogenes and M. succiniciproducens genomes were re-annotated using the fully automated, prokaryotic genome annotation service, RAST (Rapid Annotation using Subsystem Technology) . Pairwise BLAST comparisons of protein sets encoded by A. succinogenes and M. succiniciproducens genomes and predictions of the number of subsystems were performed using the sequence-based comparison tool available in RAST. Orthologous protein-coding genes in the two succinogens were manually compiled by comparing gene order, gene orientation (forward/reverse), features of intergenic regions, and protein similarity (minimum 25% identity at the protein level).
NUCmer and PROmer  whole-genome alignments were performed using an online Synteny plot tool . Clustered regularly interspaced short palindromic repeats (CRISPR) and spacers were identified using the CRISPRs web service [16–18]. Spacer sequences were then aligned against the A. succinogenes genome sequence using BLAST. 16 S rRNA phylogeny was determined using the Michigan State University Ribosomal Database Project tools [19, 20]. Hierarchical clustering of Pasteurellaceae genomes was done using tools at the JGI's Integrated Microbial Genomes (IMG) website [21, 22].
Uptake signal sequence (USS) 9-mer cores  were counted, and their surrounding sequences were reported using our perl script, 200804USS.pl. 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. A search of A. succinogenes and M. succiniciproducens genomes for Pasteurellaceae virulence genes was performed by compiling a list of known Pasteurellaceae virulence genes based on the literature, then using a custom Python script to align their sequences against the two genomes using BLAST and report the data for the top hit.
Results and Discussion
General features of the A. succinogenes genome compared to fifteena complete Pasteurellaceae genomes
Chromosome size (bp)
Gene density (genes/kb)
GC content (%)
5 S rRNAs
16 S rRNAs
23 S rRNAs
The A. succinogenes genome is most closely related to that of its succinogen relative, M. succiniciproducens  (additional file 1: Figures S1 and S2). M. succiniciproducens was also isolated from a bovine rumen, albeit on a different continent, and it shares many metabolic traits with A. succinogenes (see below). Based on genome re-annotations performed with RAST (2,223 protein-encoding genes, total), the two succinogens' genomes have 1,735 ORFs (78%) in common, 442 ORFs are found only in M. succiniciproducens, and 488 ORFs are found only in A. succinogenes. Of 2,081 automated KEGG comparisons , 1,861 (89%) A. succinogenes genes were most similar to other Pasteurellaceae genes, with 1,252 (60%) being most similar to M. succiniciproducens genes. However, A. succinogenes and M. succiniciproducens are among at least twenty-four misclassified Pasteurellaceae species that will likely be renamed, as they do not cluster with properly classified species in phylogenetic trees based on Pasteurellaceae 16 S rRNA, infB, rpoB, or atpD gene sequences . The two succinogens are often clustered together using phylogenetic approaches, but not closely enough to suggest that they belong to the same genus . Hierarchical clustering of gene function categories also places the two succinogens in a clade separate from other Pasteurellaceae (additional file 1: Figure S2). To better gauge how closely related the two organisms are, we performed whole genome NUCmer and PROmer alignments of the two succinogens with each other, as well as with eight other Pasteurellaceae (additional file 1: Figure S3). 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. Overall, though, the PROmer plots show that drastic changes in genome structure have occurred as A. succinogenes and M. succiniciproducens evolved divergently from their last common ancestor, and that the two succinogens are more distantly related than their functional traits would suggest.
A 39,489-bp prophage genome is encoded in the Asuc_1205-58 region. The presence of a prophage has biotechnological relevance for two reasons. First, it raises the possibility of using phage-based genetic engineering. Second, it suggests that A. succinogenes may be susceptible to phage lysis in an industrial bioreactor; if so, steps should be taken to eliminate this prophage from the host genome. This prophage has an organization similar to that of the Aggregatibacter actinomycetemcomitans phage, AaΦ23 , and it contains a DNA N-6-adenine-methyltransferase (Asuc_1221). The A. succinogenes prophage differs from AaΦ23, though, in that the integrase gene (Asuc_1258) is located at the opposite end of the phage genome from its location in AaΦ23. Despite sharing a similar organization, many of the A. succinogenes phage proteins are not found in AaΦ23, and they are conserved in only a few Pasteurellaceae genomes. For example, the Asuc_1233-44 proteins are not well conserved among Pasteurellaceae, but they include such crucial proteins as both terminase subunits (Asuc_1235-6), a portal protein (Asuc_1238), a prohead protease (Asuc_1239), a major capsid protein (Asuc_1240), a protein with possible DNA-packing function (Asuc_1241), and a putative head-tail adaptor (Asuc_1242). Two sets of addiction module killer and antidote proteins are also encoded (Asuc_1211-4). Another interesting feature of the A. succinogenes phage is that Asuc_1219, encoding a homolog to replication protein O (which, along with the P protein [Asuc_1220], initiates lytic replication), has an internal frame shift. It is unclear at this point whether this prophage corresponds to a functional temperate phage. Growth experiments in the presence of mitomycin C showed that 0.1 μg/mL mitomycin C started inhibiting growth after 3 h, 1 μg/mL mitomycin C inhibited growth starting at 90 min, and no growth was observed at 10 μg/mL mitomycin C. These results are similar to those observed for phage induction in E. coli K12 . More work (beyond the scope of this paper) would be needed, though, to demonstrate that the inhibitory effect of mitomycin C is associated with the release of phage particles in the culture broth.
A set of CRISPRs (located between Asuc_1293 and 1294) and a set of CRISPR-associated genes (Asuc_1284-93) are located thirty-four ORFs downstream from the prophage. Together, these CRISPRs and CRISPR-associated genes can provide resistance against phage . Homologs in the 30% to 75% identity range to A. succinogenes CRISPR-associated genes are found in M. succiniciproducens and Mannheimia haemolytica, but they are not found in other Pasteurellaceae. Short fragments of five of the ten A. succinogenes CRISPR spacers matched short sequences in A. succinogenes prophage genes. One spacer sequence showed similarity to a gene encoding a phage integrase family protein (Asuc_0030). This gene is accompanied by only one other phage gene encoding a probable capsid portal protein Q with two internal frame shifts (Asuc_0029, 58% identical to an M. haemolytica phage ΦMHaA1 protein and 73% identical to a phage protein found in several H. influenzae genomes). These two genes may be the last remnants of an ancient phage integration-excision event.
A. succinogenes, M. succiniciproducens, and nine other Pasteurellaceae species contain a homolog of Escherichia coli LamB (Asuc_0322, 54% identical), the maltoporin functioning as receptor for phage lambda. In both succinogens, the lamB homolog is part of the maltose transport operon (additional file 2: Table S1). This gene is likely functional in A. succinogenes, since A. succinogenes grows well on maltose . This result suggests that A. succinogenes may be susceptible to infection by lambda-related bacteriophages.
All Pasteurellaceae genomes contain USS repeats that feature a conserved 9-nt sequence . In most Pasteurellaceae the conserved 9-mer is AAGTGCGGT (i.e., USS1), whereas ACAAGCGGT (i.e., USS2) predominates in Actinobacillus pleuropneumoniae, H. ducreyi, H. parasuis and M. haemolytica . Each USS is followed by a conserved AT-rich region, and USS2 is additionally followed by GCAAA(A/T) 20-nt downstream of the 9-mer  (additional file 1: Figure S4). The only function yet demonstrated for USS repeats is in natural competence . Under certain conditions (e.g., starvation or the presence of elevated cAMP levels) many Pasteurellaceae preferentially internalize USS-containing DNA, perhaps being recognized as self-DNA [29, 31]. Uptake of USS-containing DNA is facilitated by a number of competency proteins, resulting in transformation frequencies that can be as high as 10-3 to 10-2 transformants per CFU  and in homologous recombination with chromosomal DNA. This DNA uptake mechanism works best with linear DNA, making it well suited for strain engineering, since constructs can be easily generated by PCR and double recombination events are needed to integrate the linear DNA into the chromosome. Genetic tools for A. succinogenes are currently limited to expression vectors [32, 33]. The ability to replace chromosomal DNA with engineered DNA is invaluable for making gene knockouts (e.g., to block unwanted fermentation pathways) and knock-ins (e.g., of a modified promoter to affect enzyme expression).
Occurrence of USS repeats in complete Pasteurellaceae genome sequences
H. influenzae Rd KW20
A. aphrophilus NJ8700
H. influenzae PittEE
H. influenzae PittGG
H. influenzae 86-028NP
A. succinogenes 130Z
H. somni 129PT
H. somni 2336
P. multocida str. Pm70
A. pleuropneumoniae str. JL03
A. pleuropneumoniae L20
A. pleuropneumoniae str. AP76
H. parasuis SH0165
H. ducreyi 35000HP
A regulon of twenty-five competency genes is known in H. influenzae . The A. succinogenes genome contains homologs of all of these genes except for two, HI0660 and HI1631, which encode hypothetical proteins in H. influenzae. Seven of the A. succinogenes competency proteins are less than 40% identical to their H. influenzae homologs (additional file 2: Table S2). In addition to twenty-three competency genes, the A. succinogenes genome also encodes the master regulator of the competence regulon (the cAMP receptor protein, Asuc_0008) and the essential competence regulatory factor, Sxy (Asuc_0283). It also encodes proteins that are not competence-induced, but that are known to participate in DNA uptake or recombination (i.e., RecA, TopA, AtpA, and DsbA, additional file 2: Table S2) . The abundance of USS repeats in A. succinogenes and the possible presence of the necessary machinery for natural competence suggested that A. succinogenes could be naturally competent. Recent experiments in our laboratory demonstrated that A. succinogenes could uptake DNA by natural transformation. These transformations led to the construction of two gene knockouts (Joshi et al., manuscript in preparation).
All the genes encoding C4 pathway enzymes were identified in the A. succinogenes genome. In contrast to M. succiniciproducens, which contains both a PEP carboxykinase (PEPCK) and a PEP carboxylase , PEPCK is the only PEP-carboxylating enzyme in A. succinogenes (Asuc_0221). The other C4 pathway enzymes (i.e., malate dehydrogenase [Asuc_1612], fumarase [Asuc_0956], and fumarate reductase [Asuc_1813-6]) make up an incomplete arm of a reductive tricarboxylic acid cycle (TCA) that also includes succinyl-CoA synthetase (Asuc_1564-5). Unlike M. succiniciproducens , A. succinogenes lacks the TCA cycle enzymes citrate synthase and isocitrate dehydrogenase, but it does have an α-ketoglutarate (αKG) dehydrogenase (Asuc_1566-7 and 0944) and a citrate lyase (Asuc_0305, 1194-6 and 1198). Similar to the genome organization of E. coli , the A. succinogenes αKG dehydrogenase E3 subunit (i.e., lipoamide dehydrogenase) is also the pyruvate dehydrogenase E3 subunit, and it is encoded in the pyruvate dehydrogenase operon (Asuc_0942-4).
Most of the C3 pathway enzymes were identified, including pyruvate dehydrogenase and formate dehydrogenase (Asuc_1261-6) (Figure 2). Fluxes through these two dehydrogenases were shown to be important sources of reducing power for succinate production and for anabolism when coupled with transhydrogenase activity [34, 35]. A single predicted membrane-bound transhydrogenase was also identified (Asuc_1021-22). The only uncertainty in the C3 pathway is the mechanism by which ethanol is produced from acetyl-CoA, but the genome contains a number of aldehyde and alcohol dehydrogenases. Among them, Asuc_0403, 0591, 1136, and 1955 belong to the iron-containing alcohol dehydrogenase protein family. Asuc_0591 is a good candidate for ethanol production. It is probably a multifunctional aldehyde/alcohol dehydrogenase , and it is 47% identical (66% similar) to E. coli AdhE (GenBank accession no. P0A9Q7), the enzyme responsible for ethanol production. Asuc_0067 encodes a class III alcohol dehydrogenase. A class III alcohol dehydrogenase functions primarily as a formaldehyde dehydrogenase (E.C. 126.96.36.1994), but it can also produce ethanol . Even though M. succiniciproducens is not known to produce ethanol, it has homologs of all the A. succinogenes alcohol dehydrogenases mentioned above. Thus, these proteins are either not functional for ethanol production in M. succiniciproducens, or they are not involved in ethanol production in A. succinogenes.
Metabolic flux distribution between the C3 and C4 pathways is known to be influenced by CO2 and H2 concentrations. Malic enzyme (Asuc_0669) and a sodium-pumping OAA decarboxylase (Asuc_0301-3), which are responsible for large reversible fluxes between the C3 and the C4 pathways , are encoded in the genome. Carbonic anhydrase (Asuc_1199), which interconverts CO2 and HCO3-, was identified, and it could be important for making CO2 available for succinate production in environments with different pH values. A single membrane-bound hydrogenase (Asuc_1277-83) was also identified.
In contrast to M. succiniciproducens, A. succinogenes does not produce lactate . The A. succinogenes genome encodes a single lactate dehydrogenase, Asuc_0005, which is 60% identical to the E. coli enzyme (EC 188.8.131.52) that couples lactate oxidation to amino acid and sugar transport . Asuc_0005 is therefore expected to oxidize, rather than generate, lactate. In contrast, the M. succiniciproducens genome does not encode an Asuc_0005 homolog. A. succinogenes was grown anaerobically in AM3 with 100 mM NaHCO3-, 50 mM glucose, and 25 mM D, L-lactate, but no lactate consumption was observed (data not shown).
A. succinogenes is also capable of gluconeogenesis, since it can grow by anaerobic respiration using H2 or electrically reduced neutral red as an electron donor and using fumarate or malate as the carbon source and electron acceptor [6, 40, 41]. The genome sequence encodes a putative type I (Asuc_1073) and a type II (Asuc_0394) fructose-1,6-bisphosphatase, but it does not encode a PEP synthase. As a result, A. succinogenes must rely on gluconeogenic flux through PEPCK to make PEP from malate and fumarate.
Production of succinate in a chemically defined medium can decrease downstream costs in product purification. A. succinogenes is known to require glutamate, cysteine, and methionine to grow in a defined medium . Glu auxotrophy is due to an inability to synthesize αKG from glucose , which is now explained by the absence of genes encoding isocitrate dehydrogenase in the genome sequence. αKG cannot be synthesized from succinate because of the unidirectional activity of αKG dehydrogenase from αKG to succinyl-CoA (ΔG°' = -30,000) and because the A. succinogenes genome does not encode the reductive-TCA cycle enzyme, αKG ferredoxin oxidoreductase. Surprisingly, A. succinogenes encodes all the enzymes required to synthesize Cys (additional file 2: Table S4). Since both Cys and Met are sulfur-containing amino acids, we wondered if these auxotrophies could be due to an inability to assimilate sulfate, the only mineral sulfur source in AM3. Indeed, the A. succinogenes genome does not encode adenylsulfate kinase (additional files 1 and 2: Figure S5 and Table S4), which is required for assimilatory sulfate reduction. A. succinogenes grew normally in AM3 once sodium sulfide or sodium thiosulfate was added in place of Cys, confirming that the Cys auxotrophy is due to an inability to reduce sulfate. Met, however, was still required for growth in the presence of reduced sulfur compounds. A. succinogenes is missing several genes necessary to synthesize Met through the L-homocysteine pathway, and it would require a source of methanethiol to produce Met from O-acetyl homoserine (additional files 1 and 2: Figure S5 and Table S4).
We determined that nicotinic acid, pantothenate, pyridoxine, and thiamine are the only four vitamins required by A. succinogenes. The A. succinogenes genome sequence is missing several genes involved in the biosynthesis of these vitamins (additional files 1 and 2: Figure S6 and Table S4). Even though it is missing several genes involved in biotin synthesis (e.g., bioA, bioF, and bioW), A. succinogenes grows repeatedly in the absence of biotin (five consecutive transfers in AM3). However, A. succinogenes grows more reliably in AM3 supplemented with biotin when inoculated from frozen stocks or from rich medium. Biotin can therefore be considered non-essential, but stimulatory, for the growth of A. succinogenes, a feature shared with M. succiniciproducens . One mechanism that could explain the growth of A. succinogenes in the absence of biotin despite the absence of a full set of biotin biosynthetic genes is that A. succinogenes might be able to use thiamine as a precursor for biotin synthesis, as has been observed with the fungus, Humicola, strain 16-1 . The A. succinogenes gene encoding a putative biotin synthase (Asuc_1132) seems to be co-transcribed with the thiamine ABC transporter genes (Asuc_1229-31).
Similar to A. succinogenes, M. succiniciproducens is auxotrophic for Cys, Met, nicotinic acid, pantothenate, pyridoxine, and thiamine, and the genetic bases underlying these auxotrophies are the same as those identified in A. succinogenes . A. succinogenes and M. succiniciproducens differ in one respect, though. M. succiniciproducens is not auxotrophic for Glu since, unlike A. succinogenes, it has genes encoding a citrate synthase (MS2371) and an isocitrate dehydrogenase (MS2370). Although the two succinogens have an incomplete assimilatory sulfate reduction pathway, they, along with various A. pleuropneumoniae strains and Actinobacillus minor NM305, have the most complete assimilatory sulfate reduction pathway among all other sequenced Pasteurellaceae. The inability of A. succinogenes and M. succiniciproducens to carry out assimilatory sulfate reduction is likely an adaptation to their natural environment. The rumen flora produces hydrogen sulfide [44, 45]. Both succinogens encode a serine acetyltransferase (Asuc_0384 and MS2212) and a cysteine synthetase (Asuc_2108 and MS1770) that may allow them to synthesize L-Cys from H2S produced in the rumen.
The insights into A. succinogenes's auxotrophies obtained by genome analyses have allowed us to modify our original defined medium. Whereas Glu and Met are two of the least expensive amino acids (~$1/kg), cysteine is more expensive (> $10/kg) . Using inexpensive inorganic reduced sulfur compounds (such as thiosulfate in place of cysteine) and eliminating several nonessential vitamins are expected to significantly reduce the cost of defined growth medium.
Dicarboxylic acid transporters
As a producer of the dicarboxylate succinate, it is interesting to note that A. succinogenes encodes twelve possible anaerobic dicarboxylate transporters (additional file 2: Table S5). Nine of them are similar to the tripartite ATP-independent periplasmic transporters (T.C. 2.A.56) encoded by dctPQM . These transporters have been characterized in Rhodobacter capsulatus and Wolinella succinogenes for their roles during fumarate respiration, where fumarate is transported by proton symport [47, 48]. The other three anaerobic dicarboxylate transporters are related to DcuA, B, and C (T.C. 2.A.13). These transporters have been characterized during fumarate respiration by E. coli and W. succinogenes [48–50]. They 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 . DcuC may have preferential succinate efflux activity, since a dcuC ,- E. coli strain has increased dicarboxylate exchange and fumarate uptake activities . During E. coli mixed acid fermentation, glucose did not repress dcuC expression, suggesting that DcuC plays a role in succinate excretion . A microarray study examining changes in H. influenzae gene expression during competency induction suggested that DcuA and B are important for Pasteurellaceae fermentation or fumarate respiration . In that study, 151 genes showed > 4-fold increase in transcript levels as H. influenzae became competent, including transcripts for the C4 pathway enzymes aspartate ammonia-lyase, malate dehydrogenase, fumarase, fumarate reductase, and a single dicarboxylate transporter. This H. influenzae transporter is 84% and 44% identical to A. succinogenes's putative DcuB-type transporters, Asuc_0142 and 1999, respectively. A. succinogenes dcuA, B, and C are therefore candidate genes to investigate the importance of dicarboxylate transport during fumarate respiration and succinate fermentation.
Many sugar transport and degradation proteins have associated regulatory proteins (e.g., for L-arabinose, maltose, ribose, xylose, lactose, sorbitol, mannitol, glucarate and galactarate, and glycerol; additional file 2: Table S1). The ORFs putatively encoding sugar transport and degradation pathways encompass all the sugars A. succinogenes is known to use, except arabitol [1, 6]. Homologs of the E. coli arabitol transporter and arabitol dehydrogenase were not found in the A. succinogenes genome. The genome also encodes transporters and degradation pathways for carbon sources A. succinogenes was not known to metabolize (e.g., ascorbate, pectin, glucarate, and galactarate). In the conditions tested, though, we were able to detect growth on galactarate and ascorbate only. Growth on ascorbate was surprising because the ascorbate PTS transporter encoded by Asuc_0235-40, appears to be missing a component. It is not clear whether this missing component is encoded elsewhere or whether another PTS system shares some specificity for ascorbate. The absence of growth on pectin was surprising, since A. succinogenes contains three separate operons encoding a full pectin degradation pathway (Asuc_0145-58, 0366-74, and 1467-75). Three of A. succinogenes's ten possible tripartite ATP-independent periplasmic transporters (Asuc_0146-48, 0156-58, and 0366-68) are found in these operons. Several other operons appear to have a role in sugar transport and degradation, but the identity of the sugars is unknown. For example, one of the four proteins that show similarity to an idonate, a gluconate, or a 5-ketogluconate transporter is in an operon that encodes enzymes to degrade an unidentified sugar (Asuc_0119-30). Also, Asuc_0585-8 encodes a fructose-like PTS system and a protein that might be a sugar kinase.
Explanation for lack of pathogenicity
The natural Pasteurellaceae ecology is in association with a host . The host is usually mammalian, with a few exceptions , such as P. multocida colonizing birds  and possibly even amoebas . Most Pasteurellaceae can be isolated from healthy hosts and are considered part of the normal flora. However, in circumstances such as host stress, many Pasteurellaceae cause disease and are considered opportunistic pathogens. Most Pasteurellaceae are isolated from the respiratory tract and cause pulmonary diseases . Others have been isolated from the oral cavity (e.g., A. actinomycetemcomitans, which causes periodontitis), the genital tract (e.g., H. ducreyi, which causes sexually-transmitted chancroid), and bovine rumen (e.g., A. lignieresii, which causes wooden tongue) . Virulence is an undesirable trait for an industrial organism, and the relatedness of A. succinogenes and M. succiniciproducens to several pathogens cannot be ignored. With no reports of disease caused by the succinogens, their genome sequences are a convenient and logical starting point to assess their potential for lack of pathogenicity.
Many Pasteurellaceae virulence factors have been characterized. We manually compiled a list of 341 Pasteurellaceae virulence proteins (with some functional redundancy), including and expanding on those compiled by Challacombe and Inzana . We then aligned their sequences against the A. succinogenes and M. succiniciproducens protein databases (additional file 3: Table S6). Our comparison focuses on gene products having functions in toxin production, synthesis of cell surface structures, and iron uptake (additional file 3: Table S6). We excluded from the comparison virulence factors in the categories of amino acid transporters, purine and pyrimidine biosynthetic enzymes, and enzymes for anaerobic metabolism. While these metabolic activities may affect host health [55–57], they are also necessary for nonvirulent processes. The major findings from our alignments are summarized in this section with more details available in additional file 4: Supplementary text. Raw data from the alignments are reported in additional file 3: Table S6.
Repeat toxins (RTX), including leukotoxins, are produced by many Pasteurellaceae, including A. pleuropneumoniae , A. actinomycetemcomitans , M. haemolytica , and P. multocida . A. actinomycetemcomitans uses a cytolethal distending toxin that is encoded near a characteristic virulence-associated region . Neither succinogen genome sequence encodes the components necessary to produce leukotoxin or cytolethal distending toxin. Additionally, neither succinogen encodes a homolog to the H. ducreyi hemolysin .
Cell surface structures
Cell surface virulence factors used by pathogenic Pasteurellaceae include pili, adhesins, lipopolysaccharide (LPS), and capsules. Adherence to respiratory epithelial cells is the first colonization stage by respiratory Pasteurellaceae pathogens. It involves a number of cell surface mechanisms . Both succinogens have possible homologs to OapA and B and all components of a type IV pilus (pilABCD), which is involved in host surface binding in H. influenzae [65, 66]. Because type IV pili are also part of the H. influenzae competence regulon , and because A. succinogenes was recently shown to be naturally competent (Joshi et al., manuscript in preparation), type IV pili might not be related to virulence in the succinogens. M. succiniciproducens has probable homologs of the A. actinomycetemcomitans pili needed for tight adherence (flp and tad loci) , whereas A. succinogenes does not. Both succinogens have several large ORFs that could encode HMW adhesins, including gene clusters that may be involved in hemagglutinin production (Asuc_1006-12 and MS1162-9). However, Asuc_1006 and 1008 have internal frame shifts. It is currently not known whether either succinogen makes an adhesin. It is also possible that the succinogens use this feature for survival in a competitive rumen environment, rather than to cause disease.
Nontypeable H. influenzae strains are able to evade host immune defenses by incorporating host sialic acid and choline into their LPS, thereby mimicking host cell surfaces [68, 69]. The succinogens' genome sequences contain many genes involved in LPS synthesis and modification but key genes for choline and sialic acid incorporation are not present. None of these genes contain variable number tandem repeats, suggesting that the succinogens are not capable of LPS phase variation.
A. succinogenes has several LPS glycosyltransferases not found in M. succiniciproducens (e.g., Asuc_0524, 1375), suggesting that its LPS could be more complex. The two succinogens' LPSs might also differ in sugar composition. A. succinogenes is one of only four Pasteurellaceae that encodes the L-rhamnose synthesis pathway. L-rhamnose is a common component of the LPS O-antigen [70, 71], and the A. succinogenes L-rhamnose biosynthetic pathway (Asuc_0826-32) is encoded just downstream of the LPS biosynthesis genes (Asuc_0821-24). Because LPS O-antigens are mostly studied in pathogenic bacteria, it is unclear how often non-pathogenic bacteria contain rhamnose in their LPS. For this reason, the possible presence of rhamnose in A. succinogenes LPS is by no means indicative of a virulence trait. In contrast, M. succiniciproducens encodes proteins likely involved in L-rhamnose transport (RhaT, MS2326) and catabolism (RhaBAD, MS2327-29), as well as the L-rhamnose-dependent regulators RhaS (MS2322) and RhaR (MS2323). None of these genes are found in A. succinogenes. Thus, the two succinogens have evolved completely different L-rhamnose pathways--a biosynthetic one in A. succinogenes and a catabolic one in M. succiniciproducens.
P. multocida, A. pleuropneumoniae, M. haemolytica, and typeable H. influenzae produce a capsule that is important for virulence [72–76]. Both succinogens have possible homologs to, at most, two of the four capsule biosynthesis and export proteins, suggesting that they are not capsulated bacteria. However, non-typeable H. influenzae are non-capsulated but they are still virulent.
Iron uptake mechanisms
Iron acquisition is a common trait in most bacteria, making it difficult to associate iron uptake with virulence. Nonetheless, some insight can be gained from the form of iron transported. Some pathogenic Pasteurellaceae can use the mammalian iron sources, transferrin and hemoglobin [72, 77], but the succinogens have possible homologs to only a few of the proteins required for transferrin, heme/hemopexin, or hemoglobin uptake. A. succinogenes does not have homologs to the hemin receptor HemR or to the heme utilization protein Hup , while M. succiniciproducens has possible homologs of each. Still, BLAST searches show that both succinogens can assimilate other forms of iron, including iron bound by various siderophores and that they contain the heme biosynthetic pathway from L-glutamate  (additional file 3: Table S6). In both succinogens the potential hemagglutinin production system mentioned above is encoded alongside genes involved in ferrous iron transport, including feoAB. FeoA and B are not encoded in any sequenced Pasteurellaceae other than the succinogens and A. minor NM305, but they have been implicated in more distantly related bacteria in virulence and colonization of mammalian intestines [79, 80]. This genetic region may be a worthwhile target for deletion, provided it does not contain essential genes for growth and succinate production.
Other virulence proteins
Both succinogens have a putative homolog to the inner membrane protein, ImpA, involved in autoaggregation . Some Pasteurellaceae have a urease, which is a known virulence factor of gastroduodenal and urinary tract pathogens , but the succinogens have no urease homologs, and A. succinogenes tested negative for urease activity . There is also no homolog in either succinogen genome sequence to the H. influenzae Iga protease, which cleaves immunoglobulin A1, helping H. influenzae avoid host defenses at mucosal surfaces [82, 83].
We want to stress that nonpathogenicity cannot be concluded from the analysis of a genome sequence. Most Pasteurellaceae species cause respiratory diseases. The virulence factors associated with a hypothetical rumen succinogen-caused disease would likely be different. For example, the FeoAB iron uptake system, which is important for the virulence of some intestinal pathogens, is unique to the succinogens among the thirty-two partially and fully sequenced Pasteurellaceae, with A. minor NM305 (part of the pig respiratory tract normal flora) an exception. This system, though, could also be important for a commensal relationship with the mammalian host.
Sequencing of the A. succinogenes genome confirms many of our earlier results based on growth experiments, enzyme assays, and metabolic flux studies [6, 9, 34, 35]. For example, A. succinogenes lacks a complete TCA cycle as well as a glyoxylate pathway, and PEP carboxykinase is the only PEP-carboxylating enzyme in this organism. The genes missing in the glutamate, cysteine, and methionine biosynthetic pathways represent possible positive markers that can be used in genetic engineering strategies. The fact that the A. succinogenes genome encodes uptake and degradation pathways for a variety of carbohydrates reflects the variety of carbohydrate substrates available in the rumen, A. succinogenes's natural habitat. It also suggests that many different carbon sources can be used as feedstock for succinate production by A. succinogenes. The abundance of USS repeats in A. succinogenes and the possible presence of the necessary machinery for natural competence suggested that A. succinogenes is naturally competent, a feature that was recently demonstrated in our laboratory. It is encouraging that the succinogens' genome sequences lack a considerable number of the virulence genes used by their relatives, and that there are no reports of disease caused by A. succinogenes or M. succiniciproducens. The lack of pathogenicity of these two succinogens is an exciting prospect not just for industrial purposes, but because comparisons with pathogenic Pasteurellaceae could lead to a better understanding of Pasteurellaceae virulence.
This work was supported by the National Science Foundation grant BES-0224596, by a grant from the Michigan State University (MSU) Research Excellence Fund, and by a grant from the Michigan Economic Development Corporation. We wish to thank Dr. J. Gregory Zeikus for allowing us to continue his work on this fascinating and useful organism. We are deeply grateful to the JGI for sequencing the A. succinogenes genome and providing us with the automatic annotation and useful genome analysis tools. The U.S. Department of Energy Joint Genome Institute work was supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. We also thank Drs. M. Bagdasarian, J.A. Breznak, C.A. Reddy, and Y. Shachar-Hill for valuable insights and discussions. We acknowledge Dr. Carlos Araya for assistance with Python programming. We are grateful to Drs. Peter Bergholz and Hector Alaya-del-Rio for expert advice on manual genome annotations. We thank Christopher B. Jambor for his valuable editing advice.
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