Construction and characterization of shuttle vectors for succinic acid-producing rumen bacteria

Abstract

Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 x 10(6) and 7.1 x 10(6) transformants/mug DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.

FIG. 1.

E. coli-rumen bacteria shuttle vectors constructed in this study. The shuttle vectors contain both ColE1 ori for E. coli and pMVSCS1 ori for rumen bacteria. Genes are represented by arrows: strAB, streptomycin resistance gene; catAIII, chloramphenicol resistance gene; sulII, sulfonamide resistance gene; bla, ampicillin resistance gene. The pckA promoter (P_pckA) and transcription terminator (TT) were introduced in pMEx for gene expression. The shuttle vector pMS3 contains the M. succiniciproducens frdA promoter and the rrnBT2 terminator (T2) for gene expression. Multiple cloning sites (MCS) of pMS3 are 5′-EcoRI-SacI-KpnI-SmaI-BamHI-XbaI-SalI-PstI-SphI-HindIII-3′.

FIG. 2.

Determination of plasmid copy numbers by qPCR. (A) Physical maps of the targeted genes for qPCR. The M. succiniciproducens fumC (gray arrow) and the A. succinogenes pckA (line arrow) genes were used as the single-copy references for M. succiniciproducens and A. succinogenes, respectively. The replication origin (black) of pMVSCS1 (or pMEx) was the target gene for qPCR. (B) Fluorescence versus cycle number curves for 0.01 (open symbol), 0.1 (semiopen symbol), and 1 ng (closed symbol) of enzyme-digested total template DNA. The lines in the graphs are as follows: black solid, plasmid; gray solid, chromosome of rumen bacteria. (C) C_T versus log concentration curves. Error bars represent the standard deviations of data obtained from the experiments performed in triplicate.

FIG. 3.

GC skew analysis and fragment elimination study of the replicon. The symbols are as follows: white arrows, iterons; black arrow heads, repeated sequence in AT-rich regions; white squares, dnaA box; H and P, HindIII and PstI sites.

FIG. 4.

Replication mechanisms of pMVSCS1 and pMEx. (A) Agarose gel electrophoresis. Southern blotting results without (B) and with (C) denaturation of the gels. Lanes 1, 3 and 5, total DNA from M. succiniciproducens harboring pMVSCS1; lanes 2, 4, and 6, total DNA form M. succiniciproducens harboring pMEx. Abbreviations: oc, open circle double-stranded DNA; ri, replication intermediates; sc, supercoiled double-stranded DNA. (D) DNA sequence of the partial replication origin of pMVSCS1 (or pMEx). The four 22-bp iteron repeats, three A+T repeats, and a dnaA box are indicated with block arrows, arrows, and a box, respectively. Dotted underlines represent the complete palindrome sequences in the iterons. The −35 and −10 boxes of the rep promoter are solid underlined. (E) The origin of replication of pMVSCS1 (or pMEx) and other theta mode plasmids in gram-negative bacteria. Symbols: white arrows, iterons; black arrow heads, repeated sequence in A+T-rich regions; white squares, dnaA box; H and P, HindIII and PstI sites.

FIG. 5.

Expression of the M. succiniciproducens fumC gene by use of the shuttle vector pME. (A) Recombinant plasmid pMEFUMC. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showing the expression of the fumC gene in M. succiniciproducens (lanes 1 and 2) and A. succinogenes (lanes 3 and 4). Lanes 1 and 3, cells harboring pME; lanes 2 and 4, cells harboring pMEFUMC.