Journal: Microbiology (Reading, England)

Article Title: Asuc_0142 of Actinobacillus succinogenes 130Z is the l-aspartate/C4-dicarboxylate exchanger DcuA.

doi: 10.1099/mic.0.001411

Figure Lengend Snippet: Fig. 2. Initial uptake of l-[14C]aspartate in A. succinogenes. A. succinogenes strains [wild-type (WT), LMB070 (Δ0142), and LMB070 complemented with plasmid harbouring Asuc_0142 (0142C)] were grown anaerobically in AM3 medium with glucose (20 mM) and l-aspartate (40 mM) (pH7) at 37°C to OD600 0.5. The initial uptake of 200 µM l-[14C]aspartate was measured for 20 s. The different superscript indicators reflect significant differences at P<0.05. SPSS PAWS Statistics, one-way ANOVA, Duncan’s multiple comparisons. All results are the averages of at least three biological replicates using independent growth cultures. Error bars indicate the standard deviation.

Article Snippet: Strains or plasmids Relevant genotype Source/Reference Strain Actinobacillus succinogenes 130Z Wild- type strain (DSM 22257) DSMZ Escherichia coli DH5α fhuA2 lac(del)U169 phoA glnV44 Φ80' lacZ(del)M15 gyrA96 recA1 relA1 endA1 thi- 1 hsdR17 Taylor et al. (1993) [30] IMW529 LJ1, but dcuA::spcR, dcuB::kanR, dcuC::Tn10(cmR), citT::kanR, ∆ttdT Kim and Unden [22] LMB070 A. succinogenes 130Z, but ΔAsuc_0142::kanR This study Plasmid pUC19 Cloning vector, ampR Enzynomics, Inc. pKD4 riR, kanR bla FRT Datsenko and Wanner (2000) [31] pBAD30 araC, araBAD promoter, pACYC184, bla, ampR Guzman et al. (1995) [32] pLS88 Shuttle vector (ATCC 86980), kanR, strR, sulR ATCC pMB31 pUC19 with complete gene sacB (1422 bp) form pDM4, ampR Rhie et al. [10] pMB61 pBAD30 with Shine- Dalgarno sequence, ampR Rhie et al. [10] pMB147 pMB61 with Asuc_0142, ampR This study pMB148 pMB61 with Ec_dcuA, ampR This study pMB177 pLS88 with Asuc_0142, sulR, strR This study pMB179 pMB31 with Asuc_0142::kanr and sacB, ampR This study 4 Cho et al., Microbiology 2023;169:001411 General procedures for uptake assay The cell suspensions of A. succinogenes, or of E. coli, respectively, were pre- incubated at 37°C, and at this stage lactose (20 mM) was added to E. coli.

Techniques: Plasmid Preparation, Standard Deviation

Journal: Microbiology (Reading, England)

Article Title: Asuc_0142 of Actinobacillus succinogenes 130Z is the l-aspartate/C4-dicarboxylate exchanger DcuA.

doi: 10.1099/mic.0.001411

Figure Lengend Snippet: Fig. 8. Scheme for l-asparagine and l-aspartate metabolism by A. succinogenes: roles as a nitrogen source, as a precursor of fumarate, and in pH homeostasis. The gene cluster Asuc_0140, Asuc_0141 and Asuc_0142 encodes a putative periplasmic asparaginase II (AsnB), a putative l-aspartase (AspA), and DcuA. The reactions and the cellular locations of the enzymes are shown. l-Aspartate is used as a precursor, building block, or source of ammonia for other nitrogen-containing components (compare Fig. 1, or [13]). Fumarate is used as an electron acceptor for fumarate respiration.

Article Snippet: Strains or plasmids Relevant genotype Source/Reference Strain Actinobacillus succinogenes 130Z Wild- type strain (DSM 22257) DSMZ Escherichia coli DH5α fhuA2 lac(del)U169 phoA glnV44 Φ80' lacZ(del)M15 gyrA96 recA1 relA1 endA1 thi- 1 hsdR17 Taylor et al. (1993) [30] IMW529 LJ1, but dcuA::spcR, dcuB::kanR, dcuC::Tn10(cmR), citT::kanR, ∆ttdT Kim and Unden [22] LMB070 A. succinogenes 130Z, but ΔAsuc_0142::kanR This study Plasmid pUC19 Cloning vector, ampR Enzynomics, Inc. pKD4 riR, kanR bla FRT Datsenko and Wanner (2000) [31] pBAD30 araC, araBAD promoter, pACYC184, bla, ampR Guzman et al. (1995) [32] pLS88 Shuttle vector (ATCC 86980), kanR, strR, sulR ATCC pMB31 pUC19 with complete gene sacB (1422 bp) form pDM4, ampR Rhie et al. [10] pMB61 pBAD30 with Shine- Dalgarno sequence, ampR Rhie et al. [10] pMB147 pMB61 with Asuc_0142, ampR This study pMB148 pMB61 with Ec_dcuA, ampR This study pMB177 pLS88 with Asuc_0142, sulR, strR This study pMB179 pMB31 with Asuc_0142::kanr and sacB, ampR This study 4 Cho et al., Microbiology 2023;169:001411 General procedures for uptake assay The cell suspensions of A. succinogenes, or of E. coli, respectively, were pre- incubated at 37°C, and at this stage lactose (20 mM) was added to E. coli.

Techniques: Blocking Assay