CpdA is involved in amino acid metabolism in Shewanella oneidensis MR-1

Figures & data

Table 1. Bacterial strains and plasmids used in this study.

Strain or plasmid	Relevant characteristic	Source or reference
E. coli strains
DH5α	F^-, φ 80dlacZΔM15, Δ(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rK^-, mK^+), phoA, supE44, λ^-, thi^–1, gyrA96, relA1	Takara
WM6026	lacI^q, rrnB3, DElacZ4787, hsdR514, DE(araBAD)567, E(rhaBAD)568, rph-1, att-lambda::pAE12-del(oriR6 K-cat::frt5), DE(endA)::frt, uidA(delMluI)::pir(wt), attHK::pJK1006-del1/2 (deloriR6 K-cat::frt5, deltrfA::frt)	William Metcalf, University of Illinois
S. oneidensis strains
MR-1	Wild type	ATCC
ΔcpdA	SO_3901 (cpdA) disrupted	This study
ΔcyaABC	SO_4312 (cyaA), SO_3778(cyaB), and SO_1329 (cyaC) disrupted	This study
ΔcyaABCΔcpdA	SO_3901 (cpdA), SO_4312 (cyaA), SO_3778(cyaB), and SO_1329 (cyaC) disrupted	This study
Plasmids
pSMV-10	9.1 kb mobilizable suicide vector; oriR6 K, mobRP4, sacB, Km^r, Gm^r	Chad Saltikov, California Inst. of Tech.
pSMV-cpdA	1.6 kb fusion PCR fragment containing ΔcpdA cloned into the SpeI site of pSMV-10	This study
pSMV-cyaA	1.6 kb fusion PCR fragment containing ΔcyaA cloned into the SpeI site of pSMV-10	This study
pSMV-cyaB	1.6 kb fusion PCR fragment containing ΔcyaB cloned into the SpeI site of pSMV-10	This study
pSMV-cyaC	1.6 kb fusion PCR fragment containing ΔcyaC cloned into the SpeI site of pSMV-10	This study
pBBR1MCS-5	Broad-host-range vector, lacZ promoter, Gm^r	20
pBBRScpdA	pBBR1MCS-5 containing S. oneidensis cpdA	This study
pBBREcpdA	pBBR1MCS-5 containing E. coli JM109 cpdA	This study

Figure 1. Growth of S. oneidensis MR-1 and its derivatives in LB and LMM. Growth curves of cells cultured in LB under aerobic conditions are shown in panel A. Growth curves of cells cultured in LMM under aerobic and anaerobic fumarate-reducing conditions are shown in panels B and C, respectively. WT(pBBR1MCS-5), ΔcpdA(pBBR1MCS-5), ΔcpdA (pBBRScpdA), and ΔcpdA (pBBREcpdA) were aerobically grown in LMM containing Gm. Error bars represent standard deviations of means that were calculated from at least triplicate cultures.

Figure 2. Intracellular cAMP and cGMP levels and growth rates of MR-1 and mutants. cAMP and cGMP levels and growth rates of cells aerobically grown in LMM are shown in panels A, B, and C, respectively. Error bars represent standard deviations of means that were calculated from at least triplicate experiments.

Table 2. Genes involved in methionine, SAM, and histidine synthesis with altered expression in ΔcpdA.

Locus tag	Gene	Description	Log2FC^a
Methionine and SAM synthesis
SO_0817	metR	Transcription regulator of methionine biosynthesis MetR	–4.02
SO_0818	metE	B12-independent 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase MetE	8.30
SO_0929	metK	S-adenosylmethionine synthetase MetK	–1.67
SO_1030	metH	B12-dependent 5-methyltetrahydrofolate-homocysteine methyltransferase MetH	–4.15
SO_1087	metT	Methionine: proton antiporter MetT	–4.44
SO_1095	metY	O-acetylhomoserine (thiol)-lyase MetY	–2.95
SO_1676	metA	Homoserine O-succinyltransferase MetA	–3.25
SO_4054	metF	5,10-Methylenetetrahydrofolate reductase MetF	–6.01
SO_4055	metL	Bifunctional aspartokinase II/homoserine dehydrogenase methionine-sensitive MetL	–6.00
SO_4056	metB	Cystathionine gamma-synthase MetB	–6.76
Histidine metabolism
SO_2067	hisIE	Bifunctional phosphoribosyl-AMP cyclohydrolase/phosphoribosyl-ATP pyrophosphatase HisIE	–1.98
SO_2068	hisF	Imidazole glycerol phosphate synthase cyclase subunit HisF	–2.33
SO_2069	hisA	1-(5-Phosphoribosyl)-5-[(5-phosphoribosylamino) methylideneamino] imidazole-4-carboxamide isomerase HisA	–2.72
SO_2070	hisH	Imidazole glycerol phosphate synthase glutamine amidotransferase subunit HisH	–2.85
SO_2071	hisB	Bifunctional imidazoleglycerol-phosphate dehydratase/histidinol-phosphatase HisB	–2.96
SO_2072	hisC	Histidinol-phosphate aminotransferase HisC	–3.20
SO_2073	hisD	Histidinol dehydrogenase HisD	–2.90
SO_2074	hisG	ATP phosphoribosyltransferase HisG	–1.71

^a Log₂-transformed fold change (ΔcpdA/WT).

Figure 3. Effects of methionine, SAM, and histidine on the growth of ΔcpdA. Cells were aerobically grown in LMM containing 0.1 mM methionine (Met), 0.1 mM histidine (His), and/or 35 μM SAM. Error bars represent standard deviations of means that were calculated from at least triplicate cultures.