8 42% — While our results are different from the report of Heliobacterium strain HY-3 [18], the authors found more acetate being produced during chemotrophic YH25448 order growth (13.6

mM) than during phototrophic growth (5.9 mM). Both our and their studies demonstrate that acetate can be produced from pyruvate-grown heliobacterial cultures during phototrophic and chemotrophic growth. Two acetate assimilation/excretion pathways are possibly employed by H. modesticaldum. One is catalyzed by acetyl-CoA synthetase (ACS, EC 6.2.1.1), proceeding through an acetyl adenylate intermediate; and the other is catalyzed by acetate kinase (ACK, EC 2.7.2.1, acetate ⇌ acetyl-phosphate) and phosphotransacetylase (PTA, EC 2.3.1.8, acetyl-phosphate ⇌ acetyl-CoA) [22]. No ACS activity was reported in the studies of Heliobacterium strain HY-3 [18], and it is possible that ACK and PTA are responsible

in the acetate assimilation/excretion pathway in Heliobacterium strain HY-3. In contrast, genes encoding ACS (acsA, HM1_0951) and ACK (ackA, HM1_2157), but not PTA (pta), have been annotated in the genome of H. modesticaldum. The relative gene expression level (the ratio of transcript level in the light/in darkness) of acsA is approximately one order of magnitude lower than that of ackA (45 versus 3; see Table 2 and Figure 4), whereas the activity of ACS can be only detected in cell extracts of phototrophic growth (Table 4). In contrast, the enzymatic activity of ACK and PTA can be detected in cell extracts of pyruvate-grown cultures during both phototrophic and chemotrophic growth. Figure 4 Relative gene expression levels during phototrophic versus see more chemotrophic growth. Only representative genes responsible for carbon metabolism, nitrogen fixation and hydrogen production are shown. Gene name (encoding enzyme): pfkA (6-phosphofructokinase), pykA (pyruvate kinase), acsA (acetyl-CoA synthase), ackA (acetate kinase),

ppdK (pyruvate phosphate dikinase), pckA (PEP carboxykinase), fdxR (Fd-NADP+ oxidoreductase, FNR), pshB (RC core polypeptide, PshB), fdx (ferredoxin for FNR), porA (pyruvate:Fd oxidoreductase), bchY (chlorophyll reductase, subunit Y), bchB (protochlorophyllide reductase, subunit B), bchE (anaerobic cyclase), and bchG (bacteriochlorophyll synthase). Table 4 Enzyme activity of enzymes and relative expression level of genes for acetate metabolism until in pyruvate-grown cultures during phototrophic and chemotrophic growth.   Specific activity (nmole/min/mg protein)   Enzyme activity tested Phototrophic growth Chemotrophic growth Relative gene expression level (light/dark) acetyl-CoA synthetase (ACS) a 100 ± 20 N/A 45 acetate kinase (ACK) a 800 ± 40 600 ± 100 3 phosphotransacetylase (PTA) a 400 ± 50 500 ± 100 — a Activity of ACS was determined by a colorimetric assay of PPi [39], and activity of ACK and PTA was determined by coupling assays reported previously [18]. Together, our studies suggest that: (i) H.