Porphyrin derivatives from a recombinant Escherichia coli grown on chemically defined medium.

We have reported previously that a recombinant Escherichia coli co-expresses aminolevulinic acid (ALA) synthase, an NADP-dependent malic enzyme, and a dicarboxylate transporter-produced heme, an iron-chelated porphyrin, in a succinate-containing complex medium. To develop an industrially plausible process, a chemically defined medium was formulated based on M9 minimal medium. Heme synthesis was enhanced by adding sodium bicarbonate, which strengthened the C4 metabolism required for the precursor metabolite, although a pH change discouraged cell growth. Increasing the medium pH buffering capacity (100mM phosphate buffer) and adding sodium bicarbonate enabled the recombinant E. coli to produce heme at rates 60% greater than those in M9 minimal medium. Adding growth factors (1 mg/l thiamin, 0.01 mg/l biotin, 5 mg/l nicotinic acid, 1 mg/l pantothenic acid, and 1.4 mg/l cobalamin) also induced positive heme production effects at levels twice of heme production in M9-based medium. Porphyrin derivatives and heme were found in the chemically defined medium, and their presence was confirmed by liquid chromatography/mass spectroscopy (LC/MS). The formulated medium allowed for the production of 0.6 microM heme, 29 microM ALA, 0.07 microM coproporphyrin I, 0.21 microM coproporphyrin III, and 0.23 microM uroporphyrin in a 3 L pH-controlled culture.

A mammalian insulysin homolog is regulated by enzyme IIA(Glc) of the glucose transport system in Vibrio vulnificus.

Vibrio vulnificus is an opportunistic human pathogen that causes severe infections in susceptible individuals. While the components of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) have been shown to regulate numerous targets, little such information is available for the V. vulnificus PTS. Here we show that enzyme IIA(Glc) of the PTS regulates the peptidase activity of a mammalian insulysin homolog in V. vulnificus. While interaction of IIA(Glc) with the insulysin homolog is independent of the phosphorylation state of IIA(Glc), only unphosphorylated IIA(Glc) activates the insulysin homolog. Taken together, our results suggest that the V. vulnificus insulysin-IIA(Glc) complex plays a role in survival in the host by sensing glucose.

Vibrio vulnificus-induced death of Jurkat T-cells requires activation of p38 mitogen-activated protein kinase by NADPH oxidase-derived reactive oxygen species.

Vibrio vulnificus, a pathogenic bacterium causing primary septicemia, exhibited cytotoxicity towards Jurkat cells of T-lymphocytes through intracellular reactive oxygen species (ROS) production. Pretreatment of Jurkat T-cells with diphenyleneiodonium chloride (DPI) abolished V. vulnificus-induced ROS generation and bacterial ability to cause cell death. Jurkat T-cells expressing dominant-negative protein of Rac subunit of NADPH oxidase (NOX) did not show increased ROS production and cell death by V. vulnificus. Vibrio vulnificus also triggered phosphorylation of mitogen-activated protein kinases (MAPKs) including p38 and ERK1/2 in Jurkat T-cells. Experiments using inhibitors or small interfering RNAs for each MAPK showed that both MAPKs are involved in V. vulnificus-induced cell death. DPI only blocked the phosphorylation of p38 MAPK in Jurkat T-cells exposed by V. vulnificus. This study demonstrates that V. vulnificus induces death of Jurkat T-cells via ROS-dependent activation of p38 MAPK, and that NOX plays a major role in ROS generation in V. vulnificus-exposed cells.

Role of NtrC in biofilm formation via controlling expression of the gene encoding an ADP-glycero-manno-heptose-6-epimerase in the pathogenic bacterium, Vibrio vulnificus.

To identify the genetic elements required for biofilm formation, we screened a pool of random Vibrio vulnificus mutants for their ability to form biofilms. One mutant displaying significantly decreased biofilm-forming activity was found to contain a transposon insertion in the ntrC gene. The ntrC gene encodes a well-known transcriptional activator. We examined how this regulator modulates a biofilm-forming process in V. vulnificus by searching for NtrC target gene(s). Comparison of the proteomes of ntrC mutant and wild-type strains grown under planktonic and biofilm stages revealed that synthesis of the protein homologous to GmhD (ADP-glycero-manno-heptose-6-epimerase) was elevated during the growth period for biofilm formation and was strongly influenced by NtrC. A luxAB-transcriptional fusion with the gmhD promoter region indicated that gmhD expression was positively regulated by both NtrC and RpoN. The function of the gmhD gene product in V. vulnificus was assessed by constructing and phenotypic analyses of an isogenic mutant. The gmhD mutant was defective in production of mature lipopolysaccharide (LPS) and exopolysaccharides (EPS), and demonstrated an attenuated ability to form a biofilm. These results suggest that NtrC acts as a key regulator of both LPS and EPS biosyntheses and, thereby, modulates critical steps in biofilm development of V. vulnificus.