Spore-Forming Lactic Acid-Producing Bacterium Bacillus coagulans Synthesizes and Excretes Spermidine into the Extracellular Space.

The effect of spermidine in extending healthy longevity has attracted attention. As people age, their ability to synthesize putrescine, the precursor of spermidine, declines, and its supplementation from the diet or gut bacteria is needed. Many bacteria synthesize spermidine, but no strains have been reported to excrete de novo synthesized spermidine from the cells. We found that Bacillus coagulans strain YF1, isolated from "nanohana-duke", excreted de novo synthesized spermidine from the cells under anaerobic conditions. This strain synthesizes spermidine from arginine via agmatine, putrescine, and carboxyspermidine in sequential reactions, and the genes encoding the enzymes responsible for these reactions have been identified. B. coagulans is a gastric acid-resistant spore-forming lactic acid-producing bacterium, known for its beneficial effects as a probiotic. It can be used to produce lactic acid fermented foods containing spermidine. The newly discovered ability to excrete de novo synthesized spermidine is the decisive feature of this bacterium.

Agmatine production by Escherichia coli cells expressing SpeA on the extracellular surface.

A plasmid was constructed to express the CapA-SpeA fusion protein from the capA gene, which encodes one of the subunits of capsular poly-γ-glutamate synthetase of Bacillus subtilis subsp. natto, and the speA gene, encoding biosynthetic arginine decarboxylase (EC 4.1.1.19) of Escherichia coli, under the control of the T5 promoter. The expression of SpeA on the extracellular surface of cells was confirmed by confocal microscopy with the anti-SpeAE. coli antibody and anti-rabbit IgG L & H conjugated with Alexa Fluor 488. The constructed strain SH2290 produced 200 mM agmatine from 200 mM arginine, 20 mM MgSO4, 0.9 % NaCl, and 0.02 mg/mL pyridoxal 5'-phosphate (initial pH 5.3) by adjusting pH of the reaction mixture to 6.8 with HCl after each sampling during the reaction. The addition of pyridoxal 5'-phosphate to the reaction mixture was required for the maximum agmatine production. The present results demonstrate that the expression of enzymes on the extracellular surface of cells is a very powerful method for enzymatic conversion.

Improvement of putrescine production through the arginine decarboxylase pathway in Escherichia coli K-12.

In the bio-based polymer industry, putrescine is in the spotlight for use as a material. We constructed strains of Escherichia coli to assess its putrescine production capabilities through the arginine decarboxylase pathway in batch fermentation. N-Acetylglutamate (ArgA) synthase is subjected to feedback inhibition by arginine. Therefore, the 19th amino acid residue, Tyr, of argA was substituted with Cys to desensitize the feedback inhibition of arginine, resulting in improved putrescine production. The inefficient initiation codon GTG of argA was substituted with the effective ATG codon, but its replacement did not affect putrescine production. The essential genes for the putrescine production pathway, speA and speB, were cloned into the same plasmid with argAATG Y19C to form an operon. These genes were introduced under different promoters; lacIp, lacIqp, lacIq1p, and T5p. Among these, the T5 promoter demonstrated the best putrescine production. In addition, disruption of the puuA gene encoding enzyme of the first step of putrescine degradation pathway increased the putrescine production. Of note, putrescine production was not affected by the disruption of patA, which encodes putrescine aminotransferase, the initial enzyme of another putrescine utilization pathway. We also report that the strain KT160, which has a genomic mutation of YifEQ100TAG, had the greatest putrescine production. At 48 h of batch fermentation, strain KT160 grown in terrific broth with 0.01 mM IPTG produced 19.8 mM of putrescine.

Effect of Spermidine on Biofilm Formation in Escherichia coli K-12.

Polyamines are essential for biofilm formation in Escherichia coli, but it is still unclear which polyamines are primarily responsible for this phenomenon. To address this issue, we constructed a series of E. coli K-12 strains with mutations in genes required for the synthesis and metabolism of polyamines. Disruption of the spermidine synthase gene (speE) caused a severe defect in biofilm formation. This defect was rescued by the addition of spermidine to the medium but not by putrescine or cadaverine. A multidrug/spermidine efflux pump membrane subunit (MdtJ)-deficient strain was anticipated to accumulate more spermidine and result in enhanced biofilm formation compared to the MdtJ+ strain. However, the mdtJ mutation did not affect intracellular spermidine or biofilm concentrations. E. coli has the spermidine acetyltransferase (SpeG) and glutathionylspermidine synthetase/amidase (Gss) to metabolize intracellular spermidine. Under biofilm-forming conditions, not Gss but SpeG plays a major role in decreasing the too-high intracellular spermidine concentrations. Additionally, PotFGHI can function as a compensatory importer of spermidine when PotABCD is absent under biofilm-forming conditions. Last, we report here that, in addition to intracellular spermidine, the periplasmic binding protein (PotD) of the spermidine preferential ABC transporter is essential for stimulating biofilm formation.IMPORTANCE Previous reports have speculated on the effect of polyamines on bacterial biofilm formation. However, the regulation of biofilm formation by polyamines in Escherichia coli has not yet been assessed. The identification of polyamines that stimulate biofilm formation is important for developing novel therapies for biofilm-forming pathogens. This study sheds light on biofilm regulation in E. coli Our findings provide conclusive evidence that only spermidine can stimulate biofilm formation in E. coli cells, not putrescine or cadaverine. Last, ΔpotD inhibits biofilm formation even though the spermidine is synthesized inside the cells from putrescine. Since PotD is significant for biofilm formation and there is no ortholog of the PotABCD transporter in humans, PotD could be a target for the development of biofilm inhibitors.