Reassessment of minimal media reveals differences in growth among Porphyromonas gingivalis standard strains.

OBJECTIVES: Porphyromonas gingivalis is one of the etiologic agents of chronic periodontitis. Our previous study showed that the use of minimal media for P. gingivalis allowed to isolate novel inhibitors of P. gingivalis growth. However, growth of P. gingivalis in minimal media was not always reproducible. METHODS: To explain this phenomenon, we analyzed the growth of seven wild-type ATCC 33277 strains and two wild-type W83 strains in 10 minimal media and three complex media. RESULTS: All nine strains grew in LF (Lactalbumin-Ferric chloride), GC (bovine γ-immunoglobulin G-Calcium chloride), and newly developed mC (milk-Casein) minimal media. Therefore, LF, GC, and mC could be used as minimal media for P. gingivalis. In contrast, other six minimal media containing bovine serum albumin (BSA) supported the growth of several less strains; among these, two media also showed lack of reproducibility in growth among ATCC 33277 strains. On the other hand, four ATCC 33277 strains grew similarly in all 13 media, but two W83 and other three ATCC 33277 strains grew differently in at least one medium. CONCLUSIONS: These results suggest that the lack of reproducibility of P. gingivalis growth on minimal media is caused by the presence of BSA, and by differences among the standard strains of P. gingivalis.

A screening system using minimal media identifies a flavin-competing inhibitor of Porphyromonas gingivalis growth.

Chronic periodontitis is caused by dysbiosis of human oral commensals and especially by increase in Porphyromonas gingivalis. Inhibitors of P. gingivalis growth are expected to serve as effective drugs for the periodontal therapy. In the present study, we isolated new growth inhibitors of P. gingivalis using minimal media for P. gingivalis. The minimal media included the previously reported Globulin-Albumin (GA) and the newly developed Lactalbumin-Ferric chloride (LF) and Globulin-Calcium chloride (GC); all supported growth of the wild-type strain of P. gingivalis but did not support the growth of a mutant defective for a type IX secretion system. GC contains CaCl2, indicating that P. gingivalis requires a calcium ion for growth. Using LF and GA, we screened about 100 000 compounds and identified 73 that strongly inhibited the growth of P. gingivalis. More than half of these candidates would not have been obtained if these minimal media had not been used in our screen. One of our candidate inhibitors was diphenyleneiodonium chloride (DPIC), which showed strong bactericidal activity against P. gingivalis. Excess amounts of flavin adenine dinucleotide or flavin mononucleotide suppressed the inhibitory activity of DPIC, suggesting that DPIC would be a novel potent growth inhibitor.

Obtaining Soluble Folded Proteins from Inclusion Bodies Using Sarkosyl, Triton X-100, and CHAPS: Application to LB and M9 Minimal Media.

This unit describes a straightforward and efficient method of using sarkosyl to solubilize and recover difficult recombinant proteins, such as GST- and His6 -tagged fusion proteins, that are overexpressed in E. coli. This protocol is especially useful for rescuing recombinant proteins overexpressed in M9 minimal medium. Sarkosyl added to lysis buffers helps with both protein solubility and cell lysis. Higher percentage sarkosyl (up to 10%) can extract >95% of soluble protein from inclusion bodies. In the case of sarkosyl-solubilized GST-fusion proteins, batch-mode affinity purification requires addition of a specific ratio of Triton X-100 and CHAPS, while sarkosyl-solubilized His6 -tagged fusion proteins can be directly purified on Ni(2+) resin columns. Proteins purified by this method could be widely used in biological assays, structure analysis and mass spectrum assay.