Historical microbiology: revival and phylogenetic analysis of the luminous bacterial cultures of M . W . B eijerinck

Abstract

Luminous bacteria isolated by M artinus W . B eijerinck were sealed in glass ampoules in 1924 and 1925 and stored under the names P hotobacterium phosphoreum and ‘ P hotobacterium splendidum ’. To determine if the stored cultures were viable and to assess their evolutionary relationship with currently recognized bacteria, portions of the ampoule contents were inoculated into culture medium. Growth and luminescence were evident after 13 days of incubation, indicating the presence of viable cells after more than 80 years of storage. The B eijerinck strains are apparently the oldest bacterial cultures to be revived from storage. Multi‐locus sequence analysis, based on the 16S rRNA , gapA , gyrB , pyrH , recA , luxA , and luxB genes, revealed that the B eijerinck strains are distant from the type strains of P . phosphoreum , ATCC 11040 T , and V ibrio splendidus , ATCC 33125 T , and instead form an evolutionarily distinct clade of V ibrio . Newly isolated strains from coastal seawater in N orway, F rance, U ruguay, M exico, and J apan grouped with the B eijerinck strains, indicating a global distribution for this new clade, designated as the beijerinckii clade. Strains of the beijerinckii clade exhibited little sequence variation for the seven genes and approximately 6300 nucleotides examined despite the geographic distances and the more than 80 years separating their isolation. Gram‐negative bacteria therefore can survive for many decades in liquid storage, and in nature, they do not necessarily diverge rapidly over time.