This is consistent with our previous recovery of a strain of urease-negative L. hongkongensis (HLHK30) from an 84-year old male with gastroenteritis. Sequencing of the urease cassette of HLHK30 showed that all eight of the component genes were present with no deletions Foretinib research buy or frame shift mutations;
although there were a number of polymorphic sites that resulted in amino acid changes compared to gene homologues present in HLHK9 (Figure 1B). On the other hand, the ADI-deficient mutant HLHK9∆arcA1/arcA2 showed marked reduction in survival abilities in acidic media and macrophages as well as in the mouse model, indicating that arc gene cassettes play a more important role than urease gene cassettes for acid resistance in L. hongkongensis. In fact, the survival abilities of the triple knockout mutant strain HLHK9∆ureA/arcA1/arcA2 were Selumetinib only marginally lower than those of the ADI-deficient double mutant strain HLHK9∆arcA1/arcA2 in acidic media and macrophages, and both mutant strains had equivalent survival abilities in the
mouse model, which further supports the conclusion that ADI play a more important role. The gene duplication of the arc gene cassettes could be a result of their functional importance in L. hongkongensis. One of the important mechanisms of virulence evolution in bacteria and fungi is gene duplication [38–40]. L. hongkongensis is the only bacterium known to possess two adjacent arc gene cassettes. The L. hongkongensis mutant strain containing deletions of the arcA genes in both arc cassettes exhibited a marked reduction in survival abilities
compared to the mutant strains containing single deletion of either one of Metformin the two arcA genes, indicating that both arc gene cassettes are functional and contribute to acid resistance. Phylogenetic analysis showed that the two copies of arc in L. hongkongensis are clustered in all the four trees 8-Bromo-cAMP constructed using arcA, arcB arcC and arcD[41]. This strongly suggests that the two arc gene cassettes result from a gene cassette duplication event. Interestingly, in our previous study on differential gene expression in L. hongkongensis at different temperatures, it was observed that the two copies of argB, encoding two isoenzymes of N-acetyl-L-glutamate kinase from the arginine biosynthesis pathway, which have distinct biochemical properties, are also clustered phylogenetically [17]. This indicates that these two copies of argB probably also arose as a result of gene duplication. Subsequent evolution enabled the two copies of argB to adapt to different temperatures and habitats. These coincidental findings of gene duplication in two different pathways of arginine metabolism, enabling the bacterium to better adapt to different environmental conditions, argB for temperature adaptation and arc gene cassette for acid resistance, is intriguing.