Abstract
Exploration of the aquatic microbiota of several circum-neutral (6.0–8.5 pH) mid-temperature (55–85°C) springs revealed rich diversities of phylogenetic relatives of mesophilic bacteria, which surpassed the diversity of the truly-thermophilic taxa. To gain insight into the potentially-thermophilic adaptations of the phylogenetic relatives of Gram-negative mesophilic bacteria detected in culture-independent investigations we attempted pure-culture isolation by supplementing the enrichment media with 50 μg ml−1 vancomycin. Surprisingly, this Gram-positive-specific antibiotic eliminated the entire culturable-diversity of chemoorganotrophic and sulfur-chemolithotrophic bacteria present in the tested hot water inocula. Moreover, it also killed all the Gram-negative hot-spring isolates that were obtained in vancomycin-free media. Concurrent literature search for the description of Gram-negative thermophilic bacteria revealed that at least 16 of them were reportedly vancomycin-susceptible. While these data suggested that vancomycin-susceptibility could be a global trait of thermophilic bacteria (irrespective of their taxonomy, biogeography and Gram-character), MALDI Mass Spectroscopy of the peptidoglycans of a few Gram-negative thermophilic bacteria revealed that tandem alanines were present in the fourth and fifth positions of their muropeptide precursors (MPPs). Subsequent phylogenetic analyses revealed a close affinity between the D-alanine-D-alanine ligases (Ddl) of taxonomically-diverse Gram-negative thermophiles and the thermostable Ddl protein of Thermotoga maritima, which is well-known for its high specificity for alanine over other amino acids. The Ddl tree further illustrated a divergence between the homologs of Gram-negative thermophiles and mesophiles, which broadly coincided with vancomycin-susceptibility and vancomycin-resistance respectively. It was thus hypothesized that thermophilic Ddls have been evolutionarily selected to favor a D-ala-D-ala bonding. However, preference for D-ala-D-ala-terminated MPPs does not singlehandedly guarantee vancomycin susceptibility of thermophilic bacteria as the large and relatively-hydrophilic vancomycin molecule has to cross the outer membrane before it can inhibit peptidoglycan biosynthesis. Literature shows that many mesophilic Gram-negative bacteria also have D-ala-D-ala-terminated MPPs, but they still remain resistant to vancomycin due to the relative impermeability of their membranes. But the global vancomycin-susceptibility phenotype of thermophilic bacteria itself testifies that the drug crosses the membrane in all these cases. As a corollary, it seems quite likely that the outer membranes of thermophilic bacteria have some yet-unknown characteristic feature(s) that invariably ensures the entry of vancomycin.
Highlights
Relatives of phylogenetically diverse mesophilic bacteria are known to be present in hot spring waters alongside the typically thermophilic and hyperthermophilic prokaryotes (Jimenez et al, 2012; Wemheuer et al, 2013; Chan et al, 2015; Menzel et al, 2015)
Over the past few years we have investigated the taxonomic diversity of the aquatic bacterial community of several circumneutral hot springs of Northern and Eastern India by analyzing amplified 16S rRNA gene fragments
**Bacteria were first grown in MST broths without any antibiotic selection; subsequently 2% of these mid-log phase (OD600 = 0.15, pH 6.5) seed cultures were transferred to MST broths, which contained vancomycin (50 μg ml−1) or not, as warranted by the test in hand
Summary
Relatives of phylogenetically diverse mesophilic bacteria are known to be present in hot spring waters alongside the typically thermophilic and hyperthermophilic prokaryotes (Jimenez et al, 2012; Wemheuer et al, 2013; Chan et al, 2015; Menzel et al, 2015). Whatever little information is available is confined to Gram-positive Firmicutes like Bacillus (Volker et al, 1992; Schumann, 2003; Endo et al, 2006; Sikorski et al, 2008), Geobacillus (Shih and Pan, 2011; Tripathy and Maiti, 2014; Wang et al, 2014) and Anoxybacillus (Burgess et al, 2009; Paul et al, 2012; Goh et al, 2014) This is despite the fact that taxonomically-diverse bacteria are known to grow facultatively at temperatures between 50 and 80◦C (often in addition to their mesophilic growths below 50◦C) (Moreira et al, 2000; Alves et al, 2003; Rainey et al, 2003). This phenomenon held true for all the hot water inocula that were tested from various geothermal districts of
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