Edited by: Octavio Luiz Franco, Universidade Catolica de Brasilia, Brazil
Reviewed by: Dmitri Debabov, NovaBay Pharmaceuticals, USA; Santi M. Mandal, Vidyasagar University, India; Celio De Faria Junior, Central Public Health Laboratory (LACEN) of the Brazilian Federal District, Brazil
*Correspondence: Wriddhiman Ghosh
This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology
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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
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.,
For any given venting point, batches of 500 ml freshly-discharged hot water were passed through sterile 0.22 μm filters (4.7 cm radius). Filters were immediately put into cryovials containing 5 ml of either 50 mM:50 mM Tris:EDTA (TE, pH 7.8) or 15% Glycerol in 0.9% NaCl and transferred to the lab in dry ice. Upon reaching the lab the cryovials were stored at −20°C until further use, which was anyhow within 2 weeks from sampling. While the filters suspended in TE were used for the isolation of total environmental DNA, those put in glycerol stocks were used in various culture-based experiments (one filter per 80 ml of any medium).
A given TE-suspended filter was cut into small pieces with sterile scissors and put back to the original cryovial. The vial was vortexed vigorously for 30 min, following which the filter shreds were discarded and the 5 ml TE distributed to five 1.5 ml microfuge tubes. All the five tubes were centrifuged at 10,800 g for 30 min, following which 900 μl TE was discarded from the top of each tube. The 100 μl TE remaining in each tube was vortexed vigorously for 15 min and the contents of all five tubes were pooled up into one microfuge tube. This pooled up 500 μl TE was again centrifuged at 10,800 g for 30 min, following which 400 μl was discarded from the top. The remaining 100 μl was presumably a suspension of all microbial cells recoverable from the 500 ml hot water sample in question. DNA was isolated from this 100 μl purported cell suspension by the QIAamp DNA Mini Kit (QIAGEN) following manufacturer's protocol.
Chemoorganoheterotrophic growth experiments were performed in oligotrophic R2A medium while chemolithoautotrophic growth was checked in a modified basal mineral salt (MMS) medium [1 gm l−1 NH4Cl, 2 gm l−1 K2HPO4, 0.75 gm l−1 KH2PO4, 0.5 gm l−1 MgSO4 and 2 ml l−1 trace metals solution (Vishniac and Santer,
V3 regions of all potential bacterial 16S rRNA genes present in an environmental or cultured metagenome were PCR-amplified by the “Fusion Primer Protocol” using
Each 50 μl PCR reaction contained 10 μl template (corresponding to ~100 ng metagenomic DNA), 5 μl 10X KOD DNA polymerase buffer, 5 μl dNTP (0.25 mM each), 2 μl MgCl2 (25 mM), 1.5 μl (3%) DMSO, 3 μl each of the forward and reverse primer (0.3 μM each), 19.5 μl dH2O and 1 μl KOD hot start polymerase enzyme (Novagen, USA). PCR products were amplified for 30 cycles as follows: 94°C for 15 s, 65°C for 30 s and 68°C for 60 s. After amplification, all PCR products were electrophoresed on 2.5% w/v agarose gel, purified by size selection, and adjusted to final concentrations of 10 ng μl−1 using molecular grade water. PCR products from multiple samples were pooled up at equal concentrations for Ion PGM sequencing.
Before Ion PGM sequencing, size distribution and DNA concentration in the pooled-up amplicon mixture was examined using a Bioanalyzer 2100 (Agilent Technologies, USA). The mixed sample was adjusted to a final concentration of 26 pM and attached to the surface of Ion Sphere Particles (ISPs) using an Ion Onetouch 200 Template kit (Life Technologies, USA) according to the manufacturer's instructions. Manual enrichment of the resulting ISPs resulted in >95% templated-ISPs, which were then sequenced on Ion 316 Chips using the Ion PGM (Ion Express Template 200 chemistry) for 500 flows that gives an expected average read length of >220 bp. Sequencing was done upto such depths which ensured plateaus in rarefaction curves. Post sequencing, individual sequence reads were filtered by the PGM software to remove low quality and polyclonal sequences. Sequences matching the PGM 3′ adaptor were also automatically trimmed. All the data quality-filtered on the PGM were exported as fastq files for downstream applications.
The sequence files generated from PCR upon environmental DNA samples were deposited to the NCBI Sequence Read Archive (SRA) with the Run and BioProject accession numbers cited in Table
Sulfur- borax spring zone, Puga valley, Ladakh, J&K | Lotus Pond |
Western part of the Puga valley, Ladakh, Jammu and Kashmir (33°13′46″ N/78°21′18″ E) | A hot water pool that is located on the bank of the Puga rivulet and has contiguous outflow with the latter | 78–85°C pH 7-8 | 331 | Ubact, 32; |
Supplementary Table |
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Lotus Pond-adjacent ebullition | A small hot water ebullition seated in between the Lotus Pond Center and the water flow of the Puga rivulet | 70–80°C pH 7-7.5 | 186 | Ubact, 54; |
Supplementary Table |
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Shivlinga |
Fountain-type geyser | 65–75°C pH 7-7.5 | 80 | Ubact, 24; |
Supplementary Table |
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PCPR_1 | South-central part of Puga valley (33°13′27″ N/78°20′ 10 E″) | Hot water pool embedded in boratic deposits and valley-fill materials | 70–75°C 7.5-8 pH | 135 | Ubact, 7; |
Supplementary Table |
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PCPR_2 | Northern part of Puga valley (33°13′25.51″ N/78°19′2.69″ E) | Fountain-type geyser | 70–75°C pH 6.8-7.5 | 188 | Ubact, 17; |
Supplementary Table |
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Eastern Indian coalfields | Paniphala fountain | Paniphala, Burdwan, West Bengal (23°45′33.03″ N/86°58′54.28″ E) | Fountain-type geyser | 55–65°C pH 6.8-8.0 | 743 | Ubact, 400; |
Supplementary Table |
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Eastern Indian lateritic belt | Agnikunda |
Bakreshwar, Birbhum, West Bengal (23°52′48.00″ N/87°22′12.00″ E) | Hot water pool partially confined by artificial embankments | 70–85°C pH 6.0-7.0 | 283 | Ubact, 75; |
Supplementary Table |
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Kharkunda |
Hot water pool partially confined by artificial embankments | 55–65°C pH 7.5-8.5 | 234 | Ubact, 55; |
Supplementary Table |
Lotus Pond Center | 0 OD600/480 h | 0.8 OD600/16 h | 0 OD600/480 h | 0.8 OD600/12 h | 7.0 pH; 0.05 OD600/168 h | 6.0 pH; 0.3 OD600/72 h | 7.0 pH; 0.05 OD600/168 h | 5.0 pH; 0.4 OD600/36 h |
Total no. of OTUs detected: 30 (SRR1951817) | Total no. of OTUs detected: 58 (SRR1951818) | Total no. of OTUs detected: 68 (SRR1951819) | Total no. of OTUs detected: 136 (SRR1951820) | |||||
Ubact, 3; |
Ubact, 12; |
Ubact, 18; |
Ubact, 5; |
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Supplementary Table |
Supplementary Table |
Supplementary Table |
Supplementary Table |
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Lotus Pond-adjacent Vent | 0 OD600/480 h | 0.8 OD600/16 h | 0 OD600/480 h | 0.8 OD600/12 h | 7.0 pH; 0.1 OD600/216 h | 5.3 pH; 0.3 OD600/48 h | 7.0 pH; 0.1 OD600/216 h | 5.5 pH; 0.3 OD600/36 h |
Total no. of OTUs detected: 85 (SRR1951983) | Total no. of OTUs detected: 128 (SRR1951984) | Total no. of OTUs detected: 134 (SRR1951988) | Total no. of OTUs detected: 62 (SRR1951992) | |||||
Ubact, 3; |
Ubact, 2; |
Ubact, 11; |
Ubact, 1; |
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Supplementary Table |
Supplementary Table |
Supplementary Table |
Supplementary Table |
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Shivlinga Vent | 0 OD600/480 h | 0.8 OD600/16 h | 0 OD600/480 h | 0.8 OD600/12 h | 7.0 pH; 0.1 OD600/120 h | 6.0 pH; 0.3 OD600/36 h | 7.0 pH; 0.1 OD600/120 h | 5.5 pH; 0.4 OD600/16 h |
Total no. of OTUs detected: 54 (SRR1952883) | Total no. of OTUs detected: 51 (SRR1952893) | Total no. of OTUs detected: 320 (SRR1952904) | Total no. of OTUs detected: 51 (SRR1952937) | |||||
Ubact, 11; |
Ubact, 21; D-T, 21; |
Ubact, 21; |
Ubact, 3; |
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Supplementary Table |
Supplementary Table |
Supplementary Table |
Supplementary Table |
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Paniphala Fountain | 0 OD600/480 h | 0.8 OD600/12 h | 0 OD600/480 h | 0.8 OD600/12 h | 7.0 pH; 0.1 OD600/216 h | 5.5 pH; 0.4 OD600/72 h | 7.0 pH; 0.1 OD600/216 h | 5.5 pH; 0.4 OD600/48 h |
Total no. of OTUs detected: 61 (SRR1952938) | Total no. of OTUs detected: 55 (SRR1954984) | Total no. of OTUs detected: 108 (SRR1954986) | Total no. of OTUs detected: 46 (SRR1954987) | |||||
Ubact, 2; |
Ubact, 2; |
Ubact, 8; |
Ubact, 1; |
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Supplementary Table |
Supplementary Table |
Supplementary Table |
Supplementary Table |
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Agnikunda | 0 OD600/480 h | 0.8 OD600/16 h | 0 OD600/480 h | 0.8 OD600/16 h | 7.0 pH; 0.05 OD600/216 h | 5.0 pH; 0.4 OD600/48 h | 7.0 pH; 0.1 OD600/216 h | 6.5 pH; 0.3 OD600/96 h |
Total no. of OTUs detected: 53 (SRR2016659) | Total no. of OTUs detected: 79 (SRR2016660) | Total no. of OTUs detected: 78 (SRR2016657) | Total no. of OTUs detected: 112 (SRR2016658) | |||||
Ubact, 3; |
Ubact, 1; |
Ubact, 2; |
Ubact, 12; |
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Supplementary Table |
Supplementary Table |
Supplementary Table |
Supplementary Table |
Raw V3 region-specific reads were first filtered for high quality value (QV 20) and length threshold of 100 bp. Selected reads were then converted to fasta from fastq using Fastx_toolkit (v0.0.13.2). Operational taxonomic units (OTUs) were created at 97% identity level using the various modules of the UPARSE (Edgar,
Peptidoglycans of various bacterial strains were prepared by methods described earlier (Komagata and Suzuki,
Cells were collected by centrifugation and the cell pellet (2 gm wet weight) was washed twice with 5 ml phosphate buffer (0.05 M, pH 7.2). Then the cell pellet was resuspended in 6 ml 0.05 M phosphate buffer (pH 7.2) and cells were disrupted by sonication on ice using 20 s pulse for 4 times. The cell lysate was centrifuged at 1800 g for 10 min and the supernatant was transferred to a fresh centrifuge tube. It was then centrifuged at 12,000 g for 1 h. Supernatant was discarded and pellet was resuspended in 5 ml phosphate buffer. 1 ml of 5% sodium dodecyl sulfate was added and incubated at 100°C for 40 min, followed by centrifugation at 12,000 g for 30 min at 30°C. Then the pellet was washed four to five times with 5 ml 60°C distilled water. It was then washed with 5 ml 0.05 M phosphate buffer (pH 7.6). The pellet was then resuspended in 2 ml phosphate buffer (0.05 M, pH 7.6) and 100 μl pronase E (1 mg ml−1) was added. The soup was then incubated at 37°C for 2 h. Pellet was collected by centrifugation at 12,000 g for 30 min. It was further washed twice 2 ml phosphate buffer (0.05 M, pH 7.6). After that, pellet was resuspended in 2 ml of 5% TCA (Trichloroacetic Acid) and boiled at 100°C for 20 min. The suspension was cooled at room temperature and transferred to glass centrifuge tubes. It was then centrifuged at 12,000 g for 30 min. Pellet was further washed thrice with 2 ml phosphate buffer (0.05 M, pH 7.6), once each with 2 ml ethanol (95%) and 2 ml diethyl ether (99%), and finally air dried at 60°C for 3 h before further analysis.
Extracted peptidoglycans were digested with lysozyme (40 mg ml−1) for 2 h at 37°C, following which deactivation was done for 20 min at 70°C. Digested products were lyophilized, resuspended in 100 μl 99% methanol, and directly used for MALDI-MS without any more purification. DHA was used as the MALDI matrix. MALDI-MS was carried out using an AutoFlex II tandem time of flight (TOF/TOF) MALDI-mass spectrometer (Bruker Daltonics) equipped with a pulsed N2 laser (λ-337 nm, 50 Hz). The machine was calibrated for reflector mode mass spectra using a mixture of standard peptides (having mass 750 to 3150) in the positive ion mode. MS spectra were analyzed using the Flex Analysis software V2.4.
ML trees were constructed using MEGA6 (Tamura et al.,
Over the past few years we have investigated the taxonomic diversity (species richness) of the aquatic bacterial community of several circumneutral hot springs of Northern and Eastern India by analyzing amplified 16S rRNA gene fragments. V3 regions of all potential bacterial 16S rRNA genes present in the total environmental DNA isolated from thermal water samples were PCR-amplified using
Notwithstanding the discrete locations and physicochemical characters of the studied springs, taxonomic structure of their hot water communities was unified by the occurrence of several such bacterial taxa that are otherwise unexpected in high-temperature habitats. While most of the communities encompassed maximum OTUs from the
In this scenario we sought to know how this large variety of purportedly-mesophilic genera survived in these high temperature habitats. First it was imperative to check whether they could at all grow at high temperatures or were only thermo-enduring entities. Alternatively, it was also plausible that many of them were stochastically introduced into these habitats in recent times and were not at all equipped to cope with thermal stress. Since only pure culture isolates could answer these queries we attempted to get the same in chemoorganoheterotrophic (R2A) as well as chemolithoautotrophic [modified minimal salts supplemented with thiosulfate, MMST (Ghosh and Roy,
Strains related to
Supplementing R2A as well as MMST with 50 μg ml−1 vancomycin caused complete destruction of the corresponding culturable-diversities of all the explored hot water communities. However, to keep it brief, the data from three North Indian and two East Indian vents will be presented in detail. As shown in Table
The above data clearly implied that all the bacteria growing in the two vancomycin-free media types, irrespective of their taxonomic identity and Gram-property, were susceptible to this so-called Gram-positive-specific antibiotic. To know the taxonomic identity of these cultured consortia we isolated their total genomes, amplified the V3 regions of all 16S rRNA genes present therein, and sequenced the amplicon pools by Ion PGM. The obtained V3 readsets were analyzed by OTU-clustering, statistics of which are given in Supplementary Table
Corroborating the outcome of the isolation experiments, almost all the vancomycin-free cultured consortia (irrespective of the media type) encompassed maximum OTUs from
The above data summarily indicated that the Gram-negative components of hot water communities were as susceptible to vancomycin as their Gram-positive counterparts. The wide taxonomic as well as geographic spread of these community analyses further hinted that the association between vancomycin-susceptibility and thermal adaptation could well be a global phenomenon. Significantly again, this comprehensive vancomycin-susceptibility of all taxonomic- and Gram-types was not detected when the above experiments were repeated with mesophilic (
All the current hot-spring isolates died (showing near-zero CFU counts) in the presence of 50 μg ml−1 vancomycin at both higher and lower incubation temperatures. Susceptible phenotype was also exhibited by the tested siblings strains of the present isolates reported from other parts of the world. Although the strains in question took negative Gram stain, their response to vancomycin challenge was exactly same as that of the
OD600 = 0.00/10 d | OD600 = 0.52/24 h | OD600 = 0.00/10 d | OD600 = 0.72/24 h | NA | NG | NA | NG | |
OD600 = 0.00/10 d | OD600 = 0.62/24 h | OD600 = 0.00/10 d | OD600 = 0.88/24 h | NA | NG | NA | NG | |
OD600 = 0.00/10 d | OD600 = 0.80/24 h | OD600 = 0.00/10 d | OD600 = 0.86/24 h | NA | NG | NA | NG | |
OD600 = 0.00/10 d | OD600 = 0.84/24 h | OD600 = 0.00/10 d | OD600 = 0.88/24 h | NA | NG | NA | NG | |
NA | NG | OD600 = 0.00/10 d | OD600 = 0.58/24 h | NA | NG | NA | NG | |
NA | NG | OD600 = 0.00/10 d | OD600 = 0.64/24 h | NA | NG | NA | NG | |
NA | NG | NA | NG | OD600 = 0.00, pH 7.0/7 d | OD600 = 0.26, pH 5.7/24 h | OD600 = 0.00, pH 7.0/7 d | OD600 = 0.29, pH 5.7/24 h | |
OD600 = 0.00/10 d | OD600 = 0.92/24 h | NA | NG | OD600 = 0.00, pH 7.0/7 d | OD600 = 0.25, pH 6.0/24 h | NA | NA | |
OD600 = 0.00/10 d | OD600 = 0.92/24 h | NA | NG | OD600 = 0.00, pH 7.0/7 d | OD600 = 0.25, pH 6.0/24 h | NA | NA | |
OD600 = 0.00/10 d | OD600 = 0.63/24 h | OD600 = 0.00/10 d | OD600 = 0.82/24 h | NA | NG | NA | NG | |
OD600 = 0.00/10 d | OD600 = 0.69/24 h | OD600 = 0.00/10 d | OD600 = 0.77/24 h | NA | NG | NA | NG | |
OD600 = 0.53/48 h | OD600 = 0.52/48 h | NA | NG | OD600 = 0.27, pH 6.0/48 h | OD600 = 0.25, pH 6.0/48 h | NA | NG | |
OD600 0.48/24 h | OD600 = 0.46/24 h | NA | NG | OD600 = 0.30, pH 6.0/24 h | OD600 0.32, pH 6.0/24 h | NA | NG | |
OD600 = 0.57/24 h | OD600 = 0.55/24 h | NA | NG | NA | NG | NA | NG | |
OD600 = 0.85/24 h | OD600 = 0.88/24 h | NA | NG | OD600 = 0.29, pH 6.0/24 h | OD600 = 0.29, pH 6.0/24 h | NA | NG | |
OD600 = 0.78/24 h | OD600 = 0.79/24 h | NA | NG | OD600 = 0.27, pH 6.0/24 h | OD600 = 0.26, pH 6.0/24 h | NA | NG |
The most remarkable of all these observations was the susceptibility of the thermo-enduring
Hot water of the Shivlinga Fountain, Northern Puga valley, Ladakh, India | 30–60°C | 55°C and 30°C (50 μg ml−1) | This study | ||
Water of a hot spring at São Gemil in Central Portugal | 30–60°C | 55°C and 30°C (50 μg ml−1) | This study, (Alves et al., |
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Hot water of the Shivlinga fountain, Northern Puga valley, Ladakh, India | 30–55°C | 55°C and 30°C (50 μg ml−1) | This study | ||
Run-off of a hot spring located at Alcafache in Central Portugal | 30–55°C | 55°C and 30°C (50 μg ml−1) | This study, Rainey et al., |
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Colored biofilms growing on paper machines and pulp dryers | 28–56°C | 56°C (Disc diffusion method) | Denner et al., |
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Hot water of the Paniphala Fountain, Eastern Coalfields, Paniphala, West Bengal | 50–60°C | 60°C (50 μg ml−1) | This study | ||
Pool water from a hot spring in the Waimangu thermal valley, New Zealand | 37–80°C | 70°C (20 μg ml−1) | Hudson et al., |
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Algal-bacterial mat from Mushroom Spring, Lower Geyser Basin, Yellowstone National Park, USA | 40–79°C | 70°C (20 μg ml−1) | Brock and Freeze, |
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Thermal water of a hot spring located at Mine, Shizuoka Prefecture, Japan. | 47–82°C | 70°C (20 μg ml−1) | Oshima and Imahori, |
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A well in the Beatrice oil field located in the British sector of the North Sea near the coast of Scotland | 50–65°C | 60°C (150 μg ml−1) | Greene et al., |
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Brine water samples of the Atlantis II deep of the Red Sea at a depth of 2000m | 30–53°C | 50°C (150 μg ml−1) | Fiala et al., |
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Hot spring water from Yumata, Nagano, Japan | 30–65°C | 55 30°C (100 μg ml−1) | Iino et al., |
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Tsuetate Hot Spring, Kumamoto Prefecture, Japan | 50–80°C | 73°C (100 μg ml−1) | Saiki et al., |
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Upper Norris Geyser basin, Yellowstone National Park, USA | 37–70°C | 55°C (1 μg ml−1) | Mohagheghi et al., |
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Runoff channel formed from flowing bore water from the geothermally-heated aquifer of Great Artesian Basin, Australia | 44–80°C | 68°C (10 μg ml−1) | Andrews and Patel, |
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Hydrothermal sediments mixed with fragments of inactive sulfide chimneys from 2891 m depth on the East Pacific Rise | 33–78°C | 55°C (25 μg ml−1) | |||
Sediment sample of a marine geothermal area near Vulcano, Italy | 55–90°C | 70°C (100 μg ml−1) | Huber et al., |
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Production fluid of the Kubiki oil reservoir in Niigata, Japan | 47–88°C | 70°C (100 μg ml−1) | Takahata et al., |
||
Production fluid of the Kubiki oil reservoir in Niigata, Japan | 48–86°C | 70°C (100 μg ml−1) | Takahata et al., |
||
Anoxic samples from production water taken from the water separator tanks on off-shore oil platforms of North Sea oil reservoir | 40–65°C | 60°C (10 μg ml−1) | Lien et al., |
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Hot water of the Lotus Pond-adjacent ebullition, Northern Puga valley, Ladakh, India | 30–50°C | 50°C and 30°C (50 μg ml−1) | This study | ||
Taketomi Island, Okinawa, Japan | 30–60°C | 55°C (50 μg ml−1) | Takai et al., |
In view of the above data it was deemed imperative to know whether the typical vancomycin mode of action that works against Gram-positive bacteria (Healy et al.,
Peptidoglycan was extracted from vancomycin-treated as well as -untreated cells of the current Gram-negative thermophilic isolates and analyzed by MALDI MS. The resultant data suggested that the organisms in question were all susceptible to this antibiotic in the same mechanistic way as the Gram-positive
Peptidoglycan was extracted from
MALDI mass spectra of the SLCR_1D peptidoglycan extracted after incubation in the presence as well as absence of vancomycin encompassed a common peak (m/z ~1008) [M-3H]+ attributable to the monomeric precursor GlcNAc-MurNAc-ala-glu-A2pm-ala-ala (having a calculated mass average of 1011.00; Figures
1008.045 and 1008.013 | 1011 | GlcNAc-MurNAc- Ala-Glu-A2Pm-Ala-Ala | -3H |
+ | + | − | − |
943.659 and 939.784 | 939.92 | GlcNAc-MurNAc- Ala-Glu-A2Pm-Ala | +4H |
+ | + | − | − |
920.721 and 920.867 | 939.92 | GlcNAc-MurNAc- Ala-Glu-A2Pm-Ala | -COOH+Na+H |
+ | + | − | − |
889.985 | 868.84 | GlcNAc-MurNAc- Ala-Glu-A2Pm | +Na+H | + | + | − | − |
845.173 | 868.84 | GlcNAc-MurNAc- Ala-Glu-A2Pm | -COOH+Na+2H |
+ | + | − | − |
697 | 696.66 | GlcNAc-MurNAc- Ala-Glu | +H |
+ | + | − | − |
1081.969 and 1081.522 | 1068.10 | GlcNAc-MurNAc- Ala-Glu-A2Pm-Ala-Lys | [M+ NH |
− | − | + | + |
949.819 and 949.802 | 939.92 | GlcNAc-MurNAc- Ala-Glu-A2Pm-Ala | -COOH+3NH |
− | − | + | + |
The most significant information revealed by the Figures
Unlike
All the present observations unanimously indicated that vancomycin-susceptibility was widespread in thermophilic (and perhaps also thermo-enduring) bacteria, irrespective of their taxonomic affiliation and Gram phenotype. It was also confirmed that this susceptibility stemmed from the predominance of alanine-terminated MPPs in the concerned organisms. In this regard it would be pertinent to recall that the D-ala-D-ala ligase (Ddl) of
When our current findings were juxtaposed with a decades-old report showing accumulation of excess DL-alanine by thermophilic bacterial cells (Matsumoto et al.,
In this context it must also be appreciated that an apparently-universal preference for D-ala-D-ala-terminated MPPs still does not guarantee vancomycin susceptibility of thermophilic bacteria as a relatively-hydrophilic molecule as large as vancomycin still has to cross the outer membrane before it can inhibit peptidoglycan biosynthesis. Notably, many mesophilic Gram-negative bacteria (unlike the case of
With regard to the apparent preference of thermophilic bacteria for D-ala-D-ala-terminated MPPs two intriguing facts remain to be clarified at length. One is the vancomycin susceptibility of the mesophilic but apparently thermo-enduring
CR anchored the whole work and participated in all experiments and manuscript writing. WG conceived the program, interpreted the results and wrote the manuscript. On site samplings were done by CR, MA, PH, TM, SKM, and WG. CR, MA, SM, PH, SB, TM, RR, MR, RC, and SKM did metagenomics and bioinformatics analyses. Pure culture microbiology was done by CR, MA, SM, SB, and TM. Organic chemistry experiments and data analyses were done by CR, MA, AM, RC, AN, and WG. AM, RC, AN, and SKM also contributed in improving the intellectual content of the work as well as the manuscript. All the authors read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The extensive field work and DNA sequencing involved in this paper would have never been possible without the pro-active espousal of the Director of Bose Institute, Professor Sibaji Raha. Financially, the work was supported by the Bose Institute as well as the Science and Engineering Research Board, Department of Science and Technology (DST), Government of India (GOI), with the latter grant having the number SR/FT/LS-204/2009. CR and MR received fellowships from the UGC, GOI. SM received a fellowship from the DST, GOI. SB was awarded a fellowship by Bose Institute, DST.
The Supplementary Material for this article can be found online at: