Increase in Bacterial Colony Formation from a Permafrost Ice Wedge Dosed with a Tomitella biformata Recombinant Resuscitation-Promoting Factor Protein.
ABSTRACT: Resuscitation-promoting factor (Rpf) is a protein that has been found in a number of different Actinobacteria species and has been shown to promote the growth of active cells and resuscitate dormant (non-dividing) cells. We previously reported the biological activity of an Rpf protein in Tomitella biformata AHU 1821(T), an Actinobacteria isolated from a permafrost ice wedge. This protein is excreted outside the cell; however, few studies have investigated its contribution in environmental samples to the growth or resuscitation of bacteria other than the original host. Therefore, the aim of the present study was to determine whether Rpf from T. biformata impacted the cultivation of other bacteria from the permafrost ice wedge from which it was originally isolated. All experiments used recombinant Rpf proteins produced using a Rhodococcus erythropolis expression system. Dilutions of melted surface sterilized ice wedge samples mixed with different doses of the purified recombinant Rpf (rRpf) protein indicated that the highest concentration tested, 1250 pM, had a significantly (p <0.05) higher number of CFUs on agar plates after 8 d, approximately 14-fold higher than that on control plates without rRpf. 16S rRNA gene sequences revealed that all the colonies on plates were mainly related to Brevibacterium antiquum strain VKM Ac-2118 (AY243344), with 98-99% sequence identity. This species is also a member of the phylum Actinobacteria and was originally isolated from Siberian permafrost sediments. The results of the present study demonstrated that rRpf not only promoted the growth of T. biformata from which it was isolated, but also enhanced colony formation by another Actinobacteria in an environmental sample.
Project description:Functional variation of Rpf, a growth factor found exclusively in Actinobacteria, is differentiated by its source and amino acid sequences. Only purified Rpf proteins from three species have been studied so far. To seek new Rpfs for use in future studies to understand their role in Actinobacteria, the objective of this study was to identify rpf gene homologs in Tomitella biformata AHU 1821(T), a novel Actinobacteria isolated from permafrost ice wedge. Amplification using degenerate primers targeting the essential Rpf domain led to the discovery of a new rpf gene in T. biformata. Gene structure and the deduced Rpf domain amino acid sequence indicated that this rpf gene was not identical to previously studied Rpf. Phylogenetic analysis placed T. biformata Rpf in a monophyletic branch in the RpfB subfamily. The deduced amino acid sequence was 44.9% identical to RpfB in Mycobacterium tuberculosis, the closest functionally tested Rpf. The gene was cloned and expressed in Escherichia coli; the recombinant Rpf protein (rRpf) promoted the growth of dividing cells and resuscitated non-dividing cells of T. biformata. Compared to other studies, this Rpf was required at higher concentrations to promote its growth and to resuscitate itself from a non-dividing state. The resuscitation function was likely due to the highly conserved Rpf domain. This study provides evidence that a genetically unique but functional Rpf can be found in novel members of Actinobacteria and can lead to a better understanding of bacterial cytokines in this phylum.
Project description:Phylogenetic analysis of bacteria preserved within an ice wedge from the Fox permafrost tunnel was undertaken by cultivation and molecular techniques. The radiocarbon age of the ice wedge was determined. Our results suggest that the bacteria in the ice wedge adapted to the frozen conditions have survived for 25,000 years.
Project description:Bacterial dormancy can take many forms, including formation of <i>Bacillus</i> endospores, <i>Streptomyces</i> exospores, and metabolically latent <i>Mycobacterium</i> cells. In the actinobacteria, including the streptomycetes and mycobacteria, the rapid resuscitation from a dormant state requires the activities of a family of cell-wall lytic enzymes called resuscitation-promoting factors (Rpfs). Whether Rpf activity promotes resuscitation by generating peptidoglycan fragments (muropeptides) that function as signaling molecules for spore germination or by simply remodeling the dormant cell wall has been the subject of much debate. Here, to address this question, we used mutagenesis and peptidoglycan binding and cleavage assays to first gain broader insight into the biochemical function of diverse Rpf enzymes. We show that their LysM and LytM domains enhance Rpf enzyme activity; their LytM domain and, in some cases their LysM domain, also promoted peptidoglycan binding. We further demonstrate that the Rpfs function as endo-acting lytic transglycosylases, cleaving within the peptidoglycan backbone. We also found that unlike in other systems, Rpf activity in the streptomycetes is not correlated with peptidoglycan-responsive Ser/Thr kinases for cell signaling, and the germination of <i>rpf</i> mutant strains could not be stimulated by the addition of known germinants. Collectively, these results suggest that in <i>Streptomyces</i>, Rpfs have a structural rather than signaling function during spore germination, and that in the actinobacteria, any signaling function associated with spore resuscitation requires the activity of additional yet to be identified enzymes.
Project description:Nowadays, much of what we know regarding the isolated cellulolytic bacteria comes from the conventional plate separation techniques. However, the culturability of many bacterial species is controlled by resuscitation-promoting factors (Rpfs) due to entering a viable but non-culturable (VBNC) state. Therefore, in this study, Rpf from Micrococcus luteus was added in the culture medium to evaluate its role in bacterial isolation and enhanced effects on cellulose-degrading capability of bacterial community in the compost. It was found that Proteobacteria and Actinobacteria were two main phyla in the compost sample. The introduction of Rpf could isolate some unique bacterial species. The cellulase activity of enrichment cultures with and without Rpf treatment revealed that Rpf treatment significantly enhanced cellulase activity. Ten isolates unique in Rpf addition displayed carboxymethyl-cellulase (CMCase) activity, while six isolates possessed filter paper cellulase (FPCase) activity. This study provides new insights into broader cellulose degraders, which could be utilized for enhancing cellulosic waste treatment.
Project description:<h4>Background</h4>Resuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new anti-tuberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs.<h4>Methodology/principal findings</h4>Here we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC(50) 1-7 microg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus (in which Rpf is essential for growth protein) at concentrations close to IC(50). The candidate compounds suppressed resuscitation of dormant ("non-culturable") cells of M. smegmatis at 1 microg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 microg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations.<h4>Conclusions/significance</h4>NPT are the first example of low molecular weight compounds that inhibit the enzymatic and biological activities of Rpf proteins.
Project description:Relict permafrost is ubiquitous throughout the Arctic coastal shelf, but little is known about it near shore. The presence and thawing of subsea permafrost are vital information because permafrost stores an atmosphere's worth of carbon and protects against coastal erosion. Through electrical resistivity imaging across a lagoon on the Alaska Beaufort Sea coast in summer, we found that the subsurface is not ice-bonded down to ~20 m continually from within the lagoon, across the beach, and underneath an ice-wedge polygon on the tundra. This contrasts with the broadly held idea of a gently sloping ice-bonded permafrost table extending from land to offshore. The extensive unfrozen zone is a marine talik connected to on-land cryopeg. This zone is a potential source and conduit for water and dissolved organic matter, is vulnerable to physical degradation, and is liable to changes in biogeochemical processes that affect carbon cycling and climate feedbacks.
Project description:BACKGROUND: In Micrococcus luteus growth and resuscitation from starvation-induced dormancy is controlled by the production of a secreted growth factor. This autocrine resuscitation-promoting factor (Rpf) is the founder member of a family of proteins found throughout and confined to the actinobacteria (high G + C Gram-positive bacteria). The aim of this work was to search for and characterise a cognate gene family in the firmicutes (low G + C Gram-positive bacteria) and obtain information about how they may control bacterial growth and resuscitation. RESULTS: In silico analysis of the accessory domains of the Rpf proteins permitted their classification into several subfamilies. The RpfB subfamily is related to a group of firmicute proteins of unknown function, represented by YabE of Bacillus subtilis. The actinobacterial RpfB and firmicute YabE proteins have very similar domain structures and genomic contexts, except that in YabE, the actinobacterial Rpf domain is replaced by another domain, which we have called Sps. Although totally unrelated in both sequence and secondary structure, the Rpf and Sps domains fulfil the same function. We propose that these proteins have undergone "non-orthologous domain displacement", a phenomenon akin to "non-orthologous gene displacement" that has been described previously. Proteins containing the Sps domain are widely distributed throughout the firmicutes and they too fall into a number of distinct subfamilies. Comparative analysis of the accessory domains in the Rpf and Sps proteins, together with their weak similarity to lytic transglycosylases, provide clear evidence that they are muralytic enzymes. CONCLUSIONS: The results indicate that the firmicute Sps proteins and the actinobacterial Rpf proteins are cognate and that they control bacterial culturability via enzymatic modification of the bacterial cell envelope.
Project description:Sublimation of ice is rate-controlled by vapor transport away from its outer surface and may have generated landforms on Mars. In ice-cemented ground (permafrost), the lag of soil particles remaining after ice loss decreases subsequent sublimation. Varying soil-ice ratios lead to differential lag development. Here we report 52 years of sublimation measurements from a permafrost tunnel near Fairbanks, Alaska, and constrain models of sublimation, diffusion through porous soil, and lag formation. We derive the first long-term in situ effective diffusion coefficient of ice-free loess, a Mars analog soil, of 9.05?×?10<sup>-6?</sup>m<sup>2</sup>?s<sup>-1</sup>, ~5× larger than past theoretical studies. Exposed ice-wedge sublimation proceeds ~4× faster than predicted from analogy to heat loss by buoyant convection, a theory frequently employed in Mars studies. Our results can be used to map near-surface ice-content differences, identify surface processes controlling landform formation and morphology, and identify target landing sites for human exploration of Mars.
Project description:Fire-induced permafrost degradation is well documented in boreal forests, but the role of fires in initiating thermokarst development in Arctic tundra is less well understood. Here we show that Arctic tundra fires may induce widespread thaw subsidence of permafrost terrain in the first seven years following the disturbance. Quantitative analysis of airborne LiDAR data acquired two and seven years post-fire, detected permafrost thaw subsidence across 34% of the burned tundra area studied, compared to less than 1% in similar undisturbed, ice-rich tundra terrain units. The variability in thermokarst development appears to be influenced by the interaction of tundra fire burn severity and near-surface, ground-ice content. Subsidence was greatest in severely burned, ice-rich upland terrain (yedoma), accounting for ~50% of the detected subsidence, despite representing only 30% of the fire disturbed study area. Microtopography increased by 340% in this terrain unit as a result of ice wedge degradation. Increases in the frequency, magnitude, and severity of tundra fires will contribute to future thermokarst development and associated landscape change in Arctic tundra regions.
Project description:Resuscitation promoting factors (Rpfs) are the proteins involved in the process of reactivation of the dormant cells of mycobacteria. Recently a new class of nitrophenylthiocyanates (NPTs), capable of inhibiting the biological and enzymatic activities of Rpfs has been discovered. In the current study the inhibitory properties of the compounds containing both nitro and thiocyanate groups alongside with the compounds with the modified number and different spatial location of the substituents are compared.New benzoylphenyl thiocyanates alongside with nitrophenylthiocyanates were tested in the enzymatic assay of bacterial peptidoglycan hydrolysis as well as against strains of several actinobacteria (Mycobacterium smegmatis, Mycobacterium tuberculosis) on in-lab developed models of resuscitation of the dormant forms.Introduction of the additional nitro and thiocyanate groups to the benzophenone scaffold did not influence the inhibitory activity of the compounds. Removal of the nitro groups analogously did not impair the functional properties of the molecules. Among the tested compounds two molecules without nitro group: 3-benzoylphenyl thiocyanate and 4-benzoylphenyl thiocyanate demonstrated the maximum activity in both enzymatic assay (inhibition of the Rpf-mediated peptidoglycan hydrolysis) and in the resuscitation assay of the dormant M. tuberculosis cells.The current study demonstrates dispensability of the nitro group in the NPT's structure for inhibition of the enzymatic and biological activities of the Rpf protein molecules. These findings provide new prospects in anti-TB drug discovery especially in finding of molecular scaffolds effective for the latent infection treatment.