Enhanced microbial dechlorination of PCBs by anaerobic digested sludge and enrichment of low-abundance PCB dechlorinators.

The slow rate of anaerobic microbial dechlorination in natural environments limits the application of polychlorinated biphenyl (PCB) bioremediation. Anaerobic digested sludge (ADS), abundant in nutrients and microorganisms, could be an effective additive to improve microbial dechlorination. This research investigates the influence of ADS on Aroclor 1260 dechlorination performance, microbial community composition, and the abundance of functional genes. Moreover, further enrichment of organohalide-respiring bacteria (OHRB) was examined using tetrachloroethene (PCE) as the electron acceptor, followed by the serial dilution-to-extinction method in conjunction with resuscitation promoting factor (Rpf) supplementation. The results demonstrated that the addition of 5 g/L ADS achieved more extensive and efficient dechlorination of PCBs. ADS enhanced the removal of meta- and para-chlorine without significantly changing the dechlorination pathways. The abundances of dechlorinators, including Dehalobium and Dehalobacter within the Chloroflexi and Firmicutes phyla, as well as non-dechlorinators from the Desulfobacterota, Euryarchaeota, and Bacteroidetes phyla, were significantly increased with ADS amendment. Similarly, an increased abundance of bacteria, OHRB, reductive dehalogenase (RDase) genes, and archaeal 16S rRNA genes was observed. Additionally, obligate OHRB, such as Dehalobacter and Dehalobium, were further enriched. These findings indicate that ADS effectively enhances microbial reductive dechlorination and highlight the potential for enriching and isolating OHRB with Rpf.

Resting for viability: Gordonia polyisoprenivorans ZM27, a robust generalist for petroleum bioremediation under hypersaline stress.

The intrinsic issue associated with the application of microbes for practical pollution remediation involves maintaining the expected activity of engaged strains or consortiums as effectively as that noted under laboratory conditions. Faced with various stress factors, degraders with dormancy ability are more likely to survive and exhibit degradation activity. In this study, a hydrocarbonoclastic and halotolerant strain, Gordonia polyisoprenivorans ZM27, was isolated via stimulation with resuscitation-promoting factor (Rpf). Long-term exposure to dual stresses of 10% NaCl and starvation induced ZM27 to enter a viable but nonculturable (VBNC)-like state, and ZM27 cells could be resuscitated upon Rpf stimulation. Notable changes in both morphological and physiological characteristics between VBNC-like ZM27 cells and resuscitated cells confirmed the response to Rpf and their robust resistance against harsh environments. Whole-genome sequencing and analysis indicated ZM27 could be a generalist degrader with dormancy ability. Subsequently, VBNC-like ZM27 was applied in a soil microcosm experiment to investigate the practical application potential under harsh conditions. VBNC-like ZM27 combined with Rpf stimulation exhibited the most effective biodegradation performance, and the initial n-hexadecane content (1000 mg kg-1) decreased by 63.29% after 14-day incubation. Based on 16S rRNA amplicon sequencing and analysis, Gordonia exhibited a positive response to Rpf stimulation. The relative abundance of genus Gordonia was negatively correlated with that of Alcanivorax, a genus of obligate hydrocarbon degrader with the greatest abundance during soil incubation. Based on the degradation profile and community analysis, generalist Gordonia may be more efficient in hydrocarbon degradation than specialist Alcanivorax under harsh conditions. The characteristics of ZM27, including its sustainable culturability under long-term stress, response to Rpf and robust performance in soil microcosms, are valuable for the remediation of petroleum pollution under stressful conditions. Our work validated the importance of dormancy and highlighted the underestimated role of low-activity degraders in petroleum remediation.

Unlocking the potential of resuscitation-promoting factor for enhancing anaerobic microbial dechlorination of polychlorinated biphenyls.

Microbial dechlorination of polychlorinated biphenyls (PCBs) is limited by the slow growth rate and low activity of dechlorinators. Resuscitation promoting factor (Rpf) of Micrococcus luteus, has been demonstrated to accelerate the enrichment of highly active PCB-dechlorinating cultures. However, it remains unclear whether the addition of Rpf can further improve the dechlorination performance of anaerobic dechlorination cultures. In this study, the effect of Rpf on the performance of TG4, an enriched PCB-dechlorinating culture obtained by Rpf amendment, for reductive dechlorination of four typical PCB congeners (PCBs 101, 118, 138, 180) was evaluated. The results indicated that Rpf significantly enhanced the dechlorination of the four PCB congeners, with residual mole percentages of PCBs 101, 118, 138 and 180 in Rpf-amended cultures being 16.2-29.31 %, 13.3-20.1 %, 11.9-14.4 % and 9.4-17.3 % lower than those in the corresponding cultures without Rpf amendment after 18 days of incubation. Different models were identified as appropriate for elucidating the dechlorination kinetics of distinct PCB congeners, and it was observed that the dechlorination rate constant is significantly influenced by the PCB concentration. The supplementing Rpf did not obviously change dechlorination metabolites, and the removal of chlorines occurred mainly at para- and meta- positions. Analysis of microbial community and functional gene abundance suggested that Rpf-amended cultures exhibited a significant enrichment of Dehalococcoides, Dehalogenimonas and Desulfitobacterium, as well as non-dechlorinators belonging to Desulfobacterota and Bacteroidetes. These findings highlight the potential of Rpf as an effective additive for enhancing PCB dechlorination, providing new insights into the survival of functional microorganisms involved in anaerobic reductive dechlorination.

Ribosomal RNA gene operon copy number, a functional trait indicating the hydrocarbon degradation level of bacterial communities.

The lack of universal indicators for predicting microbial biodegradation potential and assessing remediation effects limits the generalization of bioremediation. The community-level ribosomal RNA gene operon (rrn) copy number, an important functional trait, has the potential to serve as a key indicator of the bioremediation of organic pollutants. A meta-analysis based on 1275 samples from 26 hydrocarbon-related studies revealed a positive relationship between the microbial hydrocarbon biodegradation level and the community-level rrn copy number in soil, seawater and culture. Subsequently, a microcosm experiment was performed to decipher the community-level rrn copy number response mechanism during total petroleum hydrocarbon (TPH) biodegradation. The treatment combining straw with resuscitation-promoting factor (Rpf) exhibited the highest community-level rrn copy number and the most effective biodegradation compared with other treatments, and the initial TPH content (20,000 mg kg-1) was reduced by 67.67% after 77 days of incubation. TPH biodegradation rate was positively correlated with the average community-level rrn copy number (p = 0.001, R2 = 0.5781). Both meta and community analyses showed that rrn copy number may reflect the potential of hydrocarbon degradation and microbial dormancy. Our findings provide insight into the applicability of the community-level rrn copy number to assess bacterial biodegradation for pollution remediation.

Solicola gregarius gen. nov., sp. nov., a soil actinobacterium isolated after enhanced cultivation with Micrococcus luteus culture supernatant.

An actinobacterial strain, designated A5X3R13T, was isolated from a compost soil suspension supplemented with extracellular material from a Micrococcus luteus-culture supernatant. The strain was cultured on tenfold-diluted reasoner's 2A agar. The cells were ovoid-to-rod shaped, non-motile, Gram-stain-positive, oxidase-negative, catalase-positive and had a width of 0.5 µm and a length of 0.8-1.2 µm. The results of both 16S rRNA-based phylogenetic and whole-genome analyses indicate that A5X3R13T forms a distinct lineage within the family Nocardioidaceae (order Propionibacteriales). On the basis of the 16S rRNA gene sequence, A5X3R13T was closely related to Aeromicrobium terrae CC-CFT486T (96.2 %), Nocardioides iriomotensis IR27-S3T (96.2 %), Nocardioides guangzhouensis 130T (95.6 %), Marmoricola caldifontis YIM 730233T (95.5 %), Aeromicrobium alkaliterrae KSL-107T (95.4 %), Aeromicrobium choanae 9H-4T (95.4 %), Aeromicrobium panaciterrae Gsoil 161T (95.3 %), and Nocardioides jensenii NBRC 14755T (95.2 %). The genome had a length of 4 915 757 bp, and its DNA G+C content was 68.5 mol %. The main fatty acids were 10-methyl C17 : 0, C16 : 0, C15 : 0, C18 : 0, C17 : 0 and iso-C16 : 0. The main polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and two unidentified phospholipids. MK-9(H4) was the predominant respiratory quinone. The peptidoglycan type was A3γ (A41.1) and contained alanine, glycine, glutamic acid and ll-diaminopimelic acid in a molar ratio of 1.2 : 0.9 : 1.0 : 0.8. On the basis of the results of the phylogenetic and phenotypic analyses and comparisons with other members of the family Nocardioidaceae, strain A5X3R13T is proposed to represent a novel species within a novel genus, for which the name Solicola gregarius gen. nov., sp. nov. is proposed. The type strain is A5X3R13T (=DSM 112953T=NCCB 100840T).

Role of Resuscitation Promoting Factor-like Protein from Nocardiopsis halophila.

Resuscitation promoting factors (Rpf), a class of proteins secreted by gram-positive bacteria including actinobacteria, promote the resuscitation of dormant bacteria and spore germination. Here, we describe the reconstitution of the resuscitation promoting activity of the Rpf protein from Nocardiopsis halophila CGMCC 4.1195Tin vitro and in vivo. The Rpf protein was expressed in the host Escherichia coli BL21 codon plus (DE3) and was confirmed to have a significant resuscitation effect on the viable but non-culturable (VBNC) N. halophila. Subsequently, the rpf gene of N. halophila was knocked out. We found that the growth rate of the mutant strain (Δrpf) was slower than that of the wild strain, and the former produced significantly shorter spores than the wild-type strain. Our results confirmed the activity of the Rpf protein in N. halophila to promote dormant bacteria resuscitation. This study will lay the foundation for the application of the Rpf protein from N. halophila to exploit actinomycetes resources.

Resuscitation-Promoting Factor Accelerates Enrichment of Highly Active Tetrachloroethene/Polychlorinated Biphenyl-Dechlorinating Cultures.

The anaerobic bioremediation of polychlorinated biphenyls (PCBs) is largely impeded by difficulties in massively enriching PCB dechlorinators in short periods of time. Tetrachloroethene (PCE) is often utilized as an alternative electron acceptor to preenrich PCB-dechlorinating bacteria. In this study, resuscitation promoting factor (Rpf) was used as an additive to enhance the enrichment of the microbial communities involved in PCE/PCBs dechlorination. The results indicated that Rpf accelerates PCE dechlorination 3.8 to 5.4 times faster than control cultures. In Aroclor 1260-fed cultures, the amendment of Rpf enables significantly more rapid and extensive dechlorination of PCBs. The residual high-chlorinated PCB congeners (≥5 Cl atoms) accounted for 36.7% and 59.8% in the Rpf-amended cultures and in the corresponding controls, respectively. This improvement was mainly attributed to the enhanced activity of the removal of meta-chlorines (47.7 mol % versus 14.7 mol %), which did not appear to affect dechlorination pathways. The dechlorinators, including Dehalococcoides in Chloroflexi and Desulfitobacterium in Firmicutes, were greatly enriched via Rpf amendment. The abundance of nondechlorinating populations, including Methanosarcina, Desulfovibrio, and Bacteroides, was also greatly enhanced via Rpf amendment. These results suggest that Rpf serves as an effective additive for the rapid enrichment of active dechlorinating cultures so as to provide a new approach by which to massively cultivate bioinoculants for accelerated in situ anaerobic bioremediation. IMPORTANCE The resuscitation promoting factor (Rpf) of Micrococcus luteus has been reported to resuscitate and stimulate the growth of functional microorganisms that are involved in the aerobic degradation of polychlorinated biphenyls (PCBs). However, few studies have been conducted to investigate the role of Rpf on anaerobic microbial populations. In this study, the enhancement of Rpf on the anaerobic microbial dechlorination of PCE/PCBs was discovered. Additionally, the Rpf-responsive populations underlying the enhanced dechlorination were uncovered. This report reveals the rapid enrichment of active dechlorinating cultures via Rpf amendment, and this sheds light on massively enriching PCB dechlorinators in short periods of time. The enhanced in situ anaerobic bioremediation of PCBs could be expected by supplementing Rpf.

A novel strategy for enhancing bioremediation of polychlorinated biphenyl-contaminated soil with resuscitation promoting factor and resuscitated strain.

PCBs bioremediation is largely impeded by the reduced metabolic activity and degradation ability of indigenous and exogenous microorganisms. Resuscitation promoting factor (Rpf) of Micrococcus luteus, has been reported to resuscitate and stimulate the growth of PCB-degrading bacterial populations, and the resuscitated strains exhibited excellent PCB-degrading performances. Therefore, this study was conducted to assess the feasibility of supplementing Rpf (SR) or resuscitated strain LS1 (SL), or both (SRL) for enhanced bioremediation of PCB-contaminated soil. The results indicated that Rpf and/or LS1 amended soil microcosms achieved more rapid PCBs degradation, which were 1.1-3.2 times faster than control microcosms. Although soil-inoculated LS1 maintained the PCB-degrading activity, higher PCBs degradation was observed in Rpf-amended soil microcosms compared with SL. The order of enhancement effect on PCBs bioremediation was SRL > SR > SL. PCBs degradation in soil microcosms was via HOPDA-benzoate-catechol/protocatechuate pathways. The improved PCBs degradation in Rpf-amended soil microcosms was attributed to the enhanced abundances of PCB-degrading populations which were mainly belonged to Proteobacteria and Actinobacteria. These results suggest that Rpf and resuscitated strains serve as effective additive and bio-inoculant for enhanced bioremediation, providing new approaches to realizing large scale applications of in situ bioremediation.

The detection of mixed tuberculosis infections using culture filtrate and resuscitation promoting factor deficient filtrate.

Tuberculosis (TB) infected individuals harbor a heterogenous population of differentially culturable tubercle bacilli (DCTB). Herein, we describe how DCTB assays using culture filtrate either containing or deficient in resuscitation promoting factors can uncover mixed infections. We demonstrate that Mycobacterium tuberculosis (Mtb) strain genotypes can be separated in DCTB assays based on their selective requirement for growth stimulatory factors. Beijing mixed infections appear to be associated with a higher bacterial load and reduced reliance on growth stimulatory factors. These data have important implications for identifying mixed infections and hetero-resistance, which in turn can affect selection of treatment regimen and establishment of transmission links.

Enhanced rhizoremediation of polychlorinated biphenyls by resuscitation-promoting factor stimulation linked to plant growth promotion and response of functional microbial populations.

Rhizoremediation is acknowledged as a green technology for removing polychlorinated biphenyls (PCBs) in soil. However, rhizoremediation is limited because most soil microorganisms enter into a viable but non-culturable (VBNC) state under PCBs stress. This work was to study the effect of resuscitation-promoting factor (Rpf) on rhizoremediation efficiency of PCBs in alfalfa and rhizosphere microbiological communities. Results suggested that Rpf promoted alfalfa growth in PCB-contaminated soil by improving antioxidant enzymes and detoxification metabolites in alfalfa. After 40 d Rpf treatment, removal rate for five selected PCBs significantly increased by 0.5-2.2 times. Rpf enhanced relative abundances of bphA and bphC responsible for degrading PCBs, and enzymatic activities of metabolizing exogenous compounds in rhizosphere soil. High-throughput sequencing showed that Rpf did not change the dominant microbial population at phyla and genera levels, but caused variation of the bacterial community structures. The promoting function of Rpf was linked to the shift of various key populations having different functions depending on Rpf concentrations. Pseudomonas and Rhizobium spp. enrichment might stimulate PCB degradation and Streptomyces and Bacillus spp. primarily contributed to alfalfa growth. Predicted functions in rhizosphere soil bacterial community indicated Rpf facilitated soil nutrient cycling and environmental adaptation. This study indicated that Rpf was an active additive for strengthening rhizoremediation efficiency of PCB-contaminated soil and enhancing their in-situ remediation.

Enzymatic properties and biological activity of resuscitation-promoting factor B of Rhodococcus sp. (GX12401).

Resuscitation-promoting factor B (RpfB) is one of the five members of Rpf-like family in Mycobacteriales, which have the resuscitation-promoting activity. Most strains of Rhodococcus also have RpfB gene, but the study of rpfB gene in Rhodococcus is not thorough. Here, we amplified the rpfB gene of intact Rhodococcus sp. (GX12401) and cloned it into pET30a (+) expression vector. Then a recombinant form of soluble RpfB was expressed in Escherichia coli BL21. The soluble recombinant RpfB was purified by Ni-Sepharose affinity chromatography and molecular weight of the protein was 55 kDa, determined by 12% SDS-PAGE stained with Coomassie brilliant blue R-250. When 4-methylumbelliferyl-ß-D-N,N',N″-triacetylchitoside was used as enzyme substrate to test lysozyme activity, the recombinant protein RpfB had good stability and enzyme activity, and the lysozyme activity was low (4.74 U), among which Mg2+, Na+, Al3+ and DMSO could significantly increase the activity of RpfB. The purified recombinant protein was added to Rhodococcus VBNC cells, and the VBNC cells were resuscitated at the concentration of 1 picomolar concentrations, which increased by 18% compared with the control, while the cell resuscitation was inhibited at the concentration of 1,000 picomolar concentrations. Therefore, RpfB can improve the survival ability of Rhodococcus in extreme or harsh environment and enhance the corresponding biological activity.

Dimethyl fumarate eliminates differentially culturable Mycobacterium tuberculosis in an intranasal murine model of tuberculosis.

Tuberculosis (TB) claims nearly 1.5 million lives annually. Current TB treatment requires a combination of several drugs administered for at least 6 months. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can persist in infected humans and animals for decades. Moreover, during infection, Mtb produces differentially culturable bacteria (DCB) that do not grow in standard media but can be resuscitated in liquid media supplemented with sterile Mtb culture filtrates or recombinant resuscitation-promoting factors (Rpfs). Here, we demonstrate that, in an intranasal murine model of TB, Mtb DCB are detectable in the lungs after 4 weeks of infection, and their loads remain largely unchanged during a further 8 weeks. Treatment of the infected mice with dimethyl fumarate (DMF), a known drug with immunomodulatory properties, for 8 weeks eliminates Mtb DCB from the lungs and spleens. Standard TB treatment consisting of rifampicin, isoniazid, and pyrazinamide for 8 weeks reduces Mtb loads by nearly four orders of magnitude but does not eradicate DCB. Nevertheless, no DCB can be detected in the lungs and spleens after 8 weeks of treatment with DMF, rifampicin, isoniazid, and pyrazinamide. Our data suggest that addition of approved anti-inflammatory drugs to standard treatment regimens may improve TB treatment and reduce treatment duration.

Single-cell-level microfluidics assisted with resuscitation-promoting factor technology (SMART) to isolate novel biphenyl-degrading bacteria from typical soils in eastern China.

Soil microorganisms represent one of the largest biodiversity reservoirs. However, most low-abundance, slow-growing or dormant microorganisms in soils are difficult to capture with traditional enrichment culture methods. These types of microorganisms represent a valuable "microbial seed bank". To better exploit and utilize this "microbial dark matter", we developed a novel strategy that integrates single-cell-level isolation with microfluidics technology and culture with resuscitation-promoting factor (Rpf) to isolate biphenyl-degrading bacteria from four typical soils (paddy soil, red soil, alluvial soil and black soil) in eastern China. Multitudinous bacteria were successfully isolated and cultured; some of the identified clades have not been previously linked to biphenyl biodegradation, such as Actinotalea, Curtobacterium and Rothia. Soil microcosmic experiments validated that some bacteria are responsible for biphenyl degradation in soil. In addition, genomic sequencing and Illumina MiSeq sequencing of 16S rRNA genes indicated that exogenous Rpf mainly promotes the recovery and growth of bacteria containing endogenous Rpf-encoding genes. In summary, this study provides a novel strategy for capturing target functional microorganisms in soils, indicates potential bioresources for the bioremediation of contaminated soils, and enhances our current understanding of the mechanisms involved in the response to exogenous Rpf.

Pedomonas mirosovicensis gen. nov., sp. nov., a bacterium isolated from soil with the aid of Micrococcus luteus culture supernatant containing resuscitation-promoting factor.

An orange-golden iridescent culture, designated A1X5R2T, was isolated from a compost soil suspension which was amended with Micrococcus luteus NCTC 2665T culture supernatant. The cells were non-motile, Gram-stain-negative, 0.4-0.5 µm wide and 0.7-1.4 µm long. The 16S rRNA-based phylogenetic and whole-genome analyses revealed that strain A1X5R2T forms a distinct lineage within the family Sphingosinicellaceae and is closely related to members of the genus Sphingoaurantiacus (S. capsulatus, 93.04 % similarity, and S. polygranulatus, 92.77 %). The organism grew at 22-47 °C (optimal at 37 °C), salinity <3 % (optimal at 1.5 %) and at pH 7. The major respiratory quinone was ubiquinone-10, but a small quantity of ubiquinone-9 was also detected The major polyamine was homospermidine, but a small quantity of putrescine was also detected. The strain contained C18  :  1ω7c, C16 : 0, C16 : 1 ω7c and C18 : 0 as the major fatty acids. The main polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol, sphingoglycolipid, diphosphatidylglycerol, two unidentified phospholipids and three unidentified amino lipids. The DNA G+C content was 64.9 mol%. According to the results of phylogenetic and phylogenomic analyses, as well as its physiological characteristics, strain A2X5R2T represents the type species of a novel genus within the family Sphingosinicellaceae. The name Pedomonas mirosovicensis gen. nov., sp. nov. is proposed, with the type strain being A1X5R2T (=NCCB 100839T=DSM 112829T).

Rapid Detection of Mycobacterium tuberculosis in Pleural Fluid Using Resuscitation-Promoting Factor-Based Thin Layer Agar Culture Method.

BACKGROUND: Pleural tuberculous is difficult to diagnose. Culture is still considered the gold standard, especially in resource-limited settings where quick, cheap, and easy techniques are needed. The aim of the study was to evaluate resuscitation-promoting factors (Rpfs)-based thin layer agar (TLA) culture method for quick detection of Mycobacterium tuberculosis in pleural fluid. METHODS: Patients with suspected pleural TB were enrolled prospectively in our hospital, pleural fluid of all patients were collected, stained with Ziehl-Neelsen for acid-fast bacilli (AFB), cultured on Rpfs-TLA, TLA, and Löwenstein-Jensen (LJ) medium, and identified according to recommended procedures. RESULTS: A total of 137 suspected pleural TB were enrolled and categorized, including 103 pleural TB (49 confirmed and 54 probable pleural TB) and 34 non-TBP patients. The sensitivity of Rpfs-TLA for total pleural TB was 43.7% (34.5∼53.3%), higher than that of TLA 29.1% (21.2∼38.5%) and LJ 26.2% (18.7∼35.5%) (p < 0.01), and all specificity was 100% in the diagnosis of pleural TB. Median time to detection of a positive culture was 11.8 days (95% CI 10.4∼13.4) for Rpfs-TLA, 21.0 days (95% CI 19.1∼22.9) for TLA, and 30.5 days (95% CI 28.5∼32.5) for LJ (p < 0.001). CONCLUSION: Rpfs-TLA is an accurate, rapid, cheap, and easy culture method, which makes it promising for use in clinical laboratories.

Morphological and genomic characteristics of two novel halotolerant actinomycetes, Tomitella gaofuii sp. nov. and Tomitella fengzijianii sp. nov. isolated from bat faeces.

Four white-pigmented, Gram-staining-positive, strictly aerobic, non-spore-forming, irregular rod-shaped bacteria were isolated from the faeces of bats collected from Guangxi autonomous region (22°20'54″N, 106°49'20″E; July 28, 2011) and Chongqing city (30°02'15″N, 107°07'4″E; September 1, 2011) of South China. The strains shared 99.3-99.9% 16S rRNA gene sequence similarity by BLAST search among them, and belonged to genus Tomitella according to 16S rRNA gene and genomic sequence-based phylogenetic/phylogenomic analyses. Strains HY172T and HY188T contained meso-diaminopimelic acid as the diagnostic diamino acid, and arabinose, glucose, galactose or ribose in their whole-cell hydrolysates. Besides sharing phosphatidylethanolamine, diphosphatidylglycerol and unidentified glycolipid(s) in their polar lipid profiles, additionally HY172T had one unidentified phosphoglycolipid and three unidentified phospholipids whereas HY188T had phosphatidyl inositol mannoside and four unidentified aminolipids. The main cellular fatty acids of strains HY172T and HY188T were C16:0, C18:0 10-methyl, C18:1ω9c and summed feature 3. The genomic DNA G + C contents of both strains (HY172T and HY188T) were 70.9 %. The genus Tomitella contains 2311 core genes, and resuscitation promoting factor (rpf) genes can be found in all members of Tomitella. The digital DNA-DNA hybridization and average nucleotide identity values of the four novel strains with other members of the genus Tomitella were within the ranges of 20.1-45.2% and 74.8-91.9%, respectively, all below the respective recommended 70.0% and 95-96% cutoff point. Based on phylogenetic, chemotaxonomic and phenotypic analyses, these four strains could be classified as two novel species of the genus Tomitella, for which the names Tomitella gaofuii sp. nov. and Tomitella fengzijianii sp. nov. are proposed. The type strains are HY172T (= CGMCC 1.18701T = JCM 34231T) and HY188T (= CGMCC 1.16971T = JCM 33467T), respectively.

Promoting effect of the recombinant resuscitation promoting factors-2 of Rhodococcus erythropolis on petroleum degradation and cultivable bacterial diversities of the oil-contaminated soils.

Resuscitation-promoting factors (Rpfs) belong to peptidoglycan hydrolases, which participate in recovery of dormant cells and promoting bacteria growth. In this study, the resuscitation promoting factor rpf2 gene of Rhodococcus erythropolis KB1 was expressed in Escherichia coli and purified by Ni2+ affinity chromatography. The purified recombinant fusion protein Rpf2 showed a closely 50 kDa band on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The protein showed muralytic activity, with a specific activity of 1503 ± 123 U mg-1 when determined with 4-methylumbelliferyl-ß-d-N, N',N″-triacetotri-ylchitoside as substrate. It also showed protease activity when measured with azocasein as substrate, with a specific activity of 1528 ± 411 U mg-1 . The addition of the recombinant Rpf2 protein significantly increased petroleum degradation efficiency of the indigenous micro-organisms and the petroleum degradation rates increased from 30·86 to 43·45%, 45·20 and 49·23% in the treatment groups. The recombinant protein also increased the petroleum-degrading bacterial diversities enriched from the contaminated soils. The cultivable bacterial flora of the treatment groups supplemented with different concentrations of Rpf2 increased from 82 genera in 9 phyla to 116 genera in 16 phyla and 138 genera in 16 phyla respectively. Thirteen extra petroleum-degrading bacteria strains were isolated from the petroleum-contaminated soils in the groups containing the recombinant Rpf2.

Small RNA F6 Provides Mycobacterium smegmatis Entry into Dormancy.

Regulatory small non-coding RNAs play a significant role in bacterial adaptation to changing environmental conditions. Various stresses such as hypoxia and nutrient starvation cause a reduction in the metabolic activity of Mycobacterium smegmatis, leading to entry into dormancy. We investigated the functional role of F6, a small RNA of M. smegmatis, and constructed an F6 deletion strain of M. smegmatis. Using the RNA-seq approach, we demonstrated that gene expression changes that accompany F6 deletion contributed to bacterial resistance against oxidative stress. We also found that F6 directly interacted with 5'-UTR of MSMEG_4640 mRNA encoding RpfE2, a resuscitation-promoting factor, which led to the downregulation of RpfE2 expression. The F6 deletion strain was characterized by the reduced ability to enter into dormancy (non-culturability) in the potassium deficiency model compared to the wild-type strain, indicating that F6 significantly contributes to bacterial adaptation to non-optimal growth conditions.

Exploring the Potential of Micrococcus luteus Culture Supernatant With Resuscitation-Promoting Factor for Enhancing the Culturability of Soil Bacteria.

A bacterial species is best characterized after its isolation in a pure culture. This is an arduous endeavor for many soil microorganisms, but it can be simplified by several techniques for improving culturability: for example, by using growth-promoting factors. We investigated the potential of a Micrococcus luteus culture supernatant containing resuscitation-promoting factor (SRpf) to increase the number and diversity of cultured bacterial taxa from a nutrient-rich compost soil. Phosphate-buffered saline and inactivated SRpf were included as controls. After agitation with SRpf at 28°C for 1 day, the soil suspension was diluted and plated on two different solid, oligotrophic media: tenfold diluted Reasoner's 2A agar (R2A) and soil extract-based agar (SA). Colonies were collected from the plates to assess the differences in diversity between different treatments and cultivation media. The diversity on both R2A and SA was higher in the SRpf-amended extracts than the controls, but the differences on R2A were higher. Importantly, 51 potentially novel bacterial species were isolated on R2A and SA after SRpf treatment. Diversity in the soil extracts was also determined by high-throughput 16S rRNA amplicon sequencing, which showed an increase in the abundance of specific taxa before their successful cultivation. Conclusively, SRpf can effectively enhance the growth of soil bacterial species, including those hitherto uncultured.

Purification effect evaluation of the designed new volcanic soil adsorption material containing bioreactor for eutrophic water treatment.

The purpose of this study was to investigate the purification effect of a new adsorption material containing bioreactor and the critical role of viable but non-culturable (VBNC) bacteria in a eutrophication ecosystem. Major water quality parameters of the prepared eutrophic water were determined, and the microbial community was analyzed during 2 years. The results showed that removal rates of total phosphorus (TP), total nitrogen (TN), chlorophyll-a (Chl-a), and chemical oxygen demand (COD) were 90.7-95.9%, 84.5-92.4%, 87.9-95.8%, and 68.3-82.7%, respectively, indicating the high efficiency of the bioreactor in the eutrophic water treatment. Although the bioreactor had been operated for 2 years, water from the treatment group was much clearer and odorless than from the control group, exhibiting the long service life of the bioreactor. Stopping operation in August caused significant decrease of the removal rates of major water quality parameters (p < 0.05). This operational stop event and high temperature in summer exerted a dual effect on the bioreactor, whereas the impact could be minimized when the bioreactor was running. Moreover, the total bacteria under +Rpf (active resuscitation-promoting factor) treatment were higher than under -Rpf (inactive resuscitation-promoting factor) treatment, implying that Rpf could resuscitate VBNC bacteria in the eutrophication ecosystem. Nine strains of VBNC bacteria were isolated based on the BLAST results of the 16S rRNA gene. Also, these bacteria might contribute to the eutrophic water treatment based on their functions of phosphorus collecting and denitrification. These results provided new insights for engineering technology innovations, and consequently these findings had benefits in eutrophic water treatment.