Toxic Pyrene Metabolism in Mycobacterium gilvum PYR-GCK Results in the Expression of Mammalian Cell Entry Genes as Revealed by Transcriptomics StudyS
Toxic Pyrene Metabolism in Mycobacterium gilvum PYR-GCK Results in the Expression of Mammalian Cell Entry Genes as Revealed by Transcriptomics StudyS
Journal of Microbiology and Biotechnology. 2014. Sep, 24(9): 1170-1177
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : December 02, 2013
  • Accepted : June 09, 2014
  • Published : September 30, 2014
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About the Authors
Abimbola Comfort Badejo
Department of Molecular and Life Science, Hanyang University, Ansan 426-791, Republic of Korea
Won Hyong Chung
Korean Bioinformation Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea
Nam Shin Kim
Korean Bioinformation Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea
Se Kye Kim
Department of Molecular and Life Science, Hanyang University, Ansan 426-791, Republic of Korea
Jin Choul Chai
Department of Molecular and Life Science, Hanyang University, Ansan 426-791, Republic of Korea
Young Seek Lee
Department of Molecular and Life Science, Hanyang University, Ansan 426-791, Republic of Korea
Kyoung Hwa Jung
Department of Molecular and Life Science, Hanyang University, Ansan 426-791, Republic of Korea
Hyo Joon Kim
Department of Molecular and Life Science, Hanyang University, Ansan 426-791, Republic of Korea
Young Gyu Chai
Department of Nanobiotechnology, Hanyang University, Seoul 133-791, Republic of Korea

Mycobacterium gilvum PYR-GCK is a bacterial strain under study for its bioremediation use on heavy hydrocarbon pollutants in the environment. During the course of our study, mammalian cell entry ( mce ) genes, known to facilitate pathogenicity in M. tuberculosis , were highly expressed during a comparative and substrate-related cultural global transcriptomic study. RNA sequencing of the global transcriptome of the test strain in two different substrates, pyrene and glucose, showed high expression of the mce genes based on the differential results. After validating the expression of these genes with quantitative real-time PCR, we arrived at the conclusion that the genes were expressed based on the pyrene substrate (a phytosterol compound), and sterol metabolism is said to activate the expression of the mce genes in some actinomycetes bacteria, M. gilvum PYR-GCK in this case. This study is believed to be important based on the fact that some mycobacterial strains are undergoing a continuous research as a result of their use in practical bioremediation of anthropogenic exposure of toxic organic wastes in the environment.
The actinomycetes group of bacteria is known for diverse catabolic versatilities, and these characteristics have been observed in their annotated genome sequences. Their robust range of metabolic abilities has made them very important in numerous technological fields such as the environmental, pharmaceuticals, and chemical and energy production. The mycobacteria are of special interest owing to their unique cell wall characteristic of complex waxes and glycolipids. Mycobacterium gilvum PYR-GCK has been under intense study owing to its ability to degrade recalcitrant high molecular weight polycyclic aromatic hydrocarbons (PAHs) in the process of crude oil bioremediation. This ability is confirmed possible as a result of its cell walls and superb armamentarium of metabolic enzymes, aiding it in exceptional survival skills (such as the ability to resist unpleasant environmental conditions: extreme salinity and pH, dehydration, and exposure to toxic substances). These abilities have also been observed in its pathogenic relatives, such as Mycobacterium tuberculosis and Mycobacterium leprae . In the pathogenic mycobacterial strains, one of their major goals is to gain entrance into their respective hosts and resist the plethora of hostile compounds therein [8] . The mammalian cell entry ( mce ) genes have been characterized as important in this invasive purpose [13] . Annotated bacterial genome data have shown mce genes present in a variety of nonpathogenic actinomycetes bacteria, including mycobacteria [6] . The M. tuberculosis genome contains four mce operons ( mce1 to mce4 ) [13] ; their structure and evolution have been described by Tekaia et al . [26] . The pioneering mce genes study revealed mce1A , in the first operon, as an essential virulence property in M. tuberculosis , based on the conferment of this property to E. coli , and a subsequent invasion into macrophages [1 , 8] . With no information on the exact function of these mce genes in the pathogenic and nonpathogenic mycobacterial strains, studies have suggested that the presence of these putative virulence genes may not be an indicator for pathogenicity [6 , 10 , 13] . Further studies have shown the expression of the mce genes during cholesterol and steroid metabolism in the Rhodococcal species [13 , 28] , with the mce4 operon encoding a steroid transporter gene. Nevertheless, mutants of this uptake system were unaffected for growth on steroid compounds having shorter polar side-chains [16] .
Polycyclic aromatic hydrocarbons (PAHs) are organic compounds released into the environment mostly as anthropogenic particles/compounds [18] , and various effects of these compounds on human and wildlife health have been documented widely [4 , 7 , 23] . Studies on Mycobacterium gilvum PYR-GCK and other mycobacterial strains have been undertaken as biological removal agents of PAHs from the environment [3 , 25] , and pyrene has been used often as one of the PAH test substrates in many studies based on its simple form as a high molecular weighted PAH compound. With the aim of finding a relationship between the mce genes and toxic pyrene metabolism, and also to characterize the genes using a gene expression technique, M. gilvum PYR-GCK was subjected to a global transcriptomic analysis. A previous proteomic study on pyrene metabolism mechanics in the same strain and conditions revealed numerous expressed proteins, but we believe that a “supplementing” transcriptomics study will reveal the expression of more gene products possibly omitted during the tricky protein analysis and quantification.
Materials and Methods
- Reagents, Media, and Bacterial Strain Cultivation
Mycobacterium gilvum PYR-GCK was acquired from the American Type Culture Collection (ATCC) under the code name M. flavescens ATCC 700033 and maintained in Bacto Brain Heart Infusion Media (BD Laboratories, Sparks, USA) at 29℃ or stock preserved in the same medium supplemented with 28% glycerol, at –80℃. Pyrene substrate (confirmed >98.0% pure by Aldrich Company) and other chemicals used were purchased from Sigma-Aldrich Company (St. Louis, MO, USA) and Tokyo Chemical Industry (Tokyo, Japan). M. gilvum PYR-GCK cells were prepared and grown in 500 ml flasks containing 200 ml of basal medium, as described in Badejo et al . [3] .
- RNA Preparation, Processing, and Sequencing
Bacterial cells were harvested from all six bacterial cultures by centrifugation at 10,000 × g at 4℃ for 1 min, after the addition of RNAprotect Bacteria reagent (Qiagen, USA) to the culture broth in the ratio of 2:1. RNAiso (Takara, Japan) lysing solution was added to the cells, along with 10 μl of β-mercaptoethanol and 0.6 g of 0.1 mm zirconia/silica beads (Biospec, USA). The mix was run in mini Bead-beater (Biospec, USA) for 45 sec and immediately placed on ice. Chloroform (200 μl) was added to the solution and the lysing mix was inverted gently to mix for 5 min. The mix was centrifuged at 12,000 × g for 15 min at 4℃ and the clear top solution was gently collected into a new tube. Then 500 μl of isopropanol was added and the tubes were gently inverted to mix once again before it was finally incubated on ice for 1 h. After incubation, the lysed mix was centrifuged at 12,000 × g for 10 min at 4℃ and the isopropanol was discarded. Ice-cold 70% ethanol was added to the RNA pellet for gentle washing. After another round of centrifuging at the same speed for 10 min, the ethanol was carefully removed. RNA pellets were dried at room temperature for 3-5 min before reconstitution in 20 μl of RNase-free water. Reconstituted RNA was treated with RNase-free DNase (Promega, USA) to remove residual DNA and then purified using an RNeasy MinElute Cleanup Kit (Qiagen, USA). The quantity and quality of the purified RNA were determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA) with an RNA Integrity number (RIN) value ≥8 [21] and were afterwards aliquoted for separate analysis. A set of total RNA sample triplicates, with common induction, were combined (to avoid biased results), purified, and sequenced at Macrogen Korea using the Illumina Genome Analyzer. Prior to sequencing, total RNA was processed using a Ribo-Zero rRNA removal kit (Epicentre Biotechnologies, USA) to remove the 23S and 16S ribosomal RNAs. Enrichment was assessed using the Agilent 2100 Bioanalyzer and RNA 6000 chip. The remaining mRNA was fragmented into small pieces using divalent cations under elevated temperature and converted into a library of template molecules suitable for subsequent cluster generation using the reagents provided in the Illumina TruSeq RNA Sample Preparation Kit (Illumina Inc., USA), following the manufacturer’s instructions. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers. Second-strand cDNA was then synthesized using DNA polymerase I and RNase H. The cDNA fragments were then processed through an end repair process by the addition of a single “A” base, and then ligation of the adapters. The products were then purified and enriched with PCR to create the final cDNA library. The cDNA fragments were sequenced with HiSeq2000 (Illumina Inc., USA).
- Alignment and Filtering of Sequence Reads
The alignment and sequenced fragment read bioinformatics processing was done at the Korean Bioinformation Center (KOBIC), Daejeon. A total of 40,012,820 and 21,440,720 fragments were generated from the glucose-induced and pyrene-induced RNA samples, respectively. Differential gene and transcript expression analyses of the sequence reads were made with TopHat and Cufflinks. Data were normalized by calculating the “fragments per kilobase per million map reads” (FPKM) for each gene [17] . Briefly, data were treated with low-quality filtering and reads removal before CuffDiff analysis. Exact duplicated reads were removed using Prinseq ver. 0.19.3, with average quality >=Q20. Filtered reads were mapped with reference genomes (GenBank CP000656, CP000657, CP000658, and CP000659) using Bowtie2 ver. 2.0.0, default option. The expression levels of chromosome and plasmids were analyzed separately. Afterwards, differential expression analysis was performed using Cuffdiff (default option) of the Cufflinks 2.0.2 package [27] . Results were manually curated to identify pyrene degradation related transcripts by comparing it with the glucose-induced transcripts. The data acquired have been deposited in the Gene Expression Omnibus database (GEO: GSE44536).
- Gene Expression Analysis by Quantitative Real-Time PCR
Total RNA was isolated and purified as described earlier and the second set (10 μg), from the same template as the first set, was reverse transcribed separately, making six samples in all. The reverse transcription step was carried out using random hexamer primers and the PrimeScript 1st strand cDNA synthesis Kit (Takara, Japan), according to manufacturer’s instructions. Briefly, random hexamers and RNA templates were mixed and denatured at 65℃ for 5 min followed by cooling for 2 min on ice. Then 5× Primescript buffers, RTase, and RNAse inhibitor were added to the cooled template mix and incubated for 1 h at 50℃ before enzyme inactivation at 70℃ for 15 min. Negative controls for reverse transcription were performed to test for the presence of genomic DNA contamination in the RNA samples. Complementary DNA samples were diluted 1.5-fold and relative quantification real-time PCR was carried out in a standard fashion using SYBR Premix Ex-Taq II (Takara Bio, Japan) according to the manufacturer’s directions. The AB-7500 Real-Time PCR System (Applied Biosystems Inc., USA) was employed for the real-time PCR run. The primer3 software [19] and NCBI primer designing tool were used to design primers that would amplify a product of approximately 200 basepairs. Amplicon expected sizes and the absence of nonspecific products were confirmed by analysis of the PCR products in 2% agarose gels in TAE buffer, stained with ethidium bromide and visualized under UV light. PCRs were set up according to the manufacturer’s instructions, and three technical replicates for each sample were included. The PCR volume was 20 μl, consisting of 0.4 μM of each primer ( Table 1 ). Each PCR run included a notemplate control with water instead of cDNA, as well as a RT negative control for each gene. The amplification conditions were 95℃ for 15 sec; and 40 cycles of 95℃ for 15 sec and 60℃ for 1 min. The specificity of the reaction was confirmed by obtaining a melting curve from 55-95℃. The efficiency values of the reactions were automatically calculated by the PCR machine. To analyze the differential expressions, the obtained mRNA levels were compared in the pyrene-induced genes with respect to the glucose-induced genes as a control measure. In all cases, the expression of all genes were quantitated after normalization of their RNA levels to the expression of the rpoB gene coding for the β-subunit of bacterial RNA polymerase (Mflv_5097), as previously described [2] . The fold change was calculated using the relative quantification (2-ΔΔ CT ) method [14]. Statistical analysis was performed using the SPSS ver. 21.0 software package for Windows (SPSS Inc., USA), applying the Student’s t -test. When the differential p -value was less than 0.05, it was regarded as statistically significant.
Sequences of primers used in the analysis of the expression profile ofmcegenes expressed during the substrates metabolism study.
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Sequences of primers used in the analysis of the expression profile of mce genes expressed during the substrates metabolism study.
Results and Discussion
Mammalian cell entry proteins are found in diverse Actinobacteria ( Mycobacterium , Nocardia , Rhodococcus , and Streptomyces ), as components of ATP-binding cassette transporter systems, based on bioinformatics data systems [6 , 15] ; and as an essential virulence property in M. tuberculosis , based on gene cloning experiments [1 , 8] . The analysis of the complete sequence of the Mycobacterium tuberculosis H37Rv genome revealed the presence of four paralogous mce genes, all encoded in an operon structure, consisting of eight genes ( yrbEA , yrbEB , mceA , mceB , mceC , mceD , mceE , and mceF ). We identified seven complete and one incomplete operons from the sequenced RNA bioinformatics analysis, based on the annotated genome sequence of our test strain (GenBank Accession No. CP000656, CP000657, CP000658, and CP000659) ( Table 2 , Fig. 1 ). With unclear information on the functions of these genes other than the reported mycobacterial mammalian cell entry facilitation, rhodococcal cholesterol metabolism, and actinomycetal envelope biosynthesis process/structure maintenance, in this work, we evaluated the gene expression pattern of M. gilvum PYR-GCK during toxic pyrene metabolism as a molecular study on PAH bioremediation. Our results have suggested the possible involvement of the mce operons or genes in the toxic hydrocarbon metabolism. With the different gene expression activities in the various operons, we had cause to believe the various operons had different functions in pyrene metabolism. No gene expression activity was observed in one operon, and minimal activity in some operons, ranging from the expression of one gene to total operon activity in others ( Fig. 1 ). The expression activities of some of these genes were confirmed in a subsequent qRT-PCR analysis ( Fig. 2 ). The eight genes, chosen from the identified gene clusters, were based on their expression activity and orientation in the various operons. Two genes (Mflv_4536 and Mflv_0709) were of similar orientation but different expression activities in the pyrene-induced cells. Likewise, Mflv_2509 and Mflv_0009 were of different orientation and different expression activities ( Figs. 1 and 2 ). Three genes were negatively expressed, whereas five were expressed positively to pyrene induction. Information on the quantity of mce operons in various mycobacterial strains differ between strains, with four and six operons documented in M. tuberculosis and M. smegmatis , respectively [11] . Seven complete mce operons have been identified in our test strain, with yet another cluster of mce genes highly expressed in response to pyrene, but these failed the “complete operon” cut-off as a result of the absence of the first two integral membrane protein genes of the operon ( yrbEA , yrbEB ). These operons with homologous set of genes have been reported to have varying effects, either increasing or decreasing virulence [9 , 20] , but their specific roles are still unclear and under further study. Owing to the varying homology of these operons in the different mycobacterial species, it could not be decided or confirmed about the specific function of each operon, as done with the mce1 and mce4 operons [16 , 22] . Nevertheless, we observed pyrene-induced expression in some genes, irrespective of their gene cluster orientations ( Fig. 1 ). The pathogenic strains equipped with the mce operons have been reported to function under the control of the transcription factor mce1R , but no significant homolog of the transcriptional factor in any of the sought nonpathogenic mycobacterial species ( M. smegmatis and M. gilvum PYR-GCK) were found, except in the pathogenic strains M. tuberculosis , M. bovis , M. avium , M. africanuum , and M. canetti , with nucleotide sequence identities ranging between 81% and 100%.
List of genes present in themcegene clusters inMycobacterium gilvumPYR-GCK.
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List of genes present in the mce gene clusters in Mycobacterium gilvum PYR-GCK.
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The genes were identified based on GenBank genome annotation, and the gene IDs are of Entrez origin. The differential expression values (pyrene/glucose expression ratio) were calculated based on the NGS RNA-seq data results curated using CuffDiff from the Cufflinks 2.0.2 software package. Highly expressed fold change values are emboldened. Differential cut-off values were <0.5 and ≥2 for lowly expressed and highly expressed gene transcripts, respectively.
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The mce gene clusters of Mycobacterium gilvum PYR-GCK. All positively expressed genes as a result of pyrene induction/metabolism are in the shaded gray boxes. Averagely expressed genes are situated in the unshaded boxes. The corresponding gene codes are located at the top of the respective boxes with Entrez gene IDs. Arrowed boxes indicate transcribed genes detected in the qRT-PCR validation assay.
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Transcriptional profile of eight mce genes in Mycobacterium gilvum PYR-GCK. The relative expressions of the gene transcripts were based on pyreneinduced cells, using the glucose-induced cells as control (n ≥ 3 independent bacterial cultures; bars indicate standard deviation).
A recent study of the mce genes in a nonpathogenic soil bacterium, Streptomyces coelicolor , revealed the importance of these genes in enhanced access to soil resources and niche specialization [5] . It was proposed that over time, the genes became specialized into strong invasive tools in the pathogenic actinomycetes strains, shifting their niche from the soil to eukaryotes. PAHs (pyrene inclusive) occur naturally in coal, crude oil, and anthropogenically in fossil fuel products. Owing to the fact that all these compounds have their origin in lignified plant material [12] , the ability of M. gilvum PYR-GCK and some other members of this genus to degrade PAH efficiently may be as a result of the presence of these mce genes in their genome. Nevertheless, further experiments involving a gene-knockout study will be required to confirm their roles in this capacity. In our study, the mce genes were upregulated only in the pyreneinduced mycobacterial cells, and not a single observation was found of high expression in the glucose-induced mycobacterial cells (Supplementary material).
Moreover, based on preceding reports [13 , 16 , 28] , mce genes have been observed expressed during cholesterol and other sterol metabolism in rhodococcal species. The only report in mycobacterial species, made by Wipperman et al . [29] , stated that only two genes in the mce3 operon ( fadE17 - fadE18 ) and the mce3 transcriptional regulator ( mce3R ) were expressed in response to cholesterol metabolism and virulence in M. tuberculosis . In our results, no mce3R homolog was found, and likewise in the most commonly utilized nonpathogenic strain, M. smegmatis . Since we observed the expression of numerous mce genes in response to pyrene metabolism, coupled with the fact that our strain was a nonpathogenic strain, we did not expect the same result as Wipperman et al .’s report. Of great importance, however, was the fact that PAHs are a form of phytosterol, based on their plant origin, but differ from most sterols owing to their different polar properties [24] . As a result of this, we chose to integrate the idea of the mce genes activation in the rhodococcal species due to sterol metabolism, with the same in our mycobacterial strain.
Although no study has been done yet on the effect of PAH degradation on mce genes, save for this, a further study is only advisable to shed more light on the level of activity that can result in a rapid evolution of the nonexisting mce transcription factors mce1R and mce3R ; and the possible conversion of the mce operons to full virulence activity in the nonpathogenic strains. This idea was suggested based on the observation of some other genes highly expressed during the global transcriptomics study, such as carbon starvation protein CstA (Mflv_4322), two copper resistance D domain proteins (Mflv_2826, Mflv_1126), heat shock protein Hsp15 (Mflv_3238), stress responsive alphabeta barrel domain protein (Mflv_4555), two UspA domain proteins (Mflv_0390, Mflv_4854), and two competencerelated genes (Mflv_2662, Mflv_2663 coding for comEA and comEC genes), in response to pyrene induction (Supplementary material).
In conclusion, mammalian cell entry genes are important for host invasiveness in pathogenic mycobacteria. Based on the limited information on the functions of these genes, we sought for its expression in a nonpathogenic bacterium intended for use in toxic substrate bioremediation purposes. Our results showed a high expression of the mce genes during pyrene metabolism compared with its expression during glucose metabolism. We proposed that the pyreneinduced expression was as a result of pyrene, being a phytosterol, emulating the result of sterol metabolism in the rhodococcal species, thereby resulting in a similar expression of the mce genes; and we also repudiate the possibility of these genes functioning in the reported pathogenic invasion, based on the absence of the transcription factors required for this purpose in the M. gilvum PYR-GCK strain.
This work was supported by Hanyang University (ERICA) under the contract number HY-2013-P.
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