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Briefings in Functional Genomics and Proteomics Advance Access originally published online on February 3, 2006
Briefings in Functional Genomics and Proteomics 2006 4(4):355-362; doi:10.1093/bfgp/eli006
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© The Author 2006. Published by Oxford University Press. All rights reserved. For permissions please email: journals.permissions@oxfordjournals.org

Transcriptomics, proteomics and interactomics: unique approaches to track the insights of bioremediation

Om V. Singh and Nagathihalli S. Nagaraj

Corresponding author. Om V. Singh, Park 316, 600 N. Wolfe St, Baltimore, MD 21209, USA. Tel: 410-614-1804; Fax: 410-955-1030; E-mail: osingh1{at}jhmi.edu; ovs11{at}yahoo.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 TRANSCRIPTOMICS
 APPLICATIONS OF DNA MICROARRAY...
 LIMITATIONS OF DNA MICROARRAYS...
 PROTEOMICS
 INTERACTOMICS
 CONCLUSION
 References
 
Microbial mediated bioremediation has a great potential to effectively restore contaminated environment, but the lack of information about factors regulating the growth and metabolism of various microbial communities in polluted environment often limits its implementation. Newly seeded techniques such as transcriptomics, proteomics and interactomics offer remarkable promise as tools to address longstanding questions regarding the molecular mechanisms involved in the control of mineralization pathways. During mineralization, transcript structures and their expression have been studied using high-throughput transcriptomic techniques with microarrays. Generally however, transcripts have no ability to operate any physiological response; rather, they must be translated into proteins with significant functional impact. These proteins can be identified by proteomic techniques using powerful two-dimensional polyacrylamide gel electrophoresis (2-DE). Towards the establishment of functional proteomics, the current advances in mass spectrometry (MS) and protein microarrays play a central role in the proteomics approach. Exploring the differential expression of a wide variety of proteins and screening of the entire genome for proteins that interact with particular mineralization regulatory factors would help us to gain insights into bioremediation.

Keywords: bioremediation, transcriptomics, proteomics, interactomics, pollutants, environmental cleanup


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
TRANSCRIPTOMICS
 APPLICATIONS OF DNA MICROARRAY...
 LIMITATIONS OF DNA MICROARRAYS...
 PROTEOMICS
 INTERACTOMICS
 CONCLUSION
 References
 
Environmental pollutants have become a major global concern, given their undesirable recalcitrant and xenobiotic compounds. A variety of polynuclear aromatic hydrocarbons (PAHs), xenobiotics, chlorinated and nitro-aromatic compounds were depicted to be highly toxic, mutagenic and carcinogenic for living organisms [1, 2]. Nevertheless, as a result of their diversity, versatility and adaptability, a number of microorganisms are considered to be the best candidates among all living organisms to remediate most of the environmental contaminants into the natural biogeochemical cycle. These microorganisms display a remarkable range of contaminant degradable ability [3] that can efficiently restore natural environmental conditions [4, 5]. However, a variety of contaminants have been shown to be unusually recalcitrant, i.e. microorganisms either do not metabolize or transform them into certain other metabolites that again accumulate in the environment [6]. Therefore, it may be more productive to explore new catabolic pathways that might lead towards complete mineralization of these pollutants. One of the reasons, our knowledge of microbial degradation pathways is so incomplete is the immense complexity of microbial physiology that allows response and adaptability to various internal and external stimuli.

An understanding of these molecular and physiological mechanisms in any site-specific bacterium is fundamental for rational development, and the study of RNA and protein expression patterns has made, and continues to make, critical contributions [7–11]. Dramatic advances in the nature and throughput of molecular technologies are making a global gene expression profile possible; genome-wide analysis of DNA (genomics), RNA expression (transcriptomics) and protein expression (proteomics) as well as exploring complexes of protein aggregation such as protein–protein interaction (interactomics) create the opportunity to systemically study the physiological expressions of such organisms.

Gene microarrays and proteomics technologies have recently been refined and made commercially available. The functional genomics approach in biological science has revolutionized research at the forefront of environmental cleanup [12]. This technology was set up to elucidate the role of the many genes of unknown function that have been identified in the numerous genome sequencing projects [13–15]. In response to different environmental conditions, the transcriptome and proteome directly reflect the physiological status of an organism. Moreover, one technology being able to ‘stand-alone’ does not suffice for gaining a comprehensive understanding of any physiological pathway in an organism. This article highlights the approaches of transcriptomics, proteomics and interactomics in brief, in order to comprehend the insights they provide into bioremediation. Attempts are made to interpret some of the areas where transcriptomics and proteomics have been successfully employed together. We raise issues on how to enhance the values of relative data obtained from combining these technologies and applying them towards future studies of active bioremediation.


   TRANSCRIPTOMICS
TOP
 ABSTRACT
 INTRODUCTION
 TRANSCRIPTOMICS
APPLICATIONS OF DNA MICROARRAY...
 LIMITATIONS OF DNA MICROARRAYS...
 PROTEOMICS
 INTERACTOMICS
 CONCLUSION
 References
 
The subset of genes transcribed in any given organism is called the transcriptome, which is a dynamic link between the genome, the proteome and the cellular phenotype. The regulation of gene expression is one of the key processes for adapting to changes in environmental conditions and thus for survival. Transcriptomics describes this process in a genome-wide range. DNA microarrays are an extremely powerful platform in transcriptomics that enable determination of the mRNA expression level of practically every gene of an organism [16–18]. The most challenging issue in microarray experiments is elucidation of data [19]. Often, hundreds of genes may be up- and/or down-regulated in a particular stress condition. In this context, several statistical issues become tremendously complex, including accounting for random and systematic errors and performing poor analysis. A few of the critical issues are classified in Figure 1.


Figure 1
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  		Figure 1: Work flow of gene array analysis. Diagrammatic representation of DNA microarray data analysis and relative limitations under each category of data analysis during data mining.

 

   APPLICATIONS OF DNA MICROARRAY IN BIOREMEDIATION
TOP
 ABSTRACT
 INTRODUCTION
 TRANSCRIPTOMICS
 APPLICATIONS OF DNA MICROARRAY...
LIMITATIONS OF DNA MICROARRAYS...
 PROTEOMICS
 INTERACTOMICS
 CONCLUSION
 References
 
Even with the complete genome sequences of microorganisms with the potential for bioremediation [17, 20–23], studies are not accelerating in a rapid manner. With the completed genome sequences, it is possible to analyse the expression of all genes in each genome under various environmental conditions using whole-genome DNA microarrays [14, 24, 25]. Such genome-wide expression analysis provides important data for identifying regulatory circuits in these organisms [12, 22, 23]. In the past, DNA microarrays have been used to evaluate the physiology of pure environmental cultures [7] and to monitor the catabolic gene expression profile in mixed microbial communities [26]. More than 100 genes were found to be affected by oxygen-limiting conditions when a DNA microarray was used to study changes in mRNA expression levels in Bacillus subtilis grown under anaerobic conditions [27]. Sensitivity may often be a part of the problem in PCR-based cDNA microarrays, since only genes from populations contributing to more than 5% of the community DNA can be detected. Several parameters were evaluated to validate the sensitivity of spotted oligonucleotide DNA microarrays and their applicability for bacterial functional genomics [28]. Optimal parameters were found to be 5'-C6-amino-modified 70-mers printed on CMT-GAPS II substrates at a 40 µM concentration combined with the use of tyramide signal amplification labelling. Based on most of the known genes and pathways involved in biodegradation and metal resistance, a comprehensive 50-mer-based oligonucleotide microarray was developed for effective monitoring of biodegrading populations [9]. This type of DNA microarray was effectively used to analyze naphthalene-amended enrichment, and soil microcosms demonstrated that microflora changed differentially depending on the incubation conditions [29]. A global gene expression analysis revealed the co-regulation of several thus-far-unknown genes during the degradation of alkylbenzenes [11]. Besides this, DNA microarrays have been used to determine bacterial species, in quantitative applications of stress gene analysis of microbial genomes and in genome-wide transcriptional profiles [24, 30].


   LIMITATIONS OF DNA MICROARRAYS IN BIOREMEDIATION
TOP
 ABSTRACT
 INTRODUCTION
 TRANSCRIPTOMICS
 APPLICATIONS OF DNA MICROARRAY...
 LIMITATIONS OF DNA MICROARRAYS...
PROTEOMICS
 INTERACTOMICS
 CONCLUSION
 References
 
The task of identifying an absolute differential expression from an environmental sample using DNA microarrays is a challenging one. Such samples often contain a variety of environmental contaminants that affects the quality of RNA and DNA hybridization [31] and make it difficult to extract undegraded mRNA [32].

The specificity of the extraction method plays a central role and should vary depending on the site of sampling; as there must be sufficient discrimination between probes. One can achieve a discrimination level of a single base pair between probes by imposing a temperature gradient and recording signal intensities [33]. However, it should be borne in mind that this method is less sensitive than PCR [31], which might be crucial in sequence analysis of poor abundance. Also, there is a promising perspective for microarrays in determining the relative abundance of a microorganism bearing a specific functional gene in a complex environment. Over a range of 1–100 ng of target genomic DNA concentration Wu et al. [34] observed a linear relationship between signal intensity and target DNA from pure and mixed culture communities. However, specificity is a key issue, since one needs to distinguish the differences in hybridization signals due to population abundance from those due to sequence divergence.


   PROTEOMICS
TOP
 ABSTRACT
 INTRODUCTION
 TRANSCRIPTOMICS
 APPLICATIONS OF DNA MICROARRAY...
 LIMITATIONS OF DNA MICROARRAYS...
 PROTEOMICS
INTERACTOMICS
 CONCLUSION
 References
 
The terms ‘proteomics’ and ‘proteome’ were introduced in 1995 [35], which is a key post-genomic feature that emerged from the growth of large and complex genome sequencing datasets. Proteomic analysis is particularly vital because the observed phenotype is a direct result of the action of the proteins rather than the genome sequence. Traditionally, this technology is based on highly efficient methods of separation using two-dimensional polyacrylamide gel electrophoresis (2-DE) and modern tools of bioinformatics in conjunction with mass spectrometry (MS) [36]. However, 2-DE has been considered to be a limited approach for very basic and hydrophobic membrane proteins in compartmental proteomics. In bioremediation, the proteome of the membrane proteins is of high interest, specifically in PAH biodegradation, where many alterations in any site specific bacterium affects cell-surface proteins and receptors [37]. The improvements in 2-DE for use in compartmental proteomics have been made by introducing an alternative approach for multidimensional protein identification technology (MudPIT) [38].

MS has revolutionized the environmental proteomics towards the analysis of small molecules to peptides and proteins that has pushed up the sensitivity in protein identification by several orders of magnitude followed by minimizing the process from many hours to a few minutes [39]. The advancement in MS techniques coupled with database searching have played a crucial role in proteomics for protein identification. Matrix-associated laser desorption/ionization time-of-flight MS (MALDI-TOF-MS) is the most commonly used MS approach to identifying proteins of interest excised from 2-DE gels, by generation of peptide-mass fingerprinting [39–41]. Surface-enhanced laser-desorption-ionization MS (SELDI-TOF-MS) is the combination of direct sample fractions on a chip integrated with MALDI-TOF-MS analysis [42, 43]. A variety of differentially expressed signature proteins were analysed using SELDI-TOF-MS in blue mussels (Mytilus edulis) exposed to PAHs and heavy metals [44].

The liquid-chromatography MS (LC-MS) technique has begun to open a new analytical window for direct detection and identification of potential contaminants in water [45]. In addition, the metabolites and degradation products have been taken into account to assess the fate of organic contaminants such as pesticides, surfactants, algal and cynobacterial toxins, disinfection by-products or pharmaceuticals in the environment and during water treatment processes [45].

Tracking the insights of bioremediation using proteomics
The cellular expression of proteins in an organism varies with environmental conditions. The changes in physiological response may occur due to the organism's adaptive responses to different external stimuli, such as the presence of toxic chemicals in the environment. The advent of proteomics has allowed an extensive examination of global changes in the composition or abundance of proteins, as well as identification of key proteins involved in the response of microorganisms in a given physiological state [46, 47]. A number of reports have described sets of proteins that are up- or down-regulated in response to the presence of specific pollutants [46–48].

PAHs, ubiquitous environmental pollutants are extremely important to remove from the environment. In situ and ex situ bioremediation of PAHs has been partially achieved using natural and genetically engineered microorganisms, as reviewed by Samanta et al. [2] and Johnsen et al. [49]. Using a proteomics approach, the physiological changes in an organism during bioremediation provide further insight into bioremediation-related genes and their regulation. An 81-kDa protein similar to catalase–peroxidase that expressed in response to pyrene exposure [50] was recovered using 2-DE from Mycobacterium sp. strain PYR-1. Later, six major proteins were significantly induced and overexpressed on 2-DE when Mycobacterium sp. strain PYR-1 was exposed to phenantherene, dibenzothiophene and pyrene [51]. Several pyrene-specific polypeptides were identified by N-terminal and internal peptide sequencing as putative enzymes. Furthermore, the induction of two ring-hydroxylating dioxygenases, i.e. Pdo1 and Pdo2, in response to pyrene was proposed during pyrene catabolism by Mycobacterium sp. strain 6PY1 [8]. A composite profile for 20 PAH-induced proteins was presented when organism Mycobacterium vanbaabenii PYR-1was grown in the presence of high-molecular-weight PAHs [10].

Progress has been made towards identification of unknown genes and proteins during anaerobic biodegradation of toluene and ethylbenzene. A global expression analysis (DNA microarray and proteomics) was performed using denitrifying bacterium strain EBN1 adapted to anaerobic growth with benzoate, toluene, ethylbenzene and a mixture of toluene and ethylbenzene [11]. Besides various differentially expressed genes and related proteins, the expression of two toluene-related operons (bss and bbs) was specifically induced in toluene-adapted cells. In agreement with the sequential regulation of the ethylbenzene pathway, Ebd proteins were reported to be formed in ethylbenzene-adapted cells but not in acetophenon-adapted cells, while Apc proteins were found to be formed under both conditions [11]. The recent combined approaches of transcriptomics and proteomics have revealed new pathways for aerobic and anaerobic biodegradation of toxic wastes that will certainly pave the way for further identification of new signature proteins.

Other than PAHs, various other environmental contaminants have also been suggested for bioremediation, and related genes and proteins have been investigated via 2-DE. A soil bacterium, Acinetobacter lwoffi K24, that can use aniline as a sole carbon and nitrogen source represents more than 20 protein spots that were selectively induced on aniline-cultured bacteria [48]. Of 20 aniline-induced spots on 2-DE, the identified protein spots belonged to ß-ketoadipate pathway, malate dehydrogenase, putative hydrolase, ABC transporter, a subunit of amino group transfer and HHDD isomerase. In addition, a protein analysis of A. lwoffi K24 cultured with various other aromatic compounds provided information about biodegradation-related genes and their regulation [48]. A 2-DE protein reference map was generated to identify variations in protein expression in Pseudomonas putida KT2440 following exposure to a sublethal inhibitory concentration of phenol [13]. Out of 81 differentially expressed protein spots, 68 proteins increased in quantity, while 13 proteins decreased. Most of the up-regulated proteins were reported to be involved in the following categories: (i) oxidative stress response; (ii) general stress response; (iii) fatty acid biosynthesis; (iv) cell envelope biosynthesis; (v) energy metabolism; (vi) transport of small molecules; (vii) transcription regulation; and (viii) inhibition of cellular division. The down-regulated proteins were found to be involved in nucleotide biosynthesis and cell motility [13]. Apart from phenol, a proteomic analysis revealed the participation of energy- and stress-related proteins in the response of P. putida DOT-T1E to toluene tolerance [52]. Such detailed information is valuable for the development of bacteria with greater solvent and/or contaminant tolerance during the bioremediation process.


   INTERACTOMICS
TOP
 ABSTRACT
 INTRODUCTION
 TRANSCRIPTOMICS
 APPLICATIONS OF DNA MICROARRAY...
 LIMITATIONS OF DNA MICROARRAYS...
 PROTEOMICS
 INTERACTOMICS
CONCLUSION
 References
 
Genome-wide mRNA profiling is unable to provide any information about the activity, arrangement, or final destination of the gene products, the proteins. Various proteomic approaches, on the other hand, can successfully provide the straight answers. It is very rare that any protein molecule acts as a unique pillar during the physiological response in bioremediation process of any contaminant when cellular proteins and various other related cellular expressions are on crest [11, 14, 15, 52]. In general, cellular life is organized through a complex protein interaction network, with many proteins taking part in multicomponent protein aggregation. The detection of these aggregated proteins, i.e. ‘interactomics’, is usually based upon affinity tag/pull down/MS/MS approaches at a proteome level [53–55]. Studies on protein–protein interaction and supermolecular complex formation represent one of the main directions of functional proteomics and/or second generation proteomics.

The growing demands of genomics and proteomics for the analysis of gene and protein function from a global bioremediation perspective are enhancing the need for microarray-based assays enormously. In the past, protein microarray technology has been successfully implicated for the identification, quantification and functional analysis of protein in basic and applied proteome research [56]. Other than the DNA chip, a large variety of protein-microarray-based approaches have already been verified that this technology is capable of filling the gap between transcriptomics and proteomics [57]. However, in bioremediation, microarray-based protein–protein interaction studies still need to make progress to understand the chemotaxis phenomenon of any site specific bacterium towards the environmental contaminant.

Transcriptomics versus proteomics and interactomics for bioremediation
Based on an overall analysis of transcriptomics and proteomics, the comprehensive analysis of whole-genome sequencing is especially helpful to understand bioremediation-relevant microorganisms whose physiology has not yet been studied in detail. Global gene expression using DNA microarray technology, very much depends on the degree of coverage of the cellular mRNA and cellular proteins, whereas the coverage of the whole genome represents all the genes of an organism by definition. Cellular mRNA levels do not display as wide a dynamic range as the encoded proteins [58]. Thus, whole genome arrays are believed to provide a much more comprehensive overview of the actual gene expression pattern than proteomic studies. According to global gene expression studies, both transcriptomics and proteomics support the view that the DNA array technologies record changes in gene expression more completely than the proteomics [11, 14, 59]. Therefore, genomics data is deemed necessary to complement the proteomics approach [60]. However, proteomics would retain its central position in functional transcriptomics and/or genomics. The protein molecules, but not the mRNAs, are the key players in an on-site microbial mineralization reaction; the later are one of the highly unstable transmitters on the path from the genes to the ribosome, but each protein molecule represents the end product of gene expression [11]. Complete protein profiling provides not only information on the individual organism, but also information on the fate and destination of protein molecules inside and outside the cell that can only be discovered via a joint transcriptomics, proteomics and interactomics approach (Figure 2).


Figure 2
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  		Figure 2: Post-genomic technologies using a systematic biology approach to track the insights of bioremediation. Directly extracted DNA from contaminant environmental sites and from organisms will end up on transcriptomics (DNA microarrays). Transcriptomics is now expending towards proteomics and interactomics by extraction of protein from pure culture using 2-DE and protein microarray platforms that will allow us to explore the new molecules of interest during mineralization process.

 

   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 TRANSCRIPTOMICS
 APPLICATIONS OF DNA MICROARRAY...
 LIMITATIONS OF DNA MICROARRAYS...
 PROTEOMICS
 INTERACTOMICS
 CONCLUSION
References
 
Towards a deeper understanding of bioremediation, new techniques in molecular biology—particularly genetic engineering, transcriptomics, proteomics and interactomics offer remarkable promise as tools to study the mechanisms involved in regulation of mineralization pathways. The applications of these techniques are still in their infancy, but the amount of data that is continuously being generated by today's genomics and proteomics technocrats needs to be organized in a stepwise manner within informative databases. The strategies need to be refined in which transcriptomics and proteomics data are combined together in order to understand the mineralization process in a meaningful way. These techniques show great promise in their ability to predict organisms’ metabolism in contaminated environments and to predict the microbial-assisted attenuation of contaminants to accelerate bioremediation.


Key Points

  • Microbial mediated bioremediation of aromatic pollutants offers great potential to effectively restore the natural environment.
  • Emerging ‘omics’ technologies are able to explore new catabolic pathways in bioremediation.
  • Functional proteomics approach in environmental remediation will further pave the way towards cell free bioremediation.

 


   FOOTNOTES
 
Dr Om V. Singh has broad research experience in environmental molecular microbiology, bioremediation and fermentation bioengineering. He is currently engaged in functional proteomics research at department of pediatrics, The Johns Hopkins School of Medicine, Baltimore, MD 21287, USA. Dr Singh also has an active research interest in metabolomics.

Dr Nagathihalli S. Nagaraj is currently working in the broad research aspects of enzymology with inclusion of molecular biology and genomics. His current research interest involves functional genomic and transcriptomics at department of medicine/JG Brown Cancer center, University of Louisville School of Medicine, Louisville, KY 40202, USA.


   References
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 ABSTRACT
 INTRODUCTION
 TRANSCRIPTOMICS
 APPLICATIONS OF DNA MICROARRAY...
 LIMITATIONS OF DNA MICROARRAYS...
 PROTEOMICS
 INTERACTOMICS
 CONCLUSION
 References
 

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