the cleanup of pollutants from soil and water is becoming an important task as human activities produce large amounts of chemical compounds ( up to tens of millions ) that are frequently released into the environment .
microbial populations play an important role in this process as they have acquired the ability to metabolise these compounds using them as carbon and energy sources ( 1 ) .
decades of biochemical studies have produced a considerable wealth of knowledge about this unique metabolism , and this has recently started to be categorized and stored in structured databases .
examples of these are the university of minnesota biocatalysis / biodegradation database ( um - bbd ) ( 2 ) and the metarouter ( 3 ) .
although this formalization has permitted the development of useful applications , such as a predictor of chemical biodegradability ( 4,5 ) , the absence of information at the sequence level of the proteins required for the biodegradation process has limited further systematic studies .
questions such as the molecular basis of enzyme specificity , their catalytic mechanism , the evolutionary origin of this novel metabolism , or the spreading of such activities in the environment , are extremely difficult to address in the absence of accurate sequence and genetic information .
current biodegradation databases such as um - bbd or metarouter link reactions to protein sequences in databases that have been annotated with the corresponding enzyme commission code ( ec code ) .
for instance , many reactions share the same ec code although they use distinct substrates and generate different products . for example
, the ec code 1.13.11.- is shared by more than 40 reactions , including a large number of dioxygenases with different substrates such as styrene or pyrene .
given that the uniprot database contains more than 200 sequences annotated with this particular ec code ( 1.13.11.- ) , it is impossible to use the information provided in the biodegradation databases to accurately associate the protein that actually carries out a specific biochemical reaction . in some cases ,
the databases provide links to the kyoto encyclopedia of genes and genomes ( kegg ) , but unfortunately kegg associations between proteins and reactions , are often inaccurate , and direct to proteins electronically annotated in the course of complete genome sequencing projects . as an alternative method , um - bbd allows searches with the enzymatic activity name as query directly in genbank , but usually it is difficult to identify if the association between the protein and the biochemical activity is based on a published experimental characterization or whether it was inferred by computational methods . as an alternative to these approximations , bionemo provides accurate associations between proteins and reactions based on customized database searches , extensive literature mining and manual curation .
bionemo offers an additional level of information by adding molecular data on the transcriptional organization of biodegradation genes and their regulation .
this molecular information is placed in the proper metabolic context and thus enables the exploration of particular features that have traditionally been difficult and tedious to address . in summary
, bionemo complements the currently available information on biodegradation that focus in the biochemical aspects by adding additional layers of molecular information at the level of proteins and gene control .
these are linked to the biochemical reactions defined as the transformation of substrates into products .
these transcription units are regulated by one or more regulatory complex , comprising of a transcription factor , one or more dna - binding sites and generally an inducer compound .
all entities in the database are linked to the corresponding external databases , such as genbank or the ncbi taxonomy database ( 6 ) , uniprot ( 7 ) , pfam ( 8) and um - bbd ( 2 ) ( figure 1 ) . 

all the biological entities contained in bionemo are shown as white boxes , with black arrows indicating their relationships .
entities are sorted in two vertical axes , the one on the left showing entities related to regulation and the one on the right showing entities related to metabolism .
orange boxes are external databases to which the different entities are connected , as indicated by dashed arrows
all the biological entities contained in bionemo are shown as white boxes , with black arrows indicating their relationships .
entities are sorted in two vertical axes , the one on the left showing entities related to regulation and the one on the right showing entities related to metabolism .
orange boxes are external databases to which the different entities are connected , as indicated by dashed arrows . the first step in the manual curation process of bionemo consisted of obtaining all the pathways and reactions from the university of minnesota biocatalysis / biodegradation database
, we manually searched sequence databases , embl ( 9 ) and genbank ( 6 ) using as many query terms as was possible , including ec number , reaction name , enzyme name or original publication as reported in the um - bbd .
the resulting entries were then screened manually and the sequences were associated to a reaction only if we could find a journal article describing that particular enzymatic activity for the gene using the same name as in the sequence database , and that refers to the same organism , including the strain name .
it is important to stress that all the connections between protein complexes and biochemical reactions were established manually and were always based on the information contained in the scientific literature .
no sequence similarity information or other computational function prediction methods were used , nor were articles using those methods considered as evidence . in total
, we obtained accurate and reliable protein associations for 324 reactions of the original set of 945 ( table 1 ) .
a similar process was followed to retrieve genes and transcriptional units . in the case of regulatory proteins
, both their binding sites and the regulatory actions on regulated promoters were also obtained from the literature , starting from the reviews by tropel and van der meer ( 10 ) and diaz and prieto ( 11 ) , and following references therein . 

table 1.summary of the bionemo information contentpathways145reactions945  with associated complex324enzymatic complexes537proteins1107microbial species234transcription units212transcription factors90effectors90tf dna - binding sites128promoters100 summary of the bionemo information content
the main way to access bionemo is via its web site ( http://bionemo.bioinfo.cnio.es ) . while a full list of each entity type is provided to browse the bionemo content , the web server implements a simple search interface that allows simultaneously querying all the biological entities described above .
the results are shown categorized by tabs representing classes containing the entity types ( reactions , complexes , etc . ) . from the results page ,
the user can easily access entity - specific pages , in which all information available is summarized , including the biochemical , sequence and regulatory data . links to external databases are provided , including the original um - bbd metabolic information , genbank and uniprot , the ncbi taxonomy database for microbial species , and the pubmeb references to the original information sources . navigating the database is simple . to illustrate this
benzoate. the results page returns all the available entities in bionemo with a partial match to that query , categorized in four classes : 54 reactions , 22 pathways , 22 enzymatic complexes and 24 compounds . without navigating away from this page , the user can , for instance , access all the protein complexes that perform reactions related to benzoate or any derivative , and the gene that encode them .
the user can also access the reactions in which any of the 24 compounds take part , either as product or substrate , and the 22 pathways they belong to .
should the user select the benzoate pathway ( benz2 ) , a graphical representation is shown ( figure 2 ) that is also conveniently clickable .
transcriptional regulation , when available , can be shown on top of the pathway if required . as always ,
a link to the original source in um - bbd and connections to alternative pathways are also shown in this page , and we have made clickable all the compounds , reactions and regulators included in the graph .
for instance , selecting the halobenzoate 12 dioxygenase link will take the user to the page for that reaction .
this page includes a link to um - bbd for a full description of the reaction .
bionemo provides the list of all complexes capable to perform that reaction along with links to the articles we used to establish that association , and links to additional relevant articles describing either the reaction or the enzymatic complexes .
, links to several external databases are offered , including genbank for dna sequences and genepep and/or uniprot for the protein sequences .
in addition , if the protein sequence contains domains included in pfam , these are shown and properly connected to pfam . clicking
the domain name will retrieve the list of all proteins in bionemo containing that domain . finally ,
if available , links to resolved 3d structures in the pdb are also provided . 

figure 2.example of information retrieval using the bionemo web site . a standard case of use could start by browsing a pathway and its regulatory elements . from there
, the user can navigate to different views . then , by clicking on a reaction name , the user will be taken to a new page showing all the enzymatic complexes able to perform that reaction , and from there to one describing proteins , their coding genes , their transcriptional organization and the transcription factors involved .
a standard case of use could start by browsing a pathway and its regulatory elements .
, by clicking on a reaction name , the user will be taken to a new page showing all the enzymatic complexes able to perform that reaction , and from there to one describing proteins , their coding genes , their transcriptional organization and the transcription factors involved . regarding the transcriptional information ,
the user can continue the exploration of the system by using the corresponding link . in this case
, the gene cbda contains information about transcriptional regulation indicated by the transcriptional unit ( the cbdabc operon ) and by the transcription factor that regulates the gene : cbds ( 12 ) .
the transcription unit page contains a scaled schematic representation of the operon and its promoter region , including the coordinates of the transcription factor binding sites if known , the type of promoter that drives its expression , the type of regulation ( activation or repression ) and the molecules that can serve as effectors of the system .
in the cbds transcription factor page , in addition to the standard gene information ( links to external sequence databases , domain structure and relevant articles ) , the list of target transcription units and the molecules that act as effectors are also included .
as seen in this example , the bionemo web site has been designed to facilitate browsing and fast access to relevant information .
bionemo has been intentionally designed to avoid redundancy with related databases , and we always provide external links to all of them . for advanced users , bionemo can be downloaded as a sql dump and installed locally .
a perl api ( application program interface ) is also provided in order to facilitate programmatic access and to avoid the use of complex sql queries . the local installation process and
information contained in bionemo can be useful for cloning , primer design for pcr amplification and design of directed mutations , among other applications . for the annotation of genomes and metagenomic libraries ,
bionemo is a better - suited alternative to the non - specialized sequence databases . in the field of sequence analysis
, bionemo allows the generation of sequence alignments enriched in experimentally characterized proteins , which can be useful for finding residues related with substrate specificity or catalytic mechanism . beyond these applications ,
we envision a wide range of emerging fields in which bionemo can be useful , such as systems biology of biodegradation .
this field has started to produce relevant results ( 13 ) and can be enhanced by the availability of protein sequences and regulatory information .
this information will allow researchers to dive deeper in the functional properties of the enzymes and their particular organization .
but proteins are only a small part of the whole picture , where transcriptional events are the driving forces of evolution .
therefore , making accessible the genetic organization of biodegradation enzymes will allow a better characterization of their ability to be transferred among different organisms , a process that has been reported as key in the biogeochemical cycles of toxic compounds ( 14,15 ) . from an engineering point of view
, bionemo can also help in the designing of new pathways and regulatory circuits , in which sequence information and protein  protein and protein  dna interactions are required for the proper design of useful and independent systems ( 1618 ) . in this context
, bionemo will be useful to assist the design of new transcription factors with the desired specificity and with a predictable behaviour ( 19 ) . in order to expand the reaction coverage of bionemo
, we are currently implementing novel tools that will that will help us keep the result up to date .
our laboratory is currently working on a set of text mining tools that will scan the literature for precise biodegradation information that will be added to bionemo under expert curation .
these tools will be able to mine relevant information : not only the database entries associations but also new biochemical reactions , the species involved and particular environment in which they take place . to summarize
, bionemo is a unique resource that contains several layers of integrated information and enables rational and comprehensive access to the biochemical pathways , protein complexes , and genetic regulation of biodegradation . to the best of our knowledge
we therefore expect this tool to be used in a broad spectrum of scientific and applied research .
this work is funded by the emergence eu grants , the psysmo project within the sysmo framework , and the fundacin banco bilbao vizcaya argentaria ( fbbva - biocon-3 ) .
i.c . is a member of the ramn y cajal program of the spanish ministry of education and science .
 bionemo ( http://bionemo.bioinfo.cnio.es ) stores manually curated information about proteins and genes directly implicated in the biodegradation metabolism . 
 when possible , the database includes information on sequence , domains and structures for proteins ; and sequence , regulatory elements and transcription units for genes . 
 thus , bionemo is a unique resource that complements other biodegradation databases such as the university of minessota biocatalysis / biodegradation database , or metarouter , which focus more on the biochemical aspects of biodegradation than in the nature of the biomolecules carrying out the reactions . 
 bionemo has been built by manually associating sequences database entries to biodegradation reactions , using the information extracted from published articles . 
 information on transcription units and their regulation was also extracted from the literature for biodegradation genes , and linked to the underlying biochemical network . in its current version , bionemo contains sequence information for 324 reactions and transcription regulation information for more than 100 promoters and 100 transcription factors . 
 the information in the bionemo database is available via a web server and the full database is also downloadable as a postgressql dump . to facilitate the programmatic use of the information contained in the database , an object - oriented perl api is also provided 
 . 