318–321 Nucleic Acids Research, 2002, Vol. 30, No. 1 © 2002 Oxford University Press
AraC-XylS database: a family of positive transcriptional
regulators in bacteria
Raquel Tobes and Juan L. Ramos*
Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín,
Consejo Superior de Investigaciones Científicas, 18008 Granada, Spain
Received July 27, 2001; Revised and Accepted October 2, 2001
ABSTRACT
The AraC-XylS database contains information about
a family of positive transcriptional regulators broadly
distributed in bacteria. This specific database
focuses on protein sequences and on the biological
and functional features of each of the proteins that
belong to this family. Each entry provides information
on the protein itself, the annotated protein sequence
and, when the crystal is available, a comprehensive
representation of its three-dimensional structure.
The organization of the database is based on an
exhaustive analysis of the scientific literature. The
data are interconnected and linked with other data-
bases. Multiple alignments of the members of the
family, an extensive collection of references and a
tutorial about the family provide additional information.
The AraC-XylS database is accessible on the World
Wide Web at http://www.AraC-XylS.org.
INTRODUCTION
During the last few years, notable developments in molecular
and computational biology have yielded a huge amount of data
related to protein and gene sequences. This raises the need to
organize and represent the data in a manner that facilitates
access to the information from different research fields. In
addition, there is an ever increasing interest in obtaining
comprehensive, structured knowledge about the function of
biological molecules. Specific databases that gather information
about a particular protein family can undoubtedly help increase
knowledge in a research area where there is an obvious
imbalance between the data and knowledge. Furthermore, an
insight into a protein family broadly distributed among many
organisms opens the possibility of designing comparative
studies and further experiments that could lead to the unequivocal
assignment of functions to different domains of these proteins.
New technologies, especially those related to DNA arrays,
have emphasized the importance of transcriptional regulation
in protein expression. Consequently, one of the next major
objectives of post-genomics is to clarify gene regulatory
networks. In bacteria, the adaptation to a changing environment is
essentially mediated by systems that regulate gene expression;
hence, it is especially important to trace the interconnections
between these networks. Most of the available databases
dealing with bacteria [RegulonDB (1) and PromEC (2) of
Escherichia coli, DBTS (3) for Bacillus subtilis and TIGR
CMR (4)] focus on a specific organism. In contrast, databases
for a specific family of bacterial transcriptional regulators are
not available.
We have developed a specific database for the AraC-XylS
family of regulators. The profile that defines the AraC-XylS
family of transcriptional regulators was generated by Gallegos
et al. (5), based on previous studies by Ramos et al. (6) and
Gallegos et al. (7), who had identified a segment of 99 conserved
amino acids at the C-terminal end of the first 27 proteins recognized
as members of the family. With the new profile the family was
extended to almost 100 members. Analyses of a protein
sequence with the help of a matrix defining the family profile
assigned a value to the query sequence. The value given by the
matrix to each of the family members was between 30.74 and
12.52, with small variations between two consecutive proteins
identified as members. However, a difference of at least 4.7 points
was observed between the last member of the family and the
closest value of a protein not identified as a member of the
family. Gallegos et al. (5) proposed that a protein belongs to
the AraC-XylS family if the value after analysis with the
PS01124 profile defined in PROSITE is above 12.52. Most
members of the AraC-XylS proteins are positive transcriptional
regulators involved in the control of many important processes
related to carbon metabolism, stress responses and pathogenesis
(reviewed in 5). The AraC-XylS database is built around a
family of proteins that are broadly distributed in Gram-negative
and Gram-positive bacteria. Members of the AraC-XylS
family belong to an ancient lineage, as deduced from the great
evolutionary distance between prokaryotes with AraC-XylS
proteins, and from the existence of marked differences in their
G+C content. The proteins belonging to this family have been
found in 47 different genera and 84 different microbial species
so far.
In broad terms, proteins of the family consist of two
domains, a non-conserved one, which seems to be involved in
effector/signal recognition and dimerization, and a conserved
one, characterized by significant amino acid sequence
homology extending over a 100 residue stretch containing the
DNA-binding domain of the family members (5). The first
high-resolution structures for the DNA-binding domain of
AraC-XylS family proteins have recently become available. X-ray
diffraction analyses of the co-crystals of MarA, a 109 amino
acid protein that contains only the DNA-binding domain with
its mar promoter binding site, shows that MarA binds as a
*To whom correspondence should be addressed. Tel: +34 958 121011; Fax: +34 958 129600; Email: jlramos@eez.csic.es

Nucleic Acids Research, 2002, Vol. 30, No. 1 319
monomer with two helix–turn–helix (HTH) motifs inserted in
two adjacent major groove segments (8). The recognition
helices of the HTH motifs are held in place by a rigid 27 Å long
α-helix, which is shorter than the 34 Å that separate the two
major grooves that induce a 35° bend in the DNA. In addition to
phosphate backbone contacts, the most important determinants of
binding appear to be H-bonds made by both HTH motifs with
several bases of the DNA.
The co-crystal structure of Rob, another member of the
family, and its target micF promoter have been resolved.
Because the two HTH motifs of Rob are superimposable on the
MarA structure (9), this way of recognizing DNA may be
common to all members of the AraC-XylS family of regulators.
Furthermore, most AraC-XylS members may share a common
mechanism to enhance transcription, i.e. they may facilitate
recruitment of RNA polymerase and isomerization to open
complexes, as demonstrated for AraC and SoxS proteins (10–12).
The α-CTD end of RNA polymerase is required at several
promoters that are activated by AraC-XylS family members,
such as AraC, MelR, MarA, RhaS, Rob, XylS and SoxS
(13,14). However, direct interaction with RNA polymerase, as
opposed to interaction with DNA UP elements or factors such
as CRP, has not been demonstrated conclusively.
Taking into account the functions that the AraC-XylS family
of transcriptional proteins regulates, it is reasonable to consider
these proteins as possible targets for designing a broad
spectrum of applications in the fields of biotechnology and
medicine. Thus, their participation in the regulation of the use
of C-sources and in the metabolism of recalcitrant pollutants
establishes a relationship between this family and the catabolism
of biogenic and xenobiotic compounds. The involvement of
AraC-XylS proteins in antibiotic resistance and in pathogenesis of
common diseases such as enterocolitis, respiratory and urinary
infections, emphasizes the importance of these regulators as
targets for new drugs. In parallel, their role in abiotic stress
responses establishes a link with the ecological adaptation of
microbes to changing environmental conditions (15).
DATABASE OVERVIEW
The AraC-XylS database includes all the proteins that fulfil
two requirements: (i) the value obtained after matching with
the PROSITE profile PS01124 must be above the threshold
(12.52) set to define the family; and (ii) the protein must be
included in SWISS-PROT or TrEMBL databases. One entry is
considered different to another if its sequence is different or if
it is present in a different organism. With these criteria, the
database includes 280 entries at the time of writing.
The database is structured to cover general knowledge and
sequence information.
Knowledge database
The information on each protein is organized into 25 fields that
contain data related to the protein, its corresponding gene, the
genes that it regulates, its function and pathogenicity, the
three-dimensional structure, published mutation studies and
bibliographic references.
ID Identification number in the database.
NA Name.
AN Accession number in SWISS-PROT or TrEMBL.
OR Organism.
DA Annotation update.
GE Name of the gene encoding the family member.
OP Orientation and position in chromosome or plasmid.
PP Name, location and characteristics of the promoter of the
gene encoding the family member.
RE Regulation of the expression of the regulatory protein.
TR Type of regulation: activation or repression.
GR Genes regulated by the family member and their functions.
PR Name, location and characteristics of the promoters of the
regulated genes.
RG Other proteins or regulating mechanisms that also participate
in the regulation of the genes regulated by the family
member.
IN Interaction with RNA polymerase subunits.
EF Effector: molecules and conditions (temperature, pH, etc.)
that trigger the regulatory protein.
FU Function: overview of the bacterial function in which the
regulator is involved.
PA Involvement of the family member in pathogenicity.
MU Published data about mutations.
ST Three-dimensional structure.
DO Functions associated with the different protein domains.
OL Oligomerization.
VA Value obtained with the PROSITE PS01124 profile.
SI Similarity with other members of the family: BLAST
restricted to this family.
CO Comments.
RF References.
The knowledge data were gathered in a database management
system to help sustain the database. The basic sources of
information that constitute this knowledge database were
bibliographic references that were specifically cited for each
protein and information available in databases (SWISS-PROT,
TrEMBL, PROSITE, INTERPRO, bacterial databases, etc.). A
direct link to MEDLINE makes it possible to immediately
retrieve published articles that are related to a given member of
the family. In addition, direct links to SWISS-PROT and
TrEMBL make it possible to retrieve information that has been
specifically stored in these databases.
Sequence database
Each entry has four sequences associated with it.
1. The complete protein sequence.
2. The conserved region. The length of this region is approxi-
mately 100 amino acid residues and the limits are defined
by alignment with the profile. It contains the DNA binding
domain and critical features essential for transcription
activation.
3. The C-terminal region with respect to the conserved region.
This fragment is often short because the conserved region
is usually located at the C-terminal portion of the protein.
4. The N-terminal region with respect to the conserved
region. This region usually contains the dimerization, the
effector binding pocket and the signal transmission
domains.
We provide the sequences as independent files and as global
files available at www.AraC-XylS.org. Each global file
contains one type of associated sequence for all the entries.
This format facilitates the use of the sequences for theoretical
studies.

320 Nucleic Acids Research, 2002, Vol. 30, No. 1
In AraC-XylS proteins, the different domains can be totally
independent from a functional and evolutionary point of view.
Consequently, we detected some members of the family that
bear, in addition to the AraC-XylS conserved domain, a region
corresponding to a receiver module of a response regulator of
bacterial sensory transduction systems. We have adapted the
sequence database to the modular structure of the proteins to
facilitate specific theoretical studies to achieve insights on the
modular evolution of the different regulators. A multiple alignment
(CLUSTAL) is given for the conserved domain of all the database
proteins (www.AraC-XylS.org). In the alignment each sequence
can be ‘clicked on’ to display information about this protein entry.
GRAPHICAL USER INTERFACE
We have designed a web interface that provides an open,
interconnected representation of the data included in the AraC-XylS
database. Access to the proteins in the database is facilitated by
three different indexes: the alphabetical order index, the organism
index and the numerical index. The multiple alignment of the
conserved domain sequences is also a useful index to access
protein information. Finally, a search engine to find a particular
protein by its name has also been included.
As an introduction to the database, we provide general tutorial
information about the features of this protein family for
researchers from different scientific fields who may consult the
AraC-Xyls family web page.
The data are interconnected and each protein entry has both
external and intra-database links. Within the latter are links
that join each protein entry with its associated sequences, with
the most similar protein (BLAST) of the family, and with the
complete results of a BLAST comparison with all family
members. Each entry also includes a link with a representation
of the crystal structure if the molecule has been crystallized.
We provide a didactic description of the crystal to provide a
rapid, comprehensive view of the three-dimensional structure
of the molecule. In the three-dimensional representation of the
molecule, we have added labels with explanations to indicate
the different domains, the most critical residues for inter-
actions, the residues related to mutations and other information
of interest. This didactic presentation enables scientists and
even non-expert scientists to rapidly grasp the information
available on the molecule.
Many fields of each protein entry are hyperlinked to related
information provided in other databases. Thus, the AN field
(accession number) is linked to the corresponding entry in
SWISS-PROT or TrEMBL, the field OR (organism) is linked
to the database of the genome of the microorganism if
available, the ST field (three-dimensional structure) is linked
to the PDB entry and the VA field (profile value) is linked to
the corresponding profile in PROSITE. Each reference is
linked to its PubMed abstract.
UTILITY AND FUTURE PERSPECTIVES
The AraC-XylS database supplies information about a specific
family of transcriptional regulators and helps maintain the
right balance between data and knowledge. We are confident
that this database will be a valuable tool for experimental
researchers and for the design of theoretical studies by
bioinformaticians.
The increasing number and diversity of protein sequences
require methods to predict details regarding functions. The
broad distribution of proteins of the AraC-XylS family makes
it possible to hypothesize the function of new proteins on the
basis of sequence similarity studies.
We expect the number of new entries in this database to
increase in parallel with the analysis of available genomes.
Escherichia coli contains 38 proteins of the AraC-XylS family.
In the unfinished (∼6 Mb) genome of Pseudomonas putida
KT2440 (16), we have found 41 chromosomal open reading
frames corresponding to proteins that could be included in the
AraC-XylS protein family. Note that only two out of these
41 proteins have been deposited previously in the SWISS-PROT
database, and therefore only these two proteins are recognized
at present as members of the AraC-XylS family. If we assume
the number of regulators of the AraC-XylS family to be
approximately 40, and if we take into account the number of
microorganisms whose genomes are currently being
sequenced, together with the wide distribution of members of
this family in different genera of Gram-positive and Gram-
negative bacteria, a considerable increase in the number of
AraC-XylS proteins can be expected in the very near future.
We will update the database by frequent additions of new
family members. We anticipate an increase in the number of
orthologs of each protein family, and a new organizational
structure will then be incorporated to the database.
The body of information on the promoters that are recognized
by the AraC-XylS proteins is restricted to about 20 members of
the family. We expect to enlarge the present database with the
DNA sequences recognized by each protein, and are currently
searching for the characteristic pattern that defines sequences
recognized by these regulators. We expect to find the distin-
guishing features for the promoters that make it possible to
predict the location of the regulator binding site with respect to
that of the RNA polymerase. This information will make it
possible to predict interactions with the different subunits of
RNA polymerase.
We also envisage a project in which thorough reviews of the
most important proteins of the family are added to the
database. These reviews will include abundant graphical
information, schematic drawings and structured knowledge.
ACKNOWLEDGEMENTS
This work was supported by a grant from the Comisión
Interministerial de Ciencia y Tecnología BIO 2000-0964 and a
grant from the European Commission QLTR-2000-00170. We
thank Carmen Lorente and Karen Shashok for checking the
English of the manuscript.
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