is the major causative agent of Localized Juvenile Periodontitis (LJP), which has been named Localized Aggressive Periodontitis (LAP), as well as one of the microorganisms responsible for adult periodontitis. Periodontitis is the most prevalent chronic inflammatory diseases in humans, and it is a major cause of tooth loss (Slots and Genco, 1984
; Zambon, 1985
; Meyer and Fives-Taylor, 1997
). The genome of the clinical isolate HK1651 has been completely sequenced and is in the final stages of the annotation process (http://www.genome.ou.edu/act.html
). According to the information provided by Dr. Dyer, the genome size of this strain is 2,105,503 bp and contains 2,345 predicted open reading frames. shows some information that was obtained after the initial automated annotation of this bacterial genome was completed. It is interesting to note that about half of the genes of this dental pathogen encode proteins that were classified either as hypothetical, unclassified, or of unknown function. This information demonstrates that much remains to be done to understand the basic biology of this pathogen and the functions it expresses during the interaction with the host’s cells and systems.
Classification of A. actinomycetemcomitans HK1651 predicted genes according to their cellular role.
We and other investigators have been unable to use genomics and bioinformatics to study A. actinomycetemcomitans
because, although closed (http://www.genome.ou.edu/act.html
), the genome of this bacterium has not been completely annotated and the results made public by Dr. Dyer’s research group. However, the information already available at their web site has been instrumental to design classical molecular genetic and molecular biology experiments to test the presence and expression of genes potentially involved in iron acquisition. Analysis of the HK1651 genome with BLAST (Altschul et al., 1997
) revealed that the genome of this strain contains genes encoding functions involved in iron acquisition and iron regulation of gene expression. PCR amplification of genomic DNA isolated from other A . actinomycetemcomitans
strains, that have been used by other investigators to study different biological aspects of this oral pathogen, proved that the afe
genes, which code for periplasmic-binding protein-dependent transport (PBT) systems, are present in all strains tested (). The presence of TonB, which provides the energy required for the transport process, and HgpA, which could be involved in the acquisition of iron from human hemoglobin, were also detected in these strains. This analysis also showed that the HK1651 strain lacks genes required for the expression of siderophore-mediated iron uptake systems, an observation that agrees with the data we have obtained experimentally (unpublished observations). However, it seems that this oral pathogen has the potential to express transport systems that could use siderophore compounds produced by other bacterial species. This possibility could be confirmed once the annotation of the genome is completed and a more complete picture can be drawn with regard to its physiological and virulence properties. Another important observation we have made related to the expression of a transferrin-binding system is that the HK1651 genome contains sequences encoding some components of this system that plays a role in other pathogens such as Haemophilus influenzae
that obtain iron via siderophore-independent iron acquisition systems (Chen et al., 1993
; Sanders et al., 1994
). However, the predicted open reading frame for the transferrin-binding protein A present in the HK1651 genome contains a point mutation that impairs the expression of this activity. This finding was confirmed experimentally in our laboratory and is in agreement with the data reported in a recent publication (Hayashida et al., 2002
Detection of genes related to iron acquisition and gene regulation in different strains of A. actinomycetemcomitans.
We have also used the genomic information already available to confirm the transcriptional expression of the afe
genes, which have been reported to be associated with siderophore-independent iron acquisition in bacteria, in the strains HK1651, SUNY465, and CU1000. In addition, we proved that in the case of HK1651, these loci are polycistronic as predicted by the computer analysis of their nucleotide sequence. RT-PCR experiments also showed that all three strains express the tonB
iron transport gene and the fur
iron regulatory gene. The results obtained with the latter gene are in agreement with our previous report describing the presence and expression of fur
and iron regulated proteins in A. actinomycetemcomitans
Y4 (Graber et al., 1998
) as well as in the SUNY465 strain (unpublished observation).
We have been using the presence and expression of fur
and the genomic data available to initiate the identification and characterization of A. actinomycetemcomitans
genes that belong to the Fur and iron regulons using a genetic approach that to some extent provides genome-wide information. For this purpose, we have used the Fur titration assay (FURTA) (Stojiljkovic et al., 1994
) with a genomic library constructed by cloning ~1 kb Sau
3AI-digested DNA fragments from HK1651 into the Bam
HI site of the multi-copy plasmid vector pUC18 (). This approach resulted in the isolation of several clones that produced red colonies on Fe-containing MacConkey agar plates
because the presence of multiple copies of Fur boxes allows the expression of the reporter fusion fhu::lacZ
under iron-rich conditions. Sequence analysis of several positive clones showed that some of them contain either promoter elements or intergenic regions that appear to have sequences that resemble the Fur consensus binding site found in the promoter regions of bacterial Fur-regulated genes (Calderwood and Mekalanos, 1988
). Computer analysis of the nucleotide sequence of some clones showed that they contain sequences related to genes encoding transferrin and hemoglobin binding proteins, ferritin, formate dehydrogenase, a transmembrane protein, and some proteins with no significant matches in the GenBank database. We are currently confirming these results by subcloning the promoter regions of these genes to test their expression under different conditions, and their ability to bind the A. actinomycetemcomitans
HK1651 Fur repressor protein, which was overexpressed and purified by affinity chromatography. We are also in the process of testing the expression of these putative Fur-regulated genes by using real-time RT-PCR with RNA samples isolated from HK1651 cells cultured in the presence of either heme or inorganic iron. We are also using the available genomic information to generate isogenic derivatives to test the role of some of the loci and genes that are predicted to be involved in iron acquisition by computer analysis of their nucleotide sequences and test their differential expression under different experimental conditions.
Figure 1 Diagram depicting the basis of FURTA used to clone Fur-regulated genes from the A. actinomycetemcomitans HK1651 strain. The grey and black straight arrows represent the fhu-lacZ genetic fusion and the fur gene, respectively, located in the chromosome (more ...)
WHAT IS IN THE FUTURE?
From the standpoint of genomics and bioinformatics, a bright and promising future is ahead for dental microbes since the genomes of several of them were completed or are near completion. The scientific community is already benefiting from bacterial genomes that have been completed and published, like that of S. mutans
UA159 which is providing basic global information such as that related to gene composition, the presence of genetic elements potentially involved in genome evolution, and the mechanisms and elements involved in horizontal gene transfer (Ajdic et al., 2002
). The genomic information has also allowed the in silico
construction of the metabolic pathways of this bacterium (Ajdic et al., 2002
), a practical and convenient result that will aid in the understanding of basic biological aspects of this member of the oral flora. For instance, the analysis derived from the nucleotide sequence of this cariogenic pathogen demonstrates that it contains a large number of membrane transport systems, some of which could be involved in iron acquisition. This analysis also showed that this bacterium lacks some important genes required for the expression of siderophore-mediated iron acquisition systems in other bacteria, an observation that is in agreement with the idea that the main mechanism by which this bacterium acquires iron is either via the direct interaction of host iron-binding proteins, such as lactoferrin or transferrin, or the expression of iron reductase activity that allows the transport of ferrous iron across the bacterial cell wall. Certainly, the S. mutans
UA159 genomic information will be used to construct DNA microarrays (DNA chips) containing probes for each open reading frame found in the genome of this bacterium. Hybridization of these chips with labeled total DNA obtained from other pathogens or clinical isolates of the same pathogen should provide data regarding the genetic components that are common and unique among them, information that should give insights into the genetic variations among bacteria without the burden of sequencing the entire genome of a large number of clinical isolates. On the other hand, hybridization of DNA chips with labeled cDNA probes generated from RNA samples obtained from cells grown under different conditions could be used to study differential gene expression and understand the host-pathogen interactions at the molecular level. This type of approach has been used recently to study the effects of iron limitation (Ochsner et al., 2002
), quorum-sensing and the extracellular environment (Schuster et al., 2003
; Wagner et al., 2003
) in P. aeruginosa
, conditions and systems that are known to play a role in the pathogenesis of significant human infectious diseases.
The sequenced genomes could be used to search for the presence and expression of classical as well as novel global regulators that could play significant roles in differential gene expression, and the consequent adaptation of bacteria to different environments. Small RNAs is one of the latter elements that is being investigated intensively in different bacterial systems after the initial observation that it plays a regulatory role in gene expression in E. coli
), particularly in the expression of genes that are involved in iron metabolism (Masse and Gottesman, 2002
). A similar regulatory mechanism has been found recently in P. aeruginosa
(Wilderman and Vasil, 103rd
General Meeting of the American Society for Microbiology, abstract B404) which was detected after the genome of this bacterium was searched with algorithms designed to find intergenic chromosomal regions that can produce transcripts with structures similar to that described for the E. coli
small RNA RyhB (Masse and Gottesman, 2002
). This, in conjunction with the appropriate bioinformatic tools, will most likely produce novel and important information that should facilitate the analysis and understanding of central cellular process in bacteria.
The examples described above demonstrate how the experimental approaches to study the molecular biology, genetics, physiology, and genome evolution of microbial cells has been revolutionized with the development and application of bioinformatics and genomics. It is evident that the same approach will be applied to study different aspects of the microbial community found in the human oral cavity. With regard to the particular case of A. actinomycetemcomitans
, it is possible to predict that significant advances in basic and clinical research will be attained once the complete genome of this oral pathogen is published. Furthermore, the application of genomics and bioinformatics together with the utilization of the animal model recently described (Schreiner et al., 2003
), in which lesions similar to those found in human patients were observed after inoculating Sprague-Dawley rats with food containing this oral pathogen, should provide a more comprehensive picture of the pathogenesis of the periodontal diseases. For instance, it will be possible to identify and characterize genes that are differentially expressed in the host when compared to laboratory conditions used to maintain and propagate A. actinomycetemcomitans
cells, an approach that will provide information on the factors involved in the host-pathogen interactions that are central to microbial pathogenicity and infectious diseases.
In our particular case, the availability of the complete genome and DNA microarrays will facilitate our work in the identification of genes that belong to the iron and Fur regulon, which are differentially expressed under iron-limiting and iron-rich conditions. Another benefit would be the utilization of comparative genomics, an approach that will facilitate the comparison of the complete genomes of different dental pathogens. The information obtained by these means will allow investigators to understand different processes, such as bacterial evolution and adaptation, species relationships, and gene transfer by different means among members of a complex microbial community at the molecular and genetic levels. These achievements will bring Oral Microbiology to levels of sophistication and advancement comparable to those already achieved with other bacterial pathogens that have different targets in the human host.