Description of the genome.
The genome of P. gingivalis
is 2,343,479 bp, with an average G+C content of 48.3% (Fig. ). There are four ribosomal operons (5S-23S-tRNAAla
-16S) and 2 structural RNA genes, as well as 53 tRNA genes with specificity for all 20 amino acids. A total of 1,990 ORFs could be identified in the genome (13
). Of these, 1,075 (54%) could be assigned to biological role categories (54
), 184 (9.2%) were conserved hypothetical proteins or conserved domain proteins, 208 (10.5%) were of unknown function, and 523 (26.3%) encoded hypothetical proteins. More than 85% of the genome encodes ORFs.
FIG. 1. Circular representation of the P. gingivalis genome. The outer circle shows the predicted coding regions on the plus strand color-coded by role categories as follows: violet, amino acid biosynthesis; light blue, biosynthesis of cofactors, prosthetic groups, (more ...)
Repetitive elements occupy ca. 6% of the P. gingivalis
genome and fall into two major classes: DNA repeats and transposable elements. The DNA repeats include uninterrupted direct repeats (Table ), and a subclass of dispersed repeats known as clustered regularly interspaced short palindromic repeats (CRISPRs) (Table ). Strain W83 does not appear to contain other classes of dispersed repetitive DNA sequence elements such as ERIC and REP elements. The transposable elements include insertion sequence (IS) elements and miniature inverted-repeat transposable elements (MITEs), which are summarized in Table , and large stretches of genes that resemble remnants of conjugable and mobilizable transposons based on sequence similarity to elements previously described in Bacteroides
). The locations of transposon-associated genes are shown in Fig. . Although there are 96 complete or partial copies of IS elements and MITEs present in strain W83 that occupy more than 94 kb of the genome, the transposable elements are rarely found in a functional gene. Instead, these elements have inserted almost exclusively into intergenic regions and other copies of transposable elements, except for one insertion into a putative outer membrane protein (PG0176/PG0178) that is intact in at least four other strains of P. gingivalis
(accession numbers AB069977
). Analysis of the IS elements reveals two possible chromosomal inversions that most likely arose by homologous recombination between identical copies of elements at widely separated insertion sites. These potential DNA rearrangements are revealed by inspection of both the duplicated target site sequences (direct repeats) that flank some of the IS elements and a transposon gene disrupted by one IS insertion. The sites of these putative inversions are shown in Fig. . In one case, 821 kb (35%) of the chromosome appears to be inverted between one copy of ISPg2
(PG1746) 512 kb before the origin and another copy of ISPg2
(PG0277) 309 kb after the origin (red dots). In the second case, 103 kb (4.4%) of the chromosome appears to be inverted between two copies of ISPg4
, which are located on opposite sides of the origin (PG2194 and PG0050) (green dots). Since inversions about the origin do not invert the direction of transcription relative to replication of genes on the segment, such inversions may be selectively neutral. It will be interesting to determine whether strain W83 and other strains of P. gingivalis
share a common genetic structure, or whether the proposed chromosomal inversions are relatively recent events.
Major families of contiguous repeats and their copy numbers in the genome
There are 21 areas of the genome that display an atypical nucleotide composition identified by χ2
) and that also correspond to regions of higher or lower G+C content than the rest of the genome. The areas range in size from 11 to 68 kb and range in G+C content from 29.4 to 61.6%. A variety of genes that could possibly have been acquired by this bacterium through lateral gene transfer are encoded in these regions. The genes include three restriction system proteins (PG0971, most similar to Anabaena
sp. strain PCC 7120; PG0968, most similar to Anabaena
sp. strain PCC 7120; and PG1469 most similar to Agrobacterium tumefaciens
); hemagglutinin proteins B and C (HagB, PG1972, P. gingivalis
specific; and HagC, PG1975, P. gingivalis
specific); many capsular biosynthesis proteins, 20 transposase genes, two large mobile elements (PG1473 to PG1480, resembles only a conjugative element of Bacteroides thetaiotaomicron
; and PG0868 to PG0875, whose sequence and gene organization most closely resembles the antibiotic-resistant mobilizable transposon Tn4555
from Bacteroides fragilis
); and a thiamine biosynthesis operon (PG2107 to PG2111, which is most similar to the thiamine biosynthesis operon of Escherichia coli
). These atypical regions in the P. gingivalis
genome also encode many hypothetical and conserved hypothetical proteins, which undoubtedly contribute to the unique biology of this organism.
Comparison of the predicted proteome of P. gingivalis with that of other completely sequenced genomes confirms the close relationship of P. gingivalis to other members of the CFB, including B. fragilis and B. thetaiotaomicron. Outside of the CFB phyla, the genome most similar to that of P. gingivalis is the Chlorobium tepidum genome, supporting previous phylogenetic studies that indicated the chlorobia and CFB phyla are related, albeit distantly. The proteomes most similar to that of P. gingivalis (in terms of the number of proteins with the best scoring matches) were those of B. thetaiotaomicron and B. fragilis with 572 and 437 best-scoring matches (P < 10−5), respectively.
A total of 332 genes were identified as being putatively duplicated in the P. gingivalis
lineage. These duplicated genes are likely an indication that there is some selective evolutionary advantage to retaining these genes in the genome. Among these genes are 10 that encode DNA-binding histone-like proteins that have a distinctive domain architecture compared to HU and related histone-like proteins. These DNA-binding proteins have been designated a superfamily (i.e., a set of proteins that share a given domain architecture; TIGRFAMs family TIGR01201). Outside of P. gingivalis
, the single known example of a DNA-binding histone-like protein is found in the gut bacterium B. fragilis
. All members of this superfamily are distantly related to the bacterial DNA-binding protein HU family (Pfam family PF00216, five of which are also found in the P. gingivalis
genome) but differ in architecture, sharing both an N-terminal extension and a glycine-rich C terminus. HU has been shown, among other DNA-binding functions, to assist the unwinding of ori
C DNA by the DNA replication initiation protein DnaA (4
). Interestingly, all 10 members of the TIGR01201 family in P. gingivalis
have direct repeats upstream of their genes that may act as binding sites for the DNA-binding proteins that are encoded by the nearby gene and perhaps regulate their own expression. Alternatively, the repeats may also coordinate expression of the other chromosomal genes that they flank.
Metabolism and transport.
The microbial species that exist in supragingival plaque of the oral cavity are exposed to the host's dietary intake, and many of these bacteria, including the oral streptococci, ferment carbohydrates to acidic end products such as lactic acid for the purpose of energy production. On the other hand, anaerobic species in the subgingival plaque are exposed to crevicular fluid and to the host tissue proteins (61
). The availability of the complete genome sequence of P. gingivalis
W83 allowed for an analysis of the physiological potential of this species. Based on this analysis, the range of transport capabilities and metabolic pathways that could be identified is presented in Fig. .
FIG.2. Overview of metabolism and transport in P. gingivalis. Primary substrates for energy metabolism are capitalized and underlined. End products of fermentation are highlighted by yellow boxes. Transporters are grouped by substrate specificity and indicated (more ...)
Genome analysis suggests that P. gingivalis
possesses a limited capacity for the uptake and metabolism of organic nutrients. Glucose utilization by P. gingivalis
is known to be very poor, and carbohydrates in general do not appear to readily support growth (61
). Strain W83 does, however, contain putative ORFs for all enzymes of the glycolytic pathway, as well as ORFs for a putative glucose/galactose transporter and glucose kinase. Sequence analysis shows that the glucose kinase is encoded in a split ORF generated by a missense mutation, and this is a likely explanation for the poor utilization of glucose to support growth. Four putative ORFs for the pentose phosphate pathway were identified, and it is likely that this pathway plays a role in the generation of precursor metabolites during anaerobic growth (Fig. ).
Whole-genome analysis suggests that P. gingivalis
can metabolize several sugars, including melibiose, galactose, starch, and maltodextrin. The bacterium also possesses enzymes for the degradation of complex amino sugars in the form of hexose aminidases. It is still unclear whether these complex sugars are metabolized, but one possibility is that the removal of amino sugars from host glycoproteins likely renders these proteins more susceptible to degradation by bacterial proteinases. In addition, at least 11 amino acids may serve as substrates for energy production (Fig. ). These amino acids are most likely derived from the degradation of host tissues (see virulence section below) or from the breakdown of other bacterial cells in the oral cavity. Pathways for glutamate and aspartate utilization have been characterized by enzyme assays (65
), and ORFs coding for all of these activities were found in the W83 genome. Intracellular glutamate is deaminated to 2-oxoglutatarate by glutamate dehydrogenase and then decarboxylated to succinyl coenzyme A (succinyl-CoA) by a CoA-dependent 2-oxoglutarate oxidoreductase. The possession of this activity is somewhat unusual in bacterial species (23
). It has been established that two-thirds of the succinyl-CoA produced in this reaction is converted to butyryl-CoA and then to butyrate. The remaining third may be converted to propionate by a pathway that involves the enzymes methylmalonyl-CoA mutase and acyl-CoA:acetate-CoA transferase, as reported for other propionate-producing bacteria (18
). This pathway appears to be unique to P. gingivalis
since other anaerobes catabolize glutamate through the hydroxyglutarate, methylaspartate, and/or the aminobutyrate pathways (5
). P. gingivalis
did not possesss activities for three key enzymes of these pathways: hydroxyglutarate dehydrogenase, 3-methylaspartate ammonia lyase, and 4-aminobutyrate aminotransferase (65
). Peptide-derived aspartate is deaminated to fumarate by aspartate ammonia lyase and then either oxidized to acetate or reduced to propionate and butyrate (65
Results from Takahashi et al. (65
) suggest that P. gingivalis
prefers to utilize arginine and lysine as free amino acids rather than in peptide form; thus, carboxy-terminal arginine and lysine residues could be released from proteins by carboxypeptidase activities. Masuda et al. (45
) found such an activity in culture supernatants, and an ORF coding for an unspecified carboxypeptidase (PG0232) was identified in the genome. A report that P. gingivalis
produces citrulline and ornithine from denatured protein (14
) implies that the bacterium degrades arginine through the arginine deiminase pathway. Indeed, a gene with homology to arginine deiminase from Bacillus licheniformis
) was identified. In addition, two genes—pyrB
(PG0357 and PG0358)—were contiguous in the genome and shared homology with aspartate/ornithine transcarbamylase catalytic and regulatory chains from Vibrio
sp. strain 2693 and Pyrococcus abyssi
The lysine catabolic pathways appear to be very similar to those found in Clostridium
sp. ORFs were identified for the first steps of both l-
lysine catabolism; thus, the isomers are apparently degraded by two different pathways that yield butyric acid, acetic acid, and ammonia. Lysine 2,3-aminomutase (KamA) catalyzes the interconversion of l-
lysine and l-
β-lysine, the first step in the lysine degradation pathway in Clostridium subterminale
). In P. gingivalis
was found clustered with the genes kamD
(PG1070, PG1073, and PG1074) that encode subunits of d-
lysine 5,6-aminomutase, the first enzyme of the d-
lysine degradative pathway. Genes encoding enzymes for the subsequent conversion of lysine to butyrate and acetate were located 3′ to kamE
. It is not yet known whether these genes are transcribed as an operon.
Little is known about serine and threonine catabolism in P. gingivalis; however, an ORF was detected with homology to serine dehydratase (PG0084) that hydrolyzes serine to pyruvate, ammonia, and water. Threonine may be split to glycine and acetaldehyde by the activity of threonine aldolase, for which an ORF was detected (PG0474). In summary, P. gingivalis appears to catabolize amino acids through pathways that generate ammonia. The organism has a growth pH optimum of >7.5, and ammonia generation may have evolved as a strategy to shift the local pH to the favored alkaline range.
Several studies have shown that P. gingivalis
preferentially uses peptides as sources of carbon and nitrogen (60
) and, in addition to the previously described proteinases that are known to degrade host proteins, a number of peptidases that may be involved in the further digestion of protein fragments to smaller peptides and amino acids could be identified from the genome.
There are two carboxylate transporters possibly for lactate and formate, and no sugar transporters other than the aforementioned glucose/galactose importer. Although P. gingivalis
possesses a broad assortment of secreted peptidases and pathways for the metabolism of amino acids, the bacterium appears to rely on two predicted peptide uptake systems and has only one amino acid transporter, the characterized sodium ion-driven serine/threonine uptake protein SstT (12
). A LysE-type amino acid efflux protein is present that may protect the organism from toxic concentrations of amino acids.
The major fermentation products that can be produced based on whole-genome analysis and in vitro end product analyses are propionate, butyrate, isobutyrate, isovalerate, acetate, ethanol, and butanol (27
). Many of these end products are probably toxic to human host tissues (see virulence section below).
Nucleosides and nucleobases may represent a hitherto-unsuspected important nutrient source for P. gingivalis
and might be used either as building blocks for nucleic acid biosynthesis or may be catabolized as carbon and energy sources. There are three predicted purine uptake systems, a NupG nucleoside uptake system, and a homolog of the Salmonella enterica
serovar Typhimurium nicotinamide mononucleotide transporter PnuC. In addition, there are four homologs of E. coli
DinF, a DNA damage-induced protein related to sodium ion-driven drug efflux transporters, that are hypothesized to play a role in nucleoside and/or nucleotide efflux (8
Common to most human pathogens, iron acquisition appears to be an important priority in P. gingivalis, and there are two iron chelate ABC uptake systems, two TonB-dependent iron receptors, and two FeoB ferrous iron uptake systems. There is an array of metal ion homeostasis transporters, including three sodium ion/proton exchangers, which may be important since a significant number of P. gingivalis transporters are predicted to be sodium ion driven.
Virulence and P. gingivalis.
The availability of the complete genome sequence of P. gingivalis
facilitates the identification of putative virulence factors associated with the establishment and survival of the bacterium in the gingival crevice and subsequent penetration into host cells (Table ). Initially, the bacterium must navigate the oral cavity where, as an obligate anaerobe, it is exposed to limited amounts of oxygen before it establishes itself in an anaerobic environment. A cluster of genes (PG1582 to PG1586) was identified with high levels of similarity to the recently described aerotolerance operon of B. fragilis
). These functions promote the survival of B. fragilis
upon exposure to oxygen, and their presence in P. gingivalis
suggests that this system may also ensure tolerance to oxygen in the oral cavity. The genome also encodes a superoxide dismutase (PG1545), genes for an alkyl hydroperoxide reductase (PG0618 and PG0619) (55
), a thiol peroxidase (PG1729), and a Dps homolog (PG0090) that is involved in the repair of oxidatively damaged nucleic acids (33
Previously characterized and newly identified putative virulence agents from the P. gingivalis genome sequencea
The bacterium uses fimbriae to adhere to other bacterial species and host tissues. Hemagglutinins and various proteases (gingipains) are also involved in tissue colonization through adhesion to extracellular matrix proteins (38
). Hemagglutinins in particular may mediate the binding of bacteria to receptors on human cells (21
), and the gene sequences for six newly identified putative hemagglutinin-like proteins (PG0411, PG1326, PG1674, PG1427, PG1548, and PG2198) could be identified. Four of these are recent duplications in the genome of HagA and HagD adhesin domain-related sequences. A total of 42 proteinases were identified in the genome sequence that may enable adherence of the bacterium to host tissues, as well as to other bacterial cells, and that may also degrade host proteins (as discussed above). In vitro experiments have demonstrated that proteases attack a range of host proteins, including extracellular matrix proteins (32
) and cell adhesion molecules (29
), the destruction of which leads to a loss of cell surface receptors (59
) and tissue integrity (29
). Protease destruction of cytokines (15
) and gamma interferon (73
) can result in disruption of polymorphonuclear leukocyte function (48
) and ultimately affect the host immune response.
A single hemolysin for the release of iron and protophoryn IX (PG1875) was identified. This sequence has full-length homology only to the characterized hemolysin gene of another periodontal pathogen, Prevotella melaninogenica
). These two hemolysins show absolutely no homology to any other biochemically characterized hemolysin and have weak homology to a conserved hypothetical protein/putative hemolysin fusion protein sequence from Vibrio cholerae
In P. gingivalis
, metabolic end products from the catabolism of various substrates include short-chained carboxylic acids that can affect the host defense system in a variety of ways. When applied directly to healthy human gingiva tissue, short-chain carboxylic acids have been shown to stimulate a gingival inflammatory response and inflammatory cytokine release (50
). Short-chain carboxylic acids have also been shown to alter cell function and gene expression and may also contribute to the initiation and prolongation of gingival inflammation (50
The capsule of P. gingivalis
is most likely involved in the evasion of the host response and has been shown to be one of the important virulence determinants in this bacterium (34
). Whole-genome analysis reveals at least four capsular biosynthesis gene clusters (PG0106 to PGPG0120, PG0435 to PG0437, PG1140 to PG1149, and PG1560 to PG1565) that are located across the genome. Closer investigation of these gene clusters suggests that mannose, glucose, and rhamnose may be some of the sugars that are present in the capsule of P. gingivalis
strain W83. In several pathogens the secretion of virulence factors targeted to the host cells is mediated by type III protein secretion systems. The complete genome of P. gingivalis
was searched for the presence of a cluster of nine genes Sct (Hrc/Ysc) that are known to be components of type III protein secretion systems (24
). No BLAST matches with these motifs were found. Although several sec
gene homologs are present in the genome, including SecA, SecY, SecD, and SecF, the main terminal branch of the general secretory pathway (type II) could not be identified, suggesting that this pathway is not functional in this bacterium.