Open Access

Markedly different genome arrangements between serotype a strains and serotypes b or c strains of Aggregatibacter actinomycetemcomitans

BMC Genomics201011:489

DOI: 10.1186/1471-2164-11-489

Received: 10 May 2010

Accepted: 8 September 2010

Published: 8 September 2010

Abstract

Background

Bacterial phenotype may be profoundly affected by the physical arrangement of their genes in the genome. The Gram-negative species Aggregatibacter actinomycetemcomitans is a major etiologic agent of human periodontitis. Individual clonal types of A. actinomycetemcomitans may exhibit variable virulence and different patterns of disease association. This study examined the genome arrangement of A. actinomycetemcomitans using the genome sequences of serotypes a-c strains. The genome alignment and rearrangement were analyzed by the MAUVE and the GRIMM algorithms. The distribution patterns of genes along the leading/lagging strands were investigated. The occurrence and the location of repeat sequences relative to the genome rearrangement breakpoints were also determined.

Results

The genome arrangement of the serotype a strain D7S-1 is markedly different from the serotype b strain HK1651 or the serotype c strain D11S-1. Specific genome arrangements appear to be conserved among strains of the same serotypes. The reversal distance between D7S-1 and HK1651 by GRIMM analysis is also higher than the within-species comparisons of 7 randomly selected bacterial species. The locations of the orthologous genes are largely preserved between HK1651 and D11S-1 but not between D7S-1 and HK1651 (or D11S-1), irrespective of whether the genes are categorized as essential/nonessential or highly/nonhighly expressed. However, genome rearrangement did not disrupt the operons of the A. actinomycetemcomitans strains. A higher proportion of the genome in strain D7S-1 is occupied by repeat sequences than in strains HK1651 or D11S-1.

Conclusion

The results suggest a significant evolutionary divergence between serotype a strains and serotypes b/c strains of A. actinomycetemcomitans. The distinct patterns of genome arrangement may suggest phenotypic differences between serotype a and serotypes b/c strains.

Background

Bacterial genomes are relatively plastic and may display significant variation even among strains within the same species. The variation is often due to large scale genome deletion and/or gene acquisition by horizontal gene transfer of elements such as genomic islands [1]. Consequently, genome content can be divided into a core gene pool and a flexible gene pool [13]. The variation in genome content is thought to be a key factor in the evolution of bacterial pathogens. Moreover, the variation in genome arrangement (ie, the physical arrangement of genes) may also affect the virulence of the bacteria.

Genome rearrangement may occur via illegitimate recombination and homologous recombination among repeated elements and duplicated genes such as rDNA operons, and may also occur after horizontal gene transfer or phage infection. While genome rearrangements occurred frequently in laboratory cultures of Escherichia coli, very few were fixed since the divergence of E. coli and Salmonella enterica ~100MYA [4, 5]. Most of the rearrangements presumably have adverse effects on the bacteria due to the constraints placed by cellular processes such as replication, transcription and gene regulation [6, 7]. Consequently, the genome rearrangements between closely related bacteria commonly involve large-scale inversions along the axis of the origin (Ori) and the terminus (Ter) of replication [810]. Such changes presumably have much less deleterious effects due to preservation of the gene locations relative to replication and other cellular processes.

Gram-negative facultative Aggregatibacter actinomycetemcomitans is a member of the Pasteurellaceae family [11]. It is a recognized pathogen in periodontitis and extra-oral infections. There are 6 distinct serotypes; each serotype may represent a distinct clonal lineage of A. actinomycetemcomitans. Depending on the disease status and race/ethnicity of the subjects dominant serotypes within the study populations may include serotypes a, b, c, and e [12, 13]. Serotypes d and f are in general detected less frequently [12, 13].

Certain clonal lineages of A. actinomycetemcomitans, such as the JP-2 clone, appear to exhibit a high degree of virulence [1420]. However, other non-JP2 A. actinomycetemcomitans strains were also associated with aggressive periodontitis and are presumed to be highly virulent as well [13, 21]. Interestingly, in the study of a subgingival microbial community by Socransky et al, A. actinomycetemcomitans serotype a strains were a component of the green complex, while A. actinomycetemcomitans serotype b strains were not in association with other bacterial species [22]. It seems plausible that A. actinomycetemcomitans strains are distinct in their phenotypes, pathogenic mechanisms, and functional roles in the subgingival microbial communities, which may result in different patterns of disease association.

To understand the molecular basis of the variations of virulence in A. actinomycetemcomitans, we sequenced and compared the genome content and structure of A. actinomycetemcomitans strains recovered from different clinical settings. We have obtained initial evidence for significant genome content variations among strains [23, 24]. This study further examined the differences in the genome arrangement among A. actinomycetemcomitans strains of serotypes a-c. The results showed striking differences in the genome arrangements of serotype a strains compared to serotypes b or c strains. Such differences indicate divergent evolutionary pathways and possibly phenotypic differences between serotype a and serotype b/c strains of A. actinomycetemcomitans.

Results

Genome rearrangement between A. actinomycetemcomitans strains

The results of genome comparison by MAUVE for A. actinomycetemcomitans are shown in Figure 1. The reversal distances obtained by GRIMM (for A. actinomycetemcomitans and other bacterial species) are summarized in Table 1. For comparison between D11S-1 and HK1651 there are 9 locally collinear blocks (LCBs) with a minimum weight of 8,386 identified by the progressive MAUVE (Figure 1a), with a reversal distance of 5 (Figure 1b). The reversal distance of 5 can be viewed as a hypothetical 5-step inversion process to convert the genome arrangement of one strain to the other strain. The rearrangements involved at least one large scale genomic inversion along the axis of Ori-Ter (see later section for more explanation). For the comparison between D7S-1 and HK1651 there are 102 LCBs with a minimum weight of 35 (Figure 1c) and a reversal distance of 80, which was not only greater than that between HK1651 and D11S-1 but also greater than those between strains in other bacterial species (Table 1).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-11-489/MediaObjects/12864_2010_Article_3083_Fig1_HTML.jpg
Figure 1

Genome alignment by the progressive MAUVE between A. actinomycetemcomitans strains. The program identifies stretches of nucleotide matches and selects locally collinear blocks (LCB) that meet a minimum weight criteria. Homologous LCBs between genomes are connected with a line and identified by the same color. Blocks that are inverted are placed under the center line of the genome. (a) Serotype b strain HK1651 (upper) and serotype c strain D11S-1 (lower). Nine LCB were identified. The predicted locations of Ori are indicated with red arrows. These two genomes are largely in synteny, which is also indicated by a relatively low reversal distance of 5 (see Table 1). The rearrangement between the genomes possibly involves an inversion along the Ori-Ter axis of the two LCBs flanking the Ori. (b) An optimal scenario of genome conversion between strain HK1651 and D11S-1 based on the GRIMM analysis. The LCBs are identified as numbered block arrows and also marked if they are involved in reversal in each step. The illustration merely gives an example and does not imply that the sequence of the inversions has to be the same as depicted. The result suggests that it is possible to convert one genome to the other by 5 steps of genome reversion. (c) Genome alignment of serotype b strain HK1651 (upper) and serotype a strain D7S-1 (lower). One hundred and two LCB were identified. These two genomes show little resemblance in their arrangements, which is also reflected by a relatively high reversal distance of 80 (see Table 1).

Table 1

Genome rearrangement analysis and by progressive MAUVE and GRIMM of bacterial species.

Bacteria

No. of LCBa

Reversal distanceb

A. actinomycetemcomitans HK1651 vs D7S-1

102

80

A. actinomycetemcomitans HK1651 vs D11S-1

9

5

H. somnus 2336 vs 129PT

51

37

H. influenzae Rd KW20 vs 86-028NP

11

6

H. influenzae PittEE vs PittGG

23

17

E. coli 536 vs ATCC8739

9

5

E. coli O157:H7 EDL933 vs O12:H6 E2348/69

9

7

E. coli O157:H7 EDL933 vs ATCC8739

6

3

E. coli CFT073 vs E. coli O157:H7 EDL933

21

16

N. gonorrhoeae NCCP11945 vs FA1090

14

10

N. meningitides FAM18 vs MC58

16

10

N. meningitides Z2491 vs MC58

14

8

P. gingivalis ATCC33277 vs W83

41

29

P. aeruginosa PA7 vs PAO1

12

7

P. aeruginosa LESB58 vs PAO1

8

5

A. actinomycetemcomitans HK1651 vs A. aphrophilus NJ8700

155

127

A. actinomycetemcomitans D7S-1 vs A. aphrophilus NJ8700

192

162

aLCB; locally colinear block identify by the progressive MAUVE

bReversal distance is determined from the post-analysis of genome rearrangement by the progressive MAUVE using the GRIMM algorithms.

Seventy genome breakpoints of D7S-1 (in comparison to HK1651) were randomly selected for PCR analysis. All examined sites yielded PCR products of the expected sizes (see Additional File-1). Sixteen of the 70 PCR products were also sequenced and the results confirmed the sequences expected of the breakpoint regions (see Additional File-1).

The conservation of genome structures within serotypes

A question may arise whether the genome arrangement of D7S-1 is unique and not found in other A. actinomycetemcomitans strains. To address this question we compared the genome arrangements of D7S-1, HK1651 and D11S-1 with those in the contigs of strains D17P-3, ANH9381, and D17P-2 (serotypes a, b and c, respectively). There were significantly fewer intra-contig breakpoints in the comparisons within each serotype than between serotypes a and b/c. We identified one intra-contig break point in 267 large contigs of D17P-3 in a pair-wise comparison to D7S-1. Similarly, we found 4 intra-contig breakpoints in 3 of the 102 large contigs of ANH9381 compared to HK1651, and 4 intra-contig breakpoints in 2 of the 62 large contigs of D17P-2 compared to D11S-1. In contrast, we identified 47 breakpoints in 40 contigs of D17P-3 compared to HK1651. The results are consistent with the conservation of the genome arrangement within serotypes, but not between serotypes a and b/c.

Distribution patterns of genes and operons in the genomes of A. actinomycetemcomitans

It is possible that the relative gene locations in the genome may be preserved after large-scale genome rearrangements [6]. To address this question in A. actinomycetemcomitans, we first identified the Ori and the Ter in the genomes of D7S-1, HK1651 and D11S-1, and analyzed (i) the balance of the replichores, (ii) the gene density in the leading and the lagging strands, and (iii) the positions of the orthologous genes relative to the Ori. We further examined the preservation of the operons in strains of different genome arrangements.

The combined cumulative T-A and C-G skews of the 3rd codons peaked at the nucleotide coordinate ~1,062 Kb and declined and changed sign at the nucleotide coordinate ~67 Kb for D7S-1 (see Additional File 2, top panel). Several peaks of similar heights were identified at coordinates 760 Kb, 825 Kb and 875 Kb for HK1651, and at 437 Kb, 515 Kb and 550 Kb for D11S-1 (see Additional File 2, middle and bottom panels). The lowest points of the combined skews changed sign at coordinates ~1,450 Kb for HK1651 and 1,246 Kb for D11S-1. Based on the results we assigned the Ori and the Ter respectively to nucleotide coordinates 1,062,100 and 67,100 in D7S-1, 825,100 and 1,449,500 in HK1651, and 515,300 and 1,246,100 in D11S-1. The predicted locations of Ori and Ter in HK1651 and D11S-1 were supported by the observation that a large-scale genomic inversion between HK1651 and D11S-1 occurred along the axis of Ori-Ter as commonly observed in other species [810].

The imbalance of the replichores was analyzed as described previously (the absolute value of [the length of the replichore-half length of the genome]/half length of the genome) [25]. We found that the imbalance of D7S-1 was 13.8%, which may be considered within the normal range of deviations among many bacterial species. In contrast, the imbalance of the replichores in HK1651 and D11S-1 were 40.1% and 30.6%, respectively.

The distributions of the predicted genes in the leading and the lagging strands are shown in Table 2. The gene density is higher in the leading strand than in the lagging strand in the 3 A. actinomycetemcomitans strains of this study. Higher numbers of essential genes were found in the leading strand than in the lagging strand for D7S-1 and HK1651, but D11S-1 showed no strand preference.
Table 2

Distribution of genes in leading and lagging strands in A. actinomycetemcomitan s strains

Strain

Strand

Gene

Density

No. of Essential

Gene (%)

No. of Non-essential

gene (%)

D7S-1

Leading

46.6%

153 (59.1)

1171 (52.2)

 

Lagging

42.0%

106 (40.9)

1074 (47.8)

HK1651

Leading

46.2%

151 (56.8)

1124 (51.8)

 

Lagging

43.9%

115 (43.2)

1047 (48.2)

D11S-1

Leading

45.1%

131 (50)

1007 (51.8)

 

Lagging

43.5%

131 (50)

938 (48.2)

The distances of the orthologous genes to the Ori are presented in Figure 2a-f. In Figure 2a the distances of each pair of the orthologous genes in HK1651 and D11S-1 were similar and can be explained by an offset of ~130 Kb between the genomes (with the exception of 8 genes where the differences in the distances to Ori were ~740 Kb). In contrast, the orthologous genes in HK1651 and D7S-1 (Figure 2b) resided in different locations relative to Ori and no specific distribution patterns were found. Similar results were found for subgroups of essential/nonessential and highly expressed/nonhighly expressed genes (Figure 2 c-f). Also, there was no discernable tendency for the highly expressed genes to be closer to the Ori than the non-essential genes.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-11-489/MediaObjects/12864_2010_Article_3083_Fig2_HTML.jpg
Figure 2

Pair-wise comparisons of the distance to the origin of replication (Ori) for orthologous genes between A. actinomycetemcomitans strains. The distances for genes on the leading strands were calculated directly from the position of Ori. The distances for genes on the lagging strands were calculated as (genome length-the distance to Ori). The linear trend line by Pearson's correlation is provided. The low r2 values indicate a lack of correlation in the gene positions between D7S-1 and HK1651. (a) Comparison between HK1651 and D11S-1. There is an apparent correlation in the locations of orthologous genes in the strains. (b) Comparison between D7S-1 and HK1651. Although there is a hint of an overall linear relationship, the distribution pattern suggested that the locations of the orthologous genes were poorly correlated. (c-e) Comparisons between D7S-1 and HK1651 with genes of different categories. Again, little correlations were found in the locations of the orthologous genes between these strains.

The potential disruptions of operons in the A. actinomycetemcomitans strains were examined. The analysis of operon positions with respect to genomic rearrangement between D7S-1 and HK1651 showed that out of 564 operons in the D7S-1 genome predicted by Database of prOkaryotic OpeRons (DOOR) tool, 558 (98.9%) were found to be intact in HK1651 and were not affected by the genomic rearrangements between these strains (see Additional File 3 for the list of disrupted operons in D7S-1). Similarly, 505 of 515 (98%) predicted operons in HK1651 were found to be intact in D7S-1 (see Additional File 3 for the list of disrupted operons in HK1651). For the affected operons, the rearrangement breakpoints occurred between genes of the operons resulting in separations of genes rather than splitting the genes into two fragments. Similar results were obtained using FGENESB (http://linux1.softberry.com/berry.phtml?topic=fgenesb&group=programs&subgroup=gfindb) (data not shown).

Features of genome rearrangement breakpoints

Genome rearrangements commonly occur via recombination between repeat elements or duplicated genes. We hypothesized that there might be specific features at the inter-LCBs regions and/or the ends of the LCBs that flanked the rearrangement breakpoints. To examine this hypothesis, 50-bp sub-sequences on both strands of each genome were extracted in sliding windows of 1 bp, and compared to the entire genome to identify a perfect match in other regions. These 50-base-pair-repeat regions are summarized in Table 3. D7S-1 genome contained a higher number of repeat regions than in HK1651, D11S-1 or other bacteria species analyzed. The percentage of the overlap between the cumulative inter-LCB regions and repeat regions are presented in Table 4. Higher percentages of the genome in D7S-1 were occupied by repeat regions than in the other two strains. Figure 3 illustrates the locations of the repeat elements and the inter-LCB breakpoint regions (relative to HK1651) of D7S-1. The regions between LCBs in D7S-1 were enriched with repeated sequences (See Additional File 4 for the locations of the repeat elements and the inter-LCB breakpoint regions of HK1651).
Table 3

Positions and percent of genome occupied by 50 base pair repeat in the genomes of A. actinomycetemcomitan s and other bacterial species.

Genome

Total number of

position with 50 bp

exact matches

Genome

length (bp)

Percent of 50 bp exact

matches with respect

to the genome length

D7S-1

144,825

2,308,328

6.27%

HK1651

44,531

2,105,503

2.11%

D11S-1

47,809

2,105,764

2.27%

E. coli K12

102,642

4,639,675

2.21%

P. aeruginosa PAO1

84,973

6,264,404

1.36%

H. pylori G28

41,316

1,652,982

2.50%

Table 4

Overlap between repeat and inter-LCB region

Strain

Total

inter-LCB

length

(bp)

Total

repeat

length

(bp)

Total

overlap

(bp)

Percent of

repeat

overlapping

with inter-LCB

Percent of

inter-LCB

overlapping

with repeat

D7S-1a

251,896

159,531

105,889

66.4%

42%

HK1651b

90,658

50,603

16,729

33.1%

18.5%

D11S-1c

112,883

53,208

20,973

39.4%

18.6%

a Regions between rearranged conserved blocks (inter-LCB) for D7S-1 are obtained from D7S-1 vs HK1651 MAUVE alignment.

b Regions between rearranged conserved blocks (inter-LCB) for HK1651 are obtained from D7S-1 vs HK1651 MAUVE alignment

c Regions between rearranged conserved blocks (inter-LCB) for D11S-1 are obtained from D7S-1 vs D11S-1 MAUVE alignment

https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-11-489/MediaObjects/12864_2010_Article_3083_Fig3_HTML.jpg
Figure 3

Circular chromosome map of D7S-1 genome. This figure shows that boundaries of the rearranged regions, as suggested by whole genome alignment of D7S-1 and HK1651 genome sequences, are significantly enriched with repeated sequences.

A summary comparison of the frequency and the feature of the repeat elements among strains are shown in Table 5 (see Additional File 5 for sequences of the repeat elements). D7S-1 has more repeat elements than HK1651 or D11S-1. Several of these repeat elements are shared among strains (allowing minor sequence variations). However, many of the repeat elements found in D7S-1 are unique to this strain.
Table 5

Frequency and features of the repeat elements identified in A. actinomycetemcomitans

Repeat ID

Estimated

length

(bp)

Features

Frequency

in D7S-1

Genome

Frequency

in HK1651

Genome

Frequency

in D11S-1

Genome

REPEAT01

6505

rRNA genes operon

6

6

6

REPEAT02

1269

IS150 like element

8

7

5

REPEAT03

717

IS200 like element

0

4

5

REPEAT04

139

Unknown

1

5

5

REPEAT05

6326

FHA domain protein

2

0

0

REPEAT06

4315

WD-40 repeat, Hypothetical protein and FHA domain protein

2

0

0

REPEAT07

2156

Sel1 domain protein repeat-containing protein

4

0

0

REPEAT08

2008

Rhs element Vgr protein

6

0

0

REPEAT09

1523

Glycoside hydrolase, family 19 gene

5

0

0

REPEAT10

1254

FHA domain protein

2

0

0

REPEAT11

1227

Tra5 Protein

6

0

0

REPEAT12

1060

IS30 like element

3

0

0

REPEAT13

1225

Translation elongation factor Tu

2

2

2

REPEAT14

837

Putative FHA domain protein

4

0

0

REPEAT15

690

IS427 like element

4

0

0

REPEAT16

472

Sel1 domain protein repeat-containing protein

7

0

0

REPEAT17

387

Hypothetical protein

10

0

0

REPEAT18

219

Unknown

8

4

2

REPEAT19

162

Hypothetical protein

7

1

1

REPEAT20

100

Unknown

7

5

5

REPEAT21

135

Autotransporter adhesin Aae

4

3

2

Discussion

A number of studies have suggested variable virulence among A. actinomycetemcomitans strains [12, 15, 16, 19, 20, 26, 27]. Most of these studies examined the clinical associations of specific genotypes of A. actinomycetemcomitans with periodontal health and disease in cross-sectional and prospective studies, but did not provide insight to the molecular basis of such variations. In the present study we provided evidence for variations in the physical arrangement of genes in the genomes of A. actinomycetemcomitans, which may affect the phenotypes or virulence of the strains. Serotypes a-c of A. actinomycetemcomitans were selected for this study because they are frequently identified and may represent up to 80% of the A. actinomycetemcomitans clinical isolates in human subgingival plaque [13].

The differences in the genome arrangement between strains were visualized with the use of MAUVE and then quantified by GRIMM to calculate the reversal distance. The use of reversal distance in phylogenetic analysis is based on the premise that genomic inversion is the primary type of rearrangement event in bacteria, which was supported by several studies [8, 28]. Moreover, there is a general correlation between reversal distance and sequence-based phylogenetic analysis [28]. While low reversal distance may be relatively accurate, high reversal distance is likely to underestimate the true phylogenetic distance between genomes.

It is striking and unusual (in comparison to the variations seen in other bacterial species) that the serotype a strain D7S-1 displayed a markedly different genome arrangement relative to HK1651 or D11S-1. The potential sources of errors were examined first. We could rule out large scale sequencing or assembly errors of the contigs based on the results of the PCR analysis of the breakpoints in D7S-1. Moreover, minor sequencing errors would have little or no effect on the genome comparison by MAUVE which examines large homologous blocks between strains. We could also rule out assembly errors because the finished genomes were confirmed with optical mapping [23, 24]. The results would not have been affected by the specific locations of the Ori or the Ter in the strains. Additional supporting evidence was from the analysis of unscaffolded large contigs of 3 A. actinomycetemcomitans strains. Few intra-contig breakpoints were found between strains of the same serotypes, in contrast to the high numbers of intra-contig breakpoints between strains of serotype a and serotypes b/c. Therefore we concluded that serotype a strains exhibited markedly different genome arrangements compared to those in serotypes b/c strains, and further suggested that the genome arrangements were conserved within serotypes.

The Ori and the Ter may be identified by various in silico methods based on analyses of DNA asymmetry, distribution of DNA boxes and dnaA gene location [2932]. The methods based on DNA asymmetry appear to be the most universal and have been used to identify the Ori and Ter of H. influenzae (a member of the Pasteurellacea) [32, 33]. In the absence of experimental determination the locations of the Ori and the Ter of A. actinomycetemcomitans strains identified in this study were in agreement with the available evidence as discussed below.

We noted that in the annotation of HK1651 the origin of replication was not identified but the starting codon of the dnaA gene was assigned the first nucleotide coordinate of the genome. While in some species the location of the dnaA gene coincides with the origin of replication [29], this is not the case in H. influenzae and presumably not in A. actinomycetemcomitans either. Eriksen et al [34] showed evidence of intragenomic recombination in JP2 clone of A. actinomycetemcomitans via homologous recombination of the 6 rRNA operons and 7 IS150-like repeat elements. It is interesting to note that, with the exception of one case (recombination between IS150-2 and IS150-4), all recombinations occurred along the axis of Ori-Ter predicted in this study. In this study we also found an example of a large-scale genomic inversion between HK1651 and D11S-1 along the axis of Ori-Ter.

Genome rearrangements may occur via homologous recombination of repeat elements in the bacterial genomes [35, 36]. A. actinomycetemcomitans genomes contain diverse repeat elements that may mediate genome rearrangement. Within A. actinomycetemcomitans genomes some inter-LCB regions were occupied by composites of diverse repeat elements, which may reoccur in other inter-LCB regions but with minor variations (sequence variations, truncation, or absence) of the individual repeat elements. Some of the sequence diversity may be due to sequencing errors. For these reasons we chose to analyze the occurrence of repeat elements with a sliding 50-base window. We noted that A. actinomycetemcomitans D7S-1 has a greater number of repeat elements than HK1651 or D11S-1. The data alone, however, cannot be used to infer the ancestral genome structure of A. actinomycetemcomitans.

Some of the repeat elements are identified in all three A. actinomycetemcomitans strains. The IS150 like elements have been reported previously in the genome of HK1651 [34] and are found in both D7S-1 and D11S-1 in this study. The presence of variable copy numbers of a 135-bp repeat sequence in the autotransporter adhesion gene Aae in different A. actinomycetemcomitans strains has been reported previously [37]. There seems to be a distinction in the distribution pattern of the repeat elements in D7S-1 in comparison to that in HK1651/D11S-1. For example, 12 of the repeat elements in D7S-1 are unique and not found in HK1651 or D11S-1. Vice versa the REPEAT03 is identified in HK1651 and D11S-1 but not in D7S-1. Also, the copy numbers of some of the repeat elements (REPEAT-04, -19, -20) are identical in HK1651 and D11S-1 and different from the copy numbers of the elements found in D7S-1. Further examination of other A. actinomycetemcomitans strains is needed to determine whether such distribution pattern has any phylogenetic significance.

The results from this study appear to suggest that the genome arrangement of A. actinomycetemcomitans strains may be less constrained by cellular processes than in other bacterial species. This could be explained by several factors. The growth rate of A. actinomycetemcomitans is comparatively low (doubling time of ~3-4 hrs in optimum laboratory growth conditions). There might be little or no gene dosage effects and problem of collisions between replication fork and RNA polymerase in slow-growing bacteria, which allow the bacteria to tolerate large-scale genomic rearrangements. The effective population size of some clonal lineages of A. actinomycetemcomitans (e.g., serotypes b and c) may be small, which allow these clones to persist in the population. It is also possible that serotypes b and c, as represented by HK1651 and D11S-1, are more recently evolved and have not had sufficient time to allow the mutation pressures to exert their effects. This interpretation is supported by the imbalanced genomes of HK1651 and D11S-1, which could be a consequence of recent changes of their genome arrangements.

While there are significant differences in the genome arrangements in D7S-1 and HK1651 (or D11S-1) they essentially did not affect the operons. However, the locations of orthologous genes were significantly different between D7S-1 and HK1651 (or D11S-1). Presumably such differences will affect the phenotypes of the strains. We further noted the replichores were severely unbalanced for strains HK1651 and D11S-1 and less so for strain D7S-1. Evidence has suggested a strong selection for bacteria with a balanced genome [38]. On the contrary, no evidence of natural selection for balanced genomes was found in the analysis of eight Yersinia genomes [25]. We have detected no significant differences in growth rate and biofilm formation under laboratory growth conditions among these 3 A. actinomycetemcomitans strains (unpublished data). The significance of genome arrangement to the phenotypes of A. actinomycetemcomitans remains to be elucidated.

The differences in the genome arrangement or genome content alone may not be sufficient to determine whether some A. actinomycetemcomitans strains should be designated a subspecies or even a new species. There appear to be no universally accepted concept and definition of bacterial species. With the advancement of bacterial genomics various approaches for species definition have been proposed that combine the analyses of the 16S rRNA gene sequence identity, DNA-DNA hybridization, percentage of the shared genes in the genome, the average nucleotide identity (ANI) of the shared genes and ecological factors [39, 40]. Pair-wise comparison of the 16S rRNA gene sequences in strains D7S-1, HK1651 and D11S-1 showed >97.6% nucleotide identity, which is within the accepted working definition of all three being from the same species. We are analyzing the genome contents of the sequenced serotypes a-f strains to further address this question.

In addition to the potential biological impact of the observed large-scale genomic rearrangement between serotype a strains and serotype b/c strains, there are also implications of the rearrangement on a practical research level. In the early stages of our assembly and finishing of the D7S-1 genome we had hoped to use the HK1651 genome as a guide to assist in the ordering of contigs. However, the level of genomic rearrangement between these two strains negated the utility of HK1651 as a reference genome for the structure of D7S-1. Also, the variation in genome structure means that PCR products predicted in one strain may cross a breakpoint in another strain and hence will not be amplified in the other strain. It is unclear how frequently similar problems will arise in the sequencing and analysis of other bacterial genomes, but it is worth noting that at least in A. actinomycetemcomitans massive variation in genome structure between strains can lead to confusion in some kinds of analyses.

Conclusions

A. actinomycetemcomitans serotype a strains display markedly different physical arrangement of genes in comparison to serotype b or c strains. This likely indicates significant differences in the evolutionary history between serotype a strains and serotype b/c strains. The results have provided significant insight to the evolutionary divergence of A. actinomycetemcomitans of different serotypes. Also, the serotype-specific genome arrangement patterns have practical application for future genome sequencing of A. actinomycetemcomitans.

Methods

Bacterial strains

Serotype a strains D7S-1, D17P-3, and serotype c strains D11S-1 and D17P-2 were cultivated from subgingival plaque of patients with aggressive periodontitis [13, 41]. Serotype b strain ANH9381 was recovered from a subgingival plaque sample of a periodontally non-diseased subject. Species identity and serotypes were examined by a 16S rRNA-based PCR analysis and a serotype analysis by a PCR-method as described previously [42].

Genome sequences

The genome sequencing of D7S-1 (one contig; genome size 2,308,328 bp) and D11S-1 (circularized; genome size 2,105,764 bp) were completed as described previously [23, 24]. The genome information of the sequenced strain HK1651 (genome size 2,105,503 bp) is accessible from University of Oklahoma (http://www.genome.ou.edu/act.html) and Oralgen (http://www.oralgen.lanl.gov/oralgen/bacteria/aact/). Contigs generated by 454 sequencing of strains D17P-3 (25× coverage), ANH9381 (16X) and D17P-2 (28X) were also included in the analyses. Additional bacterial genome sequences were downloaded from Genbank for analyses that included Haemophilus somnus strains 2336 and 129PT, Haemophilus influenzae strains RdKW20, 86-028NP, PittEE and PittGG, Escherichia coli strains 536, ATCC8739, O157:H7 EDL933, O12:H6 E2348/69, CFT073 and K12, Neisseria gonorrhoeae NCCP11945 and FA1090, Neisseria meningitidis FAM18, MC58, Z2491, Porphyromonas gingivalis ATCC33277 and W83, Pseudomonas aeruginosa PA7, PAO1, LESB58, Aggregatibacter aphrophilus NJ8700, and Helicobacter pylori G28.

Annotation and comparison of A. actinomycetemcomitans genomes

A gene prediction and annotation pipeline was put together to process the genome sequence data obtained from the Roche/454 platform and strain HK1651. The gene identification and functional annotation mostly followed the protocol developed by The Institute for Genomic Research (J. Craig Venter Institute). Specifically, protein-coding genes were identified using Glimmer3 software [43] with our custom modification of the predicted results. Similarly, rRNA and tRNA coding genes were identified by using Exonerate [44] and tRNAscanSE [45] softwares, respectively. The predicted genes were annotated by first comparing them to the HK1651 annotation using the NCBI BLAST software [46]. Genes that are annotated as hypothetical as well as those that are not present in strain HK1651 were then blasted against Genbank non-redundant protein sequence database. The description of the best BLAST hit is then used as annotation for that gene. The gene orthologs among the 3 A. actinomycetemcomitans strains were identified based on all against all BLAST search. The genes that fulfill the following criteria are included as core genes with: (i) sequence similarity of at least 85% (ii) length difference of not more than 5%. Pseudogenes and genes with frameshift mutations were excluded from the analysis.

Analysis of genomic rearrangement by MAUVE

The Progressive Mauve algorithm was used to create the whole genome alignment between different strains of A. actinomycetemcomitans[47]. GRIMM genome rearrangement algorithms were used to obtain the reversal distance between genomes [48].

PCR analysis of genome breakpoints

The genome breakpoints in D7S-1 were analyzed by PCR. Briefly, 20-mer oligonucleotides were designed with the program Primer 3 [49]. A standard PCR protocol was employed under the following conditions: 5 min at 94°C for denaturation followed by 30 cycles of 94°C for 30 sec, an annealing step at 60°C for 1 min, an extension step at 72°C for 2 min and then a final extension of 10 min at 72°C [50]. PCR amplicons were analyzed by 1% agarose gel electrophoresis. For sequencing, the PCR products were purified by GIAquick PCR purification kit and GIAquick Gel Extraction kit (Qiagen, Valencia, CA) and submitted for sequencing at the USC School of Medicine Microchemical Core Facility.

Assignment of gene categories in A. actinomycetemcomitans

All open reading frames (ORFs) identified in A. actinomycetemcomitans strains D7S-1, HK1651 and D11S-1 were categorized as essential or non-essential genes. The essential genes were identified based on the Profiling of Escherichia coli chromosome (PEC) database [51]. Specifically, A. actinomycetemcomitans ORFs that are homologous to the essential genes in PEC were considered as essential (blastp with E-value < = 1e-6). The remaining ORFs in A. actinomycetemcomitans were considered non-essential by default. ORFs were also classified into highly expressed and non-highly expressed genes based on the codon adaptation index (CAI) calculated using CAIJava tool [52]. ORFs within the top 5 percent highest CAI score are assigned as highly expressed and the remaining ORFs are considered as non-highly expressed.

Analysis of gene density and gene positions

Combined G-C and T-A skews were first used to predict the locations of the Ori and the Ter with Oriloc [31]. Each genome was then divided into two replichores. The distribution of genes of different categories (essential/non-essential, highly expressed/non-highly expressed) and their densities in the leading and the lagging strands were compared between strains. The Pearson's correlation coefficient of the distance to the Ori for orthologous genes between strains was calculated.

Analysis of preservation of operons among strains

The operons in A. actinomycetemcomitans were identified by using the Database of prOkaryotic OpeRons (DOOR) tool [53, 54] and by FGENESB, a suite of bacterial operon and gene prediction programs [55]. In brief, the DOOR tool predicts bacterial gene operons using a classifier algorithm on features such as intergenic distance, neighborhood conservation, phylogenetic distance, information from short DNA motifs, similarity score between GO terms of gene pairs and length ratio between a pair of genes. The FGENESB tool predicts gene operon based on distances between open reading frames and frequencies of different genes neighboring each other in known bacterial genomes, as well as on promoter and terminator predictions. The positions of predicted operons in one strain were examined in the other strains to identify those that are affected by the genome rearrangement.

Identification of regions with repeat sequences

A 50-base pair window (each window is a sliding of 1 base pair along the genome sequence) is compared to the entire genome to identify perfect matches (or a perfect match) on any other regions of the genome.

Declarations

Acknowledgements

This study was supported by NIDCR grant R01 DE12212. The authors wish to thank Aaron Darling for helpful discussions.

The Whole Genome Shotgun projects (strains D17P-3, ANH9381, and D17P-2) have been deposited at DDBJ/EMBL/GenBank under the accession ADOA00000000 (A. actinomycetemcomitans D17P-3), ADOC00000000 (A. actinomycetemcomitans ANH9381), and ADOB00000000 (A. actinomycetemcomitans D17P-2).

Authors’ Affiliations

(1)
Department of Microbiology, University of Washington
(2)
Division of Periodontology, Diagnostic Sciences and Dental Hygiene, Herman Ostrow School of Dentistry of the University of Southern California

References

  1. Hacker J, Carniel E: Ecological fitness, genomic islands and bacterial pathogenicity. A Darwinian view of the evolution of microbes. EMBO Rep. 2001, 2: 376-381.PubMed CentralPubMedView ArticleGoogle Scholar
  2. Hacker J, Blum-Oehler G, Muhldorfer I, Tschape H: Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol Microbiol. 1997, 23: 1089-1097. 10.1046/j.1365-2958.1997.3101672.x.PubMedView ArticleGoogle Scholar
  3. Hacker J, Hentschel U, Dobrindt U: Prokaryotic chromosomes and disease. Science. 2003, 301: 790-793. 10.1126/science.1086802.PubMedView ArticleGoogle Scholar
  4. Rocha EP: Inference and analysis of the relative stability of bacterial chromosomes. Mol Biol Evol. 2006, 23: 513-522. 10.1093/molbev/msj052.PubMedView ArticleGoogle Scholar
  5. Hill CW, Harnish BW: Inversions between ribosomal RNA genes of Escherichia coli. Proc Natl Acad Sci USA. 1981, 78: 7069-7072. 10.1073/pnas.78.11.7069.PubMed CentralPubMedView ArticleGoogle Scholar
  6. Rocha EP: The organization of the bacterial genome. Annu Rev Genet. 2008, 42: 211-233. 10.1146/annurev.genet.42.110807.091653.PubMedView ArticleGoogle Scholar
  7. Rocha EP: Order and disorder in bacterial genomes. Curr Opin Microbiol. 2004, 7: 519-527. 10.1016/j.mib.2004.08.006.PubMedView ArticleGoogle Scholar
  8. Hughes D: Evaluating genome dynamics: the constraints on rearrangements within bacterial genomes. Genome Biol. 2000, 1: REVIEWS0006-10.1186/gb-2000-1-6-reviews0006.PubMed CentralPubMedView ArticleGoogle Scholar
  9. Tillier ER, Collins RA: Genome rearrangement by replication-directed translocation. Nat Genet. 2000, 26: 195-197. 10.1038/79918.PubMedView ArticleGoogle Scholar
  10. Eisen JA, Heidelberg JF, White O, Salzberg SL: Evidence for symmetric chromosomal inversions around the replication origin in bacteria. Genome Biol. 2000, 1: RESEARCH0011-10.1186/gb-2000-1-6-research0011.PubMed CentralPubMedView ArticleGoogle Scholar
  11. Tanner A, Maiden MFJ, Paster BJ, Dewhirst FE: The impact of 16S ribosomal RNA-based phylogeny on the taxonomy of oral bacteria. Periodontology 2000. 1994, 5: 26-51. 10.1111/j.1600-0757.1994.tb00017.x.PubMedView ArticleGoogle Scholar
  12. Rylev M, Kilian M: Prevalence and distribution of principal periodontal pathogens worldwide. J Clin Periodontol. 2008, 35: 346-361. 10.1111/j.1600-051X.2008.01280.x.PubMedView ArticleGoogle Scholar
  13. Chen C, Wang T, Chen W: Occurrence of Aggregatibacter actinomycetemcomitans serotypes in subgingival plaque from United States subjects. Molecular Oral Microbiology. 2010, 25: 207-214. 10.1111/j.2041-1014.2010.00567.x.PubMedView ArticleGoogle Scholar
  14. Asikainen S, Chen C, Slots J: Actinobacillus actinomycetemcomitans genotypes in relation to serotypes and periodontal status. Oral Microbiol Immunol. 1995, 10: 65-68. 10.1111/j.1399-302X.1995.tb00120.x.PubMedView ArticleGoogle Scholar
  15. Asikainen S, Lai CH, Alaluusua S, Slots J: Distribution of Actinobacillus actinomycetemcomitans serotypes in periodontal health and disease. Oral Microbiol Immunol. 1991, 6: 115-118. 10.1111/j.1399-302X.1991.tb00462.x.PubMedView ArticleGoogle Scholar
  16. DiRienzo JM, Slots J, Sixou M, Sol MA, Harmon R, McKay TL: Specific genetic variants of Actinobacillus actinomycetemcomitans correlate with disease and health in a regional population of families with localized juvenile periodontitis. Infect Immun. 1994, 62: 3058-3065.PubMed CentralPubMedGoogle Scholar
  17. DiRienzo JM, McKay TL: Identification and characterization of genetic cluster groups of Actinobacillus actinomycetemcomitans isolated from the human oral cavity. J Clin Microbiol. 1994, 32: 75-81.PubMed CentralPubMedGoogle Scholar
  18. Haubek D, Poulsen K, Asikainen S, Kilian M: Evidence for absence in Northern Europe of especially virulent clonal types of Actinobacillus actinomycetemcomitans. J Clin Microbiol. 1995, 33: 395-401.PubMed CentralPubMedGoogle Scholar
  19. Haubek D, Poulsen K, Westergaard J, Dahlen G, Kilian M: Highly toxic clone of Actinobacillus actinomycetemcomitans in geographically widespread cases of juvenile periodontitis in adolescents of African origin. J Clinic Microbiol. 1996, 34: 1576-1578.Google Scholar
  20. Haubek D, DiRienzo JM, Tinoco EM, Westergaard J, Lopez NJ, Chung CP, Poulsen K, Kilian M: Racial tropism of a highly toxic clone of Actinobacillus actinomycetemcomitans associated with juvenile periodontitis. J Clin Microbiol. 1997, 35: 3037-3042.PubMed CentralPubMedGoogle Scholar
  21. Kaplan JB, Schreiner HC, Furgang D, Fine DH: Population structure and genetic diversity of Actinobacillus actinomycetemcomitans strains isolated from localized juvenile periodontitis patients. J Clin Microbiol. 2002, 40: 1181-1187. 10.1128/JCM.40.4.1181-1187.2002.PubMed CentralPubMedView ArticleGoogle Scholar
  22. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL: Microbial complexes in subgingival plaque. J Clin Periodontol. 1998, 25: 134-144. 10.1111/j.1600-051X.1998.tb02419.x.PubMedView ArticleGoogle Scholar
  23. Chen C, Kittichotirat W, Si Y, Bumgarner R: Genome sequence of Aggregatibacter actinomycetemcomitans serotype c strain D11S-1. J Bacteriol. 2009, 191: 7378-7379. 10.1128/JB.01203-09.PubMed CentralPubMedView ArticleGoogle Scholar
  24. Chen C, Kittichotirat W, Chen W, Downey JS, Si Y, Bumgarner R: Genome sequence of a naturally competent Aggregatibacter actinomycetemcomitans serotype a strain D7S-1. J Bacteriol. 2010Google Scholar
  25. Darling AE, Miklos I, Ragan MA: Dynamics of genome rearrangement in bacterial populations. PLoS Genet. 2008, 4: e1000128-10.1371/journal.pgen.1000128.PubMed CentralPubMedView ArticleGoogle Scholar
  26. Haubek D, Ennibi OK, Poulsen K, Vaeth M, Poulsen S, Kilian M: Risk of aggressive periodontitis in adolescent carriers of the JP2 clone of Aggregatibacter (Actinobacillus) actinomycetemcomitans in Morocco: a prospective longitudinal cohort study. Lancet. 2008, 371: 237-242. 10.1016/S0140-6736(08)60135-X.PubMedView ArticleGoogle Scholar
  27. Asikainen S, Chen C, Saarela M, Saxen L, Slots J: Clonal specificity of Actinobacillus actinomycetemcomitans in destructive periodontal disease. Clin Infect Dis. 1997, 25 (Suppl 2): S227-229. 10.1086/516211.PubMedView ArticleGoogle Scholar
  28. Belda E, Moya A, Silva FJ: Genome Rearrangement Distances and Gene Order Phylogeny in {gamma}-Proteobacteria. Mol Biol Evol. 2005, 22: 1456-1467. 10.1093/molbev/msi134.PubMedView ArticleGoogle Scholar
  29. Mackiewicz P, Zakrzewska-Czerwinska J, Zawilak A, Dudek MR, Cebrat S: Where does bacterial replication start? Rules for predicting the oriC region. Nucleic Acids Res. 2004, 32: 3781-3791. 10.1093/nar/gkh699.PubMed CentralPubMedView ArticleGoogle Scholar
  30. Worning P, Jensen LJ, Hallin PF, Staerfeldt HH, Ussery DW: Origin of replication in circular prokaryotic chromosomes. Environ Microbiol. 2006, 8: 353-361. 10.1111/j.1462-2920.2005.00917.x.PubMedView ArticleGoogle Scholar
  31. Frank AC, Lobry JR: Oriloc: prediction of replication boundaries in unannotated bacterial chromosomes. Bioinformatics. 2000, 16: 560-561. 10.1093/bioinformatics/16.6.560.PubMedView ArticleGoogle Scholar
  32. Lobry JR: Asymmetric substitution patterns in the two DNA strands of bacteria. Mol Biol Evol. 1996, 13: 660-665.PubMedView ArticleGoogle Scholar
  33. Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, Kerlavage AR, Bult CJ, Tomb JF, Dougherty BA, Merrick JM: Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995, 269: 496-512. 10.1126/science.7542800.PubMedView ArticleGoogle Scholar
  34. Eriksen KT, Haubek D, Poulsen K: Intragenomic recombination in the highly leukotoxic JP2 clone of Actinobacillus actinomycetemcomitans. Microbiology. 2005, 151: 3371-3379. 10.1099/mic.0.28193-0.PubMedView ArticleGoogle Scholar
  35. Aras RA, Kang J, Tschumi AI, Harasaki Y, Blaser MJ: Extensive repetitive DNA facilitates prokaryotic genome plasticity. Proc Natl Acad Sci USA. 2003, 100: 13579-13584. 10.1073/pnas.1735481100.PubMed CentralPubMedView ArticleGoogle Scholar
  36. Mira A, Klasson L, Andersson SG: Microbial genome evolution: sources of variability. Curr Opin Microbiol. 2002, 5: 506-512. 10.1016/S1369-5274(02)00358-2.PubMedView ArticleGoogle Scholar
  37. Rose JE, Meyer DH, Fives-Taylor PM: Aae, an autotransporter involved in adhesion of Actinobacillus actinomycetemcomitans to epithelial cells. Infect Immun. 2003, 71: 2384-2393. 10.1128/IAI.71.5.2384-2393.2003.PubMed CentralPubMedView ArticleGoogle Scholar
  38. Liu GR, Liu WQ, Johnston RN, Sanderson KE, Li SX, Liu SL: Genome plasticity and ori-ter rebalancing in Salmonella typhi. Mol Biol Evol. 2006, 23: 365-371. 10.1093/molbev/msj042.PubMedView ArticleGoogle Scholar
  39. Konstantinidis KT, Ramette A, Tiedje JM: The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci. 2006, 361: 1929-1940. 10.1098/rstb.2006.1920.PubMed CentralPubMedView ArticleGoogle Scholar
  40. Richter M, Rossello-Mora R: Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA. 2009, 106: 19126-19131. 10.1073/pnas.0906412106.PubMed CentralPubMedView ArticleGoogle Scholar
  41. Wang Y, Goodman SD, Redfield RJ, Chen C: Natural transformation and DNA uptake signal sequences in Actinobacillus actinomycetemcomitans. J Bacteriol. 2002, 184: 3442-3449. 10.1128/JB.184.13.3442-3449.2002.PubMed CentralPubMedView ArticleGoogle Scholar
  42. Fujise O, Lakio L, Wang Y, Asikainen S, Chen C: Clonal distribution of natural competence in Actinobacillus actinomycetemcomitans. Oral Microbiol Immunol. 2004, 19: 340-342. 10.1111/j.1399-302x.2004.00157.x.PubMedView ArticleGoogle Scholar
  43. Delcher AL, Bratke KA, Powers EC, Salzberg SL: Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics. 2007, 23: 673-679. 10.1093/bioinformatics/btm009.PubMed CentralPubMedView ArticleGoogle Scholar
  44. Slater GS, Birney E: Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics. 2005, 6: 31-10.1186/1471-2105-6-31.PubMed CentralPubMedView ArticleGoogle Scholar
  45. Lowe TM, Eddy SR: tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997, 25: 955-964. 10.1093/nar/25.5.955.PubMed CentralPubMedView ArticleGoogle Scholar
  46. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215: 403-410.PubMedView ArticleGoogle Scholar
  47. Darling AC, Mau B, Blattner FR, Perna NT: Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 2004, 14: 1394-1403. 10.1101/gr.2289704.PubMed CentralPubMedView ArticleGoogle Scholar
  48. Tesler G: GRIMM: genome rearrangements web server. Bioinformatics. 2002, 18: 492-493. 10.1093/bioinformatics/18.3.492.PubMedView ArticleGoogle Scholar
  49. Rozen S, Skaletsky HJ: Primer3 on the www for general users and for biologist programmers. Bioinformatics Methods and Protocols: Methods in Molecular Biology. Edited by: Krawetz S, Misener S. 2000, Totowa, NJ: Humana Press, 365-386.Google Scholar
  50. Chen W, Wang Y, Chen C: Identification of a genomic island of Actinobacillus actinomycetemcomitans. J Periodontol. 2005, 76: 2052-2060. 10.1902/jop.2005.76.11-S.2052.PubMedView ArticleGoogle Scholar
  51. Profiling of Escherichia coli chromosome (PEC) database. [http://www.shigen.nig.ac.jp/ecoli/pec/index.jsp]
  52. Carbone A, Zinovyev A, Kepes F: Codon adaptation index as a measure of dominating codon bias. Bioinformatics. 2003, 19: 2005-2015. 10.1093/bioinformatics/btg272.PubMedView ArticleGoogle Scholar
  53. Database of prOkaryotic OpeRons (DOOR) tool. [http://csbl1.bmb.uga.edu/OperonDB/operon_prediction.php]
  54. Mao F, Dam P, Chou J, Olman V, Xu Y: DOOR: a database for prokaryotic operons. Nucleic Acids Res. 2009, 37: D459-463. 10.1093/nar/gkn757.PubMed CentralPubMedView ArticleGoogle Scholar
  55. FGENESB. [http://linux1.softberry.com/berry.phtml?topic=fgenesb&group=programs&subgroup=gfindb]

Copyright

© Kittichotirat et al; licensee BioMed Central Ltd. 2010

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.