Abstract

The chromosome of Bacteroides fragilis strain YCH46 was shown to be a single circular DNA molecule of about 5.3 Mb having 16 NotI, seven AscI, and six I-CeuI sites. A physical map of the chromosome was constructed by four independent experimental approaches: linking clone analysis, cross-Southern hybridization, partial restriction digestion, and two-dimensional pulsed-field gel electrophoresis. Six rRNA operons and 10 known genes were localized on the physical map.

Introduction

Gram-negative anaerobic bacteria are often opportunistic pathogens that can be isolated from many types of infection [1]. The anaerobes most frequently isolated from human clinical specimens are members of the genus Bacteroides. One species, B. fragilis, accounts for over half of these isolates and is considered to be the most virulent species in the genus Bacteroides. Although this species shows less phenotypic variability, it is comprised of genetically heterogeneous strains [2]. A DNA–DNA hybridization study by Johnson and Ault led to the distinction of two DNA homology groups, I and II [3]. We also showed the heterogeneity of this anaerobe by PCR-ribotyping using 16S–23S rDNA internal-transcribed spacer sequences [4]. It has also been reported that the pathogenic potential and antimicrobial susceptibility of B. fragilis vary among strains [5,6]. Because of the intraspecies heterogeneity, comparative genomics is essential for understanding the virulence and antimicrobial resistance of this anaerobe. Pulsed-field gel electrophoresis (PFGE) is a useful tool for analysis of differences in the genome structure of many strains, and over a hundred of genome maps have been constructed. Our previous PFGE analysis of B. fragilis revealed an apparent difference between the genome structures of type strain ATCC25285 and clinical strain YCH46 [7]. In the present study, we have constructed a physical and genetic map of B. fragilis using the clinical isolate YCH46 in order to provide useful information for comparative genomics.

Materials and methods

Bacterial strain and culture conditions

B. fragilis strain YCH46 employed in this study was isolated originally from a patient with bacteremia and has been subjected for analysis in this laboratory. The strain was grown anaerobically in 15 ml of GAM broth (Nissui Pharmaceutical Co., Tokyo, Japan) at 37°C to an OD600 of 0.8–1.0 for preparation of the genomic DNA in agarose gel blocks.

PFGE

Preparation of the genomic DNA, restriction digestion of DNA in agarose gel blocks using I-CeuI, NotI and AscI (New England Biolabs, USA), and PFGE analysis with a Bio-Rad CHEF-DRII system were performed as described previously [7]. Two-dimensional (2D)-PFGE was performed as described by Birren and Lai [8].

Preparation of NotI linking clones

The BamHI-generated cosmid library of B. fragilis strain YCH46, that was constructed in a previous study [9], was used for selection of NotI-linking clones. Of 480 cosmid clones, 11 cosmid clones containing a single NotI site were selected and used as probes.

DNA probe

To map 10 known genes of B. fragilis, DNA probes were prepared by amplifying the internal regions of these genes: atpD for the β-subunit of ATP–synthase [10], ccrA for class B β-lactamase [11], fruA for fructanase [12], nanH for neuraminidase [13], glnA for glutamine synthetase [14], leuB for β-isopropylmalate dehyrogenase [15], tuf for elongation factor [16], rprX for two-component regulatory protein [17], fim for the fimbrial subunit [18] and recA[19].

Southern hybridization

After PFGE, the macrorestriction fragments were transferred to Hybond-N+ membranes according to the manufacturer's recommendation (Amersham, UK). The restriction fragments of interest were recovered from the gel using a Geneclean II kit (Bio 101 Inc., USA). The cosmid clones and recovered restriction fragments were labeled with 32P by the random priming method (Megaprime DNA Labelling System from Amersham). Prehybridization, hybridization and washing were carried out according to the standard procedure described by Sambrook et al. [20].

Results and discussion

Resolution of restriction fragments

The rare-cutting restriction enzymes NotI and AscI digest the genomic DNA of Bacteroides species into a relatively small number of large fragments that can be resolved well by PFGE [7]. We used three enzymes for analysis in the construction of a physical map of the B. fragilis YCH46 genome. Under standard electrophoresis conditions (with a ramping time of 5–150 s at 150 V for 48 h), NotI and AscI produced 14 and 5 bands, respectively, from the genome (Fig. 1A). Densitometric analysis suggested that both 350-kb and 65-kb bands in Fig. 1, lane 1 were doublets. The 350-kb band was resolved into two fragments of 360 kb and 340 kb when a smaller amount of DNA was used for PFGE, and the 65-kb band was resolved into two closely spaced fragments of 67 kb and 63 kb by PFGE with a ramping time of 5–12 s at 150 V for 48 h (data not shown). Similarly, the first AscI band from the top in Fig. 1A was a triplet that could be resolved into three fragments of 1420, 1065, and 985 kb by PFGE with a 50–90 s pulse time at 200 V for 24 h (data not shown). Thus, NotI and AscI cleaved the genome into 16 and 7 fragments, respectively, and these fragments were designated as summarized in Table 1. Twenty-three fragments were detected by double digestion of the genomic DNA with NotI and AscI (Fig. 1A, Table 1). Of those 23 fragments, seven fragments, designated ‘a’ through ‘g’ in Table 1, were found only in the double-digestion lane, indicating that these fragments have NotI and AscI ends. The genome size of the B. fragilis strain YCH46 was estimated to be 5295 kb by summing the double-digestion fragments.

1

A: PFGE of NotI and AscI digests of the genomic DNA of B. fragilis YCH46. The following pulse conditions were used with a Bio-Rad CHEF-DRII apparatus at 150 V: pulse time ramped from 5–150 s for 48 h. Fragments of the B. fragilis YCH46 genome were digested with NotI and AscI and with double-digest combinations of these enzymes. The lanes contain B. fragilis YCH46 DNA digest with NotI (lane 1), NotI–AscI (lane 2) and AscI (lane 3). B: Hybridization of the cosmid clone 491 with B. fragilis YCH46 genomic DNA digested with NotI (lane 1), NotI–AscI (lane 2), and AscI (lane 3). Restriction fragments hybridizing with each probe are indicated on the right side of the autoradiograph, and the migration of DNA size standards is shown on the left side. C: Analysis of the partially digested NotI fragments of the B. fragilis YCH46 genome. B. fragilis YCH46 DNA was digested completely (lane 1) and partially (lane 2) with NotI. Lanes labeled M contain a Lamda-ladder PFGE marker.

1

A: PFGE of NotI and AscI digests of the genomic DNA of B. fragilis YCH46. The following pulse conditions were used with a Bio-Rad CHEF-DRII apparatus at 150 V: pulse time ramped from 5–150 s for 48 h. Fragments of the B. fragilis YCH46 genome were digested with NotI and AscI and with double-digest combinations of these enzymes. The lanes contain B. fragilis YCH46 DNA digest with NotI (lane 1), NotI–AscI (lane 2) and AscI (lane 3). B: Hybridization of the cosmid clone 491 with B. fragilis YCH46 genomic DNA digested with NotI (lane 1), NotI–AscI (lane 2), and AscI (lane 3). Restriction fragments hybridizing with each probe are indicated on the right side of the autoradiograph, and the migration of DNA size standards is shown on the left side. C: Analysis of the partially digested NotI fragments of the B. fragilis YCH46 genome. B. fragilis YCH46 DNA was digested completely (lane 1) and partially (lane 2) with NotI. Lanes labeled M contain a Lamda-ladder PFGE marker.

1

Sizes (kb) of restriction fragments of the B. fragilis YCH46 DNA chromosome

 NotNotI+AscAsc
 1255 (N1)  1420 (A1) 
   1065 (A2) 
   985 (A3) 
 750 (N2) 750  
 640 (N3)   
 590 (N4)   
  510 510 (A4) 
  490 490 (A5) 
   420 (A6) 
  370 370 (A7) 
 360 (N5) 360  
 340 (N6) 340  
 300 (N7) 300  
  260 (a)  
 240 (N8) 240  
  230 (b)  
 220 (N9) 220  
 180 (N10) 180  
  170 (c)  
  155 (d)  
 145 (N11) 145  
  115 (e)  
 100 (N12) 100  
  85 (f)  
  75 (g)  
 65 (N13) 67  
 65 (N14) 63  
 45 (N15) 45  
 25 (N16) 25  
Total 5320 5295 5260 
 NotNotI+AscAsc
 1255 (N1)  1420 (A1) 
   1065 (A2) 
   985 (A3) 
 750 (N2) 750  
 640 (N3)   
 590 (N4)   
  510 510 (A4) 
  490 490 (A5) 
   420 (A6) 
  370 370 (A7) 
 360 (N5) 360  
 340 (N6) 340  
 300 (N7) 300  
  260 (a)  
 240 (N8) 240  
  230 (b)  
 220 (N9) 220  
 180 (N10) 180  
  170 (c)  
  155 (d)  
 145 (N11) 145  
  115 (e)  
 100 (N12) 100  
  85 (f)  
  75 (g)  
 65 (N13) 67  
 65 (N14) 63  
 45 (N15) 45  
 25 (N16) 25  
Total 5320 5295 5260 

The fragment sizes represent average values calculated from at least three independent determinations on gel run under standard conditions (5–150-s pulse time at 150 V for 48 h). Letters ‘a’ through ‘g’ indicate the fragments that were observed only in double digestion with NotI and AscI.

Analysis of the fragments from genomic DNA of B. fragilis strain YCH46

The order of the 16 NotI fragments was determined by the following four strategies.

Hybridization with NotI-linking clones

A cosmid library of B. fragilis strain YCH46 constructed in Escherichia coli[9] was screened and 11 NotI-linking clones were obtained out of 480 cosmid clones. Fig. 1B shows an example of the results of hybridization analysis using one of these linking clones, clone 491, as a probe. The results of NotI-linking analysis using all 11 NotI-linking clones are summarized in Table 2. Based on this result, the NotI fragments could be arranged in the order of N10-N7-N5-N1-N6-N13, N9-N14, and N3-N2-N15-N8-N4-N11. In addition, the results also showed that N6, N13 and N11 are contained entirely in A2, and that N1 and N4 are contained partly in this AscI fragment. Thus, NotI fragments were collected into two contiguous sets, N10-N7-N5-N1-N6-N13-N11-N4-N8-N15-N2-N3 and N9-N14. However, the position of N16 could not be determined.

2

Linkage of NotI fragments in the B. fragilis chromosome

NotI fragments linked NotI-linking clone AscI and NotI–AscI fragments hybridized  
N1–N6 129 A3, N6, g  
N6–N13 72 A3, N6, N13  
N14–N9 114 A2, N9, N14  
N11–N4 257 A3, N11, 360-kb  
N4–N8 642 A1, N8, b  
N8–N15 135 A1, N8, N15  
N15–N2 244 A1, N2, N15  
N2–N3 697 A1, N2, d  
N10–N7 82 A2, N7, N10  
N7–N5 491 A2, N7, f  
N5–N1 84 A6, a, c  
NotI fragments linked NotI-linking clone AscI and NotI–AscI fragments hybridized  
N1–N6 129 A3, N6, g  
N6–N13 72 A3, N6, N13  
N14–N9 114 A2, N9, N14  
N11–N4 257 A3, N11, 360-kb  
N4–N8 642 A1, N8, b  
N8–N15 135 A1, N8, N15  
N15–N2 244 A1, N2, N15  
N2–N3 697 A1, N2, d  
N10–N7 82 A2, N7, N10  
N7–N5 491 A2, N7, f  
N5–N1 84 A6, a, c  

a, b, c, d, f, g indicate the fragments that were observed only in double digestion with Not1 and AscI.

Partial digestion with NotI

Partial digestion of the chromosomal DNA with NotI allowed us to link N7 with N10, N6 with N13, and N10 with N12. An example of the results of NotI partial digestion analysis is shown in Fig. 1C. A 480-kb band seen in PFGE gel is most likely a product of two linking NotI fragments, N7 (300 kb) and N10 (180 kb), because Southern blotting showed a hybridization of a partial digestion product to fragments N7 and N10.

Cross-hybridization with NotI and AscI fragments

NotI and AscI fragments of genomic DNA were excised from gels after PFGE and used as probes for cross-hybridization. The results are summarized in Table 3. Cross-hybridization and NotI-linking analysis revealed that AscI fragments of the B. fragilis strain YCH46 chromosome are arranged in the order of A2-A6-A(4,5)-A3-A1-A7.

3

Hybridization of restriction fragments with single- and double-digested DNA

Fragment probe Bands hybridized  
 Single Double  
N1 A3  
 A4 A4  
 A5 A5  
 A6  
N3 A1  
 A2  
 A7 A7  
N4 A1  
 A3 360-kb  
A6 N1  
 N5  
Fragment probe Bands hybridized  
 Single Double  
N1 A3  
 A4 A4  
 A5 A5  
 A6  
N3 A1  
 A2  
 A7 A7  
N4 A1  
 A3 360-kb  
A6 N1  
 N5  

Letters ‘a’ through ‘g’ indicate the fragments that were observed only in double digestion with NotI and AscI

D-PFGE with AscI and I-CeuI

To construct a more detailed physical map and to determine the rRNA genes on the chromosome, we used an intron-encoded restriction endonuclease, I-CeuI, that recognized a highly conserved 19-bp sequence in rrl genes for the large rRNA subunit (23S). Digestion of the genome of strain YCH46 with I-CeuI produced six fragments [7]. The positions of these six fragments on the NotI–AscI physical map were determined by 2D-PFGE. I-CeuI fragments were excised from the gel, digested with AscI, and electrophoresed again, and then the sizes of the resulting fragments were determined (Table 4). In a similar manner, individual AscI fragments were redigested with I-CeuI, and the resulting fragments were analyzed (Table 4). Thus, the relative positions of the I-CeuI and AscI sites were determined.

4

Sizes of the fragments observed in the 2D-PFGE with I-CeuI and AscI

I-CeuAsc
I-CeuI fragment (kb) Fragment (kb) obtained by AscI digest AscI fragment (kb) Fragment (kb) obtained by I-CeuI digest 
2080 1065 1420 845 
 575  575 
 370 1065 1065 
 70 985 985 
1950 985 510 390 
 845  120 
 120 500 185 
575 390  150 
 185  100 
450 350  65 
 100 420 350 
150 150  70 
65 65 370 370 
I-CeuAsc
I-CeuI fragment (kb) Fragment (kb) obtained by AscI digest AscI fragment (kb) Fragment (kb) obtained by I-CeuI digest 
2080 1065 1420 845 
 575  575 
 370 1065 1065 
 70 985 985 
1950 985 510 390 
 845  120 
 120 500 185 
575 390  150 
 185  100 
450 350  65 
 100 420 350 
150 150  70 
65 65 370 370 

Construction of the physical map of B. fragilis strain YCH46

All the above results were combined and the physical map of B. fragilis strain YCH46 was constructed finally as shown in Fig. 2. The positions of six rrl genes were determined also on the physical map. The mapping shown here indicated that B. fragilis strain YCH46 has a single circular genome. In the last decade, most bacterial chromosomes have been shown to be a single circular DNA of various sizes. In contrast, there are several reports showing unique genome structures in several bacteria, including a linear chromosome found in Borrelia burgdorferi[21] and two chromosomes found in Vibrio parahaemolyticus[22]. Physical and genetic mapping provides useful information not only about the features of the whole genome structure but also about the dynamics and evolution of genome organization, such as rearrangement and large deletion or insertion. We mapped 10 known genes of B. fragilis to the physical map and found that these genes are scattered all over the genome (Fig. 2). It is expected that hybridization analysis using these genetic markers against the macrorestriction fragments of the genome from various strains will enable us to determine the evolutional changes in the genome structure of this anaerobe.

2

Physical and genetic map of the chromosome of B. fragilis YCH46. The NotI, AscI, and I-CeuI fragments are indicated by N, A, and C, respectively, from the inner to outer circles. The order of N9 and N14, A4 and A5, and C5 and C6 is presumptive. The position of N16 is not determined. Locations of rRNA genes deduced from restriction analysis with I-CeuI are indicated by arrowheads. The positions of 10 known genes from B. fragilis are as indicated.

2

Physical and genetic map of the chromosome of B. fragilis YCH46. The NotI, AscI, and I-CeuI fragments are indicated by N, A, and C, respectively, from the inner to outer circles. The order of N9 and N14, A4 and A5, and C5 and C6 is presumptive. The position of N16 is not determined. Locations of rRNA genes deduced from restriction analysis with I-CeuI are indicated by arrowheads. The positions of 10 known genes from B. fragilis are as indicated.

In this study, physical mapping of the B. fragilis strain YCH46 chromosome revealed a genomic structure of a single circular chromosome with a size of 5.3 Mb. Physical mapping with the restriction endonuclease I-CeuI showed six rrn loci and their distribution on the chromosome. The whole-genome-sequencing project is becoming more widespread. However, as such projects can only cover a limited number of strains of each species, physical mapping will still have an important role to play in determination of genome structure and size in comparative genomics.

To our knowledge, this is the first report of a physical and genetic map constructed for any Bacteroides species. The availability of this map of B. fragilis strain YCH46 chromosome will contribute to an understanding of the taxonomy and virulence of Bacteroides.

Acknowledgements

We thank Dr. I. Nakamura for supplying B. fragilis and Dr. T. Ono for providing the cosmid library of B. fragilis YCH46 genome. This work was supported by a grant-in-aid from the Ministry of Education, Science and Culture of Japan.

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