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Journal of Bacteriology, August 2006, p. 6016-6019, Vol. 188, No. 16
0021-9193/06/$08.00+0     doi:10.1128/JB.00330-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Analysis of the Terminus Region of the Caulobacter crescentus Chromosome and Identification of the dif Site

Rasmus B. Jensen*

Department of Life Sciences and Chemistry, Roskilde University, Universitetsvej 1, DK-4000 Roskilde, Denmark

Received 7 March 2006/ Accepted 8 June 2006


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ABSTRACT
 
The terminus region of the Caulobacter crescentus chromosome and the dif chromosome dimer resolution site were characterized. The Caulobacter genome contains skewed sequences that abruptly switch strands at dif and may have roles in chromosome maintenance and segregation. Absence of dif or the XerCD recombinase results in a chromosome segregation defect. The Caulobacter terminus region is unusual, since it contains many essential or highly expressed genes.


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TEXT
 
Caulobacter crescentus, like most bacteria, possesses a single circular chromosome where DNA replication initiates at a unique origin of replication (Cori) and proceeds bidirectionally (4, 5). The terminus (ter) region, where the two replication forks meet resulting in termination of DNA replication, has not been identified in Caulobacter. The terminus regions in Escherichia coli and Bacillus subtilis contain several determinants involved in sister chromosome separation, including DNA replication termination sites, the dif recombination site, and polar sequences involved in proper sorting of the daughter chromosomes away from the septum (6, 20). The dif site is the chromosomal target of the XerCD site-specific recombinase that resolves chromosome dimers into monomers to allow chromosome segregation before completion of cell division (17). The Caulobacter genome does not contain obvious homologs of the E. coli or the B. subtilis termination systems, and the dif site has not been identified (15).

To define and characterize the Caulobacter ter region, a combined bioinformatics and experimental approach was used. Most bacterial chromosomes show differences in leading and lagging strand sequence composition that can be used to identify origin and terminus regions (24). An analysis of skewed oligonucleotides, G/C and A/T skew, in the Caulobacter genome (available at http://www.cbs.dtu.dk/services/GenomeAtlas/) (33) indicated that ter is located near position 2.0 Mb, the region opposite Cori on the genome map. Since certain highly skewed sequences in the E. coli genome abruptly shift from one strand to the other at the dif recombination site in the ter region (20), highly skewed oligonucleotide motifs in the Caulobacter genome were identified to accurately determine the location of Caulobacter ter and characterize the general features of this region. Based on the genome-wide most significantly skewed 1- to 8-bp motifs (available at http://www.cbs.dtu.dk/services/GenomeAtlas/) (10, 33) and using the program Winseq (Flemming G. Hansen, The Technical University of Denmark, Lyngby, Denmark), the 8-bp motifs GCGGTGGT (or GCKGTGGT) and GGGCRGGG were identified as highly skewed sequences in the Caulobacter genome that show an abrupt strand switch near the predicted ter region (Fig. 1 and 2). Expected skews and number of sites were calculated using the strand-specific base composition as described previously (19), and the same conclusions were reached using the strand-specific dinucleotide frequencies. The statistical analysis was performed using the SAS JMP program.


Figure 1
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  		FIG. 1. Distribution of GCGGTGGT sequences in the Caulobacter chromosome. (A) Vertical lines represents GCGGTGGT motifs present on the top strand (upper row) or on the complementary strand (lower row). Numbering of the Caulobacter genome sequence is according to reference 21, and initiation of DNA replication takes place near position 0 (4). (B) Percentage of GCGGTGGT sequences on the top DNA strand showing significant skew of this motif. Each bar corresponds to a 100-kb region of the chromosome.


Figure 2
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  		FIG. 2. Distribution of GGGCRGGG sequences in the Caulobacter chromosome (R is A or G). (A) Vertical lines represents GGGCRGGG motifs present on the top strand (upper row) or on the complementary strand (lower row) in either the complete genome or in the ter region of the chromosome. The GGGCRGGG motif shifts from primarily being on the top DNA strand to the complementary strand near position 1.95 Mb. (B) Percentage of GGGCRGGG sequences on the top DNA strand, showing strong polarization of this motif and increasing skew towards the terminus. Each bar corresponds to a 100-kb region of the chromosome.

The GCGGTGGT motif (Fig. 1) has a skew of 83.5%, significantly more than the expected skew of 54.5% (P = 10–40, calculated using the binominal distribution mass function), and it is present in more copies than expected (478 sites observed versus 312 expected; P = 10–21, calculated using the Poisson distribution). The GCGGTGGT motif is significantly depleted in the 1.8- to 2.2-Mb region (P = 2 x 10–7, t test of the number of sites in 100-kb regions around the chromosome), especially on the predicted leading strand. This region corresponds to the predicted ter region of the Caulobacter chromosome. The GCGGTGGT motif shows homology to and similarity in the genome distribution pattern to the experimentally identified E. coli, Haemophilus influenzae, and Lactococcus lactis Chi sites (3, 7, 25, 26) involved in DNA repair by homologous recombination, so the Caulobacter GCGGTGGT motif may have a similar function.

The GGGCRGGG motif (Fig. 2) has a genome-wide skew of 79.4%, significantly more than the expected skew of 54.3% (P = 10–74). The skew is smallest near the origin and increases gradually towards the predicted terminus, where a skew of 94.3% is observed in the 200-kb region surrounding the putative dif site (Fig. 2 and 3). The GGGCRGGG motif is present in approximately the number of copies expected on the leading strand (965 sites observed versus 1,058 expected), but the motif is significantly depleted in the lagging strand (251 sites observed versus 892 expected; P = 10–141). The distribution of distances between adjacent GGGCRGGG motifs is consistent with an exponential distribution with a mean of 3,295 bp (P = 0.01), suggesting that the GGGCRGGG motifs are randomly distributed on the Caulobacter genome. This motif is very similar to the genetically and biochemically identified DNA motifs (named KOPS and FRS) that control FtsK-dependent movement of the terminus-proximal part of the E. coli chromosome (2, 18). In that organism, the FtsK recognition sequences are highly skewed with the orientation switching abruptly at the dif resolution site. In vitro, FtsK proteins move along DNA molecules and translocation across the FtsK recognition sequence from one direction causes FtsK proteins to pause and reverse direction (18, 22). Since FtsK is anchored at the invaginating septum, the sequence-directed DNA translocation activity causes the dif-proximal region of the chromosome to be actively moved to midcell, where FtsK activates XerCD-mediated resolution of dimeric chromosomes at the dif site and topoisomerase IV-mediated decatenation of daughter chromosomes (1, 9, 23, 27). The Caulobacter chromosomal ter region appears to be positioned near the invaginating septum in predivisional cells (13, 14). FtsK is present in Caulobacter and the protein is essential, localized at the invaginating septum and involved in terminus segregation (31), so it is likely that the GGGCRGGG motifs are involved in FtsK-mediated chromosome segregation in Caulobacter.


Figure 3
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  		FIG. 3. Identification of the Caulobacter dif site in the ter region. (A) Organization of the chromosome region containing the dif site. Arrows indicate the location and orientation of annotated genes (21). Genes marked with names have a predicted function, and genes marked with CC are hypothetical genes of unknown function. The Caulobacter dif site is located in the intergenic region between CC1763 and aceA, and the sequence of the site is shown. (B and C) Normal cells and filamentous cells with multiple constrictions from exponentially growing cultures of RBJ225 (CB15N {Delta}xerD) and RBJ221 (CB15N {Delta}dif), respectively. Shown are a phase-contrast microscopy images (left) and the fluorescence signal from chromosomal DNA stained using DAPI (right). The bar equals 5 µm. The indentations are abnormally long in the filamentous cells and chromosomal DNA is present at the indentations (arrows), indicating a defect in chromosome segregation. (D) Intracellular location of the ter region of the chromosome in abnormal {Delta}xerD cells visualized using CFP-LacI binding to a lac operator cassette inserted 22 kb from the dif site. Shown are overlays of phase-contrast and CFP fluorescence images.

The analysis of skewed oligonucleotides (Fig. 1 and 2) suggests that the Caulobacter dif site is located near position 1.95 Mb, since the oligonucleotides abruptly switch strands near that position. To identify the dif site, a sequence motif was constructed based on XerCD binding sites from different bacterial chromosomes and plasmids (17) and used to search the Caulobacter genome sequence. The best match is located at position 1,946,376 bp (Fig. 3A), in the region of the chromosome expected to be the ter region. This site is located 62 kb from the position in the genome opposite Cori, where the two replication forks are expected to meet. The putative dif site is located in a 262-bp intergenic region between CC1763, a putative transcriptional regulator of unknown function, and an operon encoding the glyoxalate cycle enzymes AceA (isocitrate lyase) and AceB (malate synthase). To experimentally verify that this is the Caulobacter dif site, a plasmid with a 24-bp deletion in the putative dif site was constructed by cloning a PCR fragment, and the chromosomal site was replaced with the deletion using a two-step recombination procedure (8), resulting in strain RBJ221 (CB15N {Delta}dif). Control strains with knockouts of xerC or xerD were constructed by PCR amplifying regions of the chromosome overlapping the start of the genes, cloning them in the plasmid pCR2.1-TOPO (Invitrogen), and integrating these plasmids into the Caulobacter genome at xerC or xerD by a single recombination event, thereby insertionally inactivating the genes, resulting in strains RBJ223 (CB15N {Delta}xerC) and RBJ225 (CB15N {Delta}xerD). In exponentially growing cultures of the RBJ221, RBJ223, and RBJ225 strains, 2 to 4% of the cells are filamentous and show multiple deep and frequently elongated invaginations (Fig. 3B and C). Less than 0.1% of exponentially growing wild-type CB15N cells have multiple invaginations. The xerC and xerD deletions can be complemented by the respective genes on a plasmid, whereas the deletion of the putative dif site cannot be complemented in trans, suggesting that it is a cis-acting site. Staining of the chromosomal DNA using 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) as described previously (13) shows that DNA is present at the invagina-tions in RBJ221, RBJ223, and RBJ225 cells with multiple constrictions (Fig. 3B and C). The phenotypes of these cells are similar to the phenotypes of various Caulobacter mutants with chromosome segregation defects (including topoisomerase IV and smc mutants), Caulobacter cells lacking the C-terminal part of FtsK, and E. coli dif, xerC, and xerD mutants (11, 14, 31, 32) (data not shown). Thus, the cell division defects in the RBJ221, RBJ223, and RBJ225 strains are most likely caused by lack of DNA clearance from the division plane, indicating a defect in chromosome segregation. To examine the intracellular location of the putative dif site and ter region separation in these strains, this region of the chromosome was tagged by inserting a tandem lac operator array into the ter region and a LacI-cyan fluorescent protein (CFP) expression construct into the {Delta}xer and {Delta}dif strains by generalized transduction (8) using a {phi}CR30 lysate prepared from strain ML133 (30). When the LacI-CFP fusion protein is expressed as described previously (13), the majority of the abnormal cells with multiple or elongated constrictions have single ter foci near the constriction sites and cells with double ter foci, representing cells with separated ter regions, are rarely observed (Fig. 3D). This indicates a defect in ter region separation in the cells with multiple or elongated constrictions. Thus, deletion of the putative Caulobacter dif site at 1.95 Mb gives the same chromosome segregation, ter region separation, and cell division defect phenotypes as are given by the absence of XerC or XerD, suggesting that this site is dif. When the Caulobacter dif site is compared with dif sites from other bacterial chromosomes (17), it is observed that the XerD binding site and the central region between the XerC and XerD binding sites are highly conserved. However, the Caulobacter XerC binding site diverges very significantly from other known XerC binding sites. The Caulobacter XerCD recombinase binds poorly in vitro to the E. coli dif site (15), supporting the observation that the Caulobacter dif site has a significantly different sequence.

Terminus regions are generally poorly conserved, even between closely related species (28), and are often a target of integration of nonessential and poorly transcribed exogenous DNA. As an example, 350 kb of the E. coli terminus region can be deleted (12) and the genes in the terminus region on average have low transcript levels (29). In contrast, the Caulobacter terminus region contains many essential genes, including genes involved in central metabolism or cell cycle progression, and many genes predicted to be highly expressed (16, 24). Further characterization of the terminus region in Caulobacter and other bacteria may give important information about the functions and evolutionary constraints of this important chromosome region.

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ACKNOWLEDGMENTS
 
I thank Tove Atlung and Ole Skovgaard for critical reading of the paper and Nynne M. Forchhammer for help with the statistical analysis.

This work was supported by grants from The Danish Natural Sciences Research Council and The Carlsberg Foundation.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Life Sciences and Chemistry, Roskilde University, Universitetsvej 1, DK-4000 Roskilde, Denmark. Phone: 45 46742471. Fax: 45 46743011. E-mail: rbjensen{at}ruc.dk. Back


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Journal of Bacteriology, August 2006, p. 6016-6019, Vol. 188, No. 16
0021-9193/06/$08.00+0     doi:10.1128/JB.00330-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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