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Genome sequence and phenotypic characterization of
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Caulobacter segnis
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Sagar Patel, Brock Fletcher, Derrick C. Scott, and Bert Ely
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Department of Biological Sciences, University of South Carolina, Columbia, SC 29208
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Keywords: pseudogene, genome sequence, genome annotation, Caulobacter
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Running title: Caulobacter segnis genome
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Corresponding author: B. Ely, ely@biol.sc.edu, 803-777-2768
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Abstract
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Caulobacter segnis is a unique species of Caulobacter that was initially deemed Mycoplana segnis because it was
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isolated from soil and appeared to share a number of features with other Mycoplana. After a 16S rDNA analysis
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showed that it was closely related to Caulobacter crescentus, it was reclassified Caulobacter segnis. Because the C.
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segnis genome sequence available in GenBank contained 126 pseudogenes, we compared the original sequencing
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data to the GenBank sequence and determined that many of the pseudogenes were due to sequence errors in the
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GenBank sequence. Consequently, we used multiple approaches to correct and reannotate the C. segnis genome
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sequence. In total, we deleted 247 bp, added 14 bp, and changed 8 bp resulting in 233 fewer bases in our corrected
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sequence. The corrected sequence contains only 15 pseudogenes compared to 126 in the original annotation.
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Furthermore we found that unlike Mycoplana, C. segnis divides by fission, producing swarmer cells that have a
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single, polar flagellum.
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Introduction
Caulobacter segnis was isolated from soil samples by Takahashi and Komahara [16]. It was initially
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classified Mycoplana segnis (TK0059) by Urakami et al. [18] due to shared phenotypic characteristics with the
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genus Mycoplana. Mycoplana are soil dwelling bacteria with branching cells that have the ability to decompose
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aromatic compounds. The phenotypic comparison was considered to be accurate at the time because M. segnis was
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isolated from the soil and produced 7-amino-3-methylcephem. Yet it had low levels of DNA-DNA homology with
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the TK0055 (13%), TK 0053(6%), and TK0051 (15%) Mycoplana isolates so it was classified as a separate species
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of Mycoplana. Subsequently, M. segnis was reclassified by Abraham et al. [1] as a Caulobacter after a 16S rDNA
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analysis revealed that M. segnis was most closely related to Caulobacter vibrioides/cresentus. However, C. segnis
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was thought to be morphologically unique from others in its genus. Urakami claimed that C. segnis does not produce
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prosthecae and that it has peritrichous flagella [18]. Also, a lipid analysis showed that C. segnis does not contain the
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equivalent chain lengths 11.798, 15:0, 17:0, 17:1ω6ϲ, and 17:1ω8ϲ which were present in all other Caulobacter
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tested [1].
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In contrast to Mycoplana, the cell cycle of the genus Caulobacter relies on a dimorphic cell division that
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produces two different cells; a non-motile stem cell and a motile immature cell. The non-motile stem cells have
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prosthecae or stalks with holdfast material at the end which allows them to adhere to surfaces and form biofilms
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from which they continually produce swarmer cells. They also use the holdfast material that accumulates at the end
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their prosthecae to attach to the stalks opf other cells to form flowery structures called rosettes. The swarmer cells
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are flagellated and reproduce on a slower timeframe than the stalked cells because they must first mature by
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shedding their flagellum and synthesizing stalks. This delay allows the swarmer cells to search for sites with more
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resources. Since the aquatic environments where Caulobacter are found tend to be nutrient limited, this dimorphic
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division allows them to spread their progeny as well as keep a firm base in the immediate region [9]. Caulobacter
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are gram-negative bacteria with shapes that vary from rods, to fusiform, or vibrioid.
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The nucleotide sequence of the C. segnis genome was determined by the Joint Genome Institute, and the
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genome sequence available in the National Center for Biotechnology Information (NCBI) database contained 4.66
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Mbp, had a 67% G/C content, and contained 4139 protein coding sequences (CDS) [5]. However, this version of the
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C. segnis genome was thought to contain 126 pseudogenes. Since the other sequenced Caulobacter genomes
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contained few pseudogenes, we amplified and resequenced the frameshift-containing region of pseudogene
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Cseg_0004 and found that there was an error in the Genbank nucleotide sequence. When the error was corrected, the
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gene contained a single continuous open reading frame with a deduced amino acid sequence that was 97% identical
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to that of the corresponding C. crescentus NA1000 gene. This result led us to hypothesize that there were probably
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additional errors in the available genome sequence. Fortunately, we were able to obtain the original Roche 454
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sequencing data from the Joint Genome Institute. When we compared the Roche 454 sequencing reads with the
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available C. segnis genome sequence, we found numerous instances where the available genome sequence contained
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single or double base pair insertions or deletions that resulted in inappropriate reading frame interruptions.
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Therefore, one aim of this study was to correct the nucleotide sequence and annotation of the Caulobacter segnis
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genome. Additionally, we examined the cell morphology and growth patterns of C. segnis and showed that it divides
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by fission to produce a swarmer cell that has a single polar flagellum. Thus, the cell morphology and growth pattern
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are typical of those observed with other Caulobacter species.
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Materials and Methods
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Correction of the Caulobacter segnis genome sequence
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The annotated C. segnis genome sequence was downloaded from NCBI
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(http://www.ncbi.nlm.nih.gov/genome/1934) and compared to the original Roche 454 sequencing dataset that was
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obtained from the Department of Energy Joint Genome Institute. The original sequencing data were viewed as
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individual reads using the Tablet software [14]. Contigs compiled from the consensus of the original reads were
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compared with the annotated version of the genome using the Mauve Multiple Genome Alignment software [7]. The
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454 data were assumed to be correct when there was a clear consensus with at least five reads with identical
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sequence. When correction of the sequence resulted in the combination of two open reading frames, the predicted
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amino acid sequence of the combined reading frame was compared to those in the NCBI database using the Basic
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Local Alignment Search Tool (BLAST) [2]. If significant matches were observed, the annotation of the gene was
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corrected as well. After correcting the genome nucleotide sequence, the genome sequence was submitted to the
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Rapid Annotation using Subsystem Technology (RAST) for automated annotation [4]. The two annotations were
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then compared manually at regions of sequence change, and the Artemis: Genome Browser and Annotation Tool
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was used to update the C. segnis genome annotation that had been downloaded from GenBank [15]. When
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differences were observed, the deduced amino acid sequences of the predicted genes were compared to those in the
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NCBI database using BLAST. A consensus of five or more matches identifying the same gene function (<1e-6) was
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used as the basis for determining feature size and feature function. Finally both the RAST annotated sequence and
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our annotated sequence were analysed using the MICheck: Microbial genome checker to verify predicted CDSs [6].
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Conflicting results in MICheck were viewed and the corresponding amino acid sequence was compared to the NCBI
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database using BLAST [2]. If the predicted CDS had at least five significant matches (<1e-6), then it was retained as
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a valid gene.
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Growth of C. segnis
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Caulobacter segnis strain TK0059 was obtained from Dr. Yves Brun (Indiana University). Strain TK0059
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was grown in peptone yeast extract (PYE) medium [11] or PYE supplemented with 10 mM glucose, 30 mM
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monosodium L-glutamate. For PYE agar plates, the broth was supplemented with 1.2% agar. TK0059 was also
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grown on minimal medium (M2) plus glucose to determine the basic growth requirements [11]. To select for
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streptomycin resistant TK0059, 100 μL of a TK0059 culture was spread on a PYE plate containing 50 μg/ml
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streptomycin. The resulting colonies were purified by two rounds of single colony isolation. Cells were stained for
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holdfast using the procedure described by Janakiraman and Brun [10] using C. crescentus as a positive control.
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Pulsed Field Gel Electrophoresis (PFGE)
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TK0059 genomic DNA in agarose plugs for PFGE was isolated using the protocol of Dingwall et al. [8].
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Restriction digests using the AseI, SpeI, and SwaI enzymes were performed for 4 h according to the manufacturer’s
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specification. PFGE was carried out in a 1% agarose gel (1.5 g pulsed field gel agarose and 150 mL 1X SBA (35
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mM Boric Acid, 10 mM NaOH, pH=8.5)).
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TEM staining protocol
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To stain the C. segnis TK0059 cell sample for transmission electron microscopy (TEM), 5 mL of liquid
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culture was centrifuged at 1157 x g for 10 minutes and the supernatant was discarded. The pellet was resuspended
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gently in 2 mL of distilled water. Subsequently, 20 μL of the resuspended cells was mixed with 20 μl of a 2%
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solution of phosphotungstic acid pH 7. A copper grid was inverted on the mixture for 30 seconds and then carefully
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dried by touching filter paper to side of grid. The grid was allowed to dry for 15 minutes before use in the TEM.
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Results and Discussion
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Sequence correction and reannotation
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The C. segnis TK0059 genome is one of three Caulobacter genomes that are present as completed genomes
in the NCBI database. The genomes of the C. crescentus strains NA1000 and CB15 are two laboratory versions of
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the same isolate and contain only minor sequence differences [13]. Neither the C. crescentus genome nor the more
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distantly-related Caulobacter K31 genome contains more than 20 pseudogenes. However, 126 pseudogenes are
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present in the Genbank version of the C. segnis TK0059 genome suggesting possible errors in the annotation and/or
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the sequencing data. Consequently, the nucleotide sequences of the 74 original contigs, assembled for the TK0059
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genome by the Joint Genome Institute (JGI), were aligned with the NCBI version of the TK0059 genome nucleotide
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sequence and visually compared using Mauve. When sequence differences were observed, the original sequence
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reads were examined to identify the correct sequence information. In most cases, the sequencing reads were
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identical. When variation in the sequencing reads was observed, it usually involved only a few reads that differed
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from the majority in the number of times a base was repeated. Therefore, we were able to correct the genome
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sequence based on the accuracy of the original 454 sequencing reads. In total, this process resulted in the removal
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247 base pairs from 157 sites and the addition of 14 base pairs at 12 sites (Figure 1d). In addition, we corrected
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single base pair errors at 8 sites.
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Next we examined the original 126 pseudogenes to determine if they were true pseudogenes and found that
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11 had no significant matches in the NCBI database, suggesting that they were merely non-coding regions. Also, 12
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of the 126 pseudogenes appeared to be true genes that coded for proteins without the need of any sequence changes,
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and finally the sequence corrections described above had converted 89 of the remaining pseudogenes into intact
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coding regions (Supplementary Table S1). An example of a sequence change that converted a pseudogene into an
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intact coding region can be observed in Figure 1 in which the NCBI sequence of Cseg_3308 contains an added
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“CA” that was not present in the original 454 sequencing data. When the extra CA nucleotides were removed, the
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two reading frames were merged to form a single open reading frame that corresponded to those of the homologous
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beta-glucuronidase-like protein genes found in other Caulobacter species (Figure 1). Based on these results, we
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concluded that Cseg_3308 was not a pseudogene, and the annotation of the gene was changed to include the relevant
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information about the gene. After making these corrections, the genome sequence contained only 14 of the original
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126 pseudogenes. However, we found that Cseg_1746 is actually a pseudogene since a sequence correction reduced
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a series of seven G nucleotides to six, resulting in a shift in the reading frame that would produce a truncated version
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of the resulting protein. Thus, the corrected C. segnis TK0059 genome sequence contains a total of 15 pseudogenes.
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One of these pseudogenes codes for two segments of the translation initiation factor IF2. Since the start and stop
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codons of these two segments overlap, it is possible that the two peptides form a functional IF2 protein.
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Alternatively the larger C-terminal peptide may perform the critical functions of IF2 as has been observed in E. coli
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mutants [12]. Two other pseudogenes, Cseg_747 and Cseg_2894, contain internal stop codons that would interrupt
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translation, but neither mutation would cause the loss of a critical function. The remaining 11 pseudogenes are
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merely small portions of genes that are common in the NCBI database and may not be expressed in C. segnis.
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To check the corrected annotation of the TK0059 genome, the corrected genome nucleotide sequence was
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annotated using RAST. RAST features at locations of sequence change were then compared to the feature
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descriptions that we generated manually for verification of correct feature creation. Discrepancies between the two
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were analysed using BLAST to determine the correct annotation. Subsequently, both the RAST annotation and our
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edited version were evaluated using the Microbial Genome Checker (MICheck) to check for annotation errors.
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MICheck suggested that the newly corrected genome contained five unnecessary features and that the RAST
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annotation contained nearly 20. These discrepancies were analysed using BLAST and four of the five features
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flagged by MICheck were deleted from the corrected annotation and one of the RAST annotation genes was
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accepted into the new annotation.
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Confirmation of the C. segnis TK0059 contig assembly
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PFGE analysis was used to determine if the original 76 C. segnis TK0059 contigs were assembled in the
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correct order in the finished sequence (Figure 2). The predicted DNA fragments from restriction digests of TK0059
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genomic DNA using AseI, SpeI, and SwaI are shown in Figure 2d. Several gels were run using different conditions
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to separate specific size ranges, and all of the large predicted bands were observed in the AseI and SwaI digests and
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all four predicted fragments were observed in the SpeI digest. Since no unexplained bands were observed, the
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correlation between the predicted and observed restriction fragments confirms that the assembly of the TK0059
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genome is correct.
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Characteristics of the C. segnis genome
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The revised Caulobacter segnis TK0059 genome is a single 4,655,405 base pair chromosome with a 67.7%
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GC content and approximately 4250 protein coding genes. The TK0059 genome is larger than the 4 Mb NA1000
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genome, but smaller than the 5.48 Mb K31 genome (Table 1). It does not contain any transposase genes even though
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the other two genomes contain approximately 10 transposase genes per million base pairs. The two rRNA operons
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are flanked by the same genes as the corresponding rRNA operons in NA1000 indicating a conservation of gene
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order between these two species. The TK0059 and NA1000 genomes also have no differences in the number or
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identity of the tRNA genes whereas the more distantly related K31 genome has differences in five tRNA genes
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compared to the TK0059 genome. These data are consistent with the idea that C. segnis TK0059 and C. crescentus
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NA1000 are closely related as demonstrated by Abraham et al. [1] using 16S rRNA sequences.
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A comparison of the C. segnis TK0059 genome to the NA1000 and K31 genomes revealed four large
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regions that were found only in the TK0059 genome suggesting that these regions have been inserted into the C.
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segnis genome and are not deletions that occurred independently in the same places in both of the other genomes
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(Figure 3). Upon closer inspection, we found that two of the four regions were adjacent to a tRNA gene and a third
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was flanked by two tRNA genes. This observation suggested that tRNA genes may play a major role in the
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nonhomologous recombination that occurs after a horizontal gene transfer event. Further inspection revealed that 29
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out of the 51 C. segnis tRNA genes were adjacent to nucleotide sequences that were not present in the NA1000
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genome. Excluding the tRNAs that are located in rRNA operons that means that nearly two-thirds of the remaining
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C. segnis tRNAs have been involved in an event that created an insertion or deletion. In the NA1000 and K31
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genomes, transposase genes appear to be involved in a substantial percentage of the insertion events [3]. However,
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since the C. segnis TK0059 genome does not contain any transposase genes the role of tRNAs in insertion and
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deletion events is magnified.
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Origin comparison
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The C. segnis origin of replication (oriC) is similar to the origin region that has been described for other
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Caulobacter strains [17]. It contains four CcrM methylation sites, three CtrA binding sites, and four weak W DnaA
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binding sites. The 60 bp highly conserved region of oriC described by Taylor et al. [17] differs from that of NA1000
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at only two nucleotide positions and contains one of the W sites, one of the CtrA binding sites, and a G-box that
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binds DnaA with moderate affinity. When comparing 226 genes downstream and 56 genes upstream of the origin of
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replication in the C. segnis TK0059 genome to the corresponding region in the C. crescentus NA1000 genome, we
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found many regions of gene homology and long stretches of conserved sequences, consistent with the close
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relationship of the two species (Figure 4). However, 11 small inversions were observed in this region. Five were
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local inversions where a set of contiguous genes was inverted without changing its chromosomal location. The other
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six were inversions that included the origin of replication and varying numbers of flanking genes such that a set of
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genes ended up in the opposite orientation on the other side of the origin. In addition, we found 54 genes that were
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present in only one of the two genomes and could be attributed to insertion or deletion events when these regions of
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the TK0059 and NA1000 genomes were compared with that of K31 (Table 2). Thus about 20% of the genes in this
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region are unique to one genome or the other. Furthermore, in both genomes, insertions greatly outnumber deletions
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suggesting that the two genomes may have increased in size compared to the genome of their shared ancestor.
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Phenotypic characterization
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C. segnis TK0059 cells are crescent-shaped bacteria, and their genome contains a crescentin gene that
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codes for a protein that has 90% amino acid identity to the C. crescentus crescentin. They form colonies that are
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white in color and tend to be mucoid, probably due to extracellular polysaccharide production. Capsules were
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observed by electron microscopy around some of the cells grown in liquid cultures. In contrast to the description
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provided by Urakami et al. [18], predivisional cells typical of Caulobacter are readily observed in C. segnis TK0059
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cultures (Figure 5), suggesting that it divides by fission, producing a mature cell that functions as a stem cell and an
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immature swarmer cell after each round of cell division. This idea is supported by the presence of C. segnis genes
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coding for the cell cycle regulatory protein CtrA, CcrM, DnaA, and SciP with predicted amino acid identities
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ranging from 93 to 100%.
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TK0059 was given the species designation segnis because Urakami et al. considered it to be slow growing,
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but at 30°C, TK0059 has a doubling time of approximately 100 minutes which is similar to that of the C. crescentus
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NA1000 strain. In agreement with Urakami et al. [18], stalks were not present, although approximately 1% of the
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cells have terminal structures that resemble incipient stalks (Figure 6a). Unlike other Caulobacter, TK0059 is
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missing all but one of the genes responsible for holdfast production, and lectin binding experiments demonstrated
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that holdfast was not produced. Without holdfast production, TK0059 would not be expected to form rosettes.
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However, clumps of cells were present in TK0059 cultures (Figure 5), suggesting that Caulobacters may have some
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secondary form of adhesion. Additionally, TK0059 swarmer cells have a single polar flagellum and multiple or
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nonpolar flagella where never observed (Figure 6b) whereas Urakami et al. [18] stated that TK0059 swarmer cells
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had peritrichous flagella. However, we did confirm that riboflavin is required for growth in defined media as
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described by Urakami et al. [18], and we found that a highly conserved operon containing five genes necessary for
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riboflavin synthesis (Ash et al,. submitted for publication) has been deleted from the TK0059 genome. This loss of a
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riboflavin operon appears to be an independent event from that observed by Ash et al. [3] in the K31 genome since
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four additional genes were deleted from the TK0059 genome along with the riboflavin synthesis operon while nine
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genes plus the riboflavin operon were deleted from the K31 genome.
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Acknowledgements The authors wish to thank the DOE Joint Genome Institute for providing the original
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sequencing data for the C. segnis genome and Dr. Yves Brun for providing a culture of C. segnis TK0059 and for
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submitting the corrected genome sequence and annotation to NCBI. We also thank Dr. Aretha Fiebig for helpful
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discussions about holdfast genes.
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Figure legends
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Fig. 1 (a) A comparison of the NCBI sequence of the C. segnis genome versus the Roche 454 contig 18 nucleotide
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sequence for a portion of the Cseg_3308 gene, reveals two additional base pairs in the NCBI version of the C. segnis
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genome sequence. Deletion of the two nucleotides changed the +1 reading frame so that the four stop codons
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designated by the vertical lines in the red oval (panel b) were moved to the +2 reading frame, creating a single open
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reading frame for the Cseg_3308 gene (corresponding to the blue bar in the +1 reading frame in panel c and the red
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line in the plot of third codon position GC content at the top of panel c).
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Fig. 2 Pulsed field gel electrophoresis (PFGE) of the C. segnis T0059 genome. (a) An agarose gel subjected to a 70
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sec alternating pulse for 15 h followed by a 120 sec pulse for 11 hours at 6V with a yeast chromosome ladder in lane
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1. Lanes 2-4 contain AseI, SpeI and SwaI digests of the C. segnis chromosome, respectively. (b) An agarose gel
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subjected to a 5 sec initial pulse and ramped to a 120 sec final pulse for 40 hours at 4.5V with a yeast chromosome
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ladder in lane 1 and lambda DNA concatemers in lane 5. Lanes 2-4 contain AseI, SpeI and SwaI digests of the C.
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segnis chromosome, respectively. (c) An agarose gel subjected to a 10 sec initial pulse and ramped to a 20 sec final
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pulse for 16 hours at 6V with a lambda DNA concatemers in lane 15. Lanes 1-12 contain AseI digests of C. segnis
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chromosomes containing Tn5 insertions at random locations. Lanes 13 and 14 were overloaded. All PFGE
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experiments were conducted at 14°C. (d) A table that shows the predicted fragment sizes of the C. segnis
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chromosome after digestion with restriction enzymes AseI, SpeI, and SwaI. Highlighted fragment sizes were
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observed in the PFGE in gels (a-c).
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Fig. 3 Large insertions in the C. segnis genome. Four large insertions designated by colored bars were observed
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when the C. segnis TK0059 genome was compared to the NA1000 and K31 genomes. Notched ends indicate the
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presence of a tRNA gene.
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Fig. 4 Comparison of the C segnis and C. crescentus genomes in a 200 kb region surrounding the origin of
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replication. Homologous regions are indicated by blocks of the same color connected by vertical lines. White space
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that segments a block of color indicates the presence of genetic material that is not present in the other genome.
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White space along the edge of a color block reflects a sliding scale that indicates the degree of nucleotide identity
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between the two genomes.
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Fig. 5 A phase contrast light microscope picture of a TK0059 culture.
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Fig. 6 Electron micrographs of C. segnis cells. (a) A swarmer cell with a single polar flagellum. (b) A stalked cell
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with a short stalk.
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