Plasmids Bring Additional Capabilities to Caulobacter Isolates.

Caulobacter is a well-studied bacterial genus, but little is known about the plasmids that are found in some wild Caulobacter isolates. We used bioinformatic approaches to identify nine plasmids from seven different Caulobacter strains and grouped them based on their size and the similarity of their repABC, parAB, and mobAB genes. Protein pathway analysis of the genes on the K31p1 and K31p2 plasmids showed many metabolic pathways that would enhance the metabolic versatility of the host strain. In contrast, the CB4 plasmid contained 21 heavy metal resistance genes with the majority coding for proteins that enhance copper resistance. Growth assays of C. henricii CB4 demonstrated increased copper resistance and quantitative PCR showed an increase in the expression of eight heavy metal genes when induced with copper.

S2B, a Temperate Bacteriophage That Infects Caulobacter Crescentus Strain CB15.

The Caulobacter crescentus strain CB15 has been the basis of numerous studies designed to characterize the biphasic life cycle of this bacterium. Here we describe a newly isolated podovirus, designated S2B, which is capable of integrating into the CB15 chromosome by recombining with the 3'-end of a particular tRNA-ser gene. In addition, we show that S2B is a representative of a family of closely related prophages that are present in the genomes of characterized strains from several Alphaproteobacteria genera. In contrast, only distantly related bacteriophage genomes are present in the GenBank database. The 42,846 bp S2B genome includes 262 bp terminal repeats, and it contains 62 genes of which 45 code for proteins of unknown function. Proteins with predicted functions include a T7 DNA polymerase, a T3/T7 RNA polymerase, and a T7 helicase/primase suggesting that S2B is part of the Studiervirinae subfamily of the Autographiviridae family.

Evolutionary history of Caulobacter toxin-antitoxin systems.

Toxin-antitoxin (TA) systems have been studied in many bacterial genera, but a clear understanding of the evolutionary trajectory of TA operons has not emerged. To address this issue, I identified 42 distinct TA operons in three genomes that represent the three branches of the Caulobacter phylogenetic tree. The location of each operon was then examined to determine if the operon was present in eight additional Caulobacter genomes. Most of the 42 TA operons were present at the same chromosomal location in genomes that represent at least two different branches of the Caulobacter phylogenetic tree. This result indicates that the chromosomal location of TA operons is conserved over evolutionary time scales. One the other hand, there were 177 instances where a TA operon was not present at an expected chromosomal location and four instances where only the antitoxin gene was present. Thus, the variable number of TA operons found in each genome appears to be due primarily to the loss of TA operons, and the addition of new TA operons to a genome was relatively rare. An additional feature of the TA operons was that they seemed to accumulate mutations faster than the adjacent genes.

Novel Caulobacter bacteriophages illustrate the diversity of the podovirus genus Rauchvirus.

The podovirus BPP-1 is currently the only member of the Podovirus genus Rauchvirus. Here, we describe three new Caulobacter bacteriophages (Jess A, SR18, and RW) that show genetic similarity to BPP-1 but have many different genetic and structural features that differentiate them from BPP-1. Jess A and SR18 are closely related to each other and should be considered two members of a new species. They share a similar gene order with BPP-1. However, they do not appear to form lysogens or have the tropism switching mechanism that has been described for BPP-1. Bacteriophage RW also exhibits some homology to BPP-1. However, it is quite different from the other three phages, and we propose that it should be considered a representative of a third species of the genus Rauchvirus. Taken together, the differences among these four members of the genus Rauchvirus indicate that this divergent genus has a long evolutionary history and that there are many more rauchviruses waiting to be discovered.

Identification of proteins associated with two diverse Caulobacter phicbkvirus particles.

Genomic evolution among bacteriophages infecting Caulobacter crescentus is inevitable. However, the conservation of the proteins associated with intact phage particles has not been investigated. In this study, we compared the structural proteins associated with two genomically diverse but morphologically similar C. crescentus-infecting bacteriophages, phiCbK and CcrSC. We were able to detect more than 20 proteins that are part of the bacteriophage particle in both phages, and we were able to identify a small number of proteins that were found in only one of the two phage particles. All but one of the genes coding for these structural proteins were located in a region of the genome that had been designated a structural region, confirming the idea that the genes in these phage genomes are clustered according to their function. During the purification process, we also discovered that phiCbk has a replication complex that can be recovered from the cell lysate, and this complex allowed us to identify many of the phage proteins involved in phage genome replication.

Analyses of four new Caulobacter Phicbkviruses indicate independent lineages.

Bacteriophages with genomes larger than 200 kbp are considered giant phages, and the giant Phicbkviruses are the most frequently isolated Caulobacter crescentus phages. In this study, we compare six bacteriophage genomes that differ from the genomes of the majority of Phicbkviruses. Four of these genomes are much larger than those of the rest of the Phicbkviruses, with genome sizes that are more than 250 kbp. A comparison of 16 Phicbkvirus genomes identified a 'core genome' of 69 genes that is present in all of these Phicbkvirus genomes, as well as shared accessory genes and genes that are unique for each phage. Most of the core genes are clustered into the regions coding for structural proteins or those involved in DNA replication. A phylogenetic analysis indicated that these 16 CaulobacterPhicbkvirus genomes are related, but they represent four distinct branches of the Phicbkvirus genomic tree with distantly related branches sharing little nucleotide homology. In contrast, pairwise comparisons within each branch of the phylogenetic tree showed that more than 80 % of the entire genome is shared among phages within a group. This conservation of the genomes within each branch indicates that horizontal gene transfer events between the groups are rare. Therefore, the Phicbkvirus genus consists of at least four different phylogenetic branches that are evolving independently from one another. One of these branches contains a 27-gene inversion relative to the other three branches. Also, an analysis of the tRNA genes showed that they are relatively mobile within the Phicbkvirus genus.

Correction to: The Isolation and Characterization of Kronos, a Novel Caulobacter Rhizosphere Phage that is Similar to Lambdoid Phages.

The original version of this article unfortunately contained mistakes in Table 1 values. Some of the values in "TAY-ASD who received services" were incorrect. The corrected Table 1 is given below.

The Isolation and Characterization of Kronos, a Novel Caulobacter Rhizosphere Phage that is Similar to Lambdoid Phages.

Despite their ubiquity, relatively few bacteriophages have been characterized. Here, we set out to explore Caulobacter bacteriophages (caulophages) in the rhizosphere and characterized Kronos, the first caulophage isolated from the rhizosphere. Kronos is a member of the Siphoviridae family since it has a long flexible tail. In addition, an analysis of the Kronos genome indicated that many of the predicted proteins were distantly related to those of bacteriophages in the lambdoid family. Consistent with this observation, we were able to demonstrate the presence of cos sites that are similar to those found at the ends of lambdoid phage genomes. Moreover, Kronos displayed a relatively rare head and tail morphology compared to other caulophages but was similar to that of the lambdoid phages. Taken together, these data indicate that Kronos is distantly related to lambdoid phages and may represent a new Siphoviridae genus.

Genome Comparisons of Wild Isolates of Caulobacter crescentus Reveal Rates of Inversion and Horizontal Gene Transfer.

Since previous interspecies comparisons of Caulobacter genomes have revealed extensive genome rearrangements, we decided to compare the nucleotide sequences of four C. crescentus genomes, NA1000, CB1, CB2, and CB13. To accomplish this goal, we used PacBio sequencing technology to determine the nucleotide sequence of the CB1, CB2, and CB13 genomes, and obtained each genome sequence as a single contig. To correct for possible sequencing errors, each genome was sequenced twice. The only differences we observed between the two sets of independently determined sequences were random omissions of a single base in a small percentage of the homopolymer regions where a single base is repeated multiple times. Comparisons of these four genomes indicated that horizontal gene transfer events that included small numbers of genes occurred at frequencies in the range of 10-3 to 10-4 insertions per generation. Large insertions were about 100 times less frequent. Also, in contrast to previous interspecies comparisons, we found no genome rearrangements when the closely related NA1000, CB1, and CB2 genomes were compared, and only eight inversions and one translocation when the more distantly related CB13 genome was compared to the other genomes. Thus, we estimate that inversions occur at a rate of one per 10 to 12 million generations in Caulobacter genomes. The inversions seem to be complex events that include the simultaneous creation of indels.

Achieving Accurate Sequence and Annotation Data for Caulobacter vibrioides CB13.

Annotated sequence data are instrumental in nearly all realms of biology. However, the advent of next-generation sequencing has rapidly facilitated an imbalance between accurate sequence data and accurate annotation data. To increase the annotation accuracy of the Caulobacter vibrioides CB13b1a (CB13) genome, we compared the PGAP and RAST annotations of the CB13 genome. A total of 64 unique genes were identified in the PGAP annotation that were either completely or partially absent in the RAST annotation, and a total of 16 genes were identified in the RAST annotation that were not included in the PGAP annotation. Moreover, PGAP identified 73 frameshifted genes and 22 genes with an internal stop. In contrast, RAST annotated the larger segment of these frameshifted genes without indicating a change in reading frame may have occurred. The RAST annotation did not include any genes with internal stop codons, since it chose start codons that were after the internal stop. To confirm the discrepancies between the two annotations and verify the accuracy of the CB13 genome sequence data, we re-sequenced and re-annotated the entire genome and obtained an identical sequence, except in a small number of homopolymer regions. A genome sequence comparison between the two versions allowed us to determine the correct number of bases in each homopolymer region, which eliminated frameshifts for 31 genes annotated as frameshifted genes and removed 24 pseudogenes from the PGAP annotation. Both annotation systems correctly identified genes that were missed by the other system. In addition, PGAP identified conserved gene fragments that represented the beginning of genes, but it employed no corrective method to adjust the reading frame of frameshifted genes or the start sites of genes harboring an internal stop codon. In doing so, the PGAP annotation identified a large number of pseudogenes, which may reflect evolutionary history but likely do not produce gene products. These results demonstrate that re-sequencing and annotation comparisons can be used to increase the accuracy of genomic data and the corresponding gene annotation.

A Genome Comparison of T7-like Podoviruses That Infect Caulobacter crescentus.

Bacteriophages remain an understudied component of bacterial communities. Therefore, our laboratory has initiated an effort to isolate large numbers of bacteriophages that infect Caulobacter crescentus to provide an estimate of the diversity of bacteriophages that infect this common environmental bacterium. The majority of the new isolates are phicbkviruses, a genus of giant viruses that appear to be Caulobacter specific. However, we have also isolated several Podoviruses with icosahedral heads and small tails. One of these Podoviruses, designated Lullwater, is similar to two previously isolated Caulobacter phages, Cd1 and Percy. All three have genomes that are approximately 45 kb and contain approximately 30 genes. The gene order is conserved among the three genomes with one of the genes coding for a DNA polymerase that has homology to the family of T7 DNA polymerases. Phylogenetic trees based on either the DNA polymerase or the RNA polymerase amino acid sequences suggests that the three phages represent a new branch of the T7virus tree. Based on these similarities, we concluded that Cd1, Lullwater, and Percy comprise a new group in the T7virus genus.

Genomic Diversity of Type B3 Bacteriophages of Caulobacter crescentus.

The genomes of the type B3 bacteriophages that infect Caulobacter crescentus are among the largest phage genomes thus far deposited into GenBank with sizes over 200 kb. In this study, we introduce six new bacteriophage genomes which were obtained from phage collected from various water systems in the southeastern United States and from tropical locations across the globe. A comparative analysis of the 12 available genomes revealed a "core genome" which accounts for roughly 1/3 of these bacteriophage genomes and is predominately localized to the head, tail, and lysis gene regions. Despite being isolated from geographically distinct locations, the genomes of these bacteriophages are highly conserved in both genome sequence and gene order. We also identified the insertions, deletions, translocations, and horizontal gene transfer events which are responsible for the genomic diversity of this group of bacteriophages and demonstrated that these changes are not consistent with the idea that modular reassortment of genomes occurs in this group of bacteriophages.

Conservation of the Essential Genome Among Caulobacter and Brevundimonas Species.

When the genomes of Caulobacter isolates NA1000 and K31 were compared, numerous genome rearrangements were observed. In contrast, similar comparisons of closely related species of other bacterial genera revealed nominal rearrangements. A phylogenetic analysis of the 16S rRNA indicated that K31 is more closely related to Caulobacter henricii CB4 than to other known Caulobacters. Therefore, we sequenced the CB4 genome and compared it to all of the available Caulobacter genomes to study genome rearrangements, discern the conservation of the NA1000 essential genome, and address concerns about using 16S rRNA to group Caulobacter species. We also sequenced the novel bacteria, Brevundimonas DS20, a representative of the genus most closely related to Caulobacter and used it as part of an outgroup for phylogenetic comparisons. We expected to find that there would be fewer rearrangements when comparing more closely related Caulobacters. However, we found that relatedness was not correlated with the amount of observed "genome scrambling." We also discovered that nearly all of the essential genes previously identified for C. crescentus are present in the other Caulobacter genomes and in the Brevundimonas genomes as well. However, a few of these essential genes were only found in NA1000, and some were missing in a combination of one or more species, while other proteins were 100 % identical across species. Also, phylogenetic comparisons of highly conserved genomic regions revealed clades similar to those identified by 16S rRNA-based phylogenies, verifying that 16S rRNA sequence comparisons are a valid method for grouping Caulobacters.

Characterization of the Proteins Associated with Caulobacter crescentus Bacteriophage CbK Particles.

Bacteriophage genomes contain an abundance of genes that code for hypothetical proteins with either a conserved domain or no predicted function. The Caulobacter phage CbK has an unusual shape, designated morphotype B3 that consists of an elongated cylindrical head and a long flexible tail. To identify CbK proteins associated with the phage particle, intact phage particles were subjected to SDS-PAGE, and the resulting protein bands were digested with trypsin and analyzed using MALDI mass spectroscopy to provide peptide molecular weights. These peptide molecular weights were then compared with the peptides that would be generated from the predicted amino acid sequences that are coded by the CbK genome, and the comparison of the actual and predicted peptide masses resulted in the identification of single genes that could code for the set of peptides derived from each of the 20 phage proteins. We also found that CsCl density gradient centrifugation resulted in the separation of empty phage heads, phage heads containing material organized in a spiral, isolated phage tails, and other particulate material from the intact phage particles. This additional material proved to be a good source of additional phage proteins, and preliminary results suggest that it may include a CbK DNA replication complex.

Comparison of genome sequencing technology and assembly methods for the analysis of a GC-rich bacterial genome.

Improvements in technology and decreases in price have made de novo bacterial genomic sequencing a reality for many researchers, but it has created a need to evaluate the methods for generating a complete and accurate genome assembly. We sequenced the GC-rich Caulobacter henricii genome using the Illumina MiSeq, Roche 454, and Pacific Biosciences RS II sequencing systems. To generate a complete genome sequence, we performed assemblies using eight readily available programs and found that builds using the Illumina MiSeq and the Roche 454 data produced accurate yet numerous contigs. SPAdes performed the best followed by PANDAseq. In contrast, the Celera assembler produced a single genomic contig using the Pacific Biosciences data after error correction with the Illumina MiSeq data. In addition, we duplicated this build using the Pacific Biosciences data with HGAP2.0. The accuracy of these builds was verified by pulsed-field gel electrophoresis of genomic DNA cut with restriction enzymes.

Genome sequence and phenotypic characterization of Caulobacter segnis.

Caulobacter segnis is a unique species of Caulobacter that was initially deemed Mycoplana segnis because it was isolated from soil and appeared to share a number of features with other Mycoplana. After a 16S rDNA analysis showed that it was closely related to Caulobacter crescentus, it was reclassified C. segnis. Because the C. segnis genome sequence available in GenBank contained 126 pseudogenes, we compared the original sequencing data to the GenBank sequence and determined that many of the pseudogenes were due to sequence errors in the GenBank sequence. Consequently, we used multiple approaches to correct and reannotate the C. segnis genome sequence. In total, we deleted 247 bp, added 14 bp, and changed 8 bp resulting in 233 fewer bases in our corrected sequence. The corrected sequence contains only 15 pseudogenes compared to 126 in the original annotation. Furthermore, we found that unlike Mycoplana, C. segnis divides by fission, producing swarmer cells that have a single, polar flagellum.

The Caulobacter crescentus transducing phage Cr30 is a unique member of the T4-like family of myophages.

Bacteriophage Cr30 has proven useful for the transduction of Caulobacter crescentus. Nucleotide sequencing of Cr30 DNA revealed that the Cr30 genome consists of 155,997 bp of DNA that codes for 287 proteins and five tRNAs. In contrast to the 67 % GC content of the host genome, the GC content of the Cr30 genome is only 38 %. This lower GC content causes both the codon usage pattern and the amino acid composition of the Cr30 proteins to be quite different from those of the host bacteria. As a consequence, the Cr30 mRNAs probably are translated at a rate that is slower than the normal rate for host mRNAs. A phylogenetic comparison of the genome indicates that Cr30 is a member of the T4-like family that is most closely related to a new group of T-like phages exemplified by фM12.

Alternative mechanism for bacteriophage adsorption to the motile bacterium Caulobacter crescentus.

2D and 3D cryo-electron microscopy, together with adsorption kinetics assays of Cb13 and CbK phage-infected Caulobacter crescentus, provides insight into the mechanisms of infection. Cb13 and CbK actively interact with the flagellum and subsequently attach to receptors on the cell pole. We present evidence that the first interaction of the phage with the bacterial flagellum takes place through a filament on the phage head. This contact with the flagellum facilitates concentration of phage particles around the receptor (i.e., the pilus portals) on the bacterial cell surface, thereby increasing the likelihood of infection. Phage head filaments have not been well characterized and their function is described here. Phage head filaments may systematically underlie the initial interactions of phages with their hosts in other systems and possibly represent a widespread mechanism of efficient phage propagation.

New Genera and Species of Caulobacter and Brevundimonas Bacteriophages Provide Insights into Phage Genome Evolution.

Previous studies have identified diverse bacteriophages that infect Caulobacter vibrioides strain CB15 ranging from small RNA phages to four genera of jumbo phages. In this study, we focus on 20 bacteriophages whose genomes range from 40 to 60 kb in length. Genome comparisons indicated that these diverse phages represent six Caulobacter phage genera and one additional genus that includes both Caulobacter and Brevundimonas phages. Within species, comparisons revealed that both single base changes and inserted or deleted genetic material cause the genomes of closely related phages to diverge. Among genera, the basic gene order and the orientation of key genes were retained with most of the observed variation occurring at ends of the genomes. We hypothesize that the nucleotide sequences of the ends of these phage genomes are less important than the need to maintain the size of the genome and the stability of the corresponding mRNAs.

Codon usage methods for horizontal gene transfer detection generate an abundance of false positive and false negative results.

Bacteria acquire new DNA in a process known as horizontal gene transfer (HGT). To investigate the evolutionary impact of this transfer of DNA, various methods have been developed to detect past HGT events. For example, codon usage-based methods detect the presence of transferred genes by identifying atypical patterns of codon usage. However, some inherited genes exhibit atypical codon usage and some transferred genes have codon usage patterns similar to those of the inherited genes. In this study, we used a comparative phylogenetic approach with Methylobacterium and Caulobacter species to demonstrate that even well-designed codon usage methods fail to detect many HGT events and generate a high rate of false positives (60-75 %) and false negatives (23-61 %). Therefore, we recommend caution when employing codon usage methods to identify transferred genes and suggest that the rapidly increasing availability of bacterial genome sequences makes the phylogenetic approach the method of choice.