Caulobacter segnis strain CBR1 enhances the germination rate and total biomass of Arabidopsis plants. Since bacteriophages are usually present in the rhizosphere, we isolated three additional bacteriophages, designated TMCBR2, TMCBR3, and W2, that can replicate using CBR1 as the host strain. The genome sizes and morphologies of the new phages were similar to those of the previously isolated Kronos phage, and when we compared the nucleotide sequence of these new phage genomes, we found only minor differences among the four phage genomes. Pairwise sequence comparisons indicated that the phage genomes should be grouped into three separate species within Kronosvirus genus. Interestingly, we found that most of the genomic variation occurred among genes of unknown function within a 10 kb region of the 42 kb genomes with little variation in the remaining 32 kb which contains the genes known to be important for phage propagation.

Interactions between bacteria and bacteriophages are important for the maintenance of soil communities. In this study, we characterized the giant bacteriophages found within diverse soil bacteria and 14 additional phages isolated directly from soil samples. Based on their genome sizes and genetic composition, we concluded that these phages belong to the Dolichocephalovirinae subfamily. In addition, we used pulsed-field gel electrophoresis to show that the genomes of these phages were present as episomal pseudolysogens in the cytoplasm of their host cells. These findings suggest that episomal phages are important components of soil microbial ecosystems. Understanding the interactions between bacteriophages and bacteria is essential for microbial ecology, as they influence nutrient cycling, community composition, and host evolution. Furthermore, these phage-bacteria dynamics offer potential applications in plant disease control, as bacteriophages could serve as biocontrol agents against soilborne pathogens, promoting sustainable agricultural practices.

Members of the Dolichocephalovirinae subfamily are giant viruses with an elongated head and a flexible tail that is used to infect Caulobacter strains. In this paper, we describe the isolation and characterization of nine newly isolated phages and present evidence that seven of these phages represent a new Dolichocephalovirinae genus that has significant differences from the four previously described Dolichocephalovirinae genera. In addition, since these new phages were isolated from a single sampling site over the course of three years, a comparison of their genome sequences reveals a low level of within-population diversity resulting from both single-nucleotide polymorphisms and insertions or deletions. A comparison of the host ranges of these phages suggests that differences in host susceptibility may be an important factor in maintaining this diversity.

Bacteriophages grown on Caulobacter vibrioides strain CB15 have reduced plating efficiency on other Caulobacter strains. To determine the cause of this reduced plating efficiency, we performed a series of experiments that demonstrated that the reduced plating efficiency is due to a novel set of restriction and modification (RM) enzymes that are present in most of the Caulobacter strains that we tested. We then demonstrated that one of these RM systems recognizes the nucleotide sequence 5′-ATNNAT-3′. A careful inspection of the genome nucleotide sequences of each of the strains revealed that the genes coding for these RM enzymes have not been annotated or identified, suggesting that the proteins may differ from the common types of bacterial restriction and modification enzymes. In addition, the host strain NA1000 contains a 26 kb mobile element that provides resistance to incoming phages.

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.

To explore how microbial interactions within the rhizosphere influence the diversity and functional roles of bacterial communities, we isolated 21 bacterial strains from soil samples collected near Rocky Branch Creek on the University of South Carolina campus. Our findings revealed that a significant proportion of the isolated bacterial strains are lysogenic. Contrary to predictions of a narrow host range, most of the bacteriophages derived from these lysogenic bacteria demonstrated the ability to infect a broad range of bacterial strains. These results suggest that the bacterial community shares a complex phage community, creating an intricate web of interactions. This study enhances our understanding of the relationships between phages and their bacterial hosts in soil ecosystems, with implications for ecological balance and agricultural practices aimed at improving plant health through microbial management strategies.

Bacteriophage diversity is a relatively unknown frontier that is rapidly being explored, leading to a wealth of new information. New bacteriophages are being discovered at an astounding rate via both phage isolation studies and metagenomic analyses. In addition, a nucleotide sequence-based viral taxonomic system has been developed to better handle this wealth of new information. As a result of these developments, phage scientists are transitioning from knowing that there must be huge numbers of diverse kinds of phage particles in natural environments to identifying the actual abundance and phage diversity that is present in specific environments. This review documents the beginning of this transition, offering a glimpse into the magnitude of change unfolding in the field. It stands as a testament to the expanding frontiers of phage research, illuminating the remarkable progress made in unraveling the intricate world of bacteriophage diversity and advancing our understanding of these enigmatic viral entities.

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.

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.

Publication Info Postprint version. Published in Journal of Fish Biology, Volume 75, Issue 2, 2009, pages 442-447. This is the peer reviewed version of the following article: Complex Evolution of a Highly-conserved Microsatellite Locus in Several Fish Species, which has been published in final form at https://doi.org/ 10.1111/j.1095-8649.2009.02340.x. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. © Journal of Fish Biology 2009, Wiley

In every kingdom of life, GC->AT transitions occur more frequently than any other type of mutation due to the spontaneous deamination of cytidine. In eukaryotic genomes, this slow loss of GC base pairs is counteracted by biased gene conversion which increases genomic GC content as part of the recombination process. However, this type of biased gene conversion has not been observed in bacterial genomes, so we hypothesized that GC->AT transitions cause a reduction of genomic GC content in prokaryotic genomes on an evolutionary time scale. To test this hypothesis, we used a phylogenetic approach to analyze triplets of closely related genomes representing a wide range of the bacterial kingdom. The resulting data indicate that genomic GC content is drifting downward in bacterial genomes where GC base pairs comprise 40% or more of the total genome. In contrast, genomes containing less than 40% GC base pairs have fewer opportunities for GC->AT transitions to occur so genomic GC content is relatively stable or actually increasing. It should be noted that this observed change in genomic GC content is the net change in shared parts of the genome and does not apply to parts of the genome that have been lost or acquired since the genomes being compared shared common ancestor. However, a more detailed analysis of two Caulobacter genomes revealed that the acquisition of mobile elements by the two genomes actually reduced the total genomic GC content as well.

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.

Bacteria play an integral role in shaping plant growth and development. However, the genetic factors that facilitate plant-bacteria interactions remain largely unknown. Here, we demonstrated the importance of two bacterial genetic factors that facilitate the interactions between plant-growth-promoting (PGP) bacteria in the genus Caulobacter and the host plant Arabidopsis. Using homologous recombination, we disrupted the cytochrome ubiquinol oxidase (cyo) operon in both C. vibrioides CB13 and C. segnis TK0059 by knocking out the expression of cyoB (critical subunit of the cyo operon) and showed that the mutant strains were unable to enhance the growth of Arabidopsis. In addition, disruption of the cyo operon, metabolomic reconstructions, and pH measurements suggested that both elevated cyoB expression and acid production by strain CB13 contribute to the previously observed inhibition of Arabidopsis seed germination. We also showed that the crescent shape of the PGP bacterial strain C. crescentus CB15 contributes to its ability to enhance plant growth. Thus, we have identified specific genetic factors that explain how select Caulobacter strains interact with Arabidopsis plants.

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.

Aims Species within the Caulobacter genus have been termed 'hub species' in the plant microbiome. To understand these interactions, we assessed the interactions between several Caulobacter strains and a common host plant. Methods We identified a set of 11 Caulobacter strains that range in genetic diversity and tested them for their ability to increase the growth of Arabidopsis thaliana. In addition, biochemical assays were employed to determine if these Caulobacter strains produce common plant growth promoting (PGP) biosynthates. To identify potential PGP-related genes, genomic analyses were performed to compare the genomes of PGP Caulobacter strains to those of non-PGP Caulobacter strains. Results For the PGP Caulobacter strains, we observed that common PGP biosynthates did not contribute to the observed Caulobacter-mediated plant growth stimulation. Genomic analyses suggested that the genomes of PGP strains maintain similar metabolic pathways compared to those of non-PGP strains, and that common genes related to PGP factors do not explain the PGP mechanisms for the Caulobacter strains we analyzed. Conclusions Plant growth enhancement is not a conserved feature in the Caulobacter genus, and some Caulobacter strains even inhibit plant growth. Moreover, common PGP factors do not fully explain Caulobacter-mediated plant growth enhancement.

Bacteria can acquire new genes by incorporating environmental DNA into their genomes, yet genome sizes stay relatively constant. In nature, gene acquisition is a rare event so it is difficult to observe. However, the Caulobacter crescentus CB2A genome contains 114 insertions of genetic material from the closely-related NA1000 strain, providing a unique opportunity to analyze the horizontal transfer of genetic material. Analyses of these insertions led to a new model that involves preferential recombination at non-homologous regions that are flanked by regions of homology and does not involve any mutational processes. The net result is the replacement of segments of the recipient genome instead of the simple addition of genetic material during horizontal gene transfer. Analyses of the genomes of closely related strains of other bacterial and archaea genera, suggested that horizontal gene transfer occurs preferentially in non-homologous regions in these organisms as well. Thus, it appears to be a general phenomenon that prokaryotic horizontal gene transfer occurs preferentially at sites where the incoming DNA contains a non-homologous region that is flanked by regions of homology. Therefore, gene replacement is a common phenomenon during horizontal gene transfer.

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.

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.

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 Caulobacter Phicbkvirus 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.