Structure of the WYL-domain containing transcription activator, DriD, in complex with ssDNA effector and DNA target site.

Transcription regulators play central roles in orchestrating responses to changing environmental conditions. Recently the Caulobacter crescentus transcription activator DriD, which belongs to the newly defined WYL-domain family, was shown to regulate DNA damage responses independent of the canonical SOS pathway. However, the molecular mechanisms by which DriD and other WYL-regulators sense environmental signals and recognize DNA are not well understood. We showed DriD DNA-binding is triggered by its interaction with ssDNA, which is produced during DNA damage. Here we describe the structure of the full-length C. crescentus DriD bound to both target DNA and effector ssDNA. DriD consists of an N-terminal winged-HTH (wHTH) domain, linker region, three-helix bundle, WYL-domain and C-terminal WCX-dimer domain. Strikingly, DriD binds DNA using a novel, asymmetric DNA-binding mechanism that results from different conformations adopted by the linker. Although the linker does not touch DNA, our data show that contacts it makes with the wHTH are key for specific DNA binding. The structure indicates how ssDNA-effector binding to the WYL-domain impacts wHTH DNA binding. In conclusion, we present the first structure of a WYL-activator bound to both effector and target DNA. The structure unveils a unique, asymmetric DNA binding mode that is likely conserved among WYL-activators.

XRE transcription factors conserved in Caulobacter and φCbK modulate adhesin development and phage production.

The xenobiotic response element (XRE) family of transcription factors (TFs), which are commonly encoded by bacteria and bacteriophage, regulate diverse features of bacterial cell physiology and impact phage infection dynamics. Through a pangenome analysis of Caulobacter species isolated from soil and aquatic ecosystems, we uncovered an apparent radiation of a paralogous XRE TF gene cluster, several of which have established functions in the regulation of holdfast adhesin development and biofilm formation in C. crescentus. We further discovered related XRE TFs throughout the class Alphaproteobacteria and its phages, including the φCbK Caulophage, suggesting that members of this cluster impact host-phage interactions. Here we show that a closely related group of XRE transcription factors encoded by both C. crescentus and φCbK can physically interact and function to control the transcription of a common gene set, influencing processes including holdfast development and the production of φCbK virions. The φCbK-encoded XRE paralog, tgrL, is highly expressed at the earliest stages of infection and can directly inhibit transcription of host genes including hfiA, a potent holdfast inhibitor, and gafYZ, an activator of prophage-like gene transfer agents (GTAs). XRE proteins encoded from the C. crescentus chromosome also directly repress gafYZ transcription, revealing a functionally redundant set of host regulators that may protect against spurious production of GTA particles and inadvertent cell lysis. Deleting the C. crescentus XRE transcription factors reduced φCbK burst size, while overexpressing these host genes or φCbK tgrL rescued this burst defect. We conclude that this XRE TF gene cluster, shared by C. crescentus and φCbK, plays an important role in adhesion regulation under phage-free conditions, and influences host-phage dynamics during infection.

A cryptic transcription factor regulates Caulobacter adhesin development.

Alphaproteobacteria commonly produce an adhesin that is anchored to the exterior of the envelope at one cell pole. In Caulobacter crescentus this adhesin, known as the holdfast, facilitates attachment to solid surfaces and cell partitioning to air-liquid interfaces. An ensemble of two-component signal transduction (TCS) proteins controls C. crescentus holdfast biogenesis by indirectly regulating expression of HfiA, a potent inhibitor of holdfast synthesis. We performed a genetic selection to discover direct hfiA regulators that function downstream of the adhesion TCS system and identified rtrC, a hypothetical gene. rtrC transcription is directly activated by the adhesion TCS regulator, SpdR. Though its primary structure bears no resemblance to any defined protein family, RtrC binds and regulates dozens of sites on the C. crescentus chromosome via a pseudo-palindromic sequence. Among these binding sites is the hfiA promoter, where RtrC functions to directly repress transcription and thereby activate holdfast development. Either RtrC or SpdR can directly activate transcription of a second hfiA repressor, rtrB. Thus, environmental regulation of hfiA transcription by the adhesion TCS system is subject to control by an OR-gated type I coherent feedforward loop; these regulatory motifs are known to buffer gene expression against fluctuations in regulating signals. We have further assessed the functional role of rtrC in holdfast-dependent processes, including surface adherence to a cellulosic substrate and formation of pellicle biofilms at air-liquid interfaces. Strains harboring insertional mutations in rtrC have a diminished adhesion profile in a competitive cheesecloth binding assay and a reduced capacity to colonize pellicle biofilms in select media conditions. Our results add to an emerging understanding of the regulatory topology and molecular components of a complex bacterial cell adhesion control system.

Regulated Proteolysis in Bacteria: Caulobacter.

Protein degradation is essential for all living things. Bacteria use energy-dependent proteases to control protein destruction in a highly specific manner. Recognition of substrates is determined by the inherent specificity of the proteases and through adaptor proteins that alter the spectrum of substrates. In the α-proteobacterium Caulobacter crescentus, regulated protein degradation is required for stress responses, developmental transitions, and cell cycle progression. In this review, we describe recent progress in our understanding of the regulated and stress-responsive protein degradation pathways in Caulobacter. We discuss how organization of highly specific adaptors into functional hierarchies drives destruction of proteins during the bacterial cell cycle. Because all cells must balance the need for degradation of many true substrates with the toxic consequences of nonspecific protein destruction, principles found in one system likely generalize to others.

The Caulobacter NtrB-NtrC two-component system bridges nitrogen assimilation and cell development.

A suite of molecular sensory systems enables Caulobacter to control growth, development, and reproduction in response to levels of essential elements. The bacterial enhancer-binding protein (bEBP) NtrC and its cognate sensor histidine kinase, NtrB, are key regulators of nitrogen assimilation in many bacteria, but their roles in Caulobacter metabolism and development are not well defined. Notably, Caulobacter NtrC is an unconventional bEBP that lacks the σ54-interacting loop commonly known as the GAFTGA motif. Here we show that deletion of Caulobacter crescentus ntrC slows cell growth in complex medium and that ntrB and ntrC are essential when ammonium is the sole nitrogen source due to their requirement for glutamine synthetase expression. Random transposition of a conserved IS3-family mobile genetic element frequently rescued the growth defect of ntrC mutant strains by restoring transcription of the glnBA operon, revealing a possible role for IS3 transposition in shaping the evolution of Caulobacter populations during nutrient limitation. We further identified dozens of direct NtrC-binding sites on the C. crescentus chromosome, with a large fraction located near genes involved in polysaccharide biosynthesis. The majority of binding sites align with those of the essential nucleoid-associated protein, GapR, or the cell cycle regulator, MucR1. NtrC is therefore predicted to directly impact the regulation of cell cycle and cell development. Indeed, loss of NtrC function led to elongated polar stalks and elevated synthesis of cell envelope polysaccharides. This study establishes regulatory connections between NtrC, nitrogen metabolism, polar morphogenesis, and envelope polysaccharide synthesis in Caulobacter. IMPORTANCE Bacteria balance cellular processes with the availability of nutrients in their environment. The NtrB-NtrC two-component signaling system is responsible for controlling nitrogen assimilation in many bacteria. We have characterized the effect of ntrB and ntrC deletion on Caulobacter growth and development and uncovered a role for spontaneous IS element transposition in the rescue of transcriptional and nutritional deficiencies caused by ntrC mutation. We further defined the regulon of Caulobacter NtrC, a bacterial enhancer-binding protein, and demonstrate that it shares specific binding sites with essential proteins involved in cell cycle regulation and chromosome organization. Our work provides a comprehensive view of transcriptional regulation mediated by a distinctive NtrC protein, establishing its connection to nitrogen assimilation and developmental processes in Caulobacter.

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.

Asymmetric division yields progeny cells with distinct modes of regulating cell cycle-dependent chromosome methylation.

The cell cycle-regulated methylation state of Caulobacter DNA mediates the temporal control of transcriptional activation of several key regulatory proteins. Temporally controlled synthesis of the CcrM DNA methyltransferase and Lon-mediated proteolysis restrict CcrM to a specific time in the cell cycle, thereby allowing the maintenance of the hemimethylated state of the chromosome during the progression of DNA replication. We determined that a chromosomal DNA-based platform stimulates CcrM degradation by Lon and that the CcrM C terminus both binds to its DNA substrate and is recognized by the Lon protease. Upon asymmetric cell division, swarmer and stalked progeny cells employ distinct mechanisms to control active CcrM. In progeny swarmer cells, CcrM is completely degraded by Lon before its differentiation into a replication-competent stalked cell later in the cell cycle. In progeny stalked cells, however, accumulated CcrM that has not been degraded before the immediate initiation of DNA replication is sequestered to the cell pole. Single-molecule imaging demonstrated physical anticorrelation between sequestered CcrM and chromosomal DNA, thus preventing DNA remethylation. The distinct control of available CcrM in progeny swarmer and stalked cells serves to protect the hemimethylated state of DNA during chromosome replication, enabling robustness of cell cycle progression.

AFM Unravels the Unique Adhesion Properties of the Caulobacter Type IVc Pilus Nanomachine.

Bacterial pili are proteinaceous motorized nanomachines that play various functional roles including surface adherence, bacterial motion, and virulence. The surface-contact sensor type IVc (or Tad) pilus is widely distributed in both Gram-positive and Gram-negative bacteria. In Caulobacter crescentus, this nanofilament, though crucial for surface colonization, has never been thoroughly investigated at the molecular level. As Caulobacter assembles several surface appendages at specific stages of the cell cycle, we designed a fluorescence-based screen to selectively study single piliated cells and combined it with atomic force microscopy and genetic manipulation to quantify the nanoscale adhesion of the type IVc pilus to hydrophobic substrates. We demonstrate that this nanofilament exhibits high stickiness compared to the canonical type IVa/b pili, resulting mostly from multiple hydrophobic interactions along the fiber length, and that it features nanospring mechanical properties. Our findings may be helpful to better understand the structure-function relationship of bacterial pilus nanomachines.

The genus Caulobacter and its role in plant microbiomes.

Recent omics approaches have revealed the prevalent microbial taxa that constitute the microbiome of various plant species. Across global scales and environmental conditions, strains belonging to the bacterial genus Caulobacter have consistently been found in association with various plant species. Aligned with agroecological relevance and biotechnological advances, many scientific communications have demonstrated that several Caulobacter strains (spanning several Caulobacter species) harbor the potential to enhance plant biomass for various plant species ranging from Arabidopsis to Citrullus and Zea mays. In the past several years, co-occurrence data have driven mechanistically resolved communications about select Caulobacter-plant interactions. Given the long-standing history of Caulobacter as a model organism for cell cycle regulation, genetic studies, and the prevalence of Caulobacter species in various plant microbiomes, the genus Caulobacter offers researchers a unique opportunity to leverage for investigating plant-microbe interactions and realizing targeted biotechnological applications. In this review, recent developments regarding Caulobacter-plant interactions are presented in terms of model utility for future biotechnological investigations.

Plant-growth-promoting Caulobacter strains isolated from distinct plant hosts share conserved genetic factors involved in beneficial plant-bacteria interactions.

The genus Caulobacter encompasses several strains that can enhance the biomass of several plant species. However, for many plant-growth-promoting (PGP) Caulobacter strains, their genomic factors that facilitate positive interactions with their plant hosts remain unknown. Given that leveraging comparative genomics analyses can establish a framework to understand these plant-bacteria interactions, the genomes of three PGP Caulobacter strains that were isolated from distinct geographical locations and have been shown to associate with distinct plant species were compared. Using previously reported analyses to contextualize the genomic patterns among PGP Caulobacter strains, each of these PGP Caulobacter strains (CBR1, RHG1, and RHGG3) was observed harboring similar metabolic potentials and previously reported PGP genetic factors in their genomes. Together, these findings reinforce previous analyses that identify the cyo operon as a general PGP factor for Caulobacter strains while establishing a framework for further investigations that seek to uncover the mechanistic details that govern interactions between Caulobacter strains and diverse plant species.

Influence of Uranium Concentration and pH on U-Phosphate Biomineralization by Caulobacter OR37.

Uranium contamination of soils and groundwater in the United States represents a significant health risk and will require multiple remediation approaches. Microbial phosphatase activity coupled to the addition of an organic P source has recently been studied as a remediation strategy that provides an extended release of inorganic P (Pi) into U-contaminated sites, resulting in the precipitation of meta-autunite minerals. Previous laboratory- and field-based biomineralization studies have investigated environments with relatively high U concentrations (>20 µM). However, most contaminated sites have much lower U concentrations (<2 µM). The Environmental Protection Agency (EPA) limit for U in drinking water is 0.126 µM. Reaching this regulatory limit becomes challenging as U concentrations approach autunite solubility. We studied the precipitation of U(VI)-phosphate minerals by an environmental isolate of Caulobacter sp. (strain OR37) from an Oak Ridge, Tennessee, U-contaminated site. Abiotic U(VI) solubility experiments reveal that U(VI)-phosphate minerals do not form in the presence of excess Pi (500 µM) when U(VI) concentrations are <1 µM and pH is <5. When OR37 cells are reacted under the same conditions with Pi or glycerol-2-phosphate, U(VI)-phosphate mineral formation was observed, along with the formation of intracellular polyphosphate granules. These results show that bacteria provide supersaturated microenvironments needed for U(VI)-phosphate mineralization while hydrolyzing organic P sources. This provides a pathway to lower U concentrations to below EPA limits for drinking water.

Caulobacter endophyticus sp. nov., an endophytic bacterium harboring three lasso peptide biosynthetic gene clusters and producing indoleacetic acid isolated from maize root.

A novel Gram-stain-negative, aerobic and rod-shaped bacterium with a single polar flagellum or a stalk at the end of the cell, was isolated from maize roots in the Fangshan District of Beijing, People's Republic of China. The new strain designated 774T produced indole acetic acid (IAA). The 16S rRNA gene sequence analysis indicated that strain 774T belongs to the genus Caulobacter and is closely related to Caulobacter flavus RHGG3T, Caulobacter zeae 410Tand Caulobacter radices 695T, all with sequence similarities of 99.9%. The genome size of strain774T was 5.4 Mb, comprising 5042 predicted genes with a DNA G+C content of 68.7%.Three striking lasso peptide biosynthetic gene clusters and two IAA synthesis genes belonging to the TPM pathway were also found in the genome of strain 774T. The average nucleotide identity values and digital DNA-DNA hybridization values of the strain774T with its closely phylogenetic neighbours were less than 91.5% and 45.0%, respectively, indicating a new Caulobacter species. The major fatty acids of strain774T were identified as C16: 0 (27.7%), summed feature 3 (C16: 1ω6c and/or C16: 1ω7c) (12.6%) and summed feature 8 (C18: 1ω7c and/or C18: 1ω6c) (42.9%).The major polar lipids consisted of phosphatidyl-glycerol and glycolipids. The predominant ubiquinone was identified as Quinone 10. Based on the polyphasic characterization, strain 774T represents a novel species of the genus Caulobacter, for which the name Caulobacter endophyticus sp. nov. is proposed with 774T (= CGMCC 1.16558T = DSM 106777T) as the type strain.

Complete Genome Sequence of the Plant-Growth-Promoting Bacterium Caulobacter segnis CBR1.

Genomic sequencing has vastly expedited our understanding of bacterial functions. However, the genomes of many plant-growth-promoting bacteria (PGPB) have yet to be sequenced and contextualized. To this end, I report the sequenced genome of a PGPB-Caulobacter segnis CBR1-and contextualize its genomic features with the genomic features of sequenced Caulobacter strains. Moreover, I demonstrate that the CBR1 genome harbors genomic features that have been shown to be necessary for select Caulobacter strains to enhance the growth and development of Arabidopsis plants. Together, these findings will help guide future investigations that seek to understand the molecular factors undergirding the positive interactions between plants and microbes.

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.

Caulobacter soli sp. nov., isolated from soil sampled at Jiri Mountain, Republic of Korea.

A Gram-stain-negative, yellow-pigmented, aerobic, non-spore-forming, motile with a single polar flagellum and rod-shaped bacterium, Ji-3-8T, was isolated from a soil sample taken from Jiri Mountain, Republic of Korea. Comparative 16S rRNA gene sequence studies showed the isolate had clear affiliation with Alphaproteobacteria and the closest relatedness to Caulobacter rhizosphaerae KCTC 52515T, Caulobacter henricii ATCC 15253T, Caulobacter segnis ATCC 21756T, Caulobacter hibisci THG-AG3.4T, Caulobacter flavus RHGG3T and Caulobacter vibrioides CB51T showing 99.1, 98.9, 97.7, 97.6, 97.5 and 97.4 % 16S rRNA gene sequence similarity, respectively, and 94.7-96.5 % to the remaining species of genus Caulobacter. The predominant ubiquinone was Q-10 and the major fatty acids were C18 : 1 ω7c 11-methyl, C16 : 0, summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c) and summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c). The major polar lipids were found to be phosphatidylglycerol, two unidentified phosphoglycolipid and two unidentified glycolipids. The G+C content of the genomic DNA of strain Ji-3-8T was 68.1 mol%. Average nucleotide identity and digital DNA-DNA hybridization values of strain Ji-3-8T with C. rhizosphaerae KCTC 52515T, C. henricii ATCC 15253T, C. segnis ATCC 21756T, C. flavus RHGG3T and C. vibrioides were 79.7-87.7% and 23.0-34.3%, respectively. Based on the polyphasic evidence, it is proposed that strain Ji-3-8T forms a novel species in the genus Caulobacter, for which the name Caulobacter soli sp. nov. is proposed. The type strain is Ji-3-8T (=CCTCC AB 2019389T=KCTC 72990T).

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.

Site-directed mutagenesis of coenzyme-independent carotenoid oxygenase CSO2 to enhance the enzymatic synthesis of vanillin.

Vanillin is a popular flavoring compound and an important food additive. Owing to the consumer preference for inexpensive natural aroma flavors, vanillin production through a biotechnological pathway has become of great interest and commercial value in recent years. In this study, an enzymatic synthetic system for vanillin using a coenzyme-independent decarboxylase (FDC) and oxygenase (CSO2) cascade was reconstituted and optimized. This system produces a slightly higher production yield (40.20%) than the largest yield reported for immobilized FDC and CSO2 (35.00%) with ferulic acid as a substrate. It was previously reported that the low catalytic activity and thermal instability of CSO2 restrict the overall productivity of vanillin. In present study, site-directed mutagenesis was applied to rate-limiting oxygenase CSO2 to generate positive mutants. The production yields of mutants A49P (58.44%) and Q390A (65.29%) were 1.45- and 1.62-fold that of CSO2 wild type, respectively. The potential mechanism for enhanced vanillin production using A49P involved increased thermostability and catalytic efficiency, while that using Q390A was probably associated with a better thermostable performance and increased catalytic efficiency resulting from a larger entrance channel.

Analysis and remedy of negativity problem in hybrid stochastic simulation algorithm and its application.

BACKGROUND: The hybrid stochastic simulation algorithm, proposed by Haseltine and Rawlings (HR), is a combination of differential equations for traditional deterministic models and Gillespie's algorithm (SSA) for stochastic models. The HR hybrid method can significantly improve the efficiency of stochastic simulations for multiscale biochemical networks. Previous studies on the accuracy analysis for a linear chain reaction system showed that the HR hybrid method is accurate if the scale difference between fast and slow reactions is above a certain threshold, regardless of population scales. However, the population of some reactant species might be driven negative if they are involved in both deterministic and stochastic systems. RESULTS: This work investigates the negativity problem of the HR hybrid method, analyzes and tests it with several models including a linear chain system, a nonlinear reaction system, and a realistic biological cell cycle system. As a benchmark, the second slow reaction firing time is used to measure the effect of negative populations on the accuracy of the HR hybrid method. Our analysis demonstrates that usually the error caused by negative populations is negligible compared with approximation errors of the HR hybrid method itself, and sometimes negativity phenomena may even improve the accuracy. But for systems where negative species are involved in nonlinear reactions or some species are highly sensitive to negative species, the system stability will be influenced and may lead to system failure when using the HR hybrid method. In those circumstances, three remedies are studied for the negativity problem. CONCLUSION: The results of different models and examples suggest that the Zero-Reaction rule is a good remedy for nonlinear and sensitive systems considering its efficiency and simplicity.

Plant Growth Promotion Driven by a Novel Caulobacter Strain.

Soil microbial communities hold great potential for sustainable and ecologically compatible agriculture. Although numerous plant-beneficial bacterial strains from a wide range of taxonomic groups have been reported, very little evidence is available on the plant-beneficial role of bacteria from the genus Caulobacter. Here, the mode of action of a Caulobacter strain, designated RHG1, which had originally been identified through a microbial screen for plant growth-promoting (PGP) bacteria in maize (Zea mays), is investigated in Arabidopsis thaliana. RHG1 colonized both roots and shoots of Arabidopsis, promoted lateral root formation in the root, and increased leaf number and leaf size in the shoot. The genome of RHG1 was sequenced and was utilized to look for PGP factors. Our data revealed that the bacterial production of nitric oxide, auxins, cytokinins, or 1-aminocyclopropane-1-carboxylate deaminase as PGP factors could be excluded. However, the analysis of brassinosteroid mutants suggests that an unknown PGP mechanism is involved that impinges directly or indirectly on the pathway of this growth hormone.

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.