Bacterial species from diverse phyla contain multiple replicons, yet how these multipartite genomes are organized and segregated during the cell cycle remains poorly understood. Agrobacterium tumefaciens has a 2.8-Mb circular chromosome (Ch1), a 2.1-Mb linear chromosome (Ch2), and two large plasmids (pAt and pTi). We used this alpha proteobacterium as a model to investigate the global organization and temporal segregation of a multipartite genome. Using chromosome conformation capture assays, we demonstrate that both the circular and the linear chromosomes, but neither of the plasmids, have their left and right arms juxtaposed from their origins to their termini, generating interarm interactions that require the broadly conserved structural maintenance of chromosomes complex. Moreover, our study revealed two types of interreplicon interactions: “ori-ori clustering” in which the replication origins of all four replicons interact, and “Ch1-Ch2 alignment” in which the arms of Ch1 and Ch2 interact linearly along their lengths. We show that the centromeric proteins (ParB1 for Ch1 and RepBCh2 for Ch2) are required for both types of interreplicon contacts. Finally, using fluorescence microscopy, we validated the clustering of the origins and observed their frequent colocalization during segregation. Altogether, our findings provide a high-resolution view of the conformation of a multipartite genome. We hypothesize that intercentromeric contacts promote the organization and maintenance of diverse replicons.
SUMMARY Bacteria in the genus Brucella are important human and veterinary pathogens. The abortion and infertility they cause in food animals produce economic hardships in areas where the disease has not been controlled, and human brucellosis is one of the world’s most common zoonoses. Brucella strains have also been isolated from wildlife, but we know much less about the pathobiology and epidemiology of these infections than we do about brucellosis in domestic animals. The brucellae maintain predominantly an intracellular lifestyle in their mammalian hosts, and their ability to subvert the host immune response and survive and replicate in macrophages and placental trophoblasts underlies their success as pathogens. We are just beginning to understand how these bacteria evolved from a progenitor alphaproteobacterium with an environmental niche and diverged to become highly host-adapted and host-specific pathogens. Two important virulence determinants played critical roles in this evolution: (i) a type IV secretion system that secretes effector molecules into the host cell cytoplasm that direct the intracellular trafficking of the brucellae and modulate host immune responses and (ii) a lipopolysaccharide moiety which poorly stimulates host inflammatory responses. This review highlights what we presently know about how these and other virulence determinants contribute to Brucella pathogenesis. Gaining a better understanding of how the brucellae produce disease will provide us with information that can be used to design better strategies for preventing brucellosis in animals and for preventing and treating this disease in humans.
As with many pathogenic bacteria, agrobacterial plant pathogens carry most of their virulence functions on a horizontally transmissible genetic element. The tumor-inducing (Ti) plasmid encodes the majority of virulence functions for the crown gall agent Agrobacterium tumefaciens. This includes the vir genes which drive genetic transformation of host cells and the catabolic genes needed to utilize the opines produced by infected plants. The Ti plasmid also encodes, an opine-dependent quorum sensing system that tightly regulates Ti plasmid copy number and its conjugal transfer to other agrobacteria. Many natural agrobacteria are avirulent, lacking the Ti plasmid. The burden of harboring the Ti plasmid depends on the environmental context. Away from diseased hosts, plasmid costs are low but the benefit of the plasmid is also absent. Consequently, plasmidless genotypes are favored. On infected plants the costs of the Ti plasmid can be very high, but balanced by the opine benefits, locally favoring plasmid bearing cells. Cheating derivatives which do not incur virulence costs but can benefit from opines are favored on infected plants and in most other environments, and these are frequently isolated from nature. Many agrobacteria also harbor an At plasmid which can stably coexist with a Ti plasmid. At plasmid genes are less well characterized but in general facilitate metabolic activities in the rhizosphere and bulk soil, such as the ability to breakdown plant exudates. Examination of A. tumefaciens C58, revealed that harboring its At plasmid is much more costly than harboring it’s Ti plasmid, but conversely the At plasmid is extremely difficult to cure. The interactions between these co-resident plasmids are complex, and depend on environmental context. However, the presence of a Ti plasmid appears to mitigate At plasmid costs, consistent with the high frequency with which they are found together.
Summary Many important pathogens maintain significant populations in highly disparate disease and non‐disease environments. The consequences of this environmental heterogeneity in shaping the ecological and evolutionary dynamics of these facultative pathogens are incompletely understood. Agrobacterium tumefaciens, the causative agent for crown gall disease of plants has proven a productive model for many aspects of interactions between pathogens and their hosts and with other microbes. In this review, we highlight how this past work provides valuable context for the use of this system to examine how heterogeneity and transitions between disease and non‐disease environments influence the ecology and evolution of facultative pathogens. We focus on several features common among facultative pathogens, such as the physiological remodelling required to colonize hosts from environmental reservoirs and the consequences of competition with host and non‐host associated microbiota. In addition, we discuss how the life history of facultative pathogens likely often results in ecological tradeoffs associated with performance in disease and non‐disease environments. These pathogens may therefore have different competitive dynamics in disease and non‐disease environments and are subject to shifting selective pressures that can result in pathoadaptation or the within‐host spread of avirulent phenotypes.
About 10% of sequenced bacteria have multiple replicons, also known as multipartite genomes. How these multipartite genomes are maintained is still poorly understood.
Bacteria with multi-replicon genome organizations, including members of the family Rhizobiaceae, often carry a variety of niche-associated functions on large plasmids. While evidence exists for cross-replicon interactions and co-evolution between replicons in many of these systems, remarkable strain-to-strain variation is also observed for extrachromosomal elements, suggesting increased genetic plasticity. Here, we show that curing of the tumor-inducing virulence plasmid (pTi) of an octopine-type Agrobacterium tumefaciens lineage leads to a large deletion in the co-resident At megaplasmid (pAt). The deletion event is mediated by a repetitive IS-element, IS66, and results in a variety of environment-dependent fitness consequences, including loss of independent conjugal transfer of the plasmid. Interestingly, a related and otherwise wild-type A. tumefaciens strain is missing exactly the same large pAt segment as the pAt deletion derivatives, suggesting a similar event over its natural history. Overall, the findings presented here uncover a novel genetic interaction between the two large plasmids of A. tumefaciens and provide evidence for cross-replicon integration and co-evolution of these plasmids.
Members of the Rhizobiaceae, often carry multiple secondary replicons in addition to the primary chromosome with compatible repABC-based replication systems. Unlike secondary chromosomes and chromids, repABC-based megaplasmids and plasmids can undergo copy number fluctuations and are capable of conjugative transfer in response to environmental signals. Several Agrobacterium tumefaciens lineages harbor three secondary repABC-based replicons, including a secondary chromosome (often linear), the Ti (tumor-inducing) plasmid and the At megaplasmid. The Ti plasmid is required for virulence and encodes a conjugative transfer (tra) system that is strictly regulated by a subset of plant-tumor released opines and a well-described acyl-homoserine lactone (AHL)-based quorum-sensing mechanism. The At plasmids are generally not required for virulence, but carry genes that enhance rhizosphere survival, and these plasmids are often conjugatively proficient. We report that the At megaplasmid of the octopine-type strain A. tumefaciens 15955 encodes a quorum-controlled conjugation system that directly interacts with the paralogous quorum sensing system on the co-resident Ti plasmid. Both the pAt15955 and pTi15955 plasmids carry homologs of a TraI-type AHL synthase, a TraR-type AHL-responsive transcription activator, and a TraM-type anti-activator. The traI genes from both pTi15955 and pAt15955 can direct production of the inducing AHL (3-octanoyl-L-homoserine lactone) and together contribute to the overall AHL pool. The TraR protein encoded on each plasmid activates AHL-responsive transcription of target tra gene promoters. The pAt15955 TraR can cross-activate tra genes on the Ti plasmid as strongly as its cognate tra genes, whereas the pTi15955 TraR is preferentially biased toward its own tra genes. Putative tra box elements are located upstream of target promoters, and comparing between plasmids, they are in similar locations and share an inverted repeat structure, but have distinct consensus sequences. The two AHL quorum sensing systems have a combinatorial effect on conjugative transfer of both plasmids. Overall, the interactions described here have implications for the horizontal transfer and evolutionary stability of both plasmids and, in a broad sense, are consistent with other repABC systems that often have multiple quorum-sensing controlled secondary replicons.
1Bacteria with multi-replicon genome organizations, including members of the family 2 Rhizobiaceae, often carry a variety of niche-associated functions on large plasmids. 3 While evidence exists for cross-replicon interactions and co-evolution between replicons 4 in many of these systems, remarkable strain-to-strain variation is also observed for 5 extrachromosomal elements, suggesting increased genetic plasticity. Here, we show 6 that curing of the tumor-inducing virulence plasmid (pTi) of an octopine-type 7 Agrobacterium tumefaciens lineage leads to a large deletion in the co-resident At 8 megaplasmid (pAt). The deletion event is mediated by a repetitive IS-element, IS66, 9 and results in a variety of environment-dependent fitness consequences, including loss 10 of independent conjugal transfer of the plasmid. Interestingly, a related and otherwise 11 wild-type A. tumefaciens strain is missing exactly the same large pAt segment as the 12 pAt deletion derivatives, suggesting a similar event over its natural history. Overall, the 13 findings presented here uncover a novel genetic interaction between the two large 14 plasmids of A. tumefaciens and provide evidence for cross-replicon integration and co-15 evolution of these plasmids. 16 Bacteria exhibit a diversity of genomic architectures and contain replicons that range 18 from small, transient plasmids and megaplasmids, to chromids, to secondary and 19 primary chromosomes (diCenzo and Finan 2017). Primary chromosomes are the largest 20 replicons encoding core functions, whereas any replicating elements in addition to the 21 primary chromosome are defined as secondary replicons. Secondary chromosomes are 48 limitation to the coexistence of multiple replicons and have been observed in diverse 49 bacterial species, including species of Vibrio (Heidelberg et al. 2000; Ramachandran et 50 al. 2017) and Sinorhizobium (Ronson et al. 1987; Barnett et al. 2004; Bobik et al. 2006; 51 Galardini et al. 2015; Pini et al. 2015; diCenzo et al. 2018). In most cases, chromosomal 52 factors influence the regulation of secondary replicon factors, with limited regulation in 53 the opposite direction (Ronson et al. 1987; Barnett et al. 2004; Bobik et al. 2006; Agnoli 54 et al. 2012; Galardini et al. 2015; Pini et al. 2015; diCenzo et al. 2018), likely due to 55 intolerance of integral pathway perturbation and the suppression of costly accessory 56 functions required to stabilize secondary replicons. However, metabolic redundancy has 57 been observed between rhizobial replicons (González et al. 2005; diCenzo and Finan 58 2017), suggesting that favorable interaction and pathway integration between primary 59 and secondary replicons can occur. However, because the majority of organisms 60 possessing multi-partite genomes exist and transition between multiple environmental 61 reservoirs, and are often host-associated, the extent to which cross-replicon interactions 62 occur and their consequences are often not determined.63Here, we characterize a cross-replicon genetic interaction in ...
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