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Pectobacterium atrosepticum and Pectobacterium carotovorum Harbor Distinct, Independently Acquired Integrative and Conjugative Elements Encoding Coronafacic Acid that Enhance Virulence on Potato Stems.

Panda P, Vanga BR, Lu A, Fiers M, Fineran PC, Butler R, Armstrong K, Ronson CW, Pitman AR - Front Microbiol (2016)

Bottom Line: Much is known about the functions of the virulence determinants that ICEs harbor, but little is understood about the cryptic effects of ICEs on their host cell.As expected, deletion of HAI2 resulted in reduced blackleg symptoms in potato stems.Thus, the future spread of these ICEs via lateral gene transfer might contribute to an increase in the prevalence of blackleg-causing strains of P. carotovorum.

View Article: PubMed Central - PubMed

Affiliation: The Bio-Protection Research CentreLincoln, New Zealand; Plant Pathology, The New Zealand Institute for Plant and Food Research LimitedLincoln, New Zealand.

ABSTRACT
Integrative and conjugative elements (ICEs) play a central role in the evolution of bacterial virulence, their transmission between bacteria often leading to the acquisition of virulence factors that alter host range or aggressiveness. Much is known about the functions of the virulence determinants that ICEs harbor, but little is understood about the cryptic effects of ICEs on their host cell. In this study, the importance of horizontally acquired island 2 (HAI2), an ICE in the genome of Pectobacterium atrosepticum SCRI1043, was studied using a strain in which the entire ICE had been removed by CRISPR-Cas-mediated genome editing. HAI2 encodes coronafacic acid, a virulence factor that enhances blackleg disease of potato stems caused by P. atrosepticum SCRI1043. As expected, deletion of HAI2 resulted in reduced blackleg symptoms in potato stems. A subsequent screen for HAI2-related ICEs in other strains of the Pectobacterium genus revealed their ubiquitous nature in P. atrosepticum. Yet, HAI2-related ICEs were only detected in the genomes of a few P. carotovorum strains. These strains were notable as blackleg causing strains belonging to two different subspecies of P. carotovorum. Sequence analysis of the ICEs in different strains of both P. atrosepticum and P. carotovorum confirmed that they were diverse and were present in different locations on the genomes of their bacterial host, suggesting that the cfa cluster was probably acquired independently on a number of occasions via chromosomal insertion of related ICEs. Excision assays also demonstrated that the ICEs in both P. atrosepticum and P. carotovorum are mobilized from the host chromosome. Thus, the future spread of these ICEs via lateral gene transfer might contribute to an increase in the prevalence of blackleg-causing strains of P. carotovorum.

No MeSH data available.


Related in: MedlinePlus

Excision of ICE PbN1_GI15 in P. carotovorum subsp. brasiliensis ICMP 19477. Electrophoresis gel image showing PCR amplicons associated with mobilization of ICE PbN1_GI15. Lane 1 (odc), ornithine decarboxylase gene; lane 2 (cfa7), coronafacic acid biosynthetic gene; lane 3 (attPPbN1_GI15), the attP site formed upon the circularization of PbN1_GI15 during excision from attBspeC; lane 4 (attBspeC), 152-bp amplicon indicative of reconstitution of the chromosomal target site attBspeC upon mobilization of PbN1_GI15; lane 5 (HL IV), Hyperladder IV (Bioline, UK); lane 6 (attR), the 153 bp sequence delimiting the right end of PbN1_GI15 when integrated within the attB site adjacent the speC; lane 7 (attL), the 153 bp sequence delimiting the left end of PbN1_GI15 when integrated within the attB site adjacent to speC.
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Figure 5: Excision of ICE PbN1_GI15 in P. carotovorum subsp. brasiliensis ICMP 19477. Electrophoresis gel image showing PCR amplicons associated with mobilization of ICE PbN1_GI15. Lane 1 (odc), ornithine decarboxylase gene; lane 2 (cfa7), coronafacic acid biosynthetic gene; lane 3 (attPPbN1_GI15), the attP site formed upon the circularization of PbN1_GI15 during excision from attBspeC; lane 4 (attBspeC), 152-bp amplicon indicative of reconstitution of the chromosomal target site attBspeC upon mobilization of PbN1_GI15; lane 5 (HL IV), Hyperladder IV (Bioline, UK); lane 6 (attR), the 153 bp sequence delimiting the right end of PbN1_GI15 when integrated within the attB site adjacent the speC; lane 7 (attL), the 153 bp sequence delimiting the left end of PbN1_GI15 when integrated within the attB site adjacent to speC.

Mentions: Despite the differences between PbN1_GI15 and HAI2, PbN1_GI15 retains features of a mobile element (e.g., an integrase, direct repeats). Thus, mobilization of PbN1_GI15 was examined by PCR. Firstly, integration of PbN1_GI15 into the chromosomal target site attBspeC in P. carotovorum subsp. brasiliensis ICMP 19477 was confirmed, with amplicons indicative of both attL and attR produced (Figure 5). However, many ICEs become anchored in the genome and lose their capacity to excise. Therefore, the excision of PbN1_GI15 from the chromosome was monitored by measuring the formation of attPPbN1_GI15 and attBspeC using PCR. These reactions produced fragments indicative of both loci (Figure 5), confirming precise excision of PbN1_GI15 and reconstitution of the chromosomal target site in P. carotovorum subsp. brasiliensis ICMP 19477. The amplification of attPPbN1_GI15 was consistent with this ICE retaining the capacity to excise from the chromosome prior to transfer to donor cells. No amplicons were produced in the negative PCR controls (containing no template DNA).


Pectobacterium atrosepticum and Pectobacterium carotovorum Harbor Distinct, Independently Acquired Integrative and Conjugative Elements Encoding Coronafacic Acid that Enhance Virulence on Potato Stems.

Panda P, Vanga BR, Lu A, Fiers M, Fineran PC, Butler R, Armstrong K, Ronson CW, Pitman AR - Front Microbiol (2016)

Excision of ICE PbN1_GI15 in P. carotovorum subsp. brasiliensis ICMP 19477. Electrophoresis gel image showing PCR amplicons associated with mobilization of ICE PbN1_GI15. Lane 1 (odc), ornithine decarboxylase gene; lane 2 (cfa7), coronafacic acid biosynthetic gene; lane 3 (attPPbN1_GI15), the attP site formed upon the circularization of PbN1_GI15 during excision from attBspeC; lane 4 (attBspeC), 152-bp amplicon indicative of reconstitution of the chromosomal target site attBspeC upon mobilization of PbN1_GI15; lane 5 (HL IV), Hyperladder IV (Bioline, UK); lane 6 (attR), the 153 bp sequence delimiting the right end of PbN1_GI15 when integrated within the attB site adjacent the speC; lane 7 (attL), the 153 bp sequence delimiting the left end of PbN1_GI15 when integrated within the attB site adjacent to speC.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814525&req=5

Figure 5: Excision of ICE PbN1_GI15 in P. carotovorum subsp. brasiliensis ICMP 19477. Electrophoresis gel image showing PCR amplicons associated with mobilization of ICE PbN1_GI15. Lane 1 (odc), ornithine decarboxylase gene; lane 2 (cfa7), coronafacic acid biosynthetic gene; lane 3 (attPPbN1_GI15), the attP site formed upon the circularization of PbN1_GI15 during excision from attBspeC; lane 4 (attBspeC), 152-bp amplicon indicative of reconstitution of the chromosomal target site attBspeC upon mobilization of PbN1_GI15; lane 5 (HL IV), Hyperladder IV (Bioline, UK); lane 6 (attR), the 153 bp sequence delimiting the right end of PbN1_GI15 when integrated within the attB site adjacent the speC; lane 7 (attL), the 153 bp sequence delimiting the left end of PbN1_GI15 when integrated within the attB site adjacent to speC.
Mentions: Despite the differences between PbN1_GI15 and HAI2, PbN1_GI15 retains features of a mobile element (e.g., an integrase, direct repeats). Thus, mobilization of PbN1_GI15 was examined by PCR. Firstly, integration of PbN1_GI15 into the chromosomal target site attBspeC in P. carotovorum subsp. brasiliensis ICMP 19477 was confirmed, with amplicons indicative of both attL and attR produced (Figure 5). However, many ICEs become anchored in the genome and lose their capacity to excise. Therefore, the excision of PbN1_GI15 from the chromosome was monitored by measuring the formation of attPPbN1_GI15 and attBspeC using PCR. These reactions produced fragments indicative of both loci (Figure 5), confirming precise excision of PbN1_GI15 and reconstitution of the chromosomal target site in P. carotovorum subsp. brasiliensis ICMP 19477. The amplification of attPPbN1_GI15 was consistent with this ICE retaining the capacity to excise from the chromosome prior to transfer to donor cells. No amplicons were produced in the negative PCR controls (containing no template DNA).

Bottom Line: Much is known about the functions of the virulence determinants that ICEs harbor, but little is understood about the cryptic effects of ICEs on their host cell.As expected, deletion of HAI2 resulted in reduced blackleg symptoms in potato stems.Thus, the future spread of these ICEs via lateral gene transfer might contribute to an increase in the prevalence of blackleg-causing strains of P. carotovorum.

View Article: PubMed Central - PubMed

Affiliation: The Bio-Protection Research CentreLincoln, New Zealand; Plant Pathology, The New Zealand Institute for Plant and Food Research LimitedLincoln, New Zealand.

ABSTRACT
Integrative and conjugative elements (ICEs) play a central role in the evolution of bacterial virulence, their transmission between bacteria often leading to the acquisition of virulence factors that alter host range or aggressiveness. Much is known about the functions of the virulence determinants that ICEs harbor, but little is understood about the cryptic effects of ICEs on their host cell. In this study, the importance of horizontally acquired island 2 (HAI2), an ICE in the genome of Pectobacterium atrosepticum SCRI1043, was studied using a strain in which the entire ICE had been removed by CRISPR-Cas-mediated genome editing. HAI2 encodes coronafacic acid, a virulence factor that enhances blackleg disease of potato stems caused by P. atrosepticum SCRI1043. As expected, deletion of HAI2 resulted in reduced blackleg symptoms in potato stems. A subsequent screen for HAI2-related ICEs in other strains of the Pectobacterium genus revealed their ubiquitous nature in P. atrosepticum. Yet, HAI2-related ICEs were only detected in the genomes of a few P. carotovorum strains. These strains were notable as blackleg causing strains belonging to two different subspecies of P. carotovorum. Sequence analysis of the ICEs in different strains of both P. atrosepticum and P. carotovorum confirmed that they were diverse and were present in different locations on the genomes of their bacterial host, suggesting that the cfa cluster was probably acquired independently on a number of occasions via chromosomal insertion of related ICEs. Excision assays also demonstrated that the ICEs in both P. atrosepticum and P. carotovorum are mobilized from the host chromosome. Thus, the future spread of these ICEs via lateral gene transfer might contribute to an increase in the prevalence of blackleg-causing strains of P. carotovorum.

No MeSH data available.


Related in: MedlinePlus