Comprehensive overexpression analysis of cyclic-di-GMP signalling proteins in the phytopathogen Pectobacterium atrosepticum reveals diverse effects on motility and virulence phenotypes.

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous bacterial signalling molecule produced by diguanylate cyclases of the GGDEF-domain family. Elevated c-di-GMP levels or increased GGDEF protein expression is frequently associated with the onset of sessility and biofilm formation in numerous bacterial species. Conversely, phosphodiesterase-dependent diminution of c-di-GMP levels by EAL- and HD-GYP-domain proteins is often accompanied by increased motility and virulence. In this study, we individually overexpressed 23 predicted GGDEF, EAL or HD-GYP-domain proteins encoded by the phytopathogen Pectobacterium atrosepticum strain SCRI1043. MS-based detection of c-di-GMP and 5'-phosphoguanylyl-(3'-5')-guanosine in these strains revealed that overexpression of most genes promoted modest 1-10-fold changes in cellular levels of c-di-GMP, with the exception of the GGDEF-domain proteins ECA0659 and ECA3374, which induced 1290- and 7660-fold increases, respectively. Overexpression of most EAL domain proteins increased motility, while overexpression of most GGDEF domain proteins reduced motility and increased poly-ß-1,6-N-acetyl-glucosamine-dependent flocculation. In contrast to domain-based predictions, overexpression of the EAL protein ECA3549 or the HD-GYP protein ECA3548 increased c-di-GMP concentrations and reduced motility. Most overexpression constructs altered the levels of secreted cellulases, pectinases and proteases, confirming c-di-GMP regulation of virulence in Pe. atrosepticum. However, there was no apparent correlation between virulence-factor induction and the domain class expressed or cellular c-di-GMP levels, suggesting that regulation was in response to specific effectors within the network, rather than total c-di-GMP concentration. Finally, we demonstrated that the cellular localization patterns vary considerably for GGDEF/EAL/HD-GYP proteins, indicating it is a likely factor restricting specific interactions within the c-di-GMP network.

Phage-selected lipopolysaccharide mutants of Pectobacterium atrosepticum exhibit different impacts on virulence.

AIMS: To positively select Pectobacterium atrosepticum (Pa) mutants with cell surface defects and to assess the impact of these mutations on phytopathogenesis. METHODS AND RESULTS: Several phages were isolated from treated sewage effluent and were found to require bacterial lipopolysaccharide (LPS) for infection. Two strains with distinct mutations in LPS were obtained by transposon mutagenesis. Along with a third LPS mutant, these strains were characterized with respect to various virulence-associated phenotypes, including growth rate, motility and exoenzyme production, demonstrating that LPS mutations are pleiotropic. Two of the strains were deficient in the synthesis of the O-antigen portion of LPS, and both were less virulent than the wild type. A waaJ mutant, which has severe defects in LPS biosynthesis, was dramatically impaired in potato tuber rot assays. The infectivity of these novel phages on 32 additional strains of Pa was tested, showing that most Pa isolates were sensitive to the LPS-dependent phages. CONCLUSIONS: Native LPS is crucial for optimal growth, survival and virulence of Pa in vivo, but simultaneously renders such strains susceptible to phage infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates the power of phages to select and identify the virulence determinants on the bacterial surface, and as potential biocontrol agents for Pa infections.

Exploitation of a new flagellatropic phage of Erwinia for positive selection of bacterial mutants attenuated in plant virulence: towards phage therapy.

AIMS: To isolate and characterize novel bacteriophages for the phytopathogen, Erwinia carotovora ssp. atroseptica (Eca), and to isolate phage-resistant mutants attenuated in virulence. METHODS AND RESULTS: A novel flagellatropic phage was isolated on the potato-rotting bacterial species, Eca, and characterized using electron microscopy and restriction analysis. The phage, named PhiAT1, has an icosahedral head and a long, contractile tail; it belongs to the Myoviridae family. Partial sequencing revealed the presence of genes with homology to those of coliphages T4, T7 and Mu. Phage-resistant transposon mutants of Eca were isolated and studied in vitro for a number of virulence-related phenotypes; only motility was found to be affected. In vivo tuber rotting assays showed that these mutants were attenuated in virulence, presumably because the infection is unable to spread from the initial site of inoculation. CONCLUSIONS: The Eca flagellum can act as a receptor for PhiAT1 infection, and resistant mutants are enriched for motility and virulence defects. SIGNIFICANCE AND IMPACT OF THE STUDY: PhiAT1 is the first reported flagellatropic phage found to infect Eca and has enabled further study of the virulence of this economically important phytopathogen.

Novel quorum-sensing-controlled genes in Erwinia carotovora subsp. carotovora: identification of a fungal elicitor homologue in a soft-rotting bacterium.

Seven new genes controlled by the quorum-sensing signal molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) have been identified in Erwinia carotovora subsp. carotovora. Using TnphoA as a mutagen, we enriched for mutants defective in proteins that could play a role in the interaction between E. carotovora subsp. carotovora and its plant hosts, and identified NipEcc and its counterpart in E. carotovora subsp. atroseptica. These are members of a growing family of proteins related to Nep1 from Fusarium oxysporum which can induce necrotic responses in a variety of dicotyledonous plants. NipEcc produced necrosis in tobacco, NipEca affected potato stem rot, and both affected virulence in potato tubers. In E. carotovora subsp. carotovora, nip was shown to be subject to weak repression by the LuxR family regulator, EccR, and may be regulated by the negative global regulator RsmA.

Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors.

The bacterial family Enterobacteriaceae is notable for its well studied human pathogens, including Salmonella, Yersinia, Shigella, and Escherichia spp. However, it also contains several plant pathogens. We report the genome sequence of a plant pathogenic enterobacterium, Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043, the causative agent of soft rot and blackleg potato diseases. Approximately 33% of Eca genes are not shared with sequenced enterobacterial human pathogens, including some predicted to facilitate unexpected metabolic traits, such as nitrogen fixation and opine catabolism. This proportion of genes also contains an overrepresentation of pathogenicity determinants, including possible horizontally acquired gene clusters for putative type IV secretion and polyketide phytotoxin synthesis. To investigate whether these gene clusters play a role in the disease process, an arrayed set of insertional mutants was generated, and mutations were identified. Plant bioassays showed that these mutants were significantly reduced in virulence, demonstrating both the presence of novel pathogenicity determinants in Eca, and the impact of functional genomics in expanding our understanding of phytopathogenicity in the Enterobacteriaceae.

The hexY genes of Erwinia carotovora ssp. carotovora and ssp. atroseptica encode novel proteins that regulate virulence and motility co-ordinately.

Mutations located in a new gene, hexY, in Erwinia carotovora ssp. carotovora (Ecc) and ssp. atroseptica (Eca) cause strong upregulation of production of exoenzyme virulence factors and motility. The hexY gene encodes a novel 14.4 kDa protein with no known homologues. The hexY mRNA transcript has an unusually long (525bp) 5' untranslated region, which may be important for post-transcriptional regulation. An elevated level of transcription of two exoenzyme genes, pelCand celV, was observed in the HexY mutant background. The levels of cellulase and protease in a HexY mutant were independent of the presence of PGA, suggesting a role for HexY in the induction of these enzymes seen upon PGA addition. Electron microscopy revealed that HexY cells were hyperflagellated, perhaps contributing to the hypermotility phenotype of this mutant. The HexY mutant M5 exhibited enhanced maceration capacity on potato tubers. Therefore, the hexY gene and its gene product may define another level of regulation of virulence determinants in Ecc and Eca.

A pleiotropic reduced virulence (Rvi-) mutant of Erwinia carotovora subspecies atroseptica is defective in flagella assembly proteins that are conserved in plant and animal bacterial pathogens.

Erwinia carotovora subsp. atroseptica was mutagenized and assayed for virulence in planta. Those mutants which exhibited reduced virulence (Rvi-) were assayed for growth rate, auxotrophy and extracellular enzyme secretion and seven mutants were found to be wild type for all of these phenotypes. When screened for other phenotypes, two were found to be non-motile. One mutant was complemented for motility by a heterologous gene library. A 2.7kb XmaIII-ClaI complementing fragment was sequenced and the gene products were found to have similarity to flagella biosynthesis gene products from several bacteria. Further similarity was found to a pathogenicity protein from the plant pathogen Xanthomonas campestris pv. glycines and to the Spa pathogenicity proteins of the human pathogen Shigella flexneri, which are involved in the surface presentation of antigens. These studies highlight the emergence of common themes in the molecular strategies employed by both plant and animal bacterial pathogens for the targeting of proteins involved in the elaboration of disease.

Isolation and characterisation of transposon-induced mutants of Erwinia carotovora subsp. atroseptica exhibiting reduced virulence.

The blackleg pathogen Erwinia carotovora subsp. atroseptica (Eca) causes an economically important disease of potatoes. We selected a genetically amenable Eca strain for the genetic analysis of virulence. Tn5 mutagenesis was used to generate nine mutants which exhibited reduced virulence (Rvi-) of strain SCRI1043. Following physiological characterisation, mutants were divided into three classes: (1) auxotrophs; (2) extracellular enzyme mutants; and (3) a growth rate mutant. The isolation of these Rvi- mutants has allowed us to consider some factors that affect Eca virulence.