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Broad application of antibiotics gave rise to increasing numbers of antibiotic resistant bacteria. Therefore, effective alternatives are currently investigated. Bacteriophages, natural predators of bacteria, could work as such an alternative. Although phages can be highly effective at eliminating specific bacteria, phage resistance can be observed after application. The nature of this resistance, however, can differ depending on the phage. Exposing Erwinia amylovora CFBP 1430, the causative agent of fire blight, to the different phages Bue1, L1, S2, S6, or M7 led to transient resistance. The bacteria reversed to a phage sensitive state after the phage was eliminated. When wild type bacteria were incubated with Y2, permanently resistant colonies (1430(Y2R)) formed spontaneously. In addition, 1430(Y2R) revealed cross-resistance against other phages (Bue1) or lowered the efficiency of plating (L1, S2, and S6). Pull down experiments revealed that Y2 is no longer able to bind to the mutant suggesting mutation or masking of the Y2 receptor. Other phages tested were still able to bind to 1430(Y2R). Bue1 was observed to still adsorb to the mutant, but no host lysis was found. These findings indicated that, in addition to the alterations of the Y2 receptor, the 1430(Y2R) mutant might block phage attack at different stage of infection. Whole genome sequencing of 1430(Y2R) revealed a deletion in the gene with the locus tag EAMY_2231. The gene, which encodes a putative galactosyltransferase, was truncated due to the resulting frameshift. The mutant 1430(Y2R) was monitored for potential defects or fitness loss. Weaker growth was observed in LB medium compared to the wild type but not in minimal medium. Strain 1430(Y2R) was still highly virulent in blossoms even though amylovoran production was observed to be reduced. Additionally, LPS structures were analyzed and were clearly shown to be altered in the mutant. Complementation of the truncated EAMY_2231 in trans restored the wild type phenotype. The truncation of EAMY_2231 can therefore be associated with manifold modifications in 1430(Y2R), which can affect different phages simultaneously., Frontiers in Microbiology, 13, ISSN:1664-302X

Many antibiotic resistance genes present in human pathogenic bacteria are believed to originate from environmental bacteria. Conjugation of antibiotic resistance conferring plasmids is considered to be one of the major reasons for the increasing prevalence of antibiotic resistances. A hotspot for plasmid-based horizontal gene transfer is the phyllosphere, i.e., the surfaces of aboveground plant parts. Bacteria in the phyllosphere might serve as intermediate hosts with transfer capability to human pathogenic bacteria. In this study, the exchange of mobilisable and self-transmissible plasmids via conjugation was evaluated. The conjugation from the laboratory strain Escherichia coli S17-1, the model phyllosphere coloniser Pantoea eucalypti 299R, and the model pathogen E. coli O157:H7 to the recipient strain E. coli O157:H7::MRE103 (EcO157:H7red) in the phyllosphere of Arabidopsis thaliana was determined. The results suggest that short-term occurrence of a competent donor is sufficient to fix plasmids in a recipient population of E. coli O157:H7red. The spread of self-transmissible plasmids was limited after initial steep increases of transconjugants that contributed up to 10% of the total recipient population. The here-presented data of plasmid transfer will be important for future modelling approaches to estimate environmental spread of antibiotic resistance in agricultural production environments.

This PDF file contains all supplementary tables and supplementary figures listed below. Table S1. Primers used in this study. Figure S1. Qualitative RT-PCR of selected T6SS genes. Figure S2. Growth curves with standard deviation measured for E. amylovora CFBP 1430 WT, T6-d1, T6-d3, and T6-d1d3 for 48 h in different media. Table S2. Significantly differentially expressed genes from the in vitro experiment. Figure S3. Classes of Erwinia amylovora CFBP 1430 genes (GO terms) that were affected by the mutation of T6SS (comparing T6-d1d3 mutant to WT) representing the fire blight T6SS transcriptome. Figure S4. Immature pears inoculated with E. amylovora CFBP 1430 or one of the T6SS mutants. Figure S5. E. amylovora CFBP 1430 and T6SS mutant populations on apple flowers at 1 DPI and 2 DPI. Table S3. Significantly differentially expressed genes from the in planta experiment. Figure S6. Role of E. amylovora CFBP 1430 T6SSs in bacterial competition. (PDF 633 kb)

Accession numbers of representative Pseudomonas genomes used to generate the phylogenetical trees in Fig. 2 ďťżand Additional file 2: Figure S2. (DOCX 70 kb)

Subread length distribution after sequencing. Figure S2. Phylogenetic tree of the genus Pseudomonas highlighting the position of P. citronellolis P3B5 relative to other representative Pseudomonas species. Xanthomonas campestris pv. campestris ATCC 33913 was chosen as outgroup. The tree is based on a MLSA using four housekeeping gene sequences (16S rRNA, gyrB, rpoB, rpoD). The bar represents the number of base substitutions per site. The percentage of replicate trees in which associated taxa clustered in the bootstrap test with 1000 replicates are shown next to the respective branches. Accession numbers of the used strains are reported in Additional file 1: Table S1. (DOCX 295 kb)

Posterpräsentation

A 530-kb megaplasmid pPag3 contributing 10.8% of the total genome of Pantoea vagans biocontrol strain C9-1 was sequenced. A rare nonpigmented variant C9-1W was obtained and shown to have lost pPag3, but retained all other plasmids (pPag1, pPag2). Phenotypic characterization of the variant confirmed the function of several annotated genes that may influence ecological fitness and efficacy. Metabolic profiling revealed important plasmid-based carbon utilization phenotypes. Plasmid loss resulted in thiamine auxotrophy, absence of carotenoid pigmentation, desferrioxamine diffusible siderophore biosynthesis, inherent ampicillin resistance and expression of AI-1 quorum-sensing signaling. This confirmed the functional expression of the corresponding genes located on pPag3 in P. vagans.

Background: Type VI secretion systems (T6SS) are widespread among Gram-negative bacteria and have a potential role as essential virulence factors or to maintain symbiotic interactions. Three T6SS gene clusters were identified in the genome of E. amylovora CFBP 1430, of which T6SS-1 and T6SS-3 represent complete T6SS machineries, while T6SS-2 is reduced in its gene content. Results: To assess the contribution of T6SSs to virulence and potential transcriptomic changes of E. amylovora CFBP 1430, single and double mutants in two structural genes were generated for T6SS-1 and T6SS-3. Plant assays showed that mutants in T6SS-3 were slightly more virulent in apple shoots while inducing less disease symptoms on apple flowers, indicating that T6SSs have only a minor effect on virulence of E. amylovora CFBP 1430. The mutations led under in vitro conditions to the differential expression of type III secretion systems, iron acquisition, chemotaxis, flagellar, and fimbrial genes. Comparison of the in planta and in vitro transcriptome data sets revealed a common differential expression of three processes and a set of chemotaxis and motility genes. Additional experiments proved that T6SS mutants are impaired in their motility. Conclusion: These results suggest that the deletion of T6SSs alters metabolic and motility processes. Nevertheless, the difference in lesion development in apple shoots and flower necrosis of T6SS mutants was indicative that T6SSs influences the disease progression and the establishment of the pathogen on host plants.