Genomic delineation and description of species and within-species lineages in the genus Pantoea.

As the name of the genus Pantoea ("of all sorts and sources") suggests, this genus includes bacteria with a wide range of provenances, including plants, animals, soils, components of the water cycle, and humans. Some members of the genus are pathogenic to plants, and some are suspected to be opportunistic human pathogens; while others are used as microbial pesticides or show promise in biotechnological applications. During its taxonomic history, the genus and its species have seen many revisions. However, evolutionary and comparative genomics studies have started to provide a solid foundation for a more stable taxonomy. To move further toward this goal, we have built a 2,509-gene core genome tree of 437 public genome sequences representing the currently known diversity of the genus Pantoea. Clades were evaluated for being evolutionarily and ecologically significant by determining bootstrap support, gene content differences, and recent recombination events. These results were then integrated with genome metadata, published literature, descriptions of named species with standing in nomenclature, and circumscriptions of yet-unnamed species clusters, 15 of which we assigned names under the nascent SeqCode. Finally, genome-based circumscriptions and descriptions of each species and each significant genetic lineage within species were uploaded to the LINbase Web server so that newly sequenced genomes of isolates belonging to any of these groups could be precisely and accurately identified.

Race-specific genotypes of Pseudomonas syringae pv. tomato are defined by the presence of mobile DNA elements within the genome.

Pseudomonas syringae pv. tomato is the causal agent of bacterial speck of tomato, an important disease that results in severe crop production losses worldwide. Currently, two races within phylogroup 01a (PG01a) are described for this pathogen. Race 0 strains have avirulence genes for the expression of type III system-associated effectors AvrPto1 and AvrPtoB, that are recognized and targeted by the effector-triggered immunity in tomato cultivars having the pto race-specific resistance gene. Race 1 strains instead lack the avrPto1 and avrPtoB genes and are therefore capable to aggressively attack all tomato cultivars. Here, we have performed the complete genome sequencing and the analysis of P. syringae pv. tomato strain DAPP-PG 215, which was described as a race 0 strain in 1996. Our analysis revealed that its genome comprises a 6.2 Mb circular chromosome and two plasmids (107 kb and 81 kb). The results indicate that the strain is phylogenetically closely related to strains Max13, K40, T1 and NYS-T1, all known race 1 strains. The chromosome of DAPP-PG 215 encodes race 1-associated genes like avrA and hopW1 and lacks race 0-associated genes like hopN1, giving it a race 1 genetic background. However, the genome harbors a complete ortholog of avrPto1, which allows the strain to display a race 0 phenotype. Comparative genomics with several PG01a genomes revealed that mobile DNA elements are rather involved in the evolution of the two different races.

Genomic Identification of Two Phytobacter diazotrophicus Isolates from a Neonatal Intensive Care Unit in Singapore.

We report the draft genome sequences of two Phytobacter diazotrophicus isolates recovered from a swab specimen from the water faucet located in the Neonatal Intensive Care Unit (ICU), National University Hospital, Singapore. The isolates were misidentified as Cronobacter sakazakii and Klebsiella oxytoca using biochemical methods. Whole-genome sequencing (WGS) was performed to determine their identity.

Dissemination of NDM-producing bacteria in Southern Brazil.

BACKGROUND: The dissemination of NDM-1 carbapenemases (New Delhi Metallo-ß-lactamase) is a global public health problem, mainly in developing countries. The aim of this study was to characterize the spread of NDM-producing bacteria in the Southern Brazilian states analyzing epidemiological, molecular, and antimicrobial susceptibility aspects. METHODS: A total of 10,684 carbapenem-resistant isolates of Enterobacterales, Pseudomonas spp. and Acinetobacter spp. obtained from several hospitals in eight cities in Southern Brazil were screened, and 486 NDM-producing bacteria were selected. RESULTS: The incidence varied from 0.5 to 77 cases/100.000 habitants. ST11, ST15, ST340 and ST674 were the most common in K. pneumoniae. A total of 5 plasmids were identified in one K. pneumoniae strain: Col440I, Col440II, IncFIA(HI1), IncFIB(K), IncFIB(pQil)/ IncFII(K), and IncR. CONCLUSIONS: The number of patients with NDM-producing bacteria has increased in Southern Brazil, whose gene is present in different plasmids, explaining the expansion of this enzyme.

Comparative genomics to examine the endophytic potential of Pantoea agglomerans DAPP-PG 734.

Pantoea agglomerans DAPP-PG 734 was isolated as endophyte from knots (tumors) caused by Pseudomonas savastanoi pv. savastanoi DAPP-PG 722 in olive trees. To understand the plant pathogen-endophyte interaction on a genomic level, the whole genome of P. agglomerans DAPP-PG 734 was sequenced and annotated. The complete genome had a total size of 5'396'424 bp, containing one circular chromosome and four large circular plasmids. The aim of this study was to identify genomic features that could play a potential role in the interaction between P. agglomerans DAPP-PG 734 and P. savastanoi pv. savastanoi DAPP-PG 722. For this purpose, a comparative genomic analysis between the genome of P. agglomerans DAPP-PG 734 and those of related Pantoea spp. was carried out. In P. agglomerans DAPP-PG 734, gene clusters for the synthesis of the Hrp-1 type III secretion system (T3SS), type VI secretion systems (T6SS) and autoinducer, which could play an important role in a plant-pathogenic community enhancing knot formation in olive trees, were identified. Additional gene clusters for the biosynthesis of two different antibiotics, namely dapdiamide E and antibiotic B025670, which were found in regions between integrative conjugative elements (ICE), were observed. The in-depth analysis of the whole genome suggested a characterization of the P. agglomerans DAPP-PG 734 isolate as endophytic bacterium with biocontrol activity rather than as a plant pathogen.

Microbial diversity across compartments in an aquaponic system and its connection to the nitrogen cycle.

Aquaponics combines hydroponic crop production with recirculating aquaculture. These systems comprise various compartments (fish tank, biofilter, sump, hydroponic table, radial flow settler and anaerobic digester), each with their own specific environmental pressures, which trigger the formation of unique microbial communities. Triplicated aquaponic systems were used to investigate the microbial community composition during three lettuce growing cycles. The sampling of individual compartments allowed community patterns to be generated using amplicon sequencing of bacterial and archaeal 16S rRNA genes. Nitrifying bacteria were identified in the hydroponic compartments, indicating that these compartments may play a larger role than previously thought in the system's nitrogen cycle. In addition to the observed temporal changes in community compositions within the anaerobic compartment, more archaeal reads were obtained from sludge samples than from the aerobic part of the system. Lower bacterial diversity was observed in fresh fish feces, where a highly discrete gut flora composition was seen. Finally, the most pronounced differences in microbial community compositions were observed between the aerobic and anaerobic loops of the system, with unique bacterial compositions in each individual compartment.

Spontaneous Resistance of Erwinia amylovora Against Bacteriophage Y2 Affects Infectivity of Multiple Phages.

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.

Comparative Genomics of Prunus-Associated Members of the Pseudomonas syringae Species Complex Reveals Traits Supporting Co-evolution and Host Adaptation.

Members of the Pseudomonas syringae species complex cause symptoms that are ranging from leaf spots to cankers on a multitude of plant species, including some of the genus Prunus. To date, a total of two species of the P. syringae species complex and six different pathovars have been associated with diseases on Prunus spp., which were shown to belong to different phylogenetic units (phylogroups, PG) based on sequence similarity of housekeeping genes or whole genomes, suggesting that virulence to Prunus spp. may be the result of convergent pathoadaptation. In this study, a comparative genomics approach was used to determine genes significantly associated with strains isolated from Prunus spp. across a phylogeny of 97 strains belonging to the P. syringae species complex. Our study revealed the presence of a set of orthologous proteins which were significantly associated with strains isolated from Prunus spp. than in strains isolated from other hosts or from non-agricultural environments. Among them, the type III effector HopAY predicted to encode for a C58 cysteine protease was found to be highly associated with strains isolated from Prunus spp. and revealed patterns supporting co-evolution and host adaptation.

Resolving taxonomic confusion: establishing the genus Phytobacter on the list of clinically relevant Enterobacteriaceae.

Although many clinically significant strains belonging to the family Enterobacteriaceae fall into a restricted number of genera and species, there is still a substantial number of isolates that elude this classification and for which proper identification remains challenging. With the current improvements in the field of genomics, it is not only possible to generate high-quality data to accurately identify individual nosocomial isolates at the species level and understand their pathogenic potential but also to analyse retrospectively the genome sequence databases to identify past recurrences of a specific organism, particularly those originally published under an incorrect or outdated taxonomy. We propose a general use of this approach to classify further clinically relevant taxa, i.e., Phytobacter spp., that have so far gone unrecognised due to unsatisfactory identification procedures in clinical diagnostics. Here, we present a genomics and literature-based approach to establish the importance of the genus Phytobacter as a clinically relevant member of the Enterobacteriaceae family.

Xanthomonas hydrangeae sp. nov., a novel plant pathogen isolated from Hydrangea arborescens.

This paper describes a novel species isolated in 2011 and 2012 from nursery-grown Hydrangea arborescens cultivars in Flanders, Belgium. After 4 days at 28 °C, the strains yielded yellow, round, convex and mucoid colonies. Pathogenicity of the strains was confirmed on its isolation host, as well as on Hydrangea quercifolia. Analysis using MALDI-TOF MS identified the Hydrangea strains as belonging to the genus Xanthomonas but excluded them from the species Xanthomonas hortorum. A phylogenetic tree based on gyrB confirmed the close relation to X. hortorum. Three fatty acids were dominant in the Hydrangea isolates: anteiso-C15 : 0, iso-C15 : 0 and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c). Unlike X. hortorum pathovars, the Hydrangea strains were unable to grow in the presence of lithium chloride and could only weakly utilize d-fructose-6-PO4 and glucuronamide. Phylogenetic characterization based on multilocus sequence analysis and phylogenomic characterization revealed that the strains are close to, yet distinct from, X. hortorum. The genome sequences of the strains had average nucleotide identity values ranging from 94.35-95.19 % and in silico DNA-DNA hybridization values ranging from 55.70 to 59.40 % to genomes of the X. hortorum pathovars. A genomics-based loop-mediated isothermal amplification assay was developed which was specific to the Hydrangea strains for its early detection. A novel species, Xanthomonas hydrangeae sp. nov., is proposed with strain LMG 31884T (=CCOS 1956T) as the type strain.

Synergistic interaction between the type III secretion system of the endophytic bacterium Pantoea agglomerans DAPP-PG 734 and the virulence of the causal agent of olive knot Pseudomonas savastanoi pv. savastanoi DAPP-PG 722.

The endophytic bacterium Pantoea agglomerans DAPP-PG 734 was previously isolated from olive knots caused by infection with Pseudomonas savastanoi pv. savastanoi DAPP-PG 722. Whole-genome analysis of this P. agglomerans strain revealed the presence of a Hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To assess the role of the P. agglomerans T3SS in the interaction with P. savastanoi pv. savastanoi, we generated independent knockout mutants in three Hrp genes of the P. agglomerans DAPP-PG 734 T3SS (hrpJ, hrpN, and hrpY). In contrast to the wildtype control, all three mutants failed to cause a hypersensitive response when infiltrated in tobacco leaves, suggesting that P. agglomerans T3SS is functional and injects effector proteins in plant cells. In contrast to P. savastanoi pv. savastanoi DAPP-PG 722, the wildtype strain P. agglomerans DAPP-PG 734 and its Hrp T3SS mutants did not cause olive knot disease in 1-year-old olive plants. Coinoculation of P. savastanoi pv. savastanoi with P. agglomerans wildtype strains did not significantly change the knot size, while the DAPP-PG 734 hrpY mutant induced a significant decrease in knot size, which could be complemented by providing hrpY on a plasmid. By epifluorescence microscopy and confocal laser scanning microscopy, we found that the localization patterns in knots were nonoverlapping for P. savastanoi pv. savastanoi and P. agglomerans when coinoculated. Our results suggest that suppression of olive plant defences mediated by the Hrp T3SS of P. agglomerans DAPP-PG 734 positively impacts the virulence of P. savastanoi pv. savastanoi DAPP-PG 722.

Microbial communities in floodplain ecosystems in relation to altered flow regimes and experimental flooding.

River floodplains are spatially diverse ecosystems that respond quickly to flow variations and disturbance. However, it remains unclear how flow alteration and hydrological disturbance impacts the structure and biodiversity of complex microbial communities in these ecosystems. Here, we examined the spatial and seasonal dynamics of microbial communities in aquatic (benthic) and terrestrial habitats of three hydrologically contrasting (natural flow, residual flow, hydropeaking flow) floodplain systems. Microbial communities (alpha and beta diversity) differed more among floodplain habitats than between riverine floodplains. Microbial communities in all systems displayed congruent seasonal effects. In the residual and hydropeaking systems, an experimental flood was released from a reservoir to mimic a natural high flow event causing hydromorphological disturbance. The experimental flood caused a temporary shift in microbial communities by releasing microbes from the reservoir as well as redistributing communities among floodplain habitats. The flood-mediated shift in community structures had only a transient impact as pelagic bacteria did not persist within floodplain habitats over time after the flood. More frequent pulse disturbances might lead to an alternate structure of bacterial communities in floodplains over time.

Broad diversity of bacteria degrading 17ß-estradiol-3-sulfate isolated from river sediment and biofilm at a wastewater treatment plant discharge.

Conjugated estrogens, such as 17ß-estradiol-3-sulfate (E2-3S), can be released into aquatic environments through wastewater treatment plants (WWTP). There, they are microbiologically degraded into free estrogens, which can have harmful effects on aquatic wildlife. Here, the degradation of E2-3S in environmental samples taken upstream, downstream and at the effluent of a WWTP was assessed. Sediment and biofilm samples were enriched for E2-3S-degrading microorganisms, yielding a broad diversity of bacterial isolates, including known and novel degraders of estrogens. Since E2-3S-degrading bacteria were also isolated in the sample upstream of the WWTP, the WWTP does not influence the ability of the microbial community to degrade E2-3S.

Environmental parameters and microbial community profiles as indication towards microbial activities and diversity in aquaponic system compartments.

BACKGROUND: An aquaponic system couples cultivation of plants and fish in the same aqueous medium. The system consists of interconnected compartments for fish rearing and plant production, as well as for water filtration, with all compartments hosting diverse microbial communities, which interact within the system. Due to the design, function and operation mode of the individual compartments, each of them exhibits unique biotic and abiotic conditions. Elucidating how these conditions shape microbial communities is useful in understanding how these compartments may affect the quality of the water, in which plants and fish are cultured. RESULTS: We investigated the possible relationships between microbial communities from biofilms and water quality parameters in different compartments of the aquaponic system. Biofilm samples were analyzed by total community profiling for bacterial and archaeal communities. The results implied that the oxygen levels could largely explain the main differences in abiotic parameters and microbial communities in each compartment of the system. Aerobic system compartments are highly biodiverse and work mostly as a nitrifying biofilter, whereas biofilms in the anaerobic compartments contain a less diverse community. Finally, the part of the system connecting the aerobic and anaerobic processes showed common conditions where both aerobic and anaerobic processes were observed. CONCLUSION: Different predicted microbial activities for each compartment were found to be supported by the abiotic parameters, of which the oxygen saturation, total organic carbon and total nitrogen differentiated clearly between samples from the main aerobic loop and the anaerobic compartments. The latter was also confirmed using microbial community profile analysis.

Complete or High-Quality Draft Genome Sequences of Six Xanthomonas hortorum Strains Sequenced with Short- and Long-Read Technologies.

We report the genome sequences of six Xanthomonas hortorum species-level clade members, X. hortorum pathovars taraxaci, pelargonii, cynarae, and gardneri (complete genome sequences) and X. hortorum pathovars carotae and vitians (high-quality draft genome sequences). Both short- and long-read sequencing technologies were used.

Host-Pathogen Interactions between Xanthomonas fragariae and Its Host Fragaria × ananassa Investigated with a Dual RNA-Seq Analysis.

Strawberry is economically important and widely grown, but susceptible to a large variety of phytopathogenic organisms. Among them, Xanthomonas fragariae is a quarantine bacterial pathogen threatening strawberry productions by causing angular leaf spots. Using whole transcriptome sequencing, the gene expression of both plant and bacteria in planta was analyzed at two time points, 12 and 29 days post inoculation, in order to compare the pathogen and host response between the stages of early visible and of well-developed symptoms. Among 28,588 known genes in strawberry and 4046 known genes in X. fragariae expressed at both time points, a total of 361 plant and 144 bacterial genes were significantly differentially expressed, respectively. The identified higher expressed genes in the plants were pathogen-associated molecular pattern receptors and pathogenesis-related thaumatin encoding genes, whereas the more expressed early genes were related to chloroplast metabolism as well as photosynthesis related coding genes. Most X. fragariae genes involved in host interaction, recognition, and pathogenesis were lower expressed at late-phase infection. This study gives a first insight into the interaction of X. fragariae with its host. The strawberry plant changed gene expression in order to consistently adapt its metabolism with the progression of infection.

Phytobacter palmae sp. nov., a novel endophytic, N2 fixing, plant growth promoting Gammaproteobacterium isolated from oil palm (Elaeis guineensis Jacq.).

A novel strain S29T with high nitrogen fixing ability was isolated from surface-sterilized leaf tissues of oil palm (Elaeis guineensis) growing in Science Park II, Singapore. On the basis of 16S rRNA gene analysis and multilocus sequence typing with the rpoB, gyrB, infB and atpD genes, strain S29T was a member of the genus Phytobacter, with Phytobacter ursingii ATCC 27989T and Phytobacter diazotrophicus LS 8T as its closest relatives. Unique biochemical features that differentiated strain S29T from its closest relatives were the ability to utilize melibiose, α-cyclodextrin, glycogen, adonital, d-arabitol, m-inositol and xylitol. The major fatty acids were C14 : 0, C16 : 0, C17 : 0, C16 : 1 ω5c and summed feature 2 containing C16 : 1 ω7c and/or C16 : 1 ω6c. The polar fatty acid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid and aminolipids. The draft genome of strain S29T comprised 5, 284, 330 bp with a G + C content of 52.6 %. The average nucleotide identity and digital DNA-DNA hybridization values between strain S29T and the phylogenetically related Enterobacterales species were lower than 95 % and 70 %, respectively. Thus, the polyphasic evidences generated through the phenotypic, chemotaxonomic and genomic methods confirmed that strain S29T represents a novel species of the genus Phytobacter, for which we propose the name Phytobacter palmae sp. nov. with the type strain of S29T (=DSM 27342T=KACC 17598T).

Updated Genome Sequence and Annotation for the Full Genome of Pseudomonas protegens CHA0.

Minor differences in the previously obtained genome of Pseudomonas protegens CHA0 were detected after resequencing the strain. Based on this, the genome size slightly increased. Additionally, we performed a manual annotation of genes involved in biocontrol and insect pathogenicity. This annotation version will be the basis for upcoming genome studies.

Pseudomonas tructae sp. nov., novel species isolated from rainbow trout kidney.

This study describes the biochemical and phylogenetic characteristics of a Gram-negative strain, SNU WT1T, isolated from rainbow trout kidney. The 16S rRNA gene sequencing indicated that strain SNU WT1T was highly similar to Pseudomonas wadenswilerensis CCOS 864T and closely related to other Pseudomonas putida-related strains. Multilocus sequence analysis of concatenated partial gyrB, rpoB and rpoD sequences revealed that strain SNU WT1T was distinct from P. putida-related strains and formed a separate clade. The average nucleotide identity and Genome-to-Genome Distance Calculator values were 90.19 and 41.7 %with its closest relative P. wadenswilerensis CCOS 864T; however, it was phenotypically distinct from CCOS 864T with respect to arginine dihydrolase, glucose fermentation, aesculin hydrolysis and N-acetyl-glucosamine assimilation. The major polar lipid of the strain was phosphatidylethanolamine and the major quinone was Q-9. The genome of strain SNU WT1T had 5 685 196 bp with a G+C content of 61.83 mol%. We describe a novel species of genus Pseudomonas, for which the name Pseudomonastructae has been proposed, with SNU WT1T (=KCTC 72265=JCM 33436) as the type strain.