Enhancement of herbicolin A production by integrated fermentation optimization and strain engineering in Pantoea agglomerans ZJU23.

BACKGROUND: The lipopeptide herbicolin A (HA) secreted by the biocontrol agent Pantoea agglomerans ZJU23 is a promising antifungal drug to combat fungal pathogens by targeting lipid rafts, both in agricultural and clinical settings. Improvement of HA production would be of great significance in promoting its commercialization. This study aims to enhance the HA production in ZJU23 by combining fermentation optimization and strain engineering. RESULTS: Based on the results in the single-factor experiments, corn steep liquor, temperature and initial pH were identified as the significant affecting factors by the Plackett-Burman design. The fermentation medium and conditions were further optimized using the Box-Behnken response surface method, and the HA production of the wild type strain ZJU23 was improved from ~ 87 mg/mL in King's B medium to ~ 211 mg/mL in HA induction (HAI) medium. A transposon library was constructed in ZJU23 to screen for mutants with higher HA production, and two transcriptional repressors for HA biosynthesis, LrhA and PurR, were identified. Disruption of the LrhA gene led to increased mRNA expression of HA biosynthetic genes, and subsequently improved about twofold HA production. Finally, the HA production reached ~ 471 mg/mL in the ΔLrhA mutant under optimized fermentation conditions, which is about 5.4 times higher than before (~ 87 mg/mL). The bacterial suspension of the ΔLrhA mutant fermented in HAI medium significantly enhanced its biocontrol efficacy against gray mold disease and Fusarium crown rot of wheat, showing equivalent control efficacies as the chemical fungicides used in this study. Furthermore, HA was effective against fungicide resistant Botrytis cinerea. Increased HA production substantially improved the control efficacy against gray mold disease caused by a pyrimethanil resistant strain. CONCLUSIONS: This study reveals that the transcriptional repressor LrhA negatively regulates HA biosynthesis and the defined HAI medium is suitable for HA production. These findings provide an extended basis for large-scale production of HA and promote biofungicide development based on ZJU23 and HA in the future.

Phylogenomic analysis of the Erwiniaceae supports reclassification of Kalamiella piersonii to Pantoea piersonii comb. nov. and Erwinia gerundensis to the new genus Duffyella gen. nov. as Duffyella gerundensis comb. nov.

To better understand the taxonomy of Erwinia in the context of the Erwiniaceae family, we carried out a taxogenomic analysis of the Erwiniaceae, a family that was created following the taxonomic revision of the family, Enterobacteriaceae. There has been no systematic analysis of this family, including the agriculturally relevant genus, Erwinia. Our analyses focused on 80 strains of Erwinia along with 37 strains representing 7 other genera in the family. We identified 308 common proteins, generated a genome-level phylogeny and carried out Average Nucleotide Identity, Average Amino Acid Identity and Percentage of Conserved Protein analyses. We show that multiple strains of Erwinia cannot be assigned to established species groups and that both Erwinia gerundensis and "Erwinia mediterraneensis" are not members of Erwinia. We propose the creation of the genus Duffyella gen. nov. and the reclassification of Erwinia gerundensis to this genus as the type species, Duffyella gerundensis comb. nov. Furthermore, divergence between other species within Erwinia as measured by Average Amino Acid Identity is greater than the divergence between Erwinia and other genera, supporting the possible subdivision of the genus Erwinia into at least two genera. Our analyses also suggest that there is no basis for the establishment of the genus Kalamiella within the Erwiniaceae or the taxonomic revision of the Pantoea septica lineage. Therefore, we propose reclassifying Kalamiella piersonii as Pantoea piersonii comb. nov. Our study provides new insight into the diversity of the Erwiniaceae and provides a solid foundation for advancing taxonomic revision of this broadly relevant family.

Effects of feeding a Lactobacillus plantarum JL01 diet on caecal bacteria and metabolites of weaned piglets.

Currently, knowledge is limited concerning the impact of a Lactobacillus plantarum JL01 diet for weaned piglets on caecal bacteria and metabolite profiles. In our experiments, 24 weaned piglets were randomly divided into two groups; each piglet in the treatment groups (Cec-Lac) was fed a basic diet and administered 10 ml of L. plantarum JL01 (1·0 × 109  CFU per ml) every day. The control group (Cec-Con) was fed a basic diet. After feeding for 28 days, we analysed the parameters of the caecal digesta of weaned piglets. We used 16S rDNA gene sequencing and mass spectrometry (MS)-based metabolomics techniques to investigate the effect of a L. plantarum JL01 diet on intestinal microbial composition and its metabolite profiles in the caecum contents of weaned piglets. The results showed that the richness estimators (ACE and Chao indices) in the caecal bacteria increased in the Cec-Lac group. Prevotella_2 and Desulfovibrio decreased significantly, while Pantoea and Rectale_group increased in the caecum of weaned piglets in the Cec-Lac group. Furthermore, Pearson's correlation analysis revealed that the genus Rectale_group was positively correlated with indole-3-acetic acid (P < 0·05), and the genus Pantoea had the same correlation with 1-palmitoyl lysophosphatidic acid. The metabolomics analysis revealed that the L. plantarum JL01 diet supplementation had significant effects on tryptophan metabolism and fat digestion and absorption. The results indicated that the L. plantarum JL01 dietary supplementation not only altered the microbial composition but also mediated tryptophan metabolism and fat digestion and absorption in the caecum, factors that may further affect the health of the host.

Characterization of a novel Pantoea symbiont allows inference of a pattern of convergent genome reduction in bacteria associated with Pentatomidae.

Phytophagous stink bugs typically harbor nutritional symbiotic bacteria in their midgut, to integrate their unbalanced diet. In the Pentatomidae, most symbionts are affiliated to the genus Pantoea, and are polyphyletic. This suggests a scenario of an ancestral establishment of symbiosis, followed by multiple symbiont replacement events by akin environmental bacteria in different host lineages. In this study, a novel Pantoeaspecies ('CandidatusPantoea persica') was characterized from the gut of the pentatomid Acrosternum arabicum, and shown to be highly abundant in a specific portion of the gut and necessary for the host development. The genome of the symbiont (2.9 Mb), while presenting putative host-supportive metabolic pathways, including those for amino acids and vitamin synthesis, showed a high level of pseudogenization, indicating ongoing genome reduction. Comparative analyses with other free-living and symbiotic Pantoea highlighted a convergent pattern of genome reduction in symbionts of pentatomids, putatively following the typical phases modelized in obligate nutritional symbionts of insects. Additionally, this system has distinctive traits, as hosts are closely related, and symbionts originated multiple independent times from closely related free-living bacteria, displaying convergent and independent conspicuous genome reduction. Due to such peculiarities, this may become an ideal model to study genome evolutionary processes in insect symbionts.

Genetic diversity of Pantoea stewartii subspecies stewartii causing jackfruit-bronzing disease in Malaysia.

Jackfruit-bronzing is caused by bacteria Pantoea stewartii subspecies stewartii (P. stewartii subsp. stewartii), showing symptoms of yellowish-orange to reddish discolouration and rusty specks on pulps and rags of jackfruit. Twenty-eight pure bacterial strains were collected from four different jackfruit outbreak collection areas in Peninsular Malaysia (Jenderam, Maran, Muadzam Shah and Ipoh). Positive P. stewartii subsp. stewartii verification obtained in the study was based on the phenotypic, hypersensitivity, pathogenicity and molecular tests. Multilocus sequence analysis (MLSA) was performed using four housekeeping genes (gyrB, rpoB, atpD and infB) on all 28 bacterial strains. Single gyrB, rpoB, atpD and infB phylogenetic trees analyses revealed the bootstrap value of 99-100% between our bacterial strains with P. stewartii subsp. stewartii reference strains and P. stewartii subsp. indologenes reference strains. On the other hand, phylogenetic tree of the concatenated sequences of the four housekeeping genes revealed that our 28 bacterial strains were more closely related to P. stewartii subsp. stewartii (99% similarities) compared to its close relative P. stewartii subsp. indologenes, although sequence similarity between these two subspecies were up to 100%. All the strains collected from the four collection areas clustered together, pointing to no variation among the bacterial strains. This study improves our understanding and provided new insight on the genetic diversity of P. stewartii subsp. stewartii associated with jackfruit-bronzing in Malaysia.

Evaluation of Pantoea eucalypti FBS135 for pine (Pinus massoniana) growth promotion and its genome analysis.

AIMS: Pinus massoniana is one of the most widely distributed forest plants in China. In this study, we isolated a bacterial endophyte (designated FBS135) from apical buds and needles of P. massoniana. Investigations were performed to understand the effects of the strain on pine growth, its genomic features and the functions of the plasmids it carries. METHODS AND RESULTS: Based on its morphological features and 16S rRNA sequence, strain FBS135 was primarily identified as Pantoea eucalypti. We found that FBS135 not only promoted the growth of P. massoniana seedlings, but also significantly increased the survival rate of pine seedlings. The whole genome of FBS135 was sequenced, which revealed that the bacterium carries one chromosome and four plasmids. Its chromosome is 4 023 751 bp in size and contains dozens of genes involved in plant symbiosis. Curing one of the four plasmids, pPant1, resulted in a decrease in the size of the FBS135 colonies and the loss of the ability to synthesize yellow pigment, indicating that this plasmid may be very important for FBS135. CONCLUSIONS: Pantoea eucalypti FBS135 has a genomic basis to be implicated in plant-associated lifestyle and was established to have the capability to promote pine growth. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report that such a bacterial species, P. eucalypti, was isolated from pine trees and evidenced to have pine beneficial activities. Our results elucidate the ecological effects of endophytes on forest plants as well as endophyte-plant interaction mechanisms.

High Prevalence of Pantoea in Diaphorina citri (Hemiptera: Liviidae): Vector of Citrus Huanglongbing Disease.

As an important insect vector, Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) transmits the pathogen 'Candidatus Liberibacter asiaticus' (CLas) that is associated with citrus greening also known as Huanglongbing (HLB) disease. The bacterial endosymbionts have a potential role in shaping the host range of insect herbivores and their performance on different host plants, which might affect the endosymbiont distribution in insect populations. Here, we detected and characterized Pantoea endosymbiont in nymph and adult ACP specimens collected from Citrus reticulata Blanco and Cordia myxa L. plants. The phylogenetic tree constructed using endosymbiotic bacteria 16S ribosomal RNA sequences indicated that Pantoea sp. was closely related to Mixta calida, sharing about 98% identity and was grouped with other Mixta and Pantoea endosymbionts. Our findings showed 100% and 92.3% infection of Pantoea in adults while 61.5% and 90% infection of Pantoea in nymphs collected from C. reticulata and C. myxa plants, respectively. Understanding the interaction of endosymbiotic bacteria with ACP associated with host plants could be useful for developing an effective management strategy for both ACP and HLB disease.

Molecular validation of clinical Pantoea isolates identified by MALDI-TOF.

The Enterobacterial genus Pantoea contains both free-living and host-associating species, with considerable debate as to whether documented reports of human infections by members of this species group are accurate. MALDI-TOF-based identification methods are commonly used in clinical laboratories as a rapid means of identification, but its reliability for identification of Pantoea species is unclear. In this study, we carried out cpn60-based molecular typing of 54 clinical isolates that had been identified as Pantoea using MALDI-TOF and other clinical typing methods. We found that 24% had been misidentified, and were actually strains of Citrobacter, Enterobacter, Kosakonia, Klebsiella, Pseudocitrobacter, members of the newly described Erwinia gerundensis, and even several unclassified members of the Enterobacteriaceae. The 40 clinical strains that were confirmed to be Pantoea were identified as Pantoea agglomerans, Pantoea allii, Pantoea dispersa, Pantoea eucalypti, and Pantoea septica as well as the proposed species group, Pantoea latae. Some species groups considered largely environmental or plant-associated, such as P. allii and P. eucalypti were also among clinical specimens. Our results indicate that MALDI-TOF-based identification methods may misidentify strains of the Enterobacteriaceae as Pantoea.

Pathogenicity and a TaqMan Real-Time PCR for Specific Detection of Pantoea allii, a Bacterial Pathogen of Onions.

Bacterial diseases of onion are reported to cause significant economic losses. Pantoea allii Brady, one of the pathogens causing the center rot on onions, has not yet been reported in Canada. We report the pathogenicity of P. allii on commercially available Canadian green onions (scallions). All P. allii-inoculated plants, irrespective of the inoculum concentration, exhibited typical leaf chlorotic discoloration on green onion leaves, which can reduce their marketability. Reisolation of P. allii from infected scallion tissues and reidentification by sequencing and phylogenetic analyses of the leuS gene suggest that the pathogen can survive in infected tissues 21 days after inoculation. This is the first report of P. allii as a potential pathogen of green onions. This study also reports the development and validation of a TaqMan real-time PCR assay targeting the leuS gene for reliable detection of P. allii in pure cultures and in planta. A 642-bp leuS gene fragment was targeted because it showed high nucleotide diversity and positively correlated with genome-based average nucleotide identity with respect to percent similarity index and identity of Pantoea species. The assay specificity was validated using 61 bacterial and fungal strains. Under optimal conditions, the selected primers and FAM-labeled TaqMan probe were specific for the detection of nine reference P. allii strains by real-time PCR. The 52 strains of other Pantoea spp. (n = 25), non-Pantoea spp. (n = 20), and fungi/oomycetes (n = 7) tested negative (no detectable fluorescence). Onion tissues spiked with P. allii, naturally infested onion bulbs, greenhouse infected green onion leaf samples, as well as an interlaboratory blind test were used to validate the assay specificity. The sensitivities of a 1-pg DNA concentration and 30 CFU are comparable to previously reported real-time PCR assays of other bacterial pathogens. The TaqMan real-time PCR assay developed in this study will facilitate reliable detection of P. allii and could be a useful tool for screening onion imports or exports for the presence of this pathogen.

Isolation and identification of temperature tolerant phosphate solubilizing bacteria as a potential microbial fertilizer.

Isolation and identification of temperature tolerant phosphate solubilizing bacteria (TTPSB) and their use as microbial fertilizers was the main goal of the study. In this study, TTPSB were isolated from soil samples treated for 16 h at 55 °C. Their phosphate solubilizing activity was either evaluated in solid media by forming a clear zone (halo) or in liquid media by quantification of the soluble phosphate in the growth medium. Five colonies (RPS4, RPS6, RPS7, RPS8 and RPS9) were identified to be able to form a halo and two of the isolates (RPS9 and RPS7) tolerated a temperature of 55 °C. With tricalcium phosphate (TCP) as the sole P-source, the phosphate solubilizing capacity of RPS9 and RPS7 was determined to be 563.8 and 324.1 mg P L-1 in liquid Sperber medium, respectively. Both bacterial isolates were identified as Pantoea agglomerans by molecular and biochemical characterization. To be used as a microbial fertilizer a carrier system for the temperature tolerant bacteria consisting of rock phosphate, sulfur and bagasse was used. It could be established that the bacterial cell counts of the microbial fertilizers were acceptable for application after storage for 4 months at 28 °C. In a greenhouse experiment using pot cultures, inoculation of maize (S.C.704) with the microbial fertilizers in an autoclaved soil resulted in a significant effect on total fresh and dry weight of the plant root and shoot as well as on the P content of the root and shoot. The effects observed with RPS9 as a component of the microbial fertilizer on plant growth and P nutrition was comparable with the addition of 50% of recommended triple superphosphate (TSP) dose. Using temperature tolerant bacteria in microbial fertilizers will overcome limitations in production and storage of the microbial fertilizers and contribute to a environmentally-friendly agriculture. The temperature tolerant P. agglomerans strain RPS9 was shown to be effective as part of a microbial fertilizer in supporting the growth and P uptake in maize.

Pantoea endophytica sp. nov., novel endophytic bacteria isolated from maize planting in different geographic regions of northern China.

Four endophytic bacterial strains were isolated from root, stem and leaf of maize planted in different regions of northern China. The four strains possessed almost identical 16S rRNA gene sequences. However, REP-PCR fingerprint patterns discriminated that they were not from one clonal origin. Furthermore, the average nucleotide identity (ANI) values among them were higher than 95%, suggesting they all belong to one species. Based on 16S rRNA gene phylogeny, the four strains were clustered together with Pantoea rodasii LMG 26273T and Pantoea rwandensis LMG 26275T, but on a separate branch. Multilocus sequence analysis (MLSA) indicated that the four strains form a novel Pantoea species. Authenticity of the novel species was confirmed by ANI comparisons between strain 596T and its closest relatives, since obtained values were considerably below the proposed thresholds for the species delineation. The genome size of 596T was 5.1Mbp, comprising 4896 predicted genes with DNA G+C content of 57.8mol%. The respiratory quinone was ubiquinone-8 (Q-8) and the polar lipid profile consisted of phosphatidylethanolamin, diphosphatidylglycerol, phosphatidylglycerol, unidentified aminophospholipid and unidentified phospholipid. The major fatty acids of strain 596T were C16:0, summed feature 2 (C12:0 aldehyde), summed feature 3 (C16:1ω7c and/or C16:1ω6c) and summed feature 8 (C18:1ω7c and/or C18:1ω6c). Based on phylogenetic, genomic, phenotypic and chemotaxonomic data, the four isolates are considered to represent a novel species of the genus Pantoea, for which the name Pantoea endophytica sp. nov., is proposed, with 596T (=DSM 100,785T=CGMCC 1.15280T) as type strain.

A Real-Time PCR Differentiating Pantoea stewartii subsp. stewartii From P. stewartii subsp. indologenes in Corn Seed.

Stewart's wilt of corn caused by the bacterium Pantoea stewartii subsp. stewartii is a seed-borne disease of major phytosanitary importance. Many countries have imposed restrictions on corn seed imports from regions where the disease occurs to prevent the potential introduction of the pathogen. Current laboratory testing methods (enzyme-linked immunosorbent assay [ELISA] and polymerase chain reaction [PCR]) cannot readily distinguish P. stewartii subsp. stewartii from the closely related subspecies Pantoea stewartii subsp. indologenes. However, P. stewartii subsp. indologenes, a nonpathogen on corn, is occasionally found on corn seed as part of the resident bacterial population and can yield false positive test results. A real-time PCR targeting the cpsAB intergenic sequence was developed to specifically detect P. stewartii subsp. stewartii from corn seeds and distinguish it from P. stewartii subsp. indologenes. The assay successfully detected P. stewartii subsp. stewartii from corn seed, and P. stewartii subsp. indologenes-contaminated seed lots, which previously yielded false positives by ELISA and published PCR methods, were negative. The absence of P. stewartii subsp. stewartii and the presence of P. stewartii subsp. indologenes in this seed were confirmed by size differentiation of the cpsAB amplicons in a conventional PCR. By distinguishing the two subspecies, the assays described would avoid false positive results and help prevent unnecessary restrictions on international movement of corn seed.

Development of a New Semi-Selective Lysine-Ornithine-Mannitol-Arginine-Charcoal Medium for the Isolation of Pantoea Species from Environmental Sources in Japan.

Although Pantoea species are widely distributed among plants, water, soils, humans, and animals, due to a lack of efficient isolation methods, the clonality of Pantoea species is poorly characterized. Therefore, we developed a new semi-selective medium designated 'lysine-ornithine-mannitol-arginine-charcoal' (LOMAC) to isolate these species. In an inclusive and exclusive study examining 94 bacterial strains, all Pantoea strains exhibited yellow colonies on LOMAC medium. The performance of the medium was assessed using Pantoea-spiked soils. Percent average agreement relative to the Api20E biochemical test was 97%. A total of 24 soil spot samples and 19 plant types were subjected to practical trials. Of the 91 yellow colonies selected on LOMAC medium, 81 were correctly identified as Pantoea species using the biochemical test. The sequencing of 16S rRNA (rrs) and gyrB from these isolates confirmed that Pantoea agglomerans, P. vagans, P. ananatis, and P. deleyi were present in Japanese fields. A phylogenetic analysis using rrs enabled only the limited separation of strains within each Pantoea spp., whereas an analysis using gyrB revealed higher variability and enabled the finer resolution of distinct branches. P. agglomerans isolates were divided into 3 groups, 2 of which were new clades, with the other comprising a large group including biocontrol strains. P. vagans was also in one of the new clades. The present results indicate that LOMAC medium is useful for screening Pantoea species. The use of LOMAC medium will provide new opportunities for identifying the beneficial properties of Japanese Pantoea isolates.

Antibacterial Activities of Crude Secondary Metabolite Extracts from Pantoea Species Obtained from the Stem of Solanum mauritianum and Their Effects on Two Cancer Cell Lines.

Endophytes are microorganisms that are perceived as non-pathogenic symbionts found inside plants since they cause no symptoms of disease on the host plant. Soil conditions and geography among other factors contribute to the type(s) of endophytes isolated from plants. Our research interest is the antibacterial activity of secondary metabolite crude extracts from the medicinal plant Solanum mauritianum and its bacterial endophytes. Fresh, healthy stems of S. mauritianum were collected, washed, surface sterilized, macerated in PBS, inoculated in the nutrient agar plates, and incubated for 5 days at 30 °C. Amplification and sequencing of the 16S rRNA gene was applied to identify the isolated bacterial endophytes. These endophytes were then grown in nutrient broth for 7⁻14 days, after which sterilized Amberlite® XAD7HP 20⁻60 mesh (Merck KGaA, Darmstadt, Germany) resin was added to each culture to adsorb the secondary metabolites, which were later extracted using ethyl acetate. Concentrated crude extracts from each bacterial endophyte were tested for antibacterial activity against 11 pathogenic bacteria and two human cancer cell lines. In this study, a total of three bacterial endophytes of the Pantoea genus were identified from the stem of S. mauritianum. The antibacterial test showed that crude secondary metabolites of the endophytes and stem of S. mauritianum possessed antibacterial properties against pathogenic microbes such as Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa, with concentrations showing inhibition ranging from 0.0625 to 8.0000 mg/mL. The anticancer analysis showed an increase in cell proliferation when A549 lung carcinoma and UMG87 glioblastoma cell lines were treated with both the plant and endophytes' crude extracts. As far as we know, this is the first study of its kind on Solanum mauritianum in South Africa showing S. mauritianum endophytes having activity against some of the common human pathogenic organisms.

[Evaluation of isolation and field effect of ginseng disease resistance and growth-promoting bacteria].

This study was aimed to isolate the strains with both disease resistance and growth-promoting, and clarify the field application effects of the strain for laying the further application foundation. The strains with good antagonistic effect were isolated from the 298 strains in Panax ginseng and the soil by plate confrontation method. The nitrogen fixation potential was verified by Ashby medium. The Salkowski method was used to determine the ability of producing IAA. Silicate medium screening and flame spectrophotometry was used to determine the ability of dissolving potassium. CAS method was applied to detect the ability of producing siderophores to determine its growth characteristics. The morphological, physiological and biochemical and 16S rRNA sequences were used to identify the species. The method of root irrigation was used to determine the effects of its disease control and growth-promoting on ginseng. A strain TY15 with broad spectrum of antimicrobial effect, nitrogen fixation, potassium-dissolving and the capacity of producing IAA and siderophores was obtained by screening. And the strain TY15 was identified as Pantoea agglomerans. The control effect of TY15 on the disease of ginseng in the field was 68.02%, which was equivalent to 68.94% of 30 billion per gram of beneficial microecological bacterium agent. The fresh weight of P. ginseng treated with TY15 strain was increased by 22.73% compared with the control group treated with water. And finally a strain TY15 with good application prospects was obtained.

Sex-related differences in the thanatomicrobiome in postmortem heart samples using bacterial gene regions V1-2 and V4.

Recent studies have revealed distinct thanatomicrobiome (microbiome of death) signatures in human body sites after death. Thanatomicrobiome studies suggest that microbial succession after death may have the potential to reveal important postmortem biomarkers for the identification of time of death. We surveyed the postmortem microbiomes of cardiac tissues from 10 corpses with varying times of death (6-58 h) using amplicon-based sequencing of the 16S rRNA gene' V1-2 and V4 hypervariable regions. The results demonstrated that amplicons had statistically significant (P < 0·05) sex-dependent changes. Clostridium sp., Pseudomonas sp., Pantoea sp. and Streptococcus sp. had the highest enrichment for both V1-2 and V4 regions. Interestingly, the results also show that V4 amplicons had higher abundance of Clostridium sp. and Pseudomonas sp. in female hearts compared to males. In addition, Streptococcus sp. was solely found in male heart samples. The distinction between sexes was further supported by principle coordinate analysis, which revealed microbes in female hearts formed a distinctive cluster separate from male cadavers for both hypervariable regions. This study provides data that demonstrates that two hypervariable regions show discriminatory power for sex differences in postmortem heart samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings represent preliminary data of the first thanatomicrobiome investigation of a comparison between 16S rRNA gene V1-2 and V4 amplicon signatures in corpse heart tissues. The results demonstrated that V4 hypervariable region amplicons had statistically significant (P < 0·05) sex-dependent microbial diversity. For example, Streptococcus sp. was solely found in male postmortem heart tissues. Interestingly, the results also show that V4 amplicons had higher abundance of Clostridium sp. and Pseudomonas sp. in female heart tissues compared to males. The finding of Clostridium sp. supports the postmortem clostridium effect in corpse heart tissues.

Consistent associations with beneficial bacteria in the seed endosphere of barley (Hordeum vulgare L.).

The importance of the plant microbiome for host fitness has led to the concept of the "plant holobiont". Seeds are reservoirs and vectors for beneficial microbes, which are very intimate partners of higher plants with the potential to connect plant generations. In this study, the endophytic seed microbiota of numerous barley samples, representing different cultivars, geographical sites and harvest years, was investigated. Cultivation-dependent and -independent analyses, microscopy, functional plate assays, greenhouse assays and functional prediction were used, with the aim of assessing the composition, stability and function of the barley seed endophytic bacterial microbiota. Associations were consistently detected in the seed endosphere with Paenibacillus, Pantoea and Pseudomonas spp., which were able to colonize the root with a notable rhizocompetence after seed germination. In greenhouse assays, enrichment with these bacteria promoted barley growth, improved mineral nutrition and induced resistance against the fungal pathogen Blumeria graminis. We demonstrated here that barley, an important crop plant, was consistently associated with beneficial bacteria inside the seeds. The results have relevant implications for plant microbiome ecology and for the holobiont concept, as well as opening up new possibilities for research and application of seed endophytes as bioinoculants in sustainable agriculture.

Heavy Metal Stress and Its Consequences on Exopolysaccharide (EPS)-Producing Pantoea agglomerans.

Currently, the heavy metal pollution is of grave concern, and the part of microorganism for metal bioremediation should take into account as an efficient and economic strategy. On this framework, the heavy metal stress consequences on exopolysaccharide (EPS)-producing agricultural isolate, Pantoea agglomerans, were studied. The EPS production is a protective response to stress to survive and grow in the metal-contaminated environment. P. agglomerans show tolerance and mucoid growth in the presence of heavy metals, i.e., mercury, copper, silver, arsenic, lead, chromium, and cadmium. EDX first confirmed the metal accumulation and further, FTIR determined the functional groups involved in metal binding. The ICP-AES identified the location of cell-bound and intracellular metal accumulation. Metal deposition on cell surface has released more Ca2+. The effect on bacterial morphology investigated with SEM and TEM revealed the sites of metal accumulation, as well as possible structural changes. Each heavy metal caused distinct change and accumulated on cell-bound EPS with some intracellular deposits. The metal stress caused a decrease in total protein content and increased in total carbohydrate with a boost in EPS. Thus, the performance of P. agglomerans under metal stress indicated a potential candidate for metal bioremediation. Graphical Abstract ᅟ.

Expression and characterization of Pantoea CO dehydrogenase to utilize CO-containing industrial waste gas for expanding the versatility of CO dehydrogenase.

Although aerobic CO dehydrogenases (CODHs) might be applicable in various fields, their practical applications have been hampered by low activity and no heterologous expression. We, for the first time, could functionally express recombinant PsCODH in E. coli and obtained a highly concentrated recombinant enzyme using an easy and convenient method. Its electron acceptor spectra, optimum conditions (pH 6.5 and 30 °C), and kinetic parameters (kcat of 12.97 s-1, Km of 0.065 mM, and specific activity of 0.86 Umg-1) were examined. Blast furnace gas (BFG) containing 20% CO, which is a waste gas from the steel-making process, was tested as a substrate for PsCODH. Even with BFG, the recombinant PsCODH retained 88.2% and 108.4% activity compared with those of pure CO and 20% CO, respectively. The results provide not only a promising strategy to utilize CO-containing industrial waste gases as cheap, abundant, and renewable resources but also significant information for further studies about cascade reactions producing value-added chemicals via CO2 as an intermediate produced by a CODH-based CO-utilization system, which would ultimately expand the versatility of CODH.

Phylogenomic resolution of the bacterial genus Pantoea and its relationship with Erwinia and Tatumella.

Investigation of the evolutionary relationships between related bacterial species and genera with a variety of lifestyles have gained popularity in recent years. For analysing the evolution of specific traits, however, a robust phylogeny is essential. In this study we examined the evolutionary relationships among the closely related genera Erwinia, Tatumella and Pantoea, and also attempted to resolve the species relationships within Pantoea. To accomplish this, we used the whole genome sequence data for 35 different strains belonging to these three genera, as well as nine outgroup taxa. Multigene datasets consisting of the 1039 genes shared by these 44 strains were then generated and subjected to maximum likelihood phylogenetic analyses, after which the results were compared to those using conventional multi-locus sequence analysis (MLSA) and ribosomal MLSA (rMLSA) approaches. The robustness of the respective phylogenies was then explored by considering the factors typically responsible for destabilizing phylogenetic trees. We found that the nucleotide datasets employed in the MLSA, rMLSA and 1039-gene datasets contained significant levels of homoplasy, substitution saturation and differential codon usage, all of which likely gave rise to the observed lineage specific rate heterogeneity. The effects of these factors were much less pronounced in the amino acid dataset for the 1039 genes, which allowed reconstruction of a fully supported and resolved phylogeny. The robustness of this amino acid tree was also supported by different subsets of the 1039 genes. In contrast to the smaller datasets (MLSA and rMLSA), the 1039 amino acid tree was also not as sensitive to long-branch attraction. The robust and well-supported evolutionary hypothesis for the three genera, which confidently resolved their various inter- and intrageneric relationships, represents a valuable resource for future studies. It will form the basis for studies aiming to understand the forces driving the divergence and maintenance of lineages, species and biological traits in this important group of bacteria.