The Intermucosal Connection between the Mouth and Gut in Commensal Pathobiont-Driven Colitis.

The precise mechanism by which oral infection contributes to the pathogenesis of extra-oral diseases remains unclear. Here, we report that periodontal inflammation exacerbates gut inflammation in vivo. Periodontitis leads to expansion of oral pathobionts, including Klebsiella and Enterobacter species, in the oral cavity. Amassed oral pathobionts are ingested and translocate to the gut, where they activate the inflammasome in colonic mononuclear phagocytes, triggering inflammation. In parallel, periodontitis results in generation of oral pathobiont-reactive Th17 cells in the oral cavity. Oral pathobiont-reactive Th17 cells are imprinted with gut tropism and migrate to the inflamed gut. When in the gut, Th17 cells of oral origin can be activated by translocated oral pathobionts and cause development of colitis, but they are not activated by gut-resident microbes. Thus, oral inflammation, such as periodontitis, exacerbates gut inflammation by supplying the gut with both colitogenic pathobionts and pathogenic T cells.

Bacterial Swarmers Enriched During Intestinal Stress Ameliorate Damage.

BACKGROUND AND AIMS: Bacterial swarming, a collective movement on a surface, has rarely been associated with human pathophysiology. This study aims to define a role for bacterial swarmers in amelioration of intestinal stress. METHODS: We developed a polymicrobial plate agar assay to detect swarming and screened mice and humans with intestinal stress and inflammation. From chemically induced colitis in mice, as well as humans with inflammatory bowel disease, we developed techniques to isolate the dominant swarmers. We developed swarm-deficient but growth and swim-competent mutant bacteria as isogenic controls. We performed bacterial reinoculation studies in mice with colitis, fecal 16S, and meta-transcriptomic analyses, as well as in vitro microbial interaction studies. RESULTS: We show that bacterial swarmers are highly predictive of intestinal stress in mice and humans. We isolated a novel Enterobacter swarming strain, SM3, from mouse feces. SM3 and other known commensal swarmers, in contrast to their mutant strains, abrogated intestinal inflammation in mice. Treatment of colitic mice with SM3, but not its mutants, enriched beneficial fecal anaerobes belonging to the family of Bacteroidales S24-7. We observed SM3 swarming associated pathways in the in vivo fecal meta-transcriptomes. In vitro growth of S24-7 was enriched in presence of SM3 or its mutants; however, because SM3, but not mutants, induced S24-7 in vivo, we concluded that swarming plays an essential role in disseminating SM3 in vivo. CONCLUSIONS: Overall, our work identified a new but counterintuitive paradigm in which intestinal stress allows for the emergence of swarming bacteria; however, these bacteria act to heal intestinal inflammation.

Ethylene induced plant stress tolerance by Enterobacter sp. SA187 is mediated by 2-keto-4-methylthiobutyric acid production.

Several plant species require microbial associations for survival under different biotic and abiotic stresses. In this study, we show that Enterobacter sp. SA187, a desert plant endophytic bacterium, enhances yield of the crop plant alfalfa under field conditions as well as growth of the model plant Arabidopsis thaliana in vitro, revealing a high potential of SA187 as a biological solution for improving crop production. Studying the SA187 interaction with Arabidopsis, we uncovered a number of mechanisms related to the beneficial association of SA187 with plants. SA187 colonizes both the surface and inner tissues of Arabidopsis roots and shoots. SA187 induces salt stress tolerance by production of bacterial 2-keto-4-methylthiobutyric acid (KMBA), known to be converted into ethylene. By transcriptomic, genetic and pharmacological analyses, we show that the ethylene signaling pathway, but not plant ethylene production, is required for KMBA-induced plant salt stress tolerance. These results reveal a novel molecular communication process during the beneficial microbe-induced plant stress tolerance.

Complete genome sequence of plant growth-promoting and heavy metal-tolerant Enterobacter tabaci 4M9 (CCB-MBL 5004).

Enterobacter tabaci 4M9 (CCB-MBL 5004) was reported to have plant growth-promoting and heavy metal tolerance traits. It was able to tolerate more than 300 mg/L Cd, 600 mg/L As, and 500 mg/L Pb and still maintained the ability to produce plant growth-promoting substances under metal stress conditions. To explore the genetic basis of these beneficial traits, the complete genome sequencing of 4M9 was carried out using Pacific Bioscience (PacBio) sequencing technology. The complete genome consisted of one chromosome of 4,654,430 bp with a GC content of 54.6% and one plasmid of 51,135 bp with a GC content of 49.4%. Genome annotation revealed several genes involved in plant growth-promoting traits, including the production of siderophore, indole acetic acid, and 1-aminocyclopropane-1-carboxylate deaminase; solubilization of phosphate and potassium; and nitrogen metabolism. Similarly, genes involved in heavy metals (As, Co, Zn, Cu, Mn, Se, Cd, and Fe) tolerance were detected. These support its potential as a heavy metal-tolerant plant growth-promoting bacterium and a good genetic resource that can be employed to improve phytoremediation efficiency of heavy metal-contaminated soil via biotechnological techniques. This, to the best of our knowledge, is the first report on the complete genome sequence of heavy metal-tolerant plant growth-promoting E. tabaci.

Peptidoglycan of Bacterial Cell Wall Affects Competitive Properties of Microorganisms.

We studied the effect of bacterial wall peptidoglycan of 7 bacterial species on the competitive properties of human-associated microorganisms. Addition of peptidoglycan to the culture medium did not change the growth characteristics of the test cultures; however, an increase in the antagonism and hydrophobicity of Bifidobacterium sp. and Enterococcus sp. was observed, while the effect on enterobacteria was predominantly indifferent or inhibitory. The effect did not depend much on the source of peptidoglycan and was equally manifested on both indigenous and probiotic strains. The observed new property of peptidoglycan indicates its participation in the formation and functioning of microbiota. The obtained data on the regulation of the properties of microorganisms provide new possibilities for the correction and maintenance of host homeostasis through host-associated microbiota.

Involvement of NO3- in Ecophysiological Regulation of Dissimilatory Nitrate/Nitrite Reduction to Ammonium (DNRA) Is Implied by Physiological Characterization of Soil DNRA Bacteria Isolated via a Colorimetric Screening Method.

Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) has recently regained attention as a nitrogen retention pathway that may potentially be harnessed to alleviate nitrogen loss resulting from denitrification. Until recently, the ecophysiology of DNRA bacteria inhabiting agricultural soils has remained largely unexplored, due to the difficulty in targeted enrichment and isolation of DNRA microorganisms. In this study, >100 DNRA bacteria were isolated from NO3--reducing anoxic enrichment cultures established with rice paddy soils using a newly developed colorimetric screening method. Six of these isolates, each assigned to a different genus, were characterized to improve the understanding of DNRA physiology. All the isolates carried nrfA and/or nirB, and the Bacillus sp. strain possessed a clade II nosZ gene conferring the capacity for N2O reduction. A common prominent physiological feature observed in the isolates was NO2- accumulation before NH4+ production, which was further examined with Citrobacter sp. strain DNRA3 (possessing nrfA and nirB) and Enterobacter sp. strain DNRA5 (possessing only nirB). Both isolates showed inhibition of NO2--to-NH4+ reduction at submillimolar NO3- concentrations and downregulation of nrfA or nirB transcription when NO3- was being reduced to NO2- In batch and chemostat experiments, both isolates produced NH4+ from NO3- reduction when incubated with excess organic electron donors, while incubation with excess NO3- resulted in NO2- buildup but no substantial NH4+ production, presumably due to inhibitory NO3- concentrations. This previously overlooked link between NO3- repression of NO2--to-NH4+ reduction and the C-to-N ratio regulation of DNRA activity may be a key mechanism underpinning denitrification-versus-DNRA competition in soil.IMPORTANCE Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) is an anaerobic microbial pathway that competes with denitrification for common substrates NO3- and NO2- Unlike denitrification, which leads to nitrogen loss and N2O emission, DNRA reduces NO3- and NO2- to NH4+, a reactive nitrogen compound with a higher tendency to be retained in the soil matrix. Therefore, stimulation of DNRA has often been proposed as a strategy to improve fertilizer efficiency and reduce greenhouse gas emissions. Such attempts have been hampered by lack of insights into soil DNRA bacterial ecophysiology. Here, we have developed a new screening method for isolating DNRA-catalyzing organisms from agricultural soils without apparent DNRA activity. Physiological characteristics of six DNRA isolates were closely examined, disclosing a previously overlooked link between NO3- repression of NO2--to-NH4+ reduction and the C-to-N ratio regulation of DNRA activity, which may be a key to understanding why DNRA activity is rarely observed at substantial levels in nitrogen-rich agricultural soils.

Bloodstream infection due to Enterobacter ludwigii, correlating with massive aggregation on the surface of a central venous catheter.

We report a case of catheter associated bloodstream infection due to Enterobacter ludwigii with a massive aggregation on the outside surface of a central venous catheter (CVC). The 57 years old patient with a history of spondylodiscitis and Staphylococcus aureus-associated endocarditis was admitted to the intensive care unit for acute cerebral infarction. The patient developed signs of infections and the CVC was removed 11 days after placement. The infectious agent was identified by standard diagnostics to the genus level as belonging to the Enterobacter cloacae complex, and additional molecular testing determined the species as E. ludwigii. The catheter was selected for a study aiming to identify the influence of blood components on the formation of central venous catheter-associated biofilms. In this course a massive biofilm was recognized and is presented here.

Diet composition has a differential effect on immune tolerance in insect larvae exposed to Mesorhabditis belari, Enterobacter hormaechei and its metabolites.

In insects, diet plays an important role in growth and development. Insects can vary their diet composition based on their physiological needs. In this study we tested the influence of diet composition involving varying concentrations of macronutrients and zinc on the immune-tolerance following parasite and pathogen exposure in Spodoptera litura larvae. We also tested the insecticidal potential of Mesorhabditis belari, Enterobacter hormaechei and its secondary metabolites on Spodoptera litura larvae. The results shows macronutrient composition does not directly affect the larval tolerance to nematode infection. However, Zinc supplemented diet improved the immune tolerance. While larvae exposed to bacterial infection performed better on carbohydrate rich diet. Secondary metabolites from bacteria produced an immune response in dose dependent mortality. The study shows that the larvae maintained on different diet composition show varied immune tolerance which is based on the type of infection.

Biochemical and nutritional characterization of the medfly gut symbiont Enterobacter sp. AA26 for its use as probiotics in sterile insect technique applications.

BACKGROUND: Enterobacter sp. AA26 was recently isolated from the midgut of Ceratitis capitata (Wiedemann) and it was shown to have positive effects in rearing efficiency when used as larval probiotics. In this study, biomass production was carried out in bench-scale bioreactors to elucidate the biokinetic properties of Enterobacter sp. AA26 and its nutritional value. RESULTS: Strain AA26 is a psychrotolerant, halotolerant, facultatively anaerobic bacterium with broad pH range for growth (pH 4 to 10.2), which possessed the typical biochemical profile of Enterobacter spp. The specific oxygen uptake rate (SOUR) was calculated as 63.2 ± 1.26 and 121 ± 1.73 mg O2 g- 1 VSS h- 1, with the yield coefficients in acetate and glucose being equal to 0.62 ± 0.03 and 0.67 ± 0.003 g biomass produced/g substrate consumed, respectively. The maximum specific growth rate (µmax) of strain AA26 grown in fill-and-draw bioreactors at 20 °C and 35 °C was 0.035 and 0.069 h- 1, respectively. Strain AA26 grew effectively in agro-industrial wastewaters, i.e. cheese whey wastewater (CWW), as alternative substrate for replacing yeast-based media. Biomass of strain AA26 could provide all the essential amino acids and vitamins for the artificial rearing of C. capitata. Greater intracellular α- and ß-glucosidase activities were observed during growth of strain AA26 in CWW than in yeast-based substrate, although the opposite pattern was observed for the respective extracellular activities (p < 0.01). Low protease activity was exhibited in cells grown in yeast-based medium, while no lipase activities were detected. CONCLUSIONS: The ability of strain AA26 to grow in agro-industrial wastes and to provide all the essential nutrients can minimize the cost of commercial media used for mass rearing and large scale sterile insect technique applications.

Coculturing Bacteria Leads to Reduced Phenotypic Heterogeneities.

Isogenic bacterial populations are known to exhibit phenotypic heterogeneity at the single-cell level. Because of difficulties in assessing the phenotypic heterogeneity of a single taxon in a mixed community, the importance of this deeper level of organization remains relatively unknown for natural communities. In this study, we have used membrane-based microcosms that allow the probing of the phenotypic heterogeneity of a single taxon while interacting with a synthetic or natural community. Individual taxa were studied under axenic conditions, as members of a coculture with physical separation, and as a mixed culture. Phenotypic heterogeneity was assessed through both flow cytometry and Raman spectroscopy. Using this setup, we investigated the effect of microbial interactions on the individual phenotypic heterogeneities of two interacting drinking water isolates. Through flow cytometry we have demonstrated that interactions between these bacteria lead to a reduction of their individual phenotypic diversities and that this adjustment is conditional on the bacterial taxon. Single-cell Raman spectroscopy confirmed a taxon-dependent phenotypic shift due to the interaction. In conclusion, our data suggest that bacterial interactions may be a general driver of phenotypic heterogeneity in mixed microbial populations.IMPORTANCE Laboratory studies have shown the impact of phenotypic heterogeneity on the survival and functionality of isogenic populations. Because phenotypic heterogeneity plays an important role in pathogenicity and virulence, antibiotic resistance, biotechnological applications, and ecosystem properties, it is crucial to understand its influencing factors. An unanswered question is whether bacteria in mixed communities influence the phenotypic heterogeneity of their community partners. We found that coculturing bacteria leads to a reduction in their individual phenotypic heterogeneities, which led us to the hypothesis that the individual phenotypic diversity of a taxon is dependent on the community composition.

Enterobacter sp. AA26 gut symbiont as a protein source for Mediterranean fruit fly mass-rearing and sterile insect technique applications.

BACKGROUND: Insect species have established sophisticated symbiotic associations with diverse groups of microorganisms including bacteria which have been shown to affect several aspects of their biology, physiology, ecology and evolution. In addition, recent studies have shown that insect symbionts, including those localized in the gastrointestinal tract, can be exploited for the enhancement of sterile insect technique (SIT) applications against major insect pests such as the Mediterranean fruit fly (medfly) Ceratitis capitata. We previously showed that Enterobacter sp. AA26 can be used as probiotic supplement in medfly larval diet improving the productivity and accelerating the development of the VIENNA 8 genetic sexing strain (GSS), which is currently used in large scale operational SIT programs worldwide. RESULTS: Enterobacter sp. AA26 was an adequate nutritional source for C. capitata larvae, comprising an effective substitute for brewer's yeast. Incorporating inactive bacterial cells in the larval diet conferred a number of substantial beneficial effects on medfly biology. The consumption of bacteria-based diet (either as full or partial yeast replacement) resulted in decreased immature stages mortality, accelerated immature development, increased pupal weight, and elongated the survival under stress conditions. Moreover, neither the partial nor the complete replacement of yeast with Enterobacter sp. AA26 had significant impact on adult sex ratio, females' fecundity, adults' flight ability and males' mating competitiveness. The absence of both yeast and Enterobacter sp. AA26 (deprivation of protein source and possible other important nutrients) from the larval diet detrimentally affected the larval development, survival and elongated the immature developmental duration. CONCLUSIONS: Enterobacter sp. AA26 dry biomass can fully replace the brewer's yeast as a protein source in medfly larval diet without any effect on the productivity and the biological quality of reared medfly of VIENNA 8 GSS as assessed by the FAO/IAEA/USDA standard quality control tests. We discuss this finding in the context of mass-rearing and SIT applications.

Screening of intestinal probiotics and the effects of feeding probiotics on the growth, immune, digestive enzyme activity and intestinal flora of Litopenaeus vannamei.

In this experiment, 426 strains were isolated from the intestinal tract of Litopenaeus vannamei, and 11 strains showed strong digestive enzyme production activity and antagonistic effect against common bacterial pathogens of shrimp. After hemolysis activity test and drug sensitivity test, 2 candidate probiotics with good bacteriostatic activity, strong enzyme production ability and relatively sensitive to antibiotics were screened out, and were identified by 16s rDNA molecular identification and Biolog-System as Enterobacter hominis (E3) and lactobacillus (L3). First, the biological characteristics of 2 candidate probiotics were studied. The optimum growth conditions of E3: temperature, 30 °C; pH, 8.0; NaCl, 2.5%; bovine bile salt, 0.15%; and the optimum growth conditions of L3: temperature, 40 °C; pH, 6.0; NaCl, 0.5%; bovine bile salt, 0.0015%. Secondly, a 28-day feeding experiment was conducted using probiotic concentration of 107 CFU g-1 to determine the changes of the activities of blood related immune enzymes (SOD, PPO, ACP, POD, CAT, LZM) and intestinal digestive enzymes (NP, AL, LPS) during the feeding process of shrimp, the results showed that during the course of feeding, the activities of immune enzyme and digestive enzyme of shrimp fed with probiotics showed an increasing trend, and the growth rate of body weight of shrimp was higher than that of control group. After feeding, the cumulative mortality of probiotics groups were significantly lower than that of the control group after WSSV infection. And the mid-gut of L. vannamei was observed by electron microscope, the results showed that the intestinal mucosa was tight and the epithelium cells showed an active secretory state in probiotics group. Finally, the intestinal microbial communities of shrimp were compared and analyzed by using Biolog-ECO method in the later period of feeding, the results showed: compared with the control group, the average color change rate of the experimental group fed with probiotics increased significantly, indicating that probiotics enhanced the intestinal microorganism activity; The ability of intestinal microorganism to utilize carbon source was significantly enhanced in the experimental group, which indicated that the digestive enzyme secreted by probiotics could improve the digestion and absorption rate of prawn feed, thus promoting the rapid growth of shrimp; The Shannon index, Simpson index and McIntosh index of probiotics groups showed significant difference in 1st and 5th days, but tended to be the same in the 10th day, the results showed that probiotics could maintain in L. vannamei intestines at least 5 days.

Molecular Insights into the Involvement of a Never Ripe Receptor in the Interaction Between Two Beneficial Soil Bacteria and Tomato Plants Under Well-Watered and Drought Conditions.

Management of plant growth-promoting bacteria (PGPB) can be implemented to deal with sustainable intensification of agriculture. Ethylene is an essential component for plant growth and development and in response to drought. However, little is known about the effects of bacterial inoculation on ethylene transduction pathway. Thus, the present study sought to establish whether ethylene perception is critical for growth induction by two different PGPB strains under drought conditions and the analysis of bacterial effects on ethylene production and gene expression in tomatoes (Solanum lycopersicum). The ethylene-insensitive never ripe (nr) and its isogenic wild-type (wt) cv. Pearson line were inoculated with either Bacillus megaterium or Enterobacter sp. strain C7 and grown until the attainment of maturity under both well-watered and drought conditions. Ethylene perception is crucial for B. megaterium. However, it is not of prime importance for Enterobacter sp. strain C7 PGPB activity under drought conditions. Both PGPB decreased the expression of ethylene-related genes in wt plants, resulting in stress alleviation, while only B. megaterium induced their expression in nr plants. Furthermore, PGPB inoculation affected transcriptomic profile dependency on strain, genotype, and drought. Ethylene sensitivity determines plant interaction with PGPB strains. Enterobacter sp. strain C7 could modulate amino-acid metabolism, while nr mutation causes a partially functional interaction with B. megaterium, resulting in higher oxidative stress and loss of PGPB activity.

Norfloxacin salts of carboxylic acids curtail planktonic and biofilm mode of growth in ESKAPE pathogens.

AIMS: To enhance the antimicrobial and antibiofilm activity of norfloxacin against the planktonic and biofilm mode of growth in ESKAPE pathogens using chemically modified norfloxacin salts. METHODS AND RESULTS: Antimicrobial testing, synergy testing and time-kill curve analysis were performed to evaluate antibacterial effect of norfloxacin carboxylic acid salts against ESKAPE pathogens. In vivo efficacy to reduce bacterial bioburden was evaluated in zebrafish infection model. Crystal violet assay and live-dead staining were performed to discern antibiofilm effect. Membrane permeability, integrity and molecular docking studies were carried out to ascertain the mechanism of action. The carboxylic acid salts, relative to parent molecule norfloxacin, displayed two- to fourfold reduction in minimum inhibitory concentration against Staphylococcus aureus and Pseudomonas aeruginosa, in addition to displaying potent bacteriostatic effect against certain members of ESKAPE pathogens. In vivo treatments revealed that norfloxacin tartrate (SRIN2) reduced MRSA bioburden by greater than 1 log fold relative to parent molecule in the muscle tissue. In silico docking with gyrA of S. aureus showed increased affinity of SRIN2 towards DNA gyrase. The enhanced antibacterial effect of norfloxacin salts could be partially accounted by altered membrane permeability in S. aureus and perturbed membrane integrity in P. aeruginosa. Antibiofilm studies revealed that SRIN2 (norfloxacin tartrate) and SRIN3 (norfloxacin benzoate) exerted potent antibiofilm effect particularly against Gram-negative ESKAPE pathogens. The impaired colonization of both S. aureus and P. aeruginosa due to improved norfloxacin salts was further supported by live-dead imaging. CONCLUSION: Norfloxacin carboxylic acid salts can act as potential alternatives in terms of drug resensitization and reuse. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study shows that carboxylic acid salts of norfloxacin could be effectively employed to treat both planktonic- and biofilm-based infections caused by select members of ESKAPE pathogens.

Auto-aggregation properties of a novel aerobic denitrifier Enterobacter sp. strain FL.

Enterobacter sp. strain FL was newly isolated from activated sludge and exhibited significant capability of auto-aggregation as well as aerobic denitrification. The removal efficiencies of NO3--N, total nitrogen (TN), and TOC by strain FL in batch culture reached 94.6, 63.9, and 72.5% in 24 h, respectively. The production of N2O and N2 in the presence of oxygen demonstrated the occurrence of aerobic denitrification. The auto-aggregation index of strain FL reached 54.3%, suggesting a high tendency that the cells would agglomerate into aggregates. The production of extracellular polymeric substances (EPSs), which were mainly composed of proteins followed by polysaccharides, was considered to be related to the cell aggregation according to Fourier transform infrared (FT-IR) and confocal laser scanning microscopy (CLSM). The proteins in EPS were evenly and tightly combined to cells and altered the protein secondary structures of cell surface from random coils to ß-sheets and three-turn helices. The alteration of protein secondary structures of cell surface caused by the proteins in EPS might play a dominant role in the auto-aggregation of strain FL. To further assess the feasibility of strain FL for synthetic wastewater treatment, a sequencing batch reactor (SBR), solely inoculated with strain FL, was conducted. During the 16 running cycles, the removal efficiency of NO3--N was 90.2-99.7% and the auto-aggregation index was stabilized at 35.0-41.5%. The EPS promoted the biomass of strain FL to aggregate in the SBR.

Selecting Bacterial Antagonists for Cucurbit Downy Mildew and Developing an Effective Application Method.

To identify new bacterial antagonists for cucurbit downy mildew (CDM) caused by Pseudoperonospora cubensis, 163 bacterial isolates were recovered from different microenvironments of field-grown cucumber plants. In the greenhouse, 19 representative isolates were applied to cucumber plants as a foliar spray (FS); 7 isolates achieved the efficacy over 60% against CDM, with 5 (DS22, HS10, DP14, HP4, and DS57) identified as Bacillus pumilus, B. licheniformis, Enterobacter sp., Bacillus sp., and Stenotrophomonas maltophilia, respectively. Strains DP14, DS22, and HS10 were assessed for their biocontrol effect on naturally occurring CDM in 2-year field trials (2010 and 2011), in which their overall efficacy relative to that of propamocarb was 106.25 to 117.17% with foliar spray plus root drench (FS+RD) but only 70.98 to 84.03% with FS. Coincidently, DP14 and HS10 applied as root drench (RD) alone also significantly reduced CDM. Under field conditions, DP14, DS22, and HS10 all successfully colonized cucumber leaves and the rhizosphere, and also significantly increased fruit yield by 37.60 to 51.03%, as well as nutrient levels. Taken together, Enterobacter sp. DP14, B. licheniformis HS10, and B. pumilus DS22 are plant-growth-promoting rhizobacteria effective in controlling CDM in the field, whose efficacy increased with FS+RD compared with FS alone.

Helicoverpa zea gut-associated bacteria indirectly induce defenses in tomato by triggering a salivary elicitor(s).

Insect gut-associated microbes modulating plant defenses have been observed in beetles and piercing-sucking insects, but the role of caterpillar-associated bacteria in regulating plant induced defenses has not been adequately examined. We identified bacteria from the regurgitant of field-collected Helicoverpa zea larvae using 16S ribosomal RNA (rRNA) gene sequencing and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. A combination of biochemical, molecular, and confocal electron microscopy methods were used to determine the role of caterpillar-associated bacteria in mediating defenses in Solanum lycopersicum (tomato). Laboratory-reared H. zea inoculated with one of the bacteria identified in field-collected H. zea, Enterobacter ludwigii, induced expression of the tomato defense-related enzyme polyphenol oxidase and genes regulated by jasmonic acid (JA), whereas the salicylic acid (SA)-responsive pathogenesis-related gene was suppressed. Additionally, saliva and its main component glucose oxidase from inoculated caterpillars played an important role in elevating tomato anti-herbivore defenses. However, there were only low detectable amounts of regurgitant or bacteria on H. zea-damaged tomato leaves. Our results suggest that H. zea gut-associated bacteria indirectly mediate plant-insect interactions by triggering salivary elicitors. These findings provide a proof of concept that introducing gut bacteria to a herbivore may provide a novel approach to pest management through indirect induction of plant resistance.

Tomato ethylene sensitivity determines interaction with plant growth-promoting bacteria.

Background and Aims: Plant growth-promoting bacteria (PGPB) are soil micro-organisms able to interact with plants and stimulate their growth, positively affecting plant physiology and development. Although ethylene plays a key role in plant growth, little is known about the involvement of ethylene sensitivity in bacterial inoculation effects on plant physiology. Thus, the present study was pursued to establish whether ethylene perception is critical for plant-bacteria interaction and growth induction by two different PGPB strains, and to assess the physiological effects of these strains in juvenile and mature tomato ( Solanum lycopersicum ) plants. Methods: An experiment was performed with the ethylene-insensitive tomato never ripe and its isogenic wild-type line in which these two strains were inoculated with either Bacillus megaterium or Enterobacter sp. C7. Plants were grown until juvenile and mature stages, when biomass, stomatal conductance, photosynthesis as well as nutritional, hormonal and metabolic statuses were analysed. Key Results: Bacillus megaterium promoted growth only in mature wild type plants. However, Enterobacter C7 PGPB activity affected both wild-type and never ripe plants. Furthermore, PGPB inoculation affected physiological parameters and root metabolite levels in juvenile plants; meanwhile plant nutrition was highly dependent on ethylene sensitivity and was altered at the mature stage. Bacillus megaterium inoculation improved carbon assimilation in wild-type plants. However, insensitivity to ethylene compromised B. megaterium PGPB activity, affecting photosynthetic efficiency, plant nutrition and the root sugar content. Nevertheless, Enterobacter C7 inoculation modified the root amino acid content in addition to stomatal conductance and plant nutrition. Conclusions: Insensitivity to ethylene severely impaired B. megaterium interaction with tomato plants, resulting in physiological modifications and loss of PGPB activity. In contrast, Enterobacter C7 inoculation stimulated growth independently of ethylene perception and improved nitrogen assimilation in ethylene-insensitive plants. Thus, ethylene sensitivity is a determinant for B. megaterium , but is not involved in Enterobacter C7 PGPB activity.

Molecular characterization and antibacterial activity of a phage-type lysozyme from the Manila clam, Ruditapes philippinarum.

A phage-type lysozyme, designed as RpPLYZ, was cloned and characterized from the clam Ruditapes philippinarum. The full-length cDNA of RpPLYZ was of 699 bp with an open reading frame (ORF) of 534 bp, encoding a polypeptide of 177-amino acid with a calculated molecular mass of 19.6 kDa and an isoelectric point of 9.05. Multiple alignments and phylogenetic analysis strongly suggested that RpPLYZ was a new member of the phage-type lysozyme family. The mRNA transcript of RpPLYZ was found to be constitutively expressed in a wide range of tissues and mainly in hemocytes and mantle. The relative expression of RpPLYZ mRNA in hemocytes was significantly up-regulated at 6, 24, 48 and 72 h after Vibrio anguillarum challenge. The recombinant RpPLYZ (rRpPLYZ) showed high activity against Entherobacter cloacae and Staphyloccocus aureus, and less effective towards Entherobacter aerogenes and V. anguillarum. Moreover, the optimal pH, temperature and ionic strength for rRpPLYZ activity was determined to be 5.5, 50 °C and 5 mM, respectively. These results suggested that RpPLYZ was a member of the phage-type lysozyme family and perhaps played an important role in the immune responses against bacterial invasion.

Seed-vectored endophytic bacteria modulate development of rice seedlings.

AIM: The aim of the present study was to evaluate the effects of the removal of indigenous bacteria from rice seeds on seedling growth and development. Here we report the presence of three indigenous endophytic bacteria in rice seeds that play important roles in modulating seedling development (shoot and root lengths, and formation of root hairs and secondary roots) and defence against pathogens. METHODS AND RESULTS: Seed-associated bacteria were removed using surface sterilization with NaOCl (bleach) followed by antibiotic treatment. When bacteria were absent, growth of seedlings in terms of root hair development and overall seedling size was less than that of seedlings that contained bacteria. Reactive oxygen staining of seedlings showed that endophytic bacteria became intracellular in root parenchyma cells and root hairs. Roots containing endophytic bacteria were seen to stain densely for reactive oxygen, while roots free of bacteria stained lightly for reactive oxygen. Bacteria were isolated and identified as Enterobacter asburiae (VWB1), Pantoea dispersa (VWB2) and Pseudomonas putida (VWB3) by 16S rDNA sequencing. Bacteria were found to produce indole acetic acid (auxins), inhibited the pathogen Fusarium oxysporum and solubilized phosphate. Reinoculation of bacteria onto seedlings derived from surface-disinfected rice and Bermuda grass seeds significantly restored seedling growth and development. CONCLUSION: Rice seeds harbour indigenous bacterial endophytes that greatly influence seedling growth and development, including root and shoot lengths, root hair formation and disease susceptibility of rice seedlings. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that seeds of rice naturally harbour bacterial endophytes that play key roles in modulation of seedling development.