Evaluation of L-Asparaginase Purified from Pseudomonas Fluorescens as Inhibitor for Biofilm Producers in Dental Decays.

BACKGROUND: This study aimed to assess Pseudomonas fluorescens-purified L-Asparaginase's effectiveness as a broad-spectrum inhibitor of biofilm producers in dental decays. METHODS: The 16S rRNA sequence was used to build a phylogenetic tree to calculate the evolutionary distance between the isolated bacterial strain SW3 and other species. The evolutionary history was inferred by using the neighbor-joining approach. RESULTS: The bacteria were identified from dental decays, including Staphylococcus aureus, Streptococcus mutans, Streptococcus oralis, and Streptococcus mitis. Each one of these isolates showed different degrees of biofilm development. Purified L-Asparaginase inhibited the most potent Gram-positive biofilm-forming bacteria (biofilm producers) with higher inhibition percentages against Streptococcus oralis and Streptococcus mitis, 65 - 73.8 % and 54.7 - 63%, respectively. The inhibition percentages increased with increasing concentration and reached up to 74 - 81% with Streptococcus oralis and 66 - 74% with Streptococcus mitis, while SW3 bacteria showed (100%). This strain was suggested SW3 (Pseudomonas spp.). Pseudomonas fluorescens bacterial strain isolated from rhizosphere soil produced extracellular L-Asparaginase when grown on as a substrate. L-Asparaginase was purified to homogeneity by using ammonium sulfate at 60% saturation, followed by gel filtration chromatography on a sephadex G-100 column, with a recovery yield of 49% and a purification fold of 2.22. CONCLUSIONS: L-Asparaginase had a promising use for removing and avoiding biofilm growth, implying that it might be used in the dental industry in the future.

Investigation of presence of endofungal bacteria in Rhizopus spp. isolated from the different food samples.

Rhizopus species are opportunistic pathogens and cause infections which lead to deaths in individuals with the weakened immune system. Some strains of Rhizopus species have been detected to have a symbiotic relationship with bacteria. The toxicity of the Rhizopus species is important. Because strains harbouring endofungal bacteria are able to produce secondary metabolites and if endofungal bacteria are released from mycelium, serious problems can occur. We aimed to investigate the presence of endofungal bacteria in Rhizopus species isolated from food samples. Rhizopus species were isolated from different food samples. The presence of endofungal bacteria in the Rhizopus isolates was investigated. Rhizopus strains containing the endofungal bacteria were identified through phenotypic and genotypic methods. Universal primers amplifying bacterial 16S rRNA region were used to amplify 1.2-1.5-kb fragment from fungal metagenomic DNA. Sequence analysis of PCR products amplified from fungal metagenomic DNA was made. Fluorescence microscopy and scanning electron microscopy were used to visualize the presence of endofungal bacteria in fungal hyphae. According to our results, the Rhizopus strains is associated with Serratia marcescens, Pseudomonas fluorescens and Klebsiella pneumoniae. Until now there is no evidence that Pseudomonas fluorescens and Klebsiella pneumoniae were identified as endofungal. These species are opportunistic pathogen dangerous for humans. It is important for humans not only the presence of the fungi but also the presence of the endofungal bacteria in foods. Our work is important because it draws attention to the presence of endofungal bacteria in foods. Because there is danger releasing of a bacterium from the mycelium, it is likely to face sepsis or serious problems.

Pneumonia caused by Pseudomonas fluorescens: a case report.

BACKGROUND: Pseudomonas fluorescens (P. fluorescens) has been detected in respiratory samples from patients. However, no previous reports have been published about these P. fluorescens cultures from lung tissues. CASE PRESENTATION: Here, we report a case of pneumonia caused by P. fluorescens. P. fluorescens was identified from lung biopsy specimens for the first time in this case. According to the antibiotic susceptibility testing (AST) of P. fluorescens, the patient was given ciprofloxacin treatment. The temperature of the patient then returned to normal. Chest CT examination revealed improvements in pulmonary inflammation. CONCLUSIONS: These findings suggest that the patients with pneumonia caused by P. fluorescens should be treated in a timely manner according to the AST results.

Design and application of a portable luminometer for bioluminescence detection.

The silicon photomultiplier (SiPM) for low light detection has many advantages when compared to existing photon counting detectors, such as high sensitivity, low cost, robustness, and compact hardware. To facilitate the use of SiPM as a portable, field deployable device, an electrical circuit was designed consisting of an amplifier, comparator, and microcontroller. In addition, a 3D printing was used to create a portable cradle for housing the SiPM. To evaluate its detection ability, a laser experiment and bioluminescent experiments, including Pseudomonas fluorescens M3A detection, E. coli O157:H7 PhiV10nluc lysogen detection, and a luminescence-based detection of E. coli O157:H7 in ground meat using the engineered luminescent-based reporter phage PhiV10nluc, were conducted. In the same experimental setting, our previously developed smartphone-based luminometer called the bioluminescent-based analyte quantitation by smartphone and a conventional photomultiplier tube-based benchtop luminometer were used to compare detection levels and applicability for supporting luminescent phage-based pathogen detection. Results showed that the SiPM provides better performance in terms of time to detection and SNR and could be used as the light detection component of the PhiV10nluc phage-based detection format.

Interactions between fish isolates Pseudomonas fluorescens and Staphylococcus aureus in dual-species biofilms and sensitivity to carvacrol.

Biofilm formation is a frequent source of contamination of food products, which results in significant economic losses through microbial spoilage and poses serious health concerns. Little is known about the fate of Staphylococcus aureus in the dual-species biofilms with Pseudomonas fluorescens an important spoiler commonly found in aquatic products. This study evaluates the interactions between mono- or dual-species biofilms formed by P. fluorescens and S. aureus, as well as the sensitivity of the two tested strains to carvacrol. The biofilm cell population, expolysaccharide production, biofilm structures of P. fluorescens as mono- and dual-species with S. aureus at ratios of 1:1 and 1:0.01 were investigated with different concentrations of carvacrol (0, 0.4, 0.8 and 1.6 mM) in fish juice at 30 °C. The results show that the biofilm cell population of S. aureus in the dual-species was significantly lower (p < 0.05) than that in the mono-species, compared to no difference for P. fluorescens. In the co-culture the dominance of P. fluorescens inhibited the growing population of S. aureus in both planktonic and biofilm cells, however, two strains were stimulated to produce the large expolysaccharides and coaggregation, forming the complex spatial multibiofilm structures. The large increase in the dual-species biofilms was positively correlated with high quorum sensing autoinductor-2 (AI-2), and exogenous 4,5-dihydroxy-2,3-pentanedione (the AI-2 precursor, DPD), rather than C4-HSL, greatly stimulated the dual-species biofilm formation. In addition, carvacrol significantly reduced the tested biofilms and expolysaccharide secretion without affecting cell viability in a concentration-dependent manner, especially for S. aureus. Furthermore, the two strains as the dual-species biofilms exhibited lower sensitivity to carvacrol than the mono-culture, regardless of the level of inoculum of S. aureus, which was consistent with the decrease of AI-2 activity. The present study highlights that the interactions between P. fluorescens and S. aureus in dual-species biofilms promoted the large production of expolysaccharides and complex biofilm structures modulated by AI-2 signal, which results in the community-level resistance to carvacrol.

Fluorescent Pseudomonas and cyclic lipopeptide diversity in the rhizosphere of cocoyam (Xanthosoma sagittifolium).

Cocoyam (Xanthosoma sagittifolium (L.)), an important tuber crop in the tropics, is severely affected by the cocoyam root rot disease (CRRD) caused by Pythium myriotylum. The white cocoyam genotype is very susceptible while the red cocoyam has some field tolerance to CRRD. Fluorescent Pseudomonas isolates obtained from the rhizosphere of healthy red and white cocoyams from three different fields in Cameroon were taxonomically characterized. The cocoyam rhizosphere was enriched with P. fluorescens complex and P. putida isolates independent of the plant genotype. LC-MS and NMR analyses revealed that 50% of the Pseudomonas isolates produced cyclic lipopeptides (CLPs) including entolysin, lokisin, WLIP, putisolvin and xantholysin together with eight novel CLPs. In general, CLP types were linked to specific taxonomic groups within the fluorescent pseudomonads. Representative CLP-producing bacteria showed effective control against CRRD while purified CLPs caused hyphal branching or hyphal leakage in P. myriotylum. The structure of cocoyamide A, a CLP which is predominantly produced by P. koreensis group isolates within the P. fluorescens complex is described. Compared with the white cocoyam, the red cocoyam rhizosphere appeared to support a more diverse CLP spectrum. It remains to be investigated whether this contributes to the field tolerance displayed by the red cocoyam.

Virulence, attachment and invasion of Caco-2 cells by multidrug-resistant bacteria isolated from wild animals.

Wild animals may be considered important reservoirs for bacterial pathogens and, consequently, possible sources of infection for humans. In this study, selected multidrug-resistant bacteria (Acinetobacter spp., Aeromonas salmonicida, Klebsiella pneumoniae, Pseudomonas fluorescens and Shewanella putrefaciens) isolated from wild animals were characterized on their ability to attach and invade/internalize human colonic carcinoma (Caco-2) cells. In addition, the viability of these bacteria to survive under simulated human gastrointestinal tract conditions as well as the production of virulence factors (homoserine lactones signal molecules, gelatinases, proteases, siderophores and biofilm formation) were studied. The results suggests that all the bacteria presented the capacity to attach and internalize into Caco-2 cells. A. salmonicida and P. fluorescens exhibited the highest ability to internalize. These bacteria were also found to be the highest proteases producers. A. salmonicida and K. pneumoniae survived under simulated human gastrointestinal conditions. These were the bacteria with the highest capacity to produce biofilms. K. pneumoniae was the only bacterium producing siderophores. Taken together, the present results reinforce the need for the "One Health" initiative, underscoring the environment and the animals as important reservoirs of infectious determinants.

The stimulatory effects of plant growth promoting rhizobacteria and plant growth regulators on wheat physiology grown in sandy soil.

The present study was aimed to investigate the effects of plant growth promoting rhizobacteria (PGPR) and plant growth regulators (PGRs) on the physiology and yield of wheat grown in less fertile sandy soil. The isolated PGPR strains were identified by 16S-rRNA gene sequencing as Planomicrobium chinense (P1), Bacillus cereus (P2) and Pseudomonas fluorescens (P3). Wheat varieties (Galaxy-13 and Pak-2013) differing in sensitivity to drought were soaked in fresh cultures of bacterial isolates and the PGRs (salicylic acid and putrescine) were sprayed at 150 mg/L on seedlings at three leaf stage. PGPR and PGRs treated plants showed significant increase in the contents of chlorophyll, sugar and protein even under harsh environmental conditions. Drought stress enhanced the production of proline, antioxidant enzymes and lipid peroxidation but a decrease was noted in the biochemical content (i.e. chlorophyll, protein and sugar) of inoculated plants. PGPR inoculation also significantly enhanced the yield parameters (i.e. plant height, spike length, grain yield and weight) and improved the fertility status of sandy soil. The accumulation of macronutrient, total NO3-N and P concentration and soil moisture content of rhizosphere soil was also enhanced by PGPRs inoculation. It is concluded that the combined effects of PGPR and PGRs have profound effects on the biochemical responses and drought tolerance of wheat grown in sandy soils.

Tropical soils are a reservoir for fluorescent Pseudomonas spp. biodiversity.

Fluorescent Pseudomonas spp. are widely studied for their beneficial activities to plants. To explore the genetic diversity of Pseudomonas spp. in tropical regions, we collected 76 isolates from a Brazilian soil. Genomes were sequenced and compared to known strains, mostly collected from temperate regions. Phylogenetic analyses classified the isolates in the P. fluorescens (57) and P. putida (19) groups. Among the isolates in the P. fluorescens group, most (37) were classified in the P. koreensis subgroup and two in the P. jessenii subgroup. The remaining 18 isolates fell into two phylogenetic subclades distinct from currently recognized P. fluorescens subgroups, and probably represent new subgroups. Consistent with their phylogenetic distance from described subgroups, the genome sequences of strains in these subclades are asyntenous to the genome sequences of members of their neighbour subgroups. The tropical isolates have several functional genes also present in known fluorescent Pseudomonas spp. strains. However, members of the new subclades share exclusive genes not detected in other subgroups, pointing to the potential for novel functions. Additionally, we identified 12 potential new species among the 76 isolates from the tropical soil. The unexplored diversity found in the tropical soil is possibly related to biogeographical patterns.

Pseudomonas fluorescens Strain R124 Encodes Three Different MIO Enzymes.

A number of class I lyase-like enzymes, including aromatic ammonia-lyases and aromatic 2,3-aminomutases, contain the electrophilic 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) catalytic moiety. This study reveals that Pseudomonas fluorescens R124 strain isolated from a nutrient-limited cave encodes a histidine ammonia-lyase, a tyrosine/phenylalanine/histidine ammonia-lyase (XAL), and a phenylalanine 2,3-aminomutase (PAM), and demonstrates that an organism under nitrogen-limited conditions can develop novel nitrogen fixation and transformation pathways to enrich the possibility of nitrogen metabolism by gaining a PAM through horizontal gene transfer. The novel MIO enzymes are potential biocatalysts in the synthesis of enantiopure unnatural amino acids. The broad substrate acceptance and high thermal stability of PfXAL indicate that this enzyme is highly suitable for biocatalysis.

Effect of Origanum vulgare essential oil on biofilm formation and motility capacity of Pseudomonas fluorescens strains isolated from discoloured Mozzarella cheese.

AIMS: The study was focused on Pseudomonas fluorescens strains isolated from Mozzarella cheese, with the aim of evaluating the effects of Origanum vulgare L. essential oil (OEO) on the biofilm formation and eradication, as well as on the motility and blue pigment production at 10°C. METHODS AND RESULTS: Microdilution method was used to determine the minimum inhibitory and bactericidal concentration of the OEO, which ranged between 10 and 40 µl ml-1 . In vitro studies demonstrated that a sublethal concentration of OEO influenced not only P. fluorescens growth and motility but also the capability to form biofilm and, in a lower degree, the biofilm eradication at 10°C. Analysis by confocal microscopy revealed a dramatic reduction in biofilm formation and thickness, with scattered damage or death of cells, stained by propidium iodide. In addition, a concentration of 5 µl ml-1 of OEO affected the motility of the cells and, in particular, their ability to swim. However, the essential oil did not inhibit the blue pigment production by any of the tested strains. CONCLUSIONS: The present findings suggest that oregano essential oil inhibits the biofilm formation of P. fluorescens strains and alters their motility. Moreover, in the preformed biofilm, OEO contributes to the detachment of the cells, deteriorating the architecture of the biofilm and reducing its thickness. SIGNIFICANCE AND IMPACT OF THE STUDY: The O. vulgare L. essential oil was revealed as a promising agent against biofilm formation and for its detaching; these results suggest that oregano EO could be used in the dairy food industry to control biofilm formation, as an alternative, or in combination with conventional sanitizers.

Pseudomonas fluorescens group bacterial strains are responsible for repeat and sporadic postpasteurization contamination and reduced fluid milk shelf life.

Postpasteurization contamination (PPC) of high temperature, short time-pasteurized fluid milk by gram-negative (GN) bacteria continues to be an issue for processors. To improve PPC control, a better understanding of PPC patterns in dairy processing facilities over time and across equipment is needed. We thus collected samples from 10 fluid milk processing facilities to (1) detect and characterize PPC patterns over time, (2) determine the efficacy of different media to detect PPC, and (3) characterize sensory defects associated with PPC. Specifically, we collected 280 samples of high temperature, short time-pasteurized milk representing different products (2%, skim, and chocolate) and different fillers over 4 samplings performed over 11 mo at each of the 10 facilities. Standard plate count (SPC) as well as total GN, coliform, and Enterobacteriaceae (EB) counts were performed upon receipt and after 7, 10, 14, 17, and 21 d of storage at 6°C. We used 16S rDNA sequencing to characterize representative bacterial isolates from (1) test days with SPC >20,000 cfu/mL and (2) all samples with presumptive GN, coliforms, or EB. Day-21 samples were also evaluated by a trained defect judging panel. By d 21, 226 samples had SPC >20,000 cfu/mL on at least 1 d of shelf life; GN bacteria were found in 132 of these 226 samples, indicating PPC. Crystal violet tetrazolium agar detected PPC with the greatest sensitivity. Spoilage due to PPC was predominantly associated with Pseudomonas (isolated from 101 of the 132 samples with PPC); coliforms and EB were found in 27 and 37 samples with spoilage due to PPC, respectively. Detection of Pseudomonas and Acinetobacter was associated with lower flavor scores; coagulated, fruity fermented, and unclean defects were more prevalent in d-21 samples with PPC. Repeat isolation of Pseudomonas fluorescens group strains with identical partial 16S rDNA sequence types was observed in 8 facilities. In several facilities, specific lines, products, or processing days were linked to repeat product contamination with Pseudomonas with identical sequence types. Our data show that PPC due to Pseudomonas remains a major challenge for fluid milk processors; the inability of coliform and EB tests to detect Pseudomonas may contribute to this. Our data also provide important initial insights into PPC patterns (e.g., line-specific contamination), supporting the importance of molecular subtyping methods for identification of PPC sources.

Evaluation of the spoilage potential of bacteria isolated from chilled chicken in vitro and in situ.

Microorganisms play an important role in the spoilage of chilled chicken. In this study, a total of 53 isolates, belonging to 7 species of 3 genera, were isolated using a selective medium based on the capacity to spoil chicken juice. Four isolates, namely Aeromonas salmonicida 35, Pseudomonas fluorescens H5, Pseudomonas fragi H8 and Serratia liquefaciens 17, were further characterized to assess their proteolytic activities in vitro using meat protein extracts and to evaluate their spoilage potential in situ. The in vitro studies showed that A. salmonicida 35 displayed the strongest proteolytic activity against both sarcoplasmic and myofibrillar proteins. However, the major spoilage isolate in situ was P. fragi H8, which exhibited a fast growth rate, slime formation and increased pH and total volatile basic nitrogen (TVBN) on chicken breast fillets. The relative amounts of volatile organic compounds (VOCs) originating from the microorganisms, including alcohols, aldehydes, ketones and several sulfur compounds, increased during storage. In sum, this study demonstrated the characteristics of 4 potential spoilage bacteria on chilled yellow-feather chicken and provides a simple and convenient method to assess spoilage bacteria during quality management.

Development of a novel loop-mediated isothermal amplification assay for the detection of lipolytic Pseudomonas fluorescens in raw cow milk from north China.

Lipases secreted by psychrotrophic bacteria are known to be heat resistant and can remain active even after the thermal processing of milk products. Such enzymes are able to destabilize the quality of milk products by causing a rancid flavor. Rapid detection of a small amount of heat-resistant lipase-producing psychrotrophic bacteria is crucial for reducing their adverse effects on milk quality. In this study, we established and optimized a novel loop-mediated isothermal amplification (LAMP) assay for the detection of Pseudomonas fluorescens in raw cow milk, as the most frequently reported heat-resistant lipase-producing bacterial species. Pseudomonas fluorescens-specific DNA primers for LAMP were designed based on the lipase gene sequence. Reaction conditions of the LAMP assay were tested and optimized. The detection limit of the optimized LAMP assay was found to be lower than that of a conventional PCR-based method. In pure culture, the detection limit of the LAMP assay was found to be 4.8 × 101 cfu/reaction of the template DNA, whereas the detection limit of the PCR method was 4.8 × 102 cfu/reaction. Evaluation of the performance of the method in P. fluorescens-contaminated pasteurized cow milk revealed a detection limit of 7.4 × 101 cfu/reaction, which was 102 lower than that of the PCR-based method. If further developed, the LAMP assay could offer a favorable on-farm alternative to existing technologies for the detection of psychotrophic bacterial contamination of milk, enabling improved quality control of milk and milk products.

Phytoavailability of Cr in Silene vulgaris: The role of soil, plant genotype and bacterial rhizobiome.

Understanding the metal behavior at the soil-root interface is of utmost significance for a successful implementation of phytoremediation. In this study, we investigated the differences in chromium (Cr) uptake, chemical changes in soil solution and the shifts in rhizosphere bacterial communities of two genotypes of Silene vulgaris (SV21, SV38) with different tolerance to Cr. A greenhouse experiment was performed in two soils that differed on pH and organic matter (OM) content. An industrial sludge with high content in Cr was used as pollution source. The soil solution in the rhizosphere was sample by Rhizon Soil Moisture Samplers. The total concentration of Cr reached the highest values in soil solution samplers from calcareous soils with poor contents in OM. Plants grown in this soil also increased the Cr uptake in roots of both genotypes, but the concentration was higher in genotype SV-38 than in SV21. The clustering analysis of denaturing gradient gel electrophoresis (DGGE) of 16S rRNA fragments revealed major differences in bacterial community structure related to Cr pollution, followed by soil type and finally, plant genotype. Diversity indices based on DGGE profiles were the highest in alkaline soil, and between genotypes, values were significantly greater in SV38. Canonical correspondence analysis (CCA) showed that changes in bacterial community structure of rhizosphere were highly correlated with total Cr concentration and soil solution pH. The isolation and identification of S. vulgaris bacterial rhizosphere revealed a different composition according to soil type and plant genotype. Results suggested the potential role of Pseudomonas fluorescens on Cr mobilization and therefore, on enhanced metal bioavailability and may provide a starting point for further studies aimed at the combined use of tolerant plants and selected metal mobilizing rhizobacteria, in the microbial-assisted phytoremediation of Cr-polluted soils.

Microbiological quality of raw milk attributable to prolonged refrigeration conditions.

Refrigerated storage of raw milk is a prerequisite in dairy industry. However, temperature abused conditions in the farming and processing environments can significantly affect the microbiological quality of raw milk. Thus, the present study investigated the effect of different refrigeration conditions such as 2, 4, 6, 8, 10 and 12 °C on microbiological quality of raw milk from three different dairy farms with significantly different initial microbial counts. The bacterial counts (BC), protease activity (PA), proteolysis (PL) and microbial diversity in raw milk were determined during storage. The effect of combined heating (75 ± 0·5 °C for 15 s) and refrigeration on controlling those contaminating microorganisms was also investigated. Results of the present study indicated that all of the samples showed increasing BC, PA and PL as a function of temperature, time and initial BC with a significant increase in those criteria ≥6 °C. Similar trends in BC, PA and PL were observed during the extended storage of raw milk at 4 °C. Both PA and PL showed strong correlation with the psychrotrophic proteolytic count (PPrBC: at ≥4 °C) and thermoduric psychrotrophic count (TDPC: at ≥8 °C) compared to total plate count (TPC) and psychrotrophic bacterial count (PBC), that are often used as the industry standard. Significant increases in PA and PL were observed when PPrBC and TDPC reached 5 × 104 cfu/ml and 1 × 104 cfu/ml, and were defined as storage life for quality (S LQ), and storage life for safety (S LS) aspects, respectively. The storage conditions also significantly affected the microbial diversity, where Pseudomonas fluorescens and Bacillus cereus were found to be the most predominant isolates. However, deep cooling (2 °C) and combination of heating and refrigeration (≤4 °C) significantly extended the S LQ and S Ls of raw milk.

Diversity of Pseudomonas Genomes, Including Populus-Associated Isolates, as Revealed by Comparative Genome Analysis.

The Pseudomonas genus contains a metabolically versatile group of organisms that are known to occupy numerous ecological niches, including the rhizosphere and endosphere of many plants. Their diversity influences the phylogenetic diversity and heterogeneity of these communities. On the basis of average amino acid identity, comparative genome analysis of >1,000 Pseudomonas genomes, including 21 Pseudomonas strains isolated from the roots of native Populus deltoides (eastern cottonwood) trees resulted in consistent and robust genomic clusters with phylogenetic homogeneity. All Pseudomonas aeruginosa genomes clustered together, and these were clearly distinct from other Pseudomonas species groups on the basis of pangenome and core genome analyses. In contrast, the genomes of Pseudomonas fluorescens were organized into 20 distinct genomic clusters, representing enormous diversity and heterogeneity. Most of our 21 Populus-associated isolates formed three distinct subgroups within the major P. fluorescens group, supported by pathway profile analysis, while two isolates were more closely related to Pseudomonas chlororaphis and Pseudomonas putida. Genes specific to Populus-associated subgroups were identified. Genes specific to subgroup 1 include several sensory systems that act in two-component signal transduction, a TonB-dependent receptor, and a phosphorelay sensor. Genes specific to subgroup 2 contain hypothetical genes, and genes specific to subgroup 3 were annotated with hydrolase activity. This study justifies the need to sequence multiple isolates, especially from P. fluorescens, which displays the most genetic variation, in order to study functional capabilities from a pangenomic perspective. This information will prove useful when choosing Pseudomonas strains for use to promote growth and increase disease resistance in plants.

Interference Competition Among Household Strains of Pseudomonas.

Bacterial species exhibit biogeographical patterns like those observed in larger organisms. The distribution of bacterial species is driven by environmental selection through abiotic and biotic factors as well dispersal limitations. We asked whether interference competition, a biotic factor, could explain variability in habitat use by Pseudomonas species in the human home. To answer this question, we screened almost 8000 directional, pairwise interactions between 89 Pseudomonas strains including members of the Pseudomonas aeruginosa (n = 29), Pseudomonas fluorescens (n = 21), and Pseudomonas putida (n = 39) species groups for the presence of killing. This diverse set of Pseudomonas strains includes those isolated from several different habitats within the home environment and includes combinations of strains that were isolated from different spatial scales. The use of this strain set not only allowed us to analyze the commonality and phylogenetic scale of interference competition within the genus Pseudomonas but also allowed us to investigate the influence of spatial scale on this trait. Overall, the probability of killing was found to decrease with increasing phylogenetic distance, making it unlikely that interference competition accounts for previously observed differential habitat use among Pseudomonas species and species groups. Strikingly, conspecific P. aeruginosa killing accounted for the vast majority of the observed killing, and this killing was found to differ across the habitat type and spatial scale of the strains' isolation. These data suggest that interference competition likely plays a large role in the within-species dynamics of P. aeruginosa but not other household Pseudomonas species.

Extracellular thermostable proteolytic activity of the milk spoilage bacterium Pseudomonas fluorescens PS19 on bovine caseins.

We studied the thermostable proteolytic activity of Pseudomonas fluorescens PS19 isolated from raw bovine milk. The heat-treated cell-free supernatant (HT-CFS) contained a thermostable protease of approximately 45 kDa, as revealed by casein zymography. We assigned this enzyme to P. fluorescens AprX metalloprotease (UniProtKB Acc. No. C9WKP6). After concentration by ultrafiltration at 10 kDa, the HT-CFS showed 2 other thermostable proteolytic bands on zymogram, with molecular masses of approximately 15 and 25 kDa. The former resulted a fragment of the AprX protease, whereas the 25-kDa protease was not homologous to any known protein of Pseudomonas spp. Subsequently, we assessed the proteolytic activity of the HT-CFS on bovine αS-, ß-, and κ-casein during in vitro incubation at 7 or 22°C. By means of ultra-performance liquid chromatography-tandem mass spectrometry we identified the released peptides (n=591). Some of them resisted proteolysis during the whole incubation period at both incubation temperatures and, therefore, they could be assumed as indicators of the proteolytic action of P. fluorescens PS19 on bovine caseins.

A glimpse of the endophytic bacterial diversity in roots of blackberry plants (Rubus fruticosus).

The aim of this study was to explore the diversity of culturable bacterial communities residing in blackberry plants (Rubus fruticosus). Bacterial endophytes were isolated from plant roots, and their 16S rDNA sequences were amplified and sequenced. Our results show that the roots of R. fruticosus exhibit low colony forming units of bacterial endophytes per gram of fresh tissue (6 x 102 ± 0.5 x 102). We identified 41 endophytic bacterial species in R. fruticosus by BLAST homology search and a subsequent phylogenetic analysis, belonging to the classes Actinobacteria, Bacilli, Alfaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Predominantly, genera belonging the Proteobacteria (Burkholderia, 29.4%; Herbaspirillum, 10.7%; Pseudomonas, 4.9%; and Dyella, 3.9%), Firmicutes (Bacillus, 42.1%), and Actinobacteria (two isolates showing high identity with the Streptomyces genus, 1.9%) divisions were identified. Fifty percent of the bacterial endophytes produced the phytohormone indole-acetic acid (IAA), eleven of which exhibited higher IAA production (>5.8 mg/mL) compared to the plant growth-promoting strain, Pseudomonas fluorescens UM270. Additionally, the endophytic isolates exhibited protease activity (22%), produced siderophores (26.4%), and demonstrated antagonistic action (>50% inhibition of mycelial growth) against the grey mold phytopathogen Botrytis cinerea (3.9%). These results suggested that field-grown R. fruticosus plants contain bacterial endophytes within their tissues with the potential to promote plant growth and display antagonism towards plant pathogens.