Comparative genomic insights into the epidemiology and virulence of plant pathogenic pseudomonads from Turkey.

Pseudomonas is a highly diverse genus that includes species that cause disease in both plants and animals. Recently, pathogenic pseudomonads from the Pseudomonas syringae and Pseudomonas fluorescens species complexes have caused significant outbreaks in several agronomically important crops in Turkey, including tomato, citrus, artichoke and melon. We characterized 169 pathogenic Pseudomonas strains associated with recent outbreaks in Turkey via multilocus sequence analysis and whole-genome sequencing, then used comparative and evolutionary genomics to characterize putative virulence mechanisms. Most of the isolates are closely related to other plant pathogens distributed among the primary phylogroups of P. syringae, although there are significant numbers of P. fluorescens isolates, which is a species better known as a rhizosphere-inhabiting plant-growth promoter. We found that all 39 citrus blast pathogens cluster in P. syringae phylogroup 2, although strains isolated from the same host do not cluster monophyletically, with lemon, mandarin orange and sweet orange isolates all being intermixed throughout the phylogroup. In contrast, 20 tomato pith pathogens are found in two independent lineages: one in the P. syringae secondary phylogroups, and the other from the P. fluorescens species complex. These divergent pith necrosis strains lack characteristic virulence factors like the canonical tripartite type III secretion system, large effector repertoires and the ability to synthesize multiple bacterial phytotoxins, suggesting they have alternative molecular mechanisms to cause disease. These findings highlight the complex nature of host specificity among plant pathogenic pseudomonads.

Bacterial-Derived Plant Protection Metabolite 2,4-Diacetylphloroglucinol: Effects on Bacterial Cells at Inhibitory and Subinhibitory Concentrations.

2,4-Diacetylphloroglucinol (2,4-DAPG) is a well-known bacterial secondary metabolite, however, its mechanism of inhibitory and subinhibitory action on bacterial cells is still poorly understood. The mechanism of 2,4-DAPG action on model bacterial strains was investigated using fluorescent spectroscopy and the action of the antibiotic was found to involve a rapid increase in membrane permeability that was accompanied by a reduction in its viability in nutrient-poor medium. At the same time, antibacterial action in nutrient-rich medium developed for several hours. Atomic force microscopy demonstrated time-dependent disturbances in the outer membrane of Escherichia coli when exposed to 2,4-DAPG, while Staphylococcusaureus cells have been visualized with signs of intracellular leakage. In addition, 2,4-DAPG inhibited the metabolic activity of S. aureus and E. coli bacterial cells in mature biofilms. Observed differences in the antibiofilm activity were dependent upon antibiotic concentration. The intracellular targets of the action of 2,4-DAPG were assessed using bacterial biosensors with inducible bioluminescence corresponding to DNA and protein damage. It was unable to register any positive response from either sensor. As a result, the bactericidal action of 2,4-DAPG is believed to be associated with the destruction of the bacterial barrier structures. The subinhibitory effect of 2,4-diacetylphloroglucinol was tested on quorum-sensing mediated processes in Pectobacterium carotovorum. Subinhibitory concentrations of 2,4-DAPG were found to lower the biosynthesis of acyl-homoserine lactones in P. carotovorum in a dose-dependent manner. Further investigation elucidated that 2,4-DAPG inhibits the metabolic activity of bacteria without affecting their viability.

Aminoglycoside riboswitch control of the expression of integron associated aminoglycoside resistance adenyltransferases.

The proliferation of antibiotic resistance has its origins in horizontal gene transfer. The class 1 integrons mediate gene transfer by assimilating antibiotic-resistance genes through site-specific recombination. For the class 1 integrons the first assimilated gene normally encodes an aminoglycoside antibiotic resistance protein which is either an aminoglycoside acetyltransferase (AAC), nucleotidyltransferase - (ANT), or adenyl transferase (AAD). An aminoglycoside-sensing riboswitch RNA in the leader RNA of AAC/AAD that controls the expression of aminoglycoside resistance genes has been previously described. Here we explore the relationship between the recombinant products of integron recombination and a series of candidate riboswitch RNAs in the 5' UTR of aad (aminoglycoside adenyltransferases) genes. The RNA sequences from the 5' UTR of the aad genes from pathogenic strains that are the products of site-specific DNA recombination by class 1 integrons were investigated. Reporter assays, MicroScale Thermophoresis (MST) and covariance analysis revealed that a functional aminoglycoside-sensing riboswitch was selected at the DNA level through integron-mediated site-specific recombination. This study explains the close association between integron recombination and the aminoglycoside-sensing riboswitch RNA.

Insights into the Draft Genome Sequence of the Kiwifruit-Associated Pathogenic Isolate Pseudomonas fluorescens AHK-1.

Pseudomonas fluorescens is a physiologically diverse species of bacteria present in many habitats, which possesses multifunctional traits that provide it with the capability to exhibit biological control activities, promote plant health or cause plant disease. Here, we present the draft genome sequence of the kiwifruit-associated pathogenic isolate AHK-1 of P. fluorescens, which was isolated from the diseased leaves of kiwifruit plants. The genome size of AHK-1 was found to be 7,035,786 bp, with a G + C content of 60.88%. It is predicted to contain a total of 6327 genes, of which 3998 were homologous to genes in the other two sequenced P. fluorescens isolates (SBW25 and GcM5-1A) and 946 were unique to AHK-1 based on comparative genomic analysis. Furthermore, we identified several candidate virulence factors in the genome of AHK-1, including the fliA gene encoding flagellar biosynthetic protein for biosynthesis, and the genes for components of type VI, III, and IV secretion systems. This genomic resource will serve as a reference for better understanding the genetics of pathogenic and non-pathogenic strains, and will help to elucidate the pathogenic mechanisms of P. fluorescens associated with plant disease.

Transcriptome-based identification and validation of reference genes for plant-bacteria interaction studies using Nicotiana benthamiana.

RT-qPCR is a widely used technique for the analysis of gene expression. Accurate estimation of transcript abundance relies strongly on a normalization that requires the use of reference genes that are stably expressed in the conditions analyzed. Initially, they were adopted from those used in Northern blot experiments, but an increasing number of publications highlight the need to find and validate alternative reference genes for the particular system under study. The development of high-throughput sequencing techniques has facilitated the identification of such stably expressed genes. Nicotiana benthamiana has been extensively used as a model in the plant research field. In spite of this, there is scarce information regarding suitable RT-qPCR reference genes for this species. Employing RNA-seq data previously generated from tomato plants, combined with newly generated data from N. benthamiana leaves infiltrated with Pseudomonas fluorescens, we identified and tested a set of 9 candidate reference genes. Using three different algorithms, we found that NbUbe35, NbNQO and NbErpA exhibit less variable gene expression in our pathosystem than previously used genes. Furthermore, the combined use of the first two is sufficient for robust gene expression analysis. We encourage employing these novel reference genes in future RT-qPCR experiments involving N. benthamiana and Pseudomonas spp.

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.

Consumption of carotenoids not increased by bacterial infection in brown trout embryos (Salmo trutta).

Carotenoids are organic pigment molecules that play important roles in signalling, control of oxidative stress, and immunity. Fish allocate carotenoids to their eggs, which gives them the typical yellow to red colouration and supports their resistance against microbial infections. However, it is still unclear whether carotenoids act mainly as a shield against infection or are used up during the embryos' immune defence. We investigated this question with experimental families produced from wild-caught brown trout (Salmo trutta). Singly raised embryos were either exposed to the bacterial pathogen Pseudomonas fluorescens or sham-treated at one of two stages during their development. A previous study on these experimental families reported positive effects of egg carotenoids on embryo growth and resistance against the infection. Here, we quantified carotenoid consumption, i.e. the active metabolization of carotenoids into compounds that are not other carotenoid types, in these infected and sham-infected maternal sib groups. We found that carotenoid contents mostly decreased during embryogenesis. However, these decreases were neither linked to the virulence induced by the pathogen nor dependent on the time point of infection. We conclude that egg carotenoids are not significantly used up by the embryos' immune defence.

Role of RpoS in stress resistance, quorum sensing and spoilage potential of Pseudomonas fluorescens.

Microorganism activities are considered the main cause of most food spoilage, leading to great economic losses. Pseudomonas fluorescens is a Gram-negative bacterium that is widely found in food and has high spoilage activity. RpoS is considered an important global regulator involved in stress survival and virulence in many pathogens. Thus, it is very possible that RpoS plays an important role in spoilage regulation in P. fluorescens. In this study an in-frame deletion mutation of rpoS was constructed to explore its function in P. fluorescens. The results showed that RpoS positively regulated the resistance of P. fluorescens to H2O2, heat, ethanol and crystal violet, negatively regulated the resistance to acetic acid, and had no effect on the resistance to NaCl. Further studies indicated that acylated homoserine lactone (AHL) production and the transcription levels of five AHL-related genes were significantly decreased in the rpoS mutant compared with those in the wild-type strain. Interestingly, the two homologous genes coding for AHL synthases contained RpoS-dependent -10 elements, suggesting that AHL quorum sensing is directly regulated by RpoS. RpoS also contributed to the spoilage activities of P. fluorescens by regulating extracellular protease and total volatile basic nitrogen (TVB-N) production in sterilized salmon juice. Our results reveal that RpoS was a key regulatory factor involved in stress resistance, the AHL quorum sensing system, and spoilage potential of P. fluorescens. Our study may benefit food safety control and food preservation.

Inhibition of quorum sensing-controlled virulence factors and biofilm formation in Pseudomonas fluorescens by cinnamaldehyde.

Pseudomonas fluorescens, an important food spoiling bacteria, uses quorum sensing to control biofilm formation and motility. To date, only a few compounds targeting the LuxR-based quorum sensing system of P. fluorescens have been identified. In the present study, the quorum sensing inhibitory effect of cinnamaldehyde at sublethal concentrations was investigated in terms of inhibition of the extracellular protease, biofilm formation, and swimming and swarming motility. The total volatile basic nitrogen value was also measured to evaluate the effect of cinnamaldehyde on quality preservation of turbot fillets stored at 4 ±â€¯1 °C for 15 days. The results showed that cinnamaldehyde significantly inhibited quorum sensing-dependent factors in P. fluorescens and extended the storage life of turbot. Unexpectedly, cinnamaldehyde did not interfere with production of AHLs (N-acylhomoserine lactones) by P. fluorescens, as shown by measurement of AHL production using GC-MS. Molecular docking analysis revealed that cinnamaldehyde can interact with the LuxR-type protein of P. fluorescens, which could constitute the molecular basis of the quorum sensing inhibition observed. These findings strongly suggest that cinnamaldehyde is a quorum sensing inhibitor with great potential for the preservation of aquatic products to guarantee food safety.

Isolation and characterization of pectolytic bacterial pathogens infecting potatoes in Nakuru County, Kenya.

AIMS: Isolation and characterization of pectolytic bacteria associated with soft rot disease of potatoes in Nakuru, Kenya, to provide the basis for the development of disease control measures. METHODS AND RESULTS: Potato tubers showing symptoms of soft rot were collected from different farms in Molo and Mau Narok regions within Nakuru county. Isolation was done using crystal violet pectate medium (CVPM). Out of the 71 isolates that showed growth on CVPM, pathogenicity tests revealed that 36 of them had the ability to macerate tissues of potato tubers. All the isolates yielded a fragment of approximately 1500 bp after 16S rDNA amplification. Using the BIOLOG microbial identification system, 20 bacterial isolates were identified as Pectobacterium carotovorum subsp. carotovorum, 7 were Pseudomonas fluorescens B while 9 were Ps. fluorescens A. Y1/Y2 primers successfully amplified pectate lyase-encoding (pel) gene, approximately 434 bp, in all the 20 P. carotovorum species. The virulence of the isolated strains to cause disease, according to pectinolytic tests, varied with change in incubation temperature of the test samples. Pectobacterium carotovorum strains were the most virulent at 30°C while disease severity due to infection by Ps. fluorescens A strains was high at 20°C compared to the other isolates. CONCLUSION: This study reveals the identity of pectolytic bacterial species from two genera, Pectobacterium and Pseudomonas, as causative agents of potato soft rot in Nakuru, Kenya. SIGNIFICANCE AND IMPACT OF THE STUDY: Research findings from this study will aid in developing suitable risk mitigation methods for adoption by farmers to prevent losses due to soft rot.

Virulence of entomopathogenic bacteria in the bed bug, Cimex lectularius.

Due in part to the development of insecticide resistance, the common bed bug, Cimex lectularius, has overcome human intervention efforts to make a global resurgence. The failure of chemical pesticides has created a need for novel strategies to combat bed bugs. While a number of insect pests are susceptible to the use of entomopathogenic microbes or microbial-derived toxins, biological control methods have not been thoroughly explored in bed bugs. Here, we tested the virulence of three entomopathogenic bacterial species in C. lectularius to determine their potential for bed bug control. We examined bed bug survival after inoculation with live or heat-killed Serratia marcescens, Pseudomonas fluorescens, and Bacillus thuringiensis israelensis at varying temperatures. We also analyzed the viability and growth of the same bacteria in infected bed bugs. All three bacterial species were pathogenic to bed bugs. However, the effects of S. marcescens and P. fluorescens were temperature-dependent while the lethality of B. thuringiensis israelensis was not. In addition, bacterial virulence was partly dependent on the route of infection but was not strongly associated with proliferation. Thus, our results suggest multiple possible mechanisms of microbial pathogenicity in the bed bug and indicate that entomopathogenic bacteria, or products derived from them, may have useful applications for bed bug control.

Biofilms and Oxidizing Biocides; Evaluation of Disinfection and Removal Effects by Using Established Microbial Systems.

The formation of bacterial biofilms and their disinfection and removal have been important subjects in the maintenance of water quality in areas such as public spas, swimming pools, food processing lines, industrial water systems, and in the hygienic control of medical devices, hospital procedures, etc. Presented here is an outline of biofilm formation, as well as studies on the disinfection and removal of biofilms by oxidizing biocides using established biofilms. These studies using established biofilms may increase the understanding of the variable response of biofilms to planktonic bacteria, and the unique aspects of oxidizing biocides in the disinfection and removal of biofilms.

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.

Influence of Aeromonas hydrophila and Pseudomonas fluorescens on motility, viability and morphometry of cryostored silver barb (Barbodes gonionotus) sperm.

This objective of the study was to evaluate the effects of A. hydrophila subsp. hydrophila and P. fluorescens on sperm motility, sperm viability and sperm morphometry of cryopreserved silver barb (Barbodes gonionotus) semen and survival of tested bacteria after cryostorage. Semen was diluted in a calcium-free Hank's balanced salt solution (Ca-F HBSS) supplemented with or without 0.25% penicillin-streptomycin (PS) after which A. hydrophila subsp. hydrophila or P. fluorescens was immediately added into extended semen prior to freezing. Extended semen and cryostored semen kept for 20 min, 24 h, 7 d, 14 d and 28 d were assessed for sperm motility, sperm viability, sperm morphometry, survival of challenged bacteria and the relationship between bacteria and sperm. Bacterial-exposed semen with or without 0.25% PS supplementation showed a significant reduction (P < 0.05) in sperm motility and viability during a cryostorage of 28 d, compared to semen without bacterial supplementation (control groups). Addition of A. hydrophila subsp. hydrophila and P. fluorescens resulted in a significant (P < 0.05) alteration of sperm morphometry of cryopreserved semen, especially flagellum width. The two pathogens were detected at a level of 10(5) CFU ml(-1) in cryostored semen with or without antibiotic supplementation. There were significant correlations among bacterial number, percentage of sperm motility and viability and flagellum width. In conclusion, the presence of A. hydrophila subsp. hydrophila and P. fluorescens had a deleterious effect on cryopreserved silver barb sperm based on a reduction in sperm motility and viability and alteration of sperm morphometry, especially flagellum width.

The Pseudomonas fluorescens Siderophore Pyoverdine Weakens Arabidopsis thaliana Defense in Favor of Growth in Iron-Deficient Conditions.

Pyoverdines are siderophores synthesized by fluorescent Pseudomonas spp. Under iron-limiting conditions, these high-affinity ferric iron chelators are excreted by bacteria in the soil to acquire iron. Pyoverdines produced by beneficial Pseudomonas spp. ameliorate plant growth. Here, we investigate the physiological incidence and mode of action of pyoverdine from Pseudomonas fluorescens C7R12 on Arabidopsis (Arabidopsis thaliana) plants grown under iron-sufficient or iron-deficient conditions. Pyoverdine was provided to the medium in its iron-free structure (apo-pyoverdine), thus mimicking a situation in which it is produced by bacteria. Remarkably, apo-pyoverdine abolished the iron-deficiency phenotype and restored the growth of plants maintained in the iron-deprived medium. In contrast to a P. fluorescens C7R12 strain impaired in apo-pyoverdine production, the wild-type C7R12 reduced the accumulation of anthocyanins in plants grown in iron-deficient conditions. Under this condition, apo-pyoverdine modulated the expression of around 2,000 genes. Notably, apo-pyoverdine positively regulated the expression of genes related to development and iron acquisition/redistribution while it repressed the expression of defense-related genes. Accordingly, the growth-promoting effect of apo-pyoverdine in plants grown under iron-deficient conditions was impaired in iron-regulated transporter1 and ferric chelate reductase2 knockout mutants and was prioritized over immunity, as highlighted by an increased susceptibility to Botrytis cinerea This process was accompanied by an overexpression of the transcription factor HBI1, a key node for the cross talk between growth and immunity. This study reveals an unprecedented mode of action of pyoverdine in Arabidopsis and demonstrates that its incidence on physiological traits depends on the plant iron status.

Sequencing and Analysis of the Pseudomonas fluorescens GcM5-1A Genome: A Pathogen Living in the Surface Coat of Bursaphelenchus xylophilus.

It is known that several bacteria are adherent to the surface coat of pine wood nematode (Bursaphelenchus xylophilus), but their function and role in the pathogenesis of pine wilt disease remains debatable. The Pseudomonas fluorescens GcM5-1A is a bacterium isolated from the surface coat of pine wood nematodes. In previous studies, GcM5-1A was evident in connection with the pathogenicity of pine wilt disease. In this study, we report the de novo sequencing of the GcM5-1A genome. A 600-Mb collection of high-quality reads was obtained and assembled into sequence contigs spanning a 6.01-Mb length. Sequence annotation predicted 5,413 open reading frames, of which 2,988 were homologous to genes in the other four sequenced P. fluorescens isolates (SBW25, WH6, Pf0-1 and Pf-5) and 1,137 were unique to GcM5-1A. Phylogenetic studies and genome comparison revealed that GcM5-1A is more closely related to SBW25 and WH6 isolates than to Pf0-1 and Pf-5 isolates. Towards study of pathogenesis, we identified 79 candidate virulence factors in the genome of GcM5-1A, including the Alg, Fl, Waa gene families, and genes coding the major pathogenic protein fliC. In addition, genes for a complete T3SS system were identified in the genome of GcM5-1A. Such systems have proved to play a critical role in subverting and colonizing the host organisms of many gram-negative pathogenic bacteria. Although the functions of the candidate virulence factors need yet to be deciphered experimentally, the availability of this genome provides a basic platform to obtain informative clues to be addressed in future studies by the pine wilt disease research community.

The pathogenic potential of Pseudomonas fluorescens MFN1032 on enterocytes can be modulated by serotonin, substance P and epinephrine.

Pseudomonas fluorescens is a commensal bacterium present at low level in the human digestive tract that has also been reported in many clinical samples (blood, urinary tract, skin, lung, etc.) and sometimes associated with acute opportunistic infections. It has recently been found that the human ß-defensin-2 can enhance the pathogenic potential of P. fluorescens. In this study, we evaluated the effect of other intestinal molecules (5HT, SP and Epi) on growth and virulence of the clinical strain P. fluorescens MFN1032. We found that P. fluorescens MFN1032 growth was not mainly affected by these factors, but several modifications in the virulence behavior of this bacterium were observed. 5HT, SP and Epi were able to modulate the motility of P. fluorescens MFN1032. 5HT and SP had an effect on pyoverdin production and IL-8 secretion, respectively. Infection of Caco-2/TC7 cells with P. fluorescens MFN1032 pretreated by SP or Epi enhanced the permeability of the monolayers and led to a partial delocalization of F-actin to the cytoplasm. These findings show that some intestinal molecules can modulate the pathogenic potential of P. fluorescens MFN1032. We can hypothesize that this dialogue between the host and the human gut microbiota may participate in health and disease.

A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors.

Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain.

Assessment of the toxic effect exerted by fluorescent pseudomonads on embryos and larvae of the sea urchin Strongylocentrotus nudus.

Strains of bacteria capable of growing on artificial culture media were isolated from the fouling of brass plates submerged in Nha Trang Bay, South China Sea, and from tissues of the seastar Distolasterias nipon, caught in Peter the Great Bay, Sea of Japan. According to the complex of data of genetic and physiological/biochemical analyzes, two strains of cultivated bacteria were identified by us as the species Pseudomonas aeruginosa, two strains as Pseudomonas fluorescens, and one strain as Ruegeria sp. It was shown that the cultivated strains of P. aeruginosa released exotoxins, particularly phenazine pigments, into the environment. Production of the toxins did not depend on presence of a target organism in the system and was aimed at regulation of interactions in the microbial community. The toxicity of the studied natural isolates of fluorescent pseudomonads was analyzed by using embryos and larvae of the sea urchin Strongylocentrotus nudus, which are the sensitive and dynamic toxicological sea-urchin embryo test (SET) system. As was established, exotoxins produced by the strains of P. aeruginosa inhibit activity of cilia in sea urchin larvae, as well as disturb processes of cell differentiation in embryos and larvae. Their toxic influence is accompanied by disturbances of protein synthesis and the disruptions of cytoskeleton in the course of zygote cleavage and larval development. Unlike P. aeruginosa, the strains of P. fluorescens and Ruegeria sp. did not exert the toxic effect on SET. The obtained data allow considering objects of the environment as the natural reservoir of opportunistic microorganisms posing a potential threat to human, whereas the use of SET for determination of toxicity of isolated bacteria provides an opportunity to study the mechanisms of their interactions with organisms in marine ecosystems.

Pseudomonas fluorescens: iron-responsive proteins and their involvement in host infection.

For pathogenic bacteria, the ability to acquire iron is vital to survival in the host. In consequence, many genes involved in iron acquisition are associated with bacterial virulence. Pseudomonas fluorescens is a bacterial pathogen to a variety of farmed fish. However, the global regulatory function of iron in pathogenic P. fluorescens is essentially unknown. In this study, in order to identify proteins affected by iron condition at the expression level, we performed proteomic analysis to compare the global protein profiles of P. fluorescens strain TSS, a fish pathogen, cultured under iron-replete and iron-deplete conditions. Twenty-two differentially expressed proteins were identified, most of which were confirmed to be regulated by iron at the mRNA level. To investigate their potential involvement in virulence, the genes encoding four of the 22 proteins, i.e. HemO (heme oxygenase), PspB (serine protease), Sod (superoxide dismutase), and TfeR (TonB-dependent outermembrane ferric enterobactin receptor), were knocked out, and the pathogenicity of the mutants was examined in a model of turbot (Scophthalmus maximus). The results showed that compared to the wild type, the hemO, pspB, and tfeR knockouts were significantly impaired in the ability to survive in host serum, to invade host tissues, and to cause host mortality. Immunization of turbot with recombinant TfeR (rTfeR) and PspB induced production of specific serum antibodies and significant protections against lethal TSS challenge. Further analysis showed that rTfeR antibodies recognized and bound to TSS, and that treatment of TSS with rTfeR antibodies significantly impaired the infectivity of TSS to fish cells. Taken together, these results indicate for the first time that in pathogenic P. fluorescens, iron affects the expression of a large number of proteins including those that are involved in host infection.