Physiological Response of Lactobacillus plantarum to Salt and Nonelectrolyte Stress

In this report, we compared the effects on the growth of Lactobacillus plantarum of raising the medium molarity by high concentrations of KCl or NaCl and iso-osmotic concentrations of nonionic compounds. Analysis of cellular extracts for organic constituents by nuclear magnetic resonance spectroscopy showed that salt-stressed cells do not contain detectable amounts of organic osmolytes, whereas sugar-stressed cells contain sugar (and some sugar-derived) compounds. The cytoplasmic concentrations of lactose and sucrose in growing cells are always similar to the concentrations in the medium. By using the activity of the glycine betaine transport system as a measure of hyperosmotic conditions, we show that, in contrast to KCl and NaCl, high concentrations of sugars (lactose or sucrose) impose only a transient osmotic stress because external and internal sugars equilibrate after some time. Analysis of lactose (and sucrose) uptake also indicates that the corresponding transport systems are neither significantly induced nor activated directly by hyperosmotic conditions. The systems operate by facilitated diffusion and have very high apparent affinity constants for transport (>50 mM for lactose), which explains why low sugar concentrations do not protect against hyperosmotic conditions. We conclude that the more severe growth inhibition by salt stress than by equiosmolal concentrations of sugars reflects the inability of the cells to accumulate K⁺ (or Na⁺) to levels high enough to restore turgor as well as deleterious effects of the electrolytes intracellularly.     

Herpes Simplex Virus Type 2 ICP47 Inhibits Human TAP but Not Mouse TAP

Herpes simplex virus serotype 1 (HSV-1) expresses an immediate-early protein, ICP47, that effectively blocks the major histocompatibility complex class I antigen presentation pathway. HSV-1 ICP47 (ICP47-1) binds with high affinity to the human transporter associated with antigen presentation (TAP) and blocks the binding of antigenic peptides. HSV type 2 (HSV-2) ICP47 (ICP47-2) has only 42% amino acid sequence identity with ICP47-1. Here, we compared the levels of inhibition of human and murine TAP, expressed in insect cell microsomes, by ICP47-1 and ICP47-2. Both proteins inhibited human TAP at similar concentrations, and the K_D for ICP47-2 binding to human TAP was 4.8 × 10⁻⁸ M, virtually identical to that measured for ICP47-1 (5.2 × 10⁻⁸ M). There was some inhibition of murine TAP by both ICP47-2 and ICP47-1, but this inhibition was incomplete and only at ICP47 concentrations 50 to 100 times that required to inhibit human TAP. Lack of inhibition of murine TAP by ICP47-1 and ICP47-2 could be explained by an inability of both proteins to bind to murine TAP.Previously, we showed that herpes simplex virus serotype 1 (HSV-1) ICP47 (ICP47-1) caused major histocompatibility complex (MHC) class I proteins to be retained in the endoplasmic reticulum (ER) of cells and that antigen presentation to CD8⁺ T cells was inhibited after ICP47-1 was expressed in human fibroblasts (9). ICP47-1 blocked peptide transport across the ER membrane by TAP (2, 6), so that, without peptides, class I proteins were retained in the ER. By contrast, ICP47 did not detectably inhibit MHC class I antigen presentation in mouse cells (9) and inhibited murine TAP poorly (2, 6). ICP47-1 inhibited peptide binding to TAP without affecting the binding of ATP (1, 7) and bound with high affinity, and in a stable fashion, to human TAP (7). Peptides could competitively inhibit ICP47 binding to TAP, consistent with the hypothesis that ICP47-1 binds to a site which includes the peptide binding domain of TAP (7). Others have suggested that the present data do not exclude a distortion in TAP caused by the binding of ICP47 at a site distant from the peptide binding site (3). This seems improbable given our observations that ICP47 inhibits peptide binding and that peptides competitively inhibit ICP47 binding. In order for peptides to inhibit ICP47 binding and vice versa, one would have to invoke allosteric inhibition by both ICP47 and peptides, a highly unlikely prospect.The predicted amino acid sequence of HSV type 2 ICP47 (ICP47-2) was recently described (3), and it was of some interest that ICP47-1 and ICP47-2 share only 42% amino acid identity (see Fig. ​Fig.1A).1A). Most of the homology is near the N termini and in the central regions of the molecules. A peptide including residues 2 to 35 of ICP47-1 blocked human TAP in permeabilized cells (3). This observation was somewhat surprising given that this peptide did not include residues 33 to 51, a sequence that is most homologous between ICP47-1 and ICP47-2. Presumably, this conserved domain, and even the C-terminal third of the protein, is important in virus-infected cells for stability or for functions that are not apparent in this in vitro assay involving detergent-permeabilized cells.Open in a separate windowFIG. 1Comparison of ICP47-1 and ICP47-2 protein sequences and preparation of purified proteins. (A) The predicted amino acid sequences of ICP47-1 derived from HSV-1 strain 17 (6a) and of ICP47-2 derived from HSV-2 strain HG52 (3) are shown. The boldface, underlined letters denote identical amino acids, and the italicized letters denote conserved residues. (B) ICP47-1 and ICP47-2 were produced in Escherichia coli by expressing the proteins as GST fusion proteins by fusing the ICP47 coding sequences to GST sequences in plasmid pGEX-2T as described previously (7). Lysates from bacteria were incubated with glutathione-Sepharose and washed several times, and then ICP47-1 or ICP47-2 was eluted by incubation with thrombin, which cleaves between the GST and ICP47 sequences (7). The thrombin was inactivated with phenylmethylsulfonyl fluoride, and the ICP47 preparations were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Bradford protein analysis. The positions of GST-ICP47, GST, and ICP47 protein, as well as those of molecular weight markers 104, 80, 48, 34, 24, and 18 KDa in size, are indicated.Given the differences between the primary structures of ICP47-1 and ICP47-2, we were interested in whether ICP47-2 might inhibit the murine TAP. If this were the case, it would make possible animal studies of the effects of ICP47. Here, we have produced a recombinant form of ICP47-2 and compared the effects of ICP47-2 and ICP47-1 on human and murine TAP proteins expressed in insect cell microsomes. Like ICP47-1, ICP47-2 efficiently blocked human TAP but even at high concentrations did not effectively block murine TAP. Moreover, there was little or no significant binding of either protein to insect microsomes containing mouse TAP.The HSV-2 ICP47 gene was subcloned from plasmid pBB17, which contains a KpnI-HindIII 8,477-bp fragment derived from the genome of HSV-2 strain HG52 inserted into pUC19, by using PCR to amplify ICP47-2 coding sequences. One PCR primer hybridized with the 5′ end of the ICP47-2 coding sequences and extended 5′ to generate a new BglII site just upstream of the initiation codon. The second PCR primer hybridized with 3′ sequences of the ICP47-2 gene, then diverged to produce an EcoRI site just downstream of the translation termination codon. After PCR, the DNA fragment was digested with EcoRI and inserted into the HincII (blunt) and EcoRI sites of pUC19, producing plasmid pUC47-2, which was subjected to DNA sequencing. The ICP47-2 coding sequences were excised from pUC47-2 with BglII and EcoRI and inserted into the BamHI and EcoRI sites of pGEX-2T to generate a fusion protein with glutathione S-transferase (GST). The ICP47-GST fusion protein was expressed in bacteria and purified by using glutathione-Sepharose, and then the GST sequences were removed with thrombin as described previously for ICP47-1 (7). A comparison between the predicted amino acid sequences of ICP47-2 and ICP47-1 is shown in Fig. ​Fig.1,1, with a comparative gel (Fig. ​(Fig.1B)1B) showing the purified preparations of ICP47-1 and ICP47-2 from bacteria. Microsomes purified from Sf9 insect cells infected with baculoviruses expressing human TAP1 and TAP2 have been described previously (7, 8), as were microsomes from Drosophila cells expressing murine TAP1 and TAP2 (1). We previously estimated that approximately 2% of the protein associated with the insect microsomes was human TAP (7), and the microsomes containing mouse TAP possessed similar TAP activity (see below). Peptide translocation by these microsomes was measured by using a library of ¹²⁵I-labelled peptides (5) that are glycosylated after transport into the ER. Radioactive peptides able to bind to concanavalin A were quantified as an indirect measure of peptide transport (6). Over a range of membranes from 2.5 to 20 μl, with protein concentrations of 10 to 12 mg/ml for human TAP microsomes and 5.0 to 7.0 mg/ml for mouse TAP microsomes, there was a linear increase in peptide transport (Fig. ​(Fig.2).2). Thus, peptides and ATP were not limiting. Peptide transport was specific because the transport observed with control membranes not containing TAP amounted to less than 1% of that observed when microsomes contained TAP. The levels of peptide transport associated with microsomes containing human or mouse TAP were also compared and standardized. Thus, in subsequent assays, 7.5 to 10 μl of microsomes exhibiting similar amounts of TAP activity were used. Open in a separate windowFIG. 2Peptide transport by insect microsomes containing human or murine TAP. Microsomes were derived from insect Sf9 cells coinfected with BacTAP1 and BacTAP2 (Human TAP) (7) or from Sf9 cells infected with a control baculovirus, BacgH (Human control). Alternatively, microsomes were derived from Drosophila cells induced to express mouse TAP (Murine TAP) (1) or from Drosophila cells which were not induced to express mouse TAP (Murine control). Various concentrations of each microsome preparation were incubated with ¹²⁵I-labelled peptides and 5 mM ATP in a volume of 150 μl for 10 min at 23°C. The microsomes were washed, pelleted, and disrupted in detergent as described previously (7). Peptides able to bind to concanavalin A-Sepharose were eluted with alpha-methylmannoside and quantified (7).ICP47-2 inhibited peptide transport by human TAP, and the inhibition was similar to that of ICP47-1; the 50% inhibitory concentration (IC₅₀) for ICP47-2 was 0.24 μM and for ICP47-1 was 0.27 μM (Fig. ​(Fig.3A).3A). In other experiments the IC₅₀ values for ICP47-1 and ICP47-2 varied from 0.15 to 0.35 μM, and there were no experiments in which there was a significant difference in the abilities of the two proteins to inhibit human TAP. Moreover, the binding properties of ICP47-2 to human TAP were similar to those of ICP47-1. Binding experiments were performed as described previously for ICP47-1 (7) by using membranes containing human TAP and ¹²⁵I-labelled ICP47-2. Specific binding of ICP47-2 was calculated by subtracting the binding to control microsomes derived from insect cells infected with a baculovirus expressing HSV gH (7). The binding of ICP47-2 was saturable, so that at a protein concentration of 1 μM approximately 16 ng of protein bound to human TAP (Fig. ​(Fig.4A).4A). In previous experiments with a similar preparation of insect microsomes containing human TAP, the binding of ICP47-1 also saturated at 15 to 16 ng (7). The ICP47-2 binding data were analyzed in a standard Scatchard plot, and the K_D was calculated to be 4.8 × 10⁻⁸ M (Fig. ​(Fig.4B),4B), compared with 5.2 × 10⁻⁸ M for ICP47-1 (7). These values are greater than those of high-affinity peptides that bind to human TAP with affinities reaching 4 × 10⁻⁷ M, though the vast majority of peptides bind to TAP with much lower affinities (8). Open in a separate windowFIG. 3Inhibition of human and murine TAP-mediated peptide transport by ICP47-1 and ICP47-2. TAP assays were performed as described in the legend for Fig. ​Fig.22 by using insect microsomes containing human TAP (10 μl of membranes containing 12 mg of membrane protein per ml) (A) or murine TAP (7.5 μl of membranes containing 4.8 mg of membrane protein per ml but with equivalent levels of TAP activity compared with microsomes containing human TAP) (B) and various concentrations of ICP47-1 and ICP47-2. The results shown are combined from two separate experiments, each involving human and murine TAP.Open in a separate windowFIG. 4Binding of ICP47-2 to human TAP. (A) Microsomes (15 μl of membranes with a 7.5-mg/ml concentration of membrane protein) derived from Sf9 cells expressing TAP1 and TAP2 or expressing HSV-1 gH (control membranes not containing TAP) were incubated with various amounts of ¹²⁵I-labelled ICP47-2 for 60 min at 4°C as described previously (7). Binding to control membranes was subtracted from binding to microsomes containing TAP at each point. (B) Scatchard analysis of the data in panel A. The K_D for ICP47-2 binding to TAP was calculated to be 4.8 × 10⁻⁸ M.To determine whether ICP47-2 could inhibit the murine TAP, microsomes from insect cells expressing mouse TAP were incubated with various concentrations of ICP47-1 and ICP47-2 and TAP assays were performed. Inhibition of the mouse TAP was observed with both ICP47-1 and ICP47-2, but relatively high concentrations of both proteins were required (Fig. ​(Fig.3B).3B). The IC₅₀ values for ICP47-1 and ICP47-2 in this experiment were 10.8 and 16.2 μM, respectively. However, we were unable to reduce TAP activity beyond approximately 40% with ICP47-1 or ICP47-2 concentrations reaching 30 μM. This was 100 times the concentration required to inhibit human TAP by 50%. We attempted to measure the specific binding of radiolabelled ICP47-1 and ICP47-2 to microsomes containing mouse TAP in experiments similar to those shown in Fig. ​Fig.4.4. However, there was little specific binding of ICP47-1 and ICP47-2, and it was difficult to measure binding at lower protein concentrations. We therefore measured binding at a single, higher protein concentration (2.75 μM), one sufficient to inhibit 10 to 20% of the mouse TAP activity and all of the human TAP activity. In this experiment, specific binding to microsomes containing murine TAP was determined by subtracting the binding to microsomes from insect cells that were not induced to express murine TAP (1). The binding of ICP47-1 and ICP47-2 to human TAP was easily measured (Fig. ​(Fig.5),5), although under these conditions it is important to note that ICP47-1 and ICP47-2 were present at concentrations beyond those required to saturate the TAP (Fig. ​(Fig.4A).4A). By contrast, it was found that there was little or no significant binding of ICP47-1 or ICP47-2 to microsomes containing murine TAP when background binding to control membranes was subtracted. In the experiment shown, specific ICP47-2 binding was greater than zero, but in other experiments this binding was less than zero, and thus we concluded that there was no detectable binding overall. In every experiment, it was clear that the level of binding of ICP47-1 and ICP47-2 to murine TAP was at least 25-fold lower than to human TAP. However, the human TAP present in these microsomes was limiting in these experiments, and thus it is very likely that the 25-fold difference between the levels of binding to human and mouse TAP is an underestimate. More likely this difference is 50- to 100-fold. On the basis of the inhibitory concentrations required to block murine TAP and the binding studies described above, estimates of the binding affinities of ICP47-1 and ICP47-2 for murine TAP may fall in the range of 5 × 10⁻⁶ M. Therefore, ICP47-1 and ICP47-2 bind poorly to the murine TAP, and this largely accounts for their inability to block mouse TAP peptide transport. Open in a separate windowFIG. 5Binding of ICP47-1 and ICP47-2 to microsomes containing murine TAP. Microsomes containing human TAP or control membranes without human TAP (100 μg of membrane protein per 150-μl assay) or microsomes containing mouse TAP or control membranes without mouse TAP (50 μg of membrane protein with the same TAP activity as with the human microsomes) were incubated with ¹²⁵I-labelled ICP47-1 or ICP47-2 at 2.75 μM for 60 min at 4°C. The microsomes were washed twice, pelleted, and disrupted with detergents as described previously (7). Radioactivity associated with the microsomes was quantified by gamma counting. “ICP47 bound” refers to specific binding, calculated by subtracting the binding to control membranes (without TAP) from that observed with microsomes containing human or murine TAP.In summary, ICP47-2 and ICP47-1 could block human TAP and bound to TAP with similar high affinities. It was interesting that these two proteins, whose primary structures are only about 40% identical, inhibit human TAP with indistinguishable profiles and bind to human TAP with virtually identical affinities. Moreover, both proteins blocked murine TAP poorly and only at high protein concentrations and could not bind to murine TAP. These results, at face value, would suggest that mice will not be an appropriate model in which to test the effects of ICP47 on HSV replication or as a selective inhibitor of CD8⁺ T-cell responses in other systems. However, we recently found that an HSV-1 ICP47 mutant showed dramatically reduced neurovirulence in mice, without altering the course of disease in the cornea (4). Therefore, ICP47 may attain sufficient concentrations in certain cells in the nervous systems of mice to inhibit TAP. This may be related to the fact that TAP and class I proteins are expressed at low levels in the nervous system. Alternatively, ICP47 may have other functions in the nervous system.     

Inducer exclusion by glucose 6-phosphate in Escherichia coli

The main mechanism causing catabolite repression by glucose and other carbon sources transported by the phosphotransferase system (PTS) in Escherichia coli involves dephosphorylation of enzyme IIA^Glc as a result of transport and phosphorylation of PTS carbohydrates. Dephosphorylation of enzyme IIA^Glc leads to 'inducer exclusion': inhibition of transport of a number of non-PTS carbon sources (e.g. lactose, glycerol), and reduced adenylate cyclase activity. In this paper, we show that the non-PTS carbon source glucose 6-phosphate can also cause inducer exclusion. Glucose 6-phosphate was shown to cause inhibition of transport of lactose and the non-metabolizable lactose analogue methyl-β- D -thiogalactoside (TMG). Inhibition was absent in mutants that lacked enzyme IIA^Glc or were insensitive to inducer exclusion because enzyme IIA^Glc could not bind to the lactose carrier. Furthermore, we showed that glucose 6-phosphate caused dephosphorylation of enzyme IIA^Glc. In a mutant insensitive to enzyme IIA^Glc-mediated inducer exclusion, catabolite repression by glucose 6-phosphate in lactose-induced cells was much weaker than that in the wild-type strain, showing that inducer exclusion is the most important mechanism contributing to catabolite repression in lactose-induced cells. We discuss an expanded model of enzyme IIA^Glc-mediated catabolite repression which embodies repression by non- PTS carbon sources.     

Adjustments of the Pesticide Risk Index Used in Environmental Policy in Flanders

Indicators are used to quantify the pressure of pesticides on the environment. Pesticide risk indicators typically require weighting environmental exposure by a no effect concentration. An indicator based on spread equivalents (ΣSeq) is used in environmental policy in Flanders (Belgium). The pesticide risk for aquatic life is estimated by weighting active ingredient usage by the ratio of their maximum allowable concentration and their soil halflife. Accurate estimates of total pesticide usage in the region are essential in such calculations. Up to 2012, the environmental impact of pesticides was estimated on sales figures provided by the Federal Government. Since 2013, pesticide use is calculated based on results from the Farm Accountancy Data Network (FADN). The estimation of pesticide use was supplemented with data for non-agricultural use based on sales figures of amateur use provided by industry and data obtained from public services. The Seq-indicator was modified to better reflect reality. This method was applied for the period 2009-2012 and showed differences between estimated use and sales figures of pesticides. The estimated use of pesticides based on accountancy data is more accurate compared to sales figures. This approach resulted in a better view on pesticide use and its respective environmental impact in Flanders.     

Volumetric Response beyond Six Months of Cardiac Resynchronization Therapy and Clinical Outcome

AimsResponse to cardiac resynchronization therapy (CRT) is often assessed six months after implantation. Our objective was to assess the number of patients changing from responder to non-responder between six and 14 months, so-called late non-responders, and compare them to patients who were responder both at six and 14 months, so-called stable responders. Furthermore, we assessed predictive values of six and 14-month response concerning clinical outcome.Methods105 patients eligible for CRT were enrolled. Clinical, laboratory, ECG, and echocardiographic parameters and patient-reported health status (Kansas City Cardiomyopathy Questionnaire [KCCQ]) were assessed before, and six and 14 months after implantation. Response was defined as ≥15% LVESV decrease as compared to baseline. Major adverse cardiac events (MACE) were registered until 24 months after implantation. Predictive values of six and 14-month response for MACE were examined.ResultsIn total, 75 (71%) patients were six-month responders of which 12 (16%) patients became late non-responder. At baseline, late non-responders more often had ischemic cardiomyopathy and atrial fibrillation, higher BNP and less dyssynchrony compared to stable responders. At six months, late non-responders showed significantly less LVESV decrease, and higher creatinine levels. Mean KCCQ scores of late non-responders were lower than those of stable responders at every time point, with the difference being significant at 14 months. The 14 months response was a better predictor of MACE than six months response.ConclusionsThe assessment of treatment outcomes after six months of CRT could be premature and response rates beyond might better correlate to long-term clinical outcome.     

Urinary Proteins,Vitamin D and Genetic Polymorphisms as Risk Factors for Febrile Urinary Tract Infection and Relation with Bacteremia: A Case Control Study

Objective/Purpose

Febrile urinary tract infection (UTI) is a common bacterial disease that may lead to substantial morbidity and mortality especially among the elderly. Little is known about biomarkers that predict a complicated course. Our aim was to determine the role of certain urinary cytokines or antimicrobial proteins, plasma vitamin D level, and genetic variation in host defense of febrile UTI and its relation with bacteremia.

Methods

A case-control study. Out of a cohort of consecutive adults with febrile UTI (n = 787) included in a multi-center observational cohort study, 46 cases with bacteremic E.coli UTI and 45 cases with non-bacteremic E.coli UTI were randomly selected and compared to 46 controls. Urinary IL-6, IL-8, LL37, β-defensin 2 and uromodulin as well as plasma 25-hydroxyvitamin D were measured. In 440 controls and 707 UTI patients polymorphisms were genotyped in the genes CXCR1, DEFA4, DEFB1, IL6, IL8, MYD88, UMOD, TIRAP, TLR1, TLR2, TLR5 and TNF.

Results

IL-6, IL-8, and LL37 are different between controls and UTI patients, although these proteins do not distinguish between patients with and without bacteremia. While uromodulin did not differ between groups, inability to produce uromodulin is more common in patients with bacteremia. Most participants in the study, including the controls, had insufficient vitamin D and, at least in winter, UTI patients have lower vitamin D than controls. Associations were found between the CC genotype of IL6 SNP rs1800795 and occurrence of bacteremia and between TLR5 SNP rs5744168 and protection from UTI. The rare GG genotype of IL6 SNP rs1800795 was associated with higher β-defensin 2 production.

Conclusion

Although no biomarker was able to distinguish between UTI with or without bacteremia, two risk factors for bacteremia were identified. These were inability to produce uromodulin and an IL6 rs1800795 genotype.     

Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress

Very high ethanol tolerance is a distinctive trait of the yeast Saccharomyces cerevisiae with notable ecological and industrial importance. Although many genes have been shown to be required for moderate ethanol tolerance (i.e., 6 to 12%) in laboratory strains, little is known of the much higher ethanol tolerance (i.e., 16 to 20%) in natural and industrial strains. We have analyzed the genetic basis of very high ethanol tolerance in a Brazilian bioethanol production strain by genetic mapping with laboratory strains containing artificially inserted oligonucleotide markers. The first locus contained the ura3Δ0 mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage for LYS2, LEU2, HIS3, and MET15. Tolerance to only very high ethanol concentrations was reduced by auxotrophies, while the effect was reversed at lower concentrations. Evaluation of other stress conditions showed that the link with auxotrophy is dependent on the type of stress and the type of auxotrophy. When the concentration of the auxotrophic nutrient is close to that limiting growth, more stress factors can inhibit growth of an auxotrophic strain. We show that very high ethanol concentrations inhibit the uptake of leucine more than that of uracil, but the 500-fold-lower uracil uptake activity may explain the strong linkage between uracil auxotrophy and ethanol sensitivity compared to leucine auxotrophy. Since very high concentrations of ethanol inhibit the uptake of auxotrophic nutrients, the active uptake of scarce nutrients may be a major limiting factor for growth under conditions of ethanol stress.     

How Attention Can Create Synaptic Tags for the Learning of Working Memories in Sequential Tasks

Intelligence is our ability to learn appropriate responses to new stimuli and situations. Neurons in association cortex are thought to be essential for this ability. During learning these neurons become tuned to relevant features and start to represent them with persistent activity during memory delays. This learning process is not well understood. Here we develop a biologically plausible learning scheme that explains how trial-and-error learning induces neuronal selectivity and working memory representations for task-relevant information. We propose that the response selection stage sends attentional feedback signals to earlier processing levels, forming synaptic tags at those connections responsible for the stimulus-response mapping. Globally released neuromodulators then interact with tagged synapses to determine their plasticity. The resulting learning rule endows neural networks with the capacity to create new working memory representations of task relevant information as persistent activity. It is remarkably generic: it explains how association neurons learn to store task-relevant information for linear as well as non-linear stimulus-response mappings, how they become tuned to category boundaries or analog variables, depending on the task demands, and how they learn to integrate probabilistic evidence for perceptual decisions.     

Diversity of Aquatic Pseudomonas Species and Their Activity against the Fish Pathogenic Oomycete Saprolegnia

Emerging fungal and oomycete pathogens are increasingly threatening animals and plants globally. Amongst oomycetes, Saprolegnia species adversely affect wild and cultivated populations of amphibians and fish, leading to substantial reductions in biodiversity and food productivity. With the ban of several chemical control measures, new sustainable methods are needed to mitigate Saprolegnia infections in aquaculture. Here, PhyloChip-based community analyses showed that the Pseudomonadales, particularly Pseudomonas species, represent one of the largest bacterial orders associated with salmon eggs from a commercial hatchery. Among the Pseudomonas species isolated from salmon eggs, significantly more biosurfactant producers were retrieved from healthy salmon eggs than from Saprolegnia-infected eggs. Subsequent in vivo activity bioassays showed that Pseudomonas isolate H6 significantly reduced salmon egg mortality caused by Saprolegnia diclina. Live colony mass spectrometry showed that strain H6 produces a viscosin-like lipopeptide surfactant. This biosurfactant inhibited growth of Saprolegnia in vitro, but no significant protection of salmon eggs against Saprolegniosis was observed. These results indicate that live inocula of aquatic Pseudomonas strains, instead of their bioactive compound, can provide new (micro)biological and sustainable means to mitigate oomycete diseases in aquaculture.