Influence of an arbuscular mycorrhizal fungus on Pseudomonas fluorescens DF57 in rhizosphere and hyphosphere soil

The influence of Glomus intraradices (BEG87) on Pseudomonas fluorescens DF57 in hyphosphere and rhizosphere soil was examined. Cucumis sativus (Aminex, F₁ hybrid) was grown in symbiosis with the arbuscular mycorrhizal fungus G. intraradices in PVC tubes, consisting of a central root compartment and two lateral root-free compartments. Two Tn 5 - lux AB-marked strains of P. fluorescens DF57 were used. Strain DF57-P2, which has an insertion of Tn 5::lux AB in a phosphate starvation-inducible locus, was used as a phosphate starvation reporter. Another lux -tagged strain DF57-40E7, which carries a constitutively expressed lux AB fusion, was used as control for strain DF57-P2 and for measuring the metabolic activity of P. fluorescens DF57. A strain of P. fluorescens DF57, which carries a constitutively expressed gfp gene, was used in studies of attachment between the bacteria and the hyphae. G. intraradices decreased the culturability of P. fluorescens DF57 significantly, both in rhizosphere and hyphosphere soil, whereas the total number of P. fluorescens DF57 measured by immunofluorescence microscopy was decreased in hyphosphere soil only. G. intraradices did not induce a phosphorus starvation response in P. fluorescens DF57, and the metabolic activity of the bacteria was not affected by the fungus after 48 h. P. fluorescens DF57 did not attach to G. intraradices hyphae and was not able to use the hyphae as carbon substrate. The negative effect of G. intraradices on culturability and on number of P. fluorescens DF57 in hyphosphere soil is discussed.     

Nitrogen Availability to Pseudomonas fluorescens DF57 Is Limited during Decomposition of Barley Straw in Bulk Soil and in the Barley Rhizosphere

The availability of nitrogen to Pseudomonas fluorescens DF57 during straw degradation in bulk soil and in barley rhizosphere was studied by introducing a bioluminescent reporter strain (DF57-N3), responding to nitrogen limitation, to model systems of varying complexity. DF57-N3 was apparently not nitrogen limited in the natural and sterilized bulk soil used for these experiments. The soil was subsequently amended with barley straw, representing a plant residue with a high carbon-to-nitrogen ratio (between 60 and 100). In these systems the DF57-N3 population gradually developed a nitrogen limitation response during the first week of straw decomposition, but exclusively in the presence of the indigenous microbial population. This probably reflects the restricted ability of DF57 to degrade plant polymers by hydrolytic enzymes. The impact of the indigenous population on nitrogen availability to DF57-N3 was mimicked by the cellulolytic organism Trichoderma harzianum Rifai strain T3 when coinoculated with DF57-N3 in sterilized, straw-amended soil. Limitation occurred concomitantly with fungal cellulase production, pointing to the significance of hydrolytic activity for the mobilization of straw carbon sources, thereby increasing the nitrogen demand. Enhanced survival of DF57-N3 in natural soil after straw amendment further indicated that DF57 was cross-fed with carbon/energy sources. The natural barley rhizosphere was experienced by DF57-N3 as an environment with restricted nitrogen availability regardless of straw amendment. In the rhizosphere of plants grown in sterilized soil, nitrogen limitation was less severe, pointing to competition with indigenous microorganisms as an important determinant of the nitrogen status for DF57-N3 in this environment. Hence, these studies have demonstrated that nitrogen availability and gene expression in Pseudomonas is intimately linked to the structure and function of the microbial community. Further, it was demonstrated that the activities of cellulolytic microorganisms may affect the availability of energy and specific nutrients to a group of organisms deficient in hydrolytic enzyme activities.     

Effects of Pseudomonas fluorescens DF57 on growth and P uptake of two arbuscular mycorrhizal fungi in symbiosis with cucumber

 The effect of Pseudomonas fluorescens DF57 on growth and P uptake of two arbuscular mycorrhizal (AM) fungi in symbiosis with cucumber plants was studied in compartmentalised growth systems. Hyphae of Glomus intraradices Schenck & Smith (BEG87) or G. caledonium (Nicol. & Gerd.) Trappe & Gerdeman (BEG15) grew into lateral root-free compartments. Non-mycorrhizal plants served as control. The soil in half of the growth units of each mycorrhizal treatment was inoculated with P. fluorescens DF57. P. fluorescens DF57 enhanced hyphal length density of one of the AM fungi, G. caledonium, but this was not reflected in a higher hyphal transport of P from the root-free soil to the plant. The total P content was higher in plants grown in symbiosis with G. intraradices than in plants in the other treatments. G. caledonium and P. fluorescens DF57 had a synergistic effect in that total P content in plants inoculated with G. caledonium was higher in the presence than in the absence of P. fluorescens DF57. Accepted: 7 January 1999     

Outer Membrane Protein Heterogeneity within Pseudomonas fluorescens and P. putida and Use of an OprF Antibody as a Probe for rRNA Homology Group I Pseudomonads

The electrophoretic patterns of outer membrane proteins of strains representing the biovars of Pseudomonas fluorescens and Pseudomonas putida were analyzed by gel electrophoresis. The outer membrane protein profiles were variable, and they were not useful for assigning strains to a specific biovar. However, three or four predominant outer membrane proteins migrating at 42 to 46 kDa, 33 to 38 kDa, and 20 to 22 kDa were conserved among the strains. They could be tentatively identified as OprE (44 kDa), OprF (38 kDa), OprH (21 kDa), and OprL (20.5 kDa), which are known proteins from Pseudomonas aeruginosa. A 37-kDa OprF-like protein was purified from P. fluorescens DF57 and used to raise a polyclonal antibody. In Western blot (immunoblot) analysis, this antibody reacted with OprF proteins from members of Pseudomonas rRNA homology group I but not with proteins from nonpseudomonads. The heterogeneity in M(infr) of OprF was greater among P. fluorescens strains than among P. putida strains. Immunofluorescence microscopy of intact cells demonstrated that the antibody recognized epitopes that were accessible only after unmasking by EDTA treatment. The antibody was used in a colony blotting assay to determine the percentage of rRNA homology group I pseudomonads among bacteria from the rhizosphere of barley. The bacteria were isolated on 10% tryptic soy agar, King's B agar, and the pseudomonad-specific medium Gould S1 agar. The estimate of OprF-containing CFU in rhizosphere soil obtained by colony blotting on 10% tryptic soy agar was about 2 and 14 times higher than the values obtained from King's agar and Gould S1 agar, respectively, indicating that not all fluorescent pseudomonads are scored on more specific media. The colonies reacting with the OprF antibody were verified as being rRNA homology group I pseudomonads by using the API 20NE system.     

Suppression of bacterial wilt in Eucalyptus urophylla by fluorescent Pseudomonas spp. in China

Bacterial wilt caused by Ralstonia solanacearum race 1, biovar III has become a severe problem in Eucalyptus plantations in south China. The disease mainly attacks young eucalypt trees, and no effective control measures are available yet. To explore possibilities to develop biological control of the disease, strains of fluorescent Pseudomonas spp. that are effective in suppressing plant diseases by known mechanisms, were tested for their potential to control bacterial wilt in Eucalyptus. Pseudomonas putida WCS358r, Pseudomonas fluorescens WCS374r, P. fluorescens WCS417r, and Pseudomonas aeruginosa 7NSK2 antagonize R. solanacearum in vitro by siderophore-mediated competition for iron, whereas inhibition of pathogen growth by P. fluorescens CHA0r is antibiosis-based. No correlations were found between antagonistic activities of these Pseudomonas spp. in vitro and biocontrol of bacterial wilt in Eucalyptus in vivo. None of the strains suppressed disease when mixed together with the pathogen through the soil or when seeds or seedlings were treated with the strains one to four weeks before transfer into soil infested with R. solanacearum. However, when the seedlings were dipped with their roots in a bacterial suspension before transplanting into infested soil, P. fluorescens WCS417r significantly suppressed bacterial wilt. P. putida WCS358r was marginally effective, whereas its siderophore-minus mutant had no effect at all, indicating that siderophore-mediated competition for iron can contribute but is not effective enough to suppress bacterial wilt in Eucalyptus. A derivative of P. putida WCS358r, constitutively producing 2,4-diacetylphloroglucinol (WCS358::phl) reduced disease. Combined treatment with P. fluorescens WCS417r and P. putida WCS358::phl did not improve suppression of bacterial wilt.     

l-Lysine-2-monooxygenase production by strains of Pseudomonas fluorescens and P. putida

Twelve strains of Pseudomonas fluorescens and P. putida were grown in a synthetic medium that contained l-lysine as the only source of carbon and nitrogen, and screened for l-lysine-2-monooxygenase production. Best production was by P. putida BKM B-1458 at 30 IU/g wet wt biomass when grown in a shake-flask but 25 IU/g in a 250-l fermenter.     

Cell Surface Display of Lipase in Pseudomonas putida KT2442 Using OprF as an Anchoring Motif and Its Biocatalytic Applications

We developed a new cell surface display system in Pseudomonas putida KT2442 using OprF, an outer membrane protein of Pseudomonas aeruginosa, as an anchoring motif in a C-terminal deletion-fusion strategy. The Pseudomonas fluorescens SIK W1 lipase gene was fused to two different C-terminal truncated OprF genes, and the fusion genes were cloned into the broad-host-range plasmid pBBR1MCS2 to make pMO164PL and pMO188PL. Plasmid pMO188PL allowed better display of lipase and thus was chosen for further study. The display of lipase on the surface of P. putida KT2442 was confirmed by Western blot analysis, immunofluorescence microscopy, and measurement of whole-cell lipase activity. The whole-cell lipase activity of recombinant P. putida KT2442 harboring pMO188PL was more than fivefold higher than that of recombinant Escherichia coli displaying lipase in the same manner. Cell surface-displayed lipase exhibited the highest activity at 47°C and pH 9.0, and the whole-cell lipase activity was greater than 90% of the initial activity in organic solvents at 47°C for 1 week. In a biocatalytic application, enantioselective resolution of 1-phenyl ethanol was carried out in an organic solvent. (R)-Phenyl ethyl acetate was successfully produced with 41.9% conversion and an enantiomeric excess of more than 99% in a 36-h reaction. These results suggest that the OprF anchor can be used for efficient display of proteins in P. putida KT2442 and consequently for various biocatalytic applications.     

Isolation of lux reporter gene fusions in Pseudomonas fluorescens DF57 inducible by nitrogen or phosphorus starvation

Abstract: We have used transposon Tn 5 mutagenesis to insert a promoter-less lux AB gene-cassette into multiple locations in the chromosome of a Pseudomonas fluorescens strain, thereby bringing the lux reporter genes under the control of resident promoters. To identify reporter bacteria responsive to nutritional stresses we isolated and characterized a collection of 23 gene fusions consistently displaying bioluminescence under nitrogen starvation and 12 phosphorus starvation inducible fusions. Bioluminescence of one group of mutants was induced after 4 to 6 h of starvation and was continuously expressed at a high level, whereas a second group was induced earlier and the bioluminescence subsequently declined. Finally, a third group was induced later after 24 h of starvation. Four strains were selected for further study, namely, two Tn 5-lux containing strains which were induced by nitrogen starvation and two strains induced by phosphorus starvation. Another two strains, carrying constitutively expressed lux fusions, were included as controls. An analysis of biochemical characters, as well as LPS and protein composition, did not reveal any discernible differences between the mutants and the wild-type strain. Survival experiments with the selected Tn 5-lux containing strains showed that they all performed comparably to the wild-type under carbon and nitrogen starvation, whereas some of the strains were less resistant to phosphorus starvation. Expression of bioluminescence by the mutants during carbon, nitrogen and phosphorus starvation was detectable even after 18 days and was not affected by high osmolarity or low temperature.     

Modeling of growth kinetics for Pseudomonas spp. during benzene degradation

A modeling study was conducted on growth kinetics of three different strains of Pseudomonas spp. (Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida) during benzene degradation to determine optimum substrate concentrations for most efficient biodegradation. Batch tests were performed for eight different initial substrate concentrations to observe cell growth and associated substrate degradation using benzene-adapted cells. Kinetic parameters of both inhibitory (Haldane–Andrews, Aiba–Edwards) and noninhibitory (Monod) models were fitted to the relationship between specific growth rate and substrate concentration obtained from the growth curves. Results showed that half-saturation constant of P. fluorescens was the highest among the three strains, indicating that this strain could grow well at high concentration, while P. putida could grow best at low concentration. The inhibition constant of P. aeruginosa was the highest, implying that it could tolerate high benzene concentration and therefore could grow at a wider concentration range. Estimated specific growth rate of P. putida was lower, but half-saturation constant was higher than those from literature study due to high substrate concentration range used in this study. These two kinetic parameters resulted in substantial difference between Monod- and Haldane-type models, indicating that distinction should be made in applying those models.     

Effect of dissolved oxygen regime on growth dynamics of Pseudomonas spp during benzene degradation

We investigated the effect of different oxygen regimes on growth patterns of Pseudomonas spp. during benzene degradation in microcosm batch studies. Benzene degradation was induced by limiting oxygen available for microbial activity, which consists of three initial-dissolved oxygen (DO) levels of oxic, hypoxic, and anoxic conditions. Batch experiments were performed for cell growth and benzene degradation by inoculating three strains of Pseudomonas spp. (Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida) in mineral salt medium containing aqueous benzene. Results showed that all strains were capable to grow and degrade benzene under all oxygen regimes but in a different manner. The highest cell growth of P. aeruginosa and P. fluorescens was achieved under oxic and anoxic condition, respectively, but there was no substantial difference on benzene degradation between the oxygen treatments with about 25% reduction for both strains. P. putida showed a facultative process for both cell growth and benzene degradation. This reveals that care should be taken in selection of microorganisms with regard to environmental studies since they exhibit different responses for given environmental conditions such as DO levels.     

Identification of Genes and Proteins Necessary for Catabolism of Acyclic Terpenes and Leucine/Isovalerate in Pseudomonas aeruginosa

Geranyl-coenzyme A (CoA)-carboxylase (GCase; AtuC/AtuF) and methylcrotonyl-CoA-carboxylase (MCase; LiuB/LiuD) are characteristic enzymes of the catabolic pathway of acyclic terpenes (citronellol and geraniol) and of saturated methyl-branched compounds, such as leucine or isovalerate, respectively. Proteins encoded by two gene clusters (atuABCDEFGH and liuRABCDE) of Pseudomonas aeruginosa PAO1 were essential for acyclic terpene utilization (Atu) and for leucine and isovalerate utilization (Liu), respectively, as revealed by phenotype analysis of 10 insertion mutants, two-dimensional gel electrophoresis, determination of GCase and MCase activities, and Western blot analysis of wild-type and mutant strains. Analysis of the genome sequences of other pseudomonads (P. putida KT2440 and P. fluorescens Pf-5) revealed candidate genes for Liu proteins for both species and candidate genes for Atu proteins in P. fluorescens. This result concurred with the finding that P. fluorescens, but not P. putida, could grow on acyclic terpenes (citronellol and citronellate), while both species were able to utilize leucine and isovalerate. A regulatory gene, atuR, was identified upstream of atuABCDEFGH and negatively regulated expression of the atu gene cluster.     

Homologous Expression of the Lipase and ABC Transporter Gene Cluster, tliDEFA, Enhances Lipase Secretion in Pseudomonas spp.

The ABC transporter TliDEF was found to be an efficient secretory apparatus for extracellular lipase TliA in Pseudomonas fluorescens. For the enhanced secretion of the lipase, we tried to coexpress tliA and tliDEF in various Pseudomonas species. Whereas the coexpression of tliA and tliDEF was required for the lipase secretion in P. fragi, the expression of tliA was sufficient for the lipase secretion in P. fluorescens, P. syringae, and P. putida, indicating the existence of compatible ABC transporter in these species. However, P. fluorescens harboring tliDEFA secreted much more lipase than P. fluorescens harboring only tliA, but the tliDEF was functional only at temperatures below 30°C. The recombinant P. fluorescens overexpressing tliDEFA showed the highest secretion level, 217 U/ml · OD (optical density) (28 μg/ml · OD) of lipase in Luria-Bertani medium under microaerated conditions. With the increase of aeration, the lipase production was decreased and the lipase seemed to be degraded as the cells entered the cell death phase. These results demonstrate that P. fluorescens can be used as a host system for the secretory production of the lipase using the ABC transporter, thus producing lipase in over 14% of the total protein.     

Mechanism of cyanide and thiocyanate decomposition by an association of Pseudomonas putida and Pseudomonas stutzeri strains

The intermediate and terminal products of cyanide and thiocyanate decomposition by individual strains of the genus Pseudomonas, P. putida strain 21 and P. stutzeri strain 18, and by their association were analyzed. The activity of the enzymes of nitrogen and sulfur metabolism in these strains was compared with that of the collection strains P. putida VKM B-2187^T and P. stutzeri VKM B-975^T. Upon the introduction of CN⁻ and SCN⁻ into cell suspensions of strains 18 and 21 in phosphate buffer (pH 8.8), the production of NH ₄ ⁺ was observed. Due to the high rate of their utilization, NH₃, NH ₄ ⁺ , and CNO⁻ were absent from the culture liquids of P. putida strain 21 and P. stutzeri strain 18 grown with CN⁻ or SCN⁻. Both Pseudomonas strains decomposed SCN⁻ via cyanate production. The cyanase activity was 0.75 μmol/(min mg protein) for P. putida strain 21 and 1.26 μmol/(min mg protein) for P. stutzeri strain 18. The cyanase activity was present in the cells grown with SCN⁻ but absent in cells grown with NH ₄ ⁺ . Strain 21 of P. putida was a more active CN⁻ decomposer than strain 18 of P. stutzeri. Ammonium and CO₂ were the terminal nitrogen and carbon products of CN⁻ and SCN⁻ decomposition. The terminal sulfur products of SCN⁻ decomposition by P. stutzeri strain 18 and P. putida strain 21 were thiosulfate and tetrathionate, respectively. The strains utilized the toxic compounds in the anabolism only, as sources of nitrogen (CN⁻ and SCN⁻) and sulfur (SCN⁻). The pathway of thiocyanate decomposition by the association of bacteria of the genus Pseudomonas is proposed based on the results obtained. Original Russian Text ? N.V. Grigor’eva, T.F. Kondrat’eva, E.N. Krasil’nikova, G.I. Karavaiko, 2006, published in Mikrobiologiya, 2006, Vol. 75, No. 3, pp. 320–328.     

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.     

Chromosomal insertion of the entire Escherichia coli lactose operon,into two strains of Pseudomonas,using a modified mini-Tn5 delivery system

A 12-kb PstI fragment including the entire E. coli lactose operon (lacIPOZYA) was inserted in one copy into the chromosome of Pseudomonas putida, Pseudomonas fluorescens and an E. coli strain with lac⁻ phenotype. This was made possible by improvements of an already existing mini-Tn5 transposon delivery system (de Lorenzo et al., 1990; Herrero et al., 1990), which integrates cloned DNA fragments at random sites on the chromosome of the recipient bacteria in single copies. This has resulted in: (a) the making of two useful low copy-number cloning vectors both with extensive multi-cloning regions flanked by NotI sites needed in the mini-Tn5 delivery system; (b) the generation of E. coli nonlysogenic strains expressing the π protein thus being capable of maintaining and delivering R6K-based mini-Tn5 vectors to other E. coli strains; (c) the successful insertion of the E. coli lactose operon into the P. fluorescens chromosome giving P. fluorescens the ability to grow on lactose; (d) evidence from Southern blotting that contradicts the assumption that the mini-Tn5 delivery system always creates one-copy inserts. These improvements allow insertion of large DNA fragments encoding highly expressed proteins into the chromosome of a large variety of Gram-negative bacteria including E. coli.     

Numerical taxonomy of fluorescent Pseudomonas associated with tomato roots

The phenetic taxonomy of 110 fluorescent bacterial strains, isolated from the roots of tomatoes and other plants was numerically studied through 97 features including 69 assimilation tests. Thirty-two reference strains of various Pseudomonas spp. were additionally included. The strains clustered into 16 clusters at the 74% similarity level when using Jaccard similarity coefficients. Almost all field strains belonged to the P. fluorescens/P. putida-complex while none clustered with P. syringae and allied bacteria. The biovar II branch, as well as the newly described biovar VI of P. fluorescens, made up 55% and 20% respectively, of the field strains; two % were allocated to P. fluorescens biovar I and three % to biovar IV. Eleven % of the root associated strains were designated P. putida; six strains were biovar A, three strains biovar B while four strains could not be referred to any known biovar. The continuum within the P. fluorescens/P. putida-complex as well as the taxonomic status of the six biovars of P. fluorescens and the three biovars of P. putida are discussed.     

Unusual traits of the pyoverdin-mediated iron acquisition system in Pseudomonas putida strain BTP1

Fluorescent Pseudomonas species are characterized by the production of pyoverdin-type siderophores for Fe³⁺ acquisition in iron-limited environments. Since it produces a structurally specific pyoverdin, Pseudomonas putida strain BTP1 could represent a valuable tool in an attempt to correlate the structural features of these compounds with some specificity in their two main properties i.e. affinity for iron and recognition rate by other Pseudomonas strains. An uncommonly high affinity for iron of the pyoverdin synthetized by P. putida BTP1 was observed by comparing both the apparent stability constant and the decomplexation kinetic of its ferric complex with those of ferripyoverdins from other strains. On another hand, results from growth stimulation experiments and labeled ferripyoverdin uptake assays highlighted the very low recognition rate of BTP1 isopyoverdins by membrane receptors of foreign strains. By contrast, P. putida BTP1 was able to utilize a broad spectrum of structurally unrelated exogenous pyoverdins by means of multiple receptors that are likely constitutively expressed in its outer membrane. The unusual traits of its pyoverdin-mediated iron acquisition system should contribute to enlarge the ecological competence of Pseudomonas putida BTP1 in terms of colonization and persistence in the rhizosphere.     

Effect of inoculation of lactic acid bacteria on proteolytic activity of psychrotrophic Gram-negative bacteria in fresh farmed sea bass (Dicentrarchus labrax) fillets during storage at 4 °C under vacuum-packed conditions

The effect of two strains of lactic acid bacteria (LAB) (Lactococcus lactis and Carnobacterium piscicola) on the proteolytic activity of four strains of Psychrotrophic Gram-negative bacteria [Psy G(?)] (Pseudomonas fluorescens, Aeromonas hydrophila, Pseudomonas putida and Photobacterium damselae) has been determined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), in fresh vacuum-packed farmed sea bass (Dicentrarchus labrax) fillets artificially contaminated, during 21 days of chilled storage. The profiles of sarcoplasmic (SP) and myofibrillar (MP) proteins indicated that the major changes were produced with Pseudomonas fluorescens, Aeromonas hydrophila, and Pseudomonas putida starters. The results also showed that LAB strains presented a weak proteolytic activity against MP and SP proteins in muscle of fresh sea bass. In fact, we noted the less pronounced degradation of protein fractions in samples inoculated with LAB combination. Moreover, a significant bacteriostatic effect of LAB strains was demonstrated against all microflora, particularly mesophilic aerobic plate counts (MAPC) and psychrotrophic bacterial counts (PBC), with fillets remaining unspoiled until the end of storage, against values of 7 and 8 log CFU/g, respectively; control fish fillets exceeded the upper acceptability limit.