Luria-Bertani Research Articles

Resistance to AFN-1252 Arises from Missense Mutations in Staphylococcus aureus Enoyl-acyl Carrier Protein Reductase (FabI)

AFN-1252 is a potent antibiotic against Staphylococcus aureus that targets the enoyl-acyl carrier protein reductase (FabI). A thorough screen for AFN-1252-resistant strains was undertaken to identify the spectrum of mechanisms for acquired resistance. A missense mutation in fabI predicted to encode FabI(M99T) was isolated 49 times, and a single isolate was predicted to encode FabI(Y147H). AFN-1252 only bound to the NADPH form of FabI, and the close interactions between the drug and Met-99 and Tyr-147 explained how the mutations would result in resistant enzymes. The clone expressing FabI(Y147H) had a pronounced growth defect that was rescued by exogenous fatty acid supplementation, and the purified protein had less than 5% of the enzymatic activity of FabI. FabI(Y147F) was also catalytically defective but retained its sensitivity to AFN-1252, illustrating the importance of the conserved Tyr-147 hydroxyl group in FabI function. The strains expressing FabI(M99T) exhibited normal growth, and the biochemical properties of the purified protein were indistinguishable from those of FabI. The AFN-1252 Ki(app) increased from 4 nm in FabI to 69 nm in FabI(M99T), accounting for the increased resistance of the corresponding mutant strain. The low activity of FabI(Y147H) precluded an accurate Ki measurement. The strain expressing FabI(Y147H) was also resistant to triclosan; however, the strain expressing FabI(M99T) was more susceptible. Strains with higher levels of AFN-1252 resistance were not obtained. The AFN-1252-resistant strains remained sensitive to submicromolar concentrations of AFN-1252, which blocked growth through inhibition of fatty acid biosynthesis at the FabI step.

A comparison and optimization of methods and factors affecting the transformation of Escherichia coli

DNA manipulation routinely requires competent bacteria that can be made using one of numerous methods. To determine the best methods, we compared four commonly used chemical methods (DMSO, MgCl2–CaCl2, CaCl2 and Hanahan's methods) on frequently used Escherichia coli (E. coli) strains: DH5α, XL-1 Blue, SCS110, JM109, TOP10 and BL21-(DE3)-PLysS. Hanahan's method was found to be most effective for DH5α, XL-1 Blue and JM109 strains (P<0.05), whilst the CaCl2 method was best for SCS110, TOP10 and BL21 strains (P<0.05). The use of SOB (super optimal broth) over LB [Luria–Bertani (broth)] growth media was found to enhance the competency of XL-1 Blue (P<0.05), dampened JM109′s competency (P<0.05), and had no effect on the other strains (P>0.05). We found no significant differences between using 45 or 90 s heat shock across all the six strains (P>0.05). Through further optimization by means of concentrating the aliquots, we were able to get further increases in transformation efficiencies. Based on the optimized parameters and methods, these common laboratory E. coli strains attained high levels of TrE (transformation efficiency), thus facilitating the production of highly efficient and cost-effective competent bacteria.

2-methylbutanal, a volatile biomarker, for non-invasive surveillance of Proteus

Pathogen detection needs a paradigm shift from time-consuming conventional microbiological and biochemical tests to much simpler identification methods with higher sensitivity and specificity. In this regard, a simple detection method for frequently isolated nosocomial uropathogen, Proteus spp., was developed using the characteristic volatile 2-methylbutanal released in Luria Bertani broth. The instant reaction of the compound with 5-dimethylaminonaphthalene-1-sulfonylhydrazine (DNSH) has been adapted to develop a sensitive fluorescence assay named "ProteAl" (Prote, "Proteus" & Al, "Aldehyde"). The assay was performed by direct addition of the fluorescence reagent to the culture after 7 h of growth. The distinct green fluorescence by Proteus (other organisms show orange fluorescence) served as the simplest and quicker identification test available for Proteus. In the laboratory, it exhibited 100% specificity and 100% sensitivity during testing of 95 strains including standard and known clinical isolates representing frequently encountered uropathogens.

Amylase enzyme from Bacillus subtilis S8‐18: A potential desizing agent from the marine environment

The present study is aimed at developing an economical medium for the production of α-amylase from Bacillus subtilis S8-18, a marine sediment isolate from Palk Bay, with various agricultural by-products that are cheap and rich in starch. These products include wheat bran, wheat husk, rice bran, rice husk, and potato peel and are used to replace soluble starch present in the Luria Bertani (LB) broth (synthetic medium). The rice husk was found to be the best to influence enzyme production significantly (61,186 IU mL⁻¹) when compared with the yield of 30,026 IU mL⁻¹ obtained by commercial starch. Hence, LB broth containing rice husk was considered an economical medium. In addition, the effect of various nutritional and physiological factors on enzyme production was also investigated. Furthermore, the desizing efficiency of α-amylases produced by synthetic and economical media was evaluated through various assays like reducing sugar estimation, weight loss assay, drop absorbency assay, scanning electron microscopy, and Fourier transform infrared analyses. In addition, a commercial α-amylase from B. subtilis was also used in desizing analyses for comparative purposes. It revealed that the α-amylase from the economical medium was as effective in desizing the cotton fabrics as that of the commercial enzyme and much superior to the enzyme produced through the synthetic medium.

English

The Bacillus by their intrinsic properties have several mechanisms of procuring beneficial effects to plants. They are among the most successful bacterial communities in the solubilization of phosphates. Ten Bacillus strains were isolated from the rhizosphere of wheat from three areas in arid and semi-arid regions in Algeria. These strains were tested for their ability to solubilize phosphates on liquid and solid media: LB (Luria-Bertani), NBRIP (National Botanical Research Institute Phosphate) and PVK (Pikovskaya) supplemented with Ca3(PO4)2 (5 g/l). The highest levels of soluble phosphates were obtained on PVK and NBRIP liquid (177.68 and 173.28 µg/ml for D13 and D1 strains, respectively). Phosphates solubilization is always associated with a decrease in the pH of the medium. An inverse relationship (r = -0.4224) existed between the levels of soluble phosphate and the decrease of pH in medium. To test the solubilization capacity of theBacillus strains under salt stress, concentrations of NaCl (0, 2, 4, 6 and 8%) were added to the NBRIP medium. The solubilization capacity was negatively affected by the increase of salinity. However, the response of the Bacillus to salt was strain dependent. Strains D1 and D13, which produced the best rate of solubilization were used to study the ability of osmoprotectants (glycine-betaine, proline) to restore the ability to solubilize phosphate at high salt concentrations. However, these strains do not seem to accumulate these types of osmoprotectants. Key words: Bacillus, phosphate solubilization, NaCl, osmoprotectant.

Enhancement of growth and production of polyhydroxyalkanoates by Bacillus subtilis from agro-industrial waste as carbon substrates

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyacids naturally synthesized in bacteria as a carbon reserves. Bacillus subtilis can synthesize and accumulate PHA as carbon source under limiting conditions of nutrients. PHAs are promising candidates for the development of environment-friendly, biodegradable plastics. The aim of the present work was to study the optimization of PHAs using different carbon sources in the media and by using agro-industrial bio-wastes as a replacement for the carbon source. The study also identifies the potential of the strain to produce maximum dry cell weight and PHA from the cheap sources, i.e. waste samples taken from different industries like distillery effluent, sugarcane molasses, milk whey, and paper industry effluent. The use of these low-cost raw materials has the potential to reduce PHA production costs, because the raw material cost contributes a significant part in traditional PHA production processes. On studying the effect of different carbon sources in the medium, it was found that sucrose-supplemented Luria–Bertani medium produced maximum PHA production of 1.58 mg/ml in 72 h constituting 58.59% w/w of the total cell dry mass at a pH value of 7.0. By substituting the carbon source in the medium with biowastes from different industries, the maximal production of PHA was 2.5 mg/ml PHA from 10% sugarcane molasses at the neutral pH range. The PHA produced in all the other industrial wastes was not comparable with that of sugarcane molasses. Hence, it can be concluded that sugarcane molasses can be used as a cheap source of carbon and is economical for the production of PHA on a large-scale.

Effects of blackberry juice on growth inhibition of foodborne pathogens and growth promotion of Lactobacillus

Berries such as blueberry, blackberry and raspberry possess several biological activities including antimicrobial and nutritional effects. In this study, the antimicrobial activities of blackberry (Rubus fruticosus) juice against foodborne pathogens including Listeria monocytogenes, Salmonella Typhimurium and Escherichia coli O157:H7 were investigated. Inhibition of growth of these foodborne pathogens was measured in broth (Luria–Bertani broth for E. coli O157:H7 and S. Typhimurium, and brain heart infusion broth for L. monocytogenes), skim milk and whole milk supplemented with 10% blackberry juice at different time points (0, 24, 48 and 72 h). The effects of blackberry juice on the growth of Lactobacillus casei, Lactobacillus plantarum and Lactobacillus rhamnosus were also investigated in Man–Rogosa–Sharpe (MRS) broth and skim and whole milk supplemented with blackberry juice. The growth of L. monocytogenes, S. Typhimurium and E. coli O157:H7 were significantly inhibited by blackberry juice by 1–3 logs in both milk and broth. We also observed that the growths of Lactobacillus strains were significantly stimulated (1–4 logs CFU/mL) by blackberry juice in both milk and MRS broth. These data clearly demonstrate that diluted blackberry juice can be used as a preservative in food processing and a preventive in foodborne infections as a natural antimicrobial.

Exposure of Escherichia coli O157:H7 to soil, manure, or water influences its survival on plants and initiation of plant defense response

This study evaluated whether growth medium or exposure conditions influence the production of capsular polysaccharides (CPS) by Escherichia coli O157:H7, and whether changes in CPS impact plant defense responses, consequently affecting survival on plants. E. coli O157:H7 grown in Luria–Bertani (LB) broth supplemented with manure extracts showed an approximately 58% increase in CPS production compared to cells grown in LB medium alone. Levels of CPS were significantly higher for E. coli O157:H7 cells exposed to soil or manure extracts as compared to the non-exposed LB cultured control. Arabidopsis thaliana plants expressing β-glucuronidase (GUS) under the control of the β-1,3-glucanase (BGL2) promoter were used to investigate whether E. coli O157:H7 induces defense-related gene expression. Plants inoculated with E. coli O157:H7 grown in LB containing manure extracts or cells exposed to manure extracts exhibited 3-fold and 2-fold lower GUS activity, respectively, suggesting a limited plant defense response compared to plants inoculated with cells grown in LB. On day 5 post inoculation the population of E. coli O157:H7 grown in LB supplemented with manure on plants was significantly greater than the population of E. coli O157:H7 grown in LB medium alone. E. coli O157:H7 cells exposed to soil or manure exhibited greater survival on plants compared to LB-grown E. coli O157:H7. The results of this study underscore the need to consider medium composition and cultural conditions when conducting crop challenge studies.

Flavobacterium limnosediminis sp. nov., isolated from sediment of a freshwater lake

A Gram-stain-negative, rod-shaped, pale yellow, aerobic bacterial strain, JC2902(T), was isolated from a sediment sample of Ungok Lake in Gochang, Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain JC2902(T) belongs to the genus Flavobacterium and forms a distinct phyletic line within a clade containing four recognized species of the genus Flavobacterium. The genomic relatedness between strain JC2902(T) and closely related strains was calculated using average nucleotide identity values of whole genome sequences, which indicated that the new isolate represents a novel genomic species. Through comparison of chemotaxonomic and other phenotypic characteristics between strain JC2902(T) and the type strains of the four phylogenetically related species, a number of characteristics differentiated strain JC2902(T) from the previously described type strains. Differential characteristics of strain JC2902(T) include fatty acid profiles, cellular motility, inability to grow on Luria-Bertani and tripticase soy agar media, and absence of N-acetyl-β-glucosaminidase and flexirubin-type pigments. Based on data from this polyphasic taxonomic study, strain JC2902(T) is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium limnosediminis sp. nov. is proposed. The type strain is JC2902(T) ( = KACC 16937(T) = JCM 18661(T)).

Comparative genomics and transcriptomics analysis of experimentally evolved Escherichia coli MC1000 in complex environments

It has recently become feasible to study the basis and nature of evolutionary changes in bacteria in an experimental setting using defined media. However, assessment of adaptive changes in complex environments has been scarce. In an effort to describe the responses in such environments, we unravel, in a comparative approach, the transcriptional and genetic profiles of 19 Escherichia coli strains that evolved in Luria Bertani medium under three different oxygen regimes over 1000 generations. A positive relationship between upregulation of gene expression and the number of mutations was observed, suggesting that a number of metabolic pathways were activated. Phenotypic polymorphisms were observed in parallel cultures, of which some were related with mutations at the regulatory level. Non-parallel responses were observed at the intrapopulational level, which is indicative of diversifying selection. Parallel responses encompassed transcriptome diversity, and their effects were directly affected by differing genomic backgrounds. A fluctuating selective force produced higher phenotypic diversity compared with constant forces. This study demonstrates how phenotypic innovations may depend on the relationship between genomic changes and local ecological conditions. Using both comparative genomics and transcriptomics approaches, the results help elucidating various adaptive responses in cultures in unexplored complex environments.

Tributyl phosphate biodegradation to butanol and phosphate and utilization by a novel bacterial isolate, Sphingobium sp. strain RSMS

A Sphingobium sp. strain isolated from radioactive solid waste management site (RSMS) completely degraded 7.98g/L of tributyl phosphate (TBP) from TBP containing suspensions in 3days. It also completely degraded 20mM dibutyl phosphate (DBP) within 2days. The strain tolerated high levels of TBP and showed excellent stability with respect to TBP degradation over several repeated subcultures. On solid minimal media or Luria Bertani media supplemented with TBP, the RSMS strain showed a clear zone of TBP degradation around the colony. Gas chromatography and spectrophotometry analyses identified DBP as the intermediate and butanol and phosphate as the products of TBP biodegradation. The RSMS strain utilized both TBP and DBP as the sole source of carbon and phosphorous for its growth. The butanol released was completely utilized by the strain as a carbon source thereby leaving no toxic residue in the medium. Degradation of TBP or DBP was found to be suppressed by high concentration of glucose which also inhibited TBP or DBP dependent growth. The results highlight the potential of Sphingobium sp. strain RSMS for bioremediation of TBP and for further molecular investigation.

Engineering of recombinant Escherichia coli cells co‐expressing poly‐γ‐glutamic acid (γ‐PGA) synthetase and glutamate racemase for differential yielding of γ‐PGA

Poly-γ-glutamic acid (γ-PGA) is a promising environmental-friendly material with outstanding water solubility, biocompatibility and degradability. However, it is tough to determine the relationship between functional synthetic enzyme and the strains' yield or substrate dependency. We cloned γ-PGA synthetase genes pgsBCA and glutamate racemase gene racE from both L-glutamate-dependent γ-PGA-producing Bacillus licheniformis NK-03 and L-glutamate-independent B. amyloliquefaciens LL3 strains. The deduced RacE and PgsA from the two strains shared the identity of 84.5% and 78.53%, while PgsB and PgsC possessed greater similarity with 93.13% and 93.96%. The induced co-expression of pgsBCA and racE showed that the engineered Escherichia coli strains had the capacity of synthesizing γ-PGA, and LL3 derived PgsBCA had higher catalytic activity and enhanced productivity than NK-03 in Luria–Bertani medium containing glucose or L-glutamate. However, the differential effect was weakened when providing sufficient immediateness L-glutamate substrate, that is, the supply of substrate could be served as the ascendance upon γ-PGA production. Furthermore, RacE integration could enhance γ-PGA yield through improving the preferred d-glutamate content. This is the first report about co-expression of pgsBCA and racE from the two Bacillus strains, which will be of great value for the determination of the biosynthetic mechanism of γ-PGA.

Inactivation of the Bacterial RNA Polymerase Due to Acquisition of Secondary Structure by the ω Subunit

The widely conserved ω subunit encoded by rpoZ is the smallest subunit of Escherichia coli RNA polymerase (RNAP) but is dispensable for bacterial growth. Function of ω is known to be substituted by GroEL in ω-null strain, which thus does not exhibit a discernable phenotype. In this work, we report isolation of ω variants whose expression in vivo leads to a dominant lethal phenotype. Studies show that in contrast to ω, which is largely unstructured, ω mutants display substantial acquisition of secondary structure. By detailed study with one of the mutants, ω6 bearing N60D substitution, the mechanism of lethality has been deciphered. Biochemical analysis reveals that ω6 binds to β' subunit in vitro with greater affinity than that of ω. The reconstituted RNAP holoenzyme in the presence of ω6 in vitro is defective in transcription initiation. Formation of a faulty RNAP in the presence of mutant ω results in death of the cell. Furthermore, lethality of ω6 is relieved in cells expressing the rpoC2112 allele encoding β'2112, a variant β' bearing Y457S substitution, immediately adjacent to the β' catalytic center. Our results suggest that the enhanced ω6-β' interaction may perturb the plasticity of the RNAP active center, implicating a role for ω and its flexible state.

Lipid Segregation and Membrane Budding Induced by the Peripheral Membrane Binding Protein Annexin A2*

The formation of dynamic membrane microdomains is an important phenomenon in many signal transduction and membrane trafficking events. It is driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Here we analyzed the ability of one peripherally associated membrane protein, annexin A2 (AnxA2), to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-rich domains in giant unilamellar vesicles (GUVs) of complex lipid composition. AnxA2 is a cytosolic protein that can bind PI(4,5)P2 and other acidic phospholipids in a Ca(2+)-dependent manner and that has been implicated in cellular membrane dynamics in endocytosis and exocytosis. We show that AnxA2 binding to GUVs induces lipid phase separation and the recruitment of PI(4,5)P2, cholesterol and glycosphingolipids into larger clusters. This property is observed for the full-length monomeric protein, a mutant derivative comprising the C-terminal protein core domain and for AnxA2 residing in a heterotetrameric complex with its intracellular binding partner S100A10. All AnxA2 derivatives inducing PI(4,5)P2 clustering are also capable of forming interconnections between PI(4,5)P2-rich microdomains of adjacent GUVs. Furthermore, they can induce membrane indentations rich in PI(4,5)P2 and inward budding of these membrane domains into the lumen of GUVs. This inward vesiculation is specific for AnxA2 and not shared with other PI(4,5)P2-binding proteins such as the pleckstrin homology (PH) domain of phospholipase Cδ1. Together our results indicate that annexins such as AnxA2 can efficiently induce membrane deformations after lipid segregation, a mechanism possibly underlying annexin functions in membrane trafficking.

Ferredoxin Competes with Bacterial Frataxin in Binding to the Desulfurase IscS*

The bacterial iron-sulfur cluster (isc) operon is an essential machine that is highly conserved from bacteria to primates and responsible for iron-sulfur cluster biogenesis. Among its components are the genes for the desulfurase IscS that provides sulfur for cluster formation, and a specialized ferredoxin (Fdx) whose role is still unknown. Preliminary evidence suggests that IscS and Fdx interact but nothing is known about the binding site and the role of the interaction. Here, we have characterized the interaction using a combination of biophysical tools and mutagenesis. By modeling the Fdx·IscS complex based on experimental restraints we show that Fdx competes for the binding site of CyaY, the bacterial ortholog of frataxin and sits in a cavity close to the enzyme active site. By in vivo mutagenesis in bacteria we prove the importance of the surface of interaction for cluster formation. Our data provide the first structural insights into the role of Fdx in cluster assembly.

Improvement of Ni phytostabilization by inoculation of Ni resistant Bacillus megaterium SR28C

The use of metal tolerant plants for the phytostabilization of metal contaminated soil is an area of extensive research and development. In this study the effects of inoculation of Ni-resistant bacterial strains on phytostabilization potential of various plants, including Brassica juncea, Luffa cylindrica and Sorghum halepense, were studied. A Ni-resistant bacterial strain SR28C was isolated from a nickel rich serpentine soil and identified as Bacillus megaterium based on the morphological features, biochemical characteristics and partial 16S rDNA sequence analysis. The strain SR28C tolerated concentrations up to 1200 mg Ni L−1 on a Luria–Bertani (LB) agar medium. Besides, it showed high degree of resistance to various metals (Cu, Zn, Cd, Pb and Cr) and antibiotics (ampicillin, tetracycline, streptomycin, chloramphenicol, penicillin and kanamycin) tested. In addition, the strain bound considerable amounts of Ni in their resting cells. Besides, the strain exhibited the plant growth promoting traits, such as solubilization of phosphate and production of indole-3-acetic acid (IAA) in modified Pikovskayas medium and LB medium, respectively in the absence and presence of Ni. Considering such potential, the effects of SR28C on the growth and Ni accumulation of B. juncea, L. cylindrica and S. halepense, were assessed with different concentrations of Ni in soil. Inoculation of SR28C stimulated the biomass of the test plants grown in both Ni contaminated and non-contaminated soils. Further, SR28C alleviated the detrimental effects of Ni by reducing its uptake and translocation to the plants. This study suggested that the PGPB inoculant due to its intrinsic abilities of growth promotion and attenuation of the toxic effects of Ni could be exploited for phytostabilization of Ni contaminated site.

Stability of Staphylococcus aureus Phage ISP after Freeze-Drying (Lyophilization)

Staphylococcus aureus phage ISP was lyophilized, using an Amsco-Finn Aqua GT4 freeze dryer, in the presence of six different stabilizers at different concentrations. Stability of the lyophilized phage at 4°C was monitored up to 37 months and compared to stability in Luria Bertani broth and physiological saline at 4°C. Sucrose and trehalose were shown to be the best stabilizing additives, causing a decrease of only 1 log immediately after the lyophilization procedure and showing high stability during a 27 month storage period.

Discovery and characterization of a novel ATP/polyphosphate xylulokinase from a hyperthermophilic bacterium Thermotoga maritima

Xylulokinase (XK, E.C. 2.7.1.17) is one of the key enzymes in xylose metabolism and it is essential for the activation of pentoses for the sustainable production of biocommodities from biomass sugars. The open reading frame (TM0116) from the hyperthermophilic bacterium Thermotoga maritima MSB8 encoding a putative xylulokinase were cloned and expressed in Escherichia coli BL21 Star (DE3) in the Luria-Bertani and auto-inducing high-cell-density media. The basic biochemical properties of this thermophilic XK were characterized. This XK has the optimal temperature of 85 °C. Under a suboptimal condition of 60 °C, the k cat was 83 s⁻¹, and the K(m) values for xylulose and ATP were 1.24 and 0.71 mM, respectively. We hypothesized that this XK could work on polyphosphate possibly because this ancestral thermophilic microorganism utilizes polyphosphate to regulate the Embden-Meyerhof pathway and its substrate-binding residues are somewhat similar to those of other ATP/polyphosphate-dependent kinases. This XK was found to work on low-cost polyphosphate, exhibiting 41 % of its specific activity on ATP. This first ATP/polyphosphate XK could have a great potential for xylose utilization in thermophilic ethanol-producing microorganisms and cell-free biosystems for low-cost biomanufacturing without the use of ATP.

Fuel ethanol production using novel carbon sources and fermentation medium optimization with response surface methodology

In this study, ethanol production abilities of the novel carbon sources: sodium and calcium gluconate in different minimal and rich media were compared with glucose using Escherichia coli KO11. The strain produced higher ethanol yield in the rich medium Luria-Bertani (LB) than the other two minimal media: corn steep liquor (CSL) and M9 for two substrates (sodium and calcium gluconate). Additionally, higher ethanol yields were achieved when the strain was grown in LB and M9 medium with calcium gluconate than sodium gluconate, while the ethanol yields were similar when both sodium and calcium gluconate were added into CSL medium respectively. Response surface methodology was used to optimize the fermentation medium components for enhancing ethanol production using strain E. coli KO11 in CSL medium with calcium gluconate as the substrate in batch culture. The concentration of the potassium phosphate buffer is the only significant factor among five factors considered. A quadratic model was developed to describe the relationship between ethanol production and the factors. The optimal conditions predicted for five factors were 14.38 g/L CSL, 0.0398 g/L FeCl3•6H2O, 1.12 g/L MgSO4•6H2O, 15.41 g/L (NH4)2SO4, and 1.58/1.26 g/L KH2PO4/K2HPO4 (2:1 molar ratio). The highest ethanol concentration under optimal conditions was 31.5 g/L, which was 5.6 g/L higher than that from the same fermentation concentration of calcium gluconate in LB media. The high correlation between the predicted and experimental values confirmed the validity of the model.

Screening antibacterial activity of entomopathogenic bacteria isolated from pests of hazelnut

Thirty-seven entomopathogenic bacteria isolated from six common pests of hazelnut in the Black Sea Region of Turkey have been screened for their potential of antibacterial substance production against indicator bacteria by the agar spot assay and well diffusion assay. Results indicated that 13.5% of entomopathogenic bacteria, Pseudomonas fluorescens (Pf-Xd1), Bacillus polymyxa (Bp-Ar2), Bacillus thuringiensis (Bt-Bn1), Serratia marcescens (Sm-Mm3) and Pseudomonas flourescens (Pf-Aa4) isolated from pests of Xyleborus dispar, Anoplus roboris, Balaninus nucum, Melolontha melolontha and Agelastica alni, respectively, showed significant levels of inhibitor activities against indicator bacteria. Well diffusion assay showed that supernatants of Bp-Ar2, Bt-Bn1 and Sm-Mm3 have antibacterial activity, whereas Pf-Xd1 and Pf-Aa4 did not show any activity. Furthermore, the antibacterial activity of the substance produced by the Bp-Ar2 has a narrow spectrum, whereas those of Bt-Bn1 and Sm-Mm3 exhibit broad spectrum. The production of these antibacterial substances were similarly determined at early logarithmic phase in the growth cycle of three bacteria and continued until the beginning of the stationary phase as primer metabolite. In addition, optimal pH (at 7-9 forBt-Bn1 and 5-9 forSm-Mm3), medium (Muller Hinton broth forBt-Bn1 and Luria Bertani broth forSm-Mm3), temperature (25 ◦ Cf or Bt-Bn1 and Sm-Mm3) and production time (24h forBt-Bn1 and 72h forSm-Mm3) of these substances were determined. Our results demonstrate that entomopathogenic bacteria are a potential source of antibacterial substances.