Daptomycin (DAP) is a cyclic lipopeptide that disrupts the functional integrity of the cell membranes of Gram-positive bacteria in a Ca2؉ -dependent manner. Here we present genetic, genomic, and phenotypic analyses of an evolved DAP-resistant isolate, Dap R 1, from the model bacterium Bacillus subtilis 168. Dap R 1 was obtained by serial passages with increasing DAP concentrations, is 30-fold more resistant than the parent strain, and displays cross-resistance to vancomycin, moenomycin, and bacitracin. Dap R 1 is characterized by aberrant septum placement, notably thickened peptidoglycan at the cell poles, and pleiotropic alterations at both the transcriptome and proteome levels. Genome sequencing of Dap R 1 revealed 44 point mutations, 31 of which change protein sequences. An intermediate isolate that was 20-fold more resistant to DAP than the wild type had only three of these point mutations: mutations affecting the cell shape modulator gene mreB, the stringent response gene relA, and the phosphatidylglycerol synthase gene pgsA. Genetic reconstruction studies indicated that the pgsA(A64V) allele is primarily responsible for DAP resistance. Allelic replacement with wild-type pgsA restored DAP sensitivity to wild-type levels. The additional point mutations in the evolved strain may contribute further to DAP resistance, serve to compensate for the deleterious effects of altered membrane composition, or represent neutral changes. These results suggest a resistance mechanism by which reduced levels of phosphatidylglycerol decrease the net negative charge of the membrane, thereby weakening interaction with the positively charged Ca 2؉ -DAP complex.Daptomycin (DAP) is a cyclic lipopeptide antibiotic used to treat complicated skin and skin structure infections caused by Staphylococcus aureus or enterococci. In addition, it has been approved to treat S. aureus-induced bacteremia and infective endocarditis (21), and animal model studies suggest that it may be a useful alternative for treatment of inhalational anthrax (26). The mechanism of action involves the calcium-dependent insertion of DAP into the bacterial membrane, followed by depolarization of the membrane and extrusion of potassium ions, leading to arrest of macromolecular synthesis and to cell death (49, 51).The introduction of new antibacterial compounds seems to be followed inevitably by the emergence of resistant isolates. It is estimated that over 1 million patients have been treated with DAP (J. Silverman, personal communication). According to the SENTRY Antimicrobial Surveillance Program in the United States for the years 2002 to 2010, 99.9% of methicillin-resistant S. aureus (MRSA) isolates treated with DAP had an MIC of 1.0 g/ml or lower, with only a slight increase of MIC over time (47; http://www.gp-pathogens.com/data/default.cfm).Previous studies to define mechanisms of resistance to DAP were performed on clinical isolates and by in vitro selection (22,31). After serial passages with increasing DAP concentrations, Friedman et al. characterized three...

Plagiodera versicolora (Laicharting, 1781) (Coleoptera: Chrysomelidae) is an important forest pest which damages many trees such as willow, poplar, and hazelnut. In order to find new microbes that can be utilized as a possible microbial control agent against this pest, we investigated the culturable bacterial flora of it and tested the isolated bacteria against P. versicolora larvae and adults. We were able to isolate nine bacteria from larvae and adults. The isolates were characterized using a combination of morphological, biochemical, and physiological methods. Additionally, we sequenced the partial sequence of the 16S rRNA gene to verify conventional identification results. Based on characterization studies, the isolates were identified as Staphylococcus sp. Pv1, Rahnella sp. Pv2, Rahnella sp. Pv3, Rahnella sp. Pv4, Rahnella sp. Pv5, Pantoea agglomerans Pv6, Staphylococcus sp. Pv7, Micrococcus luteus Pv8, and Rahnella sp. Pv9. The highest insecticidal activity against larvae and adults was obtained from M. luteus Pv8 with 50 and 40 % mortalities within 10 days after treatment, respectively. Extracellular enzyme activity of the bacterial isolates such as amylase, proteinase, lipase, cellulose, and chitinase was also determined. Consequently, our results show that M. luteus Pv8 might be a good candidate as a possible microbial control agent against P. versicolora and were discussed with respect to biocontrol potential of the bacterial isolates.

Entomopathogenic fungi (EPFs) play an important role for regulating insect pest populations, as they exist in many different ecosystems. Within these fungi, Beauveria and Metarhizium spp. genera include species that are the most commercially important. The aim of this study was to determine the diversity and distribution of Beauveria and Metarhizium spp. in walnut fields of Kırşehir, Turkey, and to evaluate their pathogenicity against Cydia pomonella (L.) (Lepidoptera: Tortricidae). To perform this, 90 soil samples were collected from walnut fields where Beauveria and Metarhizium spp. were isolated from these soils, using selective media. The isolated 40 fungi were characterized based on their morphological and molecular characteristics including Bloc and β-tubulin gene sequences. Also, eight selected fungi were tested against C. pomonella larvae under laboratory conditions. The fungal isolates were identified as Beauveria pseudobassiana (15), B. bassiana (12), Metarhizium robertsii (11), and M. brunneum (13). M. brunneum ELA-38 caused 83% mortality within 2 weeks after application of 1 × 10 8 conidia/ml. Consequently, Beauveria and Metarhizium spp. are the common component of the soils collected from walnut fields and some of fungi obtained from this work might be beneficial in the future biological control programs of C. pomonella.

Fifty-five ampicillin-resistant (Amp r ) Escherichia coli strains were isolated from 51 drinking water points in Rize region containing abundant fresh water sources in Turkey during the years 2000 to 2002 and from January to February 2004. The large number of organisms (nearly 57%) exhibited resistance to three or more antibiotics commonly used in human and veterinary medicine. These strains displayed a multiresistant phenotype. Nearly half of the strains (27%) expressed resistance to ceftazidime, but these strains were not an extended-spectrum β-lactamase-producer according to the results of double-disk synergy test. All isolates were then screened for the carriage of TEM-type β-lactamase gene (bla TEM ) by polymerase chain reaction. TEM-type β-lactamase genes were found in six (11%) isolates. Sequence analysis showed TEM-1 type genes. However, isoelectric focusing analysis did not confirm the production of TEM-1 type β-lactamase except for one strain. Conjugation experiments showed that resistance to ampicillin, tetracycline or trimethoprim/sulfamethoxazole was transferable in six (11%) isolates. Emergence of transferable antibiotic resistance and bla TEM-1 gene in E. coli strains from public drinking waters possesses a significant public health risk.

AbstractToxoptera aurantii (Homoptera: Aphididae) is one of the most important pests of many agricultural plants such as camellia, cocoa and coffee worldwide. The culturable bacterial flora of the pest was determined to find new microbes that can be used as biocontrol agent against T. aurantii. A total of six bacteria were isolated and identified by a variety of morphological, physiological, biochemical and molecular tests. In addition, an approximately 1,150 bp fragment of the 16S rRNA gene region was sequenced to verify isolate identification. According to the identification studies, the isolates were identified as Bacillus tequilensis Cb1, Chryseobacterium stagni Cb2, Pseudomonas flourescens Cb3, Rahnella aquatilis Cb4, Staphylococcus sp. Cb5 and Cb6. Pathogenicity of the bacterial isolates were carried out against the last instar nymphs of T. aurantii. The highest activity was obtained from Pseudomonas flourescens Cb3 with 50% mortality within 10 days after application (p<0.05). Mortalities of other treatments ranged from 6.6 to 20%. The results presented here show that Pseudomonas flourescens Cb3 appears to be a significant candidate as a possible biocontrol agent against T. aurantii and should be beneficial in the future biocontrol programs of the pest.

This study was performed to determine the antibacterial activity of methanol extracts of 23 bryophyte species against Paenibacillus larvae isolates that cause American foulbrood diseases in honeybee larvae. The honey and larva samples were collected from nine different locations of Rize in Turkey. A total of 22 gram-positive spore-forming bacteria were isolated from the larva and honey samples. According to the results of morphological, biochemical, and molecular (16S rRNA gene sequencing) tests, 10 isolates of the 22 gram-positive spore-forming bacteria were identified as P. larvae. A total of 10 bryophyte species (Polytrichum formasum, Polytrichum commune, Calliergonella cuspitada, Calliergonella lindbergi, Metzgeria conjugata, Isothecium alopecuroides, Syntrichia calcicola, Syntrichia intermedia, Tortella densa, and Grimmia alpestris) among 23 bryophytes showed good antimicrobial activity against P. larvae isolates according the results of agar-well diffusion method and minimal inhibition concentration experiments.

D proteins from Aeribacillus pallidus AC6 and Bacillus subtilis bound specifically, albeit weakly, to promoter DNA even in the absence of core RNA polymerase. Binding required a conserved CG motif within the ؊10 element, and this motif is known to be recognized by region 2.4 and critical for promoter activity.In the course of efforts to define gene expression determinants from the thermophilic bacterium Aeribacillus pallidus AC6 (2, 18), we identified flagellin (Hag) as being among the most highly expressed proteins in this strain. To determine the basis for high-level Hag expression, we isolated and sequenced the hag gene and identified and expressed the protein required for its expression in The resulting peptide sequences displayed high similarity to B. subtilis flagellin (NAQDGISLIQTAEGALTETHAILQR had 96% identity with amino acids [aa] 65 to 89 and LEHTINNL GTSAENLTAAESR had 85% identity with aa 242 to 262). Two degenerate primers (FlaF1 and FlaR1) were used to amplify the flagellin gene (hag) from A. pallidus AC6 chromosomal DNA. An ϳ450-bp product was cloned into pGEM-T Easy (Promega) for DNA sequencing. The remainder of hag and its upstream region were obtained by inverse PCR. The 828-bp hag gene encodes a 275-amino-acid (29.7-kDa) protein having 63% identity with B. subtilis Hag and is preceded by a typical D promoter. To identify sigD, two degenerate primers were used to amplify a PCR fragment which was cloned into the pGEM-T Easy vector system and sequenced. The flanking portions of sigD were obtained by inverse PCR, and the gene was sequenced. The 771-bp sigD gene encodes a 256-amino-acid (28.7-kDa) protein having 67% overall identity with D Bs , with the highest levels of similarity concentrated in conserved regions 2 and 4, known to mediate promoter recognition.A. pallidus AC6 flagellin is expressed from a D -dependent promoter. Transcription of hag initiates from a canonical Ddependent promoter at a G residue 79 bp upstream of the start codon (Fig. 1). Analysis of hag::lacZ fusions integrated into B. subtilis CU1065 and HB4035 (sigD::kan) indicated that activity was D dependent and highest at late logarithmic phase, as previously reported for B. subtilis (19). Optimal promoter activity required an AT-rich region just upstream of the Ϫ35 element (Table 1), which has similarity with the upstream promoter (UP) element previously described for B. subtilis hag (6). Sequence inspection suggests that high-level Hag expression may also benefit from a strong ribosome-binding site and stabilization of the mRNA by a 5Ј hairpin sequence (24). Purification D of A. pallidus and B. subtilis and reconstitution of D RNAP. D proteins from A. pallidus AC6 and B. subtilis were expressed under T7 RNA polymerase (RNAP) control in Escherichia coli BL21(DE3)/pLysS (Novagen) by using pECG1 and pECB1 (Table 2). For purification, inclusion bodies were solubilized with Sarkosyl (1), refolded, and purified using DEAE-Sepharose and heparin-Sepharose chromatography as described previously (9). B. subtilis core RNAP was purified...

Symbiotic bacteria associated with insects play important roles in different physiological processes such as digestion, insect behavior, defense and providing essential nutrition in insect gut. In addition, these bacteria can be used in biocontrol of insect pests using genetic engineering techniques. The first step is to isolate and identify symbiotic bacteria from insects to elucidate their roles, and to use in the development of transgenic strains. For this purpose, we isolated and characterized the bacterial isolates from stored product pests using a combination of conventional tests and 16S rRNA sequence analysis. The bacterial flora of Callosobruchus maculatus included Bacillus pumilus, Staphylococcus sp. and Pantoea sp. Acanthoscelides obtectus flora included Staphylococcus kloosii, Staphylococcus sp., S. saprophyticus and Enterococcus faecalis. The internal flora of Sitotroga cerealella included Staphylococcus succinus, Enterococcus sp. and Staphylococcus sp. Finally, Phthorimaea operculella flora included Bacillus sp., S t a p h y l o c o c c u s s c i u r i , E n t e ro c o c c u s m u n d t i i , E. casseliflavus, Alcaligenes faecalis, Enterobacter sp., Pantoea agglomerans and Pseudomonas fluorescens.