Possible pathways for destruction of polyaromatic hydrocarbons by some oil-degrading bacteria isolated from plant endosphere and rhizosphere

Six strains of oil-degrading bacteria isolated from the endosphere and rhizosphere of plants growing on oil polluted soils of the Irkutsk region were studied to determine the pathways for biodestruction of polyaromatic oil hydrocarbons. All strains were able to efficiently degrade polyaromatic hydrocarbons with the formation of pyrocatechin as a final product; strains 90, 108, and 112 additionally formed protocathechuic acid. The culture broth of the studied strains contained ferulic, n-coumaric, n-oxybenzoic, vanillic, and lilac acids, which probably represent metabolites of cinnamic alcohol, cinnamic aldehyde, and benzoic acid presenting in oil and metabolized by bacteria.     

Phenethyl Alcohol as a Suppressor of the EnvA Phenotype Associated with the envA Gene in Escherichia coli K-12

In Escherichia coli K-12 the envA gene was previously shown to mediate chain formation and a decreased tolerance to several antibacterial agents. Phenethyl alcohol at low concentrations has now been found to increase the tolerance to actinomycin D, ampicillin, rifampin, and gentian violet in strains containing envA. The increased tolerance to gentian violet was correlated to a decreased uptake of the dye. A phenotype suppression of chain formation and colony morphology in envA mutants was also obtained. Except for an increase in palmitic acid, chemical analysis revealed no differences between an envA and its wild-type strain in the lipopolysaccharide part of the envelope. However, a decrease in the amount of phosphatidylglycerol and a C18: 1 fatty acid was observed in the extractable lipids of a strain containing envA. Growth in the presence of phenethyl alcohol reversed the changes in fatty acid and the phospholipid composition. Phenethyl alcohol was found to cause an immediate but transient inhibition of ribonucleic acid synthesis. It is suggested that this inhibition affects the penetrability barrier of the outer cell envelope layers in strains containing envA.     

A rapid screening procedure to identify mini-Tn10 insertion mutants of Escherichia coli K-12 with altered adhesion properties

Abstract A rapid screening procedure was developed for detection of Escherichia coli mutants with altered adhesion abilities using polystyrene 96-well microtiter plates as attachment surfaces. During this assay, bacterial strains grew and adhered simultaneously, and attached cells were measured after crystal violet staining. Starting with a total of 7000 W3110::Tn10 insertion mutants of E. coli K-12 W3110, 50 adhesion-deficient mutants were isolated which showed less than 40% attachment, and 22 mutants were found with an attachment of 40–75%. Motility assays were performed on these 72 mutants, and 34 displayed altered motility.     

The effect of aromatic compounds on the work of activated sludge.

The resistance of bacteria occurring in activated sludge purifying refining-petrochemical wastewaters to phenol, cresol and pirocatechin as well as the possibility of the purification of synthetic wastewaters carrying high concentrations of these compounds by sludge composed of resistant strains was studied. The most toxic of the studied compounds was phenol. Six out of 29 strains were resistant to high concentration of phenol (1000 mg/l). Activated sludge synthesized from strains resistant to phenol, cresol and pyrocatechin was tolerant to high concentrations of these compounds. Worsening of the work of the sludge, expressed by decrease of GOD, was observed at the concentrations of phenol, cresol and pyrocatechin of 2000, 400 and 300 mg/l, respectively.     

Rapid methods to assess sanitizing efficacy of benzalkonium chloride to Listeria monocytogenes biofilms

Different methods were used to investigate biofilm growth including crystal violet staining, ATP bioluminescence and total viable count. Seven strains of Listeria monocytogenes and 8 of their derivative strains were screened for their capacity to form biofilms. Both adaptation to benzalkonium chloride (BC) and curing of plasmids did not significantly affect biofilm-forming ability. The strains of L. monocytogenes belonging to serotype 1 formed biofilms significantly better as compared to serotype 4 (P=0.0003). To estimate the efficacy of BC for biofilm elimination the best and the poorest biofilm-formers were used (C719 and LJH 381). It was observed that, L. monocytogenes strain C719 in biofilms is at least 1000 times more resistant to BC than in planktonic form. Cells present in biofilms were shown to recover and grow after BC treatment thus providing a source of recontamination. It was shown that ATP bioluminescence provides good correlation with bacterial counts of L. monocytogenes in biofilms. Staining with crystal violet, on the contrary, did not correlate with bacterial growth in biofilms in the presence of high concentrations of BC but provided information on the concentration of bacterial cells, both live and dead, attached to the surface. ATP bioluminescence was found to be a reliable method for rapid estimation of the efficacy of sanitizers for biofilm disinfection. Crystal violet staining, on the other hand, was shown to be a suitable method to monitor removal of biofilms. Our investigation showed that for Listeria biofilms concentrations of BC higher then 10 mg/ml should be applied for at least 30 min to kill almost all the live cells in biofilms. However, this concentration was still not enough to remove biofilms from the surface of plastic.     

Simultaneous detection of toxin A and toxin B genetic determinants of Clostridium difficile using the multiplex polymerase chain reaction.

A multiplex polymerase chain reaction was developed to simultaneously detect the presence of toxin A and toxin B genes of Clostridium difficile. A 1050-bp fragment of the toxin B gene and a 1217-bp fragment of the toxin A gene were amplified from 42 toxic strains of C. difficile; however, from 10 nontoxic strains the toxin gene fragments were not amplified; these data demonstrate that this multiplex polymerase chain reaction procedure can be used to differentiate between toxic and nontoxic strains. This sensitive and specific multiplex polymerase chain reaction for C. difficile toxins may prove to be a valuable diagnostic procedure.     

Effect of gentian violet on the growth of the N and T RFLP types of the Aspergillus niger aggregate

Taxa included in the Aspergillus niger aggregate are difficult to distinguish by phenotypic characterization. In this work, the effect of gentian violet on the growth of the N and T RFLP types of A. niger aggregate strains has been investigated. In total, 105 strains from different sources and origins, including reference cultures and field isolates were studied. Type N and T RFLP patterns, ochratoxin A production and the effect of different concentrations of gentian violet on the growth were determined in these strains. Forty nine strains belonged to the N type and 56 strains to the T type. Sixteen out of the 105 strains assayed were OTA producers. All the OTA-producing species belonged to the RFLP type N and none of the T type strains was able to produce OTA. Approximately 90% of the N type strains grew in the presence of 25 ppm of gentian violet. Only five N type strains did not grow on this medium. One of these strains was A. niger ATCC 22343, a well documented induced mutant strain and the remaining four strains belonged to the new species A. brasiliensis. On the contrary, all the T type strains failed to grow on this medium after 3 days of incubation (sensitivity 89.79%; specificity 100%). The use of growth in gentian violet as an additional character for classification and identification purposes in this taxonomic group may be useful because no phenotypic methods have yet been found that can distinguish between these species.     

Expression of a functional Drosophila melanogaster CMP-sialic acid synthetase. Differential localization of the Drosophila and human enzymes

CMP-N-acetylneuraminic acid is a critical metabolite in the generation of glycoconjugates that play a role in development and other physiological processes. Whereas pathways for its generation are firmly established in vertebrates, the presence and function of the relevant synthetic enzyme in insects and other protostomes is unknown. In this study, we characterize the first functional CMP-sialic acid synthase (DmCSAS) from any protostome lineage expressed from a D. melanogaster cDNA clone. Homologous genes were subsequently identified in other insect species. The gene is developmentally regulated, with expression first appearing at 12-24 h of embryogenesis, low expression through larval and pupal stages, and greatly enriched expression in the adult head, suggesting a possible role in the central nervous system. Activity of the enzyme was verified by an increase in in vitro and in vivo CMP-N-acetylneuraminic acid levels when expressed in a heterologous host. Unlike all known vertebrate CMP-sialic acid synthetase (CSAS) proteins that localize to the nucleus, the D. melanogaster CSAS protein was targeted to the Golgi compartment when expressed in both heterologous mammalian and insect cell lines. Replacement of the N-terminal leader sequence of DmCSAS with the human CSAS N-terminal sequence resulted in the redirection of the chimeric CSAS protein to the nucleus but with a concomitant loss of enzymatic activity. The localization of CSAS orthologs to different intracellular organelles represents, to our knowledge, the first example of differential protein targeting of orthologs in eukaryotes and reveals how the sialylation pathway diverged during the evolution of protostomes and deuterostomes.     

hrp genes of Erwinia chrysanthemi 3937 are important virulence factors

We developed improved virulence assays for Erwinia chrysanthemi 3937 on African violet varieties and devised a new method for the construction of precise bacterial gene knockouts. These methods were tested by constructing mutations in genes suspected to be involved with plant interactions. The virulence of the hrpG and hrcC mutant strains (both gene products presumed to be involved in protein secretion) was greatly reduced on leaves of semitolerant African violet varieties. An hrpN mutant strain produced delayed symptoms on African violet leaves and an hrpN delta pel (delta pel = five major pectate lyase genes deleted) double mutant was nonpathogenic. The hrcC and hrpG mutants did not produce a rapid hypersensitive response (HR) in tobacco, unlike the wild-type bacterium, and the hrpN mutant gave a reduced HR. The results, therefore, establish the importance of hrp genes in the virulence of E. chrysanthemi and their ability to elicit HR on nonhosts. The data also suggest that other effector proteins secreted by the Hrp system are required for full virulence and HR elicitation.     

Rapid methods to assess sanitizing efficacy of benzalkonium chloride to Listeria monocytogenes biofilms

Different methods were used to investigate biofilm growth including crystal violet staining, ATP bioluminescence and total viable count. Seven strains of Listeria monocytogenes and 8 of their derivative strains were screened for their capacity to form biofilms. Both adaptation to benzalkonium chloride (BC) and curing of plasmids did not significantly affect biofilm-forming ability. The strains of L. monocytogenes belonging to serotype 1 formed biofilms significantly better as compared to serotype 4 (P = 0.0003). To estimate the efficacy of BC for biofilm elimination the best and the poorest biofilm-formers were used (C719 and LJH 381). It was observed that, L. monocytogenes strain C719 in biofilms is at least 1000 times more resistant to BC than in planktonic form. Cells present in biofilms were shown to recover and grow after BC treatment thus providing a source of recontamination. It was shown that ATP bioluminescence provides good correlation with bacterial counts of L. monocytogenes in biofilms. Staining with crystal violet, on the contrary, did not correlate with bacterial growth in biofilms in the presence of high concentrations of BC but provided information on the concentration of bacterial cells, both live and dead, attached to the surface. ATP bioluminescence was found to be a reliable method for rapid estimation of the efficacy of sanitizers for biofilm disinfection. Crystal violet staining, on the other hand, was shown to be a suitable method to monitor removal of biofilms. Our investigation showed that for Listeria biofilms concentrations of BC higher then 10 mg/ml should be applied for at least 30 min to kill almost all the live cells in biofilms. However, this concentration was still not enough to remove biofilms from the surface of plastic.     

The structure of a local population of phytopathogenic Pseudomonas brassicacearum from agricultural soil indicates development under purifying selection pressure

Among the isolates of a bacterial community from a soil sample taken from an agricultural plot in northern Germany, a population consisting of 119 strains was obtained that was identified by 16S rDNA sequencing and genomic fingerprinting as belonging to the recently described species Pseudomonas brassicacearum. Analysis of the population structure by allozyme electrophoresis (11 loci) and random amplified polymorphic DNA–polymerase chain reaction (RAPD–PCR; four primers) showed higher resolution with the latter method. Both methods indicated the presence of three lineages, one of which dominated strongly. Stochastic tests derived from the neutral theory of evolution (including Slatkin's exact test, Watterson's homozygosity test and the Tajima test) indicated that the population had developed under strong purifying selection pressure. The presence of strains clearly divergent from the majority of the population can be explained by in situ evolution or by influx of strains as a result of migration or both. Phytopathogenicity of a P. brassicacearum strain determined with tomato plants reached the level obtained with the type strain of the known pathogen Pseudomonas corrugata . The results show that a selective sweep was identified in a local population. Previously, a local selective sweep had not been seen in several populations of different bacterial species from a variety of environmental habitats.     

Further study of the genetic toxicity of gentian violet.

The genetic toxicity of gentian violet was studied with the Ames and the Rosenkranz bacterial assays as well as the cytogenetic assays (Chinese hamster ovary cells in vitro in the presence of rat-liver S-9 fractions, the chicken-embryo and mouse-bone-marrow cells in vivo). Gentian violet was found to be toxic but not mutagenic in the Ames assay. However, it was active in the Rosenkranz assay causing reparable DNA damage. The presence of S-9 in the in vitro cytogenetic assay and in the bacterial assays showed that the activity of gentian violet could be reduced or eliminated. In the in vivo assays, gentian violet was not clastogenic and failed to induce sister-chromatid exchanges. However, gentian violet proved to be highly toxic to growing chick embryos at high dosage and depressed mitotic activities in mouse bone marrow after prolonged treatment. Our study suggested that gentian violet can be inactivated by the liver detoxification system. However, it is potentially hazardous to cells that are exposed to the dye directly (e.g. skin epithelium and cell lining of the gastrointestinal tract).     

Diversity among aromatic hydrocarbon-degrading bacteria and their meta-cleavagegenes

Sixty-one strains of bacteria capable of growth on 4-methyl benzoic acid (29 isolates) ornaphthalene (32 isolates) as the sole source of carbon and energy were isolated from sedimentsand water samples from the River Tyne, UK. Random amplification of polymorphic DNA fromgenomic DNA extracted from the different strains demonstrated that 14 of the 4-methylbenzoate-degrading isolates were unique and the remainder fell into seven groups containing twoor three isolates that produced identical banding patterns. Thirteen of the naphthalene-degradingisolates were unique and nine groups with two or three identical representatives encompassed allother isolates. Screening of the bacterial strains for the presence of genes homologous to xylE , nahC and bphC by polymerase chain reaction and dot blot hybridizationdemonstrated that most strains harboured xylE - and/or nahC -like genes and only asingle isolate was found that did not harbour any of these genes. None of the isolates harboured bphC -like genes. It was concluded that, while considerable diversity existed in host strainsisolated using a single simple enrichment procedure, the extradiol dioxygenase genes involved inaromatic ring cleavage, present in these strains, were conserved to a considerable degree.     

Development of multiplex PCR for fast detection of Paenibacillus Larvae in putrid masses and in isolated bacterial colonies

The present study was performed to develop a fast and sensitive multiplex polymerase chain reaction protocol for routine diagnostics of American foulbrood. A new approach for detection of Paenibacillus larvae in putrid masses was described. Forty five samples of putrid masses obtained from bee combs suspicious for American foulbrood, a reference strain Paenibacillus larvae (NBIMCC 8478), clinical isolates and 4 strains of closely related bacterial species were included in experiments. Bacterial colonies?? DNA was isolated by heat and centrifugation method (standard procedure) and with prepGem commercial kit. DNA from putrid masses was isolated by standard and modified procedure. Three pairs of primers specific for 16S rRNA and one pair specific for 35 kDa metalloproteinase genes of Paenibacillus larvae were tested as single pair and in different combinations as multiplex PCR. The sensitivity of the multiplex PCR protocol for putrid masses, developed in study was 100%, versus 45.2% for the standard protocol. The developed multiplex PCR protocol could be successfully used for rapid and specific detection of Paenibacillus larvae in both putrid masses and isolated bacterial colonies.     

A Simple Method for Isolation of Lipopolysaccharides from Pseudomonas aeruginosa and Some Other Bacterial Strains

A simple procedure for the isolation of bacterial lipopolysaccharides was developed. The method consists of the following three steps; recovery of lipopolysaccharides in an insoluble form together with denatured proteins in a hot MgCl₂-Triton X-100 solution (step 1), solubilization with EDTA-Triton X-100 (step 2) and precipitation with MgCl₂ (step 3). A good yield of lipopolysaccharides was obtained with most of the Pseudomonas strains tested. Although the yield was lower with strains belonging to Salmonella and Escherichia, irrespective of whether they are smooth-type or rough-type strains, the amounts obtained from 100 mg of cells as dry weight were sufficient for detailed chemical characterization. The gel-electrophoretic profiles of the lipopolysaccharides prepared by the present method were essentially the same as those of the lipopolysaccharides prepared by the conventional phenol-water method. The method allowed us to prepare lipopolysac-charides from a large number of bacterial strains in one day.     

Identification and characterization of Cr-, Cd-, and Ni-tolerant bacteria isolated from mine tailings

Eleven bacterial strains were isolated from soil samples collected from mine tailings. Bacterial strains were checked for tolerance against heavy metals (Cr, Cd, Ni), using the agar dilution method. All the strains showed multiple tolerances against heavy metals, but the most promising results appeared in strains BCr3, BCd33, and BNi11: they were tolerant to 15 mM of Cr⁶⁺, 7.5 mM of Cd²⁺, and 10 mM of Ni²⁺, respectively. The effect of heavy metals on bacterial growth was tested together with their ability to grow in different pH, NaCl, and temperature values. Bacterial isolates grew well between pH 7.5 and 8.5. The optimum temperature for maximum growth was between 35 and 37°C, and no significant change in bacterial growth was observed in the presence of 2% NaCl. In addition, the bioaccumulation potential of bacterial strains was investigated. Bacterial strains BCr3, BCd33, and BNi11 showed high bioaccumulation ability of Cr (68.7%), Cd (72.4%), and Ni (69.8%), respectively. All bacterial isolates were identified by 16S rRNA gene sequencing. Analysis of plasmid content revealed that all bacterial isolates contained a single plasmid. Further, polymerase chain reaction together with DNA sequence analysis was used to screen all bacterial strains for the presence metal tolerance genes (czcD, chrA, chrB, czcB, czcC, nccA, and cadA) on both plasmid and chromosomal genomes.     

Rapid semi-quantitative fluorimetric determination of citrinin in fungal cultures isolated from cheese and cheese factories

A new rapid semi-quantitative fluorimetric assay for citrinin production testing in mould cultures has been developed. The chemical structure of the citrinin makes it a weak native fluorophore. This fluorescence can be strongly enhanced in an acidic environment. A standard curve where the concentration of HCl needed to show the yellow fluorescence signal of different concentrations of citrinin was established, thus providing a semi-quantitative method to prove the capacity of toxin production of fungal cultures. Two Penicillium strains from the Spanish National Collection of Type Cultures, were studied for the toxin production on YES broth at 25°C for 21 d. The culture was assayed daily for the presence/absence and quantification of citrinin by adding the HCl concentration set, and also quantified by RP-HPLC as a confirmation procedure. Experiments demonstrate that 5 d are necessary to show the presence of citrinin. As an illustration, a total of 48 strains of Penicillium isolated from cheese and cheese factories were analysed with the proposed method.     

Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR

A real time polymerase chain reaction (PCR) assay was developed and evaluated to detect the presence of the thermostable direct hemolysin gene (tdh), a current marker of pathogenicity in Vibrio parahaemolyticus. The real time PCR fluorogenic probe and primer set was tested against a panel of numerous strains from 13 different bacterial species. Only V. parahaemolyticus strains possessing the tdh gene generated a fluorescent signal, and no cross-reaction was observed with tdh negative Vibrio or non-Vibrio spp. The assay detected a single colony forming unit (CFU) per reaction of a pure culture template. This sensitivity was achieved when the same template amount per reaction was tested in the presence of 2.5 microl of a tdh negative oyster:APW enrichment (oyster homogenate enriched in alkaline peptone water overnight at 35 degrees C). This real time technique was used to test 131 oyster:APW enrichments from an environmental survey of Alabama oysters collected between March 1999 and September 2000. The results were compared to those previously obtained using a streak plate procedure for culture isolation from the oyster:APW enrichment combined with use of a non-radioactive DNA probe for detection of the tdh gene. Real time PCR detected tdh in 61 samples, whereas the streak plate/probe method detected tdh in 15 samples. Only 24 h was required for detection of pathogenic V. parahaemolyticus in oyster:APW enrichments by real time PCR, whereas the streak plate/probe method required 3 days and was more resource intensive. This study demonstrated that real time PCR is a rapid and reliable technique for detecting V. parahaemolyticus possessing the tdh gene in pure cultures and in oyster enrichments.     

Fluorogenic assays for immediate confirmation of Escherichia coli.

Rapid assays for Escherichia coli were developed by using the compound 4-methylumbelliferone glucuronide (MUG), which is hydrolyzed by glucuronidase to yield a fluorogenic product. The production of glucuronidase was limited to strains of E. coli and some Salmonella and Shigella strains in the family Enterobacteriaceae. For immediate confirmation of the presence of E. coli in most-probable-number tubes, MUG was incorporated into lauryl tryptose broth at a final concentration of 100 micrograms/ml. Results of both the presumptive test (gas production) and the confirmed test (fluorescence) for E. coli were obtained from a variety of food, water, and milk samples after incubation for only 24 h at 35 degrees C. Approximately 90% of the tubes showing both gas production and fluorescence contained fecal coliforms (they were positive in EC broth incubated at 45 degrees C). Few false-positive reactions were observed. The lauryl tryptose broth-MUG-most-probable-number assay was superior to violet red bile agar for the detection of heat- and chlorine-injured E. coli cells. Anaerogenic strains produced positive reactions, and small numbers of E. coli could be detected in the presence of large numbers of competing bacteria. The fluorogenic assay was sensitive and rapid; the presence of one viable cell was detected within 20 h. E. coli colonies could be distinguished from other coliforms on membrane filters and plates of violet red bile agar if MUG was incorporated into the culture media. A rapid confirmatory test for E. coli that is amenable to automation was developed by using microtitration plates filled with a nonselective medium containing MUG. Pure or mixed cultures containing E. coli produced fluorescence within 4 h (most strains) to 24 h (a few weakly positive strains).     

A combination of assays reveals biomass differences in biofilms formed by Escherichia coli mutants

Aims:  The aim of this study was to develop an assay system that can quantify the amount of biomass in biofilms formed by different isogenic mutants of an Escherichia coli K-12 strain.
Methods and Results:  The reported assay, which is based on the BacTiter-Glo™ assay from Promega, uses bioluminescence to detect the intracellular concentration of ATP, which correlates with viable bacterial cell numbers. The quantitative data obtained with this ATP assay were compared to those obtained with the conventional crystal violet assay. As a qualitative control, scanning electron microscopy was performed.
Conclusions:  The ATP assay, the crystal violet assay and scanning electron microscopy yielded similar results for six of the eight strains tested. For the remaining two strains, the images from the scanning electron microscopy confirmed the results from the ATP assay.
Significance and Impact of the Study:  The ATP assay, in combination with other quantitative and qualitative assays, will allow us to perform genetic studies on the regulatory network that underlies the early steps in E. coli biofilm formation.