Genomic and biotechnological potential of a novel oil-degrading strain Enterobacter kobei DH7 isolated from petroleum-contaminated soil.

In this study, a novel oil-degrading strain Enterobacter kobei DH7 was isolated from petroleum-contaminated soil samples from the industrial park in Taolin Town, Lianyungang, China. The whole genome of the strain was sequenced and analyzed to reveal its genomic potential. The oil degradation and growth conditions including nitrogen, and phosphorus sources, degradation cycle, biological dosing, pH, and oil concentration were optimized to exploit its commercial application. The genome of the DH7 strain contains 4,705,032 bp with GC content of 54.95% and 4653 genes. The genome analysis revealed that there are several metabolic pathways and enzyme-encoding genes related to oil degradation in the DH7 genome, such as the paa gene cluster which is involved in the phenylacetic acid degradation pathway, and complete degradation pathways for fatty acid and benzoate, genes related to chlorinated alkanes and olefins degradation pathway including adhP, frmA, and adhE, etc. The strain DH7 under the optimized conditions has demonstrated a maximum degradation efficiency of 84.6% after 14 days of treatment using synthetic oil, which comparatively displays a higher oil degradation efficiency than any Enterobacter species known to date. To the best of our knowledge, this study presents the first-ever genomic studies related to the oil degradation potential of any Enterobacter species. As Enterobacter kobei DH7 has demonstrated significant oil degradation potential, it is one of the good candidates for application in the bioremediation of oil-contaminated environments.

Zinc solubilizing Bacillus sp (SS9) and Enterobacter sp (SS7) promote mung bean (Vigna radiata L.) growth, nutrient uptake and physiological profiles.

Zinc (Zn) is a crucial micronutrient required for optimum plant growth. Zn-solubilizing bacteria (ZSB) are potential alternatives for Zn supplementation and convert applied inorganic Zn to available forms. In this study, ZSB were isolated from the root nodules of wild legumes. From a set of 17 bacteria, the isolates SS9 and SS7 were found to be efficient in tolerating 1 g (w/v) Zn. The isolates were identified as Bacillus sp (SS9, MW642183) and Enterobacter sp (SS7, MW624528) based on morphology and 16S rRNA gene sequencing. The screening of PGP bacterial properties revealed that both isolates possessed production of indole acetic acid (50.9 and 70.8 µgmL-1), siderophore (40.2% and 28.0%), and solubilization of phosphate and potassium. The pot study experiment in the presence and absence of Zn revealed that the Bacillus sp and Enterobacter sp inoculated plants showed enhanced mung bean plant growth (45.0% to 61.0% increment in shoot length and 26.9 to 30.9% in root length) and biomass compared to the control. The isolates also enhanced photosynthetic pigments such as total chlorophyll (1.5 to 6.0-fold) and carotenoids (0.5 to 3.0-fold) and 1-2-fold increase in Zn, phosphorous (P), and nitrogen (N) uptake compared to the Zn-stressed control. The present results indicated that the inoculation of Bacillus sp (SS9) and Enterobacter sp(SS7) reduced the toxicity of Zn and, in turn, enhanced the plant growth and mobilization of Zn, N, and P to the plant parts.

Whole genome analysis of Enterobacter cloacae Rs-2 and screening of genes related to plant-growth promotion.

The genus Enterobacter is widely recognized for its biotechnology potential in improving soil environment and crop growth promotion. To further explore these biotechnological potentials, we sequenced and analyzed the whole genome of Enterobacter cloacae Rs-2. The analysis showed that the total length of the Rs-2 genome was 6,965,070,514 bp, and GC content was 55.80%; the annotation results of GO and COG databases showed that the genome contains a variety of growth-promoting genes, such as iscU, glnA, glnB (nitrogen fixation); iucABCD (siderophore synthesis) and fepA, fcuA, fhuA, and pfeA, etc. (siderophore transport); ipdC (secreted IAA) and gcd, pqqBCDEF (dissolved phosphorus), etc. No pathogenic factors such as virulence genes were found. The application of Rs-2 as a soil inoculant in pot experiments showed great potential for growth promotion. This study proved the plant growth-promoting ability of Rs-2 at the molecular level through genetic screening and analysis, which provided guidance for the further improvement of the strain and laid a foundation for its application in agricultural production.

Genomic characterization of Enterobacter xiangfangensis STP-3: Application to real time petroleum oil sludge bioremediation.

Sustainable treatment of petroleum oil sludge still remains as a major challenge to petroleum refineries. Bioremediation is the promising technology involving bacteria for simultaneous production of biosurfactant and followed by degradation of petroleum compounds. Complete genomic knowledge on such potential microbes could accentuate its successful exploitation. The present study discusses the genomic characteristics of novel biosurfactant producing petrophilic/ petroleum hydrocarbon degrading strain, Enterobacter xiangfangensis STP-3, isolated from petroleum refinery oil sludge contaminated soil. The genome has 4,584,462 bp and 4372 protein coding sequences. Functional analysis using the RAST and KEGG databases revealed the presence of biosynthetic gene clusters linked to glycolipid and lipopeptide production and multiple key candidate genes linked with the degradation pathway of petroleum hydrocarbons. Orthology study revealed diversity in gene clusters associated to membrane transport, carbohydrate, amino acid metabolism, virulence and defence mechanisms, and nucleoside and nucleotide synthesis. The comparative analysis with 27 other genomes predicted that the core genome contributes to its inherent bioremediation potential, whereas the accessory genome influences its environmental adaptability in unconventional environmental conditions. Further, experimental results showed that E. xiangfangensis STP-3 was able to degrade PHCs by 82 % in 14 days during the bioremediation of real time petroleum oil sludge with the concomitant production of biosurfactant and metabolic enzymes, To the best of our knowledge, no comprehensive genomic study has been previously reported on the biotechnological prospective of this species.

Diesel degradation efficiency of Enterobacter sp., Acinetobacter sp., and Cedecea sp. isolated from petroleum waste dumping site: a bioremediation view point.

Bioremediation through biodegradation is applied for cleaning up several environmental pollutions including petroleum oil spill containing petrol, diesel, mobil, kerosene, lubricating, etc. which have devastated several endangered terrestrial and aquatic ecosystems. Therefore, the current research was aimed to isolate and identify diesel degrading bacteria from the petroleum waste dumping site and determined their degrading efficiency. The bacterial strains were isolated through a minimum salt medium supplemented with 2% diesel as the sole carbon source. The bacteria were identified by morphological, biochemical characterization, and 16S rRNA gene sequencing. The optimized growth pattern was evaluated by utilization of a wide range of temperatures (25, 30, 35, and 40 °C) and pH (5,6,7 and 8) as well as different concentrations of diesel (2, 3, 5and 7%). Finally, the degradation rate was determined by measuring the residual diesel after 7, 14, and 21 days of incubation. The study isolated Enterobacter ludwigii, Enterobacter mori, Acinetobacter baumannii, and Cedecea davisae where all are gram-negative rod-shaped bacilli. All the bacterial strains utilized the diesel at their best at 30 °C and pH 7, among them, A. baumannii and C. davisae exhibited the best degrading efficiency at all applied concentrations. Finally, the determination of degradation rate (%) through gravimetrical analysis has confirmed the potency of A. Baumannii and C. davisae where the degradation rate was around 61 and 52% respectively after 21 days of incubation period with 10% diesel. The study concludes that all of those isolated bacterial consortiums, especially A. baumannii and C. davisae could be allocated as active agents used for bioremediation to detoxify the diesel-containing contaminated sites in a cost-effective and eco-friendly way.

Microbial enhanced oil recovery through deep profile control using a conditional bacterial cellulose-producing strain derived from Enterobacter sp. FY-07.

BACKGROUND: Heterogeneity of oil-bearing formations is one of major contributors to low oil recovery efficiency globally. Long-term water flooding will aggravate this heterogeneity by resulting in many large channels during the exploitation process. Thus, injected water quickly flows through these large channels rather than oil-bearing areas, which ultimately leads to low oil recovery. This problem can be solved by profile control using polymer plugging. However, non-deep profile control caused by premature plugging is the main challenge. Here, a conditional bacterial cellulose-producing strain, namely Enterobacter sp. FY-0701, was constructed for deep profile control to solve the problem of premature plugging. Its deep profile control and oil displacement capabilities were subsequently identified and assessed. RESULTS: The conditional bacterial cellulose-producing strain Enterobacter sp. FY-0701 was constructed by knocking out a copy of fructose-1, 6-bisphosphatase (FBP) encoding gene in Enterobacter sp. FY-07. Scanning electron microscope observation showed this strain produced bacterial cellulose using glucose rather than glycerol as the sole carbon source. Bacterial concentration and cellulose production at different locations in core experiments indicated that the plugging position of FY-0701 was deeper than that of FY-07. Moreover, enhanced oil recovery by FY-0701 was 12.09%, being 3.86% higher than that by FY-07 in the subsequent water flooding process. CONCLUSIONS: To our knowledge, this is the first report of conditional biopolymer-producing strains used in microbial enhance oil recovery (MEOR). Our results demonstrated that the conditional bacterial cellulose-producing strain can in situ produce biopolymer far from injection wells and plugs large channels, which increased the sweep volume of injection water and enhance oil recovery. The construction of this strain provides an alternative strategy for using biopolymers in MEOR.

Screening of pectinase-producing bacteria from farmlands and optimization of enzyme production from selected strain by RSM.

Pectinolytic enzymes that catalyze the breakdown of substrates containing pectin are widespread. Pectinases have potential applications in various industries, including food, animal feed, textile, paper, and fuel. In this study, one hundred bacterial isolates were collected from Marand city farmlands (Azarbaijan-E-Sharqi, Iran) and screened by MP medium on the base of pectinase activity considering the significance of pectinases. The results depicted that three isolates showed the most pectinase activity (more massive halo). The biochemical and molecular test results showed that the three screened bacteria were Enterobacter and named Enterobacter sp. MF41, Enterobacter sp. MF84, and Enterobacter sp. MF90. Enterobacter sp. MF84 had the largest halo, so this strain was selected for the study of its produced pectinase. The results exhibited that the produced enzyme has optimum temperature and pH for activity at 30 °C and in 9, respectively. Finally, the enzyme production by Enterobacter sp. MF84 is optimized using response surface methodology (RSM) considering four factors (NH4Cl, K2HPO4, pectin, and incubation time) as variables. The results showed that the optimization procedure increased the enzyme production up to 12 times (from 1.16 to 14.16 U/mg). The Pareto analysis revealed that ammonium chloride has a significant role in decreasing the enzyme production, probably by inducing the nitrification pathway enzymes in the presence of organic nitrogen in Enterobacter sp. MF84.

Comparative genomics of the Erwinia and Enterobacter olive fly endosymbionts.

The pestivorous tephritid olive fly has long been known as a frequent host of the obligately host-associated bacterial endosymbiont, Erwinia dacicola, as well as other facultative endosymbionts. The genomes of Erwinia dacicola and Enterobacter sp. OLF, isolated from a California olive fly, encode the ability to supplement amino acids and vitamins missing from the olive fruit on which the larvae feed. The Enterobacter sp. OLF genome encodes both uricase and ureases, and the Er. dacicola genome encodes an allantoate transport pathway, suggesting that bird feces or recycling the fly's waste products may be important sources of nitrogen. No homologs to known nitrogenases were identified in either bacterial genome, despite suggestions of their presence from experiments with antibiotic-treated flies. Comparisons between the olive fly endosymbionts and their free-living relatives revealed similar GC composition and genome size. The Er. dacicola genome has fewer genes for amino acid metabolism, cell motility, and carbohydrate transport and metabolism than free-living Erwinia spp. while having more genes for cell division, nucleotide metabolism and replication as well as mobile elements. A 6,696 bp potential lateral gene transfer composed primarily of amino acid synthesis and transport genes was identified that is also observed in Pseudomonas savastanoii pv savastanoii, the causative agent of olive knot disease.

Purification and characterisation of a quorum quenching AHL-lactonase from the endophytic bacterium Enterobacter sp. CS66.

The quorum quenching (QQ) activity of endophytic bacteria associated with medicinal plants was explored. Extracts of the Gram-negative Enterobacter sp. CS66 possessed potent N-acylhomoserine lactone (AHL) hydrolytic activity in vitro. Using degenerate primers, we PCR-amplified an open reading frame (denoted aiiE) from CS66 that was 96% identical to the well-characterised AHL-lactonase AiiA from Bacillus thuringiensis, but only 30% was identical to AHL-lactonases from other Gram-negative species. This confirms that close AiiA homologs can be found in both Gram-positive and Gram-negative bacteria. Purified AiiE exhibited potent AHL-lactonase activity against a broad range of AHLs. Furthermore, aiiE was able to reduce the production of secreted plant cell wall-degrading hydrolytic enzymes when expressed in trans in the economically important plant pathogen, Pectobacterium atrosepticum. Our results indicate the presence of a novel AHL-lactonase in Enterobacter sp. CS66 with significant potential as a biocontrol agent.

Coliform Bacteria for Bioremediation of Waste Hydrocarbons.

Raw, domestic sewage of Kuwait City contained about 106 ml-1 colony forming units of Enterobacter hormaechei subsp. oharae (56.6%), Klebsiella spp. (36%), and Escherichia coli (7.4%), as characterized by their 16S rRNA-gene sequences. The isolated coliforms grew successfully on a mineral medium with crude oil vapor as a sole source of carbon and energy. Those strains also grew, albeit to different degrees, on individual n-alkanes with carbon chains between C9 and C36 and on the individual aromatic hydrocarbons, toluene, naphthalene, phenanthrene, and biphenyl as sole sources of carbon and energy. These results imply that coliforms, like other hydrocarbonoclastic microorganisms, oxidize hydrocarbons to the corresponding alcohols and then to aldehydes and fatty acids which are biodegraded by ß-oxidation to acetyl CoA. The latter is a well-known key intermediate in cell material and energy production. E. coli cells grown in the presence of n-hexadecane (but not in its absence) exhibited typical intracellular hydrocarbon inclusions, as revealed by transmission electron microscopy. Raw sewage samples amended with crude oil, n-hexadecane, or phenanthrene lost these hydrocarbons gradually with time. Meanwhile, the numbers of total and individual coliforms, particularly Enterobacter, increased. It was concluded that coliform bacteria in domestic sewage, probably in other environmental materials too, are effective hydrocarbon-biodegrading microorganisms.

Diminution of the gut resistome after a gut microbiota-targeted dietary intervention in obese children.

The gut microbiome represents an important reservoir of antibiotic resistance genes (ARGs). Effective methods are urgently needed for managing the gut resistome to fight against the antibiotic resistance threat. In this study, we show that a gut microbiota-targeted dietary intervention, which shifts the dominant fermentation of gut bacteria from protein to carbohydrate, significantly diminished the gut resistome and alleviated metabolic syndrome in obese children. Of the non-redundant metagenomic gene catalog of ~2 × 10(6) microbial genes, 399 ARGs were identified in 131 gene types and conferred resistance to 47 antibiotics. Both the richness and diversity of the gut resistome were significantly reduced after the intervention. A total of 201 of the 399 ARGs were carried in 120 co-abundance gene groups (CAGs) directly binned from the gene catalog across both pre-and post-intervention samples. The intervention significantly reduced several CAGs in Klebsiella, Enterobacter and Escherichia, which were the major hubs for multiple resistance gene types. Thus, dietary intervention may become a potentially effective method for diminishing the gut resistome.

Kinetic characterization of a novel acid ectophosphatase from Enterobacter asburiae.

Expression of acid ectophosphatase by Enterobacter asburiae, isolated from Cattleya walkeriana (Orchidaceae) roots and identified by the 16S rRNA gene sequencing analysis, was strictly regulated by phosphorus ions, with its optimal activity being observed at an inorganic phosphate concentration of 7 mM. At the optimum pH 3.5, intact cells released p-nitrophenol at a rate of 350.76 ± 13.53 nmol of p-nitrophenolate (pNP)/min/10(8) cells. The membrane-bound enzyme was obtained by centrifugation at 100,000 × g for 1 h at 4 °C. p-Nitrophenylphosphate (pNPP) hydrolysis by the enzyme follows "Michaelis-Menten" kinetics with V = 61.2 U/mg and K0.5 = 60 µM, while ATP hydrolysis showed V = 19.7 U/mg, K0.5 = 110 µM, and nH = 1.6 and pyrophosphate hydrolysis showed V = 29.7 U/mg, K0.5 = 84 µM, and nH = 2.3. Arsenate and phosphate were competitive inhibitors with K i = 0.6 mM and K i = 1.8 mM, respectively. p-Nitrophenyl phosphatase (pNPPase) activity was inhibited by vanadate, while p-hydroxymercuribenzoate, EDTA, calcium, copper, and cobalt had no inhibitory effects. Magnesium ions were stimulatory (K0.5 = 2.2 mM and nH = 0.5). Production of an acid ectophosphatase can be a mechanism for the solubilization of mineral phosphates by microorganisms such as Enterobacter asburiae that are versatile in the solubilization of insoluble minerals, which, in turn, increases the availability of nutrients for plants, particularly in soils that are poor in phosphorus.

Calcium carbonate precipitation by heterotrophic bacteria isolated from biofilms formed on deteriorated ignimbrite stones: influence of calcium on EPS production and biofilm formation by these isolates.

Heterotrophic CaCO3-precipitating bacteria were isolated from biofilms on deteriorated ignimbrites, siliceous acidic rocks, from Morelia Cathedral (Mexico) and identified as Enterobacter cancerogenus (22e), Bacillus sp. (32a) and Bacillus subtilis (52g). In solid medium, 22e and 32a precipitated calcite and vaterite while 52g produced calcite. Urease activity was detected in these isolates and CaCO3 precipitation increased in the presence of urea in the liquid medium. In the presence of calcium, EPS production decreased in 22e and 32a and increased in 52g. Under laboratory conditions, ignimbrite colonization by these isolates only occurred in the presence of calcium and no CaCO3 was precipitated. Calcium may therefore be important for biofilm formation on stones. The importance of the type of stone, here a siliceous stone, on biological colonization is emphasized. This calcium effect has not been reported on calcareous materials. The importance of the effect of calcium on EPS production and biofilm formation is discussed in relation to other applications of CaCO3 precipitation by bacteria.

Root colonization and growth promotion of sunflower (Helianthus annuus L.) by phosphate solubilizing Enterobacter sp. Fs-11.

An Enterobacter sp. Fs-11 was isolated from sunflower rhizosphere, identified on the basis of 16S rRNA gene sequence analysis (GeneBank accession no. GQ179978) and studied for its root colonization and growth promotion ability in sunflower. Morphologically, it was rod shaped Gram-negative, motile bacterium, producing 4.5 µg mL(-1) indole acetic acid in tryptophan-supplemented medium. It utilized 27 out of 95 substrates in BIOLOG GN2 micro plate system. It was able to convert insoluble tri-calcium phosphate to soluble phosphorus up to 43.5 µg mL(-1) with decrease in pH of the medium up to 4.5 after 10 days incubation at 28 ± 2 °C in the Pikovskaya's broth. High performance liquid chromatography of cell free supernatant showed that Fs-11 produced malic acid and gluconic acid (2.43 and 16.64 µg mL(-1), respectively) in Pikovskaya's broth. Analysis of 900 bp fragment of pyrroloquinoline quinine pqqE gene sequence showed 98 % homology with that of E. cloacae pqqE gene. Confocal laser scanning microscope revealed strong colonization of fluorescently labeled Fs-11 with sunflower roots. Sunflower inoculation with Fs-11 and its rifampicin resistant derivative in sterile sand and natural soil showed that Fs-11 colonized sunflower roots up to 30 days after transplanting in both sterile sand as well as natural soil. Moreover, Fs-11 inoculation resulted in increased plant height, fresh weight, dry weight and total phosphorus contents as compared to un-inoculated plants. The data showed that Enterobacter sp. Fs-11 is an efficient phosphate solubilizing and plant growth promoting rhizobacterium and has great potential to be used as bio-inoculant for sunflower under phosphorus deficient conditions.

Population dynamics of bacteria associated with different strains of the pine wood nematode Bursaphelenchus xylophilus after inoculation in maritime pine (Pinus pinaster).

For a long time it was thought that Bursaphelenchus xylophilus was the only agent of the pine wilt disease. Recently, it was discovered that there are bacteria associated with the nematodes that contribute to the pathogenesis of this disease, mainly through the release of toxins that promote the death of the pines. Among the species most commonly found, are bacteria belonging to the Bacillus, Pantoea, Pseudomonas and Xanthomonas genera. The main objective of this work was to study the effect of inoculation of maritime pine (Pinus pinaster) with four different nematode isolates, in the bacterial population of nematodes and trees, at different stages of disease progression. The monitoring of progression of disease symptoms was also recorded. Also, the identification of bacteria isolated from the xylem of trees and the surface of nematodes was performed by classical identification methods, by the API20E identification system and by sequencing of bacterial DNA. The results showed that for the symptoms progression, the most striking difference was observed for the pines inoculated with the avirulent isolate, C14-5, which led to a slower and less severe aggravation of symptoms than in pines inoculated with the virulent isolates. In general, it was found that bacterial population, inside the tree, increased with disease progression. A superior bacterial quantity was isolated from pines inoculated with the nematode isolates HF and 20, and, comparatively, few bacteria were isolated from pines inoculated with the avirulent isolate. The identification system API20E was insufficient in the identification of bacterial species; Enterobacter cloacae species was identified in 79% of the isolated bacterial colonies and seven of these colonies could not be identified by this method. Molecular identification methods, through bacterial DNA sequencing, allowed a more reliable identification: eleven different bacterial species within the Bacillus, Citrobacter, Enterobacter, Escherichia, Klebsiella, Paenibacillus, Pantoea and Terribacillus genera were identified. General bacterial diversity increased with the progression of the disease. Bacillus spp. were predominant at the earlier stage of disease progression and Klebsiella oxytoca at the later stages. Furthermore, bacterial species isolated from the surface of nematodes were similar to those isolated from the xylem of pines. In the present work new bacterial species were identified which have never been reported before in this type of study and may be associated with their geographical origin (Portugal). P. pinaster, the pine species used in this study, was different from those commonly grown in Japan and China. Furthermore, it was the first time that bacteria were isolated and identified from an avirulent pine wood nematode isolate.

Plasmid load adversely affects growth and gluconic acid secretion ability of mineral phosphate-solubilizing rhizospheric bacterium Enterobacter asburiae PSI3 under P limited conditions.

Effect of the metabolic load caused by the presence of plasmids on mineral phosphate-solubilizing (MPS) Enterobacter asburiae PSI3, was monitored with four plasmid cloning vectors and one native plasmid, varying in size, nature of the replicon, copy number and antibiotic resistance genes. Except for one plasmid, the presence of all other plasmids in E. asburiae PSI3 resulted in the loss of the MPS phenotype as reflected by the failure to bring about a drop in pH and release soluble P when grown in media containing rock phosphate (RP) as the sole P source. When 100 µM soluble P was supplemented along with RP, the adverse effects of plasmids on MPS phenotype and on growth parameters was reduced for some plasmid bearing derivatives, as monitored in terms of specific growth rates, glucose consumed, gluconic acids yields and P released. When 10 mM of soluble P as the only P source, was added to the medium all transformants showed growth and pH drop comparable with native strain. It may be concluded that different plasmids impose, to varying extents, a metabolic load in the phosphate-solubilizing bacterium E. asburiae PSI3 and results in diminishing its growth and P-solubilizing ability in P deficient conditions.

Base selectivity is impaired by mutants that perturb hydrogen bonding networks in the RB69 DNA polymerase active site.

To investigate the molecular basis for the selective utilization of nucleoside triphosphates complementary to templating bases, by RB69 DNA polymerase (RB69 pol), we constructed a set of mutants that we predicted would perturb the "floor" of the nascent base-pairing interface in the enzyme. We then determined the pre-steady-state kinetic parameters for the incorporation of complementary and noncomplementary dNTPs by the exo(-) form of RB69 pol and its mutants. We found that the Y567A mutant had the same K(d) and k(pol) values for incorporation of C versus G as the wild-type exo(-) enzyme; however, the k(pol)/K(d) ratio for G versus G incorporation with the Y567A mutant was 10 times higher than the k(pol)/K(d) efficiency of G versus G incorporation using the exo(-) RB69 pol. The reduced level of discrimination by the Y567A mutant against incorporation of mismatched bases was also seen with the Y391A mutant. Stopped-flow fluorescence was also employed to monitor rates of putative conformational changes with the exo(-) RB69 pol and its mutants using a primer-template complex containing 2-aminopurine. The rates of fluorescence changes were equal to or greater than the rates of the rapid chemical quench, indicating that we were monitoring a process occurring before or during the phosphoryl transfer reaction. We have interpreted our results within the context of the crystal structure of the RB69 pol ternary complex [Franklin, M. C., et al. (2001) Cell 105, 657-667].

Bacterial community dynamics during in-situ bioremediation of petroleum waste sludge in landfarming sites.

In-situ bioremediation of petroleum waste sludge in landfarming sites of Motor Oil Hellas (petroleum refinery) was studied by monitoring the changes of the petroleum composition of the waste sludge, as well as the changes in the structure of the microbial community, for a time period of 14 months. The analyses indicated an enhanced degradation of the petroleum hydrocarbons in the landfarming areas. A depletion of n-alkanes of approximately 75-100% was obtained. Marked changes of the microbial communities of the landfarms occurred concomitantly with the degradation of the petroleum hydrocarbons. The results obtained from terminal restriction fragment length polymorphism (T-RFLP) analysis of polymerase chain reaction (PCR) amplified 16S rRNA genes demonstrated that bacteria originating from the refinery waste sludge and newly selected bacteria dominated the soil bacterial community during the period of the highest degradation activity. However, the diversity of the microbial community was decreased with increased degradation of the petroleum hydrocarbons contained in the landfarms. T-RFLP fingerprints of bacteria of the genera Enterobacter and Ochrobactrum were detected in the landfarmed soil over the entire treatment period of 14 months. In contrast, the genus Alcaligenes appeared in significant numbers only within the 10 month old landfarmed soil. Genes encoding catechol 2,3-dioxygenase (subfamily I.2.A) were detected only in DNA of the untreated refinery waste sludge. However, none of the genes known to encode the enzymes alkane hydroxylase AlkB, catechol 2,3-dioxygenase (subfamily I.2.A) and naphthalene dioxygenase nahAc could be detected in DNA of the landfarmed soils.

Imipenem resistance of enterobacter aerogenes mediated by outer membrane permeability.

Multidrug-resistant Enterobacter aerogenes strains are increasingly isolated in Europe and especially in France. Treatment leads to imipenem resistance, because of a lack of porin. We studied the evolution of resistance in 29 strains isolated from four patients during their clinical course. These strains belonged to the prevalent epidemiological type observed in France in previous studies (C. Bosi, et al., J. Clin. Microbiol. 37:2165-2169, 1999; A. Davin-Regli et al., J. Clin. Microbiol. 34:1474-1480, 1996). They also harbored a TEM-24 extended-spectrum beta-lactamase-coding gene. Thirteen strains were susceptible to gentamicin and resistant to imipenem and cefepime. All of the patients showed E. aerogenes strains with this resistance after an imipenem treatment. One patient showed resistance to imipenem after a treatment with cefpirome. Twelve of these 13 strains showed a lack of porin. Cessation of treatment with imipenem for three patients was followed by reversion of susceptibility to this antibiotic and the reappearance of porins, except in one case. For one patient, we observed three times in the same day the coexistence of resistant strains lacking porin and susceptible strains possessing porin. The emergence of multidrug-resistant E. aerogenes strains is very disquieting. In our study, infection by E. aerogenes increased the severity of the patients' illnesses, causing a 100% fatality rate.

Interspecies compatibility of selenoprotein biosynthesis in Enterobacteriaceae.

Several species of Enterobacteriaceae were investigated for their ability to synthesize selenium-containing macromolecules. Seleniated tRNA species as well as seleniated polypeptides were formed by all organisms tested. Two selenopolypeptides could be identified in most of the organisms which correspond to the 80 kDa and 110 kDa subunits of the anaerobically induced formate dehydrogenase isoenzymes of E. coli. In those organisms possessing both isoenzymes, their synthesis was induced in a mutually exclusive manner dependent upon whether nitrate was present during anaerobic growth. The similarity of the 80 kDa selenopolypeptide among the different species was assessed by immunological and genetic analyses. Antibodies raised against the 80 kDa selenopolypeptide from E. coli cross-reacted with an 80 kDa polypeptide in those organisms which exhibited fermentative formate dehydrogenase activity. These organisms also contained genes which hybridised with the fdhF gene from E. coli. In an attempt to identify the signals responsible for incorporation of selenium into the selenopolypeptides in these organisms we cloned a portion of the fdhF gene homologue from Enterobacter aerogenes. The nucleotide sequence of the cloned 723 bp fragment was determined and it was shown to contain an in-frame TGA (stop) codon at the position corresponding to that present in the E. coli gene. This fragment was able to direct incorporation of selenocysteine when expressed in the heterologous host, E. coli. Moreover, the E. coli fdhF gene was expressed in Salmonella typhimurium, Serratia marcescens and Proteus mirabilis, indicating a high degree of conservation of the seleniating system throughout the enterobacteria.