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PubMed2003

Transformation of Azospirillum brasilense Cd with an ACC deaminase gene from enterobacter cloacae UW4 fused to the Tet r gene promoter improves its fitness and plant growth promoting ability.

It has been reported that PGPB, containing ACC deaminase, can cleave the plant ethylene precursor ACC and thereby lower ethylene concentration in a developing or stressed plant, protecting it against the deleterious effects of stress ethylene and facilitating the formation of longer roots. In a previous work we have demonstrated expression of the ACC deaminase gene ( acdS) from Enterobacter cloacae UW4 under the control of the lac promoter in Azospirillum brasilense Cd. With the inference that a construct including the ACC deaminase gene under the control of a constitutive promoter weaker than the lac promoter might impose less metabolic load on Azospirillum and improve its fitness, it was decided to clone acdS under the control of a tetracycline resistance gene promoter. The ACC deaminase structural gene was fused to the Tet(r) gene promoter by overlap extension using PCR, cloned in pRK415, and transferred into A. brasilense Cd. The resulting transformants showed lower ACC deaminase activity than those with the lac promoter controlled acdS gene. However, acdS under the control of the Tet(r) gene promoter imposed lesser metabolic load on Azospirillum brasilense Cd. The result was significantly increased IAA synthesis and greater bacterial growth rate, as well as increased ability to survive on the surface of tomato leaves and to promote the growth of tomato seedlings.

Azospirillum brasilense↗

PubMed2002

Use of carbon and energy balances in the study of the anaerobic metabolism of Enterobacter aerogenes at variable starting glucose concentrations.

The anaerobic metabolism of Enterobacter aerogenes was studied in batch culture at increasing initial glucose levels (9.0< S(o) <72 g l(-1)). The ultimate concentrations of fermentation products were utilized to check a metabolic flux analysis based on simple carbon mass and energy balances that promise to be suitable for the study of different fermentation processes, either under aerobic or anaerobic conditions. The stoichiometric coefficients of products collected at increasing starting glucose concentrations under anaerobic conditions suggest: (a) little influence of starting glucose level on the formation of the main fermentation products (2,3-butanediol and ethanol); (b) possible inhibition of 2,3-butanediol and lactate formations by increased ethanol concentration; (c) consequent increase in carbon flux through the remaining metabolic pathways with increased molar productions of succinate, acetate and hydrogen; (d) relative constancy of the molar production of ATP and CO(2).

Adenosine Triphosphate↗

PubMed2003

Production of L-asparaginase in Enterobacter aerogenes expressing Vitreoscilla hemoglobin for efficient oxygen uptake.

This study is the first utilizing Vitreoscilla hemoglobin in a heterologous bacterium, Enterobacter aerogenes, to determine the effect of such a highly efficient oxygen-uptake system on the production of l-asparaginase, an enzyme that has attracted considerable attention due to its anti-tumor activity. Here, we show that the Vitreoscilla hemoglobin expressing strain has from 10-fold to more than two orders of magnitude lower l-asparaginase activity than the wild type or the control without the Vitreoscilla hemoglobin gene under different aeration conditions. Aeration and agitation were also determining factors for enzyme production. The enzyme activity was reduced considerably under both full aerobic and anaerobic conditions, while the highest enzyme activity was determined in cultures under low aeration and low agitation. Also, the effect of different concentrations of glucose on enzyme production showed catabolic repression. Glucose at 1% caused almost total inhibition of enzyme activity, while at 0.1% it showed a slightly stimulatory effect on enzyme production, compared with glucose-free medium.

Aerobiosis↗

PubMed2004

Stability and activity of an Enterobacter aerogenes-specific bacteriophage under simulated gastro-intestinal conditions.

A bacteriophage, designated UZ1 and showing lytic activity against a clinically important strain (BE1) of Enterobacter aerogenes was isolated from hospital sewage. The stability and lytic activity against this strain under simulated gastro-intestinal conditions was evaluated. After addition of bacteriophage UZ1 to a liquid feed at gastric pH 2, the phage was immediately inactivated and could not be recovered. However, by use of an antacid to neutralize stomach acidity, no significant changes in phage titer were observed after 2 h incubation at 37 degrees C. After supplementing pancreatic juice and further incubation for 4 h, the phage titer remained stable. The persistence of UZ1 in a mixed microbial ecosystem that was representative for the large intestine was monitored using an in vitro simulation of the human intestinal microbial ecosystem. A pulse administration of bacteriophage UZ1 at a concentration of 10(5) plaque-forming units (PFU)/ml to reactor 3 (which simulates the ascending colon) showed that, in the absence of the host, bacteriophage UZ1 persisted for 13 days in the simulated colon, while the theoretical washout was calculated at 16 days. To assess its lytic activity in an intestinal microbial ecosystem, a green fluorescent protein (gfp)-labeled E. aerogenes BE1 strain was constructed and gfp-specific primers were designed in order to quantify the host strain using real-time PCR. It was observed that bacteriophage UZ1 was able to replicate and showed lytic activity against E. aerogenes BE1/ gfp in an intestinal microbial ecosystem. Indeed, after 17 h a 2 log unit reduction of E. aerogenes BE1/ gfp was measured as compared with the assay without bacteriophage UZ1, while the phage titer increased by 2 log units at an initial multiplicity of infection of 0.07 PFU/colony-forming unit. This is the first report of an in vitro model to study bacteriophage activity in the complex intestinal microbial community.

Antiviral Agents↗

PubMed2005

Glucose-1-phosphatase (AgpE) from Enterobacter cloacae displays enhanced phytase activity.

Using a screening procedure developed for detection of phytate hydrolysing enzymes, the gene agpE encoding glucose-1-phosphatase was cloned from an Enterobacter cloacae VKPM B2254 plasmid library. Sequence analysis revealed 78% identity on nucleotide and 79% identity on peptide level to Escherichia coli glucose-1-phosphatase characterising the respective gene product as a representative of acid histidine phosphatases harbouring the RH(G/N)RXRP motif. The purified recombinant protein displayed maximum specific activity of 196 U mg(-1) protein against glucose-1-phosphate but was also active against other sugar phosphates and p-nitrophenyl phosphate. High-performance ion chromatography of hydrolysis products revealed that AgpE can act as a 3-phytase but is only able to cleave off the third phosphate group from the myo-inositol sugar ring. Based on sequence comparison and catalytic behaviour against phytate, we propose to classify bacterial acid histidine phosphatases/phytases in the three following subclasses: (1) AppA-related phytases, (2) PhyK-related phytases and (3) Agp-related phytases. A distinguished activity of 32 U mg(-1) of protein towards myo-inositol-hexa-phosphate, which is two times higher than that of E. coli Agp, suggests that possibly functional differences in terms of phytase activity between Agp- and AppA-like acid histidine phosphatases are fluent.

6-Phytase↗

PubMed2005

Hydrogen production by Rhodobacter sphaeroides strain O.U.001 using spent media of Enterobacter cloacae strain DM11.

Combined dark and photo-fermentation was carried out to study the feasibility of biological hydrogen production. In dark fermentation, hydrogen was produced by Enterobacter cloacae strain DM11 using glucose as substrate. This was followed by a photo-fermentation process. Here, the spent medium from the dark process (containing unconverted metabolites, mainly acetic acid etc.) underwent photo-fermentation by Rhodobacter sphaeroides strain O.U.001 in a column photo-bioreactor. This combination could achieve higher yields of hydrogen by complete utilization of the chemical energy stored in the substrate. Dark fermentation was studied in terms of several process parameters, such as initial substrate concentration, initial pH of the medium and temperature, to establish favorable conditions for maximum hydrogen production. Also, the effects of the threshold concentration of acetic acid, light intensity and the presence of additional nitrogen sources in the spent effluent on the amount of hydrogen produced during photo-fermentation were investigated. The light conversion efficiency of hydrogen was found to be inversely proportional to the incident light intensity. In a batch system, the yield of hydrogen in the dark fermentation was about 1.86 mol H(2) mol(-1) glucose; and the yield in the photo-fermentation was about 1.5-1.72 mol H2 mol-1 acetic acid. The overall yield of hydrogen in the combined process, considering glucose as the preliminary substrate, was found to be higher than that in a single process.

Bioreactors↗

PubMed2005

Genetically engineered termite gut bacteria (Enterobacter cloacae) deliver and spread foreign genes in termite colonies.

Indigenous gut bacteria of the Formosan subterranean termite (Coptotermes formosanus Shiraki, Isoptera: Rhinotermitidae) were used as shuttle systems to deliver, express and spread foreign genes in termite colonies. The gut bacterium Enterobacter cloacae was transformed with a recombinant plasmid (pEGFP) containing genes encoding ampicillin resistance and green fluorescent protein (GFP). In laboratory experiments, termite workers and soldiers from three colonies were fed with filter paper inoculated with transformed bacteria. Transformed bacteria were detected in termite guts by growing the entire gut flora under selective conditions and checking the cultures visually for fluorescence. We demonstrated that (1) transformed bacteria were ingested within a few hours and the GFP gene was expressed in the termite gut; (2) transformed bacteria established a persistent population in the termite gut for up to 11 weeks; (3) transformed bacteria were efficiently transferred throughout a laboratory colony, even when the donor (termites initially fed with transformed bacteria) to recipient (not fed) ratio was low; (4) transformed E. cloacae were transferred into soil; however, they did not accumulate over time and the GFP plasmid was not transferred to other soil bacteria. In the future, transgenic bacteria may be used to shuttle detrimental genes into termite colonies for improved pest control.

Ampicillin Resistance↗

PubMed2000

Studies on nutritional and oxygen requirements for production of L-asparaginase by Enterobacter aerogenes.

The carbon and nitrogen sources most suitable for L-asparaginase production by Enterobacter aerogenes were selected and their concentrations optimized in shake-flask cultures. Sodium citrate (1.0%) and diammonium hydrogen phosphate (0.16%) proved to be the best sources of carbon and nitrogen, respectively. Nitrogen catabolite repression of enzyme formation was absent in this bacterium. Cultivation in a reactor showed that the dissolved oxygen level is the limiting factor for L-asparaginase production by E. aerogenes. Glucose was found to be a repressor of enzyme synthesis. Asparagine was absent intracellularly when the L-asparaginase level was high. An increase in the extracellular alanine level when the dissolved oxygen remained low indicated a shift from aerobic to fermentative metabolism.

Aerobiosis↗

PubMed1998

Isolation and characterization of Enterobacter cloacae capable of metabolizing asparagine.

A gram-negative, rod-shaped bacterium capable of utilizing L-asparagine as its sole source of carbon and nitrogen was isolated from soil and identified as Enterobacter cloacae. An intracellularly expressed L-asparaginase was detected and it deaminated L-asparagine to aspartic acid and ammonia. High-pressure liquid chromatography analysis of a cell-free asparaginase reaction mixture indicated that 2.8 mM L-asparagine was hydrolyzed to 2.2 and 2.8 mM aspartic acid and ammonia, respectively, within 20 min of incubation. High asparaginase activity was found in cells cultured on L-fructose, D-galactose, saccharose, or maltose, and in cells cultured on L-asparagine as the sole nitrogen source. The pH and temperature optimum of L-asparaginase was 8.5 and 37-42 degrees C, respectively. The half-life of the enzyme at 30 degrees C and 37 degrees C was 10 and 8 h, respectively.

Asparaginase↗

PubMed1999

Production of 2,3-butanediol by newly isolated Enterobacter cloacae.

Enterobacter cloacae NRRL B-23289 was isolated from local decaying wood/corn soil samples while screening for microorganisms for conversion of L-arabinose to fuel ethanol. The major product of fermentation by the bacterium was meso-2,3-butanediol (2,3-BD). In a typical fermentation, a BD yield of 0.4 g/g arabinose was obtained with a corresponding productivity of 0.63 g/l per hour at an initial arabinose concentration of 50 g/l. The effects of initial arabinose concentration, temperature, pH, agitation, various monosaccharides, and multiple sugar mixtures on 2,3-BD production were investigated. BD productivity, yield, and byproduct formation were influenced significantly within these parameters. The bacterium utilized sugars from acid plus enzyme saccharified corn fiber and produced BD (0.35 g/g available sugars). It also produced BD from dilute acid pretreated corn fiber by simultaneous saccharification and fermentation (0.34 g/g theoretical sugars).

Acids↗

PubMed1999

Fluorescence monitoring during cultivation of Enterobacter aerogenes at different oxygen levels.

On-line monitoring of NAD(P)H fluorescence and 2D fluorescence spectroscopy was performed with Enterobacter aerogenes, a bacterium sensitive to oxygen availability. The organism was grown in a reactor under low and high dissolved oxygen concentrations and circulated through a bypass attached to the reactor. Under low dissolved oxygen concentration in the reactor, NAD(P)H fluorescence in the reactor and the bypass showed a deviation, but not when the dissolved oxygen level in the reactor was high. The pattern of growth curves was identical under low and high oxygen levels. This indicates a difference in the metabolic activity of E. aerogenes in response to oxygen. The difference spectrum of the 2D fluorescence shows that growing E. aerogenes under high dissolved oxygen levels increases the NAD(P)H content of the cells.

Bioreactors↗

PubMed2002

Transformation of Enterobacter gergoviae isolated from pink bollworm (Lepidoptera: Gelechiidae) gut with Bacillus thuringiensis toxin.

Production of molecules with toxic activity by genetically transformed symbiotic bacteria of pest insects may serve as a powerful approach to biological control. The symbiont, Enterobacter gergoviae, isolated from the gut of the pink bollworm (PBW), has been transformed to express Cyt1A, a cytolytic protein toxin lethal to mosquito and black fly larvae, as a model system. These transgenic bacteria might be used to spread genes encoding insecticidal proteins to populations of agricultural insects or as replacement for chemical insecticides such as malathion used in bait formulation to control specific insect pests, because of extreme public pressure against organophosphate pesticide spraying.

Animals↗

PubMed2003

2-ketogluconic acid production and phosphate solubilization by Enterobacter intermedium.

Enterobacter intermedium, isolated from grass rhizosphere, exhibited a strong ability to solubilize insoluble phosphate. This bacterium oxidized glucose to gluconic acid and sequentially to 2-ketogluconic acid (2-KGA), which was identified using HPLC and GC-MS. The ability of E. intermedium to solubilize phosphate and produce 2-KGA produce in broth medium containing different components was monitored with air and without air supply. With an air supply, the production of 2-KGA markedly increased to about 110 g/l at day 10 in media containing 0.2 M gluconic acid, while it was about 65 g/l without gluconic acid addition. With an air supply, the concentration of soluble phosphate significantly decreased to 200-250 mg/l in media containing 1% CaCO3, whereas it was about 1000 mg/l without CaCO3 addition. Without an air supply, the concentration of 2-KGA and phosphate were negligible throughout the culture period.

Culture Media↗

PubMed2003

Cloning and expression of pyrroloquinoline quinone (PQQ) genes from a phosphate-solubilizing bacterium Enterobacter intermedium.

A grass rhizosphere bacterium, Enterobacter intermedium (60-2G), has a strong ability to solubilize insoluble phosphate. Certain phosphate-solubilizing bacteria secrete gluconic acid for this process. The gluconic acid is produced by direct extracellular oxidation of glucose by a glucose dehydrogenase equipped with pyrroloquinoline quinone (PQQ) as a cofactor. A pqq gene cluster producing PQQ was detected in E. intermedium and this sequence conferred phosphate-solubilizing activity to Escherichia coli DH5alpha. The 6,783-bp pqq sequence had six open reading frames (pqqA, B, C, D, E, and F) and showed 50-95% homology to pqq genes of other bacteria. E. coli DH5alpha expressing the E. intermedium pqq genes solubilized phosphate from hydroxyapatite after a pH drop to pH 4.0, which paralleled in time the secretion of gluconic acid. We speculate that production of PQQ in E. coli DH5alpha expressing the pqq cluster activates an endogenous glucose dehydrogenase to permit gluconic acid secretion that solubilizes the insoluble phosphate.

Cloning, Molecular↗

PubMed2004

Decolorization of azo dyes with Enterobacter agglomerans immobilized in different supports by using fluidized bed bioreactor.

Immobilized cells of Enterobacter agglomerans, able to reduce azo dyes enzymatically, were used as a biocatalyst for the decolorization of synthetic medium containing the toxic azo dye methyl red (MR). This bacterial strain exhibits high ability to completely decolorize 100 mg/L of MR after only 6 h of incubation under aerobic conditions. Cells of E. agglomerans were immobilized in calcium alginate, polyacylamide, cooper beech, and vermiculite, and were used for the decolorization of MR from synthetic water by using a fluidized bed bioreactor. The highest specific decolorization rate was obtained when E. agglomerans was entrapped in calcium alginate beads and was of about 3.04 mg MR/g cell/h with a 50% conversion time ( t(1/2)) of about 1.6 h. Moreover, immobilized cells in calcium alginate continuously decolorized MR even after seven repeated experiments without significant loss of activity, while polyacrylamide-, cooper beech-, and vermiculite-immobilized cells retained only 62, 15, and 13% of their original activity, respectively.

Acrylic Resins↗

PubMed2006

Cloning, sequencing and characterization of a novel phosphatase gene, phoI, from soil bacterium Enterobacter sp. 4.

A gene, phoI, coding for a phosphatase from Enterobacter sp. 4 was cloned in Escherichia coli and sequenced. Analysis of the sequence revealed one open reading frame (ORF) that encodes a 269-amino acid protein with a calculated molecular mass of 29 kDa. PhoI belongs to family B acid phosphatase and exhibits 49.4% identity and 62.4% homology to the hel gene from Heamophilus influenzae, which encoded an outer membrane protein (P4). The optimum pH and temperature for phosphatase activity were pH 5.5 and 40 degrees C, respectively. Its specific activity on rho-nitrophenyl phosphatate was 70 U/mg at pH 5.5 and 40 degrees C. Enzyme activity was inhibited by Al3+, EDTA, and DTT, but fivefold activated by Cu2+ ion (350 U/mg). PhoI showed a strong synergistic effect when used with a purified E. coli phytase, AppA, to estimate combination effects.

6-Phytase↗

PubMed2005

Chitinase production in solid-state fermentation by Enterobacter sp. NRG4 using statistical experimental design.

The optimization of nutrient levels for chitinase production by Enterobacter sp. NRG4 in solid-state fermentation conditions (SSF) was carried out using response surface methodology (RSM) based on central composite design (CCD). The design was employed by selecting wheat bran-to-flake chitin ratio, moisture level, inoculum size, and incubation time as model factors. The results of first-order factorial design experiments showed that all four independent variables have significant effects on chitinase production. The optimum concentrations for chitinase production were wheat bran-to-flake chitin ratio, 1; moisture level, 80%; inoculum size, 2.6 mL; and incubation time, 168 h. Using this statistical optimization method, chitinase production was found to increase from 616 U . g(-1) dry weight of solid substrate to 1475 U . g(-1) dry weight of solid substrate.

Chitin↗

PubMed2026

Genomic Characterisation of Carbapenem-Resistant Klebsiella pneumoniae and Enterobacter hormaechei Clinical Isolates from Nigeria: Evidence of Resistance, Virulence, and Putative Plasmid-Mediated Gene Sharing.

The global proliferation of carbapenem-resistant Enterobacterales (CRE) constitutes one of the most urgent public health threats, yet high-resolution genomic data from sub-Saharan Africa remain critically scarce. We applied whole-genome sequencing (WGS) and comparative phylogenomics to characterise antimicrobial resistance determinants, virulence genes, and mobile genetic elements (MGEs) in three carbapenem-resistant clinical isolates originating from three tertiary hospitals (selected from a broader surveillance collection spanning four facilities) in Osun State, southwestern Nigeria. We purposively selected three isolates, two Klebsiella pneumoniae subsp. pneumoniae (K22, ST411; K31, ST17) and one Enterobacter hormaechei subsp. steigerwaltii (K32, ST45) from a broader surveillance collection of 27 carbapenem-non-susceptible Enterobacterales, to represent phenotypically and genotypically divergent lineages. Resistome analysis revealed extensive plasmid-associated β-lactam and aminoglycoside resistance in K31 (including blaCTX-M-15, blaOXA-1, and blaTEM-1). K32 harboured an intrinsic chromosomal blaACT-17 AmpC gene, while IS26 and ISEcp1 insertion sequences, consistent with transposon-mediated mobilisation, flanked its acquired aminoglycoside and sulfonamide resistance cassettes. K22 lacked detected acquired carbapenemase, ESBL, or plasmid-mediated AmpC genes, indicating that its carbapenem-resistant phenotype may involve non-carbapenemase mechanisms such as porin alteration or efflux-mediated reduced susceptibility; however, this mechanism requires confirmation by direct ompK35/ompK36 sequence analysis and/or phenotypic outer membrane protein profiling. Virulome profiling identified a broader repertoire of siderophore, adhesion, and biofilm genes in both K. pneumoniae isolates than in E. hormaechei. Phylogenomic analysis demonstrated that K22 and K31 cluster within the broader K. pneumoniae population framework but represent distinct high-risk lineages (ST411 and ST17) rather than a single clonal outbreak. Analysis also identified a shared plasmid backbone between K31 and K32, supporting interspecies horizontal gene transfer. These descriptive genomic findings identify clinically relevant resistance and virulence determinants in three purposively selected carbapenem-resistant Enterobacterales from Nigerian tertiary-care hospitals. The detection of shared resistance elements between K. pneumoniae and E. hormaechei suggests possible plasmid-mediated gene sharing. Still, larger WGS studies with long-read sequencing and patient-level epidemiological data are required to define transmission and dissemination patterns.

Nigeria↗