Chromate-resistance in a chromate-reducing strain of Enterobacter cloacae

Resistance to toxic hexavalent chromium (chromate: CrO4(2)) in Enterobacter cloacae strain HO1, isolated from an activated sludge sample, was investigated under aerobic and anaerobic conditions. Decreased uptake of 51CrO4(2-) in E. cloacae strain HO1 was observed under aerobic conditions, when compared with a standard laboratory E. cloacae strain (IAM 1624). Under anaerobic conditions E. cloacae strain HO1 was able to reduce hexavalent chromium to the less toxic trivalent form. When E. clocacae strain HO1 was grown with nitrate anaerobically, the cells were observed to lose simultaneously their chromate-reducing ability and chromate-resistance under anaerobic conditions.     

Identification and molecular characterization of class 1 integrons in multiresistant Escherichia coli isolates from poultry litter

This study describes the prevalence of arrays of class 1 integron cassettes and Qnr determinants (A, B, and S) in 19 fluoroquinolone-resistant Escherichia coli isolates from chicken litter. qnrS and qnrA were the predominant genes in these fluoroquinolone-resistant isolates, and an uncommon array of aacA4-catB3-dfrA1 gene cassettes from a class1 integron was found. Additionally, aadA1 and dfrA1 gene cassettes, encoding resistance to streptomycin and trimethoprim, constituted the most common genes identified and was located on megaplasmids as well on the chromosome. Antibiotic resistance, pulsed-field gel electrophoresis (PFGE), and plasmid data suggest a genetically diverse origin of poultry E. coli isolates.     

Effects of oxygen stress on chromate reduction in Enterobacter cloacae strain HO1

Enterobacter cloacae strain HO1 was able to reduce toxic hexavalent chromium (chromate) anaerobically. The reduction of chromate by E. cloacae cells was sensitive to oxygen stress. Cultures under continuous aeration showed no chromate reduction. However, when released from the oxygen stress, the cultures readily resumed chromate reduction.     

Bacterial reduction of hexavalent chromium by Enterobacter cloacae strain HO1 grown on sucrose

Chromium(VI) was reduced by Enterobacter cloacae strain HO1 grown with sucrose as a carbon source and nitrate as the initial terminal electron acceptor. Under excess substrate conditions, the Cr(VI) concentration, initially at 5 and 10 mg/l, was reduced to less than 100 mg/l.     

Isolation and characterization of restriction endonuclease EclHKI from an Enterobacter cloacae strain

An Enterobacter cloacae strain, producing restriction enzyme EclHKI, was isolated from a decaying potato. The enzyme is an isoschizomer of Eam1105I, which recognizes and cleaves GACNNN!NNGTC. EclHKI was produced at high activity (4×10⁴ U/g wet cells) and was purified from contaminants which interfere with restriction digestion by passing the cell lysate through DEAE-Sephacel and heparin columns. Activity was optimal at 37°C in a medium salt buffer.H.-Y. Chan, Y.-C. Chan and P.-C. Shaw are with the Department of Biochemistry, The Chinese University of Hong Kong, Hong Kong: K.-M. Kam is with the Institute of Pathology, Sai Ying Pun Jockey Club Clinic, Hong Kong.     

New aerobactin-mediated iron uptake system in a septicemia-causing strain of Enterobacter cloacae.

Unlike the great majority of the aerobactin-producing enteric bacteria documented in the literature, Enterobacter cloacae EK33, isolated from a case of human neonatal meningitis, did not show any homology at the DNA level with the prototype aerobactin system encoded by the ColV-K30 plasmid. However, both the nuclear magnetic resonance spectrum and fast-atom bombardment mass spectrometry of the siderophore purified from EK33 confirmed its identity with aerobactin. Bioassay screening of a gene library of total DNA of EK33 led to the isolation of several aerobactin-positive clones. Under conditions of iron limitation, these clones expressed in Escherichia coli a protein of 72 kilodaltons that reacted with antiserum raised against the pColV-K30 74-kilodalton aerobactin receptor, while the original E. cloacae strain synthesized an 85-kilodalton protein which also cross-reacted with the antiserum. Restriction endonuclease analysis of the cloned DNA confirmed the structural differences between the two aerobactin genetic systems.     

Isolation and characterization of a glyphosate-degrading rhizosphere strain,Enterobacter cloacae K7

Plant-growth-promoting rhizobacteria exert beneficial effects on plants through their capacity for nitrogen fixation, phytohormone production, phosphate solubilization, and improvement of the water and mineral status of plants. We suggested that these bacteria may also have the potential to express degradative activity toward glyphosate, a commonly used organophosphorus herbicide. In this study, 10 strains resistant to a 10 mM concentration of glyphosate were isolated from the rhizoplane of various plants. Five of these strains – Alcaligenes sp. K1, Comamonas sp. K4, Azomonas sp. K5, Pseudomonas sp. K3, and Enterobacter cloacae K7 – possessed a number of associative traits, including fixation of atmospheric nitrogen, solubilization of phosphates, and synthesis of the phytohormone indole-3-acetic acid. One strain, E. cloacae K7, could utilize glyphosate as a source of P. Gas–liquid chromatography showed that E. cloacae growth correlated with a decline in herbicide content in the culture medium (40% of the initial 5 mM content), with no glyphosate accumulating inside the cells. Thin-layer chromatography analysis of the intermediate metabolites of glyphosate degradation found that E. cloacae K7 had a C–P lyase activity and degraded glyphosate to give sarcosine, which was then oxidized to glycine. In addition, strain K7 colonized the roots of common sunflower (Helianthus annuus L.) and sugar sorghum (Sorghum saccharatum Pers.), promoting the growth and development of sunflower seedlings. Our findings extend current knowledge of glyphosate-degrading rhizosphere bacteria and may be useful for developing a biotechnology for the cleanup and restoration of glyphosate-polluted soils.     

A rapid-kinetic study of the class C beta-lactamase of Enterobacter cloacae 908R.

The individual rate constants for acylation and deacylation (k2 and k3, respectively) of the class C beta-lactamase of Enterobacter cloacae 908R by ampicillin and carbenicillin have been determined. For several other beta-lactams, the value of k2 was too high to be determined and the k2/k3 ratio could be larger than 10,000. Branched pathways were also shown to occur with several penicillins and cephalosporins.     

Biodegradation of N-methylated carbamates by free and immobilized cells of newly isolated strain Enterobacter cloacae strain TA7

A bacterial strain (TA7) capable of consuming three N-methylated carbamates as sole nitrogen and carbon source was isolated and identified as “Enterobacter cloacae” on the basis of 16S rRNA, from carbamate contaminated agricultural soil by enrichment culture technique. The agar entrapment was used to immobilize the bacterial cells. Both the free as well as the immobilized cells were used to study the degradation of three carbamets viz. aldicarb, carbofuran, and carbaryl. The immobilized cells degraded all the three carbamates much faster than their free cell counterparts. The biodegradation kinetics of aldicarb, carbaryl, and carbofuran was studied using 50 ppm as initial concentration in the presence of free cells. The average values of K_s for aldicarb, carbofuran, and carbaryl were 22.6, 17.87, and 8.9 mg/L, respectively, whereas the values for µ_max were calculated as 1.35, 1.3, and 1.2 mg/l/h^?1. The results indicated that the bacterium has high affinity towards all the three carbamates. However, relatively higher affinity is for carbaryl, in comparison with carbofuran and aldicarb. Results indicate the potential of E. Cloacae TA7 to remediate N-methylated carbamates polluted water and soil.     

A toxaphene-degrading bacterium related to Enterobacter cloacae, strain D1 isolated from aged contaminated soil in Nicaragua

Enterobacter sp. strain D1 is a facultative anaerobic, Gram-negative heterotrophic bacterium isolated from toxaphene-contaminated soil. This organism was identified and characterized through phylogenetic and taxonomic studies. Based on 16S rDNA analysis, the strain D1 was clustered closely with the species Enterobacter cloacae subsp. dissolvens (LMG 2683) and E. cloacae (ATCC 13047T). Strain D1 resembled these E. cloacae strains with respect to various biochemical and nutritional characteristics, but also exhibited differences. Moreover, strain D1 is able to grow and survive with toxaphene supplied in the medium in the range 3-96 mg/L. Amongst the chemical components of toxaphene, octachlorocamphenes, nonachlorobornanes and decachlorobornanes were seen to be rapidly metabolized, although levels of hexachlorocamphenes and heptachlorobornanes were found to be slowly degraded, and subsequently accumulated during the last stage of the cultivation.     

Genome Sequence of the Plant Growth-Promoting Bacterium Enterobacter cloacae GS1

Here, we present the genome sequence of Enterobacter cloacae GS1. This strain proficiently colonizes rice roots and promotes plant growth by improving plant nutrition. Analyses of the E. cloacae GS1 genome will throw light on the genetic factors involved in root colonization, growth promotion, and ecological success of this rhizobacterium.     

Study of a new group of Enterobacteriacea (group H1) related to Enterobacter cloacae strain]

A DNA-DNA hybridization study was carried out on a new group of enterobacteria (group H1) previously studied by numerical taxonomy work on the genus Enterobacter. This group showed a very high genetic homogeneity since the average relative binding ratio of nine analysed strains is equal to 91%. The taxonomic position of this group into the family of enterobacteria is discussed with the species E. cloacae (37 to 61%), K. pneumoniae (44 to 60%), K. oxytoca (57-58%), L. malonatica (syn. Citrobacter intermedius, a:46 to 54%), L. amalonatica (syn. C. intermedius, b: 51%), and the group H3 (52-61%). The group H1 is defined on phenotypic and genetic data.     

Direct sequencing of PCR products using the Maxam-Gilbert method

Direct sequencing of polymerase chain reaction (PCR) products by using the Maxam-Gilbert method is described. In this method, one of the primers is end labeled. Thus it is possible to sequence the reaction product directly following purification using this chemical method.     

Ascorbate as an induction inhibitor of β-lactamase in a strain of Enterobacter cloacae

H.A. SHOEB, H.I. AL-SHORA AND T. ABDEL-SALAM. 1995. The effect of ascorbate and anaerobiosis of β-lactamase content (constitutive and inducible) in relation to the susceptibility of a standard strain of Enterobacter cloacae to ampicillin was studied. Enterobacter cloacae ATCC 13047 showed increasing susceptibility to ampicillin when incubated anaerobically in the presence of increasing concentrations of ascorbic acid. The inducible β-lactamase activity in the cell-free extracts of Ent. cloacae decreased when the bacterium was grown aerobically in the presence of ascorbic acid. Under anaerobic growth conditions, however, ascorbic acid abrogated the induction of the enzyme completely. On the other hand, the constitutive enzymatic activity was markedly decreased as the bacterium was grown anaerobically. Thus under these growth conditions, ascorbate-anaerobiosis, the total β-lactamase level in the presence of ampicillin as inducer fell below the basal constitutive activity observed in the absence of ampicillin.     

Direct sequencing of the human microbiome readily reveals community differences

Culture-independent studies of human microbiota by direct genomic sequencing reveal quite distinct differences among communities, indicating that improved sequencing capacity can be most wisely utilized to study more samples, rather than more sequences per sample.     

Purification,structural characterization and biotechnological potential of tannase enzyme produced by Enterobacter cloacae strain 41

Tannase production by Enterobacter cloacae strain 41 was investigated under submerged fermentation which was optimized at various circumstances such as pH, temperature, substrate, and agitation, carbon, and nitrogen sources. Tannase was purified by a two-step approach comprising of ion exchange and size exclusion chromatography, respectively. The maximum tannase production was achieved at 1.0% tannic acid concentration, incubation temperature of 50 °C, and initial pH 6.0. The molecular weight of purified tannase was 45 kDa on 10% SDS-PAGE, and it was confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). The enzymatic products of purified tannase were characterized by HPLC, TLC and FT-IR spectroscopy which showed the functional groups such as OH, CO, and CC. The purified tannase retained the activity up to 90% under the condition at 50 °C and pH 6.0 after 1 h incubation. Enzyme kinetics and inhibition studies were also investigated. Cytotoxicity studies showed that the tannase has no cytotoxic effects on Vero cell line. The results indicated the E. cloacae strain 41 would give a potential source for the efficient production of tannase and can be used in tannery effluent degradation, food, and pharmaceutical industrial applications.     

Purification and Genetic Determination of Bacteriocin Production in Enterobacter cloacae

Enterobacter cloacae (strain DF13) was found to produce a bacteriocin which could be induced by mitomycin C. In the supernatant fluid of the induced culture phagelike particles were found. The bacteriocin was partially purified from induced cultures by ammonium sulfate precipitation and gel-filtration on Sephadex G-150. Ultraviolet-absorbing material was eluted from the Sephadex column in three fractions. The biological activity was mainly present in the second fraction and is associated with a protein with a molecular weight of about 61,000. The phagelike particles were found in the first fraction and show no biological activity. Upon conjugation of E. cloacae strain DF13 with another strain of the same species and with Escherichia coli K-12S, the ability to produce bacteriocin was transferred. The new bacteriocinogenic strain produced bacteriocin, which could not be distinguished from that produced by E. cloacae strain DF13. Although transfer of the bacteriocinogenic factor often occurred together with transfer of the ability to produce phagelike particles, it was shown that these two factors are two separate genetic entities. In addition to a bacteriocinogenic factor, E. cloacae strain DF13 was found to carry two other transferable plasmids: one determining resistance against streptomycin and sulfanilamide and another determining resistance against penicillin.     

Kinetics and modeling of hexavalent chromium reduction in Enterobacter cloacae

Kinetics of bacterial reduction of toxic hexavalent chromium (chromate: CrO(4) (2-)) was investigated using batch and fedbatch cultures of Enterobacter cloacae strain HO1. In fedbatch cultures, the CrO(4) (2-) feed was controlled on the basis of the rate of pH change. This control strategy has proven to be useful for avoiding toxic CrO(4) (2-) overload. A simple mathematical model was developed to describe the bacterial process of CrO(4) (2-) reduction. In this model, two types of bacterial cells were considered: induced, CrO(4) (2-)-resistant cells and uninduced, sensitive ones. Only resistant cells were assumed to be able to reduce CrO(4) (2-). These fundamental ideas were supported by the model predictions which well approximated all experimental data. In a simulation study, the model was also used to optimize fed-batch cultures, instead of lengthy and expensive laboratory experiments. (c) 1993 John Wiley & Sons, Inc.     

Mechanism of inhibition of the class C beta-lactamase of Enterobacter cloacae P99 by phosphonate monoesters.

The class C serine beta-lactamase of Enterobacter cloacae P99 was inhibited by a series of aryl methylphosphonate monoester monoanions. The effectiveness of these inhibitors was promoted by an acylamido substituent on the methyl group and a good leaving group at phosphorus. The former preference suggests that noncovalent interaction of these inhibitors with the enzyme resembles that of substrates, while the latter suggests that nucleophilic displacement at phosphorus occurs as part of the inhibition mechanism. The truth of the latter proposition was confirmed by observation of release of 1 equiv of phenol concomitant with inhibition and of the presence of an equivalent amount of 14C-label on the enzyme after inhibition by a 14C-labeled phosphonate. The hydrolytically inert nature of the enzyme-inhibitor adduct, and its 31P chemical shift, suggested that O-phosphonylation of the enzyme had occurred. Although, by analogy with substrates, one might expect that the hydroxyl of the active site serine residue would be covalently modified by these inhibitors, successive alkali and acid treatment of the enzyme-inhibitor adduct generated no pyruvate. Instead, 1 equiv of lysinoalanine was found. This product was rationalized to arise through intramolecular capture by an adjacent lysine amine group of the dehydroalanine residue produced by alkali treatment of an O-phosphonylated serine residue. One equivalent of lysinoalanine was also produced by alkali treatment of the enzyme that had been inhibited by 6 beta-bromopenicillanic acid, a mechanism-based inhibitor known to acylate the hydroxyl group of the active site serine residue. It is therefore likely that the aryl phosphonates phosphonylate this residue. These compounds should be useful as beta-lactamase active site titrants and as sources of fresh insight into the chemical properties of the active site. The significant mechanistic features of the inhibition, in particular its strong leaving group dependence and the distinctive ability of the beta-lactamase active site to stabilize a dianionic transition state containing a pentacoordinated phosphorus, are discussed with respect to the active site structure. The comparison with phosph(or/on)yl inhibitors of serine proteinases is made, and the mechanism-based features of inhibition of serine hydrolases by phosph(on)ates are noted.