Antimicrobial action of N-(n-dodecyl)diethanolamine on Escherichia coli: effects on enzymes and growing cultures

Aims:  This study investigates the effects of N-(n-dodecyl)diethanolamine (DDA) on enzymes and growing cells of Escherichia coli NCIMB 8277. Methods and Results:  Enzyme activities in the presence of DDA were determined by measuring substrate-dependent oxygen consumption by whole cells, or of NADH formation or oxidation by cell extracts. Lysis of growing cells was followed by measuring changes in turbidity and cell count. DDA promptly arrested oxygen uptake on pyruvate and acetate, due to cofactor loss rather than to enzyme denaturation, since cell-free glyceraldehyde-3-phosphate and NADH dehydrogenases remained active. Formate and succinate oxidation by membrane-bound enzyme systems independent of cofactors was likewise unaffected. DDA lysed growing cells at rates related to drug concentration, pH, and the previous growth rate. Conclusions:  Loss of cellular enzyme activity following addition of DDA is due to cofactor leakage and not to enzyme denaturation. Whereas nongrowing cells remain intact in the presence of DDA, actively-growing organisms undergo lysis, consistent with autolysin action. Significance and Impact of the Study:  Cell lysis, not normally observed with membrane-active antimicrobials, also occurs with cetrimide, and may be dependent on the alkyl chain length in these compounds. The action on growing cells parallels that of penicillin and daptomycin, which bears a decanoyl residue that penetrates the cell membrane, causing leakage and membrane depolarization

Further evidence for the ionospheric contribution to convection within the plasmasphere

VLF goniometers have been used to study the direction of plasma convection in the evening plasmasphere. The results suggest that sometimes even for L values greater than 4 the electric field associated with the ionospheric dynamo may be the source which drives convection

Identification and sequencing of a novel insertion sequence, IS1471, in Burkholderia cepacia strain 2a

A novel insertion sequence (IS), IS1471, has been identified which is inserted into IS element IS1071 possessed by plasmid pTJB1 in Burkholderia cepacia strain 2a. Nucleotide sequencing has revealed that IS1471 is 1112 bp in length and is flanked by 22/21-bp imperfect inverted repeats with a 3-bp duplication of the target sequence. IS1471 contains a single open reading frame encoding a putative polypeptide of 345 amino acids with molecular mass of 39 406 Da. Searches of DNA and protein sequence databases did not result in the detection of any homologous IS elements, suggesting that IS1471 is novel and may not belong to any known IS groups

Complete characterisation of Tn5530 from Burkholderia cepacia strain 2a (pIJB1) and studies of 2,4-dichlorophenoxyacetate uptake by the organism.

The complete genetic characterisation of Tn5530 in Burkholderia cepacia strain 2a (pIJB1) has been accomplished, indicating that it is a Tn3-like transposon with a complex structure bearing operons for the catabolism of 2,4-dichlorophenoxyacetate (2,4-D) and malonate. Tn5530 is terminated at both ends by the IS1071::IS1471 element and the 2,4-D- and malonate-dissimilatory operons are separated by a region encoding a putA and lrp gene and a gene encoding a chloride channel protein. The chloride channel protein may have a role in the expulsion of chloride ions liberated by the dissimilation of 2,4-D. In addition, a putative transposase with a high level of sequence similarity to those of plasmid pGH1 from Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. glycinea, and a transcription factor similar to those of the TetR family with low but significant levels of sequence similarity to those identified in a number of other organisms was observed. The entire Tn5530 sequence length, including the IS1071::IS1471 elements, was found to be 40,956bp, and pIJB1 was replicon-typed and otherwise characterised as being of the IncP-1beta subgroup, bearing merA and merD genes conferring resistance to mercuric chloride. The rate of uptake of 2,4-D by B. cepacia strain 2a was observed to proceed more readily at acid pH, suggesting involvement of the undissociated form of the compound. Uptake did not show saturation kinetics, was concentration-dependent, and appeared to occur in two stages; an initial accumulation followed by a linear second phase. Uptake could be inhibited by sodium azide but not by arsenate, N,N(')-dicyclohexylcarbodi-imide (DCCD) or carbonylcyanide m-chlorophenyl-hydrazone (CCCP) suggesting that it is not energy-dependent

2,4-Dichlorophenoxyacetate/alpha-ketoglutarate dioxygenases from Burkholderia cepacia 2a and Ralstonia eutropha JMP134

2,4-Dichlorophenoxyacetate (2,4-D)/alpha -ketoglutarate (alpha -KG) dioxygenase has been purified to apparent homogeneity from Burkholderia cepacia strain 2a, which utilizes 2.4-D as sole carbon source. The enzyme required ferrous ions, and was a homodimer composed of subunits having an M-r of similar to 32,000. The reaction catalysed consumed one mol each of 2,4-D, alpha -KG and dioxygen, with the production of one mol each of succinate, 2,4-dichlorophenol and glyoxylate. Maximum activity was exhibited at pH 7.8 and 25 degreesC, and reactivity was enhanced by the presence of ascorbate and cysteine. Mn2+, Zn2+, Cu2+ Fe3+ and Co2+ were inhibitory, and chemical modification of the dioxygenase revealed that thiol groups were essential for activity. The enzyme was active towards other substituted phenoxyacetates, but reacted most rapidly with 2,4-D. The apparent Michaelis constants for 2,4-D and alpha -KG were 109 and 8.9 muM, respectively. The properties of this enzyme are compared with those of the 2,4-D/alpha -KG dioxygenase from Ralstonia eutropha JMP134, which exhibits a differing N-terminal amino-acid sequence, and a different temperature 'optimum', pH optimum, substrate specificity and sensitivity to thiol-binding reagents

A novel plasmid pIJB1 possessing a putative 2,4-dichlorophenoxyacetate degradative transposon Tn5530 in Burkholderia cepacia strain 2a

A 102-kb plasmid, pIJB1, was isolated from Burkholderia cepacia strain 2a, which is able to use 2,4-dichlorophenoxyacetate (2,4-D) as a sole carbon source, and a physical map of the plasmid has been established. It was observed that spontaneous loss of a 37-kb fragment of the plasmid after growth in nonselective medium occurred, generating a plasmid of diminished size, pIJB2. The deletion event is concomitant with the loss of the 2,4-D dissimilatory phenotype, indicating that at least some of the 2,4-D degradative genes are on the missing fragment. The missing fragment is flanked by two identical 4.3-kb insertion sequences (IS) and shows a typical composite transposon structure of 41-kb in size, designated Tn5530. The mutant plasmid pIJB2 possesses a single copy of the IS element