Transcription in Escherichia coli PHL628 biofilms.

The biofilm-specific gene expression of Escherichia coli PHL628 was compared with that from exponentially growing planktonic cells using macroarray technology. In duplicate experiments, both biofilm and planktonic cells were grown in separate continually stirred tank reactors at 57% of the maximal planktonic growth rate. When transcriptional results from planktonic cultures were compared with that of biofilm grown cells, c. 4.5% of the genome showed a significant change in expression. The results presented here point to an extremely heterogeneous biofilm wherein specific gene induction was consistent with the response of biofilm cells to gradients in electron acceptors, nutrients, carbon source and a variety of stresses. A mutant in one of the genes, gspM (pshM), that was induced in biofilms was constructed and was shown to be compromised for its ability to form mature biofilms. This analysis provides additional insight into the genes induced during biofilm development, the gradients they respond to, the contribution of one gene to biofilm development, and a comparison of this with other transcriptional profiles from E. coli biofilms.

Immunoaffinity extraction of 4-hydroxy-2-(4-methylphenyl)benzothiazole and its metabolites for determination by gas chromatography-mass spectrometry.

Immunoaffinity extraction of 4-hydroxy-2-(4-methylphenyl)benzothiazole and its metabolites, together with the corresponding meta-isomers has been achieved by the use of an antibody raised against an immunogen, an O-carboxymethyloxime-bovine serum albumin conjugate of 4-hydroxy-2-(4-formylphenyl)benzothiazole. The antibody produced exhibited a broad spectrum of affinity, not only for metabolites oxidized at the 4-methyl group of the benzene moiety but also for the corresponding meta-isomers. Up to 4 micrograms in total of these benzothiazoles could be extracted on the immunoaffinity adsorbent and recovered almost quantitatively by elution with 90% methanol. The resulting chromatogram was free from any interference. The eluted compounds were derivatized by conversion to their methyl esters and/or trimethylsilyl ethers, and subsequently separated into individual benzothiazoles by means of gas chromatography-mass spectrometry. The derivatized compounds were monitored using a characteristic ion, [M-CH3]+., and the limit of detection was 10 fmole. The peak height ratio of each metabolite to its corresponding meta-isomer internal standard was plotted against the concentration of the former and good linearity was observed over the range 0.2-5 ng/ml.