- Volume 139, Issue 4, 1993

A promoter-probe vector (pHX200) was constructed using the broad-host-range cosmid pLA2917 and a promoterless (xylE gene of Pseudomonas as the reporter gene. Insertion of the cloned promoter fragment of the methanol dehydrogenase large subunit gene moxF (methanol oxidation) in front of the xylE gene in pHX200V-47 resulted in high-level expression of the xylE gene product catechol 2,3-dioxygenase in Methylobacterium organophilum XX. The specific activity of the enzyme was four times higher in methanol-grown M. organophilum XX culture than in succinate-grown culture. Interestingly, the insertion of the same fragment in the opposite orientation in front of the xylE gene (pHX200V-74) also led to elevated catechol 2,3-dioxygenase activity. This promoter activity was also methanol regulated. A total of 21 methanol-regulated promoter clones were identified that originate from three gene clusters (groups V, VI and VII) on the M. organophilum XX chromosome involved in methanol oxidation. Vector pHX200 and its derivatives were successfully mobilized into cells of three phylogenetically diverse methylotrophic bacteria: Methylophilus methylotrophus AS1, Methylobacterium extorquens AM1 and Methylobacterium sp. DM4. The reporter gene (xylE) was functionally expressed in all three bacteria with the aid of a proper promoter. Transcriptional fusions of methanol-regulated promoters with the xylE gene were mobilized into Mox− mutants of M. organophilum XX and M. extorquens AM1 to study the roles of methanol oxidation genes, especially regulatory genes. It appeared that vector pHX200 is an efficient promoter probe with wide host-range and an excellent tool for studies of structure and function of promoters/regulators.

The gene encoding cyclohexadienyl dehydratase from Pseudomonas aeruginosa, designated pheC, was cloned in Escherichia coli and sequenced recently by Zhao et al. (Journal of Biological Chemistry 267, 2487–2493, 1992). N-Terminal sequencing of the purified cyclohexadienyl dehydratase yielded a run of 11 residues which matched perfectly with the deduced amino acid residues 26–36. This showed that a 25 residue peptide was cleaved from the N-terminus of a preprotein formed in E. coli. The amino acid composition of the 25 residue peptide was typical of signal sequences for periplasmic proteins. Most or all of the cyclohexadienyl dehydratase was released from P. aeruginosa and E. coli carrying the pheC gene following spheroplast formation, osmotic shock or chloroform treatment. The location of the enzyme in the periplasm of both E. coli and P. aeruginosa was confirmed by Western blotting analysis using antibody prepared against PheC. Electron microscopy using immunogold labelling showed an apparent localization of cyclohexadienyl dehydratase at the polar regions of the periplasmic space in E. coli.

We have studied the immunological relatedness of LamB proteins from a wide range of enterobacterial species, using antibodies directed against denatured Escherichia coli K12 and Klebsiella pneumoniae LamB proteins (LamBE.c. and LamBK.p., respectively), and anti-peptide antibodies directed against 10 distinct loops of LamB from E. coli K12 predicted to protrude either side of the outer membrane. We have shown that a protein immunologically related to LamBE.c. and LamBK.p. was present in all members of Enterobacteriaceae tested. A protein recognized by several anti-peptide antibodies was identified in E. coli, Shigella sonnei, Salmonella typhimurium and Kleb. pneumoniae, as well as in two Citrobacter species, two Enterobacter species and Kluyvera ascorbata. The recognition patterns obtained with the anti-peptide antibodies were in agreement with the LamB protein sequence data available. They indicated that the cell surface and also the periplasmic loops of LamB are subject to great antigenic variability.

Cells of the bacterium Buchnera were isolated from embryos of the pea aphid Acyrthosiphon pisum, with an intact perisymbiont membrane (the insect membrane which surrounds each bacterial cell inside the aphid). The bacterial preparations respired aerobically, consuming oxygen at an average rate of 24 nmol (mg protein)−1 min−1. The bacteria took up a range of carboxylic acids and the amino acids glutamate and aspartate from an external concentration of 0·5 mm at rates of 1–10 nmol (mg protein)−1 h−1; glucose was taken up at 0·17 nmol (mg protein)−1 h−1. Glutamate uptake was proportional to its external concentration, at all concentrations tested between 15 μm and 10 mm. Saturable systems for the uptake of succinate and aspartate were identified. The kinetic constants were: K m 0·79 mm, V max 12·6 nmol (mg protein)−1 min−1 for succinate; and K m 0·22 mm, V max 3·3 nmol (mg protein)−1 min−1 for aspartate. Succinate uptake was not inhibited by the uncoupler CCCP and was markedly stimulated by ATP, suggesting that its transport is not linked to a proton-motive force but is dependent on an energized membrane and possibly mediated by a co-transport system involving another ion.

Caesium accumulation by Chlorella salina, from buffer (pH 8.0) supplemented with 50 μm-CsCl and 137Cs, continued for approximately 15 h and displayed first-order kinetics, indicating a single rate-limiting transport process. Efflux of Cs+ from Cs+-loaded cells occurred in two distinct phases: a rapid initial loss, representing approximately 11% of total cellular Cs+, corresponded to release from the cell surface, whereas a second, slower, phase of efflux corresponded to loss from the cytoplasm and vacuole. Analysis of subcellular Cs+ compartmentation revealed that most Cs+ was accumulated into the vacuole of C. salina, with lesser amounts being associated with the cell surface or located in the cytoplasm. Uptake of Cs+ into the vacuole was correlated with a stoichiometric exchange for K+. However, no loss of K+ from the cell surface or cytoplasm was evident nor was Cs+ or K+ associated with insoluble intracellular components. Calculated values for the Cs+ flux across the vacuolar membrane were approximately equal to, or higher than, values for total cellular influx. Cs+ influx obeyed Michaelis-Menten kinetics over the lower range of external Cs+ concentrations examined (0.01−0.25 mM) and a single transport system with a K m ~ 0.5 mm was evident. The effects of other monovalent cations on Cs+ influx implied that K+ and Rb+ were competitive, and NH+ 4 non-competitive/uncompetitive inhibitors of Cs+ uptake. The order of inhibition was Rb+ > K+ > NH+ 4. We propose that a single, relatively non-selective, rate-limiting transport system for Cs+ influx is located on the cytoplasmic membrane of C. salina, while a more permeable vacuolar membrane facilitates transport of Cs+ into the vacuole.

The need for consistent nomenclature and accurate assessment of late growth phases in diauxic yeast cultures is highlighted by the substantial variation of stress tolerance in Saccharomyces cerevisiae after the exhaustion of the initial fermentable carbon source. At present, a wide variety of assessment methods and confused terminology exists in the literature, leading to difficulties in the interpretation and comparison of published results. A method based on the depletion of ethanol accumulated during the respiro-fermentative growth phase is suggested as suitable for assessing subsequent growth phases and reporting results. Consistent application of nomenclature for growth phases is recommended to assist the interpretation of published experimental results. It is suggested that the phases of growth in diauxic batch culture should be referred to using the terms (1) initial lag phase, (2) respirofermentative phase, (3) diauxic lag phase, (4) respiratory phase, (5) stationary phase, and (6) death phase.

The halogenated fluorescein derivatives: 2′,4′,5′,7′-tetrabromofluorescein isothiocyanate dextran (Br4FD) and 4′,5′-diiodofluorescein isothiocyanate dextran (I2FD), were found to be efficient photosensitizers for the production of singlet oxygen. The singlet oxygen quantum yields were determined by reaction with acceptors in aqueous solution. Their comparison showed that the singlet oxygen quantum yield of Br4FD was threefold higher than that of I2FD. Br4FD was more resistant to photobleaching than I2FD. Both derivatives were internalized by fluid-phase pinocytosis in amoebae of the cellular slime mould Dictyostelium discoideum. Subsequently, illumination of cells led to a dose-dependent loss of viability consistent with a role for singlet oxygen generated inside the endosomal compartments in the mechanism of photoinjury. Br4FD showed a three-fold higher efficiency than I2FD for photoinduced cytotoxicity.