- Volume 138, Issue 3, 1992

Summary: The mechanism by which the aminoglycoside antibiotic streptomycin permeabilizes the cytoplasmic membrane of Escherichia coli cells was reinvestigated. For this purpose, the extent of streptomycin-induced K+ loss from cells growing at low external K+ concentrations was taken as a measure of membrane permeabilization. Experiments with different K+-uptake mutants showed that the antibiotic specifically increased the passive permeability of the cell membrane to K+ and other ions. These permeability changes were small and the membrane potential of the treated cells remained high. The membrane permeabilization was not due to a direct interaction of the antibiotic with the cell membrane, since cells that carry an rpsL mutation and synthesize proteins in a streptomycin-insensitive way did not lose K+ after the addition of the antibiotic. Due to misreading and premature termination of translation the cells synthesized aberrant proteins under the conditions where membrane permeabilization occurred. Two conditions are described under which the cells both degraded these mistranslated proteins rapidly and reaccumulated K+, lending support to the hypothesis that membrane permeabilization is due to the presence of the mistranslated proteins in the cell membrane. Evidence is presented that the irreversibility of (dihydro)streptomycin uptake by cells washed free from the antibiotic might also be due to rapid degradation of the mistranslated proteins, leading to ‘caging’ of the antibiotic inside the cells.

Summary: The effects of aminoglycoside and aminocyclitol antibiotics on intact cells of Escherichia coli were compared. The aminoglycosides streptomycin, gentamicin, kanamycin and neomycin had similar, but not identical, effects. They all caused misreading during protein synthesis, permeabilization of the cell membrane, inhibition of the initiation of DNA replication, and loss of cell viability. Cells treated with these antibiotics continued to synthesize two proteins (apparent molecular masses 72 and 60 kDa) that were not made by cells treated with the aminocyclitol hygromycin B, which did not cause misreading. Cells treated with the aminoglycosides regained their membrane tightness after residual protein synthesis in these cells had been inhibited by chloramphenicol, suggesting that under these conditions the mistranslated membrane proteins were rapidly degraded. The bacteriostatic aminocyclitols spectinomycin and kasugamycin did not cause membrane permeabilization, suggesting that these compounds do not cause misreading. Hygromycin B resembled these aminocyclitols in that it inhibited protein synthesis without causing misreading, membrane permeabilization or inhibition of initiation of DNA synthesis. However, hygromycin B also decreased cell viability. In a minimal medium this lethal effect began late in comparison to the process of inhibition of protein synthesis. It is concluded that hygromycin B is an atypical bactericidal antibiotic that strongly resembles the bacteriostatic aminocyclitols spectinomycin and kasugamycin in its action.

Summary: Nisin production by Lactococcus lactis subsp. lactis NIZO 22186 was studied in batch fermentation using a complex medium. Nisin production showed primary metabolite kinetics: nisin biosynthesis took place during the active growth phase and completely stopped when cells entered the stationary phase. A stringent correlation could be observed between the expression of the prenisin gene (nisA) and the synthesis of the post-translationally enzymically modified and processed mature nisin peptide. Moreover, it seemed likely that nisin had a growth control function. A physiological link is proposed between sucrose fermentation capacity and nisin production ability. Carbon source regulation appears to be a major control mechanism for nisin production.

Summary: Siderophore-negative mutants of Azotobacter vinelandii were generated by insertional mutagenesis with a Tn5 construct containing a promoterless luxAB fusion. The use of this construct, delivered on a suicide plasmid by conjugation, allowed the selection of mutations in iron-repressible genes by virtue of the expression of iron-regulated bioluminescence. Although many iron-regulated mutants were selected, only a few could be easily identified as defective in siderophore production. These included a non-fluorescent azotobactin-negative phenotype (strain D27), and strain F196, which had lost the ability to produce the catechol siderophores azotochelin and aminochelin as well as the lower-affinity chelator 2,3-dihydroxybenzoic acid. Strain D27 had normal production of catechol siderophores, while strain F196 produced 2·5 times as much azotobactin as the wild-type. Two other mutants demonstrated normal catechol levels and either low or relatively unrepressed azotobactin levels. Transformation of the DNA from strain F196 into another spontaneously obtained azotobactin-negative strain (UA1) resulted in strain P100, which was unable to produce the known siderophores. Unlike the wild-type and other siderophore-deficient mutants, this strain was unable to grow in the presence of the iron chelator ethylenediamine di-(o-hydroxyphenylacetic acid) (EDDHA; 50 mUg ml-1) unless stored iron was carried over in the inoculum. Strain P100 did grow on iron-limited medium containing EDDHA when the catechol or azotobactin siderophores were provided exogenously. However, strain P100 gave a positive result in the chrome azurol-S assay (CAS), a non-specific assay for siderophores. The CAS activity was iron-repressible and strain P100 was able to grow and accumulate more iron from the insoluble iron minerals FeS, vivianite and Fe3O4 than was available by simple diffusion or exchange. Therefore, it appears that iron-limited A. vinelandii produces an as yet unidentified low-affinity non-conventional (non-catechol, non-hydroxamate) siderophore.

Summary: Purified [14C]aerobactin, supplied exogenously to non-growing bacteria, was translocated via the periplasm into the cytoplasm of Escherichia coli K12 strains expressing the aerobactin receptor protein IutA. No significant uptake was observed into either compartment of strains lacking the iutA gene or specifically defective in tonB. Uptake into both compartments was markedly reduced, but not abolished, in an exb mutant. Accumulation of [14C]aerobactin in the periplasm of fhuD, fhuB or fhuC mutant strains was not significantly lower than in the wild-type strain, but entry into the cytoplasm was greatly reduced in all cases. Uptake of aerobactin by strains wild-type for all transport functions occurred most efficiently in strains either lacking or specifically defective in the genetic determinants for aerobactin biosynthesis; significantly lower levels of exogenous 14C-labelled siderophore were observed in both compartments of strains producing aerobactin. Aerobactin-mediated 59Fe uptake, however, was not inhibited by the presence of endogenous aerobactin. Endogenous enterochelin did not affect aerobactin uptake.

Summary: Spontaneous variation of the level of alginate synthesis in Pseudomonas aeruginosa was associated with changes in the activity of all four enzymes leading to synthesis of GDP-mannuronic acid, the activated precursor for polymerization. For the high-alginate-producing variant 8821M, alginate yield and properties, as well as the levels of alginate enzymes, were dependent on growth temperature. In contrast, levels of alginate and enzymes in the mucoid parent strain 8821 were very low and near temperature-independent. The difference in the specific activity of GDP-mannose dehydrogenase (GMD), encoded by the algDgene, between the two strains was associated with the alginate biosynthetic ability and with the degree of activation of the algD promoter, measured using the algD-xylE transcription fusion on plasmid pVD2X. Maximal activity of the four enzymes was observed in strain 8821M grown at 30 °, a temperature below the optimum for growth (35 °). The effect of temperature on GMD activity could not be explained by the regulation of the algD promoter by temperature, since expression of pVDZX appeared to be more active at 35 °, when the decrease of pVD2X copy number with increasing temperature was taken into account. The involvement of enzymes that catalyse steps downstream from the formation of the activated precursor should also be considered, as suggested by differences in the molecular mass of alginates synthesized by the two strains at various temperatures. Acetyl content of alginates increased as temperature decreased and strain 8821M produced the highest levels of acetylated polymers. The degree of acetylation appeared to be related to growth rate and could reflect acetyl-CoA availability.

Summary: The extracellular proteinase (EPR) of Candida albicans was induced in a medium containing bovine serum albumin as sole nitrogen source. There were two intracellular forms in cells induced to produce EPR, a 43 kDa protein (EPR) and a 45 kDa protein (cross-reacting material of EPR; CRM-EPR); these were detected by immunoblotting using anti-EPR antiserum. The 43 kDa protein (EPR) may be the same as the extracellular form judging by molecular mass, and the 45 kDa protein (CRM-EPR) may be a precursor form of EPR. Many dense granules were observed by electron microscopy near the plasma membrane of the mother cells in EPR-producing cells. Both the 43 and 45 kDa proteins were recovered in a membrane fraction and were solubilized by Triton X-100. When the membrane fraction was further fractionated by sucrose density gradient centrifugation, the 43 and 45 kDa proteins were differentially fractionated. This suggests that they were located in different membrane-bound structures and is consistent with an assumption that the 45 kDa protein is a precursor for EPR.

Summary: A pleiotropic dinitrogen and nitrate assimilation mutant was obtained by mutagenesis of Nostoc sp. strain ATCC 29133 with N-methyl-N-nitro-N-nitrosoguanidine followed by penicillin counterselection in the presence of NO- 3 and N2. Mutant strain UCD 223 was capable of reducing acetylene in the free-living growth state only under anaerobic conditions, or under atmospheric conditions when in symbiotic association with Anthoceros punctatus. Heterocysts of strain UCD 223 were noticeably lacking the cohesive outer polysaccharide layer of wild-type heterocysts. Oxygen microelectrode profiles of symbiotic Anthoceros-Nostoc tissue revealed an anaerobic environment in the symbiotic cavities containing Nostoc. The acetylene-reducing activities of strain UCD 223, and of its spontaneously-arising Fix+ revertant strain UCD 236, were not repressed by the presence of 10 mm-NO- 3 when in the free-living growth state, in contrast to wild-type Nostoc ATCC 29133. However, in situ activities of acetylene reduction by symbiotically associated Nostoc ATCC 29133 and strains UCD 223 and UCD 236 were repressed by the presence of 10 mm-NO- 3. It appears that the symbiotic cavities of Anthoceros punctatus can physiologically replace the function of the heterocyst outer wall and that the repression of nitrogenase activity in symbiotic Nostoc in response to the presence of NO- 3, and probably NH+ 4, is mediated by Anthoceros.

Summary: As part of a continuing effort to assess genetic variation among isolates of Borrelia burgdorferi we have determined the 16S rRNA signature nucleotide makeup of two tick isolates from the USSR. Signature nucleotides were identified via reverse transcriptase primer extension sequencing of select regions of the 16S rRNA molecule. In addition, the near complete 16S rRNA sequence of one of the isolates, R-IP3, was determined and utilized in a phylogenetic assessment. The sequence was aligned with the 16S rRNA sequences of other B. burgdorferi isolates as well as with other Borrelia species. Distance matrix analyses were performed and a phylogenetic tree was constructed. These analyses demonstrate that these isolates belong to a third previously unidentified genomic group of B. burgdorferi.

Summary: Isoenzymes from 23 isolates belonging to the fungal genus Pleurotus, subdivision Basidiomycotina, were examined by polyacrylamide gel isoelectric focusing and protein blotting. A numerical analysis was then done based on the zymograms for acid phosphatase, diaphorase, esterases, leucine aminopeptidase, phosphoglucomutase and phenoloxidases. The resulting dendrogram of taxonomic distances demonstrated the suitability of enzyme isoelectric focusing for use in systematics, as it clearly distinguished the four well-defined species P. cornucopiae, P. cystidiosus, P. eryngii and P. flabellatus. The species P. ostreatus, whose taxonomy is controversial, was separated from P. pulmonarius, and P. columbinus was classified as a distinct taxon. The delimitation of P. sajor-caju and P. sapidus strains appeared to be more difficult as they seemed to be closely related, the former with P. pulmonarius and the latter with P. ostreatus. Isoenzymes also proved to be valuable for Pleurotus strain characterization. Their potential application for providing biochemical markers useful in taxonomy is discussed.