- Volume 138, Issue 5, 1992

SUMMARY: Transposon Tn5, on a mobilizable ColE1 plasmid, on a Ti plasmid derepressed for bacterial transfer, and on the bacteriophage fd genome, was used to construct pathogenicity mutants of the fire blight pathogen Erwinia amylovora. Eleven nonpathogenic mutants were isolated from 1600 independent mutants screened. These mutants were divided into three types: auxotrophs, exopolysaccharide (EPS)-deficient mutants and a mutant of the dsp phenotype. According to their insertion sites the Tn5 mutants were mapped into several classes. Some of the mutants could be complemented with cosmid clones from a genomic library of the parent strain for EPS production on minimal agar. EPS-deficient mutants and the dsp mutant could complement each other to produce virulence symptoms on pear slices.

SUMMARY: Bacteriophage c6A is a lytic phage that infects strains of Lactococcus lactis. Infection of L. lactis strain C6 resulted in inhibition of culture growth within 10 min, mature intracellular phage particles appeared after 17.5 min, and cell lysis occurred after 25 min. A culture of strain C6 carrying 3H-labelled DNA was infected with c6A, and the fate of the radiolabel was monitored. The results showed that degradation of host cell DNA began within 6 min of infection and that the breakdown products were incorporated into progeny c6A DNA. Quantitative DNA hybridizations indicated that synthesis of phage DNA began within 6 min of infection and continued at an approximately constant rate throughout the latent period.

SUMMARY: Two different types of plasmid were isolated from strains of Rhodococcus rhodochrous. Two plasmids, of the same type but from different strains, were combined with Escherichia coli plasmids carrying antibiotic resistance markers to develop E. coli-Rhodocdccus shuttle vectors. The ampicillin and kanamycin resistance markers served for selection in Rhodococcus. Electroporation was used to introduce recombinant plasmid DNA into R. rhodochrous ATCC 12674 at a frequency of 5 × 107transformants per μg DNA. With these host-vector and transformation systems, the nitrile hydratase and amidase genes of a Rhodococcus strain were introduced into the host strain and were efficiently expressed.

SUMMARY: The gene nagC encodes the repressor for the nag regulon. A point mutation within the gene, which confers a superrepressor phenotype and makes the repressor insensitive to the inducer, N-acetylglucosamine 6-phosphate, has been characterized. The mutation is semi-dominant since heterozygous diploids have reduced growth rates on glucosamine and N-acetylglucosamine compared to the wild-type strain.

SUMMARY: λ-Lysin was purifed from Staphylococcus aureus strains Smith 5R and PG23 (a toxic shock syndrome isolate) by a combination of heparin-agarose and hydroxylapatite chromatography. Both strains produced two haemolytic components, designated λ1, and λ2. Though each component was weakly haemolytic they acted synergistically to potentiate haemolysis on rabbit, sheep and human blood. Rabbit and sheep erythrocytes were more sensitive to lysis by λ-lysin than human erythrocytes. The molecular mass of λ1 was 32 kDa and its pI value was 9.4. λ2 had a molecular mass of 36 kDa and a pI value of 9.3. While both trypsin and papain acted synergistically with λ2 to induce increased haemolysis, no such synergism was seen with λ1,. Also, protease inhibitors acted to inhibit synergism between λ1, and λ2. These findings suggest that λ1 could be a protease.

SUMMARY: The optimum conditions for autolysis of Clostridium acetobutylicum ATCC 824 were determined. Autolysis was optimal at pH 6.3 and 55 °C in 0.1 M-sodium acetate/phosphate buffer. The ability of cells to autolyse decreased sharply at the end of the exponential phase of growth. Lysis was stimulated by monovalent cations and compounds that complex divalent cations, and inhibited by divalent cations. The autolysin of C. acetobutylicum, which was mainly cytoplasmic, was purified to homogeneity and characterized as a muramidase. The enzyme was identical to the extracellular muramidase in terms of Mr , isoelectric point and NH2-terminal amino acid sequence. The autolysin was inhibited by lipoteichoic acids and cardiolipin but not by phosphatidylethanolamine and phosphatidylglycerol. A mechanism of regulation and fixation involving lipoteichoic acid, cardiolipin and divalent cations is proposed.

SUMMARY: In addition to the two siderophores pyoverdine and pyochelin synthesized by Pseudomonas aeruginosa ATCC 15692 (strain PAO1), several siderophores produced by other bacteria or fungi, namely cepabactin, salicylic acid, desferriferrichrysin, desferriferricrocin, desferriferrioxamine B, desferriferrioxamine E and coprogen, were able to promote iron uptake with variable efficiencies into this bacterium. For most of these siderophores, these results were consistent with the growth stimulation produced by the same compounds in a plate bioassay. Desferriferrichrome A, enterobactin and desferriferrirubin, however, did not promote iron uptake, although enterobactin and desferriferrirubin stimulated bacterial growth. These paradoxical data are discussed in view of siderophore-inducible iron uptake systems, as demonstrated recently for enterobactin. Among the strains tested, including the wild-type PAO1, the pyoverdine-less mutant PAO6606 and the two porin-mutants P. aeruginosa H636 (oprF::Ω) and P. aeruginosa H673 (oprD::Tn501), only for the porin-OprF mutant were fewer siderophores able to promote iron uptake compared to the other strains. Such results suggest that beside specific routes for iron uptake P. aeruginosa is also able to take up siderophore-liganded iron through OprF.

SUMMARY: Brevibacterium ammoniagenes ATCC 6872 was grown aerobically in minimal defined glucose media of different osmolalities induced either by NaCl, other eletrolytes or non-electrolytes. Growth rate was slightly affected by elevation of medium osmolality up to 1 M-NaCl, but severely decreased at 1.5 M; however even at 2 M-NaCl, slow growth still occurred. Glycine betaine (or its precursor choline) did not stimulate growth in high osmotic media, although it accumulated intracellularly and was not metabolized. An organic solute which increased substantially in concentration during osmotic treatment with various osmolytes, was isolated and identified as pipecolic acid. No accumulation of this imino acid was observed when the medium concentration was raised by adding the permeant glycerol or when the culture medium was supplemented with glycine betaine (1 mM). While this non-proteic cyclic amino acid may play an important role in bacterial adaptation to environmental stress, it did not accumulate to a high level. Preliminary data suggest that the biosynthesis of pipecolic acid from lysine is strongly regulated by external osmolality.

SUMMARY: Veillonella parvula cannot grow with succinate as sole energy source. However, succinate decarboxylation simultaneous with malate or lactate fermentation increased growth yields by 2.4–3.5 g (mol succinate)-1. Malate was fermented stoichiometrically to acetate and propionate whereas lactate fermentation produced more acetate and considerable amounts of H2. Aspartate was utilized only in the presence of succinate as co-substrate. Methylmalonyl-CoA decarboxylase and ATP-dependent pyruvate carboxylase, but not methylmalonyl-CoA: pyruvate transcarboxylase, were detected in cell-free extracts of malate- or lactate-grown cells. The energetic aspects of these fermentation patterns are discussed.

SUMMARY: We have examined the growth and photosynthesis of liquid cultures of the eukaryotic green alga Chlamydomonas reinhardtii incubated with iso-octane extracts of diesel particulate exhaust. At concentrations up to 0.125% (v/v), the extracts led to a dose-dependent growth retardation. Higher concentrations caused cell death. After a prolonged lag phase, air-grown illuminated algae, stressed with sublethal concentrations of diesel exhaust extracts, adapted to the pollutants. Further addition of toxic extract up to a concentration of 2.5 (v/v) affected neither growth nor photosynthesis. Media of adapted and non-adapted cells were analysed by HPLC and GC-MS and the concentrations of the remaining toxic compounds were determined. The results showed that the amounts of certain components decreased during incubation with adapted cells.

SUMMARY: Batch-mode growth rates of the acetogenic anaerobe Eubacterium limosum on various substrates were dependent on the point of entry into the central metabolic network. During batch growth on glucose/methanol mixtures, simultaneous consumption of both substrates occurred at residual glucose concentrations below 6 mM following an initial period of growth on glucose alone. During this mixotrophic growth phase, significantly higher biomass yields and specific growth rates were obtained than on either substrate alone. This phenomena was confirmed in chemostat experiments and stoichiometric analysis based on specific rates indicated that carbon and energy flow was significantly altered during mixotrophic growth with little net flux through the pyruvate ferredoxin oxidoreductase. It is suggested that such an effect on both growth rate and biomass yield was due to the improved availability of both carbon and energy for anabolic reactions.

SUMMARY: A synthetic medium was developed by the pulse and medium-shift technique for the continuous cultivation of Bacillus stearothermophilus strain LLD-15 (NCIMB 12428) under anaerobic conditions. This mutant strain lacks L-lactate dehydrogenase activity, and is a promising candidate for the production of ethanol from pentoses and hexoses, using a high-temperature two-stage process. The final medium contained four amino acids and five vitamins, and growth characteristics in this medium compared well with those in complex medium containing yeast extract and tryptone. At 70 °C, the medium was capable of supporting good anaerobic and aerobic growth at 10 g input sucrose I-1. High ethanol production indicated that pyruvate metabolism probably occurred via the combined activity of the pyruvate-formate-lyase pathway and pyruvate dehydrogenase.

SUMMARY: Three mutants, which were defective in the desaturation of fatty acids, were isolated from an arachidonic-acidproducing fungus, Mortierella alpina 1S-4, after treating wild-type spores with N-methyl-N′-nitro-N-nitrosoguanidine. They were designated Mut44, Mut48 and Mut49. Mut44 was a mutant with low Δ5-desaturase activity. It accumulated a high level of dihomo-λ-linolenic acid (DGLA) (28.6%, w/w) but a low level of arachidonic acid (Ara) (10.6%), compared with the wild type, which had levels of 6.3 and 47.0%, respectively. Mut48 was unable to desaturate oleic acid (18:1) to linoleic acid (18:2), i.e. Δ12-desaturation, and therefore a large amount of 18:1 (49.5%) accumulated and no fatty acid of the ω-6 family was detected. In addition, several fatty acids of the ω-9 family, such as 5,8,11-cis-eicosatrienoic acid, were found. In Mut49, 18:2 (46%) accumulated markedly, but only small amounts of DGLA and Ara were detected. Thus, Mut49 was considered to be defective in Δ6-desaturation. These mutants showed a somewhat longer lag phase than the wild type on cultivation at both 28 and 12 °C.

SUMMARY: A mutant of Bradyrhizobium (Parasponia) sp. ANU289 affected in the regulation of nitrogen metabolism was isolated. The mutant, designated ANU293, was unable to induce ammonium transport (Amt), nitrate reductase (NR) or glutamine synthetase II (GSII) activities under conditions that induce these activities in the wild-type. However, glutamine synthetase I (GSI), which is expressed constitutively in the wild-type, was present at normal levels in the mutant. The mutant also retained the ability to fix nitrogen in vitro and in planta, although nodule development on siratro (Macroptilium atropurpureum) was retarded. Southern blot analysis showed that ntrC, the product of which is involved in regulation of nitrogen metabolism, is the site of pSUP1021 insertion in ANU293. These results indicate that the transcriptional activator NtrC is required for the expression of Amt, NR and GSII, but not GSI or nitrogenase in Bradyrhizobium (Parasponia) sp. ANU289.