- Volume 49, Issue 2, 1967

Soluble proteins extracted from mycelium of Penicillium griseofulvum were separated by acrylamide-gel electrophoresis. The pattern and overall intensity of the protein bands varied greatly with age of culture. A marked decrease in band intensity occurred at the time of exhaustion of the nitrogen source in shaken cultures, and at an earlier stage in static cultures; the total protein in whole or ultracentrifuged extracts did not decrease to the same extent. Changes in the pattern and intensity of protein bands during incubation occurred also in P. chrysogenum and P. frequentans. Mycelium of P. griseofulvum which was induced to sporulate in shaken culture yielded little protein as shown by electrophoresis at early stages of culture, in comparison with non-sporing mycelium. Each of these three Penicillium species could be distinguished by the protein pattern, which was reproducible and characteristic of the fungus at any particular stage of culture. The results indicate the need to determine the effects of age and conditions of culture when gel electrophoresis of mycelial proteins is used for taxonomic purposes.

Intraspecific and interspecific transformation in Pseudomonas has been investigated. Two isoleucine+valine requiring mutants of Pseudomonas aeruginosa have been transformed to prototrophy. By using gelatin liqui-faction as a marker character it was observed that two strains of P. fluorescens, a phytopathogen P. mangiferaeindicae and a strain of Pseudomonas originally isolated from fish were transformable. When P. solanacearum was used as a donor, the transformation frequency of P. mangiferaeindicae was very high. DNA from fish Pseudomonas I was capable of transforming two strains of P. fluorescens and P. mangiferaeindicae in addition to fish Pseudomonas 21. When P. fluorescens was treated with the DNA from P. aeruginosa transformation frequency was lower; the capacity of pigment production, however, could not be transferred to any of the strains. The highest transformation frequency was obtained towards the end of the log phase of growth of the receptor bacteria, with a DNA concentration of about 10 μg./ml. Optimum temperature for transformation was 25–30° and agitation and starvation in diluted media increased the number of transformants. The significance of the observations has been discussed from the point of view of relationships among the strains and species studied.

A computer survey was made to find the relationships of 120 strains of Gram-negative or Gram-variable non-motile coccoid rods, isolated by the author and formerly called Achromobacter. They were compared with 75 named strains of various Gram-negative genera, on the basis of morphological properties and biochemical tests. A recently developed similarity coefficient was used and strains were sorted into phenons by single linkage. Most of the strains were grouped at 72·5% similarity into one large phenon. Strains outside this phenon were: Pseudomonas aeruginosa (2), P. fluorescens (3), P. saccharophila (1), P. putrefaciens (1), Escherichia coli (1), Cellulomonas biazotiea (1), Arthrobacter globiforme (1). These were differentiated from the 72·5-phenon by various combinations of the following characters which were nearly always negative for strains within the phenon: fermentative metabolism of sugars, pigmentation, production of reducing compounds from gluconate, alkalinity from arginine, hydrolysis of starch or proteins.

Within the 72·5-phenon, five smaller phenons were distinguished, most of them grouped at 82·5 % similarity. The strains in each of these phenons and the characters possessed by a majority of strains and useful for the differentiation of these phenons were:

Phenons 1 and 5 were Gram-negative motile rods, usually peritrichous.

Phenon 1: Agrobacterium sp. (2), Achromobacter hartlebii (1), Agr. tumefaciens (1). Acid was produced oxidatively from glycerol, sucrose, fructose, lactose, arabinose, xylose, galactose and glucose; H2S produced in Kligler medium; the Kovacs oxidase test positive; nitrate reduced; growth on Simmons medium and on Paton medium; no growth on digest agar at 0° or 37°; not sensitive to 2·5 i.u. of penicillin.

Phenon 5: Alcaligenes faecalis (3), A. viscosus (1), A. denitrificans (1), A. booked (1), Alcaligenes sp. (2). Acid not produced from sugars; oxidase-positive; nitrate reduced; growth on Simmons medium; no growth on Paton medium; growth on digest agar at 37° but not at 0°; litmus milk became alkaline; not sensitive to 2·5 i.u. of penicillin.

Phenons 2, 3 and 4 were Gram-negative coccoid rods, cocci or short rods, often in pairs.

Phenon 2: Bacterium (or Acinetobacter or Achromobacter) anitratum (10), Achromobacter lacticum (2), Herellea sp. (8), Diplococcus mucosus (6), Moraxella Iwoffi (1), MJT isolate (1). Acid not produced from glycerol, sucrose or fructose; oxidative acid production from lactose, arabinose, xylose, galactose and glucose; oxidase-negative; nitrate not reduced; growth on Simmons medium and on Paton medium; growth on digest agar at 37° but not at 0°; litmus milk became acid; not sensitive to 2·5 i.u. of penicillin.

Phenon 3: Achromobacter sp. (1), MJT isolates (22). Acid not produced from glycerol, sucrose or fructose; oxidative acid production from lactose, arabinose, xylose, galactose and glucose; oxidase-positive; nitrate reduced; no growth on Simmons or Paton media; growth on digest agar at 0° but not at 37°; no pH change in litmus milk; sensitive to 2·5 i.u. of penicillin.

Phenon 4: Mima spp. (7), Achromobacter venenosum (1), Alcaligenes viscosus (2), Moraxella Iwoffi (4), Achromobacter sp. (2), Neisseria eatarrhalis (1), MJT isolates (93). Acid not produced from sugars; no growth on Simmons medium; growth on digest agar at 0° but not at 37°; no pH change in litmus milk; sensitive to 2·5 i.u. of penicillin. Other characters variable.

Phenons 4i, 4ii, 4iii: small phenons grouped at about 92·5% similarity within Phenon 4. In properties other than sugar oxidation, Phenon 4i resembled Phenon 3, and Phenons 4ii and 4iii resembled Phenon 2, but not so closely.

The minimum inhibitory concentration of penicillin was estimated for selected strains of Phenons 2, 3 and 4, and varied from <0·1 to > 100 i.u./ ml. Strains of Phenons 3 and 4i were very sensitive, those of Phenons 2 and 4iii were resistant, and those of 4ii were intermediate or resistant. Ungrouped strains of Phenon 4 varied from very sensitive to resistant.

It was concluded, on the basis of these results and the data of Mandel & Thornley (1967) on DNA composition, that the non-motile coccoid rods in Phenons 2, 3 and 4 should be placed in a separate genus from other strains in the survey. Acinetobacter Brisou & Prévot is the most suitable generic name, and strains in Phenon 2 correspond to the type species, Acinetobacter anitratus. The relationships of Phenon 3 and the sub-divisions of Phenon 4 need further study, and it is not suggested that they should be given specific rank at present.

Phenons 2, 3 and 4 included strains formerly called Alcaligenes viscosus and Achromobacter; their classification as Acinetobacter entails division of the genera Alcaligenes and Achromobacter. The genus Alcaligenes should be retained for strains resembling the motile, peritrichously flagellate type species, A. faecalis, represented in this study in Phenon 5. It is suggested that the genus Achromobacter also should be reserved for any motile peritrichous strains which may prove suitable for inclusion. The few strains in this survey which had these properties were not very similar to each other, and the borderline between these strains and Agrobacterium was not clear. The strain atcc 15716, proposed as representative of the type species Achromobacter liquefaciens (Tulecke et al. 1965) was unlike all other strains in the survey; atcc 15716 fermented sugars and contained a large proportion of Gram-positive cells, and may be closer to a Gram-positive genus than to those studied here.

The behaviour of certain intracellular components contained in the mycelium of Aspergillus terreus during autolysis were studied. Aspergillus terreus grown in an acid medium underwent an ‘acid autolysis’ in which a decrease in mycelial dry weight amounting to 14·6% occurred. The content of fat present in the autolysing mycelium continuously decreased throughout the pre-autolytic and autolytic stages. The concentration of free glucose in the mycelium continuously increased at the beginning of autolysis, whereas xylose soon disappeared when autolysis began. At the end of the log phase and during the pre-autolytic stages the content of mannitol sharply decreased, at the beginning of autolysis its concentration decreased at a slower rate, being constant at the end of the autolytic period studied. Fourteen different amino acids were found in the autolysing mycelium of A. terreus. Eighty-six% of the maximum concentration of these free amino acids disappeared from mycelium before autolysis; 81% of the remainder during autolysis.

Treatment of competent cells of Bacillus subtilis with sodium periodate reduces markedly genetic transformation, at concentrations not significantly affecting cell viability. The action of periodate is not on DNA transforming activity. A reduction of transformation by this reagent is also obtained either if cells are treated before DNA addition or if they are first exposed to donor DNA for short time and then treated with periodate; if the cells have been pre-incubated with DNA for about 30 min. the subsequent addition of NaIO4 is without effect. The action of periodate seems to be exerted on the adsorbtion and/or entry of DNA into the competent cells. The site of action of periodate is probably a component(s) of the cell wall involved in these processes.

Production of β-lactamase by nine strains of the genus Enterobacter (eight Enterobacter cloacae, one E. aerogenes) was studied to determine its inducibility. Induction was observed with benzylpenicillin (500 μg./ml.) in all except one strain. Cultures were examined to locate the enzyme; it was found that in exponential growth the enzyme was cell-bound, and in stationary phase cultures much of it was in the culture medium. Maximum enzyme activity was only demonstrated after cell-breakage. Substrate profiles of crude enzyme preparations were examined and it was observed that the enzymes were 20–80 times more active against cephalosporins than against benzylpenicillin. Evidence is presented which suggests that one strain of E. cloacae produced two β-lactamases, an inducible cephalosporinase and a constitutive peni-cillinase.

The increase in size of Trichoderma (strain IMI 110150) spores during germination has been measured using the Coulter Counter. For sizing with this instrument, the spores were transferred from nutrient medium into dilute electrolyte solution and this resulted in an apparent delay in the onset of swelling. The length of this lag period was related to the concentration of saline electrolyte and not to the rate of swelling or the size of the spores.

Treatment of the radiation-resistant bacterium Micrococcus radiodurans with ultraviolet (u.v.) radiation and N-methyl-N′-nitro-N-nitrosoguanidine resulted in the isolation of two mutants highly sensitive to u.v. radiation. They were also sensitive to ionizing radiation and to the action of N-methyl-N′-nitro-N-nitrosoguanidime. The concentrations of sulphydryl groups in bacteria of the wild type and the mutants were not significantly different. Although the mutants were more sensitive to mitomycin C than the wild type the resistance of the latter was low. It is suggested that the DNA repair mechanism in the wild type operates very efficiently for the removal of single strand damage but not for that which involves cross-linking.

The utilization of acetate by blue-green algae was examined and the activities of enzymes involved in its metabolism measured. Although acetate did not stimulate the endogenous respiration of these organisms, the oxidation of acetate was followed by the rate of release of [14C] carbon dioxide from [1-14C] and [2-14C] sodium acetate. Similarly, sodium acetate did not alter the rate of growth of Anabaena variabilis and Anacystis nidulans but in A. variabilis it was found to contribute 7·2% of the dry weight when cultures were gassed with air + CO2 (95+5, v/v), and 16·9% when gassed with air alone. The presence of acetate in the growth medium did not alter the activity of the acetate-activating enzymes, glyoxylate cycle enzymes or two tricarboxy-lic acid cycle enzymes. The failure to show enzyme adaption by these organisms when supplied with an exogeneous substrate is discussed in relation to their hitherto apparently autotrophic nature.

Studies on the activities of suspensions of Isotricha intestinalis and I. prostoma freshly isolated from sheep rumen and washed almost free from bacteria were made by using [14C]-amino acids and 14C-labelled Escherichia coli. Bacteria were taken up by the protozoa at an approximately linear rate of up to 3000 bacteria/protozoon/hr for 24 hr, after which time at least 40% of the bacterial carbon was no longer in the form of whole bacteria. Arnino acids supplied to the protozoa, either free in the medium or as intact bacteria, were incorporated unchanged into protozoal protein. Addition of individual free 12C-amino acids to the medium partially inhibited incorporation into protozoal protein of 14C from Escherichia coli labelled with the 14C form of the same amino acid.

Twenty-nine arginine auxotrophs of Proteus mirabilis strain 13 have been divided into eight groups which represent different biochemical blocks in the arginine biosynthetic pathway. The mutants were classified according to their growth requirements, syntrophism, accumulation of intermediates and enzyme deficiencies. The steps in the pathway are the same as those of Escherichia coli although mutants will not utilize N-α-acetyl-l-ornithine or N-acetyl-l-glutamate. However, these two substances were shown to be intermediates. Ornithine is synthesized via the linear route common to entero-bacteria and not by transacetylation between N-α-acetyl-l-ornithine and l-glutamate as in some other bacteria and yeasts.

Motility was transduced by phage P22 to non-motile (fla –, etc.) Salmonella typhimurium strains, and the flagellar antigens of abortive transductants inferred from the inhibition by antisera of the trails they produced in semi-solid medium. When the recipient had a fla – gene closely linked to H1 (phase-1 flagellar antigen locus) nearly all the fla + abortive transductants evoked from a recipient culture in latent phase 1 manifested the phase-1 antigens of both recipient and donor, whereas those from a culture in latent phase 2 showed neither phase-1 antigen. Thus the expression of an exo-genote H1 allele, like that of the chromosomal H1 allele, was regulated by the phase-determinant of the recipient, at or near H2 (phase-2 flagellar antigen locus). An H1 gene adjacent (cis) to ah1 – (H1 activator gene), in the chromosome or in the exogenote, was unexpressed in ah1 + or fla + abortive transductants in phase 1. This suggests that ah1 – mutants are H1-operator-negative mutants.

When the recipient was a phase-1, and therefore non-motile, culture of an ah1 – or ‘phase-1-curly’ mutant, lysates of phase-2 cultures, but not of phase-1 cultures of the same donor, evoked trails, attributable to H2 abortive transductants. They expressed the donor H2 allele, but not the recipient H2 allele—nor the previously expressed H1 allele of the ‘phase-1-curly’ recipient. It is inferred that a phase-determinant regulates the expression of the H2 gene adjacent (cis) to it (or of which it forms part) but not that of another H2 gene in the same cell; and that it controls the expression of H1 via a represser substance, not via an inducer. The exceptional H1 allele H1-1,2 determines a flagellin of antigenic character 1,2, apparently identical with that determined by a common H2 allele. H1-1,2 and the common H1 allele H1-b were simultaneously expressed in fla + abortive transductants, which suggests that neither H2 flagellin nor H2 messenger RNA functions as the repressor of H1. It is proposed that an operon, comprising H2 and the structural gene for an H1 repressor substance, has alternative metastable states, ‘on’ in phase 2 and ‘off’ in phase 1, comparable to the wild-type state and to that of an operator-negative mutant.