Soil bacterium DNA was isolated by minor modifications of previously described methods. After purification on hydroxyapatite and precipitation with cetylpyridinium bromide, the DNA was sheared in a French press to give fragments with an average molecular mass of 420,000 daltons. After repeated hydroxyapatite purification and precipitation with cetylpyridinium bromide, high-pressure liquid chromatography analysis showed the presence of 2.1% RNA or less, whereas 5-methylcytosine made up 2.9% of the total deoxycytidine content. No other unusual bases could be detected. The hyperchromicity was 31 to 36%, and the melting curve in 1 X SSC (0.15 M NaCl plus 0.015 M sodium citrate) corresponded to 58.3 mol% G+C. High-pressure liquid chromatography analysis of two DNA samples gave 58.6 and 60.8 mol% G+C. The heterogeneity of the DNA was determined by reassociation of single-stranded DNA, measured spectrophotometrically. Owing to the high complexity of the DNA, the reassociation had to be carried out in 6 X SSC with 30% dimethyl sulfoxide added. Cuvettes with a 1-mm light path were used, and the A275 was read. DNA concentrations as high as 950 micrograms ml-1 could be used, and the reassociation rate of Escherichia coli DNA was increased about 4.3-fold compared with standard conditions. C0t1/2 values were determined relative to that for E. coli DNA, whereas calf thymus DNA was reassociated for comparison. Our results show that the major part of DNA isolated from the bacterial fraction of soil is very heterogeneous, with a C0t1/2 about 4,600, corresponding to about 4,000 completely different genomes of standard soil bacteria.(ABSTRACT TRUNCATED AT 250 WORDS) Images

High diversity in DNA of soil bacteria.

The chemistry and use of lysozyme as a food preservative and a pharmaceutical are reviewed. Lysozyme inhibits the growth of deleterious organisms, thus prolonging shelf life. Chemicals used to improve the preservative effect of lysozyme and those that inhibit the enzyme are discussed, along with the stability of lysozyme in various chemical environments. Lysozyme has been used to preserve fresh fruits and vegetables, tofu bean curd, seafoods, meats and sausages, potato salad, cooked burdock with soy sauce, and varieties of semihard cheeses such as Edam, Gouda, and some Italian cheeses. Lysozyme added to infant‐feeding formulas makes them more closely resemble human milk. Lysozyme has been used clinically in the treatment of periodontitis, administered in chewing gum, and implemented to prevent tooth decay. It has also been administered to patients suffering from cancer for its analgesic effect and has been used as a potentiating agent in antibiotic therapy.

The chemistry of lysozyme and its use as a food preservative and a pharmaceutical.

A total of 23 strains of coryneform bacteria that cause ratoon stunting disease of sugarcane and Bermudagrass stunting disease were examined. These included 17 sugarcane strains from Florida, Louisiana, South Africa, Brazil, and Japan and six Bermudagrass strains from Florida and Taiwan. The sugarcane and Bermudagrass strains contained 2,4-diaminobutyric acid, rhamnose, and fucose in their cell walls, suggesting a relationship to some other phytopathogenic coryneform bacteria and related organisms with similar cell wall compositions. The cellular protein patterns of the sugarcane and Bermudagrass strains after polyacrylamide gel electrophoresis resembled the patterns of Corynebacterium michiganense subspecies more than the patterns of some other diaminobutyric acid-containing taxa. The protein patterns of the sugarcane strains were distinct from the patterns of the Bermudagrass strains examined. The guanine-plus-cytosine contents of the deoxyribonucleic acids of two sugarcane strains and one Bermudagrass strain were 66 mol%. The sugarcane and Bermudagrass strains formed a homogeneous group based on morphological characteristics and most biochemical characteristics. All of the sugarcane strains were nonpigmented (white) and grew more slowly in culture than the yellow-pigmented Bermudagrass strains. The Bermudagrass strains, unlike the sugarcane strains, hydrolyzed starch and utilized citrate and malate. Although the phytopathogenic coryneform bacteria with 2,4-diaminobutyric acid in the peptidoglycans of their cell walls are presently classified in the genus Corynebacterium, recent studies have indicated that these phytopathogens should be reclassified. These bacteria form a distinct group with characteristics different from those of all existing genera as presently circumscribed; therefore, we propose that a new genus, Clavibacter, be established for these and similar bacteria. We propose reclassification of Corynebacterium michiganense (including Corynebacterium michiganense subsp. michiganense, Corynebacterium michiganense subsp. insidiosum, Corynebacterium michiganense subsp. sepedonicum, Corynebacterium michiganense subsp. nebraskense, and Corynebacterium michiganense subsp. tessellarius), Corynebacterium iranicum, Corynebacterium tritici, and Corynebacterium rathayi in the new genus Clavibacter. We further propose that Clavibacter xyli be recognized as a new species with the following subspecies: Clavibacter xyli subsp. xyli sp. nov., subsp. nov., the species and subspecies type strain of which is sugarcane strain L1A (= ATCC 33974 = NCPPB 3152 = PDDC 7127), and Clavibacter xyli subsp. cynodontis subsp. nov., the subspecies type strain of which is Bermudagrass strain TB1A (= ATCC 33973).

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Clavibacter: a New Genus Containing Some Phytopathogenic Coryneform Bacteria, Including Clavibacter xyli subsp. xyli sp. nov., subsp. nov. and Clavibacter xyli subsp. cynodontis subsp. nov., Pathogens That Cause Ratoon Stunting Disease of Sugarcane and Bermudagrass Stunting Disease†

The phenotypic diversity of about 200 bacterial strains isolated from soil was compared with the genotypic diversity of the same population. The strains were phenotypically characterized by the API 20B test system. The results of these tests were subjected to cluster analysis, which revealed 41 biotypes at 80% similarity. The five dominating biotypes contained 43% of the strains. The phenotypic diversity as determined by the Shannon index, equitability, rarefaction, and cumulative differences was high, but indicated some dominant biotypes. The genetic diversity was measured by reassociation of mixtures of denatured DNA isolated from the bacterial strains (C0t plots). The observed genetic diversity was high. Reassociation of DNA from all bacterial strains together revealed that the population contained heterologous DNA equivalent to 20 totally different bacterial genomes (i.e., genomes that have no homology). This study showed that reassociation of DNA isolated from a collection of bacteria gave a good estimate of the diversity of the collection and that there was good agreement with different phenotypic diversity measures. The Shannon index in particular has features in common with the genetic diversity measure presented here.

Comparison of phenotypic diversity and DNA heterogeneity in a population of soil bacteria.

Nonstarter Lactobacilli in Cheddar Cheese: A Review

A method developed for the lysis of oral streptococci that employed the action of lysozyme suspended in dilute tris(hydroxymethyl)aminomethane-hydrochloride buffer containing polyethylene glycol has been adapted for use with lactobacilli, actinomycetes, propionibacteria, and pediococci Most of the cellular deoxyribonucleic acid was liberated from many strains of bacteria usually thought to be lysozyme resistant The major observations were as follows: (i) supplementation of the growth medium with L-threonine, L-lysine, or both frequently produced cells that were more susceptible to lysis by lysozyme; (ii) glucose-containing media produced cells that were more easily lysed than those from cultures grown on other substrates; (iii) polyethylene glycol not only served as an osmotic stabilizer, it also enhanced the extent of lysis; and (iv) dilute tris(hydroxymethyl)aminomethane buffer was superior to the buffer systems most commonly employed in published muramidase-based lysis techniques Stationary-phase cells of Lactobacillus casei and Streptococcus mutans were more easily lysed than those isolated from log-phase cultures The method as detailed in this report should be generally applicable for the lysis of gram-positive, asporogenous bacteria

B M Chassy

Papers

Method for the lysis of Gram-positive, asporogenous bacteria with lysozyme.