L-Form Induction, Morphology, and Development in Two Related Strains of Erysipelothrix rhusiopathiae

Two related strains of Erysipelothrix rhusiopathiae, one the parent and the other an L-form revertant, were studied for their propensity or ability to produce L-forms under the influence of penicillin. The parent strain produced L-forms in nutrient solid media in an osmolarity range between 0.85 and 5.0% NaCl concentration whereas the revertant strain did so between 0.5 and 3.0% NaCl concentration. When various hyperosmolar media were tried without penicillin, recovery of L-forms from the revertant strain was optimal at a salt concentration of 2.0%, whereas the parent strain occasionally produced a few L-forms on 3.0% salt medium only. The process of penicillin-induced transformation from bacteria to L-form followed an unusual morphological sequence, beginning with beading of the bacterial body, followed by disintegration into granules from which the L-form colony derived. No large bodies were seen during the initial process of L-form induction, but they evolved later from the original granules and had the potential to reproduce L-type growth. The spontaneous development of L-forms in hyperosmolar media had a different morphological sequence starting with elongation of the bacteria into filaments which later developed polar and central dilatations from which granules and L-type growth developed. The differences in biological behavior between these related bacterial strains suggest that the revertant strain developed new properties, probably of genetic origin. Consequently, the assumption that L-forms revert to the "parent" bacteria may not always be justified. It can be made only after the biological properties of the parent and the revertant organisms have been properly identified.     

THE ADAPTATION OF FUNGI TO FUNGICIDES: ADAPTATION TO COPPER AND MERCURY SALTS

The susceptibility of Botrytis cinerea to copper sulphate in liquid media increased when the volume, and therefore the depth, of the medium in culture bottles exceeded certain values; when the volume was 40 ml. the maximum concentration allowing growth was 300 p.p.m.
By growing mycelium in media containing progressively higher concentrations of copper sulphate a strain was produced which grew at a concentration of 750 p.p.m.
In high concentrations of copper sulphate growth always started at the edge of the liquid, and inocula grew only if they were placed in this position.
In germination tests spores from the resistant strain were more resistant to copper sulphate than were spores of the parent strain.
The resistance of mycelium, and to a lesser extent of spores, was retained after growth of the resistant strain for six months in fungicide-free media.
Spore and mycelial inocula grew in much higher concentrations of copper sulphate when nutrient media were solidified with agar.
The strain resistant in liquid media was no more resistant than the parent strain on agar media.
The resistance of the fungus was not increased after growth for long periods on agar containing high concentrations of copper sulphate. The resistance of the strain resistant in liquid media was not lost after growth on agar media for 3 months.
Attempts to produce strains more resistant than the parent to mercuric chloride were unsuccessful.
The results obtained with phenyl-mercuric acetate were essentially similar to those obtained with copper sulphate, but relatively much more resistant strains were produced.     

Morphology, growth and reversion in a stable L-form of Escherichia coli K12

An L-form isolated from Escherichia coli K12 by sequential treatment with N-methyl-N'-nitro-N-nitrosoguanidine and lysozyme was adapted to grow in hyperosmolar liquid cultures. It was stable in the absence of antibiotic when cultured in brain heart infusion (BHI) broth containing NaCl and CaCl2, the optimal concentrations being 0.34 M and 1 mM, respectively. No growth of the L-form was observed when CaCl2 was not added to BHI medium containing 0.34 M-NaCl. On the other hand, when KCl replaced NaCl as the osmotic stabilizer, growth of the L-form was repressed in the presence of CaCl2. Electron microscopy of the L-form confirmed the absence of a cell wall. A revertant strain derived from the L-form grew as a stable bacillary form in BHI medium without osmotic stabilizer. The growth characteristics of the revertant strain resembled those of the parent strain. The revertant strain produced L-forms in the presence of NaCl.     

Alterations of the L-forms of a sporebearing bacillus

A large pleomorphic gram-negative bacillus developed as a contaminant on blood-agar. Spores were formed in one culture. L-forms were produced with penicillin on blood-agar with 2.5% NaCl; they grew well when transplanted to agar with 0.5% NaCl. After several transplants and long incubation of the L-forms without penicillin, in three transplants small gram-negative pleomorphic bacilli grew, but no L-forms. This occurred once on blood-agar and twice on 30% gelatin. The growth obtained from these small bacilli was similar in morphology and in the physical properties of the organisms to the altered L-forms of Proteus and Salmonella. Multiplication of the pleomorphic organisms and development of branching filaments from the round forms was apparent. The original large gram-negative bacillus was regularly recovered from the L-forms, and was recovered several times from the descendants of the small bacilli. These observations are essentially similar to those made with L-forms of Proteus and with an L-form studied in 1952, indicating alterations in L-forms of bacteria which do not produce B type L-forms.     

Listeria monocytogenes L Forms I. Induction, Maintenance, and Biological Characteristics

L forms were induced from 15 of 16 strains of Listeria monocytogenes on penicillin gradient plates incubated under aerobic conditions. The culture medium for maintenance of these L forms must contain an electrolyte in a concentration of 1% or sucrose in a concentration of 10%. The electrolytes NaCl, KCl, or MgSO(4) were used in both induction and maintenance media. Induction of L forms occurred more rapidly on media containing KCl. Listeria L forms had the same fermentation reactions as the parent bacterium. The L-form growth in liquid medium was slow, not extensive, and appeared as clumps on the bottom of culture tubes. The morphology of Listeria L forms was similar to that reported for other bacterial L forms. The L forms derived from strain 10403, serotype 1, were stable after two or more passages on penicillin media. They did not revert to the bacterial form after 40 subcultures on penicillin-free media. Some L-form colonies derived from strain 10403 did revert to the bacterial form when transferred directly from induction plates to penicillin-free media. Studies of the growth characteristics for L forms derived from strain 10403 gave the following results: an optimal temperature of 30 C, high electrolyte or sucrose concentration necessary for induction and maintenance, and no requirement for serum.     

L-forms ofHaemophilus influenzae: Growth and reversion as influenced by a strain ofNeisseria perflava

Factors excreted into the medium by various bacteria and fungi exert a significant effect on the growth and revertibility of bacterial L-forms. Studies with L-forms ofHaemophilus influenzae have been facilitated by a strain ofNeisseria perflava, which not only promotes the growth of the L-forms on media of normal osmolarity (horse blood agar), but also enhances the revertibility of the L-forms to the parent bacterium. L-forms derived fromN. perflava also exhibit this growth-enhacing property, an effect that does not appear to be related to X and V factors. Preliminary characterization of the active component produced byNeisseria perflava indicates that it is present in broth filtrate, diffusible through agar, heat labile, and of large molecular size.     

细胞壁缺陷细菌生物氧化特性的观察

The L-forms were induced from Staphylococcus aureus,Escherichia coli and Bacdtos cereus by β-lactam anhbiohcs and then observahons on the proPenies of oxygen requlrement sugar fermentahon and sensihve tO cyanide of the Lforms were done. The resultS were shown that the Lforms derived from the obligate aerobe or the faCultative anaerobe did not ferment sugars and were highly oxygendePendent and more sensihve tD cyedde than their Parent bacteria The metabolic achvihes which were same as the Parent bacteria of …     

A mutant of Candida albicans deficient in beta-N-acetylglucosaminidase (chitobiase)

A mutant of Candida albicans ATCC 10261 was isolated that was defective in the production of beta-N-acetylglucosaminidase (chitobiase). The mutant grew normally in minimal medium supplemented with either glucose or N-acetyl-D-glucosamine (GlcNAc) as carbon and energy source, and the cells formed germ-tubes at 37 degrees C when induced to do so with GlcNAc. However, unlike the wild-type parent strain, the mutant strain did not utilize N,N'-diacetylchitobiose for growth. The mutant and parent strains had similar growth rates on glucose or GlcNAc, similar rates of uptake of these sugars and similar rates of 14C-labelled amino acid incorporation. The chitobiase mutant did, however, contain 53-85% more chitin than the wild-type strain. No reversion of the mutant phenotype was observed following induction of mitotic recombination with UV light, suggesting that the mutant allele (chi) was carried homozygously in the chitobiase-deficient mutant. Although the chitobiase-deficient mutant was pathogenic, it was not as virulent as the wild-type strain.     

Ethanol Production by Thermophilic Bacteria: Physiological Comparison of Solvent Effects on Parent and Alcohol-Tolerant Strains of Clostridium thermohydrosulfuricum

The effects of temperature, solvents, and cultural conditions on the fermentative physiology of an ethanol-tolerant (56 g/liter at 60°C) and parent strain of Clostridium thermohydrosulfuricum were compared. An ethanol-tolerant mutant was selected by successive transfer of the parent strain into media with progressively higher ethanol concentrations. Physiological differences noted in the mutant included enhanced growth, tolerance to various solvents, and alterations in the substrate range and the fermentation end product ratio. Ethanol tolerance was temperature dependent in the mutant but not in the parent strain. The mutant grew with ethanol concentrations up to 8.0% (wt/vol) at 45°C, but only up to 3.3% (wt/vol) at 68°C. Low ethanol concentration (0.2 to 1.6% [wt/vol]) progressively inhibited the parent strain to where glucose was not fermented at 2.0% (wt/vol) ethanol. Both strains grew and produced alcohols on glucose complex medium at 60°C in the presence of either 5% methanol or acetone, and these solvents when added at low concentration stimulated fermentative metabolism. The mutant produced ethanol at high concentrations and displayed an ethanol/glucose ratio (mole/mole) of 1.0 in media where initial ethanol concentrations were ≤4.0% (wt/vol), whereas when ethanol concentration was changed from 0.1% to 1.6% (wt/vol), the ethanol/glucose ratio for the parent strain changed from 1.6 to 0.6. These data indicate that C. thermohydrosulfuricum strains are tolerant of solvents and that low ethanol tolerance is not a result of disruption of membrane fluidity or glycolytic enzyme activity.     

Glutamate Metabolism in a Glutamate-producing Bacterium,Brevibacterium flavum

Brevibacterium flavum No. 2247 was found to grow with l-glutamate as the sole carbon and nitrogen source on an agar-plate medium when high concentrations of l-glutamate, FeSO₄ and biotin were added to the medium. It grew on l-glutamate in liquid medium only when yeast extract or high concentrations of FeSO₄ and glucose or organic acids of the tricarboxylic acid cycle were added to the medium. The growth on l-glutamate in liquid medium was also stimulated by high concentrations of l-glutamate, biotin and MgSO₄, and inhibited by a high concentration of (NH₄)₂SO₄.

Aspartate aminotransferase (TA)- and α-ketoglutarate dehydrogenase (KD)-defective mutants did not grow on l-glutamate, and glutamate-utilizing revertants derived from these mutants recovered TA and KD activity, respectively, whereas glutamate dehydrogenase (GD)-defective mutants grew on l-glutamate. Washed cells of strain No. 2247 grown on glutamate decomposed the amino acid, whereas those grown on glucose did not. The degradation was observed only under aerobic conditions. The former cells showed higher KD, succinate dehydrogenase and fumarase activities than the latter cells. Of 75 mutants which did not grow on glutamate but grew on succinate, three strains lacked KD but showed the same glutamate productivity as the parent strain. Four other strains with normal KD levels showed higher glutamate productivity than the parent.     

THE ADAPTATION OF FUNGI TO FUNGICIDES: ADAPTATION TO THIRAM, ZIRAM, FERBAM, NABAM AND ZINEB

The concentrations of thiram preventing germination of spores of Botrytis cinerea in drops of a 1% solution of sucrose, and on the surface of a sucrose-nitrate agar have been determined. Thiram had much less effect on germination in the agar medium, even when a purified agar was used. There was no growth on sucrose-nitrate agar if the concentration of thiram exceeded 31 p.p.m. Attempts to obtain strains able to grow at higher concentrations were unsuccessful.
Similar results were obtained with ziram, nabam and zineb.
Ferbam also was more effective in preventing spore germination in spore drops than on agar media; this effect was obtained with ordinary and with purified agar.
On a sucrose-nitrate agar generally there was no growth if the concentration of ferbam exceeded 125 p.p.m., but in one of forty-eight plates containing 250 p.p.m. ferbam, five slowly growing colonies were produced, and from these colonies arose mycelium which grew and sporulated rapidly on 500 p.p.m. ferbam agar. Agar disk inocula were transferred from these cultures to agar containing higher concentrations of ferbam and in this way, and by repeating the process, a strain was obtained which grew slowly but continuously, and sporulated on agar containing 5000 p.p.m. ferbam. However, the poor solubility of this fungicide made it difficult to assess quantitatively the degree of adaptation.
A proportion of the spores from this strain germinated in drops containing about twice the concentration of ferbam which prevented germination of parent spores.
The resistance of the mycelium of the resistant strain was not lost after repeated subculture on fungicide-free agar. The resistant strain was as susceptible as the parent strain to thiram, ziram, nabam and zineb.
Attempts to obtain strains of Venturia inaequalis resistant to thiram, ferbam, ziram and zineb were unsuccessful.     

Phialophora parasitica, an emerging pathogen

Monoconidial cultures derived from 12 clinical and environmental isolates of Phialophora parasitica were compared with respect to morphologic and physiologic characteristics and response to antifungal agents. No yeast cells were seen in 1- and 3-week-old slide culture preparations. Also, not all of the distinguishing characteristics of this species were displayed by all isolates on all media examined. Although the isolates grew on Sabouraud agar with chloramphenicol and cycloheximide, some inhibition was observed. All cultures were strongly urease-positive and hydrolyzed casein and starch; most decomposed tyrosine but not gelatin. All but one environmental isolate grew well at both 23 and 37 degrees C, but none grew at 40 degrees C. In the sensitivity testing the isolates did not vary much in their response to each drug, although some anomalies were observed. Amphotericin B and miconazole had minimum inhibitory concentrations in the low sensitivity range (2.0-8.0 and 2.5-10 micrograms m-1 respectively), for most isolates, and most isolates were resistant to both 5-fluorocytosine and ketoconazole. Limited observations were made on three other Phialophora species which might be confused with P. parasitica.     

Induction of stable L-forms of Staphylococcus aureus and Streptococcus faecalis

Stable L-forms were induced from Staphylococcus aureus and Streptococcus faecalis. These formed typical foamy L-colonies and showed large and small round bodies. They grew continuously on routine antibiotic-free nutrient broth and blood agar media for 12 passages without reversion to their parental forms. At different concentrations of penicillin various morphological forms were observed. Effect of sucrose, normal horse serum and penicillin on their adaptation and stabilization is discussed.     

Induction of Enterococcal L-Forms by the Action of Lysozyme

Suspensions of enterococci were treated with lysozyme in the presence of osmotic stabilizers. The resulting osmotically fragile bodies prepared from Streptococcus faecium strain F24 and S. faecalis strain E1 gave rise to L-forms under optimal osmotic and nutritional conditions for treatment and subsequent growth. The most critical component of the growth medium, to obtain maximum yields, was the nature and concentration of the added salt. The two most effective salts were sodium chloride and ammonium chloride in the range of 2 to 3% (w/v) added to a suitable agar base. Ammonium chloride was more versatile, because it could be used with either sucrose or polyethylene glycol 4000 as the osmotic stabilizer for preparation and dilution of the osmotically fragile bodies. Sodium chloride would not consistently support growth of S. faecium F24 as L-forms when polyethylene glycol 4000 was used as the osmotic stabilizer during lysozyme treatment. Time-course studies of concurrent cell wall removal and L-form induction suggested that maximal induction required only cell wall damage rather than complete wall removal. This method for induction of L-forms from a suspension of enterococci is a significant improvement over other presently known methods.     

Presence and synthesis of cholesterol in stable staphylococcal L-forms.

The sterol which was present in two strains of a stable staphylococcal L-form was analyzed by gas-liquid chromatography and combined gas-liquid chromatography-mass spectrometry. The retention time of the sterol on gas-liquid chromatography was the same as that of authentic cholesterol. Analysis of the sterol by mass spectrometry showed a molecular ion at an m/e of 386 and the same patterns of major ions above an m/e of 145 as those of authentic cholesterol. As a result, the sterol in staphylococcal L-form was identified as cholesterol. A parent strain and its L-forms were cultured in medium containing [14C]acetate, and the synthesis of cholesterol was examined. In the L-forms, 0.52% of the total lipid radioactivity was found in cholesterol fraction, whereas no significant radioactivity was detected in the cholesterol fraction of the parent strain, indicating that staphylococcal L-forms have acquired the capacity to synthesize cholesterol.     

Polysaccharide and protein degradation,germination, and virulence against mosquitoes in the entomopathogenic fungus Metarhizium anisopliae

From a genetically uniform wild-type strain of Metarhizium anisopliae pathogenic to mosquitoes, mutants were selected which were altered in the ability to degrade starch, gelatin, or milk. The mutants with enhanced starch degradation (dep), when grown on starch-containing media, proved hypervirulent toward the mosquito Culex pipiens pipiens in standard laboratory tests. Alterations in protein (gelatin or milk) degradation did not correlate with changes in virulence. The dep mutants appear to belong to the same class as mutants selected previously as hypervirulent and characterized by early spore germination. The relationship among polysaccharide degradation, early germination, and virulence is discussed.     

Determinants in microbial colonization of the murine gastrointestinal tract: pH, temperature, and energy-yielding metabolism of Torulopsis pintolopesii.

Torulopsis pintolopesii is an indigenous yeast that colonizes the secreting epithelia in the stomachs of mice and rats. A wild-type strain of this microbe was isolated and identified. To attempt to learn characteristics of the yeast that are advantageous to it in colonizing its natural habitat in vivo, we examined some aspects of its nutrition and energy-yielding metabolism and some environmental conditions that influence its growth in vitro. The yeast appeared to be limited in the compounds it can utilize as carbon and nitrogen sources. It grew best at 37 degrees C and did not grow at 23 or 43 degrees C. It grew optimally at neutral pH but could grow aerobically at pH values as low as 2.0 and anaerobically at pH values as low as 3.4. As assessed by measurements of growth rates and yield coefficients, it grew better aerobically than anaerobically. When grown aerobically, it had a cyanide-sensitive system for taking up O(2) and tested positively for cytochrome c oxidase activity. A petite mutant strain isolated from the wild-type strain had a growth rate and yield coefficient when incubated aerobically that were essentially the same as those of the wild-type parent grown anaerobically. Likewise similar to the wild-type parent grown anaerobically, the petite strain, though incubated aerobically, did not take up O(2). Yeast-free mice associated with either the wild-type or the petite mutant strain were colonized at essentially the same rates and to similar final population levels by both strains. The yeast's capacity to respire may be of little advantage to it in its natural environment. By contrast, its abilities to grow best at 37 degrees C and to grow at low pH values are undoubtedly advantageous characteristics in this respect. The limitations in its carbon and nitrogen nutrition are difficult to evaluate as ecological factors in its colonization of the natural habitat.     

Nitrogen nutrition in the cyanobacterium Nostoc ANTH, a symbiotic isolate from Anthoceros: uptake and assimilation of inorganic-n and amino acids

Amino acid uptake and utilization of various nitrogen sources (amino acids, nitrite, nitrate and ammonia) were studied in Nostoc ANTH and i ts mu tant (Het(-)Nif(-)) isolate defective in heterocyst formation and N2-fixation. Both parent and its mutant grew at the expense of glutamine, asparagine and arginine as a source of fixed-nitrogen. Growth was better in glutamine-and asparagine-media as compared to that in arginine media. Glutamine and asparagine repressed heterocyst formation, N2-fixation and nitrate reduction in Nostoc ANTH, but arginine did so only partially. The poor growth in arginine-medium was not due to poor uptake rates, since the uptake rates were not significantly different from those for glutamine or asparagine. The glutamine synthetase activity remained unaffected during cultivation in media containing any one of the three amino acids tested. The uptake of amino acids was substrate-inducible, energy-dependent and required de novo protein synthesis. Nitrate and ammonium repressed ammonium uptake, but did not repress uptake of amino acids. In N2-medium (BG-11(0)), the uptake of ammonium and amino acids in the mutant was significantly higher than its parent strain. This was apparently due to nitrogen limitation since the mutant was unable to fix N2 and the growth medium lacked combined-N.     

Polyacrylamide Gel Identification of Bacterial L-Forms and Mycoplasma Species of Human Origin

Polyacrylamide gel electrophoretic patterns of acidified phenol extracts prepared from whole cells can be used for the identification of bacterial L-forms and Mycoplasma species of human origin. Ten human Mycoplasma serotypes and eight L-forms belonging to five different genera were studied. The gel patterns were sufficiently distinct and reproducible that it was possible not only to identify L-forms at the genus level (group with streptococci) and different Mycoplasma serotypes but also to differentiate between the two of them. The parentage of L-forms of Streptobacillus moniliformis L1, Listeria monocytogenes, Streptococcus MG, and Staphylococcus aureus Smith strain was established by relating their gel patterns directly to parent bacteria. It was found that an L-form designated S. moniliformis An (ATCC 14220) was actually an L-form of Proteus. In addition, it was shown electrophoretically that no relationship existed between the Streptococcus MG L-form and M. pneumoniae. The applicability of this method as a diagnostic and taxonomic tool for the differentiation of L-forms and mycoplasmas is discussed.     

Effect of growth substrates on morphology of Nocardia corallina.

Cells of Nocardia corallina ATCC 4273 form multiply branched coenocytic mycelia and subsequent fragment to spherical cells when grown on solidified complex media. In liquid shake cultures using complex media the organisms grow into pleomorphic but seldomly branched rods, divide as rods and then the rods fragment to spheres as the stationary phase is reached. In a defined liquid medium with glucose as carbon source, the organisms divide entively as spheres at a doubling time of 44 hrs. The addition of L-tyrosine, some fatty acids and tricarboxylic acid cycle intermediates or fructose to the glucose medium caused the cells to grow at considerably faster growth rates (2.8-8.5 hrs doubling times) and to undergo the shphre-rod-shpere growth cycle. Other amino acids, fatty acids or surgars added singly to the glucose medium did not produce the sphere to rod morphology change. Some amino acids when added to the medium in pairs effected sphere to rod morphopoiesis. None of these amino acids alone were effectors. Some of the culture grew as rods and the remainder as spheres when isoleucine and valine were added to the glucose medium. No other amino acid combination tested gave this result. The reason for the mixed growth response was traced to inhomogeneity of the parent culture. The life cycle of N. corallina is illustrated in a series of photomicrographs of two slide cultures.