GENETIC NATURE OF STABLE L FORMS OF
            <i>SALMONELLA PARATYPHI</i>

Landman, Otto E.; Ginoza, Herbert S.

doi:10.1128/jb.81.6.875-886.1961

Cited by 28 publications

(12 citation statements)

References 30 publications

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“…The probability of reversion is very different in different organisms. For example, Salmonella paratyphi B mass conversion stable L forms revert extremely rarely (13,15), whereas those ofBacillus megaterium multiply only briefly in the naked state before reverting (12,13). B. subtilis is intermediate in this respect: depending on the plating medium, the individual L colonies are overgrown in 2 to 20 days by spontaneous revertants within the L colonies (16).…”

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Inhibitory protein controls the reversion of protoplasts and L forms of Bacillus subtilis to the walled state

DeCastro-Costa¹,

Landman²

1977

J Bacteriol

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When the cell wall of Bacillus subtilis is removed by lysozyme and the resultant protoplasts are plated on hypertonic soft agar medium, each protoplast forms an L colony. L bodies from such L colonies again plate as L-colony-forming units (CFU). However, if protoplasts or L bodies are "conditioned" by 1 h of incubation in 0.4% casein hydrolysate medium and then incubated in 25% gelatin medium for 1 h, 60 to 100% of the formerly naked cells give rise to bacillary colonies. The present experiments largely explain the mechanism responsible for the "heritable" persistence of the wall-less state in B. subtilis. It is shown that protoplasts produce a reversion inhibitory factor (RIF) which blocks reversion when the cell concentration exceeds 5 x 105 CFU/ml. This inhibitor is nondialyzable and sensitive to trypsin, heat, and detergent. Efficient reversion at 2 x 107 CFU/ml is obtained if the protoplasts are treated with trypsin after conditioning and chloramphenicol is incorporated into the gelatin reversion medium. In the presence of 500 ,ug of trypsin per ml, the requirement for gelatin is sharply reduced, and reversion occurs rapidly in liquid medium containing only 10% gelatin. Trypsin also stimulates reversion in L colonies growing on soft agar. Latent RIF is activated by ,3-mercaptoethanol. This reagent blocks reversion of protoplast suspensions at densities of 5 x 105 CFU/ ml. Comparison of the autolytic behavior ofB. subtilis and of the RIF revealed that several of the properties ofthe two activities coincide: both are inhibited by high concentrations of gelatin, both are activated by (3-mercaptoethanol, and both have high affinity for cell wall. Going on the assumption that REF is autolysin, models for protoplast reversion and L-form stability are proposed. A role of teichoic acid in reversion is suggested by the finding that mutants with altered teichoic acid show altered reversion behavior.When the cell wall is removed from Bacillus subtilis with lysozyme and the resultant protoplasts are plated on hypertonic soft-agar medium, each protoplast gives rise to an L colony consisting of wall-less, irregularly shaped L bodies (16,24). L bodies from suspended L colonies again give rise to L colonies in further platings (16). Thus, a brief transient enzyme treatment produces an alteration in each member of the treated population-namely, walllessness-which is then passed on "heritably" through an indefinite number of cell generations. We have called these naked offspring "mass conversion stable L forms" (13) to underline the distinction between these L forms and certain kinds of gene mutants blocked in peptidoglycan or peptidoglycan precursor biosynthesis, which are also sometimes called stable L forms (see, for instance, 28). We are quite certain that the persistent change induced in mass Present address:

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Inhibitory protein controls the reversion of protoplasts and L forms of Bacillus subtilis to the walled state

DeCastro-Costa¹,

Landman²

1977

J Bacteriol

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“…System 3. The S. schottmuelleri used here was identical to S. paratyphi B 777 used earlier in our laboratory (11).…”

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Retention of Episomes During Protoplasting and During Propagation in the L State

Kawakami

Landman

1966

Journal of Bacteriology

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In earlier work, it was observed that the mesosomes of Bacillus megaterium and B. subtilis are expelled from the cell interior during protoplasting and that mesosome fragments are released into the supernatant fluid as the cell wall disintegrates. Since the resultant protoplasts remain intact and capable of reproduction, the expelled contents of the mesosome 'bag" are presumably external to the protoplast membrane and nonessential to survival. Accordingly, if episomes (plasmids) were localized in the extramembrane mesosome "bag," it would be predicted that protoplasting would cure cells of their episomes. This prediction was tested in three different systems: B. subtilis W23 carrying phage SP-10, B. megaterium 216 carrying megacinogenic factors A and C, and B. megaterium C4Mcarrying megacinogenic factor C. No curing due to protoplasting was observed. Even during propagation in the L form, when septation is not functioning and the distribution of deoxyribonucleic acid to daughter cells is severely disrupted, curing was not observed in any of the above systems nor in Salmonella L forms carrying Ft lac. It is concluded that episomes are located at a position on the interior side of the cell membrane and that their distribution to daughter cells is coordinated with that of the chromosome.

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“…Past work in our laboratory has indicated that under certain special conditions bacterial multiplication may occur through a process other than the normal division mechanism." 14 Utilizing these conditions, it has now become possible to test whether the streptomycin requirement may be eliminated by bypassing the normal septation process.…”

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“…Instead, only L colonies are obtained; these can be propagated in the absence of penicillin indefinitely as L forms [except for rare (<1/109) reversions to the bacillary form]. 14 We have interpreted the above results as follows: at 20 units of penicillin per ml, penicillin inhibits the formation of the rigid layer of the cell wall34 so that the cells assume a spherical shape. To judge from the slow growth rate of the reverting L forms, cell division is also inhibited, but the inhibition is not strong enough to prevent growth in liquid media.…”

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“…Once in the stable L form, growth could continue because of the greatly reduced requirement for streptomycin. Discussion.-In an attempt to assign a unitary cause for the multiple effects of penicillin on bacteria (its interference with rigid wall formation,14' 22, 34 cell division,1' 14,16,19,24 and permeability,8' 24) Landman and Ginoza'4 have postulated that the primary site of attack of penicillin upon the cell is in the protoplast membrane. This conclusion is supported by the earlier finding that radioactive penicillin combines preferentially with the bacillary membrane.4 Damage inflicted upon the growing membrane by the blocking action of penicillin may become manifest as inhibition of cell wall formation (spheroplasting), or inhibition of cell division (filamentation), depending on the bacterial genotype.…”

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confidence: 99%

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The Mechanism of Action of Streptomycin as Revealed by Normal and Abnormal Division in Streptomycin-Dependent Salmonellae

Landman¹,

Burchard²

1962

Proc. Natl. Acad. Sci. U.S.A.

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Earlier work with streptomycin-dependent (Sd) mutants of E. coli and Salmonella demonstrated that cells grown in streptomycin medium and then deprived of antibiotic continued to make protoplasm but soon ceased to produce new colony-forming elements. Microscopic observation of such streptomycinstarved cultures revealed that the short rods which were present in the streptomy-Downloaded by guest on July 9, 2020

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GENETIC NATURE OF STABLE L FORMS OF SALMONELLA PARATYPHI

Cited by 28 publications

References 30 publications

Inhibitory protein controls the reversion of protoplasts and L forms of Bacillus subtilis to the walled state

Inhibitory protein controls the reversion of protoplasts and L forms of Bacillus subtilis to the walled state

Retention of Episomes During Protoplasting and During Propagation in the L State

The Mechanism of Action of Streptomycin as Revealed by Normal and Abnormal Division in Streptomycin-Dependent Salmonellae

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