L-phase Variants Research Articles

Role of the Eikenella corrodens pilA locus in pilus function and phase variation.

The human pathogen Eikenella corrodens expresses type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying pilus structure and function in the clinical isolate E. corrodens VA1. Earlier work defined the pilA locus which includes pilA1, pilA2, pilB, and hagA. Both pilA1 and pilA2 predict a type IV pilin, whereas pilB predicts a putative pilus assembly protein. The role of hagA has not been clearly established. That work also confirmed that pilA1 encodes the major pilus protein in this strain and showed that the phase variation involves a posttranslational event in pilus formation. In this study, the function of the individual genes comprising the pilA locus was examined using a recently developed protocol for targeted interposon mutagenesis of S-phase variant VA1-S1. Different pilA mutants were compared to S-phase and L-phase variants for several distinct aspects of phase variation and type IV pilus biosynthesis and function. S-phase cells were characterized by surface pili, competence for natural transformation, and twitching motility, whereas L-phase cells lacked these features. Inactivation of pilA1 yielded a mutant that was phenotypically indistinguishable from L-phase variants, showing that native biosynthesis of the type IV pilus in strain VA1 is dependent on expression of pilA1 and proper export and assembly of PilA1. Inactivation of pilA2 yielded a mutant that was phenotypically indistinguishable from S-phase variants, indicating that pilA2 is not essential for biosynthesis of functionally normal pili. A mutant inactivated for pilB was deficient for twitching motility, suggesting a role for PilB in this pilus-related phenomenon. Inactivation of hagA, which may encode a tellurite resistance protein, had no effect on pilus structure or function.

Eikenella corrodens phase variation involves a posttranslational event in pilus formation.

The human pathogen Eikenella corrodens synthesizes type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying the molecular basis of this phase variation in the clinical isolate E. corrodens VA1. A genomic fragment encoding the major type IV pilin was cloned from the S-phase variant of strain VA1. Sequence analysis of the fragment revealed four tandemly arranged potential open reading frames (ORFs), designated pilA1, pilA2, pilB, and hagA. Both pilA1 and pilA2 predict a type IV pilin. The protein predicted by pilB shares sequence identity with the Dichelobacter nodosus FimB fimbrial assembly protein. The protein predicted by hagA resembles a hemagglutinin. The region containing these four ORFs was designated the pilA locus. DNA hybridization and sequence analysis showed that the pilA locus of an L-phase variant of strain VA1 was identical to that of the S-phase variant. An abundant pilA1 transcript initiating upstream of pilA1 and terminating at a predicted hairpin structure between pilA1 and pilA2 was detected by several assays, as was a less abundant read-through transcript encompassing pilA1, pilA2, and pilB. Transcription from the pilA locus was nearly indistinguishable between S- and L-phase variants. Electron microscopy and immunochemical analysis showed that S-phase variants synthesize, export, and assemble pilin into pili. In contrast, L-phase variants synthesize pilin but do not export and assemble it into pili. These data suggest that a posttranslational event, possibly involving an alteration in pilin export and assembly, is responsible for phase variation in E. corrodens.

Electroporation-induced transformation of intact cells of Clostridium perfringens.

Electroporation-induced transformation of intact cells of Clostridium perfringens 3624A with plasmids pAMB1 and pHR106 resulted in 3.8 X 10(-5) and 4.2 X 10(-4) transformants per viable cell, respectively. With respect to shuttle plasmid pHR106, these values represent a greater than 100-fold increase in transformation frequency when compared with the values reported with polyethylene glycol-induced L-phase variants.

Induction of L-phase variants from protoplasts ofClostridium thermocellum

Stable protoplasts ofClostridium thermocellum were prepared by combined treatment using glycine and lysozyme as the protoplasting agents and raffinose as the osmotic stabilizer. These protoplasts gave rise to L-form colonies on an hypertonic rich medium.

Effect of nalidixic acid on the conversion of Staphylococcus aureus cells to L-forms in a liquid medium with 6-aminopenicillanic acid and lysozyme.

Microbiology and ImmunologyVolume 30, Issue 8 p. 811-818 Original ArticleFree Access Effect of Nalidixic Acid on the Conversion of Staphylococcus aureus Cells to L-Forms in a Liquid Medium with 6-Aminopenicillanic Acid and Lysozyme Kunihiko Yabu, Kunihiko Yabu Department of Biology, Hokuriku University School of Pharmacy, Kanazawa, Ishikawa, 920-11Search for more papers by this author Kunihiko Yabu, Kunihiko Yabu Department of Biology, Hokuriku University School of Pharmacy, Kanazawa, Ishikawa, 920-11Search for more papers by this author First published: August 1986 https://doi.org/10.1111/j.1348-0421.1986.tb03007.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL REFERENCES 1Bibel, D.J., and Lawson, J.W. 1975. Morphology and viability of large bodies of streptococcal L-forms. Infect. Immun. 12: 919– 930. 2Gellert, M., O'Dea, M.H., Itoh, T., and Tomizawa, J. 1976. Novobiocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase. Proc. Natl. Acad. Sci. U. S. A. 73: 4474– 4478. 3Goss, W.A., Deitz, W.H., and Cook, T.M. 1965. Mechanism of action of nalidixic acid on Escherichia coli. II. Inhibition of deoxyribonucleic acid synthesis. J. Bacteriol. 89: 1068– 1074. 4Green, M.T., Heidger, P.M., and Domingue, G. 1974. Proposed reproductive cycle for a relatively stable L-phase variant of Streptococcus faecalis. Infect. Immun. 10: 915– 927. 5Nagate, T., Komatsu, T., Izawa, A., Ohmura, S., Namiki, S., and Mitsuhashi, S. 1980. Mode of action of a new nalidixic acid derivative, AB206. Antimicrob. Agents Chemother. 17: 763– 769. 6Smith, D.H., and Davis, B.D. 1967. Mode of action of novobiocin in Escherichia coli. J. Bacteriol. 93: 71– 79. 7Staudenbauer, W.L. 1976. Replication of E. coli DNA in vitro: inhibition by oxolinic acid. Eur. J. Biochim. 62: 491– 497. 8Sugino, A., Peebles, C.L., Kreuzer, K.N., and Cozzarelli, N. 1977. Mechanism of action of nalidixic acid: purification of Escherichia coli nal A gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme. Proc. Natl. Acad. Sci. U. S. A. 74: 4767– 4771. 9Wecke, J., Lahav, M., Ginsburg, I., and Giesbrecht, P. 1982. Cell wall degradation of Staphylococcus aureus by lysozyme. Arch. Microbiol. 131: 116– 123. 10Westmacott, D., and Perkins, H.R. 1979. Effect of lysozyme on Bacillus cereus 569: rupture of chains of bacteria and enhancement of sensitivity to autolysins. J. Gen. Microbiol. 115: 1– 11. 11Yabu, K. 1985. Conversion of Staphylococcus aureus cells to stable L-forms in a liquid growth medium. Microbiol. Immunol. 29: 157– 161. 12Yabu, K. 1986. Massive induction of Staphylococcus aureus unstable L-forms in a liquid medium. Microbiol. Immunol. 30: 275– 281. 13Yabu, K., Tomizu, H., and Kanda, Y. 1984. The effect of pipemidic acid on the growth of a stable L-form of Staphylococcus aureus. Microbiol. Immunol. 28: 1057– 1064. Citing Literature Volume30, Issue8August 1986Pages 811-818 ReferencesRelatedInformation

Studies in vivo on the killing rate and refractory period of penicillin V in an experimental streptococcal infection.

The refractory period and the killing rate of beta-haemolytic streptococci after exposure to phenoxymethylpenicillin were tested in an in-vivo model. beta-Haemolytic streptococci were injected into steel net chambers implanted subcutaneously on the backs of rabbits. The rabbits were treated with infusions of phenoxymethylpenicillin, either as a single dose to measure the refractory period or as repeated doses in order to measure the killing rate of streptococci. The peak concentration of phenoxymethylpenicillin in tissue chamber fluid occurred about 16-120 min post infusion, and reached 0.4 mg/l in infected and 0.6 mg/l in uninfected tissue cage fluid. In the tissue cage fluid the phenoxymethylpenicillin concentration exceeded 0.03 mg/l, the MIC-value for the streptococcal strain used, for at least 6 h. After a single infusion there was a decline in viable count. The bacteria did not reach their original numbers until 60-70 h later. After the sixth infusion streptococci were no longer detectable in tissue cage fluid. There was a close correlation between viable counts before treatment and the time required for eradication of bacteria. L-phase variants of beta-haemolytic streptococci were not found when tissue cage fluid was plated on special media.

Transformation of Clostridium perfringens.

Clostridium perfringens 11268 CDR (Rifr Tcs), the strain transformed in our experiments, was generated by curing a spontaneous, rifampicin-resistant mutant of C. perfringens 11268 (Rifr Tcr). High-temperature growth yielded tetracycline-sensitive, rifampicin-resistant cells which no longer contained pCW3, a 42.8-kilobase plasmid. The tetracycline-sensitive, rod-shaped cell was then converted to an L-phase variant by growth in the presence of penicillin G (10 micrograms/ml) and 0.4 M sucrose. After several passages, the antibiotic was removed from the medium, and cells continued to grow as L-phase variants. Another large plasmid, pJU124 (38.8 kilobases), which confers tetracycline resistance, was used for transformation. Transformation of L-phase variants of C. perfringens 11268 CDR (Rifr Tcs) was mediated by polyethylene glycol. Transformation frequency is a nonlinear function of DNA concentration. Restriction analysis showed that the plasmid isolated from the transformants was identical to that supplied. Stable L-phase variants do not revert to rod-shaped cells, but autoplasts can be both transformed and reverted.

Shuttle plasmids for Escherichia coli and Clostridium perfringens.

Small plasmids which replicate in both Escherichia coli and Clostridium perfringens were made by recombining E. coli plasmid pBR322 with three different small (less than 4 kilobases) plasmids native to C. perfringens. Subsequently, two homologous, though distinct, tetracycline resistance determinants (tet) from other C. perfringens plasmids were cloned into them. Both tet systems made E. coli resistant to at least 5 micrograms of tetracycline per ml when resident on the shuttle plasmids. The shuttle vectors have been used to transform L-phase variants and autoplasts of C. perfringens. In the latter case, the intact transforming plasmid could be isolated from walled cells after cell wall regeneration. Reciprocal transformation experiments in which plasmid DNAs derived from E. coli or C. perfringens were used suggest that restriction barriers exist between these two organisms. The plasmids contain restriction enzyme recognition sites in locations which are useful for cloning experiments.

Mode of action and in-vitro activity of vancomycin.

Vancomycin is a unique glycopeptide structurally unrelated to any currently available antibiotic. It also has a unique mode of action inhibiting the second stage of cell wall synthesis of susceptible bacteria. There is also evidence that vancomycin alters the permeability of the cell membrane and selectively inhibits ribonucleic acid synthesis. Induction of bacterial L-phase variants from susceptible organisms with vancomycin is extremely difficult, and such variants are unstable. Stable L-phase variants induced by other agents are susceptible to vancomycin. Vancomycin is active against a large number of species of Gram-positive bacteria, such as Staphylococcus aureus (including methicillin-resistant strains), Staph. epidermidis (including multiple-resistant strains), Streptococcus pneumoniae (including multiple-resistant strains), Str. pyogenes, Str. agalactiae, Str. bovis, Str. mutans, viridans streptococci, enterococci, Clostridium species, diphtheroids, Listeria monocytogenes, Actinomyces species and Lactobacillus species. There has been no increase in resistance to vancomycin during the past three decades. Enhancement of antimicrobial activity has been demonstrated with the combination of vancomycin and an aminoglycoside against Staph. aureus, Str. bovis, enterococci and viridans streptococci. The combination of vancomycin and rifampicin are antagonistic to most strains of Staph. aureus, though indifference and occasionally synergism have been shown, but is synergistic against strains of Staph. epidermidis. It shows indifference against enterococci. Vancomycin and fusidic acid are indifferent against Staph. aureus.

Induction of L-phase variant from protoplast of Staphylococcus aureus.

Protoplasts of Staphylococcus aureus 209P and Cowan 1 were induced by treatment with lysostaphin. These protoplasts were sensitive to detergent, a low concentration of sodium chloride and low temperature. Almost all protoplast cells spread on CLYS agar medium (casein hydrolysate, yeast extract, Na-lactate, and NaCl) formed typical L-form colonies. Horse serum (0.25%) and Mg2+ (100 mM) are essential factors for formation of the L-form colonies of 209P. In the case of Cowan 1, Mg2+ was not required. The active factor(s) in horse serum was heat-resistant and protein in nature.

Recovery of microorganisms from synovial and pleural fluids of animals using hyperosmolar media

L-phase (CWD) broth and plate media were used in parallel with conventional microbiological media during a 3-year period for culturing synovial and pleural fluids of animals. Two kinds of recoveries were obtained where parallel conventional methods were negative: (1) parent or normal bacteria, in very low numbers; and (2) Type B CWD variants in equally low numbers. Organisms in group 1 were: Streptococcus zooepidemicus from horses (2X); β-hemolytic streptococci, Lancefield Gp. G (2X); Staphylococcus aureus; Actinobacillus, and Actinomyces viscosus. Group 2 consisted of Bacteroides sp., Propionibacterium acnes, and three “ Nocardia-like” sp. Catalase + Actinomyces was not recovered equally well on CWD plates as on conventional media with fluids obtained during ampicillin treatment. This occurred in spite of the fact that the CWD media was shown to support growth and reversion of laboratory induced L-phase variants of Nocardia caviae and N. asteroides, and had facilitated recovery of a Bacteroides L-phase variant from a pleural fluid. The nature of this fault in the media is under investigation in this laboratory.

Atypical colony-like structures developing in control media and in clinical L-form cultures containing serum

Solid and liquid media designed to support growth of cell wall deficient variants were evaluated for the presence of endogenous variants. The media compared in this study consisted of brain heart infusion base containing 10% sucrose, 3% NaCl + 5% sucrose, 3% NaCl, or 5% NaCl + 20% sucrose, and supplemented with filtrates of 10–20% horse serum - Sources A, B, C, suppliers of slaughterhouse (Type I) serum; and S, a supplier of standing herd (Type II) serum - and yeast extract. Three types of yeast supplements used in this study were aqueous extracts of: (1) fresh, or (2) active dry yeast; and (3) 0.5% commercial dehydrated yeast extract. Liquid control media prepared from one supplier gave rise to a pleomorphic Gram positive rod on extended incubation ( > 10 days) in spite of rigorous controls for asepsis during preparation and use. The second and more serious problem concerned repeated encounters with ‘pseudocolony’-like structures in work with clinical material. Correlation with severity of disease, different rates of appearance, and promotion of development by high osmolarity, led to a comparison of this phenomenon in control Type I- vs. Type II-BYE medium. Type I gave rise to numerous ‘pseudocolony‘- like bodies within 3 weeks, while Type II media containing standing herd serum developed < 20 per plate. Media variations known to promote growth of L-phase variants and mycoplasmas were noted to affect time of appearance and numbers of atypical colonies in control, uninoculated Type II-BYE. These included serum level, quality of yeast extract, pH, osmolar supplements, and the O 2 tension. The development of these structures in only certain Type II-BYE cultures of clinical fluids does not support the view that disturbance of an agar surface, or the presence of cells (or nuclei) promotes ‘growth’. No atypical colony-like structures or amber bodies developed in plates incubated for 7 weeks in the presence of vapors of phenol + formaldehyde, nor during an additional 10-day interval in a normal gaseous environment. It is suggested that all horse sera tested in this study contained unclassified atypical L-forms with unusual resistance to heat and chemicals.

Mode of Action of the Staphylococcin-like Peptide Pep 5 and Culture Conditions Effecting its Activity

Pep 5, a staphylococcin-like peptide produced by Staphylococcus epidermidis 5 was bactericidal for staphylococci and micrococci (Fig. 1). A stable L-phase variant of Staphylococcus aureus, but not of Streptococcus faecalis, was susceptible, too, indicating that the selectivity of Pep 5 is not due to the presence of a cell wall receptor. Addition of Pep 5 to cultures of the indicator strain caused immediate inhibition of DNA, RNA, protein, and polysaccharide synthesis (Fig. 5). The action of Pep 5 could be inhibited by increasing the cation content of the medium (e.g., to 100 mM Mg2+ or 600 mM K+) (Fig. 3), indicating a primary binding to anionic sites on the cell wall. Sublethally injured cells could be rescued by the addition of trypsin or 1 M KCl (Fig. 4).

The Antibacterial Action of Vancomycin

Vancomycin inhibits the second stage of cell wall synthesis in susceptible bacteria. Furthermore, there is evidence that vancomycin also alters the permeability of the cell membrane and selectively inhibits ribonucleic acid synthesis. Stable L-phase variants are susceptible to vancomycin, but induction of bacterial L-phase variants with vancomycin is very difficult. Vancomycin is active against a large number of species of gram-positive cocci and bacilli, such as Staphylococcus aureus (including methicillin-resistant strains), Staphylococcus epidermidis (including multiply resistant strains). Streptococcus pneumoniae (including multiply resistant strains), Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus bovis, viridans group Streptococcus, enterococcus, Clostridium species, diphtheroids, Listeria monocytogenes, Actinomyces species, and Lactobacillus species. Antimicrobial activity can be enhanced with the combination of vancomycin and an aminoglycoside against S. aureus, S. bovis, enterococcus, and viridans Streptococcus. The combination of vancomycin and rifampin also has enhanced activity against S. aureus.

Cell wall modification resulting from in vitro induction of L-phase variants of Nocardia asteroides.

The chemical composition of the cell walls of several L-form revertants derived from Nocardia asteroides 10905 was determined at different stages of growth. It was observed that each L-form revertant had a cell well that differed from that of the parental strain when grown under identical conditions. In some strains the peptidolipid and mycolic acid components were affected the most, whereas in other strains the fatty acid, sugar, and mycolic acid moieties were altered. Shifts in mycolic acid size were prominent, whereas the basic peptidoglycan structure appeared to be affected the least. Both the method used to induce the L-form of N. asteroides 10905 and the length of time these organisms were maintained in the wall-less state affected the degree of cell wall modification during the reversion process. Thus, removal of the cell wall appeared to potentiate and select for mutational alterations within the cell envelope of N. asteroides, and these changes resulted in altered cellular and colonial morphology.

Aberrant Forms of Streptococcus cremoris HP

The cheese starter strain, Streptococcus cremoris HP, produced variant colonies when streaked on the surface of solid media and incubated at 30 or 37 degrees C or in the presence of penicillin. Serial plating and incubation at 37 degrees C or in the presence of penicillin resulted in the production of variants. Subculture followed by incubation at 25 degrees C or in the absence of penicillin resulted in the reversion or partial reversion to the parent form. Colony morphology and cell morphology exhibited the characteristics of the L-phase. Evidence suggested that the aberrant forms of S. cremoris at 30 degrees C were transitional phase variants but at 37 degrees C and in the presence of penicillin they were L-phase variants. Electron micrographs showed that the cell walls of the variant cells were defective and that there were differences in the density and the organization of the cytoplasmic constituents compared with the parent cell.

The role of complement in limiting bacteremia caused by streptcoccal L-phase variants.

The role of complement in limiting bacteremia caused by streptcoccal L-phase variants.

Induction of L-phase variants of Nocardia caviae within intact murine lungs.

The data presented show that cells of Nocardia caviae 112 were converted to cell wall-deficient microbial variants within the intact murine lung after intranasal administration. At the time that these L-phase variants were recovered in large numbers from the lung, there was a correspondingly enhanced inflammation leading to alveolar consolidation and animal death. During the peak of this response (at 1 week after infection), normal nocardial cells were neither isolated from nor seen within the lung. It is suggested that the conversion of these normal nocardial cells to their L-phase variant leads to this extensive pulmonary damage. Furthermore, the L-phase organisms appear to play an active role in this pathological effect since introduction of similar amounts of killed nocardial cells into the lungs of the mice failed to produce a similar response.

Isolation of staphylococcal L-phase variants from the blood and egg yolks of normal chickens

The use of special microbiological media and modified techniques has resulted in the stimulation of growth and development of bacterial L forms in samples of blood and egg yolk free from mature bacteria and derived from normal healthy chickens. After prolonged cultivation the majority of these L form cultures reverted to staphylococci.

Activation of the alternative complement pathway by L-phase variants of gram-positive bacteria

The present studies were performed to investigate the potential role of the alternative complement pathway in the host's defense against bacterial L-phase variants and to gain insight into the subcellular component of gram-positive bacteria responsible for activation of the alternative pathway. L-phase variants of Staphylococcus aureus and Streptococcus faecalis were able to activate the alternative pathway and consume C3 in C4-deficient guinea pig serum in amounts comparable to their respective bacterial-phase parent organisms. Activation of the complement system via the alternative pathway resulted in death of the L-phase variants. Membranes prepared from S. faecalis L-phase variants, by either osmotic lysis or mechanical disruption, retained their ability to activate the alternative pathway. Treatment of the membranes by three different methods (water washes, hot trichloroacetic acid, and cold trichloroacetic acid) resulted in a greatly diminished ability of the membranes to activate the alternative pathway. In addition, the extracts derived from the membranes by water washes and by cold-trichloroacetic acid treatment were able to activate the alternative pathway. These studies indicate that these L-phase variants can activate the alternative pathway and suggest that membrane-associated factors play a role in the alternative pathway activation by S. faecalis L-phase variants.