ANNUAL REVIEWS
Further
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Copyright
1975.
All rights reserved
Annu. Rev. Microbiol. 1975.29:79-108. Downloaded from www.annualreviews.org Access provided by Texas Christian University on 12/11/14. For personal use only.
ECOLOGY, PHYSIOLOGY, AND
+1650
GENETICS OF FIMBRIAE AND PILI J. C G. Ottow Institut f1ir Bodenkunde und Standortslehre. Fachbereich Pftanzenproduktion. Universitiit Hohenheim. 7 Stuttgart-Hohenheim 70. West Germany
CONTENTS INTRODUCTION .................. . .. . ...... . ...... . ......
80
TERMINOLOGY.... . ...... ......................................................
80
OCCURRENCE AMONG BACTERIA ..............................................
81
Ecological Considerations ............ . ............................. .
. . ....
81
Distribution Among Bacterial Taxa. . ...... . ............... . ..... . ..........
82
CLASSIFICATION AND ISOLATION.............................................. Types and Properties .....................................................
Isolation of Fimbriae and Pi li.............................................. MORPHOGENESIS AND STRUCTURE . .
85
. . . . . . . . . . . . . . . . . . . . . . . ... . .. . . ... . .. . ... .
Homology in Biosynthesis with Flagella..................................... .
86
Constituents and Structure ............ . ...... . ...... . ...... . ...... . .......
87
GENETIC CONTROL OF FIMBRIAE ............ . ................................. Fimbriae and Colony Morphology......... .................................
89
Environmentally Induced Fimbrial Phase Variation ....... . ... . .......... . . ...
89
Genotypic and Phenotypic Types of Fimbriation...............................
90
Phase Variation and Competence.......... .................................
91
Mapping
Locus ..................................................
92
Genetic Models for Fimbriation ............................................
93
the
lim
ADHESIVE PROPERTIES OF FIMBRIAE ........ . ................................. Infection and Pathogenicity............................................... '.
94
Hemagglutinating Activity of Fimbriae......................................
95
Antigenic Properties of Fimbriae...................... ......................
96
Aggregatian and Pellicle Formation by Fimbriae .. . . ... . ........ . . . . ...... . . ..
97
PHYSIOLOGICAL PROPERTIES OF FIMBRIAE.. . ........................... ...... Influence of Fimbriae on Metabolic Activity. . . . . . . .. . .. . .. . . .. . ... . .. . . ......
86
89
94
99
99
SEX PILI AND THEIR PROPERTIES.. . Definition and Functions.. .
82
82
........................................... 100 ...............................................
Types of Sex Pi":' F- and I-like Pili.........................................
101
Pili and Their Genetic Control............................................. Sex Pili in Male-Specific Phage Infection .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
101 102
Role in Conjugation........... . . .... . . . ................... ........ . ......
104
CONCLUDING REMARKS ..................... . .................................
100
105 79
80
OTTOW
INTRODUCTION Bacterial cell morphology was one of thl! early aspects of microbiology and has been
the subject of intensive studies ever since the days of Antonie van Leeuwenhoek. However, despite much progress in optical and preparation techniques, even the
most powerful light microscope was unable to reveal the cell appendages known as
"fimbriae" or "pili," because their small size (ranging from 30-250 A) is beyond the
Annu. Rev. Microbiol. 1975.29:79-108. Downloaded from www.annualreviews.org Access provided by Texas Christian University on 12/11/14. For personal use only.
resolving power (limited to about
250
nm) of even the best optical lenses. Unlike
flagella, the filamentous, nonflagellar appendages termed fimbriae or pili can not be seen even after specific staining methods such as the Leifson flagella stain. Not until
the electron microscope became widely used could these ultramicroscopic structures
be discovered and studied in detail. Anderson independently in
1949 the
(4) and Houwink (61) reported
observation of distinct filamentous appendages different
from the known flagella. Since then, a great number of publications have appeared
on the various properties of these structures. In the author's opinion, this new area
has developed sufficiently to allow a coherent review of its various aspects.
Nonftagellar appendages of bacteria that have a rigid cell wall differ from flagella
in that they are less rigid and in most cases thinner and straighter, and usually outnumber the motility organs. A more precise definition of these nonflagellar appendages is impossible because of the great variation in morphology and func
tions, as shown later. Nevertheless, a cRarifying statement should be made about all those bacterial appendages that are not the object of this review. Excluded are the
broad, ribbon-like appendages observed on spores of certain clostridia
as the flexible rodlike structures that are found on
(125), as well
Azotobacter chroococcum
and
resemble normal flagella coated with capsular polysaccharide material (70). Neither the peculiar appendages of some
Streptomyces
spore-coats (146) nor the prosthecate
(129) are Bdellovibrio bacteri
cellular extensions of some unusual ba 107
144
Structure
electron micrographs show
12,101
only to distal tip
13,64,121, 127
helical nature Composition of
Protein sensitivity to
pilin
trypsin and other proteases;
Axial hole
25-31
Hydrophobic
sensitive to organic solvents;
be h avior
aggregate side to side
F, R, or
13,15,76
I types of pilin
A in diameter
Antigenicity
F-specific, four serotypes;
F pilus
different between ends and
12,15,132 13,15 75,96, 100, 110
shafts of lube Antigenicity of
F and R antigens on
"mixed" F and
single pilus
R pili
76
FIMBRIAE AND PILI
85
filamentous structures observed with Caulobacter ( 1 29), the K88 filaments of E. coli (135), and the PF pili of Pseudomonas aeruginosa (to) form another group with functions similar to those ascribed to sex pili. Thus, the fimbriae of Caulobacter and P. aeruginosa may act as specific receptors for RNA phages, whereas the K88 filaments of E. coli (responsible for a particular surface antigen) are determined by a specific transmissible episome. 3 The thick, hollow tubes observed on Agrobacterium sp., measuring 400-600 A wide and up to 3 iJ-m long, are infundibuliformly attached to the cell surface. These features correspond to a new group of nonfiagellar, filamentous appendages (107). This hollow type has been observed on various unusual, unidenti fied soil bacteria (109, 1 34) and may occur more widely than thought, but its nature and function are not yet understood.
Annu. Rev. Microbiol. 1975.29:79-108. Downloaded from www.annualreviews.org Access provided by Texas Christian University on 12/11/14. For personal use only.
GROUP
GROUP 4 These flexible, rodlike, polarly inserted fimbriae were observed on mem bers of the genus Pseudomonas and Vibrio (36, 143). These fimbriae in polar position differ from the peritrichously arranged fimbriae with adhesive properties (36) in their ability to promote bacterial motion as opposed to acting as attachment organelles ( 1 43). Further, it appears that most of these polar fimbriae have a hollow core (48), alth ough this trait was not confirmed in general (143).
This class comprises the polarly arranged, contractable tubules observed on star-forming soil inhabitants such as Pseudomonas "rhodos," P. "echinoides," Agrobacterium spp., and Rhizobium lupini (92-94, 107). These structures pull
GROUP 5
bacteria together by contraction into star-forming cell clusters, allowing competent cells to conjugate
(53-55). Fimbriae of this group thus serve in recognition, contact, (55), rather than acting as true transfer
and irreversible joining of competent cells ring bridges.
GROUP 6
Cells of gram-positive
Corynebacterium renale
(responsible for bovine
pyelonephritis and cystitis) produce characteristic bundles of fimbriae. So far, such .
bundles have not yet been described amon� gram-negative bacteria. Each bundle
contains several filaments of about 25-30 A in diameter. The number of bundles varies considerably according to the strain. These filaments act as specific antigens, but neither hemagglutination nor pellicle formation could be detected with these structures ( 1 49, 1 50). Isolation of Fimbriae and Pili Isolation and concentration of nontlagellar appendages for chemical analysis, for the preparation of antisera, and for electron microscope studies are easily achieved by mechanical agitation (blending) and high speed centrifugation of cultures in the logarithmic growth phase
(7, 1 2, 1 5, 21, 1 1 1 , 1 49).
If the sediment is resuspended
in neutral saline solution and left for several days, the aggregated fimbriae can be separated from remaining impurities by recentrifugation
( 1 5, 2 1 ).
In particular, the capacity of defimbriated cells to produce nontlagellar append ages is used in isolation studies. With these types of experiments, synthesis and
Annu. Rev. Microbiol. 1975.29:79-108. Downloaded from www.annualreviews.org Access provided by Texas Christian University on 12/11/14. For personal use only.
86
OTTOW
growth can be studied, and a correlation made between the functions reacquired as the fimbriae reappear (111). Sex pili usually reappear rapidly, reaching full length in 4-5 min, whereas fimbriae increase in length much more slowly and constantly throughout the generation time of a cell (91, 111). In contrast, sex pili reach a constant length and number per cell in about one seventh of the generation time. This characteristic way of growing observed in sex pili is strikingly similar to the mode of outgrowth observed with DNA male phages ( 1 2, 1 14). For certain investigations, the separation of sex pili from fimbriae is necessary. Ippen & Valentine (64) developed a filtration assay in order to determine the relative quantity of sex pili in a given culture. This method is based on the knowledge that free male-specific RNA phages pass through a cellulose filter, whereas sex pili-phage complexes are retained on the filter pad. Using 32P-or 35S-labeled phages, the relative quantity of sex pili present in the mixture can be determined by counting the radioactivity of the filter (7, 15, 142). The purification of sex pili could be facilitated if the host strain could be freed of flagella and fimbriae. Attempts to produce nonfimbriate mutants from nonflagel lar variants of E coli K12 were unsuccessful (7), despite the fact that fimbriae and pili are differently determined genetically. To overcome this difficulty, Beard and co-workers (7) blended fla- E coli K 1 2 cells at the peak time of sex pilus produc tion, concentrated the appendages by c(:ntrifugation, and dialyzed and finally clari fied the material in an ultrafiltration system. After additionally blending clearing, and spinning, the sediment was centrifuged at 70,000 X g for 72 hr in a CsCI gradient. Electron micrographs showed the material at a density of 1.309 g/cm3. The peak of phage-binding activity (determined with the radioactive male-specific phage assay) contained both sex pili and fimbriae. Control experiments with a nonpiliated but fimbriated strain showed that fimbriae alone banded at the same density of 1. 309 g/cm3. With the aid of isoelectric focusing, the peak from the CsCI gradient was separated into two main protein peaks, the first of which showed phage-binding activity. With this procedure, about 5 mg of intact pili of about 98% purity were obtained from approximately 100 g wet weight of cell material (7). Purification of sex pili is a prerequisite for the elucidation of their specific properties.
MORPHOGENESIS AND STRUCTURE Homology in Biosynthesis with Flagella
Fimbriae and pili are similar to flagella in several aspects. Because several excellent reviews on the biology of flagella (5,63,69,133) have appeared within the last years, the reader is referred to those publications for a detailed survey on biosynthesis and properties of flagella. In summary, flagella originate from the cytoplasm (1, 93, 133, 140) at a circular portion of the cytoplasmic membrane (5,23,28, 106) and penetrate through the peptidoglycan layer(s) of the cell wall, without being covered by the latter. Like flagella, fimbriae and pili can be regarded as cytoplasmic extrusions through the cell wall (60, 93). Further, both flagella as well as nonflagellar append ages can originate either from cellula,r poles or from random positions on the
Annu. Rev. Microbiol. 1975.29:79-108. Downloaded from www.annualreviews.org Access provided by Texas Christian University on 12/11/14. For personal use only.
fIMBRIAE AND PILI
87
bacterial cell (33, 36,48, 139, 143). However, bacterial fimbriae and pili differ from flagella in one characteristic aspect: they do not display sinuous waves with a characteristic wavelength ("'2.5 nm), although some fimbriae of Pseudomonas aeruginosa may show some coiling and bending (143). The homology between flagella and fimbriae or pili is supported by several other observations. Thus, motility and flagellar-specific antigens reappear, whereas the generation of flagella on defla gellated bacteria proceeds with time. Similarly, specific fimbrial antigens are reac quired as soon as these filamentous structures reappear on artificially defimbriated organisms (12). It is generally accepted that flagella are synthesized from amino acids in a two-step process (5, 63, 88, 89). In the first step, a flagellin pool is filled inside the cell. Second, flagellin is transported to the growing, distal ends of the flagellum. The fact that cells, prelabeled with radioactive amino acids, still produced radioactive flagella (only in one trial) in the presence of chloramphenicol, suggests that the biosynthesis of amino acids rather than the polymerization of flagella from flagellin is affected by the drug. Again, similar experimental results were obtained with defimbriated bacteria. When defimbriated cells of E. coli were treated with growth-inhibiting concentrations of chloramphenicol, a regeneration of the nonfla gellar appendages took place, even after being artificially defimbriated several times (12, 15, 89, 10 \ ). Apparently, a considerable pool of fimbrial precursor protein can be assumed. In the presence of such a large flagellin or fimbrial protein poo\' both types of filamentous appendages can be polymerized even in the presence of chlor amphenicol. Of particular interest for the biosynthesis of fimbriae is the discovery of basal bodies on Proteus mirabilis fimbriae that arise either from the cytoplasm or at the base of the cytoplasmic membrane (60). Thus, even in this respect, flagella and fimbriae (at least those of Proteus mirabilis) are similarly composed. The cytoplas mic origin of both flagella and fimbriae is additionally supported by the observation that both structures are retained even after conversion of E. coli and Proteus mirabilis cells into their spheroplasts (87). On the other hand, it has been observed that flagellation may be suppressed at relatively high growth temperatures through effects exerted on the cell surface structures (95). Proteus mirabilis cells ceased flagella production after one generation time, when the organisms were transferred from 37 to 42.5°C. Neither flagellar hooks nor basal structures were observed in Iysates when viewed through the electron microscope. The cells regained their flagellin synthesis with the production of flagella after one generation, by transfer ring them back to 37°C. However, penicillin, when added to nonflagellated cells during the first 30 min after the shift from 42.5 to 37°C, also inhibited by 98% the generation of flagellin. These findings imply that the early processes of flagella regeneration require either the presence or the concurrent synthesis of intact cell wall structures. Experiments with fimbriate Proteus mirabilis strains may decide whether these findings are also true for fimbrial synthesis.
I
Constituents and Structure
Lowry and ninhydrin assays of electron microscopically and electrophoretically pure fimbriae showed that E. coli fimbriae consist of nearly 100% protein (17). Such
Annu. Rev. Microbiol. 1975.29:79-108. Downloaded from www.annualreviews.org Access provided by Texas Christian University on 12/11/14. For personal use only.
88
OTTOW
E. coli fimbriae contained 4.6% N (Kjeldahl). They were strongly positive in the xanthoproteic or biuret tests, and could be precipitated easily with (NH4)2S04 (11). DNA, RNA, phosphate, polysaccharides, and reducing sugars were each present at less than 0.6%. The UV adsorption spectrum was typical for proteins, and the optical density of fimbriae suspensions could be reduced considerably by trypsin and pepsin, but remained unaffected by chernotrypsin, DNase, and RNase (15, 21, 150). For the fimbrial protein (fimbrilin), a molecular weight of approximately 16,600 was found (flagellin has a molecular weight of about 40,(00). A chemical analysis showed that the fibrous components of this protein contained at least 163 amino acids, most of them occurring in the L form with a large quantity of hydrocarbon side chains. The nonpolar side chains of these amino acids could be responsible for the hydrophobic property of these filaments as concluded by their tendency to escape from an aqueous milieu (12). Whether there is any difference in composition of fimbrilin and pilin (15) has not yet been determined. Information from X-ray diffraction patterns as well as from geometrical consider ations of electron microscopic examina.tions of E coli fimbriae suggest that these structures have a rod-shaped, rigid, helicai structure similar to that of bacterial flagella. This structural model was proposed by Brinton & Lee Huang (19) and completed later by Brinton (12,), Lawn (72), and Mayer (91). In shadowed electron micrographs of single fimbriae, the presence of regularly arranged subunits was seen (12). Chemical agents, such as urea, glacial acetic acid, and guanidine HCl, all known to break weaker bonds of tertiary protein structures but to be unable to disrupt polypeptide chains, fractured single fimbriae into smaller units. The exact size of the subunit has not yet been determined, but a model with 3 Ys subunit per right turn of the helix makes the best filt to the experimental position of the reflec tions observed (12, 136). Fimbrial subunits were also found in negatively contrasted preparations of Pseudomonas testosteroni, P. 'tragi" (48), P. "echinoides" (56), and Salmonella typhimurium (WI). From their observations with pseudomonads, Fuerst & Hayward (48) proposed a fimbrial model, consisting of two helically wound strands, each composed of rep
Further
Quick links to online content
Copyright
1975.
All rights reserved
Annu. Rev. Microbiol. 1975.29:79-108. Downloaded from www.annualreviews.org Access provided by Texas Christian University on 12/11/14. For personal use only.
ECOLOGY, PHYSIOLOGY, AND
+1650
GENETICS OF FIMBRIAE AND PILI J. C G. Ottow Institut f1ir Bodenkunde und Standortslehre. Fachbereich Pftanzenproduktion. Universitiit Hohenheim. 7 Stuttgart-Hohenheim 70. West Germany
CONTENTS INTRODUCTION .................. . .. . ...... . ...... . ......
80
TERMINOLOGY.... . ...... ......................................................
80
OCCURRENCE AMONG BACTERIA ..............................................
81
Ecological Considerations ............ . ............................. .
. . ....
81
Distribution Among Bacterial Taxa. . ...... . ............... . ..... . ..........
82
CLASSIFICATION AND ISOLATION.............................................. Types and Properties .....................................................
Isolation of Fimbriae and Pi li.............................................. MORPHOGENESIS AND STRUCTURE . .
85
. . . . . . . . . . . . . . . . . . . . . . . ... . .. . . ... . .. . ... .
Homology in Biosynthesis with Flagella..................................... .
86
Constituents and Structure ............ . ...... . ...... . ...... . ...... . .......
87
GENETIC CONTROL OF FIMBRIAE ............ . ................................. Fimbriae and Colony Morphology......... .................................
89
Environmentally Induced Fimbrial Phase Variation ....... . ... . .......... . . ...
89
Genotypic and Phenotypic Types of Fimbriation...............................
90
Phase Variation and Competence.......... .................................
91
Mapping
Locus ..................................................
92
Genetic Models for Fimbriation ............................................
93
the
lim
ADHESIVE PROPERTIES OF FIMBRIAE ........ . ................................. Infection and Pathogenicity............................................... '.
94
Hemagglutinating Activity of Fimbriae......................................
95
Antigenic Properties of Fimbriae...................... ......................
96
Aggregatian and Pellicle Formation by Fimbriae .. . . ... . ........ . . . . ...... . . ..
97
PHYSIOLOGICAL PROPERTIES OF FIMBRIAE.. . ........................... ...... Influence of Fimbriae on Metabolic Activity. . . . . . . .. . .. . .. . . .. . ... . .. . . ......
86
89
94
99
99
SEX PILI AND THEIR PROPERTIES.. . Definition and Functions.. .
82
82
........................................... 100 ...............................................
Types of Sex Pi":' F- and I-like Pili.........................................
101
Pili and Their Genetic Control............................................. Sex Pili in Male-Specific Phage Infection .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
101 102
Role in Conjugation........... . . .... . . . ................... ........ . ......
104
CONCLUDING REMARKS ..................... . .................................
100
105 79
80
OTTOW
INTRODUCTION Bacterial cell morphology was one of thl! early aspects of microbiology and has been
the subject of intensive studies ever since the days of Antonie van Leeuwenhoek. However, despite much progress in optical and preparation techniques, even the
most powerful light microscope was unable to reveal the cell appendages known as
"fimbriae" or "pili," because their small size (ranging from 30-250 A) is beyond the
Annu. Rev. Microbiol. 1975.29:79-108. Downloaded from www.annualreviews.org Access provided by Texas Christian University on 12/11/14. For personal use only.
resolving power (limited to about
250
nm) of even the best optical lenses. Unlike
flagella, the filamentous, nonflagellar appendages termed fimbriae or pili can not be seen even after specific staining methods such as the Leifson flagella stain. Not until
the electron microscope became widely used could these ultramicroscopic structures
be discovered and studied in detail. Anderson independently in
1949 the
(4) and Houwink (61) reported
observation of distinct filamentous appendages different
from the known flagella. Since then, a great number of publications have appeared
on the various properties of these structures. In the author's opinion, this new area
has developed sufficiently to allow a coherent review of its various aspects.
Nonftagellar appendages of bacteria that have a rigid cell wall differ from flagella
in that they are less rigid and in most cases thinner and straighter, and usually outnumber the motility organs. A more precise definition of these nonflagellar appendages is impossible because of the great variation in morphology and func
tions, as shown later. Nevertheless, a cRarifying statement should be made about all those bacterial appendages that are not the object of this review. Excluded are the
broad, ribbon-like appendages observed on spores of certain clostridia
as the flexible rodlike structures that are found on
(125), as well
Azotobacter chroococcum
and
resemble normal flagella coated with capsular polysaccharide material (70). Neither the peculiar appendages of some
Streptomyces
spore-coats (146) nor the prosthecate
(129) are Bdellovibrio bacteri
cellular extensions of some unusual ba 107
144
Structure
electron micrographs show
12,101
only to distal tip
13,64,121, 127
helical nature Composition of
Protein sensitivity to
pilin
trypsin and other proteases;
Axial hole
25-31
Hydrophobic
sensitive to organic solvents;
be h avior
aggregate side to side
F, R, or
13,15,76
I types of pilin
A in diameter
Antigenicity
F-specific, four serotypes;
F pilus
different between ends and
12,15,132 13,15 75,96, 100, 110
shafts of lube Antigenicity of
F and R antigens on
"mixed" F and
single pilus
R pili
76
FIMBRIAE AND PILI
85
filamentous structures observed with Caulobacter ( 1 29), the K88 filaments of E. coli (135), and the PF pili of Pseudomonas aeruginosa (to) form another group with functions similar to those ascribed to sex pili. Thus, the fimbriae of Caulobacter and P. aeruginosa may act as specific receptors for RNA phages, whereas the K88 filaments of E. coli (responsible for a particular surface antigen) are determined by a specific transmissible episome. 3 The thick, hollow tubes observed on Agrobacterium sp., measuring 400-600 A wide and up to 3 iJ-m long, are infundibuliformly attached to the cell surface. These features correspond to a new group of nonfiagellar, filamentous appendages (107). This hollow type has been observed on various unusual, unidenti fied soil bacteria (109, 1 34) and may occur more widely than thought, but its nature and function are not yet understood.
Annu. Rev. Microbiol. 1975.29:79-108. Downloaded from www.annualreviews.org Access provided by Texas Christian University on 12/11/14. For personal use only.
GROUP
GROUP 4 These flexible, rodlike, polarly inserted fimbriae were observed on mem bers of the genus Pseudomonas and Vibrio (36, 143). These fimbriae in polar position differ from the peritrichously arranged fimbriae with adhesive properties (36) in their ability to promote bacterial motion as opposed to acting as attachment organelles ( 1 43). Further, it appears that most of these polar fimbriae have a hollow core (48), alth ough this trait was not confirmed in general (143).
This class comprises the polarly arranged, contractable tubules observed on star-forming soil inhabitants such as Pseudomonas "rhodos," P. "echinoides," Agrobacterium spp., and Rhizobium lupini (92-94, 107). These structures pull
GROUP 5
bacteria together by contraction into star-forming cell clusters, allowing competent cells to conjugate
(53-55). Fimbriae of this group thus serve in recognition, contact, (55), rather than acting as true transfer
and irreversible joining of competent cells ring bridges.
GROUP 6
Cells of gram-positive
Corynebacterium renale
(responsible for bovine
pyelonephritis and cystitis) produce characteristic bundles of fimbriae. So far, such .
bundles have not yet been described amon� gram-negative bacteria. Each bundle
contains several filaments of about 25-30 A in diameter. The number of bundles varies considerably according to the strain. These filaments act as specific antigens, but neither hemagglutination nor pellicle formation could be detected with these structures ( 1 49, 1 50). Isolation of Fimbriae and Pili Isolation and concentration of nontlagellar appendages for chemical analysis, for the preparation of antisera, and for electron microscope studies are easily achieved by mechanical agitation (blending) and high speed centrifugation of cultures in the logarithmic growth phase
(7, 1 2, 1 5, 21, 1 1 1 , 1 49).
If the sediment is resuspended
in neutral saline solution and left for several days, the aggregated fimbriae can be separated from remaining impurities by recentrifugation
( 1 5, 2 1 ).
In particular, the capacity of defimbriated cells to produce nontlagellar append ages is used in isolation studies. With these types of experiments, synthesis and
Annu. Rev. Microbiol. 1975.29:79-108. Downloaded from www.annualreviews.org Access provided by Texas Christian University on 12/11/14. For personal use only.
86
OTTOW
growth can be studied, and a correlation made between the functions reacquired as the fimbriae reappear (111). Sex pili usually reappear rapidly, reaching full length in 4-5 min, whereas fimbriae increase in length much more slowly and constantly throughout the generation time of a cell (91, 111). In contrast, sex pili reach a constant length and number per cell in about one seventh of the generation time. This characteristic way of growing observed in sex pili is strikingly similar to the mode of outgrowth observed with DNA male phages ( 1 2, 1 14). For certain investigations, the separation of sex pili from fimbriae is necessary. Ippen & Valentine (64) developed a filtration assay in order to determine the relative quantity of sex pili in a given culture. This method is based on the knowledge that free male-specific RNA phages pass through a cellulose filter, whereas sex pili-phage complexes are retained on the filter pad. Using 32P-or 35S-labeled phages, the relative quantity of sex pili present in the mixture can be determined by counting the radioactivity of the filter (7, 15, 142). The purification of sex pili could be facilitated if the host strain could be freed of flagella and fimbriae. Attempts to produce nonfimbriate mutants from nonflagel lar variants of E coli K12 were unsuccessful (7), despite the fact that fimbriae and pili are differently determined genetically. To overcome this difficulty, Beard and co-workers (7) blended fla- E coli K 1 2 cells at the peak time of sex pilus produc tion, concentrated the appendages by c(:ntrifugation, and dialyzed and finally clari fied the material in an ultrafiltration system. After additionally blending clearing, and spinning, the sediment was centrifuged at 70,000 X g for 72 hr in a CsCI gradient. Electron micrographs showed the material at a density of 1.309 g/cm3. The peak of phage-binding activity (determined with the radioactive male-specific phage assay) contained both sex pili and fimbriae. Control experiments with a nonpiliated but fimbriated strain showed that fimbriae alone banded at the same density of 1. 309 g/cm3. With the aid of isoelectric focusing, the peak from the CsCI gradient was separated into two main protein peaks, the first of which showed phage-binding activity. With this procedure, about 5 mg of intact pili of about 98% purity were obtained from approximately 100 g wet weight of cell material (7). Purification of sex pili is a prerequisite for the elucidation of their specific properties.
MORPHOGENESIS AND STRUCTURE Homology in Biosynthesis with Flagella
Fimbriae and pili are similar to flagella in several aspects. Because several excellent reviews on the biology of flagella (5,63,69,133) have appeared within the last years, the reader is referred to those publications for a detailed survey on biosynthesis and properties of flagella. In summary, flagella originate from the cytoplasm (1, 93, 133, 140) at a circular portion of the cytoplasmic membrane (5,23,28, 106) and penetrate through the peptidoglycan layer(s) of the cell wall, without being covered by the latter. Like flagella, fimbriae and pili can be regarded as cytoplasmic extrusions through the cell wall (60, 93). Further, both flagella as well as nonflagellar append ages can originate either from cellula,r poles or from random positions on the
Annu. Rev. Microbiol. 1975.29:79-108. Downloaded from www.annualreviews.org Access provided by Texas Christian University on 12/11/14. For personal use only.
fIMBRIAE AND PILI
87
bacterial cell (33, 36,48, 139, 143). However, bacterial fimbriae and pili differ from flagella in one characteristic aspect: they do not display sinuous waves with a characteristic wavelength ("'2.5 nm), although some fimbriae of Pseudomonas aeruginosa may show some coiling and bending (143). The homology between flagella and fimbriae or pili is supported by several other observations. Thus, motility and flagellar-specific antigens reappear, whereas the generation of flagella on defla gellated bacteria proceeds with time. Similarly, specific fimbrial antigens are reac quired as soon as these filamentous structures reappear on artificially defimbriated organisms (12). It is generally accepted that flagella are synthesized from amino acids in a two-step process (5, 63, 88, 89). In the first step, a flagellin pool is filled inside the cell. Second, flagellin is transported to the growing, distal ends of the flagellum. The fact that cells, prelabeled with radioactive amino acids, still produced radioactive flagella (only in one trial) in the presence of chloramphenicol, suggests that the biosynthesis of amino acids rather than the polymerization of flagella from flagellin is affected by the drug. Again, similar experimental results were obtained with defimbriated bacteria. When defimbriated cells of E. coli were treated with growth-inhibiting concentrations of chloramphenicol, a regeneration of the nonfla gellar appendages took place, even after being artificially defimbriated several times (12, 15, 89, 10 \ ). Apparently, a considerable pool of fimbrial precursor protein can be assumed. In the presence of such a large flagellin or fimbrial protein poo\' both types of filamentous appendages can be polymerized even in the presence of chlor amphenicol. Of particular interest for the biosynthesis of fimbriae is the discovery of basal bodies on Proteus mirabilis fimbriae that arise either from the cytoplasm or at the base of the cytoplasmic membrane (60). Thus, even in this respect, flagella and fimbriae (at least those of Proteus mirabilis) are similarly composed. The cytoplas mic origin of both flagella and fimbriae is additionally supported by the observation that both structures are retained even after conversion of E. coli and Proteus mirabilis cells into their spheroplasts (87). On the other hand, it has been observed that flagellation may be suppressed at relatively high growth temperatures through effects exerted on the cell surface structures (95). Proteus mirabilis cells ceased flagella production after one generation time, when the organisms were transferred from 37 to 42.5°C. Neither flagellar hooks nor basal structures were observed in Iysates when viewed through the electron microscope. The cells regained their flagellin synthesis with the production of flagella after one generation, by transfer ring them back to 37°C. However, penicillin, when added to nonflagellated cells during the first 30 min after the shift from 42.5 to 37°C, also inhibited by 98% the generation of flagellin. These findings imply that the early processes of flagella regeneration require either the presence or the concurrent synthesis of intact cell wall structures. Experiments with fimbriate Proteus mirabilis strains may decide whether these findings are also true for fimbrial synthesis.
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Constituents and Structure
Lowry and ninhydrin assays of electron microscopically and electrophoretically pure fimbriae showed that E. coli fimbriae consist of nearly 100% protein (17). Such
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E. coli fimbriae contained 4.6% N (Kjeldahl). They were strongly positive in the xanthoproteic or biuret tests, and could be precipitated easily with (NH4)2S04 (11). DNA, RNA, phosphate, polysaccharides, and reducing sugars were each present at less than 0.6%. The UV adsorption spectrum was typical for proteins, and the optical density of fimbriae suspensions could be reduced considerably by trypsin and pepsin, but remained unaffected by chernotrypsin, DNase, and RNase (15, 21, 150). For the fimbrial protein (fimbrilin), a molecular weight of approximately 16,600 was found (flagellin has a molecular weight of about 40,(00). A chemical analysis showed that the fibrous components of this protein contained at least 163 amino acids, most of them occurring in the L form with a large quantity of hydrocarbon side chains. The nonpolar side chains of these amino acids could be responsible for the hydrophobic property of these filaments as concluded by their tendency to escape from an aqueous milieu (12). Whether there is any difference in composition of fimbrilin and pilin (15) has not yet been determined. Information from X-ray diffraction patterns as well as from geometrical consider ations of electron microscopic examina.tions of E coli fimbriae suggest that these structures have a rod-shaped, rigid, helicai structure similar to that of bacterial flagella. This structural model was proposed by Brinton & Lee Huang (19) and completed later by Brinton (12,), Lawn (72), and Mayer (91). In shadowed electron micrographs of single fimbriae, the presence of regularly arranged subunits was seen (12). Chemical agents, such as urea, glacial acetic acid, and guanidine HCl, all known to break weaker bonds of tertiary protein structures but to be unable to disrupt polypeptide chains, fractured single fimbriae into smaller units. The exact size of the subunit has not yet been determined, but a model with 3 Ys subunit per right turn of the helix makes the best filt to the experimental position of the reflec tions observed (12, 136). Fimbrial subunits were also found in negatively contrasted preparations of Pseudomonas testosteroni, P. 'tragi" (48), P. "echinoides" (56), and Salmonella typhimurium (WI). From their observations with pseudomonads, Fuerst & Hayward (48) proposed a fimbrial model, consisting of two helically wound strands, each composed of rep
