JOURNAL OF BACTERIOLOGY, Sept. 1990, p. 5425-5431 0021-9193/90/095425-07$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 172, No. 9
Identification of Genes Affecting Production of the Adhesion Organelle of Caulobacter crescentus CB2 DAVID MITCHELLt AND JOHN SMIT*
Department of Microbiology, University of British Columbia, Vancouver, British Columbia V6T I W5 Canada Received 12 January 1990/Accepted 6 June 1990
Transposon (Tn5) mutagenesis was used to identify regions in the genome involved with production, regulation, or attachment to the cell surface of the adhesive holdfast of the freshwater bacterium Caulobacter crescentus CB2. A total of 12,000 independently selected transposon insertion mutants were screened for defects in adhesion to cellulose acetate; 77 mutants were detected and examined by Southern blot hybridization mapping methods and pulsed-field gel electrophoresis. Ten unique sites of TnS insertion affecting holdfast function were identified that were clustered in four regions of the genome. Representative mutants of the 10 TnS insertion sites were examined by a variety of methods for differences in their phenotype leading to the loss of adhesiveness. Four phenotypes were identified: no holdfast production, production of a smaller or an altered holdfast, production of a holdfast that was unable to remain attached to the cell, and a fourth category in which a possible alteration of the stalk was related to impaired adhesion of the cell. With the possible exception of the last class, no pleiotropic mutants (those with multiple defects in the polar region of the cell) were detected among the adhesion-defective mutants. This was unexpected, since holdfast deficiency is often a characteristic of pleiotropic mutants obtained when selecting for loss of other polar structures. Overall, the evidence suggests that we have identified regions containing structural genes for the holdfast, genes involved with proper attachment or positioning on the caulobacter surface, and possibly regions that regulate the levels of holdfast production.
Caulobacters are gram-negative, chemoheterotrophic bacteria that during their growth cycle express two distinct cell types: a dispersive, flagellated swarmer cell and a proliferative, sessile stalked cell (20, 24). Swarmer cells express several temporally and spatially controlled organelles at one pole of the cell: a cluster of pili, a single flagellum, a caulophage receptor, and an adhesive holdfast. Differentiation into the stalked cell involves loss of the flagellum, disappearance of the pili, and localized growth of the cell envelope at the same polar region to form a stalk. The holdfast, first produced in the swarmer cell, is retained and finally resides at the tip of the stalk (8, 24, 32). Because of this adhesion organelle, caulobacters are epiphytes of surfaces and members of the bacterial consortia in both freshwater and marine environments that participate in the initial stages of biofouling of submerged objects. Attachment may be promoted by swarmer-cell motility (that is, by producing a strong initial contact with the surface) and may be assisted by the polar pili but is chiefly mediated by the holdfast. The stalk that develops lengthens in response to nutrient-deficient conditions, possibly enabling the main cell body to remain at the surface of biofilms, where the cell can preferentially acquire nutrition that arrives at the surface and have better access to oxygen for efficient metabolism. Together, the polar organelles probably contribute significantly to the competitiveness of caulobacters on surfaces, which may explain their nearly ubiquitous representation in oligotrophic environments (8, 19, 20, 24). The only function of the caulobacter holdfast seems to be adhesion, and as such it mediates cell attachment to a variety of biological and nonbiological substrata (19, 24). The bound
holdfast can remain intact under conditions of high shear force for many generations, suggesting that the holdfast mediates strong, stable adhesion (19, 24). These factors argue that the holdfast is an appropriate model for the analysis of chemical and structural principles used by bacteria to accomplish generalized adhesion. The composition or structure of the holdfast material has not been elucidated, but all available evidence points to the holdfast being a complex polysaccharide. Lectin binding and enzyme sensitivity studies suggest that N-acetylgalactosamine (22) and stretches of two or more units of N-acetylglucosamine are present in the holdfast material of Caulobacter crescentus CB2 (19, 24). Positive histochemical staining (32) suggests that some caulobacter holdfasts may contain acidic components such as uronic acids, as has been suggested for other putative adhesive polymers (5, 6, 13, 30, 31). Lipids or proteins have not yet been detected (19). Assuming that the holdfast is a complex polysaccharide, a number of genes must be involved with the production of the adhesive structure. In addition, analysis of holdfast mutants suggests that there is a specific site or membrane component at which the holdfast material is anchored to the stalk (22). Finally, production of the holdfast is temporally regulated during the caulobacter cell cycle and is spatially restricted to the polar region of the cell. In sum, we predicted the presence of a number of genes involved with holdfast production and proper functioning and that not all would be structural genes. In this report we describe our initial findings in evaluating the location and complexity of the genes related to the holdfast of the freshwater bacterium C. crescentus CB2A.
* Corresponding author. t Present address: Quadra Logic Technologies, Inc., Vancouver, BC V5Z 4H5, Canada.
MATERIALS AND METHODS Strains, phage, plasmids, and growth conditions. C. crescentus CB2A, a variant of strain CB2 that lacks the paracrys5425
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MITCHELL AND SMIT
talline surface protein layer (24, 28), was used for transposon mutagenesis. A spontaneous rifampin-resistant strain was used for the conjugal matings. Strains CB2A-B5 and CB2A-B9 are holdfast-negative and holdfast-shedding mutants, respectively, produced by UV mutagenesis (22). Escherichia coli SM10 (26) was used for conjugal mating experiments. CbK is a caulophage that only attaches to the polar region of swarmer caulobacter cells (1, 17). T7 is a lytic coliphage (4). C. crescentus strains were grown at 30°C in a peptoneyeast extract medium (PYE) (23) supplemented with CaCl2 . 2H20 (0.1 g/liter) or Hutner mineral base (24), supplemented with 0.05% NH4C1 and 0.2% glucose (HMG). As appropriate, antibiotics were added at the following concentrations: chloramphenicol, 2 ,ug/ml; kanamycin, 50 p.g/ml; rifampin, 5 j.g/ml; streptomycin, 50 jig/ml; and trimethoprim, 150 ,ug/ml. E. coli SM10 was grown at 37°C in Luria broth (18) with antibiotics at the following concentrations: chloramphenicol, 30 ,ug/ml and kanamycin, 50 ,ug/ml. Coliphage T7 and caulophage CbK were propagated by standard methods (25). The delivery vehicle for TnS mutagenesis was the narrowhost-range (ColEl replicon) plasmid pSUP2021 (26), which can be transferred to C. crescentus by conjugal mating but is not maintained. pBR322 (3) was used as a hybridization control is some experiments. pBR322-Neo contains the 1.8-kilobase (kb) HindIII-BamHI fragment of TnS inserted into the corresponding sites of pBR322 (John Swindle, unpublished data). The insert was used as hybridization probe for TnS. The TnS mutant library. (i) Production of the library. Conjugal mating of C. crescentus CB2A (RFI) with SM10 (pSUP2021) was done by using a variation of previous methods (2). After overnight mating of 108 donor and recipient cells, the cells were suspended in PYE and 108 PFU of coliphage T7 was added. This mixture was incubated for 2 h at 37°C, and then samples were plated onto PYE containing drugs selective against the SM10 cells and selective for caulobacters containing Tn5 (trimethoprim, rifampin, kanamycin, streptomycin) (21). Then 16,000 colonies were pooled (TnS library) and frozen at -70°C. Standard calculations (18) suggested a probability of >98% that all genes were interrupted at least once, assuming completely random transposition, an average gene size of 1 kb, and a genome size for C. crescentus strains of 4,000 kb (10). (ii) Analysis of the library. The proportion of mutants in the Tn5 library spontaneously resistant to kanamycin and streptomycin selection was determined by colony hybridization on filter paper with probing for the presence of TnS. Probes were prepared by incorporation of 32P-labeled nucleotides by nick translation (18) and were purified by using Geneclean (Bio 101, Inc.) according to the instructions of the manufacturer. The degree of randomness of TnS insertion of the TnS library was investigated by determining the frequency of auxotrophic mutation, done by replica plating onto PYE and HMG media. Also, Southern blots of chromosomal DNA derived from the entire pooled TnS library were probed with
TnS. To determine at what frequency the entire plasmid was integrated into the chromosome by recombination rather than transposon transposition, TnS library mutants were tested for Cmr; a Southern blot of genomic DNA from the entire TnS library was probed with pBR322, and Cmr colonies selected from the TnS library were probed with pBR322.
J. BACTERIOL.
A similar analysis was performed on subsequent holdfastdefective mutants. Approximately 12,000 TnS library isolates were assayed for aberrant colony adhesiveness to cellulose acetate, as previously described (22). Colonies that did not stick to the cellulose acetate (acetate negative) were then evaluated by phase-contrast microscopy for the ability to form rosettes (24). A separate screening of about 2,600 TnS library isolates was done to accumulate mutants that were unable to stick to cellulose acetate but able to make nearly normal rosettes. Analysis of holdfast-defective mutants. (i) Determining the sites of Tn5 insertion. Chromosomal DNA extractions, restriction enzyme digestions, and Southern blot hybridizations were done by standard methods (18). TnS-mutated chromosomal DNA was digested with restriction enzymes that cut once (BamHI) or twice (BgIII) within the TnS element and with enzymes that do not cut within the Tn5 element (ClaI, EcoRI, KpnI, and SstI) (7, 15). Nucleic acid preparations were also separated by using pulsed-field gradient gel electrophoresis by published procedures (10) after digestion with AseI. This procedure was done by Bert Ely (University of South Carolina). Holdfast mutants that were found by Southern blot analysis to have the same site of TnS insertion were collectively referred to as a mutant group (gx, where x is equal to 1 to 10), and subsequent experiments were performed on a representative mutant. (ii) Testing for pleiotropic mutants. To determine whether the holdfast mutants were pleiotropic for defects in the expression of other polar organelles, all were tested for other
pole-related characteristics: motility, polar phage CbK attachment, and presence of a stalk. Motility was tested by examining liquid cultures of all holdfast mutant isolates by phase-contrast microscopy and for the ability of cells to swarm through semisolid (0.3% agar) PYE medium (14). To ensure that variability in the results of the swarm assay was not due to differences in growth rate, specific growth rates were determined. The stalk character of the 10 mutant groups was evaluated by negative-stain electron microscopy (29) with 2% ammonium molybdate (pH 7.5). (iii) Determining the adhesion phenotypes. The holdfastdefective mutant groups were differentiated phenotypically by the degree of lectin binding to holdfast and holdfast adhesiveness to glass, to cellulose acetate, and to itself (rosette formation). Lectin binding to the holdfast was assayed using seven fluorescein isothiocyanate (FITC)-conjugated lectins (concanavalin A, Dolichos biflorus agglutinin, peanut agglutinin, Ricinus communis agglutinin, soybean agglutinin, Ulex europaeus agglutinin, and wheat germ agglutinin [WGA]; Vector Laboratories, Inc.) as previously described (19, 33). The relative adhesiveness of holdfast material to glass and cellulose acetate was tested by using cover slips. Glass culture flasks were treated with 2% dimethyl-dichlorosilane solution in 1,1,1-trichloroethane to reduce holdfast-mediated binding of cells to the flask. Ethanol-washed glass and cellulose acetate cover slips were added to PYE, and cells were then inoculated to an optical density at 600 nm of 0.005. Cultures were grown with shaking to an optical density at 600 nm of about 0.5. The cover slips were then rescued and labeled with FITC-conjugated WGA. RESULTS Detection and analysis of holdfast-defective mutants. Holdfast mutants detected in the TnS library as aberrant in
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GROUP:
g5
g7g9g8
g1g2
93g4glog6
KB 14.3
10.3
-
..
8.0
do
.. ..
:p F
6.5-
4 .8 4-
3.7
..:
.X.:
.... :. .:::
3.0
::
::....
FIG. 1. Comparison of Tn5-containing fragments among holdfast-defective mutant groups with Southern blot hybridization and BamHI digestions of chromosomal DNA. The blot was hybridized with the 1.8-kb HindIII-BamHI fragment of Tn5. Holdfast-defective groups gl through glO are indicated above lanes.
mutant
adhesiveness by failing to bind to cellulose acetate or produce rosettes (colony acetate negative, rosette negative) occurred at a frequency of 0.6% and as colony acetate negative, rosette positive at a frequency of 0.2%. A total of 77 mutants were isolated, and the failure to bind to cellulose acetate was confirmed twice. The Tn5-containing restriction fragments in each of the 77 holdfast mutants were identified on Southern blots of chromosomal DNA digested with enzymes that cut once (BamHI; Fig. 1) or twice (BglII) within the TnS element. By using low-percentage (0.45%) agarose gels, size differences of 0.1 kb could be resolved between most TnS-containing fragments. Also, the HindIII-BamHI fragment used as a probe spans most of the TnS sequence to the left of its BamHI site, including a small part of the left insertion sequence of TnS (15). Because of the sequence similarity in the right insertion sequence, light and dark bands were produced when hybridized to BamHI digests of TnS-containing DNA (Fig. 1). This provided another means of determining the position and similarity of the TnS insertions. Thus the TnS insertions among the mutants could be compared with significant precision. Only 10 unique sites of TnS insertion were identified among the 77 holdfast-defective mutants, and all mutants were classified as belonging to one of these 10 mutant groups (Table 1, gl to glO). Of the 77 holdfast mutants, 30 were the result of a single TnS insertion (g3). By comparing the size of hybridizing fragments produced by several restriction enzymes that cut outside of the TnS element (Table 2), it could be determined by Southern
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hybridization methods that certain holdfast-related TnS insertion groups were clustered: gl and g2; g3, g4, g6, and glO; and g7, g8, and g9. This left g5 as a separate locus for a total of four distinct regions. Probing AseI-digested and pulsedfield gel electrophoresis-separated genomic fragments (Fig. 2) with TnS produced results that agreed with the above interpretation and indicated that the four genomic regions related to holdfast production could not all be linked in one region of the genome. General analysis of the TnS mutant library. The frequency of positive selection for CB2A::TnS mutants was lo-5 per initial recipient in the conjugation. Spontaneous resistance of CB2A to the double antibiotic (kanamycin, streptomycin) selection was undetectable. Several experiments were done in an attempt to establish the degree of randomness of TnS insertion into CB2A. The frequency of auxotrophy in the TnS library was 3.3%. This was somewhat higher than but comparable to frequencies of 1 to 2% reported for other bacteria and for C. crescentus CB15, a related strain (9, 26). Southern blots of DNA from the combined TnS library probed with TnS produced an expected smear but also a particularly noticeable hot-spot region on autoradiographs that was not correlated with an unusual concentration of DNA fragments (data not shown). The region did not hybridize with pBR322 (data not shown) and so was not a site of fortuitous homology and recombination-mediated insertion of the plasmid vector. This apparently preferential site of transposition was not the same as any of the holdfast-encoding sites of TnS insertion. Among the TnS library isolates, 2.6% were chloramphenicol resistant and hybridized with pBR322, indicating plasmid integration into the chromosome. No bands were detected when Southern blots of TnS library chromosomal DNA were probed with pBR322 (data not shown), indicating that the sites of plasmid integration were relatively random. All of the holdfast-defective mutants probed negative with pBR322, indicating that plasmid insertion was not the means of gene inactivation. Testing for pleiotropic mutants. All 77 holdfast-defective mutants were equally motile. All holdfast-defective mutants and wild-type CB2A were fully sensitive to caulophage CbK. Mutants from all 10 mutant groups produced stalks. No major defects (for example, multiple or improperly positioned flagella or stalks) were observed by electron microscopy. However, in group g6 cultures labeled with FITCconjugated WGA, curved or weak stalks were commonly observed, such that the labeled tip of the stalk was typically seen about midway on the lateral portion of the cell body. Negative-stain electron microscopy of g6 mutants did not show a characteristic stalk curvature, but there was some indication that the stalk may be a weaker structure, because a greater angle of stalk curvature was observed more frequently in g6 mutants than in the wild-type CB2A (data not shown). Phenotypic evaluation of the holdfast mutants. Four mutant phenotypes were identified (Table 1): no holdfast produced (gl, g2, g3, g4, and glO); a low amount or altered holdfast produced (g5); an apparently normal holdfast produced but shed into the medium (g7, g8, and g9; Fig. 3); and holdfast produced but abnormal in adhesiveness, either due to an alteration in the composition of the holdfast material or to a physical abnormality of the stalk (g6). All holdfast mutants failed to bind to cellulose acetate in the colony assay, but mutant groups g7, g8, and g9 did score positive in the cellulose acetate cover slip assay because of their holdfast-shedding character (Table 1, Fig. 3); that is,
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MITCHELL AND SMIT
TABLE 1. Phenotype analysis of holdfast-defective mutantsa Holdfast adhesion to: Cellulose
Lectin binding
Colony cellulose toadhesion acetate
Rosette formation
Yes Yes
+++ +++
No
+
No
-
Tn5-derived holdfast mutantsb I, g5 (6)c
No
++
++
-
-
-
++
++
Less or altered holdfast
II, g7 (2) Il, g8 (2) II, g9 (2)
No No No
++ +
++++ ++++ +++
++ +++ ++
+ + +
+ ++ +
++++ ++++ +++
++++ ++++ +++
Shed holdfast Shed holdfast Shed holdfast
III, gl (6) III, g2 (16)
No No
-
-
-
-
-
-
-
No holdfast
IV, g3 (30) IV, g4 (3) IV, glO (1) IV, g6 (9)
No No No No
-
++
-
-
-
+
+
No holdfast No holdfast No holdfast Less or altered holdfast, curved or weak stalk
Strain
Control C. crescentus strains C132A (wild type) CB2A::TnS (random isolate from Tn5 library) CB2A-B9 (characterized holdfast-shedding mutant) CB2A-B5 (characterized holdfast-negative mutant)
phenotype Holdfast summary
acetate
DBA
SBA
UEA
Glass
+++ +++
++ ++
++ ++
++ ++
+++
+++
+++ +++
Wild type Wild type
++++
++
+
++
++++
+++
Shed holdfast
WGA
No holdfast
No holdfast
a See text for a description of the methods used to evaluate each of the parameters. Plus (+) values are relative scores, ranging from + + + + (maximum amount possible) to - (not detectable). DBA, D. biflorus agglutinin; SBA, soy bean agglutinin; UEA, U. europaeus I agglutinin. b Mutants are listed by cluster (I through IV) and group, with the number of isolates given within parentheses. c This number was detected in a separate screening effort, where 2,600 TnS library mutants were screened for inability to bind to cellulose acetate but ability to still produce nearly normal rosettes. Thus the number obtained is not a directly comparable reflection of the relative abundance of this mutant group.
because the holdfast material did not remain attached to the stalk, the cells did not remain on the cellulose acetate and nothing remained to be stained with Coomassie blue. However, the holdfast material could be detected by FITCconjugated WGA labeling. Mutant groups g5 and g6 also scored positive in the cellulose acetate cover slip assay. For these mutant groups, the lower levels of FITC-conjugated WGA labeling, compared with that in wild-type CB2A (Table 2), suggested that the colony adhesion assay was not sensitive enough to detect a lesser degree of adhesiveness to cellulose acetate. Although all mutants could be classified according to a general phenotype, some variability among the members of a group often remained (Table 1). For example, although mutants in groups g7, g8, and g9 were all classified as
holdfast-shedding mutants, they exhibited somewhat differing abilities to adhere to various substrata. All mutant groups except g6 showed that the amount of holdfast produced, assessed by the FITC-conjugated WGA labeling assay (Table 1), could be correlated to the extent of holdfast adhesiveness to glass or cellulose acetate. Group g6 showed poor binding and absence of rosette formation, even though a significant amount of holdfast material was produced. This may be due to an altered holdfast composition or a defective stalk structure, such that the stalk readily breaks and thereby prevents firm attachment to surfaces. DISCUSSION We used TnS mutagenesis procedures to identify holdfastencoding regions of the genome. Insertions in two regions
TABLE 2. Size comparison of TnS-containing DNA fragments produced in Southern blot hybridizations with restriction enzymes that cut outside of the TnS elementa Size of TnS-containing fragment (kb)
Enzyme(s)
AseI ClaI SstI
EcoRI SstI-EcoRI SstI-KpnI ClaI-KpnI EcoRI-KpnI
g5 350 12 11 10 10 11 12 10
g7
310 22 15 ND 13 15 22 ND
g8
g9
gl
g2
g3
g4
glO
g6
310 22 15 14 13 15 22 14
ND 22 15 ND 13 15 22 ND
950 14 16 HM 16 16
950 14 16 HM 16 16 14 HM
90 20 27 17 16 22 14 11
ND ND ND ND ND 22 14 ND
ND ND ND ND ND 22 14 ND
90 20 27 7 7 22 14 7
a ND, Not determined; HM, too high a molecular weight to resolve a size.
14 HM
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CB g3 g6 g9 g7 gl
A
g2 g5
B
CB g3 g6 g9 g7 gi
5429
g2 g5
KB 950 820 750
400
350,330
310 140
90 40 20
950
350 310 gO
FIG. 2. Positioning of Tn5-interrupted, holdfast-encoding DNA in the CB2 genome with pulsed-field gradient gel electrophoresis. C. CB2A (wild type) (CB) and holdfast-defective mutant groups gl through g9 were digested with AseI. (A) Ethidium bromide-stained gel; (B) Southern blot of the same gel probed for Tn5. Both were prepared by Bert Ely (University of South Carolina). crescentus
(clusters III and IV) mainly resulted in a null (no holdfast) phenotype. We expect that one or both of these regions are responsible for the actual synthesis and transport of the holdfast molecule(s). A third region (cluster I) resulted in a probable reduction in the amount of holdfast material produced, since, in addition to poor adhesion, a reduced signal was recorded for the lectins that bind the wild-type holdfast. This suggests that the insertions'disrupt a positive regulator or a component that promotes efficient transport or assembly of holdfast subunits. However, in the absence of more complete informatio'n about holdfast composition, we cannot rule out the possibility that the defect lies in a latter stage of synthesis of the holdfast polysaccharide structure and that the lectin binding profile truly reflects an altered holdfast. A similar argument of holdfast reduction or alteration is applied to the g6 class within cluster IV. This group is under study to discern the exact nature of the defects, but the possible altered stalk structure may indicate that this is a type of polar pleiotropic mutant. This suggests that the holdfast defect may be the consequence of a defect in the formation or functioning of the entire polar region (see references 12 and 27 for proposed models of polar region formation and structure) and not a specific defect in holdfast formation. If this is true, it is intriguing that this group is physically linked in the genome with holdfast-negative mutants that do not exhibit other polar defects. This allows the possibility that diverse components or controls on the polar region are clustered within the caulobacter genome. Transposon insertions in a fourth region (cluster II) resulted in a holdfast-shedding phenotype. We have previously noted this type of mutant with UV mutagenesis techniques (22); the evidence points to the existence of a second molecule or set of molecules that account for the ability of
the holdfast material to adhere tightly and specifically to the end of the stalk. In such a mutation apparently normal holdfast material is produced in normal quantities, but the holdfast structure adheres inefficiently to the stalk end. Thus, even though cells can occasionally be seen by phasecontrast microscopy to be attached to glass surfaces, even a gentle flow of water will remove the cells. Subsequent treatment of the glass with FITC-conjugated WGA revealed that a holdfast is left behind in the detachment process (data not shown). This is the first report of the use of Tn5 as a mutagenesis tool in strain CB2. In several ways the transposon was appropriate for use in this strain. Transposition occurred at a relatively high frequency, resulting in a high effective rate of mutant production in the suicide conjugation procedure. The streptomycin resistance character of TnS was expressed at high levels, permitting selection with two drugs, thereby eliminating spontaneous drug-resistant mutants. This is in comparison with E. coli, in which streptomycin resistance is not expressed, and C. crescentus CB15, in which expression is poor (21). There were indications, however, that TnS did not transpose with complete randomness in CB2. The hot spot of insertion in the library, the high frequency of a single type of holdfast mutant, and the detection of only 10 sites of insertion leading to the adhesion-defective phenotype argued that there was a degree of transposition site preference. In addition, the absence of pleiotropic mutants among the holdfast-defective mutants (with the possible exception of the g6 class) was surprising, since they are a common occurrence when selecting for the loss of other polar characteristics (11, 16). This, too, might reflect a degree of nonrandomness of transposition. Overall, it seems possible that there may be genetic
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J. BACTERIOL.
FIG. 3. FITC-conjugated WGA labeling of the caulobacter holdfast. Shown are combined fluorescence and low-light phase-contrast microscopy images. (A) C. crescentus CB2A (wild type) cells showing holdfast material (appearing as white spots) attached to stalks of single cells or in the center of rosettes. (B) Mutant group g8 cells (holdfast-shedding CB2A mutant) show holdfast material that is detached from
cells. Bar, 5 ,um.
regions that are not accessible to interruption by TnS, and the question of whether we have identified all genomic regions responsible for holdfast production is therefore unresolved. Nevertheless, the regions we have identified seemingly span aspects of production, assembly or attach-
ment, and regulation of holdfast production and the TnS element readily provided the means for cloning these regions (D. Mitchell and J. Smit, unpublished studies). A direct assessment of the genes directing synthesis of this distinctive polysaccharide should prove to be an important part in
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learning how bacteria accomplish strong generalized adhesion to surfaces in the environment. ACKNOWLEDGMENTS We thank Bert Ely of the University of South Carolina for performing the pulsed-field gel electrophoresis analysis. We also thank Wade Bingle for his assistance at numerous stages in this project, including a review of the manuscript. We also thank Harry Kurtz for a review of the manuscript. The work was supported by grants from the U.S. Office of Naval Research (N00014-87-J-1127, N00014-89-J-1749) to J.S. LITERATURE CITED 1. Agabian, N., and L. Shapiro. 1970. Stalked bacteria: properties of deoxyribonucleic acid bacteriophage CBK. J. Virol. 5:795800. 2. Anast, N., and J. Smit. 1988. Isolation and characterization of marine caulobacters and assessment of their potential for genetic experimentation. Appl. Environ. Microbiol. 54:809-817. 3. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heyneker, and H. W. Boyer. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95-113. 4. Brunovskis, I., and W. C. Summers. 1972. The process of infection with coliphage T7. Virology 50:322-327. 5. Costerton, J. W., K.-J. Cheng, G. G. Geesey, T. I. Ladd, J. C. Nickel, M. Dasgupta, and T. J. Marrie. 1987. Bacterial films in nature and disease. Annu. Rev. Microbiol. 41:435-464. 6. Costerton, J. W., R. T. Irvin, and K.-J. Cheng. 1981. The bacterial glycocalyx in nature and disease. Annu. Rev. Microbiol. 35:299-324. 7. De Bruin, F. J., and J. R. Lupski. 1984. The use of transposon TnS mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids-a review. Gene 27:131-149. 8. Dworkin, M. 1985. "Caulobacter," p. 50-67. In Developmental biology of the bacteria. The Benjamin/Cummings Publishing Co., Menlo Park, Calif. 9. Ely, B., and R. Croft. 1982. Transposon mutagenesis in Caulobacter crescentus. J. Bacteriol. 149:620-625. 10. Ely, B., and C. Gerardot. 1988. Use of pulsed-field-gradient gel electrophoresis to construct a physical map of the Caulobacter crescentus genome. Gene 68:323-333. 11. Fukuda, A., K. Miyakawa, H. Iida, and Y. Okada. 1976. Regulation of polar surface structures on Caulobacter crescentus: pleiotropic mutations affect the coordinate morphogenesis of flagella, pili, and phage receptors. Mol. Gen. Genet. 149:167-173. 12. Huguenel, E., and A. Newton. 1982. Localization of surface structures during procaryotic differentiation: role of cell division in Caulobacter crescentus. Differentiation 21:71-78. 13. Isaac, D. H. 1985. Bacterial polysaccharides, p. 141-184. In E. D. Atkins (ed.), Polysaccharides, topics in structure and morphology. VCH Publishers, Deerfield Beach, Fla. 14. Johnson, R., and B. Ely. 1979. Analysis of nonmotile mutants of the dimorphic bacterium Caulobacter crescentus. J. Bacteriol. 137:627-634. 15. Jorgensen, R. A., S. J. Rothstein, and W. S. Reznikoff. 1979. A
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restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol. Gen. Genet. 177:65-72. 16. Kurn, N., S. Ammer, and L. Shapiro. 1974. A pleiotropic mutation affecting expression of polar development events in Caulobacter crescentus. Proc. Natl. Acad. Sci. USA 71:31573161. 17. Lagenaur, C., S. Farmer, and N. Agabian. 1977. Adsorption properties of stage-specific Caulobacter phage-CBK. Virology 77:401-407. 18. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 19. Merker, R. I., and J. Smit. 1988. Characterization of the adhesive holdfast of marine and freshwater caulobacters. Appl. Environ. Microbiol. 54:2078-2085. 20. Newton, A. 1989. Differentiation in Caulobacter: flagellum development, motility, and chemotaxis, p. 199-220. In D. A. Hopwood and K. F. Chater (ed.), Genetics of bacterial diversity. Academic Press, Inc., Toronto. 21. O'Neill, E., G. Keily, and R. Bender. 1984. Transposon TnS encodes streptomycin resistance in nonenteric bacteria. J. Bacteriol. 159:388-389. 22. Ong, C., M. L. Y. Wong, and J. Smit. 1990. Analysis of the attachment of the adhesive holdfast organelle to the cellular stalk of Caulobacter crescentus. J. Bacteriol. 172:1448-1456. 23. Poindexter, J. S. 1964. Biological properties and classification of the Caulobacter group. Bacteriol. Rev. 28:231-295. 24. Poindexter, J. S. 1981. The Caulobacters: ubiquitous unusual bacteria. Microbiol. Rev. 45:123-179. 25. Quigley, N. B., and P. R. Reeves. 1987. Chloramphenicol resistance cloning vector based on pUC9. Plasmid 17:54-57. 26. Simon, R., U. Priefer, and A. Puhler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Technology 1:784790. 27. Smit, J., and N. Agabian. 1982. Cell surface patterning and morphogenesis: biogenesis of a periodic surface array during Caulobacter development. J. Cell Biol. 95:41-49. 28. Smit, J., and N. Agabian. 1984. Cloning the major protein of the Caulobacter crescentus periodic surface layer: detection and characterization of the cloned peptide by protein expression assays. J. Bacteriol. 160:1137-1145. 29. Smit, J., D. A. Grano, R. M. Glaeser, and N. Agabian. 1981. Periodic surface array in Caulobacter crescentus: fine structure and chemical analysis. J. Bacteriol. 146:1135-1150. 30. Sutherland, I. W. 1983. Microbial exopolysaccharides-their role in microbial adhesion in aqueous systems. Crit. Rev. Microbiol. 10:173-201. 31. Sutherland, I. W. 1985. Biosynthesis and composition of Gram negative bacterial extracellular and cell wall polysaccharides. Annu. Rev. Microbiol. 39:243-270. 32. Umbreit, T. H., and J. L. Pate. 1978. Characterization of the holdfast region of wild type cells and holdfast mutants of Asticcacaulis biprosthecum. Arch. Microbiol. 118:157-168. 33. Wu, A. M. 1985. A table of lectin carbohydrate specificities, p. 629-636. In T. C. Bog-Hansen and J. Breborowicz (ed.), Lectins: biology, biochemistry, clinical biochemistry, vol. 4. Walter de Gruyter and Co., New York, N.Y.
Vol. 172, No. 9
Identification of Genes Affecting Production of the Adhesion Organelle of Caulobacter crescentus CB2 DAVID MITCHELLt AND JOHN SMIT*
Department of Microbiology, University of British Columbia, Vancouver, British Columbia V6T I W5 Canada Received 12 January 1990/Accepted 6 June 1990
Transposon (Tn5) mutagenesis was used to identify regions in the genome involved with production, regulation, or attachment to the cell surface of the adhesive holdfast of the freshwater bacterium Caulobacter crescentus CB2. A total of 12,000 independently selected transposon insertion mutants were screened for defects in adhesion to cellulose acetate; 77 mutants were detected and examined by Southern blot hybridization mapping methods and pulsed-field gel electrophoresis. Ten unique sites of TnS insertion affecting holdfast function were identified that were clustered in four regions of the genome. Representative mutants of the 10 TnS insertion sites were examined by a variety of methods for differences in their phenotype leading to the loss of adhesiveness. Four phenotypes were identified: no holdfast production, production of a smaller or an altered holdfast, production of a holdfast that was unable to remain attached to the cell, and a fourth category in which a possible alteration of the stalk was related to impaired adhesion of the cell. With the possible exception of the last class, no pleiotropic mutants (those with multiple defects in the polar region of the cell) were detected among the adhesion-defective mutants. This was unexpected, since holdfast deficiency is often a characteristic of pleiotropic mutants obtained when selecting for loss of other polar structures. Overall, the evidence suggests that we have identified regions containing structural genes for the holdfast, genes involved with proper attachment or positioning on the caulobacter surface, and possibly regions that regulate the levels of holdfast production.
Caulobacters are gram-negative, chemoheterotrophic bacteria that during their growth cycle express two distinct cell types: a dispersive, flagellated swarmer cell and a proliferative, sessile stalked cell (20, 24). Swarmer cells express several temporally and spatially controlled organelles at one pole of the cell: a cluster of pili, a single flagellum, a caulophage receptor, and an adhesive holdfast. Differentiation into the stalked cell involves loss of the flagellum, disappearance of the pili, and localized growth of the cell envelope at the same polar region to form a stalk. The holdfast, first produced in the swarmer cell, is retained and finally resides at the tip of the stalk (8, 24, 32). Because of this adhesion organelle, caulobacters are epiphytes of surfaces and members of the bacterial consortia in both freshwater and marine environments that participate in the initial stages of biofouling of submerged objects. Attachment may be promoted by swarmer-cell motility (that is, by producing a strong initial contact with the surface) and may be assisted by the polar pili but is chiefly mediated by the holdfast. The stalk that develops lengthens in response to nutrient-deficient conditions, possibly enabling the main cell body to remain at the surface of biofilms, where the cell can preferentially acquire nutrition that arrives at the surface and have better access to oxygen for efficient metabolism. Together, the polar organelles probably contribute significantly to the competitiveness of caulobacters on surfaces, which may explain their nearly ubiquitous representation in oligotrophic environments (8, 19, 20, 24). The only function of the caulobacter holdfast seems to be adhesion, and as such it mediates cell attachment to a variety of biological and nonbiological substrata (19, 24). The bound
holdfast can remain intact under conditions of high shear force for many generations, suggesting that the holdfast mediates strong, stable adhesion (19, 24). These factors argue that the holdfast is an appropriate model for the analysis of chemical and structural principles used by bacteria to accomplish generalized adhesion. The composition or structure of the holdfast material has not been elucidated, but all available evidence points to the holdfast being a complex polysaccharide. Lectin binding and enzyme sensitivity studies suggest that N-acetylgalactosamine (22) and stretches of two or more units of N-acetylglucosamine are present in the holdfast material of Caulobacter crescentus CB2 (19, 24). Positive histochemical staining (32) suggests that some caulobacter holdfasts may contain acidic components such as uronic acids, as has been suggested for other putative adhesive polymers (5, 6, 13, 30, 31). Lipids or proteins have not yet been detected (19). Assuming that the holdfast is a complex polysaccharide, a number of genes must be involved with the production of the adhesive structure. In addition, analysis of holdfast mutants suggests that there is a specific site or membrane component at which the holdfast material is anchored to the stalk (22). Finally, production of the holdfast is temporally regulated during the caulobacter cell cycle and is spatially restricted to the polar region of the cell. In sum, we predicted the presence of a number of genes involved with holdfast production and proper functioning and that not all would be structural genes. In this report we describe our initial findings in evaluating the location and complexity of the genes related to the holdfast of the freshwater bacterium C. crescentus CB2A.
* Corresponding author. t Present address: Quadra Logic Technologies, Inc., Vancouver, BC V5Z 4H5, Canada.
MATERIALS AND METHODS Strains, phage, plasmids, and growth conditions. C. crescentus CB2A, a variant of strain CB2 that lacks the paracrys5425
5426
MITCHELL AND SMIT
talline surface protein layer (24, 28), was used for transposon mutagenesis. A spontaneous rifampin-resistant strain was used for the conjugal matings. Strains CB2A-B5 and CB2A-B9 are holdfast-negative and holdfast-shedding mutants, respectively, produced by UV mutagenesis (22). Escherichia coli SM10 (26) was used for conjugal mating experiments. CbK is a caulophage that only attaches to the polar region of swarmer caulobacter cells (1, 17). T7 is a lytic coliphage (4). C. crescentus strains were grown at 30°C in a peptoneyeast extract medium (PYE) (23) supplemented with CaCl2 . 2H20 (0.1 g/liter) or Hutner mineral base (24), supplemented with 0.05% NH4C1 and 0.2% glucose (HMG). As appropriate, antibiotics were added at the following concentrations: chloramphenicol, 2 ,ug/ml; kanamycin, 50 p.g/ml; rifampin, 5 j.g/ml; streptomycin, 50 jig/ml; and trimethoprim, 150 ,ug/ml. E. coli SM10 was grown at 37°C in Luria broth (18) with antibiotics at the following concentrations: chloramphenicol, 30 ,ug/ml and kanamycin, 50 ,ug/ml. Coliphage T7 and caulophage CbK were propagated by standard methods (25). The delivery vehicle for TnS mutagenesis was the narrowhost-range (ColEl replicon) plasmid pSUP2021 (26), which can be transferred to C. crescentus by conjugal mating but is not maintained. pBR322 (3) was used as a hybridization control is some experiments. pBR322-Neo contains the 1.8-kilobase (kb) HindIII-BamHI fragment of TnS inserted into the corresponding sites of pBR322 (John Swindle, unpublished data). The insert was used as hybridization probe for TnS. The TnS mutant library. (i) Production of the library. Conjugal mating of C. crescentus CB2A (RFI) with SM10 (pSUP2021) was done by using a variation of previous methods (2). After overnight mating of 108 donor and recipient cells, the cells were suspended in PYE and 108 PFU of coliphage T7 was added. This mixture was incubated for 2 h at 37°C, and then samples were plated onto PYE containing drugs selective against the SM10 cells and selective for caulobacters containing Tn5 (trimethoprim, rifampin, kanamycin, streptomycin) (21). Then 16,000 colonies were pooled (TnS library) and frozen at -70°C. Standard calculations (18) suggested a probability of >98% that all genes were interrupted at least once, assuming completely random transposition, an average gene size of 1 kb, and a genome size for C. crescentus strains of 4,000 kb (10). (ii) Analysis of the library. The proportion of mutants in the Tn5 library spontaneously resistant to kanamycin and streptomycin selection was determined by colony hybridization on filter paper with probing for the presence of TnS. Probes were prepared by incorporation of 32P-labeled nucleotides by nick translation (18) and were purified by using Geneclean (Bio 101, Inc.) according to the instructions of the manufacturer. The degree of randomness of TnS insertion of the TnS library was investigated by determining the frequency of auxotrophic mutation, done by replica plating onto PYE and HMG media. Also, Southern blots of chromosomal DNA derived from the entire pooled TnS library were probed with
TnS. To determine at what frequency the entire plasmid was integrated into the chromosome by recombination rather than transposon transposition, TnS library mutants were tested for Cmr; a Southern blot of genomic DNA from the entire TnS library was probed with pBR322, and Cmr colonies selected from the TnS library were probed with pBR322.
J. BACTERIOL.
A similar analysis was performed on subsequent holdfastdefective mutants. Approximately 12,000 TnS library isolates were assayed for aberrant colony adhesiveness to cellulose acetate, as previously described (22). Colonies that did not stick to the cellulose acetate (acetate negative) were then evaluated by phase-contrast microscopy for the ability to form rosettes (24). A separate screening of about 2,600 TnS library isolates was done to accumulate mutants that were unable to stick to cellulose acetate but able to make nearly normal rosettes. Analysis of holdfast-defective mutants. (i) Determining the sites of Tn5 insertion. Chromosomal DNA extractions, restriction enzyme digestions, and Southern blot hybridizations were done by standard methods (18). TnS-mutated chromosomal DNA was digested with restriction enzymes that cut once (BamHI) or twice (BgIII) within the TnS element and with enzymes that do not cut within the Tn5 element (ClaI, EcoRI, KpnI, and SstI) (7, 15). Nucleic acid preparations were also separated by using pulsed-field gradient gel electrophoresis by published procedures (10) after digestion with AseI. This procedure was done by Bert Ely (University of South Carolina). Holdfast mutants that were found by Southern blot analysis to have the same site of TnS insertion were collectively referred to as a mutant group (gx, where x is equal to 1 to 10), and subsequent experiments were performed on a representative mutant. (ii) Testing for pleiotropic mutants. To determine whether the holdfast mutants were pleiotropic for defects in the expression of other polar organelles, all were tested for other
pole-related characteristics: motility, polar phage CbK attachment, and presence of a stalk. Motility was tested by examining liquid cultures of all holdfast mutant isolates by phase-contrast microscopy and for the ability of cells to swarm through semisolid (0.3% agar) PYE medium (14). To ensure that variability in the results of the swarm assay was not due to differences in growth rate, specific growth rates were determined. The stalk character of the 10 mutant groups was evaluated by negative-stain electron microscopy (29) with 2% ammonium molybdate (pH 7.5). (iii) Determining the adhesion phenotypes. The holdfastdefective mutant groups were differentiated phenotypically by the degree of lectin binding to holdfast and holdfast adhesiveness to glass, to cellulose acetate, and to itself (rosette formation). Lectin binding to the holdfast was assayed using seven fluorescein isothiocyanate (FITC)-conjugated lectins (concanavalin A, Dolichos biflorus agglutinin, peanut agglutinin, Ricinus communis agglutinin, soybean agglutinin, Ulex europaeus agglutinin, and wheat germ agglutinin [WGA]; Vector Laboratories, Inc.) as previously described (19, 33). The relative adhesiveness of holdfast material to glass and cellulose acetate was tested by using cover slips. Glass culture flasks were treated with 2% dimethyl-dichlorosilane solution in 1,1,1-trichloroethane to reduce holdfast-mediated binding of cells to the flask. Ethanol-washed glass and cellulose acetate cover slips were added to PYE, and cells were then inoculated to an optical density at 600 nm of 0.005. Cultures were grown with shaking to an optical density at 600 nm of about 0.5. The cover slips were then rescued and labeled with FITC-conjugated WGA. RESULTS Detection and analysis of holdfast-defective mutants. Holdfast mutants detected in the TnS library as aberrant in
ADHESION-RELATED GENES OF CAULOBACTER CRESCENTUS
VOL. 172, 1990
GROUP:
g5
g7g9g8
g1g2
93g4glog6
KB 14.3
10.3
-
..
8.0
do
.. ..
:p F
6.5-
4 .8 4-
3.7
..:
.X.:
.... :. .:::
3.0
::
::....
FIG. 1. Comparison of Tn5-containing fragments among holdfast-defective mutant groups with Southern blot hybridization and BamHI digestions of chromosomal DNA. The blot was hybridized with the 1.8-kb HindIII-BamHI fragment of Tn5. Holdfast-defective groups gl through glO are indicated above lanes.
mutant
adhesiveness by failing to bind to cellulose acetate or produce rosettes (colony acetate negative, rosette negative) occurred at a frequency of 0.6% and as colony acetate negative, rosette positive at a frequency of 0.2%. A total of 77 mutants were isolated, and the failure to bind to cellulose acetate was confirmed twice. The Tn5-containing restriction fragments in each of the 77 holdfast mutants were identified on Southern blots of chromosomal DNA digested with enzymes that cut once (BamHI; Fig. 1) or twice (BglII) within the TnS element. By using low-percentage (0.45%) agarose gels, size differences of 0.1 kb could be resolved between most TnS-containing fragments. Also, the HindIII-BamHI fragment used as a probe spans most of the TnS sequence to the left of its BamHI site, including a small part of the left insertion sequence of TnS (15). Because of the sequence similarity in the right insertion sequence, light and dark bands were produced when hybridized to BamHI digests of TnS-containing DNA (Fig. 1). This provided another means of determining the position and similarity of the TnS insertions. Thus the TnS insertions among the mutants could be compared with significant precision. Only 10 unique sites of TnS insertion were identified among the 77 holdfast-defective mutants, and all mutants were classified as belonging to one of these 10 mutant groups (Table 1, gl to glO). Of the 77 holdfast mutants, 30 were the result of a single TnS insertion (g3). By comparing the size of hybridizing fragments produced by several restriction enzymes that cut outside of the TnS element (Table 2), it could be determined by Southern
5427
hybridization methods that certain holdfast-related TnS insertion groups were clustered: gl and g2; g3, g4, g6, and glO; and g7, g8, and g9. This left g5 as a separate locus for a total of four distinct regions. Probing AseI-digested and pulsedfield gel electrophoresis-separated genomic fragments (Fig. 2) with TnS produced results that agreed with the above interpretation and indicated that the four genomic regions related to holdfast production could not all be linked in one region of the genome. General analysis of the TnS mutant library. The frequency of positive selection for CB2A::TnS mutants was lo-5 per initial recipient in the conjugation. Spontaneous resistance of CB2A to the double antibiotic (kanamycin, streptomycin) selection was undetectable. Several experiments were done in an attempt to establish the degree of randomness of TnS insertion into CB2A. The frequency of auxotrophy in the TnS library was 3.3%. This was somewhat higher than but comparable to frequencies of 1 to 2% reported for other bacteria and for C. crescentus CB15, a related strain (9, 26). Southern blots of DNA from the combined TnS library probed with TnS produced an expected smear but also a particularly noticeable hot-spot region on autoradiographs that was not correlated with an unusual concentration of DNA fragments (data not shown). The region did not hybridize with pBR322 (data not shown) and so was not a site of fortuitous homology and recombination-mediated insertion of the plasmid vector. This apparently preferential site of transposition was not the same as any of the holdfast-encoding sites of TnS insertion. Among the TnS library isolates, 2.6% were chloramphenicol resistant and hybridized with pBR322, indicating plasmid integration into the chromosome. No bands were detected when Southern blots of TnS library chromosomal DNA were probed with pBR322 (data not shown), indicating that the sites of plasmid integration were relatively random. All of the holdfast-defective mutants probed negative with pBR322, indicating that plasmid insertion was not the means of gene inactivation. Testing for pleiotropic mutants. All 77 holdfast-defective mutants were equally motile. All holdfast-defective mutants and wild-type CB2A were fully sensitive to caulophage CbK. Mutants from all 10 mutant groups produced stalks. No major defects (for example, multiple or improperly positioned flagella or stalks) were observed by electron microscopy. However, in group g6 cultures labeled with FITCconjugated WGA, curved or weak stalks were commonly observed, such that the labeled tip of the stalk was typically seen about midway on the lateral portion of the cell body. Negative-stain electron microscopy of g6 mutants did not show a characteristic stalk curvature, but there was some indication that the stalk may be a weaker structure, because a greater angle of stalk curvature was observed more frequently in g6 mutants than in the wild-type CB2A (data not shown). Phenotypic evaluation of the holdfast mutants. Four mutant phenotypes were identified (Table 1): no holdfast produced (gl, g2, g3, g4, and glO); a low amount or altered holdfast produced (g5); an apparently normal holdfast produced but shed into the medium (g7, g8, and g9; Fig. 3); and holdfast produced but abnormal in adhesiveness, either due to an alteration in the composition of the holdfast material or to a physical abnormality of the stalk (g6). All holdfast mutants failed to bind to cellulose acetate in the colony assay, but mutant groups g7, g8, and g9 did score positive in the cellulose acetate cover slip assay because of their holdfast-shedding character (Table 1, Fig. 3); that is,
5428
J. BACTERIOL.
MITCHELL AND SMIT
TABLE 1. Phenotype analysis of holdfast-defective mutantsa Holdfast adhesion to: Cellulose
Lectin binding
Colony cellulose toadhesion acetate
Rosette formation
Yes Yes
+++ +++
No
+
No
-
Tn5-derived holdfast mutantsb I, g5 (6)c
No
++
++
-
-
-
++
++
Less or altered holdfast
II, g7 (2) Il, g8 (2) II, g9 (2)
No No No
++ +
++++ ++++ +++
++ +++ ++
+ + +
+ ++ +
++++ ++++ +++
++++ ++++ +++
Shed holdfast Shed holdfast Shed holdfast
III, gl (6) III, g2 (16)
No No
-
-
-
-
-
-
-
No holdfast
IV, g3 (30) IV, g4 (3) IV, glO (1) IV, g6 (9)
No No No No
-
++
-
-
-
+
+
No holdfast No holdfast No holdfast Less or altered holdfast, curved or weak stalk
Strain
Control C. crescentus strains C132A (wild type) CB2A::TnS (random isolate from Tn5 library) CB2A-B9 (characterized holdfast-shedding mutant) CB2A-B5 (characterized holdfast-negative mutant)
phenotype Holdfast summary
acetate
DBA
SBA
UEA
Glass
+++ +++
++ ++
++ ++
++ ++
+++
+++
+++ +++
Wild type Wild type
++++
++
+
++
++++
+++
Shed holdfast
WGA
No holdfast
No holdfast
a See text for a description of the methods used to evaluate each of the parameters. Plus (+) values are relative scores, ranging from + + + + (maximum amount possible) to - (not detectable). DBA, D. biflorus agglutinin; SBA, soy bean agglutinin; UEA, U. europaeus I agglutinin. b Mutants are listed by cluster (I through IV) and group, with the number of isolates given within parentheses. c This number was detected in a separate screening effort, where 2,600 TnS library mutants were screened for inability to bind to cellulose acetate but ability to still produce nearly normal rosettes. Thus the number obtained is not a directly comparable reflection of the relative abundance of this mutant group.
because the holdfast material did not remain attached to the stalk, the cells did not remain on the cellulose acetate and nothing remained to be stained with Coomassie blue. However, the holdfast material could be detected by FITCconjugated WGA labeling. Mutant groups g5 and g6 also scored positive in the cellulose acetate cover slip assay. For these mutant groups, the lower levels of FITC-conjugated WGA labeling, compared with that in wild-type CB2A (Table 2), suggested that the colony adhesion assay was not sensitive enough to detect a lesser degree of adhesiveness to cellulose acetate. Although all mutants could be classified according to a general phenotype, some variability among the members of a group often remained (Table 1). For example, although mutants in groups g7, g8, and g9 were all classified as
holdfast-shedding mutants, they exhibited somewhat differing abilities to adhere to various substrata. All mutant groups except g6 showed that the amount of holdfast produced, assessed by the FITC-conjugated WGA labeling assay (Table 1), could be correlated to the extent of holdfast adhesiveness to glass or cellulose acetate. Group g6 showed poor binding and absence of rosette formation, even though a significant amount of holdfast material was produced. This may be due to an altered holdfast composition or a defective stalk structure, such that the stalk readily breaks and thereby prevents firm attachment to surfaces. DISCUSSION We used TnS mutagenesis procedures to identify holdfastencoding regions of the genome. Insertions in two regions
TABLE 2. Size comparison of TnS-containing DNA fragments produced in Southern blot hybridizations with restriction enzymes that cut outside of the TnS elementa Size of TnS-containing fragment (kb)
Enzyme(s)
AseI ClaI SstI
EcoRI SstI-EcoRI SstI-KpnI ClaI-KpnI EcoRI-KpnI
g5 350 12 11 10 10 11 12 10
g7
310 22 15 ND 13 15 22 ND
g8
g9
gl
g2
g3
g4
glO
g6
310 22 15 14 13 15 22 14
ND 22 15 ND 13 15 22 ND
950 14 16 HM 16 16
950 14 16 HM 16 16 14 HM
90 20 27 17 16 22 14 11
ND ND ND ND ND 22 14 ND
ND ND ND ND ND 22 14 ND
90 20 27 7 7 22 14 7
a ND, Not determined; HM, too high a molecular weight to resolve a size.
14 HM
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CB g3 g6 g9 g7 gl
A
g2 g5
B
CB g3 g6 g9 g7 gi
5429
g2 g5
KB 950 820 750
400
350,330
310 140
90 40 20
950
350 310 gO
FIG. 2. Positioning of Tn5-interrupted, holdfast-encoding DNA in the CB2 genome with pulsed-field gradient gel electrophoresis. C. CB2A (wild type) (CB) and holdfast-defective mutant groups gl through g9 were digested with AseI. (A) Ethidium bromide-stained gel; (B) Southern blot of the same gel probed for Tn5. Both were prepared by Bert Ely (University of South Carolina). crescentus
(clusters III and IV) mainly resulted in a null (no holdfast) phenotype. We expect that one or both of these regions are responsible for the actual synthesis and transport of the holdfast molecule(s). A third region (cluster I) resulted in a probable reduction in the amount of holdfast material produced, since, in addition to poor adhesion, a reduced signal was recorded for the lectins that bind the wild-type holdfast. This suggests that the insertions'disrupt a positive regulator or a component that promotes efficient transport or assembly of holdfast subunits. However, in the absence of more complete informatio'n about holdfast composition, we cannot rule out the possibility that the defect lies in a latter stage of synthesis of the holdfast polysaccharide structure and that the lectin binding profile truly reflects an altered holdfast. A similar argument of holdfast reduction or alteration is applied to the g6 class within cluster IV. This group is under study to discern the exact nature of the defects, but the possible altered stalk structure may indicate that this is a type of polar pleiotropic mutant. This suggests that the holdfast defect may be the consequence of a defect in the formation or functioning of the entire polar region (see references 12 and 27 for proposed models of polar region formation and structure) and not a specific defect in holdfast formation. If this is true, it is intriguing that this group is physically linked in the genome with holdfast-negative mutants that do not exhibit other polar defects. This allows the possibility that diverse components or controls on the polar region are clustered within the caulobacter genome. Transposon insertions in a fourth region (cluster II) resulted in a holdfast-shedding phenotype. We have previously noted this type of mutant with UV mutagenesis techniques (22); the evidence points to the existence of a second molecule or set of molecules that account for the ability of
the holdfast material to adhere tightly and specifically to the end of the stalk. In such a mutation apparently normal holdfast material is produced in normal quantities, but the holdfast structure adheres inefficiently to the stalk end. Thus, even though cells can occasionally be seen by phasecontrast microscopy to be attached to glass surfaces, even a gentle flow of water will remove the cells. Subsequent treatment of the glass with FITC-conjugated WGA revealed that a holdfast is left behind in the detachment process (data not shown). This is the first report of the use of Tn5 as a mutagenesis tool in strain CB2. In several ways the transposon was appropriate for use in this strain. Transposition occurred at a relatively high frequency, resulting in a high effective rate of mutant production in the suicide conjugation procedure. The streptomycin resistance character of TnS was expressed at high levels, permitting selection with two drugs, thereby eliminating spontaneous drug-resistant mutants. This is in comparison with E. coli, in which streptomycin resistance is not expressed, and C. crescentus CB15, in which expression is poor (21). There were indications, however, that TnS did not transpose with complete randomness in CB2. The hot spot of insertion in the library, the high frequency of a single type of holdfast mutant, and the detection of only 10 sites of insertion leading to the adhesion-defective phenotype argued that there was a degree of transposition site preference. In addition, the absence of pleiotropic mutants among the holdfast-defective mutants (with the possible exception of the g6 class) was surprising, since they are a common occurrence when selecting for the loss of other polar characteristics (11, 16). This, too, might reflect a degree of nonrandomness of transposition. Overall, it seems possible that there may be genetic
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MITCHELL AND SMIT
J. BACTERIOL.
FIG. 3. FITC-conjugated WGA labeling of the caulobacter holdfast. Shown are combined fluorescence and low-light phase-contrast microscopy images. (A) C. crescentus CB2A (wild type) cells showing holdfast material (appearing as white spots) attached to stalks of single cells or in the center of rosettes. (B) Mutant group g8 cells (holdfast-shedding CB2A mutant) show holdfast material that is detached from
cells. Bar, 5 ,um.
regions that are not accessible to interruption by TnS, and the question of whether we have identified all genomic regions responsible for holdfast production is therefore unresolved. Nevertheless, the regions we have identified seemingly span aspects of production, assembly or attach-
ment, and regulation of holdfast production and the TnS element readily provided the means for cloning these regions (D. Mitchell and J. Smit, unpublished studies). A direct assessment of the genes directing synthesis of this distinctive polysaccharide should prove to be an important part in
VOL. 172, 1990
ADHESION-RELATED GENES OF CAULOBACTER CRESCENTUS
learning how bacteria accomplish strong generalized adhesion to surfaces in the environment. ACKNOWLEDGMENTS We thank Bert Ely of the University of South Carolina for performing the pulsed-field gel electrophoresis analysis. We also thank Wade Bingle for his assistance at numerous stages in this project, including a review of the manuscript. We also thank Harry Kurtz for a review of the manuscript. The work was supported by grants from the U.S. Office of Naval Research (N00014-87-J-1127, N00014-89-J-1749) to J.S. LITERATURE CITED 1. Agabian, N., and L. Shapiro. 1970. Stalked bacteria: properties of deoxyribonucleic acid bacteriophage CBK. J. Virol. 5:795800. 2. Anast, N., and J. Smit. 1988. Isolation and characterization of marine caulobacters and assessment of their potential for genetic experimentation. Appl. Environ. Microbiol. 54:809-817. 3. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heyneker, and H. W. Boyer. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95-113. 4. Brunovskis, I., and W. C. Summers. 1972. The process of infection with coliphage T7. Virology 50:322-327. 5. Costerton, J. W., K.-J. Cheng, G. G. Geesey, T. I. Ladd, J. C. Nickel, M. Dasgupta, and T. J. Marrie. 1987. Bacterial films in nature and disease. Annu. Rev. Microbiol. 41:435-464. 6. Costerton, J. W., R. T. Irvin, and K.-J. Cheng. 1981. The bacterial glycocalyx in nature and disease. Annu. Rev. Microbiol. 35:299-324. 7. De Bruin, F. J., and J. R. Lupski. 1984. The use of transposon TnS mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids-a review. Gene 27:131-149. 8. Dworkin, M. 1985. "Caulobacter," p. 50-67. In Developmental biology of the bacteria. The Benjamin/Cummings Publishing Co., Menlo Park, Calif. 9. Ely, B., and R. Croft. 1982. Transposon mutagenesis in Caulobacter crescentus. J. Bacteriol. 149:620-625. 10. Ely, B., and C. Gerardot. 1988. Use of pulsed-field-gradient gel electrophoresis to construct a physical map of the Caulobacter crescentus genome. Gene 68:323-333. 11. Fukuda, A., K. Miyakawa, H. Iida, and Y. Okada. 1976. Regulation of polar surface structures on Caulobacter crescentus: pleiotropic mutations affect the coordinate morphogenesis of flagella, pili, and phage receptors. Mol. Gen. Genet. 149:167-173. 12. Huguenel, E., and A. Newton. 1982. Localization of surface structures during procaryotic differentiation: role of cell division in Caulobacter crescentus. Differentiation 21:71-78. 13. Isaac, D. H. 1985. Bacterial polysaccharides, p. 141-184. In E. D. Atkins (ed.), Polysaccharides, topics in structure and morphology. VCH Publishers, Deerfield Beach, Fla. 14. Johnson, R., and B. Ely. 1979. Analysis of nonmotile mutants of the dimorphic bacterium Caulobacter crescentus. J. Bacteriol. 137:627-634. 15. Jorgensen, R. A., S. J. Rothstein, and W. S. Reznikoff. 1979. A
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