Program in Cell, Molecular, and Developmental Biology, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA
Correspondence
Philip Silverman
silvermanp{at}omrf.ouhsc.edu
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ABSTRACT |
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Present address: Center for Global Health and Diseases, Case Western Reserve University, Wolstein Research Building 4-4301, 10900 Euclid Avenue, Cleveland, OH 44106-7286, USA.
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INTRODUCTION |
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F-pili are 89 nm in diameter and of indeterminate length. Structurally, they are hollow cylinders with a hydrophilic axial lumen that is accessible to the aqueous medium (Folkhard et al., 1979; Paranchych & Frost, 1988
; Silverman, 1997
). Functionally, F-pili make initial, SDS-sensitive contacts between donor and recipient cells (Achtman & Skurray, 1977
; Manning & Achtman, 1979
). Thereafter, F-pili retract so that donor and recipient cells are in direct surface-to-surface contact (Durrenberger et al., 1991
). DNA transfer between closely apposed cells appears to be general (Samuels et al., 2000
; Lawley et al., 2002
), arguing against transfer through extended pili. In fact, once stable surface-to-surface contacts are formed, extended F-pili are no longer required for DNA transfer (Panicker & Minkley, 1985
), although a role for very short filaments cannot be excluded. F-pili also serve as adsorption organelles for several classes of bacteriophage, notably the f2 and Q
classes of icosahedral RNA bacteriophage and the F1/M13 class of filamentous DNA, or Ff, bacteriophage. RNA bacteriophage bind to the sides of F-pili, whereas DNA bacteriophage bind to the tip (Valentine et al., 1969
).
F-pilin (70 amino acids) first accumulates as an inner-membrane protein. It is derived from the traA gene product (121 amino acids), beginning with proteolytic cleavage between TraA amino acids A51 and A52 and catalysed by host leader peptidase B (Moore et al., 1981a; Frost et al., 1984
; Majdalani & Ippen-Ihler, 1996
; Majdalani et al., 1996
). N
-Acetylation of A1 is required to yield mature F-pilin, although unacetylated subunits still assemble and function (Grossman & Silverman, 1989
; Grossman et al., 1990
; Moore et al., 1993
; Maneewannakul et al., 1995
). Unlike T- and RP4-pilins (Eisenbrandt et al., 1999
), F-pilin is not circular.
Ippen-Ihler and colleagues showed that formation of membrane F-pilin requires only the traA and traQ genes (Moore et al., 1981b; Maneewannakul et al., 1993
) and provided evidence that membrane F-pilin is the precursor to filament F-pilin (Sowa et al., 1983
). Our studies showed that TraQ interacts directly with the C-terminal domain IV of F-pilin (Paiva et al., 1992
; Harris et al., 1999
), and we proposed that TraQ acts catalytically to escort TraA into the inner membrane.
Once formed, membrane F-pilin is stable as such in cells unable to elaborate F-pili (Sowa et al., 1983). F-pilus assembly from membrane F-pilin substrate requires numerous additional Tra proteins that act at or in association with the cell envelope (Firth et al., 1996
; Harris et al., 2001
; Harris & Silverman, 2004
). Altogether, about half the F DNA transfer (tra) genes essential for DNA transfer are required for F-pilus assembly and function (Grossman & Silverman, 1989
; Firth et al., 1996
). These additional proteins form an envelope-associated secretion machine (R. Harris and others, unpublished data), as is also true of other type IV systems (Thorstenson et al., 1993
; Grahn et al., 2000
; Gilmour et al., 2001
; Kumar et al., 2000
; Krall et al., 2002
).
Several indirect tests exist for the presence or absence of functional F-pili, including conjugal DNA donor activity and sensitivity to bacteriophage that use F-pili as adsorption sites. Direct assays for F-pili have included electron microscopy (Valentine et al., 1969; Curtiss et al., 1969
), binding of RNA bacteriophage labelled with 32P (Valentine et al., 1969
), and competitive ELISA (Frost et al., 1985
). The last two assays are now rarely used, electron microscopy, though poorly suited to kinetic or other studies requiring high throughput, having by default become the assay of choice for F-pili. Here we describe alternative assays that employ fluorescent bacteriophage that bind specifically to F-pili. We show that fluorescence microscopy can be used to analyse F-pili number and length distributions within and between populations, whereas fluorescence measurements can be used for rapid, quantitative assays of cell cultures. We illustrate these advantages using a set of F-pilin missense mutants.
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METHODS |
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Bacteriophage R17 and the tet transducing bacteriophage F1fus2 (Parmley & Smith, 1988) were from our laboratory stocks. R17 titres were measured by standard agar overlay with HfrH or JC3272/JCFL0 as host. F1fus2 titres were measured as transductant-forming units (t.f.u.) (tetracycline-resistance) with strain K91 as host (Parmley & Smith, 1988
).
Where indicated, plasmid pMR119, encoding DsRed-Express (Otto et al., 2004), was introduced by transformation.
Bacteria were grown routinely in LuriaBertani (LB) medium supplemented with antibiotics as necessary. For bacteriophage binding, the medium was also supplemented with 10 mM CaCl2. Incubation was at 37 °C with vigorous aeration. Growth was monitored by optical density at 600 nm. When used, nutrient broth contained, per litre: 10 g tryptone, 1 g yeast extract, 8 g NaCl and 0·2 % (w/v) glucose.
Preparation of fluorescent R17.
R17 was prepared from crude lysates by liquid polymer phase partition and isopycnic banding in step CsCl gradients (Yamamoto et al., 1972). After dialysis against P buffer [50 mM TrisHCl (pH 7·6)/0·1 M NaCl/5 mM MgCl2/0·1 mM EDTA], the suspension was made 50 % (w/v) glycerol and stored at 20 °C. The final titre of the preparation used in these studies was 2·5x1012 p.f.u. ml1 and has been stable for several years.
For conjugation with fluorescent dyes, a portion of the R17 suspension (1215 ml) was dialysed at 4 °C for 48 h against 2 litres of a solution containing 0·1 M NaHCO3 (pH 8·5)/1 mM MgCl2. The dialysis solution was changed once after 24 h. Alexa 488 carboxylic acid (succinimidyl ester; Molecular Probes) (1 mg) was dissolved in 0·1 ml anhydrous DMSO and added in 10 µl aliquots to the R17 suspension with gentle stirring and at ambient temperature over a 2030 min period. After an additional 60 min with gentle stirring, the R17 suspension was loaded onto two linear CsCl gradients (30 ml; =1·6631·226 g ml1) in P buffer. Centrifugation in the Beckman SW28 rotor was at 25 000 r.p.m. for 18 h at 5 °C. The fluorescent band visible under UV illumination (
1·45 g ml1) was collected, dialysed against P buffer, diluted with an equal volume of 80 % (w/v) glycerol, and stored at 20 °C. Titres were generally about 5x1011 p.f.u. ml1 and Alexa 488-labelling corresponded to
105 fluorescence intensity units (FIU) ml1. In earlier experiments, glycerol was removed by dialysis before the preparation was used, but this proved to be unnecessary.
R17 binding and fluorescence measurements.
Cells (0·51 ml) and R17 (1540 µl) were mixed at 4 °C and incubated at that temperature for 10 min. Formaldehyde (50 µl of a 16 % solution) was added and the samples incubated for 10 min at ambient temperature. Cells and bound bacteriophage were harvested by sedimentation for 4 min in a microcentrifuge at 10 000 g. Supernatant fractions were carefully removed by aspiration and discarded. Cell pellets were suspended in 1 ml 0·1 % (w/v) SDS. Cells were sedimented, suspended in 1 ml 0·1 % SDS, and sedimented a last time. Supernatant fractions were combined.
Fluorescence was measured with an excitation wavelength of 490 nm and an emission wavelength of 520 nm. A blank value consisting of the fluorescence from 0·1 % SDS (40 FIU) or, depending on the experiment, from F cells in 0·1 % SDS (5070 FIU) was subtracted from all readings.
Fluorescence microscopy.
Cells were grown to an OD600 of 0·30·4 in Medium E salts (Vogel & Bonner, 1956) containing 1 % Casamino acids/1 % tryptophan/1 % glucose. Cultures were diluted to an OD600 of 0·1 with PBS and 50 µl applied to a microscope dish (35 mmx12 mmx0·17 mm; World Precision Instruments). Before use, dishes were incubated for 10 min in normal goat serum diluted 100-fold in PBS and then rinsed three times with PBS. After 10 min incubation to allow cells to adhere, excess liquid was carefully removed and replaced with 50 µl R17 diluted fivefold in PBS. After 10 min, liquid was removed and an agar disc (2 % in PBS, 0·160·19 mm thick) immediately placed over the cells. Fixation was with 2 % formaldehyde in PBS (10 µl added to the surface of the agarose disc). After 2 min, excess liquid was removed and the cells were examined with a Zeiss LSM 510META laser scanning confocal microscope.
DNA donor activity and F1fus2 sensitivity.
Cultures of TOP10/pOX38traA : : cat containing traA genes cloned into a pBAD vector (Guzman et al., 1995) (see below) were grown to an OD600 of 0·4. Where appropriate, traA expression was induced by addition of L-arabinose (final concentration 0·2 %, w/v) 2·53 h before the cells were used. For donor activity, 0·5 ml of each culture was subjected to centrifugation. Supernatant fractions were aspirated and the cells suspended in 0·5 ml of a recipient cell culture (AE2248 grown to OD600 0·23). After incubation for 60 min at 37 °C, the cell samples were diluted and 10 µl aliquots plated on media selective for transconjugants (chloramphenicol+tetracycline) and for donor cells (ampicillin+chloramphenicol).
For F1fus2 infectivity, we measured transduction of the bacteriophage tet gene. A portion (90 µl) of the cultures used to measure donor activity was mixed with 10 µl of an F1fus2 preparation containing 108 t.f.u. Samples were incubated at 4 °C for 15 min, 37 °C for 15 min, and subjected to centrifugation at 10 000 g for 2 min. Cell pellets were suspended in nutrient broth and incubated for 45 min at 37 °C. Portions (10 µl) of serial dilutions were then spotted on LB/tetracycline plates.
Construction of F-pilin cysteine mutants by site-directed mutagenesis.
With the exception of G64C, which was obtained by mutagenic PCR, cysteine mutations were introduced into traA of plasmid pWP901 using the USE Mutagenesis kit (Amersham Pharmacia Biotech). The primers used for the different mutations are listed in Table 1. We also found it necessary to introduce a third, wild-type primer for all mutations except A33C (Table 1
). The reaction product was used to transform XK1200/pOX38traA : : cat, selecting for CamR AmpR transformants. Plasmid DNA was isolated and traA inserts sequenced in both directions. Following Manchak et al. (2002)
, we transferred the mutant and wild-type traA genes into the vector pBAD/Myc-His A (Invitrogen). These plasmids were introduced by transformation into TOP10/pOX38traA : : cat.
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RESULTS |
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Fluorescent R17 (20003000 FIU) was incubated with F'lac or isogenic F cells (0·40·7 OD600 units) to allow bacteriophage adsorption. Cells and bound R17 were collected by sedimentation. The pellets were suspended in 0·1 % SDS, which dissolves F-pili but leaves cells intact. Cells were removed by sedimentation and washed once with SDS. Fluorescence of the combined supernatant fractions was then measured. In preliminary assays, fluorescence in supernatant fractions from F' or Hfr cells corresponded to 500800 FIU per OD600 unit, depending on the strain. Fluorescence in equivalent fractions from F cells was 5070 FIU per OD600 unit. Fluorescence increased linearly with increasing amounts of F+ cells except at very low levels (< 0·1 OD600 unit) (Fig. 3) and it was proportional to culture density during exponential growth (Fig. 4
). As the culture left exponential growth (OD600>1·5), the ratio FIU/OD600 unit began to diminish (Fig. 4
); a stationary-phase culture of the F'lac strain used in this experiment (OD600 3·2) measured 450 FIU per OD600 unit. This reduced level of F-piliation undoubtedly reflects the well-established F phenocopy effect.
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F-pilin, which is only 70 amino acids in length, normally lacks cysteines (Frost et al., 1984). We constructed several mutant traA genes, each with a cysteine codon at a different site (Table 2
). Four of the mutations were in domain I, one in domain II, one in domain III and three in domain IV (Table 2
) (Paiva et al., 1992
). (Note that the traAS25C gene also contained a second mutation, A55V.) These mutant traA genes were cloned into a pBAD vector as described in Methods.
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DISCUSSION |
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As with other assays for F-pili, the ones we describe here rely on the lateral binding of RNA bacteriophage to F-pili. This can be misleading. As we have shown here for the G69C F-pilin mutant, and as Grossman & Silverman (1989) and Manchak et al. (2002)
have also shown, altered F-pilins can be incorporated into filaments that fail to bind RNA bacteriophage. Such ambiguities might be minimized by using cysteine-reactive fluorescent dyes, rather than fluorescent bacteriophage, in conjunction with cysteine-containing F-pili.
Manchak et al. (2002), examining the effects of single missense mutations of F-pilin, found that in general DNA donor activity and sensitivity to filamentous DNA bacteriophage tracked together. In contrast, several mutations abolished RNA bacteriophage sensitivity with less of an effect on the other two functions. The G69C mutation we describe here evidently belongs in this class. The effects of the G69C mutation can not be attributed to the presence of cysteine at this locus since the G69D mutation had much the same effects (Frost & Paranchych, 1988
). Interestingly, neither we nor Manchak et al. (2002)
, nor the more limited study by Frost & Paranchych (1988)
, identified F-pilin missense mutants that significantly reduced Ff bacteriophage sensitivity and DNA donor activity without also reducing or abolishing RNA bacteriophage sensitivity. One explanation for these data is that donor activity and DNA bacteriophage sensitivity are relatively robust functions with respect to modest alterations to F-pilin structure, whereas RNA bacteriophage infection is more sensitive.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 27 April 2005;
revised 8 June 2005;
accepted 12 June 2005.
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