H-pilus assembly kinetics determined by electron microscopy

Abstract

The kinetics of pilus outgrowth were examined for Escherichia coli containing pDT1942, a TnkacZ insertion derivative of the IncHIl plasmid R27, which was derepressed for transfer. IncHI1 plasmids are thermosensitive for transfer. The pili specified by pDT1942 were examined by transmission electron microscopy after the pilus had been labeled with the H-pilus-specific bacteriophage Hgal, which had been inactivated with RNase A. H pili were extended by extrusion from the cell surface and not by the addition of pilin subunits to the pilus tip. After pili were removed by vortexing, the outgrowth of full-length pili (2 ;Lm long) required 20 min. H pili expressed at the transfer optimal temperature (270C) remained stable after incubation at the transfer inhibitory temperature (370C), but the formation of mating aggregates was inhibited at 370C. Within 1 min of exposure of the host cell to a heat stimulus of 50'C, pili vanished. Pili were observed in straight and flexible forms with a field emission scanning electron microscope, which may indicate a dynamic role for the pilus in conjugation.H pili are conjugation appendages specified by incompatibility group HI and HII plasmids of the family Enterobacteriaceae (for reviews of H plasmids, see references 15 and 27). With the exception of the rigid pilus specified by the HI3 plasmid MIP233 (7), all plasmids belonging to the HIl, HI2, and HII groups specify thick, flexible pili (3). Thick, flexible pili are also specified by plasmids of the incompatibility groups FI, FII, FV, C, D, J, T, V, and X (3, 5, 6). Members of these plasmid incompatibility groups transfer to recipient cells with equal proficiency by both liquid and surface mating (9). HI group plasmids differ from those in the other groups in being thermosensitive for conjugation, i.e., transferring optimally below 30'C and negligibly (<10-8) at 370C (30). The basis for thermosensitive conjugation is not understood. H-pilus biochemistry is also unknown, and the basic pilin subunit(s) has not been characterized.Plasmid pDT1942, also known as R27::TnlacZ (16) Two single-stranded RNA bacteriophage, pilHa and Hgal (10, 16, 21), adsorb uniformly along the H-pilus shaft. The goal of this study was to exploit phage adsorption, constitutive pilus production, and the thermosensitive conjugation system of R27::TnlacZ to examine several aspects of H-pilus assembly, stability, and morphology by transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM). * Corresponding author. MATERIALS AND METHODSBacterial strains, plasmid, and bacteriophage. The donor strain, Escherichia coli DT1944, is a rifampin-resistant mutant of JE2571 (leu thr str fla pit) (4) harboring plasmid pDT1942 (R27::TnlacZ) (16). This derivative of the tetracycline-resistant HIU plasmid R27 (28) mediates constitutive pilus production and kanamycin resistance. The recipient strain was E. coli K-12 strain J53-1 (pro met F-Nal) (2). Bacteriophage Hgal (21) is an H-pilus-specific RNA phage, 18.7 nm in size (16), which adsorbs to ...

Subcellular localization and functional domains of the coupling protein, TraG, from IncHI1 plasmid R27

“…A unique feature of the IncHI family of plasmids is that their conjugative transfer is significantly reduced at 37 uC (Taylor & Levine, 1980). Electron microscopic analysis of cells harbouring R27 plasmid grown at 37 uC revealed an absence of H-pili on the cell surface (Maher et al, 1993). As pili are required to bring the donor and recipient cells into close proximity, the lack of pili explains the inhibition of transfer at 37 uC.…”

Towards a structural biology of bacterial conjugation

“…Maher et al (1993) presented evidence showing that H-pili are asembled by addition of subunits to the cell-proximal (basal) end of the filament. It is obviously important to see if their interpretation can be confirmed and extended to other conjugative pili.…”

Genetic and nucleotide sequence analysis of the gene htdA, which regulates conjugal transfer of IncHI plasmids

“…For plasmid cloning experiments involving the vectors pUC13 and pUC1 19, transformants were plated on LB medium containing 20 jig of 5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside (X-Gal) per ml, 20 ,ug of isopropyl-3-D-thiogalactopyranoside (IPTG) per ml, and 100 ,ug of ampicillin per ml. For cloning experiments using the vector pOK12, transformants were plated on 30 ,ug of X-Gal, 20 ,g of IPTG, and 100 ,ug of kanamycin per ml. A higher concentration of X-Gal was used for pOK12, as this vector has a modified lacZ promoter structure which results in a fainter blue color on X-Gal medium than that produced by strains containing the vector pUC119 or pUC13 (39).…”

“…Pili produced by the sole member of the IncHI3 group, MIP233, are dissimilar in morphology to the pili produced by the other IncH groups (7). Recent studies on H-pilus formation suggest that H pili are assembled from the base and that full-length pili are formed after 20 min during conditions which favor the production of these pili (20).…”

“…Complementation experiments between R27 and pHH1508a have shown that there is some relatedness between the transfer systems of these plasmids (22). The conjugative pili produced by IncHIl, IncHI2, and IncHII plasmids are referred to as H pili (6,20). Pili produced by the sole member of the IncHI3 group, MIP233, are dissimilar in morphology to the pili produced by the other IncH groups (7).…”