Matthew Hobbs

Common components in the assembly of type 4 fimbriae, DNA transfer systems, filamentous phage and protein-secretion apparatus: A general system for the formation of surface-associated protein complexes

The Pseudomonas aeruginosa genes pilB‐D and pilQ are necessary for the assembly of type 4 fimbriae. Homologues of these genes and of the subunit (pilin) gene have been described in various different bacterial species, but not always in association with type 4 fimbrial biosynthesis and function. Pil‐like proteins are also involved in protein secretion, DNA transfer by conjugation and transformation, and morphogenesis of filamentous bacteriophages. It seems likely that the Pil homologues function in the processing and export of proteins resembling type 4 fimbrial sub‐units, and in their organization into fimbrial‐like structures. These may either be true type 4 fimbriae, or components of protein complexes which act in the transport of macromolecules (DNA or protein) into or out of the cell. Some PilB‐like and PilQ‐like proteins are apparently also involved in the assembly of non‐type 4 polymeric structures (filamentous phage virions and conjugative pili). The diverse studies summarized in this review are providing insight into an extensive infrastructural system which appears to be utilized in the formation of a variety of cell surface‐associated complexes.

Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria.

Type-4 fimbriae (pili) are associated with a phenomenon known as twitching motility, which appears to be involved with bacterial translocation across solid surfaces. Pseudomonas aeruginosa mutants which produce fimbriae, but which have lost the twitching motility function, display altered colony morphology and resistance to fimbrial-specific bacteriophage. We have used phenotypic complementation of such mutants to isolate a region of DNA involved in twitching motility. This region was physically mapped to a SpeI fragment around 20 min on the P. aeruginosa PAO chromosome, remote from the major fimbrial locus (around 75 min) where the structural subunit-encoding gene (fimA/pilA) and ancillary genes required for fimbrial assembly (pilB, C and D) are found. A gene, pilT, within the twitching motility region is predicted to encode a 344-amino acid protein which has strong homology to a variety of other bacterial proteins. These include the P. aeruginosa PilB protein, the ComG ORF-1 protein from the Bacillus subtilis comG operon (necessary for competence), the PulE protein from the Klebsiella oxytoca (formerly K. pneumoniae) pulC-O operon (involved in pullulanase export), and the VirB-11 protein from the virB operon (involved in virulence) which is located on the Agrobacterium tumefaciens Ti plasmid. We have also identified other sets of homologies between P. aeruginosa fimbrial assembly (Pil) proteins and B. subtilis Com and K. oxytoca Pul proteins, which suggest that these are all related members of a specialised protein export pathway which is widespread in the eubacteria.

PilS and PilR, a two-component transcriptional regulatory system controlling expression of type 4 fimbriae in Pseudomonas aeruginosa

Transposon mutagenesis was used to identify genes necessary for the expression of Pseudomonas aeruginosa type 4 fimbriae. In a library of 12 700 mutants, 147 were observed to have lost the spreading colony morphology associated with the presence of functional fimbriae. Of these, 28 had also acquired resistance to the fimbrial‐specific bacteriophage P04. The mutations conferring this phage resistance were found to have occurred at at least six different loci, including the three that had been previously shown to be required for fimbrial biosynthesis or function: the structural subunit (pilA) and adjacent genes (pilB,C,D), the twitching motility gene (pilT), and the Sigma 54 RNA polymerase initiation factor gene (rpoN). One novel group of phage‐resistant mutants was identified in which the transposon had inserted near sequences that cross‐hybridized to an oligonucleotide probe designed against conserved domains in regulators of RpoN‐dependent promoters. These mutants had no detectable transcription of pilA and did not produce fimbriae. A probe derived from inverse polymerase chain reaction was used to isolate the corresponding wild‐type sequences from a P. aeruginosa PAO cosmid reference library, and two adjacent genes affected by transposon insertions, pilS and pilR, were located and sequenced. These genes were shown to be capable of complementing the corresponding mutants, both at the level of restoring the phenotypes associated with functional fimbriae and by the restoration of pilA transcription. The pilSR operon was physically mapped to Spel fragment 5 (corresponding to about 72–75/0 min on the genetic map), and shown to be located approximately 25 kb from pilA–D. PilS and PilR clearly belong to the family of two‐component transcriptional regulatory systems which have been described in many bacterial species. PilS is predicted to be a sensor protein which when stimulated by the appropriate environmental signals activates PilR through kinase activity. PilR then activates transcription of pilA, probably by interacting with RNA polymerase containing RpoN. The identification of pilS and pilR makes possible a more thorough examination of the signal transduction systems controlling expression of virulence factors in P. aeruginosa.

Characterization of a complex chemosensory signal transduction system which controls twitching motility in Pseudomonas aeruginosa

Virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of a wide range of virulence factors including type IV pili which are required for colonization of host tissues and are associated with a form of surface translocation termed twitching motility. Twitching motility in P. aeruginosa is controlled by a complex signal transduction pathway which shares many modules in common with chemosensory systems controlling flagella rotation in bacteria and which is composed, in part, of the previously described proteins PilG, PilH, PilI, PilJ and PilK. Here we describe another three components of this pathway: ChpA, ChpB and ChpC, as well as two downstream genes, ChpD and ChpE, which may also be involved. The central component of the pathway, ChpA, possesses nine potential sites of phosphorylation: six histidine‐containing phosphotransfer (HPt) domains, two novel serine‐ and threonine‐containing phosphotransfer (SPt, TPt) domains and a CheY‐like receiver domain at its C‐terminus, and as such represents one of the most complex signalling proteins yet described in nature. We show that the Chp chemosensory system controls twitching motility and type IV pili biogenesis through control of pili assembly and/or retraction as well as expression of the pilin subunit gene pilA. The Chp system is also required for full virulence in a mouse model of acute pneumonia.

Characterization of pilQ, a new gene required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa

Type 4 fimbriae are produced by a variety of pathogens, in which they appear to function in adhesion to epithelial cells, and in a form of surface translocation called twitching motility. Using transposon mutagenesis of Pseudomonas aeruginosa, we have identified a new locus required for fimbrial assembly. This locus contains the gene pilQ which encodes a 77 kDa protein with an N‐terminal hydro‐phobic signal sequence characteristic of secretory proteins, pilQ mutants lack the spreading colony morphology characteristic of twitching motility, are devoid of fimbriae, and are resistant to the fimbrial‐specific bacteriophage PO4. The pilQ gene was mapped to Spel fragment 2, which is located at 0–5 minutes on the P. aeruginosa PAO1 chromosome, and thus it is not closely linked to the previously characterized pilA‐D, pilS,R or pilT genes. The pilQ region also contains ponA, aroK and aroB‐like genes in an organization very similar to that of corresponding genes in Escherichia coli and Haemophilus influenzae. The predicted amino acid sequence of PilQ shows homology to the PulD protein of Kleb‐siella oxytoca and related outer membrane proteins which have been found in association with diverse functions in other species including protein secretion, DNA uptake and assembly of filamentous phage. PilQ had the highest overall homology to an outer membrane antigen from Neisseria gonorrhoeae, encoded by omc, that may fulfil the same role in type 4 fimbrial assembly in this species.

Gene sequences and comparison of the fimbrial subunits representative of Bacteroides nodosus serotypes A to I: class I and class II strains

We have determined the nucleotide sequences of the genes encoding the fimbrial subunits representative of the known Bacteroides nodosus serogroups. All of the genes are preceded by a highly conserved region which includes the likely promoter and transcriptional regulator sites as well as the ribosome‐biding site, and are followed within a short but variable distance by a sequence with the characteristics of a transcription termination or attenuation signal. Based on sequence and organization, the subunits can be divided into two major classes called I (serogroups A, B, C., E, F, G, and I) and II (serogroups D and H). All contain the same seven‐amino‐acid positively charged leader sequence and conserved hydrophobic amino‐terminal sequence typical of type 4 fimbriae. Beyond this point the class II subunits are quite different from class I and share features more in common with those from other type 4 fimbriate bacteria, such as Moraxella bovis and Pseudomonas aeruginosa. The larger class I may be further subdivided into two subsets: (i) ((A, E, F)(B, I)) and (ii) (C, G). These proteins exhibit three major clusters of variation, at either end of the presumptive disulphide loop which spans the central third of the protein, and near the carboxy‐terimus, with dispersed changes in between. The length of the mature subunits varies from 152–156 amino acids, and the variation Includes small insertions or deletions in the variable clusters between more conserved domains. The class II subunits are 149 amino acids in length and contain two pairs of cysteine residues: one is at the end of the amino‐terminal conserved region, and the other is at the end of the protein. The major variation occurs in the central region of the molecule, and again small insertions or deletions are required to align adjacent conserved domains. There is also a striking absence of silent codon changes in the 5′ coding region of all of these genes, indicating that these sequences have a secondary genetic function, probably in recombi‐national exchange.

Organization of the fimbrial gene region of Bacteroides nodosus: class I and class II strains

The fimbrial subunit genes of Bacteroides nodosus may be divided into two distinct classes, based on the sequence of the major subunit gene fimA (accompanying paper — Mattick et al., 1991). The genetic organization of the fimbrial gene region in these two classes is also distinct. Upstream of fimA in both classes in opposite transcriptional orientation is the gene aroA which encodes amino acid biosynthetic enzyme 5‐enolpyruvylshikimate‐3‐phosphate synthase. However, downstream of fimA the two classes are quite different until homology is restored at a bidirectional transcription termination signal separating the fimbrial operon from a gene clpB, which appears to encode the regulatory subunit of an ATP‐dependent protease. Between aroA and clpB class I strains contain, apart from fimA, only one other gene (fimB). Sequence and polymerase chain reaction analyses indicate that fimB does not have a separate promoter but rather is co‐transcribed with fimA at a level attenuated by the strength of the transcription termination signal in the intergenic region. In class II strains fimA is followed by a more extended region containing three genes, which appear to have the same transcriptional arrangement as fimB. The second of these genes (fimD) may represent a functional analogue of fimB although there is no close sequence homology. The first gene (fimC) has no obvious similarity to either fimB or fimD. Beyond fimO, at the 3 end of the class ll‐specific region, is a variant fimbrial subunit gene (fimZ) which is virtually identical in serogroups 0 and H and which appears to represent a duplicate, possibly redundant, gene closely related to the progenitor of the more divergent structural subunit fimA gene found in these strains. Comparisons of the predicted ftmZ product with those of fimA in class I and class II strains, as well as of the boundaries of the class‐specific regions, suggest that the class II sequences evolved in another type 4 fimbriate species and were subsequently substituted in the 8. nodosus genome by lateral transfer. Analysis of the sequences flanking fimA in different strains indicates that recombinational exchange of both fimA and the entire operon has also occurred between strains, and is possibly a mechanism for disseminating structural diversity in the population.

Molecular Biology of the Fimbriae of Dichelobacter (Previously Bacteroides) nodosus

Dichelobacter nodosus is a gramnegative anaerobe and the essential causative agent of ovine footrot (Egerton, 1977). Virulent isolates of this organism contain large numbers of fine surface filaments termed fimbriae (or common pili) (Figure 39.1), which have a diameter of about 6 nm and may extend up to several micrometers (µm) in length (Stewart, 1973). In other bacteria such fimbriae have been shown to have adhesive properties (Paranchych and Frost, 1988; Moore and Rutter, 1989; see also following) and to represent a primary mechanism for colonization of animal cell surfaces. Although the exact function of D. nodosus fimbriae has not yet been clearly defined, they appear to play a central role in the invasion by the bacterium of the epidermal matrix of the hoof (see Mattick et al., 1985a).