In this paper, we report the results of a saturating mini-Tn5 screen for Caulobacter mutants with adhesion defects. These mutants can be grouped into three classes: developmental mutants (pleC and podJ), holdfast synthesis or export mutants (hfs), and mutants that disrupt the attachment of the holdfast to the tip of the stalk (hfa).
The developmental mutants displayed additional polar defects, in addition to a lack of adhesion and an absence of a holdfast. The pleC
mutants were deficient in stalk synthesis and flagellar rotation and were resistant to caulophage
CbK as shown previously (33
). PleC is a histidine kinase that localizes to the flagellated pole of swarmer cells and of late predivisional cells and is involved in coordinating many aspects of polar morphogenesis (35
). The podJ
mutants had defects in chemotaxis and were resistant to
CbK, as shown previously (33
), in addition to their deficiency in surface adhesion and holdfast synthesis. We have recently shown that PodJ is localized to the flagellated pole of swarmer cells, disappears from that pole in stalked cells, and localizes to the opposite pole, where it remains for the rest of the cell cycle (8a
). PodJ is required for PleC localization (8a
), suggesting that the holdfast synthesis deficiency of podJ
mutants may be due to PleC delocalization (8a
genes were identified previously (11
) and are transcribed from a promoter upstream of hfaA
; J. Cole, D. Bodenmiller, and Y. V. Brun, unpublished observations). Nonpolar mutants of hfaA
, and hfaD
produce holdfasts, as shown by their binding to fluorescently labeled lectins, but do not attach them to the stalk at wild-type levels (Cole et al., unpublished). This leads to an overall defect in adhesion. HfaB shares some similarity with the curli attachment gene, csgG
, from E. coli
and has been experimentally proven to be a lipoprotein (Cole et al., unpublished). Curli are proteinaceous fibers that mediate attachment to surfaces. hfaA
share no significant sequence similarity with any characterized proteins. Both HfaB and HfaD have been shown to localize to the stalk (Cole et al., unpublished), suggesting that they are directly involved in attaching the holdfast to the stalk. The hfa
genes appear to be the only nonessential genes required for the attachment of the holdfast to the tip of the stalk based on the fact that 10 independent insertions have been isolated at this locus (10
; this work).
genes are likely to be involved in holdfast export. hfsA
, and hfsD
mutants all show a dramatic defect in cellular adhesion to a variety of surfaces. Lectin binding studies also show that hfs
mutants do not shed holdfasts in the medium, further separating this new class of mutants from the hfa
mutants. Lectin binding experiments and electron microscopy show the absence of holdfast material at the tip of the stalk. The gene products of the hfs
operon have a high degree of sequence similarity to polysaccharide export components. HfsD resembles an oligomeric secretin, Wza, from E. coli
. An outer membrane lipoprotein, Wza functions as a hexameric channel for export of capsular polysaccharide to the surface (4
). Wza belongs to the outer membrane auxiliary, or OMA, family of proteins (19
). HfsD also carries a potential lipoprotein sorting signal sequence at its N-terminal portion (15
). In addition, based upon the presence of noncharged residues at the +2 and +3 positions of the predicted mature lipoprotein, HfsD is predicted to reside in the outer leaflet of the outer membrane (15
HfsA has sequence similarity in its 400-aa periplasmic loop to the polysaccharide transport protein, GumC, from X. campestris
. GumC has been shown to be involved in the export of the EPS xanthan gum from the cytoplasm to the cellular exterior (32
). Export of high-molecular-weight EPS is required for the invasion of plant hosts by X. campestris
. GumC is a member of membrane periplasmic auxiliary (MPA-1) family of polysaccharide export proteins (19
). One of the defining characteristics of the genes encoding the MPA-1 is the nearby presence of an OMA gene, which participates in EPS export (19
). Normally, members of the MPA-1 protein family share an inner membrane topology and possess a large cytoplasmic domain containing a Walker A (GXXXXGKT/S) ATP binding motif, which is responsible for providing the energy necessary for driving the export process in the form of ATP hydrolysis (19
). Although HfsA possesses the required two-transmembrane-helix topology, with a large periplasmic loop, it lacks a sizeable cytoplasmic domain (30a
) and the necessary Walker A motif (1
). However, two other members of the MPA-1 family also lack the relatively well-conserved C-terminal cytoplasmic domain: GumC and OtnB (19
). In these cases, energy may be provided by an ABC cassette transport system. Members of the MPA-2 family, such as KpsE and CtrB, work in concert with these cytoplasmic membrane transporters (19
). It is unknown how the 400-aa periplasmic loop of the MPA-1 proteins functions to facilitate transport of polysaccharide residues from the cytoplasmic membrane face of the periplasm to the outer membrane face.
HfsC has sequence similarity to ExoQ from R. melliloti
. HfsC adopts a similar membrane topology to ExoQ, with 11 (rather than 12) predicted transmembrane helices spanning the inner membrane (30a
). The region of highest similarity between the two proteins occurs in an ~75-aa periplasmic loop (30a
) in which they are located (33% identical and 46% similar). ExoQ has been shown to be directly involved in the polymerization of a high-molecular-weight EPS, succinoglycan, which is required for nodule formation (22
). An hfsC
deletion mutant does not have an adhesion-deficient phenotype to surfaces such as borosilicate glass, polyvinylchloride, polypropylene, and polystyrene and binds lectin at wild-type levels. The data do not rule out an effect on binding to different surfaces, but clearly hfsC
is not required for holdfast synthesis.
Finally, HfsB has no significant similarity to available protein sequences. Combined with the absence of a signal sequence or hydrophobic regions (15
), it is difficult to propose a role for HfsB in holdfast biogenesis.
No biosynthetic genes were isolated in the cellulose acetate screen; however, the possibility remains that hfsB functions in this capacity. One explanation for the absence of biosynthetic mutants is that the screen is not saturated. However, we have identified multiple insertions at every locus (seven at hfa, seven at hfs, two at pleC, and four at podJ). On the basis of this evidence, we believe the screen has been saturated for knockout mutations that abolish adhesion. Another possibility is that mutations in some genes required for optimal adhesion have been missed in this screen. For example, mutations in pilus and flagellar structural genes reduce, but do not completely abolish adhesion (D. Bodenmiller and Y. V. Brun, unpublished data; Cole et al., unpublished). Similarly, it is possible that mutations that eliminate some yet unknown component of the holdfast reduce but do not abolish adhesion to the cellulose acetate used in the screen. Isolation of these potential mutants would require a screen for binding to materials with different surface chemistries. Finally, since N-acetylglucosamine is a component of both the holdfast and peptidoglycan, mutations that abolish its synthesis would be lethal and would not be represented among our collection of insertional knockouts. EPS subunits are synthesized in the cytoplasm from precursors. The EPS subunits are then attached to a lipid carrier residing in the inner membrane, which is energized to flip across the membrane to the periplasm. In a poorly understood process, the EPS subunits are oligomerized into their final form, modified if necessary, and exported to the outer membrane, where they are transported to the exterior matrix via a secretin. Such a system would take advantage of the fact N-acetylglucosamine subunits are present in the periplasm for peptidoglycan biosynthesis, where the hfs gene products could utilize N-acetylglucosamine for holdfast biogenesis.
In conclusion, we have identified the first nonregulatory genes known to be required for holdfast synthesis. Based on their sequence, we hypothesize that the gene products of the hfs cluster are involved in EPS export from the periplasmic space to the cellular exterior. This particular area of the EPS biosynthesis pathway has not been fully elucidated. HfsA might provide periplasmic export functions for holdfast polysaccharide subunits with the energy provided by an ABC transport complex. It is also possible that HfsA plays a role in the processing of holdfast subunits, which could be required for export. HfsD probably terminates the export branch of holdfast biogenesis by serving as the site of secretion to the eventual site of holdfast localization, the tip of the stalk.