(Received for publication, August 7, 1995; and in revised form, November 6, 1995)
From the
XpsD is an outer membrane lipoprotein, required for the
secretion of extracellular enzymes by Xanthomonas campestris pv. campestris. Our previous studies indicated that when
the xpsD gene was interrupted by transposon Tn5,
extracellular enzymes were accumulated in the periplasm (Hu, N.-T.,
Hung, M.-N., Chiou, S.-J., Tang, F., Chiang, D.-C., Huang, H.-Y. and
Wu, C.-Y.(1992) J. Bacteriol. 174, 2679-2687). In this
study, we constructed a series of substitutions and deletion mutant xpsD genes to investigate the roles of NH- and
COOH-terminal halves of XpsD in protein secretory function. Among these
secretion defective xpsD mutations, one group (encoded by
pCD105, pYL4, pKdA6, and pKD2) caused secretion interference when
co-expressed with wild type xpsD, but the other (encoded by
pMH7, pKdPs, and pKDT) did not. Cross-linking studies and gel
filtration chromatography analysis indicated that the wild type XpsD
protein forms a multimer in its native state. Similar gel filtration
analysis of xpsD mutants revealed positive correlations
between multimer formation and secretion interfering properties exerted
by the mutant XpsD proteins in the parental strain XC1701. Those mutant
XpsD proteins (encoded by pCD105, pYL4, pKdA6, and pKD2) that caused
secretion interference formed multimers that are similar to the wild
type XpsD multimers and those (encoded by pMH7, pKdPs, and pKDT) that
did not formed smaller ones. Furthermore, gel filtration and anion
exchange chromatography analyses indicated that the wild type XpsD
protein co-fractionated with XpsD(
29-428) or
XpsD(
448-650) protein but not with XpsD(
74-303)
or XpsD(
553-759) protein. We propose that the mutant
XpsD(
29-428) protein caused secretion interference primarily
by forming mixed nonfunctional multimers with the wild type XpsD
protein in XC1701(pCD105), whereas the mutant XpsD(
74-303)
did so by competing for unknown factor(s) in XC1701(pYL4).
XpsD is an outer membrane (OM) ()protein of Xanthomonas campestris pv. campestris required for
the secretion of extracellular proteins with a cleavable
NH
-terminal signal
peptide(1, 2, 3) . It is likely to be
involved in the second step of a two-step secretory pathway, the type
II pathway(4, 5, 6) . Mutations in the xpsD gene caused the accumulation of extracellular enzymes in
the periplasm(1) . These enzymes are probably exported from
cytoplasm in the first step via a Sec-like pathway(7) .
Homologues of XpsD are widespread in Gram-negative bacteria. Among
them, the PulD protein of Klebsiella oxytoca(8) , the
OutD protein of Erwinia chrysanthemi(9, 10) ,
the OutD protein of Erwinia carotovora(11) , the XcpQ
protein of Pseudomonas aeruginosa(12) ,and the ExeD
protein of Aeromonas hydrophila(13) have been shown
to be involved in the same type of secretion pathway. In addition,
between 11 to 13 more genes in each of these bacteria are required for
the second step of secretion(5, 6) . Some other XpsD
homologues, however, are involved in a different type of secretory
pathway, the type III pathway, for proteins without a cleavable
NH
-terminal signal peptide. These XpsD homologues, which
include the YscC protein of Yersinia
enterocolitica(14) , the HrpH protein of Pseudomonas
syringae(15) , the MxiD protein of Shigella flexneri(16) , and the InvG protein of Samonella
typhimurium(17) , work with a different set of accessory
proteins(6) . There are other proteins which share significant
sequence homolgy with XpsD, such as the pIV proteins of the filamentous
phages f1, I2-2, and Ike of Escherichia
coli(18, 19, 20) , the pIV protein of
the filamentous phage pf3 of P. aeruginosa(21) , the
PilQ protein of P. aeruginosa(22) , and the OrfE
protein of Haemophilus influenzae(23) . These proteins
are required, respectively, for filamentous phage
release(24, 25) , pili assembly (26) , and
genetic transformation(23, 26) . Alignment of all
these protein sequences revealed the strongest homology at their COOH
termini for approximately 200 amino acid
residues(22, 27) .
Recent studies by Kázmierczak et al.(28) indicated that pIV, which is required for filamentous phage release, appeared to form a stable complex composed of 10-12 monomers. Moreover, the OutD protein of E. chrysanthemi, an XpsD homologues required for type II secretion, was co-precipitated with pIV by antibody against pIV (28) , suggesting a complex formation between the two proteins. OutD is also highly homologous to pIV at the COOH-terminal end, implicating the involvement of this region in the mixed complex formation(28) .
Secondary structure prediction indicated that XpsD is rich in
-structures. At least 14 amphipathic
-strands were predicted
based upon Jähnig's algorism(29) .
Near the middle of XpsD, there is a region of more than 100 amino acids
that is longest compared with other XpsD homologues. It is rich in Gly
and Ser, which is observed in only one other XpsD homologue, i.e. the OutD protein of E. carotovora(11) . This long
loop divides the entire XpsD into two domains: the upstream
NH
-terminal domain terminates at the 330th amino acid
residue and the downstream COOH-terminal domain begins at the 440th
residue.
In this report, we show that XpsD also forms multimeric
complex as pIV does. In order to find out what roles do NH-
and COOH-terminal domains play in the complex formation, we constructed
a series of deleted xpsD mutants, examined each for secretion
interference in presence of wild type xpsD gene and conducted
gel filtration and anion exchange chromatography analyses on them.
Figure 1:
Summary of the xpsD mutations
and their secretion and multimer formation properties. The structural
gene of xpsD on pKC118 plasmid was shown on top with its derivatives shown below. Truncated regions were
left blank. The numbers in parentheses next to the
restriction sites used indicate the nucleotide numbers. The open
box represents the phoA gene without its
NH
-terminal signal peptide sequence. The designations
listed under the column ``XpsD'' show the deleted
residues (
), missense substitutions, and fusion.
``+'' indicates positive secretion, multimer
formation, or positive interaction with wild type XpsD.
``-'' indicates secretion negative, no
multimer formation, or no interaction with wild type XpsD.
``±'' indicates intermediate level of
secretion with clear zones of diameters between those of XC1701 and
XC1708. ND, not determined.
Figure 3: Immunoblot of DE52-purified XpsD. DE52-purified XpsD was analyzed on SDS-polyacrylamide gel followed by immunoblotting. Samples were loaded as follows: lane 1, XC17433(pKC118); lane 2, XC17433(pYL4); lane 3, XC17433(pMH7); lane 4, XC17433(pKdPs); lane 5, XC17433(pKdA6); lane 6, XC17433(pKDT); lane 7, XC17433(pKD2).
Figure 5:
FPLC gel filtration chromatography
analysis of XpsD and its mutant XpsD proteins. Triton
X-100 membrane extract was passed through a DE52 anion exchanger.
Samples were eluated with 0.2 M NaCl and precipitated with 70%
ammonium sulfate. After centrifugation, precipitates were solublized in
a small volume of Buffer B and chromatographed on an FPLC gel
filtration column (Superdex HR-200, 25 ml), pre-equilibrated with the
same buffer. Proteins in the eluated fractions were separated on
SDS-polyacrylamide gel, followed by immunoblot using antibody against
XpsD (A) or XpsD(
29-428) (B). Each mutant
XpsD protein was produced in XC17433
(
xpsE,F,G,H,I,J,K,L,M,N,D) harboring the respective
plasmid.
Figure 4: Glutaraldehyde cross-linking of XpsD in vitro and in vivo. Glutaraldehyde (GA) (final concentrations indicated above each lane) was added to a Triton X-100 membrane extract of XC1701 (A); B, growing XC17433(pKC118) cells. XpsD was detected by immunoblotting. The arrows indicate cross-linked XpsD.
Next, we wanted to find out if those mutant XpsD proteins that did
not exhibit negative dominance could not form multimeric complex with
the wild type XpsD protein. As expected, the XpsD(553-759)
protein appeared between 21 and 24 min on FPLC Superdex HR-200 column
when it was co-expressed in XC1701(pKDT) culture with the wild type
XpsD protein, which appeared in near void volume (15-16 min) (Fig. 6A). This result indicated that the
XpsD(
553-759) protein probably did not form a complex with
the wild type XpsD protein, consistent with our model of negative
dominance based on protein-protein interaction (Fig. 8B). However, the XpsD(
448-650)
protein synthesized in XC1701(pKdPs) co-migrated with the wild type
XpsD protein (Fig. 6B). This result suggested that the
XpsD(
448-650) protein formed a complex, either with the wild
type XpsD protein, or with other protein (including itself). The former
possibility contradicts with the observation that the
XpsD(
448-650) protein did not interfere with secretion in
XC1701, unless we assume that the heteromultimer of wild type XpsD and
XpsD(
448-650) is functional in protein secretion or enough
wild type XpsD homomultimers were formed to support normal protein
secretion in XC1701(pKdPs) (Fig. 8D).
Figure 6:
Analysis of the interaction between XpsD
and XpsD(553-759) or XpsD(
448-650) by FPLC gel
filtration chromatography. Samples were prepared from XC1701(pKDT) (A)
and XC1701(pKdPs) (B), following the same procedures as
described in the legend to Fig. 7.
Figure 8:
Diagrammatic presentations for the
interference of normal protein secretion by mutant XpsD proteins.
Co-expressed with the wild type XpsD protein encoded by the chromosomal
DNA of XC1701 are mutant XpsD proteins: A,
XpsD(29-428); B, XpsD(
553-759); C, XpsD(
74-303); and D,
XpsD(
448-650).
Figure 7:
Analysis of the interaction between
XpsD protein and XpsD(
29-428) or
XpsD(
74-303) protein by FPLC anion exchange chromatography.
Triton X-100 extracts of the membranes prepared from French press
disrupted cells were passed through an FPLC anion exchange column (Mono
Q, HR 5/5, 1 ml), eluted with a 0-1.0 M NaCl linear
gradient. Fractions 8-18 were analyzed separately on
SDS-polyacrylamide gel, followed by immunoblot analysis with antibody
against XpsD. Samples were prepared from XC17433(pKC118),
XC17433(pCD105), XC17433(pYL4) (A); XC1701(pCD105) (B); and XC1701(pYL4) (C).
All the xpsD truncated mutants inspected in this
study were defective in the secretion of -amylase and protease,
causing accumulation of
-amylase in the periplasm. These XpsD
proteins can be divided into two groups: Group I, including
XpsD(
29-428) (pCD105), XpsD(
74-303) (pYL4), and
XpsD(
545-553) (pKdA6), displayed negative dominance over the
wild type XpsD protein, and Group II, including
XpsD(
414-759) (pMH7), XpsD(
448-650) (pKdPs), and
XpsD(
553-759) (pKDT), did not. In this study we found good
correlation between negative dominance and the formation of XpsD
multimers: all Group I proteins formed multimers that appeared in the
near void volume, whereas all Group II proteins did not. This indicates
that Group I proteins exerted negative dominance by interacting with
the wild type XpsD protein. This was supported by our observations that
the XpsD(
29-428) protein co-fractionated with the wild type
XpsD protein, whereas the XpsD(
553-759) protein did not.
The amino acid sequences common in the Group I proteins are residues
1-28, 429-544, and 554-759. At the NH terminus, a conserved lipoprotein signal peptidase cleavage site
(-LLAG
C-) (33) is located between residues 21 and 22.
Fatty acylation of XpsD has been demonstrated with
[
H]palmitate labeling of wild type
XpsD(3) . This suggested that wild type XpsD is probably
cleaved between residues 21 and 22, followed by fatty acylation at the
NH
-terminal cysteine residue. It is thus unlikely that the
NH
-terminal amino acid residues 1-28 play any
significant role in multimer formation. On the other hand, in the amino
acid sequences spanning residues 429-544 and 554-759, which
comprise almost half of the entire XpsD, eight amphipathic
-strands were predicted. The region between residues 545 and 553
was predicted to be within a loop based upon
Jähnig's algorism(29) . The former
two regions are either absent entirely in the XpsD(
414-759)
protein or partially in XpsD(
448-650) and
Xps(D
553-759) proteins, none of which formed functional
multimers by themselves or affect protein secretion in XC1701. We
propose that these two regions (429-544 and 554-759) are
required for multimer formation. Although the mutant
XpsD(
29-428), XpsD(
74-303),
XpsD(
545-553), and XpsD(A553L/I554D) proteins formed
multimers by themselves, all of them are not functional in protein
secretion. We suggest that the regions covered within residues
74-303 and the amino acid residues 553A, 554I, albeit unrelated
with multimer formation, is important for protein secretion.
Due to the small size of deoxycholate micells(28, 34) , we carried out gel filtration chromatography in the presence of deoxycholate. Wild type XpsD eluted in the near void volume with an estimated molecular mass of approximately 1,000 kDa, suggesting that it forms stable multimeric complexes. At this stage we do not know whether XpsD forms homomultimers or heteromultimers. A homomultimer of XpsD of this size would contain at least 12 mature XpsD polypeptides (77 kDa each). This is comparable with the filamentous phage pIV multimers, estimated to be 10-12-mers(28) .
A gated channel
formed by pIV was proposed for releasing of assembled filamentous phage
particles(25) . The COOH-terminal 200 amino acid sequence of
XpsD are highly homologous to pIV and other analogous
proteins(22, 27) . Moreover, XpsD co-expressed with
pIV was precipitated with antibody against pIV, which does not
cross-react with XpsD. ()When we introduced pIV gene into XC1701, we clearly observed secretion interference. (
)Both results suggested complex formation between XpsD and
pIV. In this study we provide evidences for the first time to show that
an OM component of one of the terminal branches of general secretion
pathway (5) probably forms stable complex of a minimal 12-mer.
An OM secretion channel is anticipated for secreted proteins that are
translocated across OM as disulfide-bonded
molecules(35, 36, 37, 38, 39) .