Microbiology (2004), 150, 21–31
DOI 10.1099/mic.0.26684-0
The importance of the Tat-dependent protein
secretion pathway in Streptomyces as revealed
by phenotypic changes in tat deletion mutants
and genome analysis
Kristien Schaerlaekens, Lieve Van Mellaert, Elke Lammertyn,
Nick Geukens and Jozef Anné
Laboratory of Bacteriology, Rega Institute, Katholieke Universiteit Leuven,
Minderbroedersstraat 10, B-3000 Leuven, Belgium
Correspondence
Jozef Anné
[email protected]
Received 4 August 2003
Revised 2 October 2003
Accepted 8 October 2003
Streptomyces are Gram-positive soil bacteria that are used industrially, not only as a source
of medically important natural compounds, but also as a host for the secretory production of a
number of heterologous proteins. A good understanding of the different secretion processes in this
organism is therefore of major importance. The functionality of the recently discovered bacterial
twin-arginine translocation (Tat) pathway has already been shown in Streptomyces lividans.
Here, the aberrant phenotype of S. lividans DtatB and DtatC single mutants is described. Both
mutants are characterized by a dispersed growth in liquid medium, an impaired morphological
differentiation on solid medium and growth retardation. To reveal the extent to which the Tat
pathway is used in Streptomyces, putative Tat-dependent precursor proteins of Streptomyces
coelicolor, a very close relative of S. lividans, and of Streptomyces avermitilis, of which the
genomes have been completely sequenced, were identified by a modified version of the TATFIND
computer program designed by Rose and colleagues [Rose, R. W., Brüser, T., Kissinger, J. C. &
Pohlschröder, M. (2002). Mol Microbiol 45, 943–950]. A list of 230 precursor proteins was
obtained; this is the highest number of putative Tat substrates found in any genome so far. In
addition to the Streptomyces antibioticus tyrosinase, it was also demonstrated that the secretion
of the S. lividans xylanase C is Tat-dependent. The predicted Tat substrates belong to a variety
of protein classes, with a high number of proteins functioning in degradation of macromolecules,
in binding and transport, and in secondary metabolism. Only a minor fraction of the proteins
seem to bind a cofactor. The aberrant phenotype of the DtatB and DtatC mutants together with
the high number of putative Tat-dependent substrates suggests that the Streptomyces Tat
pathway has a distinct and more important role in protein secretion than in most other bacteria.
INTRODUCTION
Protein secretion is a universal process that occurs in all
organisms. In bacteria, the major routes for secretion
through the cytoplasmic membrane are the Sec and SRP
secretion pathways, which have already been known for
a long time and have been investigated in great detail in
several organisms. Proteins to be secreted via these two
pathways are tagged by a signal peptide that directs
them to the secretion apparatus. Signal peptides consist of
three domains, i.e. an N-terminal domain having one or
more positive charges, a hydrophobic core region and a
C-terminal end containing the recognition site for cleavage
by a signal peptidase (von Heijne, 1984).
A different secretion route for protein transport across
Abbreviation: Tat, twin-arginine translocation.
0002-6684 G 2004 SGM
the cytoplasmic membrane has been described, the twinarginine translocation (Tat) pathway (Berks, 1996; Santini
et al., 1998). This pathway seems to be fundamentally
different from the other two pathways for several reasons.
In contrast to the Sec and SRP pathways, the energy to drive
protein translocation is provided by the proton motive
force and not by nucleoside triphosphates (Santini et al.,
1998). Furthermore, although a signal peptide that routes
a protein to the Tat pathway has the same overall structure
as the signal peptides for Sec- and SRP-dependent transport, a characteristic twin-arginine motif is located at the
border of the N-terminal domain and the hydrophobic
region. Moreover, in contrast to the Sec and SRP pathways for which proteins need to be unfolded to cross the
membrane, the Tat pathway allows the secretion of fully
folded proteins and binding of a cofactor does not inhibit
translocation (Halbig et al., 1999b; Santini et al., 1998). In
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21
K. Schaerlaekens and others
fact, the secretion of cofactor-containing, folded proteins
is regarded as a specific characteristic of the Tat pathway.
However, recent data suggest that the role of the Tat
pathway in protein secretion might be broader than originally thought from Escherichia coli studies (Bolhuis, 2002;
Ding & Christie, 2003; Ochsner et al., 2002; Rose et al., 2002;
Voulhoux et al., 2001).
In this report, we discuss the possible role of the Tat pathway in Streptomyces, a soil-dwelling filamentous bacterium
responsible for the production of the majority of natural
antibiotics used in medicine. Mainly thanks to its high
secretion capacity, it is an interesting host for the secretory
production of heterologous proteins (Van Mellaert et al.,
1994; Binnie et al., 1997; Lammertyn et al., 1997). In this
respect, the Sec-dependent protein secretion pathway in
Streptomyces lividans has already been investigated to some
extent, including the characterization of the signal peptidases (Geukens et al., 2001) and the importance of the
signal peptide and its charge in heterologous protein
secretion (Lammertyn et al., 1997, 1998). Recently, the
genes encoding TatA, TatB and TatC, the three components constituting the Tat machinery, were identified and
the functionality of the Tat pathway has been illustrated
by comparing the secretion efficiency of the chimeric
preTorA23K and the Streptomyces antibioticus tyrosinase
in the wild-type and a DtatC mutant (Schaerlaekens et al.,
2001). In this work, a DtatB mutant is constructed and the
aberrant phenotype of both tat deletion mutants is discussed. Because of the availability of the genome sequences
of Streptomyces coelicolor and of Streptomyces avermitilis
(Bentley et al., 2002; Ikeda et al., 2003), a prediction of
putative Tat substrates could be made. For this computer
analysis, a modified version of the program TATFIND 1.2,
designed by Rose et al. (2002), was used. The result of
this analysis will be discussed and related to the aberrant
phenotype of the S. lividans tat deletion mutants.
METHODS
Bacterial strains and growth conditions. E. coli strain TG1 was
used as host for cloning purposes. Cultures were grown at 37 uC
(300 r.p.m.) in Luria–Bertani medium in the presence of ampicillin
(50 mg ml21) or apramycin (50 mg ml21) when applicable. S.
lividans TK24 and derivatives thereof were routinely precultured
at 27 uC with continuous shaking at 300 r.p.m. in phage medium
(Korn et al., 1978). Subsequently, the strains were inoculated in NM
medium (Van Mellaert et al., 1994) or minimal medium (Kieser
et al., 2000). Regeneration of S. lividans protoplasts and selection of
transformants occurred on MRYE medium (Anné et al., 1990).
Where appropriate, apramycin (50 mg ml21), kanamycin (50 mg ml21)
or thiostrepton (10 mg ml21) were added. Protoplast formation
and subsequent transformation of S. lividans were carried out as
described by Kieser et al. (2000).
DNA techniques and vector constructions. DNA manipulations
were performed using standard techniques (Sambrook et al., 1989).
All PCR fragments were checked by DNA sequence analysis according to the dideoxy chain termination method with the Thermo
Sequenase Primer Cycle Sequencing Kit 7-Deaza-dGTP on an
ALFexpress apparatus (Amersham Biosciences).
22
For the construction of a DtatB mutant, the E. coli–Streptomyces
shuttle vector pGM160 (Muth et al., 1989), containing a temperaturesensitive ori, was used. The neo gene of plasmid pBSKAN
(Schaerlaekens et al., 2001) was removed by digestion with PstI/
HindIII and a fragment containing the aac(3)IV gene, encoding
resistance to apramycin, was inserted in the same sites. Then, the
surrounding regions of the tatB gene were cloned on both sides of
aac(3)IV. An upstream fragment of 399 bp was amplified with
primers TatB1 (59-ATCTCGAGTGCCAAGGGCGGCGACGGCG-39)
and TatB2 (59-ATAAGCTTGCCTATGTCATTGAACACCT-39) with
an XhoI and HindIII restriction site, respectively (underlined). A
downstream fragment of 500 bp was amplified using primers TatB3
(59-ATGGATCCGCCCTGAAGGCGACGCCCGC-39) and TatB4 (59TATCTAGAGGCCTCCAGCAGGCCGGTGC-39), with a BamHI and
XbaI restriction site, respectively (underlined). All PCR reactions
were performed with SuperTaq polymerase (HTBiotechnology) in
the presence of 10 % DMSO. The obtained fragments were cloned
in pGEM-T Easy (Promega) and subsequently digested with the
appropriate restriction enzymes. The aac(3)IV-containing derivative
of pBSKAN was digested first with XhoI and HindIII to allow insertion of the 59 fragment, and then with BamHI and XbaI for the
insertion of the 39 region. To obtain the complete cassette, the resulting vector was partially digested with XbaI and XhoI. The obtained
fragment was blunted and ligated into a HindIII-digested and bluntended pGM160, giving rise to plasmid pGMDtatB. After PEG6000mediated transformation of S. lividans protoplasts with pGMDtatB,
a temperature shift to 39 uC promoted the integration of the
temperature-sensitive replicon into the chromosomal DNA. The
tatB deletion resulting from double homologous recombination was
confirmed by PCR and Southern blot analysis.
For DtatB complementation tests in S. lividans, a derivative of the
integrative plasmid pSET152 (Bierman et al., 1992) containing the
tatB gene was constructed. To achieve this, the aac(3)IV gene was
removed by SacI restriction and replaced by the neo gene of pBSKAN
(Schaerlaekens et al., 2001). Then, the tatB gene with its putative
promoter region was amplified by PCR using primers TatB1 and
TatBNNID
(59-ATCCATGGCCGCGCGGGCGTCGCCTTCA-39).
After cloning of the fragment in the EcoRI site of pBluescript KS(+)
(Stratagene), restriction with EcoRV and XbaI allowed subsequent
ligation of the fragment in the modified pSET152, resulting in the
complementation plasmid pSETtatB.
To construct a Streptomyces vector overexpressing xylanase C, the
S. lividans xylanase C gene was amplified by PCR with chromosomal
DNA as template. The reaction was carried out in the presence of
primer Xyl1 (59-ATCTGCAGAGAAAGGAGAACGCATGCAGCAGG39), including an optimized Shine–Dalgarno sequence (bold) and a
PstI restriction site (underlined), primer Xyl2 (59-ATAAGCTTAGAGGTCAACCGCTGACCG-39), with a HindIII restriction site (underlined), Pfu polymerase (Promega) and 10 % DMSO. After cloning in
pGEM-T Easy, the PstI–HindIII fragment was ligated in pBS-CBSS,
a pBluescript KS(+) derivative containing the vsi promoter
(Lammertyn et al., 1997), such that the xlnC sequence was preceded
by the vsi promoter. The resulting pBSvsixyl plasmid was digested
with BamHI and HindIII to obtain a 1?1 kb fragment with the vsi
promoter and the xlnC coding sequence that was subsequently
ligated in BamHI/HindIII-digested pIJ486 (Ward et al., 1986), giving
rise to pIJvsixyl.
Activity assays. Xylanase activity was measured using the dinitrosalicylic acid assay (Miller, 1959). We used 48 h precultures grown
in phage medium to inoculate 50 ml NM medium and cultures
were subsequently incubated for 24–48 h. After centrifugation, extracellular fractions were diluted in assay buffer and the amount of
reducing sugar was quantified. The intracellular amount of xylanase
was determined on cell lysates obtained by sonication (2 min,
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Microbiology 150
Streptomyces Tat-dependent protein secretion
20 000 Hz, 0 uC) of the mycelium suspended in assay buffer. One
unit of xylanase was defined as the amount of enzyme that produces 1 mg reducing sugar in 10 min at 60 uC from a saturated
xylan solution.
The inhibitory activity of subtilisin inhibitor was determined in
the presence of the substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-pnitroanilide as described by Kojima et al. (1990). Precultures grown
in phage medium (48 h) were used to inoculate 50 ml NM medium
and cultures were subsequently cultivated for 24 h. After centrifugation, extracellular fractions were diluted in assay buffer and the
percentage of subtilisin inhibition was measured. The intracellular
amount of subtilisin inhibitor was measured on cell lysates obtained
by sonication (2 min, 20 000 Hz, 0 uC) of the mycelium suspended in
assay buffer. One unit was defined as the amount of enzyme that
inhibited 1 mg subtilisin during 10 min incubation at 25 uC.
Immunoblot analysis. Western blot analysis was performed to
check the translocation of xylanase C in S. lividans. Extracellular
fractions of 24 or 30 h recombinant S. lividans cultures in NM
medium after inoculation with a 48 h preculture in phage medium
were obtained by centrifugation (10 min, 4200 g). Proteins in the
growth medium were precipitated with trichloroacetic acid (20 %
final concn) and separated by SDS-PAGE (Laemmli, 1970). Transfer
of proteins onto a nitrocellulose Porablot membrane (Macherey–
Nagel) was performed using a Bio-Rad Transblot semidry transfer
cell (Bio-Rad), according to the manufacturers’ recommendations.
Xylanase C was detected using rabbit anti-XlnC antibodies (kindly
provided by J. Dusart, University of Liege, Belgium), followed by
alkaline phosphatase-conjugated goat anti-rabbit antibodies (Sigma).
RESULTS AND DISCUSSION
Phenotype of S. lividans DtatB and DtatC single
mutants
S. lividans DtatB and DtatC single mutants clearly showed
phenotypic differences in comparison with wild-type
S. lividans TK24. In liquid medium, the mycelium grew
very dispersed, while wild-type S. lividans formed mycelial
aggregates. In addition, growth of DtatB and DtatC was
much slower and mycelial mass remained lower than that
of the wild-type in rich medium (Fig. 1a) as well as in
minimal medium (data not shown). Also on solid MRYE
medium, growth of the mutant strains was hampered in
comparison with that of the wild-type (Fig. 1b). Morphological differentiation of the mutant strains from vegetative
to aerial mycelium, although delayed, could be observed,
but the mutant strains never sporulated. Also the red
pigment undecylprodigiosin could not be produced in the
mutant strains (Fig. 1c). On solid MS medium, on the
contrary, both mutant strains became greyish, indicating
the formation of spores (Fig. 1d). Interestingly, growth
and differentiation were more affected in DtatC than in
DtatB, suggesting a more important role for TatC in the
translocation process.
The S. coelicolor and S. avermitilis translated
genomes contain an extremely high number of
putative Tat substrates
To find an explanation for the differential phenotype of the
S. lividans tat deletion mutants, it was interesting to predict
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the number and nature of the proteins secreted in a Tatdependent way and therefore likely to be mislocalized in
these mutants. The availability of the genome sequences
of S. coelicolor and S. avermitilis allowed us to make a list
of all putative Tat-dependent precursor proteins for these
organisms. Because S. coelicolor has been shown to belong
to the same taxon at the species level as S. lividans
(Kawamoto & Ochi, 1998), its genome analysis is regarded
as representative for S. lividans.
In the first instance, TATFIND version 1.2 (Rose et al., 2002)
was used. For both S. coelicolor and S. avermitilis, 145
Tat-dependent candidates were found. This is the highest
number of putative Tat substrates found in any genome
so far. However, because the number of predicted ORFs in
S. coelicolor is the highest so far recorded among bacteria
(Bentley et al., 2002), we reasoned that a comparison of
the number of Tat-dependent substrates between different
bacteria should be expressed as a percentage of the total
number of predicted ORFs. These numbers for Streptomyces and some other micro-organisms are shown in
Table 1. The archaeon Halobacterium sp. NRC-1 leads
with 2?7 % putative Tat-dependent proteins. Moreover,
the vast majority of haloarchaeal preproteins seem to be
predicted substrates of the Tat pathway (Rose et al., 2002).
S. avermitilis and S. coelicolor have the second highest
percentage of putative Tat-dependent precursor proteins.
Mycobacterium tuberculosis and Corynebacterium glutamicum, two other bacteria belonging to the Actinomycetales
group, do not seem to have the same high number of Tat
substrates, an indication that a high number of putative Tatdependent proteins is not a general feature of this group of
bacteria. A more extensive list of bacteria with their number
of putative Tat substrates is given by Dilks et al. (2003).
The ratio between the estimated number of secreted Tat
substrates and the total number of secreted proteins can
give an idea about the contribution of the Tat pathway to
the total secretome. Of the 145 predicted Tat substrates, 127
proteins are putative secreted proteins. The other proteins
identified by TATFIND are putative membrane proteins.
Indeed, it is generally believed that the Tat pathway also
serves for the targeting and translocation of certain membrane proteins. In E. coli, for example, sequence analysis
suggests that one-quarter of all traffic on the Tat pathway
is inner-membrane proteins (Sargent et al., 2002). The
annotated genome database of S. coelicolor contains 819
potentially secreted proteins; this is 10?5 % of all predicted
proteins (Bentley et al., 2002). If 127 of these 819 proteins
are predicted to be secreted via the Tat pathway, then 15?5 %
of all signal-peptide-dependent transport occurs via the
Tat pathway. For comparison, the seven predicted Tat
substrates in Bacillus subtilis represent only 3?9 % of its
predicted 180 secretory proteins (Tjalsma et al., 2000).
A modified version of the
TATFIND
program
version 1.2 searches for the following pattern
between residues 2 and 35 of the predicted proteins:
TATFIND
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K. Schaerlaekens and others
(a)
5
_
Dry weight (mg ml 1)
4
3
2
1
0
0
6
12
18
24
30
36
42
48
Incubation time (h)
Fig. 1. Growth of S. lividans DtatB and DtatC compared to wild-type S. lividans TK24. (a) NM medium (50 ml) was
inoculated with 1 ml of a 48 h preculture in phage medium and cultured for another 48 h. Every 6 h, samples were taken to
determine the dry weight. Filled squares, S. lividans TK24; grey circles, S. lividans DtatB; open triangles, S. lividans DtatC.
(b) Top of an MRYE plate inoculated with S. lividans TK24, DtatB and DtatC after 7 days growth. (c) Bottom of an MRYE
plate inoculated with S. lividans TK24, DtatB and DtatC after 7 days growth. (d) Top of an MS plate inoculated with S.
lividans TK24, DtatB and DtatC after 7 days growth.
(X21)R0R+1(X+2)(X+3)(X+4), where the amino acid at
position X21 has a hydrophobicity score ¡0?26; X+2 and
X+3 have a hydrophobicity score ¡0?02 and ¢20?77
(positively charged residues were excluded from this
position), respectively, and X+4 is one of the residues
ILVMF. This pattern was taken from literature in combination with a list of putative secreted proteins from
Halobacterium sp. NRC-1, and then refined with residues
24
found in putative Tat substrates of other Halobacteriaceae.
Proteins corresponding to the above pattern and containing
a hydrophobic region following the twin-arginine motif,
were designated as Tat-dependent (for further details, see
Rose et al., 2002).
So far, the only Streptomyces protein experimentally proven
to be secreted in a Tat-dependent way is S. antibioticus
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Microbiology 150
Streptomyces Tat-dependent protein secretion
Table 1. Number of ORFs and putative Tat substrates of some micro-organisms as identified
by TATFIND version 1.2 (Rose et al., 2002)
Organism
Halobacterium sp. NRC1
Streptomyces avermitilis
Streptomyces coelicolor
Escherichia coli K-12
Mycobacterium tuberculosis
Corynebacterium glutamicum
Bacillus subtilis
No. of
ORFs
No. of putative
Tat substrates
Putative Tat substrates
(percentage of total no. of ORFs)
2446
7574
7825
4279
3924
3041
4112
68
145
145
34
31
15
7
2?7
1?9
1?8
0?79
0?79
0?49
0?17
MelC1, the transactivator protein of tyrosinase (Schaerlaekens
et al., 2001). The twin-arginine motif in its signal peptide
is TRRQVM. Although this motif fulfils the criteria of
TATFIND version 1.2, we reasoned that this program was
not ideally adapted for recognition of putative Tat substrates of Streptomyces. Because little is known about the
Streptomyces Tat pathway, we preferred to change the
program to search for the more theoretically based motif
RRx[FLIVAM][FLIVAM], because only the twin arginines
and the hydrophobic residues at the +3 and +4 positions
have been shown experimentally to be important for Tatdependent transport (Brink et al., 1998; Halbig et al., 1999a;
Gross et al., 1999; Stanley et al., 2000). The additional
criteria for the identification of an uncharged region
following RR were not changed. When the modified
TATFIND program was used to analyse the S. coelicolor
genome, a list of 230 putative Tat substrates was obtained.
This modified TATFIND program is designed to recognize
every possible Tat substrate and might therefore give an
overestimation of the number of Tat substrates. Therefore, Tat dependence should be experimentally confirmed
for every protein. In B. subtilis for example, it was shown
that the extracellular accumulation of 13 proteins with
potential RR/KR-signal peptides was Tat-independent
(Jongbloed et al., 2002). In this bacterium, however, there
was no obvious phenotypic difference between the wildtype strain and the tatCd-tatCy double mutant (Jongbloed
et al., 2000).
Characteristics of the putative twin-arginine
signal peptides of S. coelicolor
The list of 230 putative Tat signal peptides was analysed by
the SIGNALP-HMM program which can predict the position
and length of the three signal peptide domains and the
position of the signal peptidase cleavage site (Nielsen et al.,
1997). This analysis revealed a mean Tat signal peptide
length of 39 aa and a mean N region length of 13 aa. The
mean positive charge of these N regions was 4?2. The H
region had a mean length of 15 aa and a mean hydrophobicity of 1?95 as determined by the algorithms of Kyte
& Doolittle (1982) using the PROTPARAM tool (http://us.
expasy.org/tools/protparam). In addition, 33 % of the Tat
signal peptides contained within the C region a positive
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charged amino acid that might serve as a Sec avoidance
signal (Cristóbal et al., 1999; Blaudeck et al., 2003).
Following analysis of the whole S. coelicolor genome with
(Nielsen et al., 1997), a mean signal peptide length
of 42 aa was found. Since the mean length of the Tat
signal peptides was calculated to be 39, it seems that in the
case of Streptomyces, the Tat signal peptides are not longer
than the Sec signal peptides. Tat signal peptides from
Gram-negative bacteria on the contrary, are on average
14 aa longer than Sec signal peptides, with most of this
additional length being caused by an extended N-region
(Cristóbal et al., 1999).
SIGNALP
The only other Gram-positive bacterium for which these
values are available is B. subtilis. The mean length of its
predicted Tat signal peptides is 36, which is 8 aa longer
than the mean length of its Sec signal peptides (van Dijl
et al., 2002). This extra length of the Tat signal peptides
could be attributed to an N region with a mean length of
13–14 aa. Concerning length (19?2 on average) and hydrophobicity (1?9 on average) of the H region, however, no
significant difference with the Sec signal peptides was
observed (van Dijl et al., 2002). This contrasts to the
situation in E. coli where the H region of Tat signal peptides
is significantly less hydrophobic than the H region of Sec
signal peptides (Cristóbal et al., 1999).
Putative Streptomyces Tat substrates belong to
a variety of protein classes
The 230 proteins identified by the modified version of the
TATFIND program were classified according to the protein
classification scheme on the S. coelicolor genome project
website (http://www.sanger.ac.uk/Projects/S_coelicolor).
From this classification (Table 2), it is clear that the Tat
substrates do not belong to a few specified protein classes,
but instead are members of a variety of classes. However,
a number of groups are overrepresented while other
groups are completely absent. Besides the expected groups
of membrane proteins (II.C.1), lipoproteins (II.C.2) and
putative secreted proteins (II.C.2), a high percentage of
the putative Tat substrates function in the degradation
of macromolecules (II.B) and in transport and binding
(III.A). Ten proteins functioning in secondary metabolism
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TATFIND
1.2
I. Small molecule metabolism
I.A Degradation
I.A.1 Carbon compounds
SCO6665, putative secreted glucosidase
I.A.2 Amino acids
gad, SCO3416, putative glutamate decarboxylase
I.A.3 Fatty acids
lipA, SCO0713, lipase
SCO4368, putative lipase
SCO6691, putative phospholipase C
I.B Energy metabolism
I.B.1 Aerobic respiration
cox, SCO2156, putative cytochrome c oxidase subunit II
I.C Central intermediary metabolism
SCO2015, putative nucleotidase
SCO0828, putative alkaline phosphatase
gabT, SCO5676, putative 4-aminobutyrate aminotransferase
I.D Amino acid biosynthesis
proB, SCO2587, glutamate 5-kinase
I.E Biosynthesis of cofactors
SCO6407, putative c-glutamyltranspeptidase
I.F Fatty acid biosynthesis
SCO3502, putative short-chain dehydrogenase
I.G Broad regulatory functions
SCO0551, putative histidine kinase protein
SCO6621, putative serine/threonine protein kinase
II. Macromolecule metabolism
Microbiology 150
II.A Protein modification
SCO1639, putative secreted peptidyl-prolyl cis-trans isomerase protein
II.B Degradation of macromolecules
II.B.1 RNA
SCO2766, putative secreted ribonuclease
II.B.2 Polysaccharides
xlnC, SCO0105, endo-1,4-b-xylanase
cel1, SCO0765, secreted endoglucanase
SCO0766, putative secreted b-galactosidase
SCO1879, putative secreted pectinesterase
SCO2226, putative bi-functional protein (secreted a-amylase/dextrinase)
SCO2427, putative secreted arabinase
SCO2821, putative secreted pectate lysase
SCO2838, putative secreted endoglucanase
dagA, SCO3471, extracellular agarase precursor
SCO5673, secreted chitinase
SCO6300, putative secreted hydrolase
SCO6345, putative secreted chitinase
SCO7225, secreted chitinase
SCO7637, putative secreted endoglucanase
II.B.3 Proteins, peptides, glycoproteins
SCO5821, undefined product
SCO5913, undefined product
csn, SCO0677, secreted chitosanase
SCO2446, putative secreted peptidase
nagA, SCO2758, b-N-acetylglucosaminidase
SCO5043, putative hydrolase
SCO5447, putative neutral zinc metalloprotease
SCO6736, putative metallopeptidase
SCO7256, putative protease
endoH, SCO7633, putative secreted endo-b-N-acetylglucosaminidase
II.C Exported/membrane/lipoproteins
II.C.1 Membrane proteins
25 putative membrane proteins
II.C.2 Exported/lipoproteins
46 putative secreted proteins
21 putative lipoproteins
SCO0505, putative multi-domain protein (fragment)
SCO0732, putative secreted protease
SCO1049, putative secreted oxidoreductase
SCO1290, putative secreted phosphatase (fragment)
SCO1356, putative iron–sulphur protein
SCO1565, putative glycerophosphoryl diester phosphodiesterase (fragment)
SCO1734, putative secreted cellulose-binding protein
SCO1741, putative secreted serine protease
SCO1763, putative iron–sulphur protein
SCO1955, putative iron–sulphur-binding protein
SCO5534, putative secreted lyase
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K. Schaerlaekens and others
26
Table 2. Functional classification of putative S. coelicolor Tat substrates as identified by a modified version of
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Table 2. cont.
II. Macromolecule metabolism
SCO0716, putative glycosyl hydrolase
salO, SCO0752, protease precursor
SCO0787, putative secreted hydrolase
SCO0829, putative serine protease
SCO6272, putative secreted FAD-binding protein
II.C.3 Peptidoglycan
SCO3811, putative D-alanyl-D-alanine carboxypeptidase
III. Cell processes
SCO7399, possible binding-protein-dependent transport lipoprotein
SCO7503, putative extracellular solute-binding protein
SCO7504, putative integral membrane binding-protein-dependent transport protein
SCO7563, putative ABC transporter solute-binding lipoprotein
malE, SCO2231, putative maltose-binding protein
SCO5658, putative polyamine-binding lipoprotein
SCO2828, probable amino acid ABC transporter protein, solute-binding component
SCO0065, putative extracellular binding protein
III.B Detoxification
SCO1172, putative amidase
SCO6712, putative copper oxidase
III.C Secondary metabolism
SCO3244, hypothetical protein
SCO0333, putative dioxygenase
SCO0494, putative iron-siderophore-binding lipoprotein
SCO0497, putative iron-siderophore permease transmembrane protein
ssp, SCO1824, secreted subtilisin-like protease
melC1, SCO2701, tyrosinase co-factor
SCO5799, putative aminotransferase
SCO6281, putative FAD-binding protein
SCO5317, polyketide b-ketoacyl synthase b
SCO5318, polyketide b-ketoacyl synthase a
IV. Others
27
IV.A Others
SCO0236, putative DNA-binding protein
SCO0370, possible DNA-binding protein
SCO4755, putative transcriptional regulator
IV.B Not classified
SCO0486, putative monooxygenase
SCO0529, probable oxidoreductase
pstS, SCO4142, phosphate-binding protein precursor
SCO4168, putative oxidoreductase
SCO5286, putative secreted hydrolase
SCO5941, aminotransferase
SCO5948, putative oxidoreductase
SCO6032, putative hydrolase
SCO7069, putative secreted hydrolase
SCO7179, putative secreted amidase
IV.C Conserved hypotheticals
9 conserved hypothetical proteins
IV.D Unknown function, no known homologues
8 hypothetical proteins
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Streptomyces Tat-dependent protein secretion
III.A Transport/binding proteins
11 putative solute-binding proteins
SCO0538, probable sugar transporter sugar-binding lipoprotein
SCO1056, putative sugar transport sugar-binding protein
SCO1065, putative sugar transport sugar-binding protein
SCO1854, putative integral membrane protein (fragment)
SCO2404, putative sugar-binding receptor
SCO2434, putative sugar transporter sugar-binding protein
SCO2795, putative sugar-binding secreted protein
SCO3417, putative ABC transporter transmembrane component
SCO3484, putative secreted sugar-binding protein
SCO3505, putative secreted substrate-binding protein
SCO4024, putative integral membrane protein
SCO4404, putative ABC transport system integral membrane protein
SCO5428, putative integral membrane transport protein
SCO5429, putative integral membrane transport protein
SCO5667, putative ABC-transporter polyamine-binding lipoprotein
SCO6114, putative secreted peptide-binding transport
SCO6179, putative nucleotide-sugar dehydratase
SCO6250, putative transport protein
SCO6295, putative ABC transporter ATP-binding protein
SCO7170, putative secreted sugar hydrolase
K. Schaerlaekens and others
Tat-dependent secretion of tyrosinase and
xylanase C
So far, the only Streptomyces protein experimentally
proven to be secreted in a Tat-dependent way is S.
antibioticus MelC1, the transactivator protein of tyrosinase,
which directs the secretion of the MelC1/apotyrosinase
complex to the Tat translocon (Schaerlaekens et al., 2001).
The modified TATFIND program identified its S. coelicolor
homologue as a putative Tat-dependent substrate (Table 2,
III.C). Because of the similarity between S. antibioticus
MelC1 and S. coelicolor MelC1 (25 % identity), it can
be assumed that secretion of S. coelicolor MelC1 is also
Tat-dependent.
For experimental confirmation of the Tat dependency of a
second predicted protein, xylanase C (XlnC, II.B.2), containing the motif RRGFL, was chosen. Only small amounts
of xylanases were detectable in the extracellular fraction
of the S. lividans wild-type strain. Therefore, xylanase C
was overexpressed in S. lividans. This was achieved by
cloning the coding sequence of XlnC downstream from
the strong constitutive promoter of the S. venezuelae subtilisin inhibitor gene vsi (Lammertyn et al., 1997) on the
multicopy plasmid pIJ486 (Ward et al., 1986). Selected
transformants were grown for 24 h in NM medium and the
presence of XlnC in the culture medium was checked by
Western blot analysis (Fig. 2a). A strong immunoreactive
band of 20 kDa could be detected in the wild-type strain
containing the XlnC expression vector (lane 1). This band
represents the mature XlnC protein of 25?7 kDa because
purified recombinant XlnC migrates at the same distance
(lane 8). In the extracellular fraction of the DtatB mutant
containing the XlnC expression vector, only a faint band was
detected, while no band was visible in the DtatC mutant
28
(a)
kDa
25
20
15
(b)
Extracellular xylanase activity
_
[units (mg dry wt) 1]
10
8
6
4
2
0
wt
dB
dC
wt
dB
dC
(c)
Xylanase secretion efficiency (%)
(III.C) form a third important group. Although an
important fraction of the proteins in this list have only
putatively assigned functions, it is clear that only a minority
are cofactor-binding proteins. Examples are a putative
cytochrome c oxidase subunit II (I.B.1), a putative copper
oxidase (III.B) and the tyrosinase cofactor MelC1 (III.C).
This contrasts to the situation in E. coli where the presence
of a twin-arginine motif in the signal peptide is strongly
correlated to the binding of a redox cofactor (Berks, 1996),
and where it is believed that the transportation of cofactorcontaining folded proteins is a fundamental feature of the
Tat apparatus (Berks et al., 2000). The same overall features
apply to the list of putative Tat-dependent substrates of
Streptomyces avermitilis predicted by the modified TATFIND
program. In the case of Halobacterium, it has been suggested that the extensive use of the Tat pathway could
be an evolutionary adaptation to high-salt conditions by
allowing cytoplasmic folding of secreted proteins before
their secretion (Bolhuis, 2002; Rose et al., 2002). The reason
why the secretion of many precursor proteins in Streptomyces would occur via the Tat pathway is not yet clear.
However, it can be postulated that the Tat pathway has a
distinct role in Streptomyces compared to E. coli or B. subtilis.
100
80
60
40
20
0
Fig. 2. Secretion of xylanase C is blocked in DtatB and DtatC
single mutants. (a) Western blot analysis with anti-XlnC antibodies to detect XlnC in extracellular fractions of 24 h cultures.
Lanes: 1, S. lividans TK24(pIJvsixyl); 2 and 3, S. lividans
DtatB(pIJvsixyl); 4 and 5, S. lividans DtatC(pIJvsixyl); 6, S. lividans DtatB(pIJvsixyl)(pSETtatB); 7, molecular mass marker;
8, 0?5 ng purified recombinant XlnC. (b) Xylanase activities in
extracellular fractions of 24 h cultures measured using the dinitrosalicylic acid assay (Miller, 1959). Given values are the mean
of values obtained from three different transformants. wt, S.
lividans TK24(pIJvsixyl); dB, S. lividans DtatB(pIJvsixyl); dC,
S. lividans DtatC(pIJvsixyl). (c) Secretion efficiencies of xylanase
(extracellular activity as a percentage of total activity) in 24 h
cultures. wt, S. lividans TK24(pIJvsixyl); dB, S. lividans
DtatB(pIJvsixyl); dC, S. lividans DtatC(pIJvsixyl).
containing the same vector. These results indicated that
secretion of XlnC was partially blocked in DtatB and
completely blocked in DtatC. After complementation of
the deleted tatB with a chromosomally integrated tatB, a
strong immunoreactive band reappeared (lane 6), indicating that the secretion defect in this mutant was a specific
consequence of the tatB deletion. Next, the xylanase activity
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Microbiology 150
Streptomyces Tat-dependent protein secretion
in the different strains was measured using the dinitrosalicylic acid assay (Miller, 1959). Activities measured in
24 h culture filtrates are shown in Fig. 2(b). Values of about
8 units xylanase activity (mg dry wt)21 could be measured
for the wild-type strain, while hardly any activity could be
measured in the DtatB strain and no activity in the DtatC
strain. Because the Dtat mutants showed hampered growth,
activities were also measured in 30, 36 and 48 h cultures.
Even at these later stages of growth, extracellular XlnC
activity did not increase in DtatB and DtatC (data not
shown). Upon measurement of intracellular activities,
secretion efficiencies could be calculated. While 98 %
XlnC was secreted in the wild-type strain, this amount
was reduced to about 20 % in DtatB and 0 % in DtatC
(Fig. 2c) mutants. The remaining secretion efficiency in the
DtatB mutant is due to the relatively low intracellular
accumulation of XlnC. Others have also observed that
depletion of tat genes results in inactivation or degradation
of Tat substrate proteins in the cytoplasm (Angelini et al.,
2001; Santini et al., 2001; DeLisa et al., 2003). In the
complemented DtatB mutant, XlnC secretion was completely restored to 8 units (mg dry wt)21. Taken together, these
results are evidence that secretion of S. lividans XlnC is
dependent on intact TatB and TatC proteins and, therefore, occurs via the Tat pathway. Because of the very high
homology between S. lividans XlnC and S. coelicolor XlnC
(98 % identity), it can be postulated also that the secretion
of S. coelicolor XlnC is Tat-dependent.
Tat-independent secretion of subtilisin inhibitor
As already demonstrated, the translocation of the highly
secreted S. lividans trypsin/subtilisin inhibitor (Sti1;
Strickler et al., 1992) is not affected in the DtatC mutant
(Schaerlaekens et al., 2001). To investigate the effect of the
DtatB mutation on Sti1 secretion, extracellular Sti1 activity
was measured according to Kojima et al. (1990) in 24 h
cultures. While wild-type S. lividans and the DtatC mutant
gave equal values of about 19 units (mg dry wt)21, the value
in the DtatB mutant reached 29 units (mg dry wt)21.
Secretion efficiencies of the subtilisin inhibitor could be
calculated after measurement of intracellular activity. In
S. lividans TK24, about 97 % of this protein was secreted.
In the DtatB and DtatC mutants the secretion efficiency
reached values of 98?5 %. Although the reason for the
observed positive effect of DtatB on Sti1 secretion is not
clear yet, these results show that the secretion of subtilisin
inhibitor is not dependent on either intact TatB or TatC
proteins and, therefore, occurs in a Tat-independent way.
(III.A). The absence of spore formation on MRYE could be
the consequence of the Tat dependency of two polyketide
b-ketoacyl synthases encoded by the whiE locus (III.C).
Also the impaired growth rate might be explained by
the absence of a number of solute-binding and sugartransporting proteins in the culture medium (III.A). On
the other hand, although two amidases (III.B and IV.B) are
predicted substrates of the Tat pathway, the DtatB and
DtatC mutants did not show hypersensitivity to SDS (data
not shown). This contrasts with the situation in E. coli,
where the Tat dependency of two amidases has been shown
to cause the highly defective cell envelope phenotype of the
tat mutant strains (Ize et al., 2003).
Future experiments will need to focus on testing the Tatdependent secretion of the proteins putatively assigned to
be Tat-dependent by the modified TATFIND program, on
construction of a DtatA mutant and on analysing possible
differential secretion in the different tat mutants. This
will lead to a better understanding of the importance and
mechanism of the Streptomyces Tat pathway.
ACKNOWLEDGEMENTS
We thank Mechthild Pohlschröder for kindly providing the TATFIND
program. Also thanks to Gert Sclep for aid with computer analysis.
Kristien Schaerlaekens is a research fellow of IWT (Vlaams Instituut
voor de Bevordering van het Wetenschappelijk-Technologisch
Onderzoek in de Industrie). N. G. and E. L. are postdoctoral fellows
of Katholieke Universiteit Leuven (PDM/02/200, PDM/00/168 and
PDM/01/163). This study was further supported by grants G40271.98
and 1. 5. 107. 01 from Fonds voor Wetenschappelijk Onderzoek –
Vlaanderen (FWO), OT/00/37 from Onderzoeksfonds Katholieke
Universiteit Leuven and QLK3-2000-00122 from the European
Commission.
REFERENCES
Angelini, S., Moreno, R., Gouffi, K., Santini, C., Yamagishi, A.,
Berenguer, J. & Wu, L. (2001). Export of Thermus thermophilus
alkaline phosphatase via the twin-arginine translocation pathway in
Escherichia coli. FEBS Lett 506, 103–107.
Anné, J., Van Mellaert, L. & Eyssen, H. (1990). Optimum conditions
for efficient transformation of Streptomyces venezuelae protoplasts.
Appl Microbiol Biotechnol 32, 431–435.
Bentley, S. D., Chater, K. F., Cerdeno-Tarraga, A. M. & 40 other
authors (2002). Complete genome sequence of the model actino-
mycete Streptomyces coelicolor A3(2). Nature 417, 141–147.
Berks, B. C. (1996). A common export pathway for proteins binding
complex redox cofactors? Mol Microbiol 22, 393–404.
Relationship between aberrant phenotype and
predicted Tat substrates
Berks, B. C., Sargent, F. & Palmer, T. (2000). The Tat protein export
Because a list of putative Tat-dependent substrates is now
available, hypotheses to explain the aberrant phenotype
of the DtatB and DtatC single mutants can be made. The
absence of undecylprodigiosin production in the tat
mutants, for example, might be explained by the Tat
dependency of six proteins involved in ABC transport
Bierman, M., Logan, R., O’Brien, K., Seno, E. T., Rao, R. N. &
Schoner, B. E. (1992). Plasmid cloning vectors for the conjugal
http://mic.sgmjournals.org
pathway. Mol Microbiol 35, 260–274.
transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116,
43–49.
Binnie, C., Cossar, J. D. & Stewart, D. I. (1997). Heterologous
biopharmaceutical protein expression in Streptomyces. Trends
Biotechnol 15, 315–320.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 19:57:04
29
K. Schaerlaekens and others
Blaudeck, N., Kreutzenbeck, P., Freudl, R. & Sprenger, G. A. (2003).
Kojima, S., Obata, S., Kumagai, I. & Miura, K. (1990). Alteration
Genetic analysis of pathway specificity during posttranslational
protein translocation across the Escherichia coli plasma membrane.
J Bacteriol 185, 2811–2819.
of the specificity of the Streptomyces subtilisin inhibitor by gene
engineering. Biotechnology 8, 449–452.
Korn, F., Weingärtner, B. & Kutzner, H. J. (1978). A study of
Halobacterium sp. NRC-1: a major role for the twin-arginine
translocation pathway? Microbiology 148, 3335–3346.
twenty actinophages: morphology, serological relationship and
host range. In Genetics of the Actinomycetales, pp. 251–270. Edited
by E. Freechsen, I. Tarnak & J. H. Thumin. Stuttgart: Fisher.
Brink, S., Bogsch, E. G., Edwards, W. R., Hynds, P. J. & Robinson, C.
(1998). Targeting of thylakoid proteins by the delta pH-driven
Kyte, J. & Doolittle, R. F. (1982). A simple method for displaying
the hydropathic character of a protein. J Mol Biol 157, 105–132.
twin-arginine translocation pathway requires a specific signal in the
hydrophobic domain in conjunction with the twin-arginine motif.
FEBS Lett 434, 425–430.
Laemmli, U. K. (1970). Cleavage of structural proteins during the
Bolhuis, A. (2002). Protein transport in the halophilic archaeon
Cristóbal, S., de Gier, J.-W., Nielsen, H. & von Heijne, G. (1999).
Competition between Sec- and TAT-dependent protein translocation
in Escherichia coli. EMBO J 18, 2982–2990.
assembly of the head of bacteriophage T4. Nature 227, 680–685.
Lammertyn, E., Van Mellaert, L., Schacht, S., Dillen, C., Sablon, E.,
Van Broekhoven, A. & Anné, J. (1997). Evaluation of a novel
DeLisa, M. P., Tullman, D. & Georgiou, G. (2003). Folding quality
subtilisin inhibitor gene and mutant derivatives for the expression
and secretion of mouse tumor necrosis factor alpha by Streptomyces
lividans. Appl Environ Microbiol 63, 1808–1813.
control in the export of proteins by the bacterial twin-arginine
translocation pathway. Proc Natl Acad Sci U S A 100, 6115–6120.
Lammertyn, E., Desmyter, S., Schacht, S., Van Mellaert, L. &
Anné, J. (1998). Influence of charge variation in the Streptomyces
Dilks, K., Rose, R. W., Hartmann, E. & Pohlschröder, M. (2003).
Prokaryotic utilization of the twin-arginine translocation pathway: a
genomic survey. J Bacteriol 185, 1478–1483.
venezuelae alpha-amylase signal peptide on heterologous protein
production by Streptomyces lividans. Appl Microbiol Biotechnol
49, 424–430.
Ding, Z. & Christie, P. J. (2003). Agrobacterium tumefaciens
Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for deter-
twin-arginine-dependent translocation is important for virulence,
flagellation, and chemotaxis but not type IV secretion. J Bacteriol
185, 760–771.
Muth, G., Nussbaumer, B., Wohlleben, W. & Pühler, A. (1989). A
mination of reducing sugars. Anal Chem 31, 426–428.
Geukens, N., Lammertyn, E., Van Mellaert, L. & 7 other authors
(2001). Membrane topology of the Streptomyces lividans type I signal
vector system with temperature-sensitive replication for gene
disruption and mutational cloning in streptomycetes. Mol Gen
Genet 219, 341–348.
peptidases. J Bacteriol 183, 4752–4760.
Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997).
Gross, R., Simon, J. & Kröger, A. (1999). The role of the twin-
Identification of prokaryotic and eukaryotic signal peptides and
prediction of their cleavage sites. Protein Eng 10, 1–6.
arginine motif in the signal peptide encoded by the hydA gene of
the hydrogenase from Wolinella succinogenes. Arch Microbiol 172,
227–232.
Halbig, D., Hou, B., Freudl, R., Sprenger, G. A. & Klösgen, R. B.
(1999a). Bacterial proteins carrying twin-R signal peptides are
specifically targeted by the delta pH-dependent transport machinery
of the thylakoid membrane system. FEBS Lett 447, 95–98.
Halbig, D., Wiegert, T., Blaudeck, N., Freudl, R. & Sprenger, G. A.
(1999b). The efficient export of NADP-containing glucose-fructose
oxidoreductase to the periplasm of Zymomonas mobilis depends both
on an intact twin-arginine motif in the signal peptide and on the
generation of a structural export signal induced by cofactor binding.
Eur J Biochem 263, 543–551.
Ikeda, H., Ishikawa, J., Hanamoto, A., Shinose, M., Kikuchi, H.,
Shiba, T., Sakaki, Y., Hattori, M. & Omura, S. (2003). Complete
genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nat Biotechnol 21, 526–531.
Ize, B., Stanley, N. R., Buchanan, G. & Palmer, T. (2003). Role of
the Escherichia coli Tat pathway in outer membrane integrity. Mol
Microbiol 48, 1183–1193.
Jongbloed, J. D. H., Martin, U., Antelmann, H., Hecker, M.,
Tjalsma, H., Venema, G., Bron, S., van Dijl, J. M. & Müller, J.
(2000). TatC is a specificity determinant for protein secretion via the
Ochsner, U. A., Snyder, A., Vasil, A. I. & Vasil, M. L. (2002). Effects of
the twin-arginine translocase on secretion of virulence factors, stress
response, and pathogenesis. Proc Natl Acad Sci U S A 99, 8312–8317.
Rose, R. W., Brüser, T., Kissinger, J. C. & Pohlschröder, M. (2002).
Adaptation of protein secretion to extremely high-salt conditions
by extensive use of the twin-arginine translocation pathway. Mol
Microbiol 45, 943–950.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning:
a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory.
Santini, C. L., Ize, B., Chanal, A., Muller, M., Giordano, G. & Wu, L. F.
(1998). A novel sec-independent periplasmic protein translocation
pathway in Escherichia coli. EMBO J 17, 101–112.
Santini, C. L., Bernadac, A., Zhang, M., Chanal, A., Ize, B., Blanco, C.
& Wu, L. F. (2001). Translocation of jellyfish green fluorescent
protein via the Tat system of Escherichia coli and change of its
periplasmic localization in response to osmotic up-shock. J Biol
Chem 276, 8159–8164.
Sargent, F., Berks, B. C. & Palmer, T. (2002). Assembly
of membrane-bound respiratory complexes by the Tat proteintransport system. Arch Microbiol 178, 77–84.
twin-arginine translocation pathway. J Biol Chem 275, 41350–41357.
Schaerlaekens, K., Schierova, M., Lammertyn, E., Geukens, N.,
Anné, J. & Van Mellaert, L. (2001). Twin-arginine translocation
Jongbloed, J. D. H., Antelmann, H., Hecker, M. & 7 other authors
(2002). Selective contribution of the twin-arginine translocation
Stanley, N. R., Palmer, T. & Berks, B. C. (2000). The twin-arginine
pathway to protein secretion in Bacillus subtilis. J Biol Chem 277,
44068–44078.
pathway in Streptomyces lividans. J Bacteriol 183, 6727–6732.
Kawamoto, S. & Ochi, K. (1998). Comparative ribosomal protein
consensus motif of the Tat signal peptides is involved in Secindependent protein targeting in Escherichia coli. J Biol Chem 275,
11591–11596.
(L11 and L30) sequence analyses of several Streptomyces spp.
commonly used in genetic studies. Int J Syst Bacteriol 48, 597–600.
Strickler, J. E., Berka, T. R., Gorniak, J., Fornwald, J., Keys, R.,
Rowland, J. J., Rosenberg, M. & Taylor, D. P. (1992). Two novel
Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F. & Hopwood, D. A.
(2000). Practical Streptomyces Genetics. Norwich: John Innes Foundation.
Streptomyces protein protease inhibitors. Purification, activity,
cloning, and expression. J Biol Chem 267, 3236–3241.
30
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 19:57:04
Microbiology 150
Streptomyces Tat-dependent protein secretion
Tjalsma, H., Bolhuis, A., Jongbloed, J. D. H., Bron, S. & van Dijl,
J. M. (2000). Signal peptide-dependent protein transport in Bacillus
Von Heijne, G. (1984). How signal sequences maintain cleavage
subtilis: a genome-based survey of the secretome. Microbiol Mol Biol
Rev 64, 515–547.
Voulhoux, R., Ball, G., Ize, B., Vasil, M. L., Lazdunski, A., Wu, L. F. &
Filloux, A. (2001). Involvement of the twin-arginine translocation system
specificity. J Mol Biol 173, 243–251.
van Dijl, J. M., Braun, P. G., Robinson, C. & 7 other authors (2002).
in protein secretion via the type II pathway. EMBO J 20, 6735–6741.
Functional genomic analysis of the Bacillus subtilis Tat pathway for
protein secretion. J Biotechnol 98, 243–254.
Ward, J. M., Janssen, G. R., Kieser, T., Bibb, M. J. & Buttner, M. J.
(1986). Construction and characterisation of a series of multi-copy
Van Mellaert, L., Dillen, C., Proost, P. & 7 other authors (1994).
promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator. Mol Gen
Genet 203, 468–478.
Efficient secretion of biologically active mouse tumor necrosis factor
alpha by Streptomyces lividans. Gene 150, 153–158.
http://mic.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 19:57:04
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