Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, SE-750 07 Uppsala, Sweden
Correspondence
Hans Jonsson
Hans.Jonsson{at}mikrob.slu.se
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ABSTRACT |
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INTRODUCTION |
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Extracellular and transmembrane proteins are synthesized in the cytoplasm and exported to their site of action, either to the plasma membrane or to the outside of the cell. The protein precursor of such proteins usually contains an amino-terminal signal peptide, which is identified by the cellular sorting and translocation machinery. Examples of protein sorting pathways in bacteria are the general secretory (Sec) pathway, the twin-arginine translocation (Tat) pathway and the ATP-binding cassette (ABC) transporter system (reviewed by Tjalsma et al., 2000). There is considerable variation in length and amino acid sequence of signal peptides (Martoglio & Dobberstein, 1998
), but a classical signal sequence consists of three different domains. These are the amino-terminal (N-) region with positively charged amino acids; the central hydrophobic (H-) region; and the carboxy-terminal (C-) region containing the signal-peptide cleavage-site. After translocation, signal peptidases separate the signal peptide from the protein (van Wely et al., 2001
), or alternatively the signal peptide is inserted into the plasma membrane as a signal anchor (Martoglio & Dobberstein, 1998
).
In a recently developed method, Rosander et al. (2002) utilized the function of signal peptides for identification of genes encoding extracellular proteins in bacterial genomes. This method is based on phage display technology, in which foreign peptides or proteins are expressed in fusion with a coat protein on the surface of a filamentous phage. Usually, the fusion protein is expressed with a phagemid vector, i.e. a plasmid containing a filamentous phage intergenic region, and a helper phage (Bass et al., 1990
). The vector encodes the signal peptide required for direction of the fusion protein to the cell membrane, where phage assembly occurs. The vector pG3DSS constructed by Rosander et al. (2002)
does not contain a signal sequence. Thus, a signal peptide encoded by the foreign DNA inserted into pG3DSS is required for the export of fusion proteins to the cell surface. Consequently, only proteins containing a signal peptide, i.e. extracellular proteins, are incorporated into the phage coat and displayed as fusion proteins. In addition, the phagemid vector pG3DSS encodes an E-tag epitope, a short peptide recognized by monoclonal antibodies. Therefore, anti-E-tag antibodies can be utilized to isolate phages displaying fusion proteins by affinity selection.
Studies of the commensal bacterium Lactobacillus reuteri, which commonly occurs in the human gastrointestinal tract, imply that it has positive health effects (reviewed by Casas & Dobrogosz, 2000). However, only a few of the extracellular proteins of this bacterium have been described. These include: a collagen-binding protein, CnBP (Roos et al., 1996
); an autoaggregation-promoting protein, AggH (Roos et al., 1999
); a mucus-binding protein, Mub (Roos & Jonsson, 2002
); and a fructosyltransferase (van Hijum et al., 2002
). The aim of this study was to search for additional genes encoding extracellular proteins from L. reuteri. Therefore, a phage display library was constructed by inserting randomly fragmented L. reuteri DNA into the vector pG3DSS. Screening of the library identified 52 novel genes encoding putative extracellular and transmembrane proteins. Future studies and characterization of these genes may reveal mechanisms important for the probiotic properties of L. reuteri.
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METHODS |
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Isolation of chromosomal DNA from L. reuteri DSM 20016T.
For construction of the library, chromosomal DNA was isolated from L. reuteri. Ten millilitres of an overnight culture of the bacterium was added to 300 ml MRS broth and grown to an OD600 of approximately 1·5. Cells were harvested by centrifugation at 4000 g for 15 min, washed in 50 ml 10 mM Tris/HCl (pH 8·0)/0·3 M sucrose (T-sucrose) and resuspended in 50 ml T-sucrose supplied with 20 mg lysozyme ml-1 and 160 U mutanolysin ml-1 (Sigma). The suspension was incubated for 2 h at 37 °C with slow shaking; the cells were then pelleted by centrifugation and resuspended in 9 ml 50 mM Tris/HCl (pH 8·0)/5 mM EDTA (5x TE). After addition of 1 ml 10 % (w/v) SDS, the suspension was carefully mixed and incubated at 65 °C for 15 min. Next, RNase was added to a final concentration of 100 µg ml-1 and the incubation was continued for 30 min at 37 °C. Proteinase K was added to a final concentration of 200 µg ml-1 and the suspension was incubated at 55 °C for 30 min. Finally, the DNA was precipitated by addition of 1 ml 3 M sodium acetate (pH 5·2) and 20 ml 95 % ethanol, washed in 70 % ethanol, and dissolved in 5 ml 1x TE. After repeated phenol and chloroform extractions, the DNA was precipitated again, washed in 70 % ethanol and dissolved in an appropriate volume of 1x TE.
For DNA sequencing with chromosomal DNA as template, L. reuteri DNA was isolated with Qiagen Genomic-tips 500/G according to the manufacturer's instructions, with one modification. For lysis of the bacteria, in addition to lysozyme (final concentration 10 mg ml-1), mutanolysin was added to the cell suspension to a final concentration of 80 U ml-1. After incubation for 30 min at 37 °C with gentle shaking, Proteinase K (final concentration 0·9 mg ml-1) was added and the incubation was continued for 30 min.
Determination of the size of the genome of L. reuteri DSM20016T.
Chromosomal DNA was cleaved with NotI or SgrAI and the fragments were separated by PFGE with a Gene Navigator (Amersham Pharmacia Biosciences) as described by Lindmark et al. (1999).
Construction of the library.
The library was constructed as described by Jacobsson et al. (2003) with minor modifications. Chromosomal DNA (400 µl, 150 µg ml-1) was sonicated for 10 s at maximum power with a microprobe (Soniprep 150, MSE). The fragments obtained varied in size between 0·5 and 2 kb, with the majority between 0·7 and 1·3 kb. Blunt ends were achieved by treatment with T4 DNA polymerase. The phagemid vector pG3DSS (Rosander et al., 2002
) was digested with the restriction enzyme SnaBI and dephosporylated with calf intestine alkaline phosphatase. The DNA manipulations were performed according to standard methods (Sambrook et al., 1989
).
Five tubes of Ready-To-Go T4 DNA Ligase (Amersham Pharmacia Biotech) were utilized to ligate approximately 1 µg (0·2 µg per tube) of L. reuteri fragments with approximately 3 µg (0·6 µg per tube) of the vector pG3DSS. After phenol and chloroform extraction, the ligated DNA was ethanol-precipitated, washed with 70 % ethanol and dissolved in 10 µl H2O. The ligation mix was transformed into E. coli TG1 by electroporation (2·5 kV, 25 µF, 400 ) in 2-mm-gap cuvettes. The transformed cells were transferred to 100 ml LB and incubated at 37 °C for 1 h with gentle shaking. Then, a 2 ml aliquot was taken from the culture to determine the number of transformants by plating on LB/Amp agar. After addition of ampicillin to a final concentration of 50 µg ml-1, the remaining cell culture was grown overnight at 37 °C with vigorous shaking.
An 8 ml aliquot of the overnight culture was infected with helper phage R408 (m.o.i. 5), mixed with 50 ml soft agar (LB broth with 0·5 %, w/v, agarose) and poured onto 10 LB/Amp agar plates. The plates were incubated overnight at 37 °C, then the phages were extracted from the soft agar. Finally, the titre of the phage library was determined by infection of E. coli TG1.
Panning procedures.
In order to isolate phages displaying fusion proteins, the library was panned against anti-E-tag antibodies (Amersham Biosciences). Nunc-immuno modules (MaxiSorp) were coated with 200 µl anti-E-tag antibodies (final concentration 25 µg ml-1), or as a control with 0·1 % (w/v) BSA, in 50 mM sodium carbonate (pH 9·7) and the wells were blocked with 400 µl phosphate-buffered saline (Sambrook et al., 1989)/0·05 % Tween 20 (v/v) (PBS-T). After addition of 200 µl of the phage library to the wells, the phagemid particles were allowed to bind to the anti-E-tag antibodies during 4 h incubation with slow agitation at room temperature (22 °C). The wells were rinsed thoroughly with PBS-T (30 times), bound phages were eluted by addition of 200 µl 50 mM sodium citrate/140 mM NaCl (pH 2·1) and the eluate was neutralized with 30 µl 2 M Tris/HCl (pH 8·0). An overnight culture of E. coli TG1 was infected with the eluate and the infected cells were plated on LB/Amp agar. After resuspension in LB, the clones obtained were infected with helper phage R408 (as described under Construction of the library) to produce a phage stock for repanning. To enrich for positive clones (i.e. those displaying fusion proteins), this stock was utilized for a second panning, which was performed as described above. The procedure of panning and repanning was repeated in two separate experiments.
Nucleotide sequencing of L. reuteri inserts and of L. reuteri chromosomal DNA.
After panning, 120 clones from the first panning cycles and 143 clones from the repannings were randomly selected for nucleotide sequencing of the L. reuteri DNA inserts. The phagemid vectors from these clones were isolated with the QIAprep Spin Miniprep Kit (Qiagen). The DNA fragments inserted in the vector were sequenced with the primer G3DelRev (5'-cattgacaggaggttgaggc-3') or the primer pG3DR2 (5'-gcggttccagtgggtcc-3'), with the DYEnamicET terminator cycle sequencing premix kit (Amersham Biosciences) and an ABI PRISM 377 DNA Sequencer (PerkinElmer Instruments).
Since the L. reuteri fragments encoded the 5' end of genes and not complete genes, additional sequence information was required for the majority of the genes. Therefore, primers were designed for sequencing the DNA downstream of the inserts using L. reuteri chromosomal DNA as template. The sequencing of chromosomal DNA was performed as described by Heiner et al. (1998). Specifically, in each reaction, 16 µl BigDye Terminator mix v3.0 or v3.1 (Applied Biosystems), 32 pmol primer and 2·53·1 µg L. reuteri DNA were added to a total volume of 40 µl. The sequencing reaction was performed with an initial denaturation step at 95 °C for 5 min, followed by 90 cycles at 95 °C for 30 s, 55 °C for 20 s and 60 °C for 4 min.
Sequence analysis.
The sequences obtained were analysed with Vector NTI software (InforMax). For prediction of signal sequences, the SignalP V2.0 program for Gram-positive bacteria (Nielsen et al., 1997) (http://www.cbs.dtu.dk) was utilized. The signal sequences were verified manually to confirm the presence of the N-, C- and H-regions (van Wely et al., 2001
). In addition, translation initiation sequences (reviewed by Kozak, 1999
) were searched for manually. Similarity studies and searches for conserved domains were performed with BLASTP (Altschul et al., 1990
) at the National Centre for Biotechnology Information (NCBI) web page (http://www.ncbi.nlm.nih.gov), using an e-value lower than e-4 as a cut-off for notable similarity. In addition, motifs were searched for with InterProScan (http://www.ebi.ac.uk/interpro/scan.html) (Mulder et al., 2003
), transmembrane domains with TMHMM 2.0 (Krogh et al., 2001
) (http://www.cbs.dtu.dk/services/TMHMM-2.0/) and the G+C content of the genes was determined with the program Geecee from EMBOSS (Rice et al., 2000
) at the Pasteur Institute website (http://bioweb.pasteur.fr/seqanal/interfaces/geecee.html).
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RESULTS AND DISCUSSION |
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Portions of the library were utilized for affinity panning against anti-E-tag antibodies. The clones obtained in the first panning were used to produce a phage stock for a second panning to increase the number of positive clones. The number of phagemid particles in the eluate after the panning and repanning is presented in Table 1. In the first experiment, there was a 2500-fold increase in eluted phages after the repanning compared to after the first panning. In the second experiment, the number of phages in the eluate had increased 800-fold between the first panning and the repanning. As a control, portions of the library and the phage stocks for repanning were panned against BSA. After the first pannings, the differences in enriched phages between the controls and the affinity selections against anti-E-tag antibodies were small. In contrast, the repannings against anti-E-tag antibodies contained markedly higher numbers of phagemid particles than the BSA controls. This indicated that positive clones were enriched in the eluate after panning against the anti-E-tag antibodies. It is a common feature of the phage display method that the first panning results in small differences from the control because of an inherent background. However, a clear enrichment is usually established after repanning (Bjerketorp et al., 2002
; Jacobsson & Frykberg, 1996
).
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The majority of the 53 genes encoding extracellular and transmembrane proteins expressed a classical N-terminal signal peptide recognized by the Sec pathway. In addition, some genes appeared to encode lipoproteins, a subclass of secreted proteins. The precursors of lipoproteins contain signal peptides with a conserved motif called lipobox and the peptides are cleaved by SPase II in front of an invariable cysteine residue (reviewed by Tjalsma et al., 2000). For Gram-positive bacteria the motif varies and the consensus is continually refined. Based on the analyses of lipoboxes from Gram-positive bacteria (Sutcliffe & Russell, 1995
) and from Bacillus subtilis (Tjalsma et al., 2000
) in particular, the consensus sequence has been extracted respectively as (LVAI)-(SALIMTGFV)-(AGLS)-C-(SGNATYWQP) with the C at positions 1729 and (LVFTIMG)-(ASTIVGMLCPFL)-(AGLISVTFP)-C-(GSAITFWKL) with the C at positions 1432. By comparing the L. reuteri proteins with these sequences and with the proteins to which they have high similarities, six putative lipoproteins were found in this study. These are presented in Table 3
.
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Transmembrane and extracellular proteins.
The proteins with an N-terminal signal peptide were screened for transmembrane domains with TMHMM 2.0 (Krogh et al., 2001). Proteins with more than one membrane-spanning domain were classified as transmembrane proteins (17 proteins), while proteins predicted to have one or no transmembrane domain were classified as extracellular proteins (36 proteins) (Table 2
). In the case of extracellular proteins, the transmembrane parts probably consisted of the signal peptides. Since the hydrophobicity and length of signal peptides vary, the TMHMM program recognizes them either as transmembrane or as extracellular parts of proteins.
Initiation of translation.
To identify ShineDalgarno sequences (reviewed by Kozak, 1999), the translation starts of the genes encoding the extracellular and transmembrane proteins and their upstream regions were analysed manually (data not shown). The manual screening revealed eight genes with alternative start codons: three genes started with GTG (lre0020, lre0024 and lre0037) and five with TTG (lre0004, lre0023, lre0025, lre0048 and lre0050). The utilization of alternative start codons is widespread in prokaryotic organisms (Kozak, 1999
). Further, six genes (lre0042lre0047) encoding transmembrane proteins were probably incomplete at the 5' end. The reading frames of the inserts encoding these genes were open, without stop codons. Probably, these fragments still contained sequences able to initiate translation and the membrane-spanning domains were functioning as signal peptides. Consequently, these inserts were able to function in the phage display system and direct fusion proteins to the phage surface. With the exception of lre0008, ShineDalgarno sequences were identified upstream of the 5' end of the remaining genes.
In several genes two or three different putative initiation codons with adjacent ShineDalgarno sequences were identified. Genes containing dual translational initiation signals and thus generating signal sequences with various lengths have been described previously. Taguchi et al. (1991) suggested that dual starts could affect gene expression and Graschopf & Blasi (1999)
reported that the different starts result in mature proteins of different functions. In L. reuteri, the gene encoding the mucus-binding protein Mub has two possible start motifs generating signal peptides of different lengths (Roos & Jonsson, 2002
). In the present study, the two genes lre0005 and lre0026 have a dual-start motif where the start codons are located close to each other and the two possible signal peptides would be very similar in length. The genes lre0033, lre0037 and lre0040, on the other hand, have possible translation initiation signals that are clearly separated and the encoded signal peptides might thus differ significantly in length. Although not thoroughly studied, the presence of genes with more than one potential translation initiation motif, generating different signal peptides, indicates that signal peptides have a more complicated function than just directing the proteins to the cell membrane.
G+C content.
The G+C content of the genes was determined with the Geecee program from EMBOSS (Rice et al., 2000) (Table 2
). For the majority of the partial and complete genes the G+C content was between 38 and 42 mol%. This was expected, since the G+C content of L. reuteri is 41 mol% (Kandler et al., 1980
). However, two genes showed a notable difference: the G+C contents of lre0048 and lre0052 were 31 mol%. The G+C contents of the corresponding genes in Streptococcus thermophilus and Streptococcus agalactiae are 30 and 35 mol%, respectively. This indicates that lre0048 may originate from another bacterium and has been obtained by L. reuteri by horizontal gene transfer.
Relative efficiency of the method.
Since the genome sequence of L. reuteri is not available, the number of proteins with N-terminal signal sequences encoded by this organism is unknown. By comparison with the relationship between genome size and number of extracellular proteins of other Gram-positive bacteria (Bolotin et al., 2001; Kleerebezem et al., 2003
; Saleh et al., 2001
), L. reuteri could be estimated to encode approximately 100200 extracellular proteins. After sequencing 263 clones, 36 putative extracellular proteins of L. reuteri were identified in this study. In the first round of sequencing the majority of the positive clones represented a new gene, while at the end of the experiment most positive clones were copies of already found genes. Although additional genes clearly would have been detected by further screening, the new information obtained per sequencing round was not considered sufficient to continue the experiments.
The capacity of this method to describe the complete secretome of an organism is not fully evaluated. The number of sequenced genes from Staphylococcus aureus in the study by Rosander et al. (2002) is limited and therefore gives no indications of the potential of the method. However, the authors suggest that the phage display screening method is more efficient for Gram-negative than for Gram-positive bacteria. In addition, the efficiency may depend on the promoters and signal sequences of the organism studied as well as the size of the phage library. This method has also been utilized to screen for extracytoplasmic proteins in the Gram-negative bacterium Bradyrhizobium japonicum (Rosander et al., 2003
). Analysis of DNA inserts from 182 clones showed that 167 clones, originating from 132 genes, encoded extracytoplasmic proteins. This supports the hypothesis that the phage display screening system is more effective for Gram-negative bacteria.
Prediction of the protein functions
The functions of the putative extracellular and transmembrane proteins were predicted by similarity studies with BLASTP (Altschul et al., 1990) at the NCBI web site. To be classified as similar to previously known proteins, the hits had to obtain an e-value lower than e-4. The stringency of this cut-off value was set low, bearing in mind that the sequence was not complete for the majority of the detected genes. When the searches revealed several hits with comparable, high similarities to the query protein, hits with an assigned function were preferred to proteins with an unknown function. The similarity studies resulted in the identification of six categories of proteins (Fig. 1
): transport proteins (6 extracellular and 11 transmembrane proteins); enzymes (11 extracellular and 4 transmembrane proteins); sensorregulator proteins (1 extracellular protein); proteins involved in host/microbial interactions (3 extracellular proteins); unconserved hypothetical proteins (10 extracellular and 1 transmembrane protein); and conserved hypothetical proteins (5 extracellular and 1 transmembrane protein). For the 18 hypothetical proteins, there were no matches in the databases that could lead to a prediction of their function. Even if the complete genomes of the relatively closely related L. plantarum (Kleerebezem et al., 2003
) and Lactobaccillus gasseri (http://genome.ornl.gov/microbial/lgas/) are known, 11 of the hypothetical proteins lacked notable similarities to any known proteins. These proteins are of interest since they could be unique for L. reuteri and involved in interactions between the bacterium and its environment. The remaining seven hypothetical proteins were similar to hypothetical proteins and proteins of unknown function.
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There was a clear difference between the distribution of extracellular and transmembrane proteins (Fig. 1). Although transmembrane proteins were expected to represent a larger part of the genome than extracellular proteins, approximately 68 % of the genes detected here encoded extracellular proteins. Also, in the previous studies utilizing this method (Rosander et al., 2002
, 2003
) the majority of the identified signal sequences were from extracellular proteins, while a smaller proportion originated from transmembrane proteins. Probably, the phage display screening method is better for selection of extracellular than transmembrane proteins.
Transport proteins.
Of the 53 potential proteins identified here, 17 were predicted to be transport proteins. The sequencing of complete genomes has revealed that a large fraction of the genes in bacterial genomes encodes proteins of this type. More than 13 % of the proteins in L. plantarum (Kleerebezem et al., 2003) and 10 % in Lactococcus lactis (Bolotin et al., 2001
) are involved in transport. The large proportion of transport proteins identified here implies that this is also valid for L. reuteri. Further, transmembrane proteins predominated among the transport proteins. This was expected, since transport proteins often have several membrane-spanning domains.
Enzymes.
The second-largest category of proteins with an assigned function was enzymes. This diverse class included, for example, putative autolysins, hydrolases and peptidases. It also contained a gene, lre0051, similar to a gene encoding a putative cobalamin biosynthesis protein in Listeria monocytogenes. Recent findings indicate that L. reuteri indeed may produce cobalamin (vitamin B12) or a cobalamin-like substance, especially in the presence of glycerol (Hugenholtz et al., 2002).
Sensorregulator proteins.
The gene lre0020 encoded a protein similar to a histidine protein kinase from L. plantarum. Probably, Lre0020 is involved in signal transduction as a sensor protein. Further analyses are required to investigate to what signals this signalling system is responding.
Host/microbial interaction proteins.
Three proteins possibly involved in specific interaction between L. reuteri and its host were identified: Lre0019 with similarities to the S. pneumoniae PspC; Lre0018 with similarities to the Lactobacillus johnsonii Apf1; and the previously described CnBP (Roos et al., 1996). The cell surface adhesin PspC is involved in colonization of mucosal surfaces in the nasopharynx (Balachandran et al., 2002
; Rosenow et al., 1997
). The protein interferes with the complement pathway by binding to the third component, C3 (Cheng et al., 2000
), and to factor H (Dave et al., 2001
). Balachandran et al. (2002)
demonstrated that intranasal immunization of mice with PspC reduced the level of pneumococcal carriage in the nasopharynx, suggesting that PspC is indeed important for colonization. Like the pathogen S. pneumoniae, L. reuteri has to colonize mucosal surfaces. Although the similarities between Lre0019 and PspC were most notable in the peptidoglycan-binding domain, Lre0019 could still have a role in colonization and interactions with the immune system.
The protein encoded by lre0018 was similar to Apf1 in Lactobacillus johnsonii. A homologous APF protein was originally identified in L. gasseri and suggested to be an aggregation-promoting factor (Reniero et al., 1992). However, Apf1 was recently described as an S-layer protein (Ventura et al., 2002
). The function of Lre0018 is therefore uncertain. So far, no S-layer proteins have been found in L. reuteri.
During this work one previously described extracellular protein was identified: the collagen-binding protein CnBP. In addition to being an adhesin, CnBP is probably part of an ABC transporter (Roos et al., 1996). This demonstrates that proteins may have more than one function or a function differing from what is predicted in similarity studies. The identification of a known, confirmed extracellular protein from L. reuteri also implied that the phage display screening method was functioning successfully in the search for extracellular proteins.
Conclusion
In this study, 52 novel L. reuteri genes encoding extracellular and transmembrane proteins were identified with a phage display screening method. This method has notable potential to screen bacterial genomes for genes encoding proteins with an N-terminal signal sequence and it enables screening of a large set of genes during a short period of time. Although these genes do not cover the complete secretome of L. reuteri, they do contribute to the knowledge of this organism. Several of the detected genes were predicted to have functions related to the probiotic properties of L. reuteri, for example genes involved in colonization. For approximately 30 % of the genes no function could be assigned. The proteins encoded by these genes could be mediators of host/bacterial interactions with an equally essential role as extracellular proteins have in pathogenic bacteria. The function of the identified genes will be further examined by mutation and expression analysis in order to reveal their possible probiotic significance.
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ACKNOWLEDGEMENTS |
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Received 30 May 2003;
revised 27 August 2003;
accepted 9 September 2003.