Department of Microbiology and Immunology, University of Maryland School of Medicine, 655 West Baltimore Street, BRB: 13-009, Baltimore, MD 21201, USA
Correspondence
M. Sayeedur Rahman
mrahm001{at}umaryland.edu
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ABSTRACT |
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The GenBank accession numbers of the secA genes reported in this work are AY312572 for Rickettsia rickettsii and AY386402 for Rickettsia typhi.
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INTRODUCTION |
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Protein secretion is a significant aspect of bacterial pathogenesis, and the molecular mechanism of protein secretion in rickettsiae remains an important subject of research. Research on various bacterial pathogens has demonstrated that the majority of virulence factors are either secreted into the extracellular environment or attached to the cell surface (Finlay & Falkow, 1997). The importance of protein export pathways has been demonstrated by the identification of specialized secretion pathways (types IV) in bacteria (Lee & Schneewind, 2001
; Pallen et al., 2003
). In bacteria, the majority of extracytoplasmic proteins transported by the specialized secretion pathways (except type III) are synthesized as preproteins with an amino-terminal extension known as the signal or leader peptide, and are translocated by the Sec-dependent protein export pathways (Desvaux et al., 2004
). During translocation, the signal or leader sequence is cleaved off by a signal peptidase (Economou, 1999
; Pugsley, 1993
). These precursor secretory preproteins are selectively targeted to the translocon by interactions with SecB or the signal recognition particle (which is composed of Ffh, 4·5S RNA and FtsY). These pathways converge at the translocon, which consists of the ATPase SecA, which acts as a molecular motor, and the integral membrane proteins (SecD, SecE, SecF, SecG, SecY, YidC and YajC) (Paetzel et al., 2000
, 2002
). SecA plays a central and essential role in Sec-dependent protein export. It is an ATPase that provides energy for the transmembrane movement of the preprotein. Through cycles of ATP binding and hydrolysis, SecA delivers bound preproteins to the translocase, and undergoes cycles of membrane insertion and retraction to mediate the stepwise translocation of the preprotein (de Keyzer et al., 2003
).
In order to elucidate the protein secretion pathways, and their involvement in rickettsial pathogenesis, we set out to characterize the genes involved in rickettsial protein secretion. As in our previous work examining the rickettsial lepB gene encoding signal peptidase I (Rahman et al., 2003), in this paper we report a detailed molecular and functional characterization of the putative secA genes from R. rickettsii and R. typhi.
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METHODS |
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Genomic DNA extraction.
African green monkey kidney cells (Vero cells, ATCC CRL-1586) were cultured in Dulbecco's modified Eagle's medium with 4·5 g glucose l1 and glutamine (Biofluids), supplemented with 10 % fetal bovine serum (Gemini). Propagation of R. rickettsii and R. typhi in Vero cells, and their partial purification from the host cells, were previously described (Rahman et al., 2003). Genomic DNA of R. rickettsii and R. typhi was extracted using the Wizard genomic DNA purification kit (Promega,).
Cloning of the rickettsial secA gene.
Primers were designed based on the published genome sequences of Rickettsia conorii (Ogata et al., 2001) and R. prowazekii (Andersson et al., 1998
) and used for cloning the secA genes from R. rickettsii and R. typhi, respectively. Primers used in PCR reactions are shown in Table 1
. The secA gene from R. rickettsii was amplified in two different fragments (5' end and 3' end) by PCR. The primers AZ1940 and AZ1676 were used to amplify the 1149 bp fragment of the 5' end of the secA gene of R. rickettsii (first fragment) using Herculase DNA polymerase (Stratagene). The PCR product (1149 bp) was cloned into the pPCR-Script Amp SK(+) vector (Stratagene), and was transformed into E. coli TOP10 cells (Invitrogen Life Technologies). The cloned 5' end of the secA region of R. rickettsii was sequenced by the dye termination method, using a model 373 automated fluorescent sequencing system (Applied Biosystems). The 3' end of the secA gene (second fragment of 1785 bp) was PCR amplified from R. rickettsii DNA using the primers AZ1668 and AZ1667 by following the same procedure as described for the 5' end of the secA gene (first fragment of 1149 bp). The R. rickettsii secA 5'- and 3'-end sequences were combined using MacVector 6.5.3 software (Genetic Computer Group).
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The sequences of the secA genes of R. rickettsii and R. typhi, and the deduced amino acid sequences, were analysed using MacVector 6.5.3 software. Sequences were compared with those available in GenBank by using BLAST analysis (http://www.ncbi.nlm.nih.gov).
Isolation of RNA and RT-PCR.
R. rickettsii and R. typhi were purified from Vero cells (>90 % infection), as described above. Total RNA from the partially purified rickettsiae was isolated by use of Trizol reagent (Invitrogen Life Technologies), and treated with RQ1 RNase-free DNase (Promega) by following the manufacturer's recommendations. RT-PCR was performed with 500 ng total RNA in a 50 µl reaction using SuperScript One-Step RT-PCR with Platinum Taq (Invitrogen Life Technologies). The thermal cycling conditions were one cycle of 45 °C for 30 min, 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 48 °C for 30 s, 72 °C for 3 min, and a final extension of 72 °C for 10 min.
Northern analysis.
Northern blotting of total RNA isolated from R. rickettsii was performed by using the NorthernMax kit (Ambion). Total RNA (8 µg) was separated on a 1 % agarose gel, and transferred to a positively charged nylon membrane (Ambion). The membrane was hybridized with a radiolabelled probe specific to the R. rickettsii secA-coding sequence corresponding to the region from the start codon to the primer AZ1649. The 476 nt probe was prepared by linear amplification with primer AZ1649 in the presence of [-32P]dATP (Amersham Pharmacia Biotech) using the Strip-EZ PCR probe synthesis kit (Ambion). The hybridized membrane was exposed (16 h) to Kodak Biomax MS film for autoradiography.
RACE (rapid amplification of cDNA ends).
The transcriptional start point (TSP) of R. rickettsii secA gene expression was determined by using the 5'RACE kit (Invitrogen Life Technologies) by following the manufacturer's instructions. First-strand cDNA was prepared from DNase-treated total RNA (5 µg) of R. rickettsii using Superscript II reverse transcriptase with the secA-gene-specific primer AZ1649. The cDNA was tailed by adding cytosine residues at the 3' end using terminal deoxynucleotidyl transferase. The dC-tailed cDNA was PCR amplified with a primer set consisting of a second secA-specific primer AZ1793 and the 5'RACE abridged anchor primer. The primary PCR product was further amplified by nested PCR with the primer AZ1792 and the 5'RACE abridged universal amplification primer. The nested PCR product was cloned and sequenced to determine the transcriptional start point.
Construction of full-length and chimeric secA genes.
PCR was used to construct plasmids carrying the full-length ORFs of the secA genes from R. rickettsii, R. typhi and E. coli MC4100. To introduce the required restriction sites, nucleotides were changed in the primer design. The primers AZ2116 (BglII) and AZ2115 (EcoRI) were used for cloning the full-length ORF (2729 bp fragment) of R. rickettsii secA at the BglII and EcoRI sites of the pTrcHisB vector, which contains an N-terminal His6 tag under the trc (trplac) promoter (Invitrogen Life Technologies), to generate the plasmid pTrcRR73. To clone the full-length ORF (2729 bp fragment) of R. typhi secA, a PCR-amplified product obtained by using the primers AZ2454 (BamHI) and AZ2456 (EcoRI) was ligated at the BamHI and EcoRI sites of the pTrcHisB vector to generate the plasmid pTrcRT3. The full-length ORF (2725 bp fragment) of secA from E. coli MC4100, amplified by primers AZ2259 (BamHI) and AZ2258 (XhoI), was cloned at the BamHI and XhoI sites into the pTrcHisA vector, which contains an N-terminal His6 tag under the trc (trplac) promoter (Invitrogen Life Technologies), to generate the plasmid pTrcEC85.
Based on strong sequence similarity in the N-terminal ATP binding domains of the SecA protein, the chimeric secA gene was constructed. This gene encoded the N-terminal 408 aa of R. rickettsii SecA, and the C-terminal 480 aa of E. coli MC4100 SecA. To construct the chimeric secA gene, the third nucleotide of the codon for Leu and Asp residues of R. rickettsii SecA protein (chimeric site, shown in Fig. 1) was changed in the primer design, to create a BglII site whilst retaining the Leu and Asp amino acids. E. coli secA has a natural BglII site at the same region. The primers AZ2352 (BamHI) and AZ2351 (BglII) were used for PCR amplification of R. rickettsii to produce (1250 bp fragment) the 5' end of the chimeric secA. For the 3' end of the chimeric secA from E. coli MC4100, a 2059 bp fragment (which has a natural BglII site) amplified by primers AZ2349 and AZ2348 (EcoRI) was used for fusion. The 5' end (BamHI- and BglII-digested 1230 bp fragment) of the chimeric secA from R. rickettsii, and the 3' end (BglII- and EcoRI-digested 1462 bp fragment) of the chimeric secA from E. coli MC4100, were ligated together into the BamHI and EcoRI sites of the pTrcHisB vector to generate the plasmid pTrcRE208. The accuracy of the constructed plasmids pTrcRR73, pTrcRT3, pTrcEC85 and pTrcRE208 was checked by sequencing.
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The synthesis of the recombinant SecA proteins in the transformed E. coli MM52 secA51(ts) was detected by Western blot analysis using a monoclonal antibody to the N-terminal His6 tag, as described previously (Rahman et al., 2003).
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RESULTS |
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Transcriptional analysis of rickettsial secA
Total RNA isolated from R. typhi and R. rickettsii was analysed by RT-PCR to detect the expression of the secA gene. Amplification products (confirmed by sequencing) of 543 bp (lane 1) and 366 bp (lane 2), as shown in Fig. 2, were detected, thereby indicating the expression of secA mRNA in these rickettsial species.
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The TSP of the R. rickettsii secA gene was determined by 5'RACE as described in Methods. The TSP corresponding to an Ala residue was located 32 nt upstream of the secA start codon (GenBank accession no. AY312572). Sequence analysis of the TSP region revealed the presence of sequences similar to the putative 10 (TATATT) and 35 (TTGATA) consensus sequences separated by 17 nt of the canonical prokaryotic promoter recognized by the general sigma factor (70) (Lewin, 1997
). The sequence analysis also showed the presence of sequences similar to the putative ribosome-binding site (AAGGA) on the upstream of the secA initiation codon (Lewin, 1997
).
Complementation assay with rickettsial secA
The functional activity of rickettsial secA was assayed by genetic complementation in the temperature-sensitive E. coli MM52 secA51(ts). A point mutation (T to C) in the secA gene changing amino acid residue Leu43 to Pro (Fig. 1) renders E. coli MM52 unable to grow at 42 °C (Schmidt et al., 1988
). Strain MM52 has been used to demonstrate the functional activity of secA homologues from many bacterial species (McNicholas et al., 1995
; Owens et al., 2002
).
The temperature-sensitive growth of the transformed E. coli MM52 (secA51) was assayed as described in Methods. From the growth curve in Fig. 3, it was observed that E. coli MM52 transformed with plasmid pTrcEC85 (positive control, carrying the full-length ORF of E. coli secA) grew faster than E. coli MM52 with or without control plasmids (pTrcHisA or pTrcHisB) at the non-permissive temperature 42 °C. However, E. coli MM52 transformed with pTrcRR73 (carrying the full-length ORF of R. rickettsii secA) or pTrcRT3 (carrying the full-length ORF of R. typhi secA) failed to restore the growth of strain MM52 at 42 °C (Fig. 3
). For quantitative detection of the growth of the transformed E. coli MM52 at the non-permissive temperature, survival was also determined by the c.f.u. assay. In this assay, E. coli MM52 showed almost no growth at 42 °C, and the control plasmids pTrcHisA and pTrcHisB were unable to restore growth. However, E. coli MM52 transformed with the pTrcEC85 (positive control, carrying full-length ORF of E. coli secA gene) showed a restoration of growth at 42 °C by 99·12±0·96 % (compared to that at 30 °C). Expression of the recombinant SecA protein was detected in E. coli MM52 carrying the positive control plasmid pTrcEC85 (Fig. 4
, lane 3, a band of 106 kDa); this was not detected for the negative control (Fig. 4
, lanes 1 and 2). E. coli MM52 transformed with pTrcRR73 (carrying the full-length ORF of R. rickettsii secA) or pTrcRT3 (carrying the full-length ORF of R. typhi secA) was still unable to grow at 42 °C as judged by the c.f.u. assay, even though the expression of recombinant SecA proteins of R. rickettsii and R. typhi were detected in E. coli by Western blotting as shown in Fig. 4
(lanes 4 and 6, 107 kDa bands). The complementation assay with the full-length ORF of rickettsial secA indicated that the full-length SecA protein from these rickettsial species is not functional in E. coli for preprotein translocation.
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The functional complementation analysis performed by using the c.f.u. assay in the temperature-sensitive E. coli MM52 transformed with pTrcRE208 showed a restoration of growth at 42 °C by 97·44±2·16 % (compared to that at 30 °C). Complementation was also assayed by growth curves. Transformation of E. coli MM52 with pTrcRE208 led to an approximately 3·5-fold increase in growth at 42 °C after 5·5 h incubation in comparison with E. coli MM52 carrying control plasmids pTrcHisA and pTrcHisB (Fig. 3). The expression of the recombinant chimeric SecA protein was detected in E. coli MM52 carrying pTrcRE208 by Western blotting, as shown in Fig. 4
(lane 5, a band of 104 kDa).
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DISCUSSION |
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The analysis of E. coli secA gene expression revealed that secA is the second gene in the polycistronically transcribed secMsecA operon. Translation of secA is coupled to the translation of secM (secretion monitor), since ribosomes translating the distal portion of secM are needed to disrupt the secondary structure of mRNA formed in the secMsecA intergenic region that normally blocks secA translation initiation (Nakatogawa et al., 2004; Sarker & Oliver, 2002
). However, analysis of the published genome sequences of rickettsiae (Andersson et al., 1998
; Ogata et al., 2001
; McLeod et al., 2004
) showed no SecM homologue. The R. rickettsii transcription analysis data presented here show the monocistronic expression of the secA gene with a transcript size of 2·8 kb, and indicate that its translation does not appear to be coupled with any other gene translation.
SecA, an essential component of the general protein translocation pathway, has been found in a wide variety of bacteria (den Blaauwen & Driessen, 1996). Sequence analysis of the deduced amino acids of rickettsial SecA suggests the presence of highly conserved amino acid residues and motifs that are considered to be essential for the ATP catalytic activity of SecA in preprotein translocation (de Keyzer et al., 2003
; Mitchell & Oliver, 1993
). However, the functional complementation monitored by the c.f.u. assay and the growth curve plot show that the full-length SecA proteins of R. typhi and R. rickettsii fail to restore growth in E. coli MM52 secA51(ts) at the non-permissive temperature (42 °C), despite the expression of recombinant SecA protein, as detected in E. coli by Western blotting. This failure may explain the inability of recombinant rickettsial SecA to interact with the complex multisubunit translocon of E. coli cells for in vivo preprotein translocation, or it could result from the failure of the recombinant rickettsial SecA to deliver preprotein to the E. coli Sec pathway for translocation.
The high-affinity ATP binding sites (motifs A0 and B0) confined to the N-terminal domain of SecA function as the ATP hydrolytic site. The low-affinity ATP binding site (motif A3) and the C-terminal domain regulate the ATP hydrolysis at the high-affinity ATP binding site of the SecA preprotein translocase (de Keyzer et al., 2003; Karamanou et al., 1999
). Based on strong sequence similarity in the N-terminal ATP binding domains of SecA protein, we constructed a chimeric (R. rickettsiiE. coli) secA gene to test the functional complementation in the temperature-sensitive E. coli strain MM52 secA51. The expression of the chimeric secA gene (encoding the N-terminal 408 aa of R. rickettsii SecA fused with the C-terminal 480 aa of E. coli SecA) in E. coli produced a functional and active preprotein translocase subunit SecA, as demonstrated by a complementation assay. The positive correlation between the functional complementation in the temperature-sensitive secA mutant of E. coli and the ATP catalytic activity of the amino-terminal domain of SecA has been used to demonstrate in vivo preprotein translocon ATPase activity of the putative SecA from other bacteria (McNicholas et al., 1995
). Studies with different domains of the SecA indicate that the C-terminal end mediates SecA dimerization, binds with SecB, and interacts with the membrane and the intramolecular regulator of ATP hydrolysis, and, thus, the C-terminal end confers the species-specific characteristic to SecA for preprotein translocation activity (de Keyzer et al., 2003
; Karamanou et al., 1999
; Owens et al., 2002
).
The functionally conserved high-affinity ATP binding sites in the N-terminal end of SecA are the critical regions for functional complementation of the SecA homologue from other bacteria. Our complementation data presented here indicate that R. rickettsii SecA carries a functional N-terminal ATP catalytic domain for preprotein translocon in E. coli. Biochemical analysis of rickettsial SecA, and its role in the secretion of virulence factors, are currently under investigation.
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ACKNOWLEDGEMENTS |
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Received 12 August 2004;
revised 5 October 2004;
accepted 18 October 2004.
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