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1 1st Department of Internal Medicine, University of Cologne, 50924 Cologne, Germany
2 Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, 50924 Cologne, Germany
3 Department of Microbiology, University of Tennessee, Knoxville, TN 37996, USA
4 Department of Internal Medicine 1, Division of Infectious Diseases, University of Regensburg, 93042 Regensburg, Germany
Correspondence
Jan Rybniker
jan.rybniker{at}uk-koeln.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Pedulla et al., 2003). The functions of the vast majority of these mycobacteriophage ORFs remain unknown and only a few proteins have been expressed and examined in detail. Even more striking is the genomic diversity of mycobacteriophages, and exchange of genes among the phages themselves and among their hosts has led to a vivid and probably highly influential evolutionary relationship between virus and bacterial host.
Extensive comparison of the mycobacteriophage genomes and amino acid sequences and their organization in so-called ‘phamilies’ (putative proteins with amino acid identity of 25 % or more) showed that almost half of the phage-derived sequences match non-phage and predominantly bacterial genes (Hatfull et al., 2006). Furthermore, some of these non-phage-associated genes are homologues of suspected mycobacterial virulence genes and genes that are involved in bacterial cell-wall synthesis (Pedulla et al., 2003). On the host's side, the sequencing of mycobacterial genomes led to the identification of many prophages, which presumably influence the phenotype and virulence of some mycobacteria (Stinear et al., 2007).
Mycobacteriophage L5, a temperate phage isolated from Mycobacterium smegmatis, was the first mycobacteriophage to be sequenced (Hatfull & Sarkis, 1993). L5 forms stable lysogens in M. smegmatis and has a broad host range among the pathogenic mycobacteria (Rybniker et al., 2006). Although L5 is a temperate phage, the following evidence suggests that the phage switches off host protein synthesis during early lytic growth. First, a quantitative decrease of host proteins is seen minutes after induction of a thermo-inducible L5 lysogen (Hatfull & Sarkis, 1993), and second, it has been reported that a genetic element spanning ORF83 to ORF77 does not transform in M. smegmatis (Donnelly-Wu et al., 1993). These ORFs are situated on the right arm of the L5 genome, which encodes primarily early regulatory proteins.
The potential presence of host shut-off proteins in the temperate phage L5 implies an extremely tight silencing of the genes responsible by the L5 repressor. The repressor encoded by ORF71 is responsible for the temperate phenotype and the superinfection immunity of lysogens. Transcriptional silencing is achieved by binding of the repressor protein within or downstream of the four early promoter sequences identified so far (Brown et al., 1997; Donnelly-Wu et al., 1993).
The host shut-off phenomenon is well known for many lytic bacteriophages as well as for viruses of the eukaryotic cell (Fenwick & Clark, 1982; Sharma et al., 2004; Svenson & Karlstrom, 1976). It has recently been shown that the systematic exploration of staphylococcal bacteriophage derived shut-off proteins and their targets in Staphylococcus aureus can lead to the identification of new antibiotic chemical compounds (Liu et al., 2004).
For the identification of phage-derived toxic proteins, the existence of tightly regulated inducible expression vectors is a prerequisite. The recent development of inducible mycobacterial shuttle plasmids carrying the acetamidase promoter of M. smegmatis enabled us to screen for potentially toxic ORFs within the regulatory region of mycobacteriophage L5 (Daugelat et al., 2003).
Candidate genes were amplified from the L5 genetic region that does not transform in M. smegmatis and were inserted in a set of modified acetamidase-inducible expression plasmids. Here we present data showing that the L5 gene products (gp) 77, 78 and 79 are toxic to its natural host. Furthermore, we provide new insight into the regulation of these proteins by the description of a new promoter element and transcription control through the L5 repressor gp71.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Since the pSD24 acetamidase promoter shows intrinsic activity in Escherichia coli, amplification of some plasmid constructs carrying toxic proteins was not possible. To address this problem, the two lac operator elements of plasmid pQE80 (Qiagen) were amplified using the primers lacOfor (5'-TTTTATCCATGGATCGTTGTGAGCGGATAA-3') and lacOrev (5'-TCTGCAGCTGGATCCGTGATGGTGATGGTG-3') and cloned between the acetamidase promoter and multiple cloning site of pSD24, giving pJR5 (Table 1, Fig. 1). This allowed the propagation of otherwise toxic plasmids in E. coli strains constitutively expressing the lac repressor such as Fusion-Blue (Clontech). Insertion of this DNA fragment had no influence on the function of the acetamidase promoter in M. smegmatis.
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DNA manipulation.
DNA manipulation, electrophoresis and PCRs were carried out using standard techniques (Sambrook & Russell, 2001). All PCRs for subsequent cloning were performed using Phusion high fidelity polymerase (Finnzymes) according to the instructions provided by the manufacturer.
DNA cloning.
All cloning steps were done by homologous recombination using the In-Fusion PCR cloning kit (Clontech). All primers had 15 bp homology with each end of the linearized vector at their 5'-end followed by 15 bp specific sequence homologues to the L5-ORF amplified as recommended by the In-Fusion protocol. Primers for the amplification and cloning of ORF77 in pJR5 are given as examples: forward primer 5'-TCACCATCACGGATCCatgATCGACGGCAA-3', reverse primer 5'-TCTGCAGCTGGATCCtcaGCCCGCCAGCGC-3'; sequence with homology to the linearized vector is underlined whereas sequence with homology to ORF77 is shown in italics (atg, start codon; tca, stop codon). With regard to pSD24, pJR5, pJR6, pJR7, pJR8 and pJR10 the single BamHI restriction site was used for all insertional cloning steps with subsequent expression, whereas pQE80 was linearized with BamHI and HindIII. Sequence identity was confirmed for all clones by sequencing the first 250–500 bp downstream of the restriction enzyme site that was used for the cloning procedure.
Electroporation of mycobacteria.
Electrocompetent M. smegmatis cells were prepared by growing the bacteria in 7H9 broth (containing OADC and Tween 80) to an OD600 of 1.0. The cells were kept on ice for 90 min and harvested by centrifugation at 4000 g for 15 min. The cells were washed three times in ice-cold 10 % glycerol and resuspended in a 1/100 volume of 10 % glycerol. Settings for electroporation were 2.5 kV, 25 µF and a resistance of 200 
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Identification of toxic phage ORFs.
Fig. 2 illustrates the plasmids that were created for the identification of toxic phage ORFs. M. smegmatis clones were subcultured on 7H10 agar containing 0.1 % acetamide (Sigma) and E. coli DH5
clones on LB agar supplemented with 0.2 mM IPTG (Roth). Clones showing growth inhibition on selective media were further examined using serial dilutions of acetamide for M. smegmatis and IPTG for E. coli followed by OD600 readings after 4 h for E. coli and after 12 h for M. smegmatis. Also, standard survival curves were created for the clones of interest (see Fig. 4). For survival curves and OD600 readings, subculturing of M. smegmatis clones with derivatives of pSD24 carrying toxic ORFs was kept to a minimum since pSD24 plasmid propagation is not fully stable over several subculturing steps.
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). In brief, individual colonies were scraped off 7H10 agar plates and washed in 1x PBS. Then 180 µl 0.1 M NaHCO3 and 20 µl FITC (100 mg ml–1; Sigma) were added to approximately 25 mg bacteria (wet wt). After incubation for 30 min at 37 °C the stained cells were washed three times with PBS and mounted on a glass slide. Microscopy was done on an Olympus IX81 inverted fluorescence microscope.
Insertion of the repressor gene ORF71 in pJR5 and co-expression studies.
A sequence spanning bp 44331 to bp 45045 of the L5 genome was amplified by PCR using the forward primer 5'-GCGCGGCCGCGGTACCTTAGGGCTCCGAGA-3' and the reverse primer 5'-CACTTCTTTATCTAGATAACGCCAGTCGAT-3'. This amplified sequence covers ORF71, which codes for the L5 repressor, and the two promoters P1 and P2, which are situated upstream of ORF71. The PCR product was cloned into pJR5 linearized with XbaI and Acc65I, giving pJR6 (Fig. 1
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A PCR product spanning ORF83 to ORF77 was cloned in pJR5 and pJR6 respectively (Fig. 2). Plasmid DNA was amplified in E. coli Fusion-Blue and 250 ng of each construct was electroporated into 80 µl competent M. smegmatis cells. Transformants were plated onto 7H10 plates containing 50 µg hygromycin B ml–1.
Generation of mycobacterial promoter-probe vectors and promoter identification.
The lacZ gene of pMC1871 was amplified by PCR using the primers pSDlacFO (5'-TTTTATCCATGGATCCGTCGTTTTACAACG-3') and pSDlacRE (5'-TCTGCAGCTGGATCTCCCCTGCCCGCTTAT-3'). Primer pSDlacRE deletes the second BamHI site, leaving only one functional BamHI site upstream of the lacZ gene. The PCR product was purified and cloned into BamHI-linearized pSD24, giving pJR7 (Fig. 1). The acetamidase promoter of pJR7 was deleted by digestion with BamHI and XbaI and subsequent religation of the vector ends using an oligo-linker made by annealing the oligos LinkXbaBamFO (5'-CTAGCCCGAGGTATCTAGATATCTTTG-3') and LinkXbaBamRE (5'-GATCCAAAGATATCTAGATACCTCGGG-3'), resulting in the plasmid pJR10 (Fig. 1). To generate pJR8, which is pJR7 constitutively expressing the L5 phage repressor gp71, a similar procedure was chosen as described for pJR5 and pJR6. These three plasmids, pJR7, pJR8 and pJR10, gave the basis for several constructs used for the identification of promoter activity in ORF83, which was amplified by PCR together with 12 bp of the 5'-end of ORF82, leading to a fusion protein of the gene product of ORF82 and the β-galactosidase protein (see Fig. 5a). For pJR7 : : 8382 and pJR8 : : 8382 the genetic element spanning ORF83, ORF82, the intergenic region of ORF82/ORF81 and the start codon of ORF81 was amplified and cloned in-frame with the lacZ gene. The 21 bp sequence between the stop codon of ORF82 and the start codon of ORF81 contains a repressor-binding site (Brown et al., 1997).
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Primer extension.
Five picomoles of primer Seclaq (5'-TTGGGTAACGCCAGGGTTTTC-3') was end-labelled with [
-32P]ATP using 20 units T4-polynucleotide kinase (Fermentas). Labelled primer was passed through a Sephadex-G50 NICK-column (GE-Healthcare) to remove unincorporated nucleotides. Total RNA was isolated from M. smegmatis clones carrying either pJR10 : : 83 or pJR10 as a control. Five micrograms of total RNA was incubated with approximately 50 fmol labelled primer for 5 min at 65 °C in a total volume of 10 µl. Two microlitres of a 10 mM dNTP mix, 1 µl 0.1 M DTT, 4 µl 5x cDNA buffer, 2 µl water and 15 units Thermoscript reverse transcriptase (Invitrogen) were added to each sample and the tubes were incubated at 50 °C for 45 min. After heating the samples to 85 °C for 5 min, phenol/chloroform extraction and ethanol precipitation were performed and the pellets were resuspended in 5 µl water together with 5 µl stop solution (T7-sequencing kit). A sequencing ladder was generated using the T7-sequencing kit (USB) following the instructions of the manufacturer. In brief, 2 µg plasmid DNA derived from pJR10 : : 83 were denatured with 2 M NaOH, ethanol precipitated and resuspended in 10 µl water. Primer Seqlac (10 pmol in 2 µl) and 2 µl annealing buffer were added and incubated for 5 min at 65 °C followed by 10 min at 37 °C and 5 min at room temperature. Then 3 µl labelling mix, 1 µl [-32P]dCTP and 2 µl diluted T7-DNA polymerase were added. After 5 min incubation at room temperature, 4.5 µl aliquots were added to 2.5 µl of the A, C, G and T termination mixes, respectively. The reaction was stopped after 5 min by the addition of 5 µl stop solution, and loaded next to the primer-extended samples on a denaturing sequencing gel (6 % acrylamide : bis-acrylamide 19 : 1, 7 M urea, 0.9xTBE). The bands were visualized on storage phosphor screens using a Typhoon imager (GE-Healthcare).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). We could confirm this finding using the construct pJR5 : : 8377 (Fig. 2), which could not be transformed into M. smegmatis mc2155. We then deleted ORF83 from this construct, leading to the plasmid pJR5 : : 8277, which was transformable, giving relatively small M. smegmatis colonies on 7H10 agar after 5 days incubation at 37 °C. When transformants were subcultured on 7H10 agar containing 0.1 % acetamide there was no visible growth, suggesting that the phenotype of clones carrying pJR5 : : 8277 is due to one or more inhibitory ORFs within the phage sequence spanning ORF82 to ORF77. One could also assume that in pJR5 : : 8277 these toxic ORFs are decoupled from a promoter sequence upstream of ORF82 (Fig. 2). To define the exact ORF responsible for toxicity, six plasmids were designed, individually carrying ORF82, 81, 80, 79, 78 or 77 under control of the acetamidase promoter (Fig. 2). Using these plasmids we were able to show that gp77, gp78 and gp79 possess growth-inhibitory properties, with gp77 being the most potent (Fig. 3a, b).
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). This is most likely due to the expression of minute amounts of gp77 from an incompletely repressed acetamidase promoter. This incomplete silencing of the acetamidase promoter in pSD24 was also shown in the ONPG assay as described further below.
Acetamide titration and survival curves of clones carrying toxic phage ORFs
To identify the minimum amount of acetamide necessary for the growth-inhibitory effect, individual clones were grown to an OD600 of 0.1 and acetamide titration was performed. Here, acetamide concentrations as low as 0.01 % were sufficient to inhibit the growth of clones containing pJR5 : : 77, pJR5 : : 78 and pJR5 : : 79. The growth inhibition was directly proportional to the concentration of acetamide as determined by OD600 measurements after 12 h in a shaking incubator at 37 °C (Fig. 4a). The M. smegmatis clone carrying pJR7, which expresses LacZ under control of the acetamidase promoter, was used as a control.
To further study the effects of L5 toxic gene expression in M. smegmatis (bacteriostatic vs bacteriocidal) survival curves of induced (0.05 % acetamide) and uninduced clones grown in 7H9 broth were created by plating serial dilutions every 6 h for a period of 18 h. Expression of gp77 led to a bacteriostatic effect on M. smegmatis, with growth arrest for all time points examined and recovery of the bacteria after plating on 7H10 agar without acetamide for enumerating c.f.u. (Fig. 4c). Expression of gp78 and gp79 allowed growth of M. smegmatis, although at a significantly lower rate compared to the uninduced clones, gp78 being the least potent inhibitor.
Expression of gp79 alters cell morphology of M. smegmatis
Growing induced M. smegmatis clones carrying pJR5 : : 79 to stationary phase in liquid medium led to the development of non-homogeneous turbidity and the formation of small clumps despite the fact that Tween 80 was added to the medium (Fig. 3b). When grown on solid medium containing 0.1 % acetamide these clones had a rough surface morphology compared to that of wild-type M. smegmatis. Fluorescence microscopy of induced pJR5 : : 79 clones showed filamentation, with most of the bacteria being more than three times longer than uninduced clones and M. smegmatis expressing LacZ, indicating impaired cell division (Fig. 3c). Interestingly, some pJR5 : : 79-bearing cells showed a branching phenotype (Fig. 3c).
Activity of phage ORFs in E. coli
Since some toxic proteins of bacteriophages infecting Gram-positive bacteria show a growth-inhibitory effect on E. coli, we were interested in the phenotype of E. coli DH5 clones expressing L5 proteins encoded by ORF79 to ORF77. In contrast to pSD24 or pJR5, the expression vector pQE80 is tightly regulated, allowing normal growth of uninduced clones. Growth of clones carrying pQE80 : : 77, pQE80 : : 78 and pQE80 : : 79 in the presence of IPTG was clearly inhibited (Fig. 4b, d). With respect to gp79, the effect of expression in E. coli was different from that observed in M. smegmatis. Expression of gp79 is clearly bactericidal to E. coli, with almost a 2 log loss of viable cells after 3 h growth in the presence of 0.1 mM IPTG (Fig. 4d). The expression of gp77 in E. coli led to a bacteriostatic effect whereas the expression of gp78 resulted in a significantly lower growth rate (Fig. 4b, d).
Co-expression of the L5 repressor allows transformation of ORF83 to ORF77
In contrast to pJR5 : : 8377, which does not transform competent M. smegmatis cells, electroporation of pJR6 : : 8377, which constitutively expresses the L5 repressor gp71, resulted in approximately 2.4x104 transformants per µg plasmid DNA. Interestingly, the colonies obtained varied widely in colony size and morphology. Subcultures of these clones on media containing acetamide led to growth inhibition, showing that the extremely strong acetamidase promoter may overcome transcriptional silencing of the L5 repressor (data not shown).
ORF83 contains a putative promoter that is functional in M. smegmatis but not in E. coli
Transformability of pJR5 : : 8277 but not of pJR5 : : 8377 indicates the presence of a positive regulatory element within ORF83, which possibly induces the transcription of the three cytotoxic genes. To confirm this hypothesis two new mycobacterial promoter-probe vectors were created using pSD24 as a backbone. The insertion of lacZ from pMC1871 downstream of the acetamidase promoter led to pJR7. This construct allows the insertion of candidate promoter sequences between the acetamidase promoter and the lacZ gene using the single BamHI site (Fig. 1). pJR7 grown without acetamide shows a relatively low but measurable β-galactosidase activity whereas addition of acetamide to the medium results in an extremely high activity (Fig. 5a). This is consistent with recently published data indicating that the acetamidase promoter in pSD24 is about three times stronger than the mycobacterial hsp60 promoter (Daugelat et al., 2003). Induced pJR7 was used as a positive control in subsequent experiments for the evaluation of the promoter activity of L5 genes. The presence of a functional promoter inserted between the acetamidase promoter and lacZ gene results in an increased β-galactosidase activity compared to uninduced clones carrying pJR7.
To abolish any background activity of the acetamidase promoter the complete element was excised, resulting in pJR10 (Fig. 1). This construct had no detectable β-galactosidase activity, like wild-type M. smegmatis mc2155.
The insertion of ORF83 together with a small part of the 5'-end of ORF82 into the linearized promoter-probe vectors (leading to a fusion protein of gp82 and LacZ) gave pJR7 : : 83 and pJR10 : : 83 (Fig. 5a). M. smegmatis transformants harbouring these plasmids showed an increase of β-galactosidase activity compared to clones containing pJR10 and uninduced pJR7 (Fig. 5a). These constructs mimic the situation seen in pJR5 : : 8377, with the toxic ORFs being exchanged for the lacZ gene. Compared to fully induced pJR7, promoter activity was about three times lower in (uninduced) pJR7 : : 83 and about four times lower in pJR10 : : 83, suggesting that the promoter within ORF83 is of moderate strength (Fig. 5a).
Uninduced pJR7 had measurable activity, indicating incomplete suppression of the acetamidase promoter in pSD24 and related constructs (Fig. 5a). This explains the slower growth of uninduced clones containing pJR5 : : 77.
Interestingly, E. coli DH5 containing pJR10 : : 83 (which has shuttle-vector properties) or pMC1871 : : 83, an E. coli promoter-probe vector carrying ORF83, did not show any β-galactosidase activity in the ONPG assay (data not shown). This indicates that promoter activity within ORF83 is restricted to mycobacteria.
Detection of the transcriptional start site within ORF83
Primer extension analysis was performed on M. smegmatis RNA containing either pJR10 : : 83 or pJR10 as a negative control. As shown in Fig. 5(b), a transcriptional start point was detected mapping to position 37 of ORF83. A putative core promoter sequence with –35 (GTGACC) and –10 (TCAAAT) hexamers is situated upstream of this position. The –35 site is homologous with the –35 site of promoter P4 of mycobacteriophage L1, a closely related homoimmune phage of L5 (Chattopadhyay et al., 2003). The experiment was performed with three individual sets of total RNA, which showed identical results.
Transcriptional regulation of the putative promoter is achieved through repressor binding between ORF82 and ORF81
Lysogeny of mycobacteriophage L5 is achieved through binding of the repressor gp71 to asymmetric DNA sites with the consensus sequence GGTGG(C/A)TGTCAAG, which are situated either within some promoter sites or downstream of promoters (Brown et al., 1997). The binding sites within promoters represent true operators that negatively regulate transcription initiation whereas the downstream ones act as stoperators inhibiting transcription elongation, leading to a downregulation of gene expression (Brown et al., 1997). One such stoperator is situated downstream of ORF83 between the stop codon of ORF82 and the start codon of ORF81 (Fig. 2). To show that gp71 and its binding site have a regulatory influence on the promoter element in ORF83, the repressor gene 71 was cloned into the promoter-probe plasmid pJR7, leading to pJR8 (Fig. 1). Insertion of ORF83 upstream of the lacZ gene of pJR8 led to similar β-galactosidase activity as seen for pJR7 : : 83 in the ONPG assay whereas integration of ORF83 together with the intergenic region of 82 and 81 (pJR8 : : 8382) led to a 10-fold reduction of β-galactosidase activity (Fig. 5a). Addition of acetamide to the medium abolished gp71 activity. A single stoperator site downstream of the extremely strong acetamidase promoter seems to have only a slight negative regulatory effect on promoter activity and thus may not be detectable in this assay.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Thus tight regulation of these early gene products is a prerequisite to achieve successful L5 lysogeny. Our co-expression results are in favour of the explanation that transcriptional silencing of the downstream genes during lysogeny is exerted by the phage repressor gp71. These results emphasize the major role of gp71 for globally silencing prophage gene expression, a crucial mechanism for a temperate virus. However, co-expression of gp71 only led to suppression of promoter activity to values just above the background activity of the leaky acetamidase promoter but not to complete silencing of expression. Also, M. smegmatis clones carrying pJR6 : : 8377, a plasmid containing the repressor gene, the putative promoter sequence in ORF83, the repressor-binding site and the downstream toxic ORFs, showed variable colony size and morphology. This may reflect unstable expression or a variable half-life of the repressor protein, which could be one explanation for the high rate of L5 infections which lead to lytic growth (80 %) rather than lysogeny (Sarkis et al., 1995). However, it is not clear how the promoter within ORF83 is regulated in true L5 lysogeny. In contrast to Pleft, a promoter upstream of ORF88 that has a gp71-binding site reaching into its –35 sequence controlling the initiation of transcription, there are no such sites in ORF83 (Brown et al., 1997). There may be other, so far undescribed regulatory genes in L5, not present in our plasmid construct, which control transcription during lysogeny.
Although the amino acid sequences of gp77, gp78 and gp79 do not show any sequence homologies with gene products of phages infecting E. coli, we showed that these L5 proteins are inhibitory to the growth of this Gram-negative bacterium. This is not unprecedented since other phage-derived shut-off genes such as e3 of phage SPO1, a bacteriophage infecting the Gram-positive Bacillus subtilis, also lead to growth inhibition when expressed in E. coli (Wei & Stewart, 1993).
The distinct interaction of the three proteins with the host remains unclear. The predicted 28.2 kDa protein encoded by ORF77, which possesses the strongest activity in M. smegmatis, does not contain an excess of positively charged amino acids and we were unable to identify any DNA-binding motifs such as helix–turn–helix structures enabling DNA-binding activity. Membrane association of gp77 is unlikely since there is a lack of hydrophobic regions, signal peptides or predicted transmembrane helices. Using p-BLAST to compare the amino acid sequence of gp77 with the available database revealed sequence homology not only to other mycobacteriophages but also to hypothetical proteins of actinomycetes such as Frankia spp., Streptomyces spp. and Mycobacterium spp. (Table 2). This indicates that L5 or an L5-related phage may have acquired gp77 from a former host by illegitimate recombination. The toxic protein may have been retained, conferring a selective advantage upon the host.
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). PBPs are highly conserved across slow- and fast-growing species and interact with several other cell-wall-associated proteins such as FtsZ and FtsW. These proteins form a structure called Z-ring, a cytoskeletal scaffold mediating cell division (Datta et al., 2006). Alteration of Z-ring proteins also leads to growth inhibition and impaired cell division in both E. coli and M. smegmatis (Belhumeur & Drapeau, 1984; Dziadek et al., 2003). Furthermore, simultaneous deletion of several low-molecular-mass PBPs of E. coli leads to elongation and branching of individual mutants (Nelson & Young, 2000). This study presents evidence that gp79 interferes with the cell membrane or cell-wall synthesis of M. smegmatis. First, colonies of clones carrying pJR5 : : 79 grown for several days on 7H10 agar containing 0.1 % acetamide have a rough surface compared to the smooth phenotype of wild-type M. smegmatis and clones expressing other L5 proteins. Second, the same clones grown in 7H9 broth containing acetamide form relatively large clumps even in the presence of Tween 80, again in contrast to wild-type M. smegmatis (Fig. 3b). Third, microscopy of induced clones shows elongated rods compared to the uninduced control, and in some bacterial cells branching can be observed, indicating that gp79 interferes with cell septation (Fig. 3c).
Expression of only the N-terminal hydrophobic part (pJR5 : : 79A, Fig. 2) or the C-terminal part (pJR5 : : 79B, Fig. 2) of gp79 eliminates the toxic effect (data not shown). This indicates that the complete polypeptide is necessary for proper function despite the fact that there is a predicted cleavage site between the alanine at position 34 and the aspartic acid at position 35.
In addition to the fact that PBP knockout mutants in several bacterial species show a similar phenotype, the homology of the hydrophobic N-terminal part of gp79 to a PBP signal peptide may indicate an interaction of gp79 with proteins or metabolites involved in the peptidoglycan synthesis of M. smegmatis.
The synthesis of the outer cell envelope lipids seems not to be affected by gp79 since their analysis by TLC revealed no difference between induced and uninduced M. smegmatis clones (data not shown).
The reason for L5 interfering with the cell wall of its host in the early stage of phage propagation is not clear. The genes responsible for lysis of the cell wall after completion of the lytic cycle are situated in the far left arm of the phage genome.
The smallest and least active of the toxic proteins described is gp78. This polypeptide, with a predicted molecular mass of 5.5 kDa, has detectable sequence homology only to other mycobacteriophage-derived proteins (Table 2), leaving little option for the interpretation of its function.
The data presented here address the intriguing question of the mechanism by which mycobacteriophages ‘take over’ the host machinery during early lytic growth to replicate to higher levels or to evade host defence. With gp77 and gp79, neighbouring L5 proteins have been described that interfere with the host biogenesis, most likely through two completely different mechanisms.
A potential application of the inducible expression of toxic phage proteins could be as counter-selectable markers for the genetic manipulation of mycobacteria. Furthermore, we note that gp71 expression and binding features combined on a single plasmid provide powerful and convenient applications for the controlled expression of foreign genes in mycobacteria.
In view of the large number of mycobacteriophages in the ecosystem and their extraordinarily high diversity, the approach described in this paper may also allow the further identification of proteins targeting important metabolic pathways of mycobacteria. Target identification is a major step in the drug-discovery process (Terstappen & Reggiani, 2001). With regard to the pathogenic mycobacteria such as Mycobacterium tuberculosis, determination of the interacting partner proteins on the host's side may lead to the identification and exploitation of as yet unknown key proteins of mycobacterial metabolism and biogenesis.
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ACKNOWLEDGEMENTS |
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Edited by: P. R. Herron
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 25 January 2008;
revised 16 April 2008;
accepted 1 May 2008.
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) between ORF82 and ORF81 (pJR8 : : 8382) leads to a 10-fold reduction of promoter activity, which can be overcome by the strong acetamidase promoter. (b) Primer extension analysis. Lanes A, C, G, T, sequencing ladder generated with pJR10 : : 83 DNA. Lanes 1 and 2, primer extension products obtained with RNA isolated from M. smegmatis clones harbouring pJR10 (promoterless control) and pJR10 : : 83, respectively. A single band was detected matching position 38 (*) of the sequencing ladder and position 37 of ORF83 (one base upstream due to use of [