Insights into the function of the WhiB-like protein of mycobacteriophage TM4 – a transcriptional inhibitor of WhiB2


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WhiB-like proteins of actinomycetes are known to co-ordinate iron-sulfur (Fe-S) clusters and are believed to have regulatory functions in many essential bacterial processes. The systematic determination of the genome sequences of mycobacteriophages has revealed the presence of several whiB-like genes in these viruses. Here we focussed on the WhiB-like protein of mycobacteriophage TM4, WhiBTM4. We provide evidence that this viral protein is capable of co-ordinating a Fe-S cluster. The UV-visible absorption spectra obtained from freshly purified and reconstituted WhiBTM4 were consistent with the presence of an oxygen sensitive [2Fe-2S] cluster. Expression of WhiBTM4 in the mycobacterial host led to hindered septation resembling a WhiB2 knockout phenotype whereas basal expression of WhiBTM4 led to superinfection exclusion. The quantification of mRNA-levels during phage infection showed that whiBTM4 is a highly transcribed early phage gene and a dominant negative regulator of WhiB2. Strikingly, both apo-WhiB2 of Mycobacterium tuberculosis and apo-WhiBTM4 were capable of binding to the conserved promoter region upstream of the whiB2 gene indicating that WhiB2 regulates its own synthesis which is inhibited in the presence of WhiBTM4. Thus, we provide substantial evidence supporting the hypothesis of viral and bacterial WhiB proteins being important Fe-S containing transcriptional regulators with DNA-binding capability.


Fe-S clusters represent one of the simplest and most functionally versatile prosthetic groups (Beinert et al., 1997). By undergoing oxidation-reduction reactions they play an important role in metabolic pathways and regulatory processes across all kingdoms of life. In these clusters Fe ions are linked to each other through sulphide bridges on a cysteine rich protein scaffold. The family of WhiB proteins of actinomycetes are putative transcription factors which have been identified in all actinomycetes sequenced so far, but not in other organisms. The majority of the WhiB-like proteins contain four perfectly conserved cysteines. WhiD, a protein of Streptomyces coelicolor which is required for late stages of sporulation, was the first WhiB protein shown to co-ordinate a Fe-S cluster with the help of these essential cysteines (Jakimowicz et al., 2005).

The determination and annotation of the Mycobacterium tuberculosis genome sequence revealed the presence of seven whiB-like genes (whiB1-whiB7) (Cole et al., 1998; Soliveri et al., 2000). The proteins are characterized by the presence of four invariant cysteine residues and a C-terminal helix-turn-helix (HTH) motif with a GV/IWGG amino acid sequence signature in the putative β-turn. Though these motifs are conserved in all seven WhiB proteins, their cellular functions seem to differ substantially and are believed to involve, pathogenesis, cell division, stress response as well as antibiotic resistance (Gomez and Bishai, 2000; Steyn et al., 2002; Morris et al., 2005; Alam et al., 2007). Fe-S cluster co-ordinating properties have recently been shown for all M. tuberculosis derived WhiB proteins (Alam et al., 2007; 2009; Singh et al., 2007). Though several reports on mycobacterial WhiB proteins give insight into their possible function as regulatory proteins, only WhiB3 has been examined in detail with regard to regulation and transcription (Singh et al., 2009). This protein was shown to bind DNA with high affinity in its clusterless and oxidized apo-form (WhiB3-SS) and with lower affinity in the cluster carrying holo-form (WhiB3-[4Fe-4S]).

The systematic sequencing and comparison of bacteriophage genomes revealed the presence of WhiB-like proteins in several mycobacteriophages and streptomyces phages (Pedulla et al., 2003; Van Dessel et al., 2005; Morris et al., 2008). Mycobacteriophages have proven to be useful tools for the exploration of mycobacterial genetics. Furthermore, the recent sequencing of more than 30 mycobacteriophage genomes revealed an extraordinary high genetic diversity among these phages (Pedulla et al., 2003; Hatfull et al., 2006). The variety and high number of phage genes of bacterial origin reflect the frequent recombination events which were probably evolutionarily important for both virus and host. However, the interaction of bacteriophage and mycobacterium during the phage replication cycle is not yet fully understood and only a few phage proteins have been examined in detail.

In this study we have focussed on mycobacteriophage TM4. This dsDNA-tailed phage is fully sequenced and has a broad host range among the fast and slow growing mycobacteria including M. tuberculosis, Mycobacterium bovis bacille Calmette-Guérin (BCG), Mycobacterium ulcerans and Mycobacterium smegmatis (Ford et al., 1998; Rybniker et al., 2006). The annotation of the TM4 genome showed a single putative WhiB-like protein situated in a genomic region which encodes primarily for regulation- and DNA metabolism-associated proteins. We termed this protein WhiBTM4. The alignment of the 76 amino acid sequence of WhiBTM4 with the seven mycobacterial WhiB proteins identified so far revealed high sequence identity in 66 amino acids of the C-terminal region of WhiB2 (Fig. 1). The N-terminal 56 amino acids of WhiB2, which are of unknown function, are missing in the short bacteriophage protein. In M. smegmatis WhiB2 was shown to be an essential gene for cell division and a conditional WhiB2 mutant exhibited irreversible filamentous and branched growth with aberrant septum formation (Gomez and Bishai, 2000). This conditional mutant could be complemented by both WhiB2 of M. smegmatis (WhiB2Ms) and WhiB2 of M. tuberculosis (WhiB2Mtb), showing that these proteins are functionally equivalent (WhiB2Ms has also been termed WhmD in some publications, but to maintain consistency in this publication we use the term WhiB2Ms) (Raghunand and Bishai, 2006a). The role of WhiB2 in mycobacterial cell division as well as its essential nature and uniqueness to mycobacteria makes this protein an interesting drug target.

Figure 1.

Alignment of WhiBTM4 with M. smegmatis (Ms) WhiB2 and M. tuberculosis (Mtb) WhiB2 amino acid sequences. The conserved cysteines are highlighted. The conserved β-turn of the C-terminal HTH motif is marked at position 117–121.

Here we show that overexpression of WhiBTM4 leads to a WhiB2 knockout phenotype in M. smegmatis by downregulating WhiB2 expression. Basal expression from the leaky mycobacterial shuttle vector leads to a phage-resistant phenotype of the host. This phenomenon is known as superinfection exclusion and can be observed in both lytic and lysogenic bacteriophages as well as in many eukaryotic viruses (Lu and Henning, 1994; Tscherne et al., 2007).

The spectroscopic analysis of the purified protein suggests that WhiBTM4 co-ordinates a [2Fe-2S] cluster which is oxygen sensitive and can be restored under anaerobic conditions. We present data showing that WhiB2 as well as WhiBTM4 are capable of binding regulatory DNA of the host, a key feature in the establishment of WhiB proteins as regulatory proteins. A mutational analysis revealed that both the Fe-S cluster co-ordinating cysteines as well as amino acid residues of the C-terminal HTH motif are essential for the observed defect in septation. We also provide evidence that this C-terminal HTH motif is involved in DNA-binding of WhiBTM4. Furthermore, we show that in contrast to its homologue in the host, WhiBTM4 is a non-essential phage protein under laboratory conditions.


Overexpression of WhiBTM4 in M. smegmatis leads to a WhiB2 knockout phenotype

Conditional expression of WhiBTM4 in liquid media with the acetamidase inducible promoter in pSD24 led to the formation of elongated and highly branched cells (Fig. 2E and G). Furthermore, these cells had lost their acid-fast properties in the Ziehl-Neelsen stain (Fig. 2B). Fluorescence microscopy with SYTO11, which specifically stains nucleic acids, revealed the presence of multiple nucleoids in the filamented cells (Fig. 2F and H). Thus, overexpression of WhiBTM4 in liquid medium leads to a phenotype similar to the WhiB2Ms knockout mutant with hindered septum formation and fragmentation as previously shown by Gomez and Bishai (2000). Induction of WhiBTM4 expression on agar plates containing 0.2% acetamide was not tolerated by M. smegmatis (not shown).

Figure 2.

Microscopic analysis. M. smegmatis cells containing pSD24WhiBTM4 were grown in the presence of 0.02% acetamide (B, E–H) or in the absence of acetamide (A, C, D). Ziehl-Neelson stained cells (A, B) show a loss of acid fast properties of cells expressing WhiBTM4. After induction for 12 h, cells become highly filamentous and branched (E–H). SYTO11-stained induced clones show nucleoids throughout the elongated filament suggesting multiple chromosomes per cell (F, H). Magnification of light and fluorescence microscopy: ×1000. Scale bars: 20 µm.

WhiBTM4 is highly expressed during the early phase of phage replication and leads to a transcriptional downregulation of WhiB2Ms

The fact that expression of WhiBTM4 leads to morphological changes comparable to a WhiB2 knockout phenotype directed our interest towards the expression pattern of WhiBTM4 and WhiB2Ms during phage infection. Mycobacterium smegmatis cells were infected with TM4 and mycobacterial RNA was extracted at different time points. mRNA levels of whiBTM4 and three host genes (whiB2Ms, hsp60 and 16S rRNA) were determined using qRT-PCR. The phage gene was highly transcribed 20 min after phage infection and expression levels gradually declined at later time points (Fig. 3A). This establishes WhiBTM4 as an early phage gene. whiB2-mRNA levels decreased in the presence of WhiBTM4 whereas 16S rRNA levels were unchanged until the late phase of infection. HSP60 was slightly downregulated after 40 min of infection but levels increased during the late phase of phage replication. These findings are supported by similar data in M. smegmatis cells harbouring pSD24WhiBTM4. Here, induction of WhiBTM4 expression with acetamide for 4 h led to a 90% decrease in whiB2-specific RNA (Fig. 3B).

Figure 3.

A. Quantitative RT-PCR of M. smegmatis infected with mycobacteriophage TM4. All data were obtained as triplicates. For better visualization and comparability for each gene at the time point of highest expression the middle value of the triplicate was defined as 10 (arbitrary unit). All other values for this particular gene were calculated as the ratio of 10. Infection of M. smegmatis with TM4 leads to detection of whiBTM4 RNA after 20 min. Presence of WhiBTM4 (inline image) leads to a decline of whiB2 (inline image) and HSP60 (inline image) specific RNA. Values for HSP60 increase during later time points whereas whiB2 levels remain low.
B. Quantitative RT-PCR of M. smegmatis carrying pSD24WhiBTM4. Induction of pSD24WhiBTM4 with different concentrations of acetamide leads to a decrease of whiB2Ms levels which is not seen in cells carrying pSD24 only. Here HSP60 RNA seems to increase independently of the presence of WhiBTM4 (no acetamide: □; 0.2% acetamide: inline image; 0.02% acetamide: inline image). Error bars indicate the standard error of the mean, n = 3. Three individual cultures were used to extract RNA which was then used for RT-PCR experiments. The actual qRT-PCR values determined in the experiments are given in Table S1.

Purification of WhiBTM4 and WhiB2Mtb

As the results obtained so far suggested that WhiBTM4 is a transcriptional inhibitor of WhiB2, we were interested to study the biophysical properties of the protein. Overexpression of 6x His tagged WhiBTM4 in Escherichia coli BL21 with subsequent loading of cell lysate to a Ni-NTA column led to a deep brown colouring of the column. When purified and eluted under native conditions, total yield of protein was low and most of the brown substance remained on the column, indicating the formation of protein aggregates. However, the soluble fraction had a light brown colour and the eluates could be further examined by SDS-PAGE and UV-visible absorption spectroscopy. Purification and elution in the presence of 8 M urea led to a rapid clearance of the column and elution of a dark brown fluid. On SDS-PAGE the eluates of both natively purified protein and protein isolated in the presence of 8 M urea showed the presence of highly pure WhiBTM4 with prominent bands at approximately 9 kDa (Fig. 4A). Protein samples loaded without reducing agents such as dithiothreitol (DTT) revealed a second band at 18 kDa which disappeared in the presence of 1 mM DTT. All visible bands were examined by peptide mass fingerprint (PMF). The peptide pattern was consistent with his-tagged WhiBTM4. The second larger band at 18 kDa, which resolves in the presence of reducing agents, indicates that WhiBTM4 is capable of forming protein dimers. Furthermore, in the presence of DTT the prominent bands at 9 kDa migrated slower through the gel, most likely through the reduction of intramolecular disulfide bonds formed between the four cysteine residues of the protein leading to a less compact protein formation (Fig. 4A). This phenomenon has also been observed in other WhiB proteins (Raghunand and Bishai, 2006b). Gelfiltration analysis of native WhiBTM4 mirrored these results; two peaks were visible at 5.6 and 11.2 kD showing that WhiBTM4 is capable of forming a protein dimer (Fig. 4B). The monomer eluted after the 6.5 kD aprotinin standard showing that the protein forms a highly compact tertiary structure under native conditions. Dimerization has also been shown for other WhiB proteins; the role of these dimers is not yet understood (Alam et al., 2007; Crack et al., 2009).

Figure 4.

A. Coomassie blue stained SDS-PAGE. A total of 5 µg of WhiBTM4 were loaded in each lane. Lane 1 and 2, native WhiBTM4; lane 3 and 4 WhiBTM4 purified under denaturing conditions. Samples of lane 2 and 4 were treated with 1 mM DTT leading to a loss of the second higher band at approximately 18 kDa and slower migration of the protein. The bands at approximately 6 kDa in lane 1 and 2 are most likely truncated protein samples due to proteolytic digest [note the weak additional band between the 9 and 18 kDa bands (lane 1), indicating the presence of a dimer formed by the truncated form of the protein]. Molecular mass is given as determined by MALDI-TOF. All prominent bands were excised and examined with PMF.
B. Gelfiltration of native WhiBTM4. Two peaks can be detected showing that in native buffer the protein is present as a dimer and monomer. The monomer has a calculated mass of 5.6 kD showing that the folded protein is highly compact most likely through the formation of disulfide bonds between the four cysteines.

Samples loaded onto SDS gels were also examined by MALDI-TOF for an accurate analysis of protein mass (labels of Fig. 4A). Here the major peak was detected at 9220 Dalton, which corresponds to the calculated protein mass of the 6xHIS-tagged WhiBTM4.

For spectroscopic analysis and DNA-binding experiments we also purified WhiB2Mtb which is a functional homologue of WhiB2Ms. Interestingly, the purification of WhiB2Mtb could readily be performed in native buffer without the formation of protein aggregates.

Evidence for WhibTM4 co-ordinating an oxygen sensitive [2Fe-2S] cluster

The brown appearance of purified WhiBTM4 suggests the presence of an iron-sulfur chromophore. Samples were examined for their UV-visible absorption spectra which showed peaks at 322 and 423 nm (Fig. 5A). The highly concentrated cluster extracted with buffers containing 8 M urea showed two additional shoulders at 462 and 590 nm. This pattern is characteristic for mycobacterial proteins carrying a [2Fe-2S] cluster as well as for [2Fe-2S] ferredoxins from other organisms (Messerschmidt, 2001; Alam et al., 2007). The A423/A280 ratio of freshly purified WhiBTM4 was 0.25. With 0.34 atoms of iron per WhiBTM4 monomer the amount of protein containing the cluster was substoichiometric (Table 1). We note that the presence of 8 M urea had no effect on cluster stability indicating that the WhiBTM4 cluster is resistant to chaotropic agents.

Figure 5.

A. UV-visible absorption spectra of WhiBTM4 purified under native conditions (red line) and purified in the presence of 8 M urea (blue line). Numbers indicate the peak at the specified wavelength. This peak pattern is characteristic for proteins carrying a [2Fe-2S] cluster.
B. Effect of air exposure on the UV-visible absorption spectrum of WhiBTM4. The sample was kept under atmospheric oxygen at 25°C and measured every 10 min. The WhiBTM4 cluster is sensitive to oxygen.
C. Kinetics of [2Fe-2S] cluster loss determined by the decrease of absorbance at A423 upon treatment with different oxidizing and reducing agents. Samples were incubated with 10 mM of DTT (dithiothreitol) (▴), GSSG (oxidized glutathione) (▾) or DTH (dithionite) (◆) and the absorbance recorded every 30 min (untreated control: inline image). WhiBTM4 senses the presence of reducing and oxidizing agents in vitro. Experiments were performed in triplicates.

Table 1.  A423/A280 ratio and total iron content of freshly purified and reconstituted WhiBTM4. Shown are the means of three individual experiments and the standard deviation in parenthesis.
SampleA423/A280Atoms of iron per WhiBTM4 monomerAtoms of iron per WhiBTM4 dimer
Freshly purified WhiBTM40.25 (+/−0.05)0.34 (+/−0.09)0.69
Reconstituted WhiBTM40.39 (+/−0.04)1.13 (+/−0.12)2.26

Iron-sulfur clusters are known to sense the presence of various oxidants and reductants making them intracellular redox sensing molecules. We were interested in similar properties of the mycobacteriophage TM4 derived Fe-S cluster. We found that the intensity of the characteristic UV-visible absorption peaks decreased when the protein was exposed to atmospheric oxygen. The peaks as well as the intense brown colour of the protein were lost after 3 h, indicating that the cluster is oxygen sensitive (Fig. 5B). This rapid cluster degradation observed in WhiBTM4 stands in contrast to recently published data on WhiB proteins of the host where cluster loss through atmospheric oxygen is achieved within days (Alam et al., 2009). In WhiBTM4 cluster disassembly could be arrested in the presence of 10 mM DTT which protects Fe-S clusters as an antioxidant and inhibits the formation of intramolecular disulphide bonds (Fig. 5C). On the contrary, the decay of the WhiBTM4 Fe-S cluster could be enhanced by the addition of 10 mM oxidized glutathione (GSSG) indicated by a faster drop of the peak at A423 (Fig. 5C). The characteristic light absorptions of [2Fe-2S] clusters are generated by sulfur-Fe3+ charge transfer transitions (Messerschmidt, 2001). Thus, a brown coloured [2Fe-2S]2+ cluster can be reduced with the reducing agent dithionite to a [2Fe-2S]+ cluster which results in visible bleaching of protein and a characteristic change in the UV-visible absorption spectrum (rapid loss of the peak at A423 and appearance of a peak at A322). This UV-visible change of charge rather than disassembly of cluster could also be observed in WhiBTM4 (Fig. 5C).

Recently it was shown that fully oxygenated WhiB4 (apo-WhiB4) of M. tuberculosis is capable of co-ordinating an [4Fe-4S] cluster under semi-anaerobic conditions in the presence of DTT (Alam et al., 2007). During anaerobic incubation of colourless apo-WhiBTM4 with FeCl3, Na2S and DTT, a characteristic brown colour developed within three to 4 hours. The peak pattern of the UV-visible absorption spectra of this reconstituted WhiBTM4 was similar to that of freshly purified protein indicating that apo-WhiBTM4 can be reconstituted to a [2Fe-2S] cluster (Fig. S1). During anaerobic incubation and dialysis, a substantial amount of protein had aggregated. However, the A423/A280 ratio of reconstituted WhiBTM4 was higher than the ratio of the freshly purified cluster and with 0.39 this ratio indicates the presence of an intact [2Fe-2S] cluster (Rouhier et al., 2007). This was mirrored by the high number of iron atoms per monomer/dimer of WhiBTM4 which was 1.13 and 2.26, respectively, as determined by atom absorption spectroscopy (Table 1).

Cluster degradation and anaerobic reconstitution of WhiB2Mtb

In contrast to data on host derived WhiB proteins (Alam et al., 2009), cluster degradation through atmospheric oxygen in WhiBTM4 is a matter of minutes rather than hours. We therefore studied the kinetics of the host derived WhiB2-cluster when exposed to oxygen. WhiB2 of M. tuberculosis was isolated under native conditions. In our hands, freshly purified WhiB2Mtb showed a WhiBTM4-like spectroscopic behaviour with a relatively rapid cluster loss under atmospheric oxygen (Fig. S2B). Furthermore, reconstitution of this protein in an anaerobic atmosphere led to the formation of a [2Fe-2S]-like UV-visible absorption spectrum indicating that, like apo-WhiBTM4, apo-WhiB2Mtb can be reconstituted to a [2Fe-2S] cluster protein (Fig. S2C).

WhiB2Mtb and WhiBTM4 specifically bind WhiB2Mtb promoter DNA

Since the presence of WhiBTM4 leads to a downregulation of WhiB2, we were interested in DNA binding properties of the proteins. In both M. smegmatis and M. tuberculosis a highly identical promoter sequence can be found 126 bp (M. smegmatis) or 226 bp (M. tuberculosis) upstream of the respective whiB2-gene-start codon (Fig. 6A). Apo-WhiB2Mtb and apo-WhiBTM4 were generated and complete loss of cluster was confirmed by UV-Vis spectroscopy. Proteins were pre-incubated with biotin-labelled WhiB2-promoter DNA and protein-DNA complexes were examined by electrophoretic mobility shift assays (EMSA). Figure 6B and C shows that apo-WhiB2Mtb as well as apo-WhiB2TM4 generated a DNA complex with retarded mobility. No shift was observed when the proteins were incubated with biotin-labelled Epstein-Barr virus promoter DNA or M. tuberculosis Ag85A coding DNA showing that binding is sequence specific (Fig. 6D and E). We note that increasing the protein concentration and decreasing the concentration of poly-dI-dC DNA leads to the formation of a faint shifted band in these controls; however, in these experiments the whiB2-promoter protein/DNA complex is clearly more distinct than the controls (not shown). DNA-binding of both proteins could be competed by the addition of a 200-fold molar excess of unlabeled promoter DNA (competitor 1) (Fig. 6A–C). This phenomenon was used to identify the protein binding site within the promoter sequence by competing DNA binding with small unlabeled oligo duplexes 15–16 bp in size (competitor 2–4, Fig. 6A). The addition of a 200-fold molar excess of competitor 2, but not competitor 3 or 4 inhibited the formation of a DNA complex with retarded mobility showing that WhiB2Mtb and WhiBTM4 specifically bind to the 5′ end of the WhiB2 promoter (Fig. 6F and G).

Figure 6.

Electrophoretic mobility shift assay (EMSA).
A. Promoter sequence upstream of the whiBMtb/whiB2Ms genes. Sequence in bold letters was biotinylated, annealed and used for EMSA experiments, the line segments below show the sequences of unlabeled oligo duplex competitors used in competition experiments.
B. Different concentrations of apo-WhiB2Mtb were incubated with 20 fmol of biotin labelled WhiB2Mtb promoter DNA. The mobility of promoter DNA is retarded in the presence of WhiB2 protein, the underlying Protein/DNA complex can be resolved in the presence of a 200-fold molar excess of unlabeled promoter DNA.
C. A similar shift of labelled DNA can be observed in the presence of apo-WhiBTM4. D/E. WhiB2Mtb and WhiBTM4 were incubated with 20 fmol of biotin labelled WhiB2Mtb promoter DNA, Epstein-Barr nuclear antigen promoter DNA (EBNA) and M. tuberculosis Ag85A coding sequence as controls. Only in the presence of WhiB2Mtb promoter DNA a shifted DNA complex can be observed.
F/G. DNA binding of the two proteins is inhibited in the presence a 200-fold molar excess of unlabeled competitor 2 but not through the addition of competitor 3 or 4 showing that the specific binding site is situated within the 5′-end of the promoter sequence. Individual experiments were performed at least three times to rule out pipetting errors.

Both the conserved cysteines and the C-terminal HTH motif are involved in the WhiBTM4 growth inhibitory effect

To show that growth inhibition induced by WhiBTM4 is not due to a nonspecific effect such as the accumulation of large amounts of protein aggregates, we tried to identify amino-acid substitutions allowing normal growth of M. smegmatis cells. Spontaneous resistance towards the growth inhibitory effect in M. smegmatis clones expressing WhiBTM4 occurred at a rate of 5x10−3. We created a plasmid library expressing mutated WhiBTM4 proteins and screened for clones with uninhibited growth when expressing proteins harbouring sensitive mutations. Cells transformed with the pSD24WhiBTM4M-mutant library and plated on 7H10 containing 0.2% acetamide resulted in an acetamide resistant phenotype at a rate of 1:3. Two dominant phenotypes could be observed on these plates: approximately 50% of the colonies were relatively large and white resembling wild-type M. smegmatis colonies and approximately 50% were somewhat smaller colonies with a red to orange colour (Fig. 7A). Colony-PCR of 100 individual clones amplifying the insert of pSD24 and subsequent sequencing of the products showed that approximately 50% of the white colonies had been selected for re-ligated plasmids without insert. Most of the remaining white colonies had mutations resulting in the insertion of stop codons upstream or within the cluster co-ordinating cysteines (not shown). However, two mutants had single amino acid exchanges of the cysteine at position 39 (Fig. 7B). All plasmids of the red colonies had mutated WhiBTM4 proteins and approximately 50% of these showed single amino acid exchanges resulting in cytotoxicity resistance. These mutations clustered in the C-terminal HTH motif of WhiBTM4 (Fig. 7B). However, we note that six of the 17 single amino acid mutations inducing cytotoxicity resistance affected non-cysteine and non-HTH motif amino acids (Fig. 7B). The role of these amino acids in the function of WhiBTM4 and other WhiB proteins is not clear. Among the orange colonies we found a C45S substitution of the most C-terminal cysteine and C36S/Y substitution. No cytotoxicity resistant clone harbouring mutated C13 (the most N-terminal cysteine) could be recovered from the screen. In WhiB2 this cysteine was shown to be non-essential for the complementation of a WhiB2 mutant which might be an explanation for the lack of this mutation in our cytotoxicity resistance screening (Raghunand and Bishai, 2006b).

Figure 7.

A. Macroscopic appearance of M. smegmatis colonies expressing mutated WhiBTM4 species plated on 7H10 agar plates containing 0.2% acetamide. The red/orange phenotype of the V58E mutation is shown as an example. Cells expressing WhiBTM4 with a C39F mutation have the wild-type appearance of cells carrying pSD24 only.
B. Single amino acid mutations allowing WhiBTM4 expression in M. smegmatis and their colour (O = orange, W = white). The C-terminal HTH motif and conserved cysteines are highlighted.

Mutated Genes containing the C36S, C39F or C45S amino acid substitution were amplified by PCR and cloned into pQE80 for expression and UV-visible absorption spectroscopy. Only small amounts of protein carrying the C39F mutation could be purified and there was no UV-visible absorption indicating the presence of a Fe-S cluster (Fig. S3A and B). This mutation seems to destabilize the protein to an extent that prevents sufficient expression indicating the vital role of this amino acid and explaining the white colour of M. smegmatis clones expressing this mutated gene. Protein destabilization is a common observation in Fe-S proteins carrying sensitive cysteine mutations (Klinge et al., 2007). The C45S and C36S mutations allowed protein expression and purification comparable to wild-type protein and the eluate of these mutated proteins showed a faint brown colour. UV-visible absorption spectra indicated the presence of a Fe-S cluster with a low A423/A280 ratio (Fig. S3A). Compared with the wild-type spectrum, the shoulder at 462 nm was more prominent in the C45S mutant and less prominent in the C36S mutant. Disassembly of the mutated clusters under atmospheric oxygen was approximately twice as fast compared to wild-type protein (not shown).

Mutations of the C-terminal HTH motif affect WhiBTM4 DNA-binding

Since amino acid substitutions of WhiBTM4 allowing normal growth of M. smegmatis clustered in the C-terminal part of the protein we were interested in DNA-binding properties of these mutated proteins. Six of these proteins were purified and examined in EMSA-experiments using labelled WhiB2 promoter DNA. Four proteins showed low binding affinity compared to the wild-type protein whereas the V56D substitution abolished DNA binding of WhiBTM4 (Fig. 8). WhiBTM4 with a A49V substitution seemed to be a better DNA-binder than the wild-type protein; however, this protein expressed poorly in E. coli and low expression may also be the reason for unaffected growth of M. smegmatis in the presence of this altered protein. We note that WhiBTM4 proteins with substitutions of the cysteines at position 36 or 45, which were also tested in this experiment, had no effect on DNA binding (Fig. 8).

Figure 8.

Electrophoretic mobility shift assay (EMSA) of mutated WhiBTM4. WhiBTM4 proteins with single amino acid substitutions situated in the C-terminal HTH motif were incubated with labelled WhiB2Mtb promoter DNA. The L55Q, V58E and R67C substitutions led to decreased binding affinity compared with the wild-type protein whereas the V56D substitution abolished DNA binding of WhiBTM4. WhiBTM4 with a A49V substitution seemed to be a better DNA-binder than the wild-type protein. This protein expressed poorly in E. coli (SDS-PAGE below EMSA film). Substitutions of the cysteines at position 36 and 45 had no effect on DNA binding. The experiment was performed three times to rule out pipetting errors.

WhiBTM4 is dispensable for plaque formation of phage TM4

In the host the WhiBTM4 homologue WhiB2 is an essential protein. To further characterize WhiBTM4 we were interested in the phenotype of a WhiBTM4 mutant. A whiBTM4-gene deletion was performed by cloning the whiBTM4 flanking regions carrying MfeI recognition sites into MfeI cut TM4 genomic DNA (Fig. 9A and B). After transformation the gene deletion was confirmed by plaque-PCR using the forward primer I30 which anneals to gene 48 and the reverse primer I31 (Table S3) which anneals to gene 50. The deletion leads to smaller PCR products compared with PCR performed on wild-type phage (Fig. 9B). Furthermore, primers 49for and 49rev (Table S3) were used to show that whiBTM4 is missing in the deletion mutant (Fig. 9B). Mutant and wild-type phage showed similar plaque morphology indicating that WhiBTM4 is dispensable for phage growth (Fig. 9C). We also determined the average number of particles per plaque for both, mutant and wild-type phage (n = 10 plaques). In the mutant we counted 5.4 × 107 (standard deviation 3.1 × 107) particles per plaque and 7.4 × 107 (standard deviation 3.7 × 107) particles per plaque for the wild-type phage. Using student's t-test this difference was not significant (P = 0.195, 95% confidence interval) indicating that there is no decrease in phage fitness caused by the whiBTM4 deletion.

Figure 9.

A. Strategy for creating a whiBTM4 deletion mutant. PCR products of primers I25/I26 and I28/I29 were sequentially cloned in pQE80 giving pJRI25I28. Phage DNA and pJRI25I28 were MfeI cut, DNA pieces were gel purified, ligated and transformed in M. smegmatis.
B. Phage TM4 DNA (lane 1) and pJRI25I28 (lane 2) digested with MfeI, the labelled bands were excised, purified and ligated. The gene deletion was confirmed by PCR using wild-type TM4 (lanes 3, 6), mutant (lanes 4, 7) and pJRI25I28 (lanes 5, 8) as templates. Primers I30/I31 (lanes 3–5) anneal to the flanking genes of whiBTM4 thus the PCR product of the mutant phage is approximately 260 bp smaller compared with the wild-type PCR product. Primers 49for/49rev (lanes 6–8) amplify whiBTM4.
C. Plaque morphology of mutated and wild-type phage.

The presence of WhiBTM4 induces superinfection exclusion

Since WhiBTM4 is non-essential for proper plaque formation in top-agar containing M. smegmatis cells we were wondering whether the expression of sub-toxic levels of WhiBTM4 allows TM4 an exclusive utilization of its host. Interestingly, we have recently identified another early phage protein of mycobacteriophage L5 that leads to a similar phenotye when expressed in the host bacterium M. smegmatis (Rybniker et al., 2008). The alteration of cell wall components early in the infectious cycle may prevent infection of M. smegmatis with additional TM4 particles or other mycobacteriophage species, a phenomenon known as superinfection exclusion. Full expression of WhiBTM4 was lethal for the host. Uninduced cells grew relatively slow due to leakiness of pSD24 (Rybniker et al., 2008) which leads to basal transcription of whiBTM4-specific RNA (Table S1B). Spotting phage lysate on topagar containing these cells did not lead to the formation of single clear plaques indicating a TM4 resistant phenotype (Fig. 10A). Thus, a function of the early cell shape altering protein WhiBTM4 may be found in superinfection exclusion. This mechanism seems not to involve altered binding of phage particles. An adsorption assay performed with wild-type M. smegmatis, M. smegmatis carrying pSD24WhiBTM4 or bacteria expressing mutated WhiBTM4 species gave similar results for the different hosts (Fig. 10B).

Figure 10.

A. Plaque formation on M. smegmatis mc2155 carrying no plasmid, pSD24, pSD24WhiBTM4 or the mutants pSD24WhiBTM4V58E and pSD24WhiBTM4C36S. In contrast to TM4 grown on the native host, there is no zone of clear plaques visible in cells expressing basal levels of WhiBTM4. Only at the lowest dilution some turbid plaques are visible. Phage grown on bacteria expressing mutated proteins show single turbid plaques at the highest dilution. Mycobacteriophage D29, another lytic mycobacteriophage, forms small clear plaques at the highest dilution.
B. Adsorption assay of phage TM4 incubated with different M. smegmatis transformants. Cells in the exponential growth phase were incubated with TM4 at a MOI (multiplicity of infection) of 0.0002. Unbound phage particles were rescued at different time points. There is no significant difference in phage adsorption between the examined transformants. Experiments were performed in triplicates.


This work identifies and characterizes the bacteriophage derived Fe-S protein WhiBTM4. To our knowledge this is the first description of a viral protein capable of co-ordinating a Fe-S cluster. Since the sequence of WhiBTM4 is highly identical to the WhiB-protein family of actinomycetes, functional interpretation of this protein also requires substantial knowledge of its bacterial counterparts.

Sequence comparison of WhiBTM4 with the seven WhiB proteins of mycobacteria revealed high sequence identity in 66 amino acids of the C-terminal region of WhiB2 of M. tuberculosis and M. smegmatis, a protein that is essential for proper septation and cell division (Fig. 1). This C-terminal part represents the active part of WhiB2 which is capable of rescuing a WhiB2Ms deletion mutant (Raghunand and Bishai, 2006b). Our data show that WhiBTM4 expression leads to downregulation of WhiB2 with subsequent filamentation and growth inhibition. The presence of the Fe-S co-ordination cysteines is mandatory for this phenotype. Furthermore, cells expressing the TM4-protein showed a loss of acid fast properties indicating a WhiBTM4 mediated modulation of cell wall lipids (Fig. 2).

We show here, for the first time, that a phage derived WhiB protein and the essential host protein WhiB2 are capable of binding DNA. Both proteins specifically bound the promoter DNA upstream of the whiB2 gene, strengthening the hypothesis that WhiB proteins are important transcriptional regulators (Fig. 6). Mycobacteriophage TM4 has a broad host range and is lytic in most of the mycobacteria it infects. In contrast to other mycobacteriophages such as L5 and Bxb1, TM4 infection does not lead to a rapid and global shut-off of host protein synthesis immediately after infection (Ford et al., 1998). SDS-PAGE of mycobacterial proteins after infection of the cells with TM4 showed that host gene expression is unchanged throughout the late phase of the cycle which starts approximately 60 min after infection (Ford et al., 1998). This observation is supported by our qRT-PCR experiments where 16S rRNA and HSP60-mRNA-levels were stable during TM4 infection (Fig. 3A). However, whiB2-mRNA levels clearly declined. Since whiB2 is an essential host gene and WhiBTM4 expression is growth inhibitory, the protein functions as a more subtle shut-off protein. Our findings clearly establish WhiBTM4 as a dominant negative regulator of WhiB2.

We also found that host cells expressing WhiBTM4 at a basal, sub-toxic level are resistant to TM4 infection. This implies a function of WhiBTM4 in superinfection exclusion. Though well known in T-even phages of E. coli, superinfection exclusion genes have not yet been identified in lytic mycobacteriophages. In the E. coli phage T4 the Imm protein induces resistance to most T-even phages but not to phage lambda (Lu and Henning, 1994). The extremely lipophilic Imm protein is associated with the cell membrane and is believed to inhibit the transfer of phage-DNA across the cytoplasmic membrane. It is likely that WhiBTM4 alters the host cell wall in such a way that superinfection by TM4 or other mycobacteriophages is impossible. Since WhiB proteins do not contain membrane spanning domains and are believed to be cytoplasmic proteins, this alteration is most likely achieved by gene regulation rather than direct interaction with the bacterial membrane. Here WhiBTM4 could represent a truncated, non-functional homologue of WhiB2 which acts as a transcriptional antagonist displacing WhiB2 from its promoter region and leading to down regulation of the host protein with subsequent alterations of the cell wall composition. Superinfection exclusion guarantees genetic diversity of phage populations by decreasing the degree of selection for dominant phage species co-infecting hosts or lysogens (Lu and Henning, 1994). Thus in mycobacteriophages, the presence of genes causing superinfection exclusion is consistent with their extraordinarily high genetic diversity.

However, the observed septation deficiency and superinfection exclusion could also be caused by the perturbation of protein levels due to the presence of a fully functional WhiB2 homologue (WhiBTM4). Wild-type bacteria seem to be highly sensitive towards the alteration of WhiB2 levels and the overexpression of WhiB2Ms in M. smegmatis leads to filamentation and a decrease in viability (Raghunand and Bishai, 2006a).

Another mycobacterial WhiB protein involved in the regulation of cell wall components is WhiB3. This protein has been shown to co-ordinate an oxygen sensitive [4Fe-4S] cluster with the help of the M. tuberculosis cysteine desulfurase IscS (Singh et al., 2007). We provide evidence that WhiBTM4 and WhiB2 co-ordinate a [2Fe-2S] cluster when kept in an oxygen depleted atmosphere (Figs 5, S1, S2B/C). We cannot rule out that WhiBTM4 may build up a [4Fe-4S] cluster in vivo, possibly with the help of a host protein such as IscS. Despite several trials under strict anaerobic conditions using different reducing agents such as dithionite and sodium aspartate, we were not able to generate an electroparamagnetic resonance (EPR) signal from either freshly purified or reconstituted WhiBTM4. However, the UV-visible absorbance of WhiBTM4 contained features that are entirely consistent with those of other freshly purified [2Fe-2S] cluster carrying WhiB proteins of the host and those from well-defined [2Fe-2S] cluster co-ordinating proteins such as the ferredoxins (Jakimowicz et al., 2005; Alam et al., 2007; 2009).

A recent publication identified WhiB3 as a regulator of virulence lipid synthesis, most likely by binding to promoter regions upstream of polyketide synthases (Singh et al., 2009). Here, apo-WhiB3 was a much better DNA-binder than the cluster carrying holo-WhiB3 indicating that WhiB3 is a redox-responsive regulatory protein. Additionally, in WhiB3 the conserved cysteines are not only mandatory for Fe-S cluster formation but also for the formation of intramolecular disulfide-bonds after cluster loss. It is the formation of these bonds that enables apo-WhiB3 to form a strong protein-DNA complex (Singh et al., 2009). This mechanism stands in contrast to that of other well described Fe-S proteins such as FNR of E. coli. In FNR it rather is the presence of a [4Fe-4S] cluster that activates the expression of genes through binding of the C-terminal HTH motif to specific DNA sequences (Lazazzera et al., 1996; Khoroshilova et al., 1997). Cluster loss through oxygenation leads to the formation of apo-FNR, a biologically inactive molecule without DNA-binding properties (Lazazzera et al., 1996).

It is an intriguing finding that a viral protein expressed early in the infectious cycle is capable of sensing hypoxia, normoxia and the presence of reducing or oxidizing agents with the help of its Fe-S cluster. It is rather unlikely that WhiBTM4 undergoes the cycle of cluster assembly, loss and re-assembly as part of a redox sensing machinery during the brief presence of the protein in the early stage of phage infection. Redox sensing is not considered to be an essential part of the viral infectious cycle. Since it is the apo-forms of WhiB3, WhiB2 and WhiBTM4 that are capable of binding DNA, the Fe-S cluster in WhiBTM4 could rather be a by-product engendered by the presence of four highly conserved cysteines. Here, the cysteine mediated formation of intramolecular disulfide bonds is the prerequisite for DNA-binding and transcriptional regulation. In this scenario the formation of the observed Fe-S cluster is secondary, which may be mirrored in the relative fast cluster loss we observed. Recently published data on the WhiB2 kinetics of cluster degradation through atmospheric oxygen showed a relatively slow cluster loss over a period of 48 h (Alam et al., 2009). This stands in contrast to WhiBTM4 where cluster loss is a matter of minutes. Thus, we hypothesized that WhiB2 downregulation by WhiBTM4 may be achieved through a faster transformation of [2Fe-2S]-WhiBTM4 to apo-WhiBTM4. However, in our hands WhiB2Mtb showed a WhiBTM4-like spectroscopic behaviour under atmospheric oxygen (Fig. S2).

Eight of the 17 mutations leading to normal bacterial growth during WhiBTM4 expression were situated in the predicted HTH DNA-binding motif represented by the most C-terminal α-helices and an exposed β-turn. We were able to show a decrease in promoter DNA-binding affinity of WhiBTM4 proteins carrying amino acid substitutions in this conserved HTH motif (Fig. 8). These facts strengthen the theory that WhiB proteins transmit their information using this C-terminal structure (Soliveri et al., 2000). In contrast to the highly conserved β-turn, the α-helices are less well conserved among the different WhiB proteins and might have different targets explaining the versatile functions of these otherwise nearly identical proteins.

It was surprising to us that the whiBTM4 gene is non-essential for proper plaque formation (Fig. 9). It was speculated by others that large portions of the TM4 genome are non-essential due to the relative ease with which foreign genetic elements can be introduced into the TM4 genome (Ford et al., 1998). However, these dispensable genes were expected to be situated downstream of gene 71 and not in the whiBTM4 (gene 49) region. It is well known that up to 50% of the genes of extensively studied bacteriophages such as T7 are non-essential for plaque formation. These genes, although dispensable for phage growth under laboratory conditions, bear meaningful functions and some of them are known to interact with and inhibit host proteins (Kim and Chung, 1996).

The systematic sequencing of more than 30 mycobacteriophages identified a large amount of interesting genetic data spanning a diverse range of gene families such as modified host-derived genes, clearly bacteriophage derived genes and a substantial number of genes with no match in the database (Pedulla et al., 2003; Hatfull et al., 2006). The analysis of phage genes that are homologous to, but not necessarily identical to host genes is a pioneering way to dissect the function of poorly understood genes of the host. Here we show that the expression and analysis of a phage derived protein provides information not only on the phage itself, but also on essential bacterial proteins. Particularly with regard to the potential drug target WhiB2, we believe that our findings may provide a new tool to target the major pathogen M. tuberculosis.

Experimental procedures

Plasmids, primers and bacterial strains

A description of the plasmids, primers and bacterial strains used in this study is given in Tables S2 and S3. A description of the bacteriophage used in this study is given in Table S2.

Bacteriophage methods

Propagation of phage and spot assays were performed as described recently (Rybniker et al., 2006). For spot assays on bacteria carrying pSD24 (Daugelat et al., 2003) or its derivatives cells were grown in 7H9 medium with 50 µg ml−1 hygromycin B. Cells were then harvested by centrifugation, washed and plated in top agar without hygromycin B.

Expression of WhibTM4 in M. smegmatis and microscope techniques

whibTM4 (ORF49) was amplified by PCR using the primer pair 49for and 49rev (Table S3) and TM4-DNA as a template. The product was cloned into BamHI cut pSD24 using the In-Fusion PCR cloning technique (Clontech). After transformation into the E. coli Fusion Blue strain the plasmid pSD24WhibTM4 was isolated, sequenced and transformed into M. smegmatis mc2155. Clones were grown on Middlebrook 7H10 agar containing 10% ADC and 50 µg ml−1 hygromycin B. For microscopy studies single colonies were picked and grown in 7H9 broth supplemented with 10% ADC, 0.05% Tween 80 and 50 µg ml−1 hygromycin B until an OD of 1 at A600 was reached, then cells were diluted 1:10 v/v in 7H9 broth containing 0.02% acetamide. Cells were collected at different time points, washed in sterile water and stained.

The TB stain kit (Becton Dickinson) was used for Ziehl-Neelsen acid fast staining. For fluorescence microscopy cells were washed in ultra pure water, the pellets were resuspended in 500 µl SYTO 11 (Molecular Probes) at a final concentration of 5 µM and incubated at 37°C for 30 min. Cells were then washed several times in water until the supernatant was colourless. A total of 20 µl of cells were mounted on microscope slides in ProLong Gold Antifade Reagent (Invitrogen). Images were acquired with an inverted Olympus IX81 microscope (Olympus) equipped with a F-View II Trigger camera and then analysed by analySISD software (Soft Imaging Software, Muenster, Germany).

RT-PCR for the quantification of gene expression during phage infection

Mycobacterium smegmatis mc2155 was grown to late log phase in 7H9 medium containing 10% ADC and 1 mM CaCl2 without Tween 80. The cells were centrifuged and the pellets resuspended in warm 7H9 medium with 1 mM CaCl2 but without ADC. Larger clumps were allowed to settle for 5 min and cells in the supernatant were adjusted to an OD of 0.1 at A600 in a total volume of 0.8 litres 7H9 medium with 1 mM CaCl2. (approximately 2 × 1010 cells). Mycobacteriophage TM4 was added at a multiplicity of infection (MOI) of 10 and the cells were shaken slowly at 60 rpm at 37°C. Cells were harvested in 100 ml portions at different time points by centrifugation for 3 min (4000 × g) at 4°C, the pellets were shock frozen and stored at –80°C. Mycobacterial RNA was isolated using the RNeasy MIDI-kit (Qiagen) including the on-column DNAse digestion step. RNA (1 µg) was reverse transcribed with the QuantiTect Reverse Transcription kit (Qiagen). Real-time PCR was performed using the QuantiFast SYBR green PCR kit (Qiagen) on a GeneAmp 5700 sequence detection system (Applied Biosystems). Primers were H27/H28 for the detection of whiBTM4, H23/H24 for the mycobacterial 16S rRNA, H25/H26 for HSP60 and H31/H32 for the detection of whiB2 of M. smegmatis (primer sequences are listed in Table S3). Genomic DNA was used to generate a standard for the mycobacterial genes and pSD24WhiBTM4 to standardize the amount of whiBTM4.

Purification of WhiBTM4 and WhiB2Mtb

whiBTM4 and whiB2Mtb were PCR-amplified using the primer set Q8049for/Q8049rev or Q80WBfor/Q80WBrev (Table S3), respectively, and cloned into the expression plasmid pQE80 (Qiagen) in frame with the hexa-histidine tag. Escherichia coli BL21 (DE3) cells were transformed with the respective plasmids and grown in LB-broth containing 100 µg ml−1 ampicillin to an OD of 0.6 at A600. Expression was induced with 0.5 mM IPTG for 2 h at 30°C without shaking. For purification under native conditions, a cell pellet from a 800 ml culture was resuspended in 20 ml buffer A (200 mM NaCl, 50 mM NaH2PO4, 30 mM imidazol, 10% glycerol, pH 8.0) plus EDTA-free protease inhibitor cocktail (Roche). Cells were disrupted in a FRENCH press (Thermo scientifc) at 1250 psi; the soluble lysate was separated by centrifugation at 17 000 × g for 15 min at 4°C and loaded on a Ni2+-NTA affinity column (HisTrap FF crude, GE-Helthcare). The column was washed with 15 volumes of buffer A and eluted with buffer B (200 mM NaCl, 50 mM NaH2PO4, 300 mM imidazole, 10% glycerol, pH 8.0) on an ÄKTA-FPLC system using the UNICORN workstation software (GE-Healthcare). For purification under denaturing conditions the lysis/wash-buffer was buffer C (8 M urea, 100 mM NaH2PO4, 10 mM Tris-HCL, 30 mM imidazole, pH 8.0) and elution-buffer D (8 M urea, 100 mM NaH2PO4, 10 mM Tris-HCL, 500 mM imidazole, pH 8.0). All buffers were extensively vacuum degassed and purged with nitrogen. Eluted protein was immediately examined by spectroscopy or frozen in liquid nitrogen for storage at −80°C. Protein concentrations were determined with the Compat-Able BCA protein assay and the Pierce 660 nm protein assay (Thermo scientific). Colorimetric tests were performed with apo-WhiBTM4 or apo-WhiBMtb.

Electrophoretic mobility shift assays (EMSA)

Electrophoretic mobility shift assays were performed using the lightshift assay (Pierce) following the recommendations of the manufacturer. In brief, native WhiB2Mtb or WhiBTM4 were desalted using zeba spin columns (Pierce) and incubated with 20 fmol of 5′-biotinylated and annealed WhiB2Mtb promoter DNA (Sigma Aldrich) in binding buffer supplemented with 2% glycerol, 5 mM MgCl2, 0.5% NP-40, 10 mM diamide, 0.5% Tween 20 and 10 ng µl−1 poly dI-dC-DNA for 30 min. The reactions were separated using a 4–20% gradient TBE PAGE gel (Invitrogen) and 0.5x TBE buffer as running buffer. DNA was transferred on a nylon membrane and biotinylated DNA was detected by chemoluminescence through exposure to an X-ray film for approximately 2 min.

Reconstitution of the Fe-S cluster

Fully oxygenized protein (0.8 mg ml−1) purified under denaturing conditions was dialysed against buffer E (150 mM NaCl, 50 mM Tris-HCL, 10 mM DTT, 100 µM FeCl3, 100 µM Na2S, pH 7.5) for 12 h at 22°C. The dialysis cassette was rinsed with buffer F (200 mM NaCl, 50 mM Tris-HCL, 10 mM DTT, pH 7.5) and dialysed against buffer F at 4°C for 12 h. Again, all buffers were degassed and nitrogen purged. The dialysis steps were performed under strict anaerobic conditions using anaerobic jars that were depleted from oxygen with an Anoxomat system (Mart Microbiology, Netherlands) which evacuates atmospheric oxygen from the jar and refills with an anaerobic gas mixture (nitrogen 85%, CO2 10%, hydrogen 5%).

Random mutagenesis of WhibTM4

Low fidelity PCR was performed using the primer pair 49for/49rev (Table S3) and pSD24WhiBTM4 as a template. A 50 µl standard PCR reaction was modified by the addition of 320 µM MgSO4 and 80 µM dGTP (final concentration) and 1 µl of the diversify dNTP-mix (Clontech). Cycle conditions were 94°C for 1 min followed by 25 cycles of 30 s at 94°C and 1 min at 68°C. Using this setting, the mutational bias is approximately 2.7 mutations per 1000 bp. The PCR products were In-Fusion-cloned into BamHI linearized pSD24 and transformed into E. coli Fusion-Blue. Approximately 104 individual clones were pooled and plasmids isolated (pSD24WhiBTM4M). Approximately 0.5 µg of this randomly mutagenized plasmid library was transformed into M. smegmatis mc2155 and clones were plated on 7H10 plates with and without 0.2% acetamide. Insertions from mutants growing on 0.2% acetamide were sequenced and the sequences were aligned using ClustalW software. To confirm the acetamide resistant phenotype, all mutated whiBTM4 genes were re-amplified under stringent PCR conditions and the products were cloned into pSD24. The resulting plasmids were transformed into M. smegmatis and the transformants plated on 7H10 plates containing 0.2% acetamide. The inserts of these plasmids were sequenced again as a further proof for a relevant mutation.

Construction of a whiBTM4 gene deletion mutant

The TM4 genome carries two MfeI recognition sites at position 28.578 and 36.370 (Fig. 9A). These sites flank the whiBTM4 gene. Two PCR products were created: one product [primers I25 and I26 (Table S3)] spanning the recognition site at position 28.578 and the gene upstream of whiBTM4 (gene 48) and a second product [primers I28 and I29 (Table S3)] spanning gene 50 and the downstream recognition site at position 36.370. These products were sequentially cloned into pQE80, the insert was excised and ligated into gel purified MfeI cut TM4 genomic DNA. The ligation reaction was transformed in M. smegmatis mc2155. The resulting plaques were plaque purified and the gene deletion was confirmed by PCR using primers 49for/49rev and I30/I31 (Table S3).

Other analytical techniques

For the quantification of iron by atomic absorption spectrometry (AAS) the WhiBTM4 samples were treated with HNO3 and diluted in an appropriate amount of ultrapure water. Samples were analysed on an AAnalyst 800 (Perkin Elmer).

UV-visible absorption spectra were recorded on a Lambda 40 spectrophotometer (Perkin Elmer).

The PMF was performed as described recently (El Mourabit et al., 2004). In brief, proteins were pre-treated and trypsin digested at 37°C over night. The digest was stopped by the addition of 5–20 µl 1% trifluoraceticacid (TFA) in water and peptides were extracted for 30 min at 37°C. Positive ion spectra were acquired on a Reflex IV MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) in the reflectron mode. A peptide calibration standard (Bruker Daltonics, Bremen, Germany) was used for external calibration of the mass range from m/z 1046 to m/z 3147. The FlexAnalysis postanalysis software was used for optional internal recalibration on trypsin autolysis peaks and the generation of peaklists. Biotools 3.0 (Bruker Daltonics, Bremen, Germany) was used for interpretation of mass spectra with regard to the expected sequence of recombinant 6xHIS-tagged WhiBTM4.

Gelfiltration was performed using a superdex 75 column (GE Healthcare) operated at a flow rate of 0.05 ml min−1 with 100 mM NaCl, 50 mM Tris pH 8.0. The column was calibrated using a low molecular weight protein standard (GE Healthcare).


J.R. and P.H. are supported by the German Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF) grant #01 KI 0771. We thank Stefan H. E. Kaufmann at the Max Planck Institute for Infection Biology Berlin for providing pSD24. Mycobacteriophage TM4 and D29 were kind gifts of Graham F. Hatfull at the University of Pittsburgh. Also we thank Werner Falk at the University of Regensburg and Eckhard Bill of the Max Plank Institute for Bioanorganic Chemistry for their valuable technical advice. We thank Frank Seifert of the IMMIH for help with the AAS. Eva Glowalla of the IMMIH was of great help during protein purification steps. We are in debt of Elizabeth Schell-Frederick and Karin Schnetz for valuable critical comments on the manuscript.