Mycobacterium smegmatis pafbc is involved in regulation of dna damage response

ABSTRACT Two genes, _pafB_ and _pafC_, are organized in an operon with the Pup-ligase gene _pafA_, which is part of the Pup-proteasome system (PPS) present in mycobacteria and other

actinobacteria. The PPS is crucial for _Mycobacterium tuberculosis_ resistance towards reactive nitrogen intermediates (RNI). However, _pafB_ and _pafC_ apparently play only a minor role in

RNI resistance. To characterize their function, we generated a _pafBC_ deletion in _Mycobacterium smegmatis (Msm)_. Proteome analysis of the mutant strain revealed decreased cellular levels

of various proteins involved in DNA damage repair, including recombinase A (RecA). In agreement with this finding, _Msm_ Δ_pafBC_ displayed increased sensitivity to DNA damaging agents. In

mycobacteria two pathways regulate DNA repair genes: the LexA/RecA-dependent SOS response and a predominant pathway that controls gene expression via a LexA/RecA-independent promoter, termed

P1. PafB and PafC feature winged helix-turn-helix DNA binding motifs and we demonstrate that together they form a stable heterodimer _in vitro_, implying a function as a heterodimeric

transcriptional regulator. Indeed, P1-driven transcription of _recA_ was decreased in _Msm_ Δ_pafBC_ under standard conditions and induction of _recA_ expression upon DNA damage was strongly

impaired. Taken together, our data indicate an important regulatory function of PafBC in the mycobacterial DNA damage response. SIMILAR CONTENT BEING VIEWED BY OTHERS NOVEL WYL

DOMAIN-CONTAINING TRANSCRIPTIONAL ACTIVATOR ACTS IN RESPONSE TO GENOTOXIC STRESS IN RAPIDLY GROWING MYCOBACTERIA Article Open access 02 December 2023 AN RNA-BINDING PROTEIN ACTS AS A MAJOR

POST-TRANSCRIPTIONAL MODULATOR IN _BACILLUS ANTHRACIS_ Article Open access 21 March 2022 INTRACELLULAR LOCALIZATION OF THE MYCOBACTERIAL STRESSOSOME COMPLEX Article Open access 12 May 2021

INTRODUCTION _Mycobacterium tuberculosis_ (_Mtb_), the causative agent of tuberculosis, is one of the most successful human pathogens and represents a major global health problem. _Mtb_

features a multitude of mechanisms that allow the phagocytozed bacteria to withstand the host’s innate and adaptive immune response and enable them to persist inside the host macrophages1,2.

After successful immune evasion the pathogen can persist in the human for decades. A hallmark of activated macrophages is the production of reactive oxygen species and nitric oxide, which

damage bacterial proteins, lipids and nucleic acids3,4. Thus, in order to maintain the integrity of its genome, _Mtb_ relies on robust mechanisms of DNA damage repair. Whereas in most

bacteria the genes involved in these processes are regulated as part of the so-called SOS response, mycobacteria were shown to make use of an additional, independent regulatory

mechanism5,6,7,8. In the SOS response pathway the key regulators are LexA and RecA. Under standard growth conditions the repressor LexA prevents transcription of DNA repair genes by binding

to a specific sequence within their promoter, the SOS box9. RecA senses DNA damage by binding to single-strand DNA that is generated as a result, thereby forming nucleoprotein filaments.

This in turn activates RecA to promote auto-catalytic cleavage of the repressor LexA9. As a consequence LexA levels initially decrease and transcription of DNA damage repair genes is

de-repressed10,11. With only few exceptions, _Mtb_ harbours the genes found in _Escherichia coli_ to mediate a fully functional SOS response12,13. Early studies demonstrated that the _Mtb

recA_ gene, which itself is regulated by LexA, contains a second, LexA-independent promoter (usually referred to as P1) located more proximal to the _recA_ start codon5,6,7. Mutations in the

Lex-dependent promoter P2 that abolish its activity do not abrogate basal _recA_ transcription, and RecA expression remains inducible by the DNA damaging agent mitomycin C6. Strikingly, the

majority of DNA damage inducible genes in _Mtb_ are controlled in a LexA/RecA-independent manner, including also many members of the LexA-regulon8. In an _Mtb recA_ deletion strain,

induction of many DNA repair genes upon exposure to mitomycin C is either the same as in wild type or is only partly decreased. Bioinformatic analysis identified a consensus sequence in the

upstream region of these genes (tTGTCRgtg - 8 nt - TannnT) that resembles the _recA_ P1 promoter and is responsible for the observed LexA-independent regulation14. A recent study in _Msm_

showed that the _clp_ gene regulator (ClgR; MSMEG_2694/Rv2745c) recognizes the P1 promoter region15. Moreover, deletion of _clgR_ in _Msm_ results in impaired induction of various genes

related to DNA repair mechanisms under DNA damaging conditions. This indicates a regulatory function of ClgR in the LexA/RecA-independent DNA damage repair pathway in mycobacteria. A

post-translational protein modification pathway akin to ubiquitination contributes to the survival of _Mtb_ in host macrophages. In this modification pathway termed pupylation, mycobacteria

(and other actinobacteria) make use of the small ubiquitin-like protein Pup to target proteins for degradation by the bacterial proteasome16,17. Pup is covalently attached via its C-terminal

glutamate to lysine side chains of proteasomal substrates by action of the Pup ligase PafA18,19. Before ligation can occur, Pup, which in all mycobacteria is encoded with a C-terminal

glutamine, must be rendered competent for ligation through deamidation of its C-terminal glutamine residue by the enzyme Dop (deamidase of Pup)19,20. Toward pupylated substrates Dop can act

as a depupylase21,22, thereby counteracting the ligase activity. Pupylated proteins are recognized by the proteasomal ATPase ring, termed Mpa (mycobacterial proteasomal ATPase) in

mycobacteria, which unfolds the substrate and translocates it into the proteolytic chamber of the proteasome complex23,24,25. The genes encoding the core proteins involved in pupylation and

proteasomal degradation are clustered together in the so-called Pup-proteasome gene locus. In all mycobacterial species the gene _pafA_ encoding the Pup ligase is organized in an operon

together with two other genes, _pafB_ and _pafC_ 26. _Mtb_ PafB and PafC were shown to copurify, suggesting that they might form a complex26. Deletion of the coding genes does not affect

pupylation in _Mtb_ and therefore has no influence on the degradation of proteasomal substrates. Unlike an _Mtb pafA_ deletion mutant, a strain lacking _pafBC_ displays only a slight

decrease in resistance against nitrosative stress _in vitro_ 26,27. However, PafB and PafC were linked to conjugal DNA transfer in _Msm_ 28. Deletion of the corresponding genes does not

impair the donor strain’s ability to transfer DNA. In contrast, the corresponding recipient strain is strongly compromised in DNA uptake. A recent study indicates that PafC contributes to

the intrinsic resistance of mycobacteria to fluoroquinolone antibiotics29. However, the underlying mechanism for any of the observed phenotypes remained elusive. In order to understand the

biological function of PafB and PafC, we generated a _pafBC_ deletion mutant of _Msm SMR5_ and employed a comparative proteomic approach using iTRAQ (Isobaric Tags for Relative and Absolute

Quantitation in mass spectrometry). Notably, a large part of the proteins that displayed decreased cellular levels in the deletion mutant are associated with DNA repair, including RecA.

Accordingly, deletion of _pafBC_ resulted in increased sensitivity towards DNA damaging agents, and induction of _recA_ transcription in _Msm_ Δ_pafBC_ was strongly impaired under DNA

damaging growth conditions. Strikingly, whereas the LexA/RecA-dependent _recA_ promoter P2 remained inducible to wild type levels, induction of P1-driven transcription was completely

blocked. Our findings establish _pafBC_ as a new regulatory element in the LexA/RecA-independent DNA damage response and provide new insights into RecA regulation as well as the control of

DNA repair mechanisms in mycobacteria. RESULTS _M. SMEGMATIS_ PAFB AND PAFC FORM A STABLE HETERODIMER AND FEATURE DNA BINDING MOTIFS The genes encoding PafB and PafC are located downstream

of the Pup ligase gene _pafA_. Like in its pathogenic relative _Mtb_ 26, these three genes are co-transcribed in _M_. _smegmatis_ as evidenced by the presence of a single mRNA encoding both

PafC and PafB in the _Msm_ wild type strain (Fig. 1A). Sequence analyses of PafB and PafC showed that they exhibit similarities to a multitude of bacterial transcription factors featuring a

so-called winged helix-turn-helix (wHTH) DNA-binding motif in their N-terminal region (Supplementray Fig. S1). The wHTH motif is typically comprised of three helices forming a compact

helical bundle, which is capped by an adjacent hairpin (“wing”) (Supplementray Fig. S1)30. Binding to DNA is usually mediated by insertion of helix α3 into the major groove (Supplementray

Fig. S1). Furthermore, PafB and PafC contain a C-terminal WYL domain. This domain is often found in association with wHTH motifs and is possibly involved in protein dimerization and/or the

binding of ligands, which thereby trigger a specific transcriptional response31. Indeed, our _in vitro_ analysis of the assembly state of _Msm_ PafB and PafC demonstrates that the two

proteins form a heterodimer (Fig. 1B). Analytical gel filtration profiles of PafB and PafC expressed and purified separately (Fig. 1B, upper panel) indicate that PafB (36 kDa) with an

apparent molecular weight of 40 kDa runs as a monomer, while PafC (34 kDa) runs at an apparent molecular weight of 84 kD, possibly due to homodimer formation in absence of PafB. However,

when PafB and PafC are co-expressed and co-purified (Fig. 1B, lower panel), a single main elution peak shifted away from either of the positions of the individual proteins is detected at an

apparent molecular weight in agreement with a PafBC heterodimer. Consistent with this interpretation, analysis of the elution peak in SDS-PAGE reveals that it contains equal amounts of PafB

and PafC. Taken together, bioinformatic analysis along with the observed stable heterodimer formation between PafB and PafC imply that the two proteins might act as a heterodimeric

transcriptional regulator in mycobacteria. GENERATION OF A _M. SMEGMATIS PAFBC_ DELETION STRAIN As an initial step towards understanding the role of PafBC in mycobacteria we generated a

markerless deletion of the _pafBC_ coding genes in the model organism _Mycobacterium smegmatis_ (_Msm_). The mutant was obtained by transformation of _Msm SMR5_, the streptomycin resistant

parent strain carrying a point mutation in the ribosomal protein S12 coding gene (_rpsL_) with the suicide plasmid _pMCS_Δ_pafBC-hyg-rpsL_ carrying the _rpsL_ + gene and subsequent

application of the _rpsL_ counterselection strategy (Fig. 2A)32,33. Disruption of _pafBC_ was confirmed by Southern blot analysis, and the parent strain is hereafter referred to as _Msm_

wild type, the deletion strain as _Msm_ Δ_pafBC_. The used probe hybridized to a 2.6-kbp-fragment in the wild type strain and to a 6.5-kbp-fragment in the _pafBC_ deletion mutant (Fig. 2B).

A complemented strain was generated by transforming _Msm_ Δ_pafBC_ with the integrative plasmid _pMV361_-_hyg_-_pafBC_ carrying a fragment consisting of the presumable promoter region of the

_pafABC_ operon fused to _Msm pafBC_. The complementation vector re-established cellular levels of PafBC similar to wild type (Fig. 2C). _In vitro_ growth of the _pafBC_ deletion mutant did

not differ from _Msm_ wild type when grown under standard conditions at 37 °C (Supplementray Fig. S2). Since _pafB_ and _pafC_ form an operon with the Pup-ligase encoding gene _pafA_ (Fig. 

1A) we investigated whether pupylation is affected by the absence of PafBC. Anti-Pup immunoblot analysis of cell-free lysates deriving from _Msm_ wild type or _Msm_ Δ_pafBC_ grown under

standard conditions revealed no significant differences in the band pattern or band intensities of the pupylated proteome (pupylome), suggesting that PafA pupylation activity remains

unaffected (Fig. 3A). In accordance with this, we found that deletion of _pafB_ and _pafC_ did not alter the relative cellular amount of _pafA_ mRNA (Fig. 3B). Similar findings were made for

other proteins involved in the Pup-proteasome pathway, namely Dop, Mpa, PrcA and PrcB, which all displayed unchanged cellular protein levels in the _Msm_ Δ_pafBC_ mutant strain

(Supplementray Fig. S3). DELETION OF _PAFBC_ AFFECTS THE CELLULAR LEVELS OF PROTEINS INVOLVED IN DNA REPAIR PATHWAYS, TRANSCRIPTION AND RECOMBINATION In order to gain insights into the

cellular function of PafBC, a quantitative proteomic analysis of the _Msm_ Δ_pafBC_ mutant in comparison to the wild type strain was performed using iTRAQ. We identified a total of 2554

proteins (corresponding to 38.2% of all annotated _Msm_ proteins) in three biological replicates deriving from _Msm_ wild type and Δ_pafBC_ cultures grown to late exponential phase. In _Msm_

Δ_pafBC_ 54 proteins were found to be down-regulated and 17 proteins to be up-regulated at least 1.5-fold compared to _Msm_ wild type (Table 1). Among the affected proteins, 11 can be

assigned to DNA-related functions, namely DNA repair, recombination and transcription. The strongest decrease was observed for RecA, with a relative reduction of about 2.5 fold in the

_pafBC_ deletion mutant. Accordingly, when cell-free lysates deriving from cells grown under the conditions of the iTRAQ experiment were analysed by an anti-RecA immunoblot, the signal for

RecA, in the Δ_pafBC_ background was significantly reduced compared to either _Msm_ wild type or the complemented deletion strain (Fig. 4). Further analysis showed that RecA levels are

controlled by concomitant action of PafB and PafC. Expression of either _pafB_ or _pafC_ alone in _Msm_ Δ_pafBC_ did not affect RecA expression as compared to the mutant strain, while

co-expression of _pafB_ and _pafC_ resulted in a significant increase of cellular RecA levels (Supplementray Fig. S4). DOWN-REGULATION OF RECA IN _MSM_ Δ_PAFBC_ OCCURS AT THE TRANSCRIPTIONAL

LEVEL The changes in cellular levels of various proteins in _Msm_ Δ_pafBC_ implied a transcriptional regulation of the corresponding coding genes. It should be noted, however, that some of

the down-regulated proteins found in our iTRAQ analysis were previously identified as possible or, like RecA, genuine targets of the pupylation pathway. In addition, we find that RecA

accumulates in a proteasome-deficient strain of _Msm_ (Fig. 5A, bottom blot), also indicating that RecA is degraded via the Pup-proteasome pathway in _Msm_. However, the significantly

reduced levels of RecA in the _Msm_ Δ_pafBC_ mutant strain (Fig. 4, second lane) cannot be due to increased degradation of the protein, since _Msm_ wild type and _Msm_ Δ_pafBC_ exhibit

similar turn-over rates for a C-terminally His6-tagged RecA variant expressed in either of the strains (Fig. 5A, upper and middle blot). This result strongly supports the notion that

transcription of _recA_ in _Msm_ is either directly or indirectly regulated by PafBC. Carrying out relative quantification by qRT-PCR, we found _recA_ transcript levels decreased by about

36% in the _Msm_ Δ_pafBC_ mutant strain as compared to the parental strain (Fig. 5B, upper panel). In a concomitant control, we checked the transcriptional levels of another player involved

in DNA damage repair, namely _uvrB_ 34, whose corresponding gene product was not covered by the analysed iTRAQ samples. In analogy to _recA_, expression _uvrB_ decreased by 37% in _Msm_

Δ_pafBC_ compared to wild type (Fig. 5B, lower panel). Next, we examined whether PafBC influences transcription of _recA_ by binding to its promoter region. To this end, an electrophoretic

mobility shift assay (EMSA) was performed using purified _Msm_ PafBC and a DNA fragment comprising the 300 bp upstream of the _recA_ gene. Indeed, PafBC was found to bind to the _recA_

promoter region (Supplementray Fig. S5). However, PafBC displays a rather promiscuous binding behaviour _in vitro_ and comparable interaction with different promoter regions used as negative

controls in the assay was observed (Supplementray Fig. S5). Various attempts to increase specificity by testing different buffer conditions were not successful. Therefore, the EMSA did not

allow for a final conclusion concerning the binding of PafBC to the _recA_ promoter region. DELETION OF _PAFBC_ RENDERS _M. SMEGMATIS_ MORE SUSCEPTIBLE TO DNA DAMAGING CONDITIONS Like in

other prokaryotes, RecA in mycobacteria holds a key role in homologous recombination and was shown to be a crucial element in regulating the SOS response upon DNA damage10,35,36. Thus, the

observed decrease of RecA prompted us to perform a phenotypical analysis of the _Msm_ Δ_pafBC_ mutant strain under DNA-damaging conditions. In the growth experiments DNA stress was emulated

by exposing the bacterial cultures to the DNA interstrand crosslinking agent mitomycin C. In both, _Mtb_ and _Msm_, _recA_ was shown to be upregulated upon exposure to mitomycin C5,37,38. In

an initial approach we determined the minimal inhibitory concentration (MIC) of mitomycin C for _Msm_ wild type to be 160 ng/ml and 4 ng/ml for the _pafBC_ deletion strain. We subsequently

performed a survival assay using 50% of the MIC for _Msm_ wild type. The _Msm_ wild type strain, _Msm_ Δ_pafBC_ and the complemented strain were grown in shaking cultures and mitomycin C was

added at an OD of 0.1. After 8 h, serial dilutions of the respective cultures were spotted onto LB agar plates without mitomycin C. _Msm_ wild type and the complemented strain were only

mildly affected by mitomycin C, when compared to the controls grown in absence of the agent (Fig. 6A). In contrast, the number of viable cells in cultures of the _pafBC_ deletion mutant

decreased by about two orders of magnitude upon exposure to mitomycin C. Similar observations were made when _Msm_ Δ_pafBC_ was exposed to UV irradiation. After a UV dose of 25 mJ/cm2

viability of the deletion strain decreased by approximately two orders of magnitude as compared to the _Msm SMR5_ parental strain (Fig. 6B). INDUCTION OF _RECA_ UNDER DNA-DAMAGING CONDITIONS

IS IMPAIRED IN _MSM_ Δ_PAFBC_ The increased sensitivity of the _Msm_ Δ_pafBC_ mutant towards mitomycin C and UV radiation strongly implied that the induction of _recA_, which is usually

observed under DNA damaging conditions5, is hampered in the _Msm_ Δ_pafBC_ mutant. Indeed, when we performed an anti-RecA immunoblot analysis, levels of RecA increased significantly in _Msm_

wild type after the exposure to mitomycin C (Fig. 7). In contrast, the _pafBC_ deletion mutant exhibited significantly impaired upregulation of RecA levels under the same DNA damaging

growth conditions (Fig. 7A), which could be rescued by complementation with _pafBC_. In addition, we addressed the question whether induction of _recA_ under DNA damaging conditions is

accompanied by upregulation of PafBC. Therefore, _Msm_ wild type grown in the presence of mitomycin C was analysed by an anti-PafBC Western Blot (Fig. 7B). However, no alterations in PafBC

levels were observed compared to cells grown under standard conditions. PAFBC AFFECTS TRANSCRIPTIONAL CONTROL OF _RECA_ VIA THE LEXA/RECA-INDEPENDENT PROMOTER In contrast to other bacteria,

_recA_ is controlled by two promoters in mycobacteria: a LexA/RecA-dependent promoter (P2), which is activated as part of the classical SOS response, and a LexA/RecA-independent promoter

(P1). Transcriptional activity of both, the P1 and the P2 promoter, was previously shown to be inducible by mitomycin C6,7. As we observed a decrease of RecA levels in _Msm_ Δ_pafBC_ under

both standard and DNA damaging conditions, we addressed the question through which of the two promoters PafBC affects transcription of the corresponding gene. We constructed reporter strains

of _Msm_ wild type and _Msm_ Δ_pafBC_ that allowed us to monitor transcriptional activities of P1 and P2 independently. A DNA fragment encompassing the entire _recA_ promoter region and the

first 11 codons of _recA_ was fused to GFP (Fig. 8A). Either P1 or P2 was subsequently mutated in order to determine the individual contributions of the two promoter sites with respect to

the presence of PafBC. The constructs were introduced into the genome of the respective strain by an integrative vector. In the _Msm_ wild type strain reporting on the activity of the entire

P1/P2 promoter region, GFP fluorescence was observed to be about 6-fold higher in presence of mitomycin C than in the absence of the agent (Fig. 8B). In the Δ_pafBC_ strain the mitomycin

C-induced response was markedly reduced and resulted only in an approximately 3-fold increase reaching less than half of the wild type fluorescence value. When P2 was inactivated, the

response to mitomycin C stress could only be observed for the wild type strain. In contrast, mutation of P1 retained the response to mitomycin C in both strains, although the Δ_pafBC_ strain

displayed only about three-fourths of the wild type level. The weaker P2 response of the Δ_pafBC_ strain may be explained by the fact that P2 activity is dependent on RecA6, whose level was

shown is significantly reduced in the Δ_pafBC_ strain (Figs 4A and 7A). In absence of mitomycin C, GFP fluorescence in the Δ_pafBC_ strain was approximately half of the wild type level for

the reporter constructs exhibiting an intact P1 promoter, while GFP fluorescence reached the same level under standard conditions when P1 was mutated. Together, these findings clearly

demonstrate that P1-driven transcription of recA is absent in the pafBC deletion mutant while P2-driven transcription remains largely unaffected. DISCUSSION The response to DNA damage in

mycobacteria is regulated by two independent mechanisms. Besides the ubiquitous LexA/RecA-dependent SOS response, most DNA damage-inducible genes in mycobacterial species are controlled in a

LexA/RecA-independent manner via the P1 promoter5,6,7. Notably, some genes, like e.g. _recA_, are regulated by both mechanisms8,14. The presented study broadens the scope of P1-driven

transcription of DNA repair genes by adding PafBC as a novel regulatory player to the DNA damage response in mycobacteria. The goal of our study was to gain insights into the physiological

function of _pafB_ and _pafC_ in mycobacteria. Since these genes form an operon with the Pup-ligase coding gene _pafA_ (Fig. 1A)26 in mycobacteria, they were generally considered to be

associated with the Pup-proteasome gene locus. The formation of a heterodimeric complex (Fig. 1B) and the presence of a winged helix-turn-helix DNA-binding motif at the N-termini of PafB and

PafC (Fig. S1) indicated a regulatory function at the transcriptional level. However, under the conditions tested we found no influence of PafBC on any of the components known to be

involved in pupylation or proteasomal degradation (Fig. 3, Supplementray Fig. S3). Rather, quantitative proteome analyses revealed that levels of various proteins related to DNA damage

repair were altered in a PafBC-dependent manner (Table 1). Accordingly, _Msm_ Δ_pafBC_ displayed an increased susceptibility to DNA damaging agents (Fig. 6). Finally, using the example of

RecA, we demonstrated that PafBC exerts its influence on transcription of DNA-damage inducible genes at the level of the LexA/RecA-independent P1-promoter (Figs 5B and 8). To our knowledge,

this is the first study showing a role of PafBC in DNA damage response. Disruption of _pafB_ or _pafC_ in _Msm_ was previously shown to hamper a recipient strain’s ability to take up and/or

integrate DNA via homologous recombination (HR) into its genome during conjugation28. RecA is a key element in HR, as it binds to the DNA strands and catalyzes strand exchange, which

eventually results in recombination. In the iTRAQ analysis RecA displayed the highest decrease in cellular levels of those proteins that could be identified in the _Msm pafBC_ deletion

strain (Table 1). Thus, against the background of this observation the impact of disrupted _pafB_ or _pafC_ on conjugation can be attributed to impaired RecA-dependent HR rather than

impaired DNA uptake. Very recently, a _M. smegmatis_ mutant that carries a transposon insertion in _pafC_ was shown to be hypersensitive to various fluoroquinolones, like moxifloxacin or

ciprofloxacin29. The data presented here provide an explanation for the increased sensitivity. Quinolones target topoisomerase IV and DNA gyrase and their action on these enzymes eventually

results, amongst others, in multiple DNA strand breaks39. Thus, while _Msm_ wild type is able to at least partially compensate the action of quinolones by a functional DNA damage repair, the

impaired response of Δ_pafBC_ to DNA damaging conditions (Figs 6 and 7) leads to increased sensitivity to these drugs. At least under _in vitro_ conditions PafBC binding lacks specificity

as reflected by the results of our EMSA approach (Supplementray Fig. S4). It is possible that additional mechanisms might exist _in vivo_ to bring about specificity. Many transcription

factors are targeted by post-translational modifications like phosphorylation or acetylation that modulate their binding affinity40,41. For example, VirS, the transcription factor that

regulates the mycobacterial monooxygenase (_mymA_) operon, is phosphorylated by the kinase PknK42. Phosphorylation increases the affinity of VirS for the _mymA_ promoter. Furthermore, PafB

and PafC feature a WYL domain in their C-terminal half. WYL domains were previously postulated to act in ligand sensing and binding31. It is therefore conceivable that PafBC relies on an

additional interaction partner in order to specifically bind its genuine target, which is absent in the _in vitro_ EMSA experiment. It could be argued that PafBC does not act as a specific

transcriptional regulator but might function analogous to members of the Dps (DNA binding protein from starved cells) protein family43. Dps proteins are upregulated under various stress

conditions and in several species were shown to bind DNA without apparent sequence specificity, thereby shielding the DNA molecule from damaging agents44,45,46. The fact that PafBC did not

exhibit a change in cellular levels under DNA damaging conditions, makes such a mode of function very unlikely (Fig. 7B). Furthermore, most factors with wHTH motifs interact with specific

DNA sequences, while DNA binding of Dps is mediated simply by the positive charge of N-terminal lysine residues47,48. The involvement of PafBC in regulating the DNA damage response in _Msm_

is clearly supported by our data. However, it remains elusive how the signal of DNA damage is eventually translated into PafBC-dependent transcriptional control. Regulation of DNA

repair-related genes by PafBC could occur directly by binding to the P1 promoter, or indirectly by e.g. affecting the transcription of other regulators. Recently, the _clp_ gene regulator

ClgR was shown to recognize the LexA/RecA-independent P1 promoter15. Analogous to our observation with the _Msm_ Δ_pafBC_ mutant, upregulation of _recA_ and other genes involved in DNA

repair is impaired in an _Msm_ Δ_clgR_ mutant upon exposure to DNA damaging conditions15. The coding genes of 15 proteins that displayed altered levels in _Msm_ upon deletion of _pafBC_

either harbour the P1 consensus motif recognized by ClgR in the promoter region or are part of operons which bear the motif in their promoter region (Table S1). Based on these observations

different scenarios regarding P1-driven transcription are conceivable: i) PafBC and ClgR act independently on the P1 promoter, but share certain targets, like e.g. _recA_, which are

regulated by one or the other protein, depending on distinct cellular signals and/or additional transcriptional factors ii) transcription is regulated by concerted binding to the P1 promoter

by both, PafBC and ClgR iii) the impact of PafBC could be indirect by affecting transcriptional activity of _clgR_. The function of PafBC is apparently not restricted to DNA damage. This is

clearly indicated by the large number of proteins (about 80% of the proteins identified in our iTRAQ analysis) whose expression is influenced by PafBC independent of the P1 promoter and

that are not related to DNA repair (Table 1).This points towards a more global impact of PafBC in the cell’s regulatory networks. In accordance with an earlier study we did not find any

influence of PafBC on pupylation or proteasomal degradation at least under the conditions tested (Fig. 3 and Supplementray Fig. S3)26. Nevertheless, the organization of _pafB_ and _pafC_ in

an operon with _pafA_ implies functional coupling and therefore a role for PafBC in the Pup-proteasome system (PPS). Interestingly, conditional expression of ClgR in _Mtb_ results in the

induction of _prcB_ and _prcA_ 49. Given the possible link of PafBC and ClgR indicated above, it is therefore tempting to speculate that these two regulators together might modulate levels

of the PPS upon a particular cellular signal. In summary, our study assigns the first known function to the genes _pafB_ and _pafC_ in _Msm_. The elucidation of the function of PafBC as a

heterodimeric transcriptional regulator involved in the LexA/RecA-independent mycobacterial DNA damage response adds a new facet to the intricate system of DNA damage control in

mycobacteria. Yet, the co-occurrence of _pafBC_ with the Pup-proteasome gene locus remains elusive. This association could have evolved since both systems - DNA repair and

Pup-proteasome-dependent protein degradation - might play a role under specific stress conditions and are therefore regulated by overlapping regulatory mechanisms. Whether PafBC and the

Pup-proteasome system indeed share also a more direct functional link remains to be further scrutinized. METHODS BACTERIAL STRAINS, OLIGONUCLEOTIDES, MEDIA AND GROWTH CONDITIONS The

oligonucleotides used in this study are summarized in table S2. Strains used were _Mycobacterium smegmatis_ (_Msm_) SMR5, _Msm_ Δ_pafBC_ and the complemented strain _Msm_ Δ_pafBC_-_pafBC_

(see following section). Bacteria were grown in LB medium supplemented with 0.05% (v/v) TWEEN-80 (LB-T) or 7H9 medium (Difco Laboratories) without OADC supplement. When appropriate,

hygromycin B was added to a final concentration of 100 µg ml−1. Growth curves of _Msm_ SMR5, _Msm_ Δ_pafBC_ and _Msm_ Δ_pafBC_-_pafBC_, were measured in 24-well plates in a total volume of

0.5 ml. OD600 measurements were carried out in a Synergy H1 Hybrid Multi-Mode Microplate Reader (Biotek). For proteomic analyses, strains were grown in 7H9 medium without OADC. Cells were

routinely grown at 37 °C in a shaking incubator at 140 rpm. DISRUPTION OF PAFBC IN M. SMEGMATIS To generate a markerless deletion of _pafBC_ in _Msm_ the knock-out plasmid

pMCS-_hyg-rpsL_-Δ_pafBC_ was generated as follows: a 1.5 kbp _Nco_I/_Hind_III fragment encompassing the 5’ _pafBC_ flanking sequence and a 1.5 kbp _Hind_III/_Nde_I fragment encompassing the

3’ flanking sequence were amplified from _Msm_ chromosomal DNA. The PCR products were cut with the respective restriction enzymes and were cloned into the vector pMCS-_hyg-rpsL_. Competent

cells of streptomycin-resistant _Msm_ mc2155 SMR5 were transformed with the knock-out plasmid and the _rpsL_ counterselection strategy32,33 was applied to delete a 1.9 kbp fragment

comprising _pafB_ and _pafC_. Deletion of the genes was confirmed by PCR and Southern blot analysis, using a _pafA_ specific 0.5 kbp probe. _Msm_ Δ_pafBC_ was complemented with the

integrative plasmid pMV361-_hyg-amp_ containing the genes encoding wild type PafB and PafC fused to 300 bp upstream sequence of the _pafABC_ operon, encompassing the native promoter.

PREPARATION OF CELL-FREE LYSATES AND IMMUNOBLOT ANALYSES Cells were harvested by centrifugation and were washed once with 1 Vol. PBS supplemented with 0.05% (v/v) TWEEN-80. Pellets were then

resuspended in lysis buffer containing 100 mM Tris-HCl pH 8, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM MgCl2, 1 mM PMSF, supplemented with 1x protease inhibitor cocktail (Sigma-Aldrich), DNase

and RNase. Cells were lysed using a FastPrep-24 Bead Beater (MP Biomedicals; 2 × 30 s at 6 m/s, 0.15mm zirconia beads). Cell debris and insoluble material was removed by centrifugation

(16,000 × g, 5 min, 4 °C). Protein concentration of cell-free lysates was determined by the Bradford assay. Lysates were routinely separated by SDS-PAGE and were subjected to immunoblot

analyses, using polyclonal anti-PafBC antiserum (1:1,000) raised against purified _Msm_ PafBC (Eurogentec), anti-Pup antiserum (1:10,000)19 or anti-His6-tag antibody (1:10,000; Bethyl

Laboratory, Montgomery, TX, USA). For detection a goat anti-rabbit IgG alkaline phosphatase antibody-horseradish peroxidase (HRP) (BioRad) or a rabbit anti-mouse IgG alkaline phosphatase

antibody-HRP (Promega) was used together with Amersham ECL Prime Western Blotting Detection Reagent (1:1 ratio; GE Healthcare Life Sciences) as a substrate for the horseradish peroxidase. To

control for equal loading, membranes were stripped and control immunoblots were performed using a monoclonal antibody that was raised against the RNA polymerase subunit β (RpoB) of _E.

coli_ (1:7,500; GeneTex). For immunoblotting of RecA, cells were harvested by centrifugation and were washed once with 700 µl PBS. Pellets were then resuspended in lysis buffer (PBS

supplemented with 1 mM PMSF, 1x c0mplete (Roche) protease inhibitor), bead beaten and centrifuged as above. Lysates were separated by SDS-PAGE and were subjected to immunoblot analyses,

using commercially available anti-RecA antibody (1:1,000, MBL International, clone ARM414) raised in mouse and commercially available anti-RpoB antibody (1:1,000, BioLegend, clone 8RB13)

raised in mouse. For detection, HRP-conjugated anti-mouse IgG antibody (1:250,000, abcam #ab6789) together with Amersham ECL Select Western Blotting Detection Reagent (GE Healthcare Life

Sciences) was used. DETERMINATION OF THE MINIMAL INHIBITORY CONCENTRATION (MIC) MICs were determined as described50. Briefly, cultures of _Msm_ SMR5 and _Msm_ Δ_pafBC_ were grown to mid-log

phase in LB-T. Cultures were diluted to an OD600 of 0.01 and aliquots of 50 µl were transferred to 96-well plates containing two-fold serial dilutions of mitomycin C, resulting in a final

concentration ranging from 500 to 1 ng ml−1. Each strain was tested in triplicates in two independent experiments. Plates were incubated at 37 °C. The MIC was defined as the mitomycin C

concentration at which no visible growth was observed after three days of incubation. ASSESSING SURVIVAL AFTER MITOMYCIN C AND UV EXPOSURE _Msm_ SMR5, _Msm_ Δ_pafBC_ (both carrying the empty

plasmid pMV361-_hyg-amp_) and the complemented strain _Msm_ Δ_pafBC_-_pafBC_ were grown in LB-T to OD600 of 1.0 and were subsequently diluted to an OD600 of 0.1. Cultures were supplemented

with 80 ng ml−1 mitomycin C. Controls were grown in the absence of mitomycin C. After 8 h of incubation, serial dilutions of the cultures were spotted onto LB agar containing 100 µg ml−1

hygromycin B. Plates were incubated at 37 °C for 3 days and survival was assessed by comparing control samples to the samples deriving from mitomycin C-treated cultures. For the UV

irradiation assay, serial dilutions of freshly grown cultures (OD600 = 1.0) were spotted onto LB agar plates containing 100 µg ml−1 hygromycin B. Plates were exposed to a UV dose of 25

mJ/cm2 using a Stratalinker UV Crosslinker 2400 (Stratagene). Control samples were not exposed to UV. Plates were incubated for 3 days at 37 °C. Survival was assessed by comparing colonies

grown on the respective treated and untreated plates. GFP-REPORTER ASSAY TO MONITOR RECA P1 AND P2 TRANSCRIPTIONAL ACTIVITY To monitor activities of the _recA_ P1 and P2 promoter, a 419 bp

DNA fragment, comprising 386 bp upstream sequence and the first 11 codons of _recA_ was fused to the GFP gene and was inserted in a pMyNT-derived integrative plasmid. pMyNT was kindly

provided by A. Geerlof (EMBL Hamburg). To assess the individual activity of each promoter, the P1 or P2 promoter motif was replaced with a stretch of the coding sequence of mouse histone

H2Ab2 of identical size and similar GC content. The vectors were transformed into _Msm_ SMR5 and _Msm_ Δ_pafBC_. Three individual colonies of each reporter strain were grown in 7H9

supplemented with hygromycin B (50 µg ml−1) to an OD600 of 0.7 at 37 °C. Cultures were exposed to DNA-damaging stress by the addition of mitomycin C (80 ng/ml) and were grown for an

additional 4 h. Control cultures were grown in the absence of mitomycin C. Cells were harvested by centrifugation and cell-free lysates were prepared by resuspension of the pellet in 700 µl

ice-cold lysis buffer (PBS supplemented with 1 mM PMSF, 1x c0mplete EDTA-free (Roche) protease inhibitor, 1 mM EDTA) and bead beating as described above for the RecA immunoblots. Protein

content was determined by the Bradford assay. GFP fluorescence was measured in a black 96-well half-area plate (30 µl lysate/well) with a Synergy2 microplate reader (BioTek) instrument

(excitation filter 485/20, emission filter 528/20). RECA DEGRADATION ASSAY The gene encoding an N-terminally His6-tagged variant of _Msm recA_ was cloned into the pMyC plasmid under the

control of an acetamide-inducible promoter. _Msm_ SMR5, _Msm_ Δ_pafBC_ and _Msm_ ΔprcB carrying pMyC-His6-_recA_ were grown in 7H9 medium to an OD600 of 1.0. Acetamide (final concentration

0.02% w/v) was added to the cultures to induce _recA_ expression for 1 h. Cells were washed three times with PBS supplemented with 0.05% TWEEN-80 (v/v). Then cells were resuspended in 7H9

medium without acetamide and incubation of cultures continued for 9 h. Samples were withdrawn at the given time points. Cell-free lysates were prepared as described above. Equal protein

concentrations were loaded and separated by SDS-PAGE for subsequent immunoblot analysis with an anti-His6-tag antibody in order to monitor His6-RecA degradation. RNA ISOLATION AND

QUANTITATIVE REAL-TIME PCR For RNA isolation strains were grown to an OD600 of 2.6 in 7H9 medium without OADC. Cells were pelleted by centrifugation and were resuspended in an equal volume

of Trizol-reagent (Life Technologies). The samples were subsequently lysed by bead beating using Lysing MatrixB tubes and a Fast Prep-24 device (MP Biomedicals). Supernatants were used for

isolation of RNA with the RNA mini kit (Bio & Sell) according to the manufacturer’s instructions. DNA contaminations were removed by treating isolated RNA with DNase I (Thermo Fisher

Scientifc). Total RNA concentration was determined by using a NanoDrop spectrophotometer (Thermo Fisher Scientific). To synthesize cDNA from _Msm_ RNA the High Capacity RNA-to-DNA Kit

(Applied Biosystems) was used according to the manufacturer’s instructions. Primers for qRT-PCR were designed with the Primer3 software51. An equivalent of ~10 ng cDNA was analysed by

qRT-PCR using 5x QPCR MIX EvaGreen (Bio&Sell) in a 7500 Fast System (Applied Biosystems). Cycling conditions were as follows: 1 cycle of 95 °C for 15 min, 40 cycles of 95 °C for 15 s, 60

°C for 20 s, and 72 °C for 30 s, followed by a melting curve for each sample. cDNA derived from at least three independent biological replicates and was measured in technical triplicates,

respectively. The relative expression levels were calculated according to Pfaffl52 using the 16 S rRNA gene as the reference. PROTEOMIC ANALYSIS BY ITRAQ Cell lysates were prepared as

described above from cultures of _Msm_ SMR5 and _Msm_ Δ_pafBC_ grown to an OD600 of 2.5. 100 µg of proteins were digested and labelled according to the iTRAQTM manufacturing protocol (AB

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Scholar  Download references ACKNOWLEDGEMENTS We would like to thank Peter Sander for fruitful discussions and critical comments on the manuscript. We thank Jonas Grossmann, Christian

Trachsel and Claudia Fortes from the FGCZ proteomics team for technical support and advice. Research in the lab of FI is supported by grants of the University of Zurich. Research in the lab

of EWB is supported by the Swiss National Science Foundation (31003A_163314). AUTHOR INFORMATION Author notes * Begonia Fudrini Olivencia and Andreas U. Müller contributed equally to this

work. AUTHORS AND AFFILIATIONS * University of Zurich, Institute of Medical Microbiology, Zurich, Switzerland Begonia Fudrini Olivencia, Sibylle Burger & Frank Imkamp * ETH Zurich,

Institute of Molecular Biology and Biophysics, Zurich, Switzerland Andreas U. Müller & Eilika Weber-Ban * Functional Genomic Center, University of Zurich/ETH, Zurich, Switzerland Bernd

Roschitzki Authors * Begonia Fudrini Olivencia View author publications You can also search for this author inPubMed Google Scholar * Andreas U. Müller View author publications You can also

search for this author inPubMed Google Scholar * Bernd Roschitzki View author publications You can also search for this author inPubMed Google Scholar * Sibylle Burger View author

publications You can also search for this author inPubMed Google Scholar * Eilika Weber-Ban View author publications You can also search for this author inPubMed Google Scholar * Frank

Imkamp View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS B.F.O., A.U.M., E.W.-B. and F.I. designed research; B.F.O., A.U.M., S.B., B.R.,

E.W.-B. and F.I. performed experiments and analysed data; B.F.O., A.U.M., E.W.-B. and F.I. wrote the paper. CORRESPONDING AUTHOR Correspondence to Frank Imkamp. ETHICS DECLARATIONS COMPETING

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smegmatis_ PafBC is involved in regulation of DNA damage response. _Sci Rep_ 7, 13987 (2017). https://doi.org/10.1038/s41598-017-14410-z Download citation * Received: 18 November 2016 *

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