Molecular Microbiology (2014) 93(3), 391–402 ■

doi:10.1111/mmi.12689 First published online 10 July 2014

MicroReview The expression of antibiotic resistance genes in antibiotic-producing bacteria Stefanie Mak, Ye Xu† and Justin R. Nodwell* Department of Biochemistry, University of Toronto, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8.

Summary Antibiotic-producing bacteria encode antibiotic resistance genes that protect them from the biologically active molecules that they produce. The expression of these genes needs to occur in a timely manner: either in advance of or concomitantly with biosynthesis. It appears that there have been at least two general solutions to this problem. In many cases, the expression of resistance genes is tightly linked to that of antibiotic biosynthetic genes. In others, the resistance genes can be induced by their cognate antibiotics or by intermediate molecules from their biosynthetic pathways. The regulatory mechanisms that couple resistance to antibiotic biosynthesis are mechanistically diverse and potentially relevant to the origins of clinical antibiotic resistance.

Introduction More than 70% of antibiotics used in medicine are derived from secondary metabolites produced by the actinomycete bacteria – these compounds are responsible for much of our success in managing bacterial infectious diseases. The accumulation of antibiotic-resistant pathogens in hospitals around the world is therefore of serious concern and the origin of the genes that confer resistance is an important question. It is clear that there is a global ensemble of antibiotic resistance genes called the ‘resistome’ (Wright, 2007) and while the distribution of these genes in nature is not well understood, the actinomycete genomes themselves are a large reservoir (D’Costa et al., 2007). Indeed, of the resistance determinants we will discuss in this Accepted 22 June, 2014. *For correspondence. E-mail [email protected]; Tel. (+1) 416 978 2696; Fax (+1) 416 978 8548. †Present address: Department of Bacteriology, University of Wisconsin, 1550 Linden Dr. Madison, WI 53706, USA.

© 2014 John Wiley & Sons Ltd

review, at least six (AphD, OtrA, OtrB, ActA, ActB, GyrBR) are mechanistically similar or identical to antibiotic resistance genes and mutations identified in clinical pathogens (Levy, 1992; Sundin and Bender, 1996; Aminov et al., 2001; Vickers et al., 2007). The evolutionary link between resistance mechanisms in antibiotic producers and clinical pathogens is beyond the scope of this review; nevertheless, whether antibiotic resistance in one bacterium evolved convergently or divergently, it can be readily passed on to the progeny via vertical transfer or mobilized into other unrelated bacteria through horizontal gene transfer (Barlow, 2009). The enzymatic pathways that generate the antibiotics are encoded in contiguous biosynthetic gene clusters. Typically, each gene cluster encodes at least one resistance gene for its cognate antibiotic. Just as there are many mechanisms of antibiotic resistance (target modification and protection, antibiotic inactivation, sequestration, and efflux) (Cundliffe and Demain, 2010), there is remarkable diversity of regulatory mechanisms controlling resistance gene expression. Some resistance genes are expressed constitutively regardless of whether their cognate biosynthetic genes are being expressed – an example of this is the erythromycin resistance gene ermE in Saccharopolyspora erythraea (Bibb et al., 1985). Constitutive resistance is relatively rare; most of these genes are tightly regulated. Two themes have emerged from the studies on pathwayencoded resistance that have been conducted so far. First, the expression of resistance is wired such that it precedes or accompanies biosynthesis gene expression. Second, some cluster-encoded resistance genes can be induced by the antibiotics themselves or by biochemical intermediates from their biosynthetic pathways. These additional regulatory links could upregulate resistance in response to increasing antibiotic titres, thus preventing the cell from overwhelming its own capacity for resistance. Alternatively, these cell–cell signalling mechanisms might serve to activate resistance in cells that are not producing the antibiotic, thereby preventing antibiotic-producing cells from killing their non-producing siblings. These regulatory mechanisms ensure that the biosynthesis of antibiotics is not deleterious to the producer

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organisms, but instead, provides an evolutionary advantage. In this review, we will discuss specific examples that illustrate these themes. Our focus has been on those processes where both biosynthesis and resistance have been investigated in some detail – this is a relatively small subset of the known antibiotics.

ArpA adpA A-factor = OH O

Streptomycin Streptomycin is an aminoglycoside antibiotic that binds the 30S subunit of the bacterial ribosome, causing misreading of the genetic code during translation. It is produced by Streptomyces griseus as one of the defining features of the A-factor signalling cascade (Fig. 1) (Horinouchi, 2002). A-factor is a secreted γ-butyrolactone signalling molecule that binds the Arp protein, a member of the TetR-family of repressors (TFR) (Onaka et al., 1995; Onaka and Horinouchi, 1997; Cuthbertson and Nodwell, 2013), and causes it to release its target promoter adpA (Ohnishi et al., 1999). AdpA is a master regulator of sporulation and secondary metabolic genes, including the str cluster which encodes the biosynthetic machinery for streptomycin (Ohnishi et al., 2005). AdpA’s target in the str cluster is the aphD P1 promoter of the strR-aphD operon, resulting in the expression of StrR (a transcription factor that activates the biosynthetic genes) and AphD (which confers streptomycin resistance) (Vujaklija et al., 1991; 1993). The streptomycin resistance determinant aphD (also known as strA) encodes a streptomycin 6phosphotransferase (Tohyama et al., 1984; Distler et al., 1987). This enzyme transfers the γ-phosphate from ATP to the C6 hydroxyl of the streptidine moiety of the mature drug to generate the inactive compound streptomycin-6phosphate and prevent toxicity to the cells through metabolic shielding. The inactive streptomycin can be exported by the StrVW transporters (Beyer et al., 1996), and the phosphate group can be subsequently removed by the StrK phosphatase (Mansouri and Piepersberg, 1991). It has been suggested that AphD can also phosphorylate the streptidine precursor before this 6-O-phosphoryl subunit is assembled with the dTDP-dihydrostreptose and UDP-Nmethyl-L-glucosamine subunits to generate the prodrug, thereby ensuring that the mature antibiotic is produced in its inert form (Cundliffe and Demain, 2010). This is an appealing model; however, to our knowledge, it is unsupported by direct evidence. Nonetheless, the fact that the AphD resistance gene is co-transcribed with strR, which is required to activate the biosynthetic genes, ensures that the acquisition of streptomycin resistance precedes the appearance of the streptomycin biosynthetic machinery in the S. griseus cytoplasm. Thus, the resistance enzyme is available in the cytoplasm to inactivate the antibiotic and this is sufficient to prevent cells from poisoning them-

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Fig. 1. The A-factor regulatory cascade controls the onset of streptomycin biosynthesis and resistance in S. griseus. The γ-butyrolactone signalling molecule A-factor binds to ArpA to derepress the expression of adpA. AdpA, in turn, activates the streptomycin pathway-specific regulator gene strR as well as the streptomycin resistance gene aphD. Since strR and aphD are co-transcribed from the aphD P1 promoter, this ensures that resistance is established before the antibiotic accumulates in the cell. It should be noted that the aphD gene can also be expressed from a second promoter aphD P2, which does not depend on the A-factor signalling cascade. The mechanism of regulation from this promoter has not been fully elucidated yet.

selves. Furthermore, because the str gene cluster is activated by a diffusible signal (A-factor), it is likely that the pathway is expressed in most or all cells in the culture.

Novobiocin, clorobiocin and coumermycin A1 The aminocoumarin antibiotics novobiocin, clorobiocin, and coumermycin A1 are produced by Streptomyces sphaeroides, Streptomyces roseochromogenes, and Streptomyces rishiriensis respectively. All three antibiotics act by interfering with DNA gyrase and topoisomerase IV, © 2014 John Wiley & Sons Ltd, Molecular Microbiology, 93, 391–402

Antibiotic resistance in antibiotic-producing bacteria 393

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Fig. 2. The biosynthetic gene clusters of novobiocin (nov), clorobiocin (clo), and coumermycin A1 (cou) all share a similar genomic arrangement. Resistance determinants for each aminocoumarin are shown in red, while regulators of aminocoumarin biosynthesis are shown in blue. Promoters responsible in the expression of the novobiocin resistance gene gyrBR are highlighted. Inset: Novobiocin triggers the transcription of the novobiocin gyrBR gene. The promoter of gyrBR is sensitive to changes in DNA topology; when negative superhelical density is reduced due to the action of novobiocin, the promoter becomes activated and expression of gyrBR occurs. RNAP: RNA polymerase.

with the former enzyme being the primary target (Maxwell, 1997). By interfering with these enzymes, aminocoumarins cause alterations in DNA topology, affecting DNA replication and transcription. The structural similarities of the aminocoumarin antibiotics are reflected in their biosynthetic gene clusters, which encode each biosynthetic pathway in a single polycistronic transcript (Fig. 2). Two transcriptional regulators (novE & novG in the novobiocin gene cluster; cloE & cloG in the clorobiocin gene cluster; couE & couG in the coumermycin A1 gene cluster) are encoded at the 5′ end of each gene cluster and act as positive regulators of the cognate biosynthetic genes (Li and Heide, 2006). In addition, each cluster encodes the self-resistance genes at its 3′ end. The nov cluster encodes a novobiocin-resistant allele of gyrase subunit B (gyrBR) – this replaces the novobiocin-sensitive B subunit of DNA gyrase and is the primary resistance determinant against these molecules (Thiara and Cundliffe, 1988). The clorobiocin and coumermycin A1 biosynthetic gene clusters encode a resistant gyrase subunit B and, immediately downstream, a resistant subunit of topoisomerase IV (parYR) to provide additional self-protection that is consistent with their broader target range (Schmutz et al., 2003). © 2014 John Wiley & Sons Ltd, Molecular Microbiology, 93, 391–402

The expression of the self-resistance genes is tightly linked to the biosynthesis of the aminocoumarins themselves – this is best understood for novobiocin. In S. sphaeroides, gyrBR can be expressed via two promoters. One of these is upstream of novH and directs transcription of all the novobiocin biosynthetic genes in addition to gyrBR (Fig. 2) (Dangel et al., 2009). A second promoter is immediately upstream of gyrBR (Dangel et al., 2009). At the onset of novobiocin production, gyrBR is thought to be co-transcribed with the novobiocin biosynthetic genes from the upstream novH promoter (Dangel et al., 2009). The fact that the resistance-conferring gyrase subunit is produced at the same time as the biosynthetic genes indicates that resistance appears concomitantly with the enzymes of the antibiotic’s biosynthetic pathway, presumably at around the same time when the compound begins to accumulate in the cytoplasm. It is interesting to note that the novobiocin-sensitive form of DNA gyrase (gyrBS) is still expressed during novobiocin production; however, it is believed this expression does not affect the overall resistance phenotype of the novobiocin producer (Thiara and Cundliffe, 1989). In contrast, the gyrBR-specific promoter is activated at a later point in biosynthesis (Dangel et al., 2009). Interest-

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ingly, this promoter can be activated as a result of reduced negative supercoiling brought on by novobiocin’s inhibition of DNA gyrase (Fig. 2, inset) (Thiara and Cundliffe, 1989). This is important for two reasons. First, by elevating levels of the resistance enzyme, it is thought to provide enhanced resistance as the local titre of the antibiotic rises, providing further protection to the producer. Second, it means that the resistance gene can be induced by exogenous aminocoumarins. Thus, S. sphaeroides cells that are not producing novobiocin can be rendered resistant to the antibacterial activity produced by their siblings. Additional aminocoumarin resistance determinants have been identified in S. sphaeroides and S. rishiriensis. In S. sphaeroides, novA encodes a type III ATP-binding cassette (ABC) transporter for novobiocin (Steffensky et al., 2000; Méndez and Salas, 2001), while in S. rishiriensis, couR5 is predicted to be a major facilitator superfamily (MFS) transporter for coumermycin A1 (Pao et al., 1998). These export proteins confer moderate resistance to aminocoumarins independently of the topoisomerase subunits (Schmutz et al., 2003).

Tylosin Tylosin is a macrolide antibiotic produced by Streptomyces fradiae that binds and inhibits the 50S ribosomal subunit. The tylosin biosynthetic gene cluster encodes four tylosin resistance determinants: tlrA, tlrB, tlrC, and tlrD. Three of these genes, tlrA, tlrB, and tlrD, encode ribosomal RNA methyltransferases that protect the ribosome from tylosin action (Zalacain and Cundliffe, 1989; 1991; Liu et al., 2000), whereas tlrC encodes a putative efflux pump that exports the antibiotic and prevents its build up in the cytoplasm (Rosteck et al., 1991). As is the case for tylosin biosynthesis, the regulation of these resistance genes is complex. TlrD is produced constitutively, thus the 23S rRNA is, at least to some extent, protected against the antibiotic regardless of whether it is being synthesized (Zalacain and Cundliffe, 1991). In contrast, tlrA, tlrB, and tlrC are inducible genes. The regulation of tlrA is proposed to occur through ribosome-mediated transcriptional attenuation (Fig. 3) (Kelemen et al., 1994). In the absence of antibiotic, the ribosome moves quickly over the nascent transcript. This results in the formation of a transcriptional attenuation site within the mRNA leader sequence, leading to premature termination of transcription and preventing the production of a complete tylA mRNA. The action of tylosin and its biosynthetic precursors (Fig. 3, inset) causes the ribosomes to stall on the tylA leader sequence, inducing the formation of an anti-termination form of the tylA mRNA leader sequence. This, in turn, permits the production of a full-length tylA transcript.

Interestingly, tlrA induction depends on the methylation state of the ribosomes caused by TlrD; in a tlrD-deficient S. fradiae strain, tlrA expression cannot be induced by tylosin (Kelemen et al., 1994). Therefore, the co-ordination of these two resistance determinants provides a switch from a basal level (due to constitutive tlrD expression) to a high level of tylosin resistance (due to the induction of tlrA) during tylosin biosynthesis. Likewise, S. fradiae cells that are not engaged in tylosin production are also protected from the exogenous molecules that are being synthesized by other cells including most likely, their siblings.

Oxytetracycline Oxytetracycline is a bacteriostatic antibiotic produced by Streptomyces rimosus that binds the 30S ribosomal subunit and blocks the access to aminoacyl-tRNAs during translation. Two resistance genes, otrA and otrB (formerly tetA and tetB respectively), are encoded in the oxytetracycline biosynthetic gene cluster (Fig. 4) (Ohnuki et al., 1985). Their products provide two forms of self-resistance: target protection and oxytetracycline efflux. OtrA shares sequence similarity with the translation elongation factors EF-Tu and EF-G, which co-ordinate the actions of the ribosome, mRNA and aminoacyl-tRNAs during translation (Doyle et al., 1991). Like the translation factors, OtrA has GTPase activity that is vital for its action. It confers oxytetracycline resistance by interacting with the ribosome and dislodging the bound oxytetracycline in a GTP-binding and hydrolysis-dependent manner. The otrA gene is expressed via two promoter sequences otrAp1 and oxySp1 (Fig. 4, top inset) (McDowall et al., 1999). When cells are growing exponentially, otrA is expressed as a monocistronic mRNA from otrAp1. Once the cells enter stationary phase and commence oxytetracycline production, otrAp1 is shut off and oxySp1 is activated. The oxySp1 promoter then drives expression of a three-gene operon that includes otrA and two genes involved in maturation of the oxytetracycline polyketide: oxyT (also known as otcZ; encoding a methyltransferase) and oxyS (also known as otcC; encoding a hydroxylase). It has been suggested that the recognition of these different promoters is carried out by RNA polymerase holoenzymes bearing different sigma factors (McDowall et al., 1999). Importantly, this dual promoter arrangement ensures that OtrA is always present in cells prior to the mature antibiotic, which can only be produced once OxyS and OxyT are present. The second oxytetracycline mechanism that S. rimosus confers is the MFS transport protein OtrB (Ohnuki et al., 1985; Reynes et al., 1988). This gene is apparently regulated by a MarR-family transcriptional regulator, OxyTA1 (also known as OtrR) that might also respond to oxytetracycline (Fig. 4) (Petkovic et al., 2006; Perera and © 2014 John Wiley & Sons Ltd, Molecular Microbiology, 93, 391–402

© 2014 John Wiley & Sons Ltd, Molecular Microbiology, 93, 391–402

ribosome

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Fig. 3. Transcriptional attenuation of the tylosin resistance gene tylA in S. fradiae. In the absence of inducer molecule, the mRNA leader sequence forms a secondary structure with the attenuator sequence (bolded in purple) that acts to prevent transcription of the entire tylA gene. In the presence of tylosin or its biosynthetic intermediates (shown in inset), the ribosome stalls in the leader sequence due to the action of these molecules. This results in the conformational change within the mRNA leader and allows transcription to continue beyond the attenuation site. RNAP: RNA polymerase; OMT: O-mycaminosyltylonolide.

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Antibiotic resistance in antibiotic-producing bacteria 395

396 S. Mak, Y. Xu and J. R. Nodwell ■

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Fig. 4. Oxytetracycline biosynthesis and resistance. The oxytetracycline biosynthetic gene cluster contains two resistance determinants, otrA and otrB (shown in red). Promoters important in the regulation of the ribosomal protection protein OtrA are highlighted. OxyTA1, the regulator for the oxytetracycline efflux pump OtrB, is shown in blue. Top inset: Regulation of otrA expression during the growth cycle of S. rimosus. (A) During exponential phase, otrA is expressed from its cognate promoter otrAp1. (B) When cells reach stationary phase and begin to produce oxytetracycline, otrAp1 is silenced and oxySp1 is activated to drive expression of oxyS, oxyT, and otrA as a polycistronic mRNA. Bottom inset: Proposed model of regulation of otrB expression by the OxyTA1 repressor in S. rimosus. In the absence of oxytetracycline, OxyTA1 binds upstream of the otrB gene and inhibits its expression. When oxtetracycline is present, this ligand binds to OxyTA1 and relieves repression of otrB, allowing for the expression of the exporter gene.

Grove, 2010; Pickens and Tang, 2010). While the proposed mechanism requires further investigation, this mode of regulation offers potential advantages for cells. In addition to providing additional protection for the oxytetracycline producer, S. rimosus cells that are not expressing the oxytetracycline biosynthetic genes would be protected against the compound produced by their oxytetracycline-producing sibling cells.

Actinorhodin Actinorhodin is a benzoisochromanequinone antibiotic produced by Streptomyces coelicolor that exhibits potent antibacterial activity against Gram-positive cells including many streptomycetes (S. Mak and J.R. Nodwell, unpub-

lished). The biosynthetic gene cluster for actinorhodin encodes three putative integral membrane proteins. These proteins are ActA (also known as ActII-ORF2), ActB (also known as ActII-ORF3), and ActVA-ORF1 (which we refer to as ActC). To date, there has been no systematic investigation of the capacity of these proteins to confer resistance to actinorhodin; however, ActA and ActC are predicted to be MFS pumps, and ActB is similar to the resistancenodulation-cell division (RND) transporters (Tseng et al., 1999). Many members from both of these families have been implicated in narrow- and broad-spectrum antibiotic resistance (Poole, 2005). The actA and actB genes are expressed as a two-gene operon from a promoter upstream of actA. This promoter is repressed by ActR (also known as ActII-ORF1), the © 2014 John Wiley & Sons Ltd, Molecular Microbiology, 93, 391–402

Antibiotic resistance in antibiotic-producing bacteria 397

General biosynthetic pathway scheme: tailoring

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Fig. 5. Model of the feed-forward mechanism as seen in actinorhodin resistance. In this type of regulatory system, the intermediate molecule (generated during the early stages of antibiotic production), as well as the final mature antibiotic (tailored from the biosynthetic intermediates) act as inducer ligands to activate expression of self-resistance genes. A. The actinorhodin biosynthetic intermediate (S)-DNPA binds to the regulator to relieve repression of the resistance gene, priming S. coelicolor for self-resistance. B. Once antibiotic resistance has been established, the mature antibiotic actinorhodin behaves similarly to maintain resistance within the producer cells, as well as to protect non-producers from any exogenous antibiotic.

product of the adjacent and divergently expressed gene. ActR is a member of the TFR family of transcription factors (Ahn et al., 2012; Cuthbertson and Nodwell, 2013). Repression by ActR can be relieved not only by actinorhodin or but also by its biosynthetic intermediate (S)-DNPA (Tahlan et al., 2007; 2008). Indeed, this intermediate was found to be a more potent inducer than the mature compound itself, leading to the suggestion that the intermediates might ‘feed forward’ to activate the expression of actA and actB before actinorhodin accumulates inside cells. However, a growing body of evidence suggests that ActA and/or ActB are also important for efficient actinorhodin production. A mutant lacking of these genes produced reduced levels of actinorhodin relative to the wild type, whereas an actR deletion mutant (in which the actAB © 2014 John Wiley & Sons Ltd, Molecular Microbiology, 93, 391–402

promoter is constitutively active) exhibited enhanced yields of actinorhodin (Xu et al., 2012). Furthermore, in a strain expressing a mutant ActR (ActR*) which can only respond to the (S)-DNPA intermediate and not the mature antibiotic, actinorhodin yields were normal in spite of the fact that the expression of actAB was very low (Xu et al., 2012). This suggested that intermediate-mediated activation of actAB expression might be sufficient to support normal actinorhodin biosynthesis. It was observed, however, that after prolonged growth, the cultures of the actR* mutant exhibited widespread cell death (Xu et al., 2012), suggesting that low level expression of the pumps was not sufficient for actinorhodin resistance. These studies suggested a two-step mechanism for the activation of the actAB operon (Fig. 5). In actinorhodin-

398 S. Mak, Y. Xu and J. R. Nodwell ■ microbisporicin intermediate

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producing cells, pathway intermediates could trigger actAB expression, priming the organism for resistance and efficient actinorhodin production (Fig. 5A). As actinorhodin begins to accumulate in the culture medium, it could act as a signal to sustain expression and perhaps activate the operon in cells that are not expressing the actinorhodin biosynthetic genes (Fig. 5B) (Xu et al., 2012).

Microbisporicin Another antibiotic biosynthesis/resistance process that has been described in terms of an intermediate-mediated ‘feed-forward’ mechanism is the production of microbisporicin by Microbispora corallina (Foulston and Bibb, 2011). Microbisporicin is a potent lantibiotic having unusual chlorotryptophan and dihydroxyproline residues that acts by blocking peptidoglycan biosynthesis (Castiglione et al., 2008; Foulston and Bibb, 2010). The mib biosynthetic gene cluster consists of six operons. While the initial stages of biosynthesis are not well understood, it is clear that there is some expression of several of these operons. Low-level expression of mibXW generates the extracytoplasmic function (ECF) sigma factor, σMibX and its cognate membrane-associated anti-sigma factor, MibW. An unknown process results in the expression of mibR, which encodes a transcription factor. MibR directly activates mibABCDTUV, which generates the microbisporicin pre-peptide (mibA), some of the enzymes involved in the antibiotic’s maturation (mibB, mibC and mibD) and an export protein (mibTU).

As a result, during this early phase of biosynthesis, a microbisporicin intermediate is produced and exported out of the cytoplasm by the MibTU exporter, while σMibX is held in an inactive state through its association with MibW (Fig. 6, left panel). A transition then occurs that is believed to result from either a direct or an indirect interaction between the exported intermediate and MibW (Fig. 6, right panel). This results in the release of σMibX and the activation of its target promoters upstream of mibJYZO, mibQ, mibXW and mibEFHSN. Two things now happen. First, a second microbisporicin exporter, MibEF, is deployed and this is thought to confer-high level resistance to, presumably, the mature antibiotic. Second, the enzymes involved in the final microbisporicin maturation steps are produced, including the biogenesis of the chlorotryptophan and dihydroxyproline residues by MibHS and MibO respectively (Castiglione et al., 2008). At this point, the cell is committed to high-level production of microbisporicin. How important is this two-stage interplay of regulators and biosynthetic enzymes? The available evidence suggests that it is essential; otherwise, microbisporicin production is lethal to the cell. Intriguingly, efforts to inactivate mibW proved to be unsuccessful except in cells that also had null mutations in mibX (Foulston and Bibb, 2011). If both MibTU and MibEF are not deployed, then the mature antibiotic has the capacity to overpower the cell and kill it. Indeed, MibQ, another target gene of σMibX, may also serve as a microbisporicin immunity function (Foulston and Bibb, 2011). Thus, as is the case with actinorhodin, the existence of multiple layers of regulation serves the vital function of © 2014 John Wiley & Sons Ltd, Molecular Microbiology, 93, 391–402

Antibiotic resistance in antibiotic-producing bacteria 399

protecting the producer from its own antibiotic biosynthetic capacity.

Exceptions and outliers The diversity, and in some cases, the complexity of these regulatory mechanisms is remarkable and it is an outstanding subject for continued investigation. Indeed, in spite of the fact that the first biosynthetic gene clusters for antibiotics were cloned and sequenced 30 years ago or more, there is still relatively little known about the regulation of resistance as it manifests during antibiotic production in living cells. The cases we have cited are among the most extensively investigated; however, it is unclear how universally conserved they are. For instance, there are several mechanisms, related to those described above, that do not fit well with either (i) linked resistance and biosynthesis model or (ii) induction by antibiotics or pathway intermediates. In the 5′-hydroxystreptomycin producer Streptomyces glaucescens, the resistance determinant sph and its biosynthetic activator strR, are obvious orthologues of their counterparts in the str cluster of S. griseus. However, these genes are separated by four genes that are not arranged in a way where strR and sph can be coexpressed, unlike the case in S. griseus (Distler et al., 1992). How this resistance gene is regulated is unclear – perhaps it is expressed constitutively like the erythromycin resistance gene? The biosynthetic gene cluster for simocyclinone D8, encoded in Streptomyces antibioticus, includes a TFR/ exporter gene pair (simR/simX) similar to actR/actAB. The mature antibiotic and its biosynthetic intermediate (simocyclinone D4) can both relieve repression of simX by SimR; however, simocyclinone D8 is a more potent inducer compared to simocyclinone D4 (Le et al., 2009). Furthermore, structural information concerning the recognition of the antibiotics by SimR strongly argues that this mature molecule plays the critical role in the induction of resistance (Le et al., 2011). In spite of the similarity of these genes to those that mediate export/resistance to actinorhodin, the feed-forward model does not fit. The biosynthetic gene cluster for the landomycin antibiotics, encoded in Streptomyces cyanogenus S136, also encodes a TFR/export pump gene pair (lanK/lanJ). S. cyanogenus S136 can generate landomycins having chains of two to six sugar residues. The short-chained compounds (such as landomycin E) are the most potent antibacterials, while the long-chained landomycin A is much weaker due to its possible compromised entry into prokaryotes (Matseliukh et al., 1998). Repression of lanJ by LanK can be relieved by either landomycin A or its shorter-chained biosynthetic intermediates landomycins B and E (Ostash et al., 2008). If this occurs in a ‘feedforward’ manner, then the most lethal short-chained land© 2014 John Wiley & Sons Ltd, Molecular Microbiology, 93, 391–402

omycin intermediates must first accumulate in the cytoplasm to induce resistance – how cells are able to survive this event without killing themselves is unclear. In addition, many antibiotic producers encode other resistance genes whose relevance and mode of regulation are not well understood. For instance, S. rimosus encodes a third oxytetracycline resistance protein OtrC, which is an ABC transport protein. Although this gene is located outside of the oxytetracycline biosynthetic gene cluster, it has been shown to confer high level resistance of oxytetracycline when expressed heterologously in other bacteria (Yu et al., 2012). It is unclear why a third resistance mechanism is needed or whether it is in fact important for conferring resistance.

Antibiotic resistance and biosynthesis go hand-in-hand In spite of these caveats and the fairly limited data set that is available for many antibiotic biosynthesis-linked antibiotic resistance determinants, a striking picture emerges from the examples we have described. First, in many cases, antibiotic resistance precedes or is mechanistically coupled to biosynthesis such that it is expressed prior to or concomitantly with the appearance of the antibiotic molecule in the cell. This includes constitutively expressed resistance mechanisms for erythromycin (ermE), oxytetracycline (otrAp1-dependent expression of otrA) and tylosin (tylD). In the streptomycin producer S. griseus, the aphD gene is expressed along with the pathway-specific activator of the str biosynthetic cluster, indicating that, again, resistance precedes biosynthesis. Additionally, resistance determinants (or putative resistance determinants) for novobiocin (gyrBR) and oxytetracycline (oxySp1-dependent expression of otrA), are expressed as parts of operons along with biosynthetic enzymes, meaning that resistance appears concurrently (or is upregulated at the same time) with the biosynthetic machinery. Indeed, in the case of actinorhodin, it is known that resistance itself is important for efficient biosynthesis. Second, many antibiotics can act as cell–cell signals to activate resistance in non-producing cells. The mechanisms are equally diverse: the compound can act by deactivating a transcriptional repressor as shown for actinorhodin (actR) and landomycin (lanJ), by releasing the repression of an alternative sigma factor as shown for microbisporicin (σMibX), or through the action of a transcriptional attenuation mechanism as shown for tylosin (tylA). Moreover, the antibiotic action of the aminocoumarins can induce the expression of resistant alleles of gyrase subunits (gyrBR). The advantages to the antibiotic producer are obvious. First, resistance must be expressed in a timely manner or antibiotic production would be a high-risk activity for producer organisms – the advantage of resistance preceding

400 S. Mak, Y. Xu and J. R. Nodwell ■

antibiotic producer

antibiotic non-producer

antibiotic biosynthesis

exogenous antibiotic

antibiotic resistance

antibiotic resistance

Fig. 7. Resistance determinants that can be induced by antibiotics can protect both antibiotic producers as well as non-producers. In an antibiotic producer, resistance (gene shown in red) appears prior to or concurrently with the production of the antibiotic (biosynthetic gene shown in blue) to protect the organism from its output. Antibiotic non-producers are also protected from any exogenous antibiotics that produced by their antibiotic-producing siblings.

or being tightly coupled to biosynthesis is therefore clear. Second, the cell–cell signalling mechanisms means that if there are cells in a culture that are not producing the antibiotic, it would be possible for producing cells to trigger resistance in non-producing siblings (Fig. 7). It is known that during growth, streptomycetes generate differentiated cell types having distinct programmes of gene expression including most notably, the spore-forming aerial hyphae and the vegetative substrate hyphae (Claessen et al., 2006; McCormick and Flärdh, 2012). Evidence also supports the existence of distinct vegetative cell types, again having distinct gene expression profiles (Dalton et al., 2007). Therefore, it is plausible that in nature, cells of the same organism, living in close proximity to one another, might not all produce antibiotics at the same time. Induction of resistance by antibiotic-mediated cell–cell signalling would mean that non-producing cells would also be protected. While the details are lacking for a great number of cluster-encoded antibiotic resistance genes, it seems probable that linked biosynthesis and resistance, as well as antibiotic- or intermediate-inducible resistance, will turn out to be widespread in nature.

Acknowledgements S.M. was supported by a grant from the Canadian Institutes for Health Research (MOP-57684).

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