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Conjugative transfer is a major contributor to the dissemination of antibiotic resistance and virulence genes in the human and animal pathogen, Clostridium perfringens. The C. perfringens plasmid pCW3 is the archetype of an extensive family of highly related conjugative toxin and antibiotic resistance plasmids found in this bacterium. These plasmids were thought to constitute the only conjugative plasmid family in C. perfringens. Recently, another series of C. perfringens plasmids, the pCP13-like family, have been shown to harbour important toxin genes, including genes that encode the novel binary clostridial enterotoxin, BEC. Based on early bioinformatics analysis this plasmid family was thought to be non-conjugative. Here we demonstrate that pCP13 is in fact conjugative, transfers at high frequency and that the newly defined Pcp conjugation locus encodes putative homologues of a type 4 secretion system (T4SS), one of which, PcpB4, was shown to be essential for transfer. The T4SS of pCP13 also appears to be evolutionarily related to conjugative toxin plasmids from other clostridia-like species, including Paeniclostridium (formerly Clostridium) sordellii, Clostridioides (formerly Clostridium) difficile and Clostridium botulinum. Therefore, it is clear that there are two distinct families of conjugative plasmids in C. perfringens: the pCW3 family and the pCP13 family. This study has significant implications for our understanding of the movement of toxin genes both within C. perfringens, but also potentially to other pathogenic clostridia., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Bacterial pathogens have adopted numerous mechanisms for acquiring iron from host proteins during an infection, including the direct acquisition of ferric iron from heme-associated proteins or from iron-scavenging siderophores. Ferric iron then is transported into the cytosol, where it can be utilized by the bacterial pathogen. Under anaerobic conditions bacteria can also transport ferrous iron using the transmembrane complex FeoAB, but little is known about iron transport systems in anaerobic bacteria such as the pathogenic clostridia. In this study we sought to characterize the iron acquisition process in Clostridium perfringens. Bioinformatic analysis of the Clostridium perfringens strain 13 genome sequence revealed that it has seven potential iron acquisition systems: three siderophore-mediated systems, one ferric citrate uptake system, two heme-associated acquisition systems and one ferrous iron uptake system (FeoAB). The relative level of expression of these systems was determined using quantitative real-time RT-PCR assays that were specific for one gene from each system. Each of these genes was expressed, with the feoAB genes generating the most abundant iron-uptake related transcripts. To further examine the role of this system in the growth of C. perfringens, insertional inactivation was used to isolate a chromosomal feoB mutant. Growth of this mutant in the presence and absence of iron revealed that it had altered growth properties and a markedly reduced total iron and manganese content compared to the wild type; effects that were reversed upon complementation with the wild-type feoB gene. These studies suggest that under anaerobic conditions FeoB is the major protein required for the uptake of iron into the cell and that it may play an important role in the pathogenesis of C. perfringens infections., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The ability of a pathogenic bacterium to scavenge iron from its host is important for its growth and survival during an infection. Our studies on C. perfringens gas gangrene strain JIR325, a derivative of strain 13, showed that it is capable of utilizing both human hemoglobin and ferric chloride, but not human holo-transferrin, as an iron source for in vitro growth. Analysis of the C. perfringens strain 13 genome sequence identified a putative heme acquisition system encoded by an iron-regulated surface gene region that we have named the Cht (Clostridium perfringens heme transport) locus. This locus comprises eight genes that are co-transcribed and includes genes that encode NEAT domain-containing proteins (ChtD and ChtE) and a putative sortase (Srt). The ChtD, ChtE and Srt proteins were shown to be expressed in JIR325 cells grown under iron-limited conditions and were localized to the cell envelope. Moreover, the NEAT proteins, ChtD and ChtE, were found to bind heme. Both chtDE and srt mutants were constructed, but these mutants were not defective in hemoglobin or ferric chloride utilization. They were, however, attenuated for virulence when tested in a mouse myonecrosis model, although the virulence phenotype could not be restored via complementation and, as is common with such systems, secondary mutations were identified in these strains. In summary, this study provides evidence for the functional redundancies that occur in the heme transport pathways of this life threatening pathogen., Competing Interests: The authors have declared that no competing interests exist.

The clostridia cause many human and animal diseases, resulting in significant morbidity and mortality. Host damage results from the action of potent exotoxins, an important group of which is the large clostridial toxins (LCTs) produced by Clostridium difficile, Clostridium sordellii, Clostridium perfringens and Clostridium novyi. Knowledge of the structure and function of these toxins has been attained, however, apart from C. difficile, the regulatory pathways that control LCT production remain largely unknown. Here we show that LCT production in C. sordellii and C. perfringens is temporally regulated and repressed by glucose in a similar manner to C. difficile. Furthermore, we show that the TpeL-encoding gene of C. perfringens is located in an uncharacterized Pathogenicity Locus (PaLoc), along with accessory genes predicted to encode a bacteriophage holin-type protein and a TcdR-family alternative sigma factor, TpeR. Inactivation of tpeR demonstrated that TpeR is critical for C. perfringens TpeL production, in a similar manner to C. difficile TcdR and C. sordellii TcsR, but cross-complementation showed that TpeR is not functionally interchangeable with TcdR or TcsR. Although conserved mechanisms are employed by the clostridia to control LCT production there are important functional differences that distinguish members of the TcdR-family of clostridial alternative sigma factors., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Gas gangrene is a potentially fatal disease that is primarily caused by the ubiquitous, anaerobic bacteria Clostridium perfringens and Clostridium septicum. Treatment is limited to antibiotic therapy, debridement of the infected tissue, and, in severe cases, amputation. The need for new treatment approaches is compelling. Opioid-based analgesics such as buprenorphine and morphine also have immunomodulatory properties, usually leading to faster disease progression. However, here we show that mice pretreated with buprenorphine and morphine do not die from clostridial myonecrosis. Treatment with buprenorphine after the onset of infection also arrested disease development. Protection against myonecrotic disease was specific to C. perfringens-mediated myonecrosis; buprenorphine did not protect against disease caused by C. septicum infection even though infections due to both species are very similar. These data provide the first evidence of a protective role for opioids during infection and suggest that new therapeutic strategies may be possible for the treatment of C. perfringens-mediated myonecrosis., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

TnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn4451 and Tn4453 in Clostridium perfringens and Clostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes; however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids in Escherichia coli and from marked chromosomal C. perfringens mutants. This methodology enabled the construction of a C. perfringens plc virR double mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic to E. coli. We show that TnpX represses expression from its own promoter, PattCI, which can be exploited to facilitate the cloning of recalcitrant genes in E. coli for subsequent expression in the heterologous host C. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.

Clostridium perfringens uses its arsenal of >16 toxins to cause histotoxic and intestinal infections in humans and animals. It has been unclear why this bacterium produces so many different toxins, especially since many target the plasma membrane of host cells. However, it is now established that C. perfringens uses chromosomally encoded alpha toxin (a phospholipase C) and perfringolysin O (a pore-forming toxin) during histotoxic infections. In contrast, this bacterium causes intestinal disease by employing toxins encoded by mobile genetic elements, including C. perfringens enterotoxin, necrotic enteritis toxin B-like, epsilon toxin and beta toxin. Like perfringolysin O, the toxins with established roles in intestinal disease form membrane pores. However, the intestinal disease-associated toxins vary in their target specificity, when they are produced (sporulation vs vegetative growth), and in their sensitivity to intestinal proteases. Producing many toxins with diverse characteristics likely imparts virulence flexibility to C. perfringens so it can cause an array of diseases.

Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and produces many different extracellular toxins and enzymes, including the cysteine protease α-clostripain. Mutation of the α-clostripain structural gene, ccp, alters the turnover of secreted extracellular proteins in C. perfringens, but the role of α-clostripain in disease pathogenesis is not known. We insertionally inactivated the ccp gene C. perfringens strain 13 using TargeTron technology, constructing a strain that was no longer proteolytic on skim milk agar. Quantitative protease assays confirmed the absence of extracellular protease activity, which was restored by complementation with the wild-type ccp gene. The role of α-clostripain in virulence was assessed by analysing the isogenic wild-type, mutant and complemented strains in a mouse myonecrosis model. The results showed that although α-clostripain was the major extracellular protease, mutation of the ccp gene did not alter either the progression or the development of disease. These results do not rule out the possibility that this extracellular enzyme may still have a role in the early stages of the disease process.

Clostridium perfringens has been associated with necrotizing enterocolitis (NEC), which is a serious disease of neonates. Our study describes the novel use of selective tryptose sulfite cycloserine with egg yolk agar (TSC-EYA) during a nursery outbreak. This medium provides a rapid, sensitive, and accurate presumptive identification of C. perfringens.

The essential toxin in Clostridium perfringens-mediated gas gangrene or clostridial myonecrosis is alpha-toxin, although other toxins and extracellular enzymes may also be involved. In many bacterial pathogens extracellular sialidases are important virulence factors, and it has been suggested that sialidases may play a role in gas gangrene. C. perfringens strains have combinations of three different sialidase genes, two of which, nanI and nanJ, encode secreted sialidases. The nanI and nanJ genes were insertionally inactivated by homologous recombination in derivatives of sequenced strain 13 and were shown to encode two functional secreted sialidases, NanI and NanJ. Analysis of these derivatives showed that NanI was the major sialidase in this organism. Mutation of nanI resulted in loss of most of the secreted sialidase activity, and the residual activity was eliminated by subsequent mutation of the nanJ gene. Only a slight reduction in the total sialidase activity was observed in a nanJ mutant. Cytotoxicity assays using the B16 melanoma cell line showed that supernatants containing NanI or overexpressing NanJ enhanced alpha-toxin-mediated cytotoxicity. Finally, the ability of nanI, nanJ, and nanIJ mutants to cause disease was assessed in a mouse myonecrosis model. No attenuation of virulence was observed for any of these strains, providing evidence that neither the NanI sialidase nor the NanJ sialidase is essential for virulence.

Toxin production in Clostridium perfringens is controlled by the VirSR two-component signal transduction system, which comprises the VirS sensor histidine kinase and the VirR response regulator. Other studies have concentrated on the elucidation of the genes controlled by this network; there is little information regarding the phosphorelay cascade that is the hallmark of such regulatory systems. In this study, we have examined each step in this cascade, beginning with autophosphorylation of VirS, followed by phosphotransfer from VirS to VirR. We also have studied the effects of gene dosage and phosphorylation in vivo. We have used random and site-directed mutagenesis to identify residues in VirS that are important for its function and have identified a region in the putative sensory domain of VirS that appeared to be essential for function. In vitro phosphorylation studies showed that VirSc, a truncated VirS protein that lacked the N-terminal sensory domain, was capable of autophosphorylation and could subsequently act as a phosphodonor for its cognate response regulator, VirR. Conserved residues of both VirS and VirR, including the D57 residue of VirR, were shown to be essential for this process. By use of Targetron technology, we were able to introduce a single copy of virR or virR(D57N) onto the chromosome of a virR mutant of C. perfringens. The results showed that in vivo, when virR was present in single copy, the production of wild-type levels of perfringolysin O was dependent on the presence of virS and an unaltered D57 residue in VirR. These results provide good evidence that phosphorylation is critical for VirR function.

Reduced tissue perfusion leading to tissue ischemia is a central component of the pathogenesis of myonecrosis caused by Clostridium perfringens. The C. perfringens alpha-toxin has been shown capable of inducing these changes, but its potential synergy with perfringolysin O (theta-toxin) is less well understood. Similarly, Clostridium septicum is a highly virulent causative agent of spontaneous gas gangrene, but its effect on the microcirculation has not been examined. Therefore, the aim of this study was to use intravital microscopy to examine the effects of C. perfringens and C. septicum on the functional microcirculation, coupled with the use of isogenic toxin mutants to elucidate the role of particular toxins in the resultant microvascular perfusion deficits. This study represents the first time this integrated approach has been used in the analysis of the pathological response to clostridial toxins. Culture supernatants from wild-type C. perfringens induced extensive cell death within 30 min, as assessed by in vivo uptake of propidium iodide. Furthermore, significant reductions in capillary perfusion were observed within 60 min. Depletion of either platelets or neutrophils reduced the alteration in perfusion, consistent with a role for these blood-borne cells in obstructing perfusion. In addition, mutation of either the alpha-toxin or perfringolysin O structural genes attenuated the reduction in perfusion, a process that was reversed by genetic complementation. C. septicum also induced a marked reduction in perfusion, with the degree of microvascular compromise correlating with the level of the C. septicum alpha-toxin. Together, these data indicate that as a result of its ability to produce alpha-toxin and perfringolysin O, C. perfringens rapidly induces irreversible cellular injury and a marked reduction in microvascular perfusion. Since C. septicum induces a similar reduction in microvascular perfusion, it is postulated that this function is central to the pathogenesis of clostridial myonecrosis, irrespective of the causative bacterium.

The Clostridium perfringens alpha-toxin has previously been implicated as the major virulence factor in necrotic enteritis in chickens, although definitive proof has not been reported. In this study an alpha-toxin mutant was constructed in a virulent chicken isolate and shown to retain full virulence in a chicken disease model. These results demonstrated that alpha-toxin is not an essential virulence factor in the pathogenesis of necrotic enteritis in chickens.

Clostridium perfringens is a Gram-positive, anaerobic spore-forming bacterium commonly found in soil, sediments, and the human gastrointestinal tract. C. perfringens is responsible for a wide spectrum of disease, including food poisoning, gas gangrene (clostridial myonecrosis), enteritis necroticans, and non-foodborne gastrointestinal infections. The complete genome sequences of Clostridium perfringens strain ATCC 13124, a gas gangrene isolate and the species type strain, and the enterotoxin-producing food poisoning strain SM101, were determined and compared with the published C. perfringens strain 13 genome. Comparison of the three genomes revealed considerable genomic diversity with >300 unique "genomic islands" identified, with the majority of these islands unusually clustered on one replichore. PCR-based analysis indicated that the large genomic islands are widely variable across a large collection of C. perfringens strains. These islands encode genes that correlate to differences in virulence and phenotypic characteristics of these strains. Significant differences between the strains include numerous novel mobile elements and genes encoding metabolic capabilities, strain-specific extracellular polysaccharide capsule, sporulation factors, toxins, and other secreted enzymes, providing substantial insight into this medically important bacterial pathogen.

The VirSR two-component signal transduction pathway regulates virulence and toxin production in Clostridium perfringens, the causative agent of gas gangrene. The response regulator, VirR, binds to repeat sequences located upstream of the promoter and is directly responsible for the transcriptional activation of pfoA, the structural gene for the cholesterol-dependent cytolysin, perfringolysin O. Comparative sequence analysis of the 236 amino acid residue VirR protein revealed a two-domain structure: a typical N-terminal response regulator domain and an uncharacterised C-terminal domain. Database searching revealed that over 40 other proteins, many of which appeared to be response regulators or transcriptional activators, had homology with the VirR C-terminal domain (VirRc). Multiple sequence alignment of this VirRc family revealed a highly conserved region that was designated the FxRxHrS motif. By deletion analysis this motif was shown to be essential for the functional integrity of the VirR protein. Alanine scanning mutagenesis and subsequent phenotypic analysis indicated that conserved residues located within the motif were required for activity. These residues extended from L179 to N194. More detailed site-directed mutagenesis showed that amino acid residues R186, H188 and S190 were essential for activity since even conservative substitutions in these positions resulted in non-functional proteins. Three of the mutant proteins, R186K, S190A and S190C, were purified and shown by in vitro gel shift analysis to be unable to bind to the specific target DNA with the same efficiency as the wild-type protein. These data reveal for the first time that VirRc functions as a DNA binding domain in which the highly conserved FxRxHrS motif has a functional role. These studies have important implications for this new family of transcriptional factors since they imply that the conserved FxRxHrS motif may be involved in DNA binding in all of these proteins, irrespective of their biological role.

The pathogenesis of Clostridium perfringens-mediated gas gangrene or clostridial myonecrosis involves the extracellular toxins alpha-toxin and perfringolysin O. Previous studies (T. Shimizu, A. Okabe, J. Minami, and H. Hayashi, Infect. Immun. 59:137-142, 1991) carried out with Escherichia coli suggested that the perfringolysin O structural gene, pfoA, was positively regulated by the product of the upstream pfoR gene. In an attempt to confirm this hypothesis in C. perfringens, a pfoR-pfoA deletion mutant was complemented with isogenic pfoA(+) shuttle plasmids that varied only in their ability to encode an intact pfoR gene. No difference in the ability to produce perfringolysin O was observed for C. perfringens strains carrying these plasmids. In addition, chromosomal pfoR mutants were constructed by homologous recombination in C. perfringens. Again no difference in perfringolysin O activity was observed. Since it was not possible to alter perfringolysin O expression by mutation of pfoR, it was concluded that the pfoR gene product is unlikely to have a role in the regulation of pfoA expression in C. perfringens.

Background: Clostridium sordellii can cause severe infections in animals and humans, the latter associated with trauma, toxic shock and often-fatal gynaecological infections. Strains can produce two large clostridial cytotoxins (LCCs), TcsL and TcsH, related to those produced by Clostridium difficile, Clostridium novyi and Clostridium perfringens, but the genetic basis of toxin production remains uncharacterised. Results: Phylogenetic analysis of the genome sequences of 44 strains isolated from human and animal infections in the UK, US and Australia placed the species into four clades. Although all strains originated from animal or clinical disease, only 5 strains contained LCC genes: 4 strains contain tcsL alone and one strain contains tcsL and tcsH. Four toxin-positive strains were found within one clade. Where present, tcsL and tcsH were localised in a pathogenicity locus, similar to but distinct from that present in C. difficile. In contrast to C. difficile, where the LCCs are chromosomally localised, the C. sordellii tcsL and tcsH genes are localised on plasmids. Our data suggest gain and loss of entire toxigenic plasmids in addition to horizontal transfer of the pathogenicity locus. A high quality, annotated sequence of ATCC9714 reveals many putative virulence factors including neuraminidase, phospholipase C and the cholesterol-dependent cytolysin sordellilysin that are highly conserved between all strains studied. Conclusions: Genome analysis of C. sordellii reveals that the LCCs, the major virulence factors, are localised on plasmids. Many strains do not contain the LCC genes; it is probable that in several of these cases the plasmid has been lost upon laboratory subculture. Our data are consistent with LCCs being the primary virulence factors in the majority of infections, but LCC-negative strains may precipitate certain categories of infection. A high quality genome sequence reveals putative virulence factors whose role in virulence can be investigated. [ABSTRACT FROM AUTHOR]

Protein secretion is generally mediated by a series of distinct pathways in bacteria. Recently, evidence of a novel bacterial secretion pathway involving a bacteriophage-related protein has emerged. TcdE, a holin-like protein encoded by toxigenic isolates of Clostridioides difficile, mediates the release of the large clostridial glucosylating toxins (LCGTs), TcdA and TcdB, and TpeL from C. perfringens uses another holin-like protein, TpeE, for its secretion; however, it is not yet known if TcdE or TpeE secretion is specific to these proteins. It is also unknown if other members of the LCGT-producing clostridia, including Paeniclostridium sordellii (previously Clostridium sordellii), use a similar toxin-release mechanism. Here, we confirm that each of the LCGT-producing clostridia encode functional holin-like proteins in close proximity to the toxin genes. To characterise the respective roles of these holin-like proteins in the release of the LCGTs, P. sordellii and its lethal toxin, TcsL, were used as a model. Construction and analysis of mutants of the P. sordellii tcsE (holin-like) gene demonstrated that TcsE plays a significant role in TcsL release. Proteomic analysis of the secretome from the tcsE mutant confirmed that TcsE is required for efficient TcsL secretion. Unexpectedly, comparative sample analysis showed that TcsL was the only protein significantly altered in its release, suggesting that this holin-like protein has specifically evolved to function in the release of this important virulence factor. This specificity has, to our knowledge, not been previously shown and suggests that this protein may function as part of a specific mechanism for the release of all LCGTs. [ABSTRACT FROM AUTHOR]

Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and produces many different extracellular toxins and enzymes, including the cysteine protease &agr;-clostripain. Mutation of the a-clostripain structural gene, ccp, alters the turnover of secreted extracellular proteins in C. perfringens, but the role of &agr;-clostripain in disease pathogenesis is not known. We insertionally inactivated the ccp gene C. perfringens strain 13 using TargeTron technology, constructing a strain that was no longer proteolytic on skim milk agar. Quantitative protease assays confirmed the absence of extracellular protease activity, which was restored by complementation with the wild-type ccp gene. The role of aclostripain in virulence was assessed by analysing the isogenic wild-type, mutant and complemented strains in a mouse myonecrosis model. The results showed that although &agr;-clostripain was the major extracellular protease, mutation of the ccp gene did not alter either the progression or the development of disease. These results do not rule out the possibility that this extracellular enzyme may still have a role in the early stages of the disease process. [ABSTRACT FROM AUTHOR]

The pathogenesis of clostridial myonecrosis, or gas gangrene, involves the growth of the anaerobic bacterium Clostridium perfringens in the infected tissues and the elaboration of numerous extracellular toxins and enzymes. The precise role of each of these toxins in tissue invasion and necrosis has not been determined. To enable genetic approaches to be used to study C. perfringens pathogenesis we developed an Allelic exchange method which involved the transformation of C. perfringens cells with a suicide plasmid carrying a gene insertionally inactivated with an erythromycin-resistance determinant. The frequency with which double reciprocal cross-over events were observed was increased to a workable level by increasing the amount of homologous DNA located on either side of the inactivated gene. Allelic exchange was used to isolate mutations in the 'chromosomal pfoA gene, which encodes an oxygen-labile haemolysin known as θ-toxin or perfringolysin O. and in the chromosomal plc gene, which encodes the α-toxin or phospholipase C. The resultant mutants failed to produce detectable θ-toxin or θ-toxin activity, respectively, and could be complemented by recombinant plasmids that carried the respective wild-type genes. The resultant strains were virulence tested in a mouse myonecrosis model. The results showed that the plc mutants had demonstrably reduced virulence and therefore provided definitive genetic evidence tor the essential role of α-toxin in gas gangrene or clostridial myonecrosis. [ABSTRACT FROM AUTHOR]

The anaerobic bacterium Clostridium perfringens mediates clostridial myonecrosis. or gas gangrene, by producing a number of extracellular toxins and enzymes. Transposon mutagenesis with Tn916 was used to isolate a pleiotropic mutant of C. perfringens that produced reduced levels of phospholipase C, protease and sialidase, and did not produce any detectable perfringolysin O activity. Southern hybridization revealed that a single copy of Tn916 had inserted into a 2.7 kb Hindlll fragment in the C. perfringens chromosome. A 4.3 kb Pstl fragment, which spanned the Tn916 insertion site, was cloned from the wild-type strain. When subcloned into a shuttle vector and introduced into C. perfringens this fragment was able to complement the Tn916-derived mutation. Transformation of the mutant with plasmids containing the 2.7 kb HindllL fragment, or the 4.3 kb Pstl fragment, resulted In toxin and enzyme levels greater than or equal to those of the wild-type strain. The Pstl fragment was sequenced and found to potentially encode seven open reading frames, two of which appeared to be arranged in an operon and shared sequence similarity with members of two-component signal transduction systems. The putative virR gene encoded a protein with a deduced molecular weight of 30140, and with sequence similarity to activators in the response regulator family of proteins. The next gene, virS, into which Tn916 had inserted, was predicted to encode a membrane-spanning protein with a deduced molecular weight of 51 274. The putative VirS protein had sequence similarity to sensor proteins and also contained a histidine residue highly conserved in the histidine protein kinase family of sensor proteins. Virulence studies carried out using a mouse model implicated the virS gene in the pathogenesis of histotoxic C. perfringens infections. It was concluded that a two-component... [ABSTRACT FROM AUTHOR]