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Transient blockade of glycine decarboxylase (GLDC) can restrict de novo pyrimidine synthesis, which is a well-described strategy for enhancing the host interferon response to viral infection and a target pathway for some licenced anti-inflammatory therapies. The aminothiol, cysteamine, is produced endogenously during the metabolism of coenzyme A, and is currently being investigated in a clinical trial as an intervention in community acquired pneumonia resulting from viral (influenza and SARS-CoV-2) and bacterial respiratory infection. Cysteamine is known to inhibit both bacterial and the eukaryotic host glycine cleavage systems via competitive inhibition of GLDC at concentrations, lower than those required for direct antimicrobial or antiviral activity. Here, we demonstrate for the first time that therapeutically achievable concentrations of cysteamine can inhibit glycine utilisation by epithelial cells and improve cell-mediated responses to infection with respiratory viruses, including human coronavirus 229E and Influenza A. Cysteamine reduces interleukin-6 (IL-6) and increases the interferon-λ (IFN-λ) response to viral challenge and in response to liposomal polyinosinic:polycytidylic acid (poly I:C) simulant of RNA viral infection., Competing Interests: Declaration of competing interest The authors declare a conflict of interest. DFP, DKM, M-LF, DTA, and DWS were all paid employees of NovaBiotics Ltd at the time of this study. DAO is CEO, CSO and a shareholder at NovaBiotics Ltd. DFP and DAO are named on patents related to cysteamine. This does not alter our adherence to Elsevier and journal policies., (Copyright © 2023 NovaBiotics Ltd. Published by Elsevier Inc. All rights reserved.)

In this age of antimicrobial resistance (AMR) there is an urgent need for novel antimicrobials. One area of recent interest is in developing antimicrobial effector molecules, and even cell-based therapies, based on those of the immune system. In this review, some of the more interesting approaches will be discussed, including immune checkpoint inhibitors, Interferons (IFNs), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), Chimeric Antigen Receptor (CAR) T cells, Antibodies, Vaccines and the potential role of trained immunity in protection from and/or treatment of infection., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology.)

Pseudomonas aeruginosa is a major opportunistic human pathogen which employs a myriad of virulence factors. In people with cystic fibrosis (CF) P. aeruginosa frequently colonises the lungs and becomes a chronic infection that evolves to become less virulent over time, but often adapts to favour persistence in the host with alginate-producing mucoid, slow-growing, and antibiotic resistant phenotypes emerging. Cysteamine is an endogenous aminothiol which has been shown to prevent biofilm formation, reduce phenazine production, and potentiate antibiotic activity against P. aeruginosa , and has been investigated in clinical trials as an adjunct therapy for pulmonary exacerbations of CF. Here we demonstrate (for the first time in a prokaryote) that cysteamine prevents glycine utilisation by P. aeruginosa in common with previously reported activity blocking the glycine cleavage system in human cells. Despite the clear inhibition of glycine metabolism, cysteamine also inhibits hydrogen cyanide (HCN) production by P. aeruginosa , suggesting a direct interference in the regulation of virulence factor synthesis. Cysteamine impaired chemotaxis, lowered pyocyanin, pyoverdine and exopolysaccharide production, and reduced the toxicity of P. aeruginosa secreted factors in a Galleria mellonella infection model. Thus, cysteamine has additional potent anti-virulence properties targeting P. aeruginosa , further supporting its therapeutic potential in CF and other infections., Competing Interests: DF-P, DT-A, JH, DS, and DM are all employees of NovaBiotics Ltd. DAO is the CEO, CSO and a shareholder of NovaBiotics Ltd. DF-P and DO are authors of patent; WO2016198842A1, IL256162D0 - An amino thiol for use in the treatment of an infection caused by the bacterium mycobacterium spp. DO is also author of the following patents; US9364491B2 (and other territories) - Antimicrobial compositions with cysteamine/A composition comprising an antibiotic and a dispersant; WO2016046524A1 - Use of cysteamine in treating infections caused by yeasts/moulds; US9782423B2 (and other territories) - Antibiotic compositions comprising an antibiotic agent and cysteamine; US9339525B2 (and other territories) - Inhibition of biofilm organisms; and US20190175501A1 pending (and other territories) - Microparticles comprising a sulphur-containing compound. This does not alter our adherence to Frontiers policies on sharing data and materials. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fraser-Pitt, Dolan, Toledo-Aparicio, Hunt, Smith, Lacy-Roberts, Nupe Hewage, Stoyanova, Manson, McClean, Inglis, Mercer and O’Neil.)

The need for optimized as well as standardized test systems of novel antimicrobial peptides (AMPs) was discussed by experts in the field at the International Meeting on Antimicrobial Peptides (IMAP) 2017 and the 2019 Gordon Research Conference (GRC) on Antimicrobial Peptides, and a survey related to this topic was circulated to participants to collate opinions. The survey included questions ranging from the relevance of susceptibility testing for understanding the mode of action of AMPs, to the importance of optimization and a degree of standardization of test methods and their clinical relevance. Based on the survey results, suggestions for future improvements in the research field are made.

Se is a micronutrient essential for human health. Sub-optimal Se status is common, occurring in a significant proportion of the population across the world including parts of Europe and China. Human and animal studies have shown that Se status is a key determinant of the host response to viral infections. In this review, we address the question whether Se intake is a factor in determining the severity of response to coronavirus disease 2019 (COVID-19). Emphasis is placed on epidemiological and animal studies which suggest that Se affects host response to RNA viruses and on the molecular mechanisms by which Se and selenoproteins modulate the inter-linked redox homeostasis, stress response and inflammatory response. Together these studies indicate that Se status is an important factor in determining the host response to viral infections. Therefore, we conclude that Se status is likely to influence human response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and that Se status is one (of several) risk factors which may impact on the outcome of SARS-CoV-2 infection, particularly in populations where Se intake is sub-optimal or low. We suggest the use of appropriate markers to assess the Se status of COVID-19 patients and possible supplementation may be beneficial in limiting the severity of symptoms, especially in countries where Se status is regarded as sub-optimal.

NP213 (Novexatin®) is a novel antifungal peptide specifically designed for the topical treatment of onychomycosis. NP213 was designed using host defense peptides (HDP), essential components of the innate immune response to infection, as a template. NP213 is a water-soluble cyclic fungicidal peptide that effectively penetrates human nail. NP213 demonstrated a promising preclinical and clinical safety profile, with no evidence of systemic exposure following topical application to the skin and nails. NP213 was efficacious in two phase IIa human trials with 43.3% of patients having no fungi detectable by culture of fragments from NP213-treated nails after 180 days in the first study and likewise 56.5% of patients were culture negative for dermatophytes after 360 days in the second phase IIa study. In both trials, NP213 was applied daily for only 28 days in marked contrast to other topical onychomycosis treatments that require application for up to 52 weeks. Patient reported outcomes from the phase IIa studies were positive with participants recording an improved appearance of their nails after only 14 days of application. All fungi identified in these studies were Trichophyton spp. NP213 (Novexatin®) is a promising, highly differentiated peptide-based candidate for the topical treatment of onychomycosis, addressing the infectious cause and cosmetic issues of this very common condition., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

The purpose of this review is to describe antifungal therapeutic candidates in preclinical and clinical development derived from, or directly influenced by, the immune system, with a specific focus on antimicrobial peptides (AMP). Although the focus of this review is AMP with direct antimicrobial effects on fungi, we will also discuss compounds with direct antifungal activity, including monoclonal antibodies (mAb), as well as immunomodulatory molecules that can enhance the immune response to fungal infection, including immunomodulatory AMP, vaccines, checkpoint inhibitors, interferon and colony stimulating factors as well as immune cell therapies. The focus of this manuscript will be a non-exhaustive review of antifungal compounds in preclinical and clinical development that are based on the principles of immunology and the authors acknowledge the incredible amount of in vitro and in vivo work that has been conducted to develop such therapeutic candidates., (Copyright © 2020 Mercer and O'Neil.)

During the development of antimicrobial peptides (AMP) as potential therapeutics, antimicrobial susceptibility testing (AST) stands as an essential part of the process in identification and optimisation of candidate AMP. Standard methods for AST, developed almost 60 years ago for testing conventional antibiotics, are not necessarily fit for purpose when it comes to determining the susceptibility of microorganisms to AMP. Without careful consideration of the parameters comprising AST there is a risk of failing to identify novel antimicrobials at a time when antimicrobial resistance (AMR) is leading the planet toward a post-antibiotic era. More physiologically/clinically relevant AST will allow better determination of the preclinical activity of drug candidates and allow the identification of lead compounds. An important consideration is the efficacy of AMP in biological matrices replicating sites of infection, e.g., blood/plasma/serum, lung bronchiolar lavage fluid/sputum, urine, biofilms, etc., as this will likely be more predictive of clinical efficacy. Additionally, specific AST for different target microorganisms may help to better predict efficacy of AMP in specific infections. In this manuscript, we describe what we believe are the key considerations for AST of AMP and hope that this information can better guide the preclinical development of AMP toward becoming a new generation of urgently needed antimicrobials., (Copyright © 2020 Mercer, Torres, Duay, Lovie, Simpson, von Köckritz-Blickwede, de la Fuente-Nunez, O'Neil and Angeles-Boza.)

Onychomycosis is a common, difficult-to-treat nail infection that is mainly caused by dermatophytes. Current therapies are not wholly effective and are associated with manifold side effects. The development of treatments for onychomycosis is challenging because standard in vitro tests are not predictive of antifungal efficacy within the nail. We have developed a new antifungal agent, NP213, for the treatment of onychomycosis. NP213 is based on endogenous host defense peptides produced within the nail. We compared the in vitro activity of NP213 and existing antifungal agents using conventional antimicrobial susceptibility test (AST) systems and more physiologically relevant models based on the human nail. We observed that the standard in vitro AST methodologies failed to predict the efficacy of antifungal agents within the nail. To address that, we present a more physiologically relevant modified AST method. This method, alongside other standard in vitro assessments of activity (including mechanism-of-action and time-of-kill studies), better reflected the activity of NP213 and other antifungal agents within the nail than standard in vitro AST methods. NP213 is a rapidly acting, fungicidal peptide that is superior to existing antifungal agents in vitro It penetrated the nail more effectively than other antifungals, as confirmed by using an optimized in vitro nail infection model. The data presented here support the current clinical development status of NP213 as a novel agent for treating onychomycosis. We propose that the modified tests developed and applied for NP213 characterization are the most relevant to use for screening any potential therapeutic candidates for onychomycosis., (Copyright © 2019 Mercer et al.)

Dermatophytes are the most common cause of superficial fungal infections (tinea infections) and are a specialized group of filamentous fungi capable of infecting and degrading keratinised tissues, including skin, hair, and nail. Essential to their pathogenicity and virulence is the production of a broad spectrum of proteolytic enzymes and other key proteins involved in keratin biodegradation and utilization of its breakdown products. The initial stage of biodegradation of native keratin is considered to be sulfitolysis, in which the extensive disulfide bridges present in keratin are hydrolyzed, although some secreted subtilisins can degrade dye-impregnated keratin azure without prior reduction (Sub3 and Sub4). Sulfitolysis facilitates the extracellular biodegradation of keratin by the dermatophytes' extensive array of endo- and exoproteases. The importance of dermatophyte proteases in infection is widely recognized, and these enzymes have also been identified as important virulence determinants and allergens. Finally, the short peptide and amino acid breakdown products are taken up by the dermatophytes, using as yet poorly characterised transporters, and utilized for metabolism. In this review, we describe the process of keratin biodegradation by dermatophytes, with an especial focus on recent developments in cutting edge molecular biology and '-omic' studies that are helping to dissect the complex process of keratin breakdown and utilization.

Cysteamine is an endogenous aminothiol produced in mammalian cells as a consequence of coenzyme A metabolism through the activity of the vanin family of pantetheinase ectoenzymes. It is known to have a biological role in oxidative stress, inflammation, and cell migration. There have been several reports demonstrating anti-infective properties targeting viruses, bacteria, and even the malarial parasite. We and others have previously described broad-spectrum antimicrobial and antibiofilm activities of cysteamine. Here, we go further to demonstrate redox-dependent mechanisms of action for the compound and how its antimicrobial effects are, at least in part, due to undermining bacterial defenses against oxidative and nitrosative challenges. We demonstrate the therapeutic potentiation of antibiotic therapy against Pseudomonas aeruginosa in mouse models of infection. We also demonstrate potentiation of many different classes of antibiotics against a selection of priority antibiotic-resistant pathogens, including colistin (often considered an antibiotic of last resort), and we discuss how this endogenous antimicrobial component of innate immunity has a role in infectious disease that is beginning to be explored and is not yet fully understood., (Copyright © 2018 Fraser-Pitt et al.)

Staphylococcus aureus is a clinically significant human pathogen that causes infectious diseases ranging from skin and soft tissue infections (SSTI) and health care-associated infections (HAI) to potentially fatal bacteremia and endocarditis. Nasal carriage of S. aureus , especially for persistent carriage, is associated with an increased risk of subsequent infection, particularly nosocomial and surgical site infections (SSI), usually via autoinfection. NP108 is a cationic antimicrobial polymer composed of generally recognized as safe (GRAS) amino acid building blocks. NP108 is broad spectrum and rapidly bactericidal (3-log kill in ≤3 h), killing bacteria by membrane disruption and cell lysis. NP108, contrary to many antibiotics, shows equally effective antimicrobial activity against a variety of S. aureus (MIC 100 = 8 to 500 mg/liter) and S. epidermidis (MIC 100 = 4 to 8 mg/liter) isolates, whether exponentially growing or in stationary phase. NP108 is antimicrobially active under nutrient-limiting conditions similar to those found in the anterior nares (MIC 100 = 8 mg/liter) and kills antibiotic-resilient small colony variants (MIC 100 = 32 mg/liter) and S. aureus biofilms (prevention, MIC 100 = 1 to 4 mg/liter; eradication, MIC 100 ≥ 31.25 mg/liter). NP108 is active against isolates of S. aureus resistant to the current standard-of-care decolonization agent, mupirocin, with no significant increase in the MIC 100 NP108 is water soluble and has been formulated into compatible aqueous gel vehicles for human use in which antimicrobial efficacy is retained (2.0% [wt/vol]). NP108 is a potential nonantibiotic antimicrobial alternative to antibiotics for the nasal decolonization of S. aureus , with clear advantages in its mechanism of action over the existing gold standard, mupirocin., (Copyright © 2017 Mercer et al.)

Superficial mycoses are fungal infections of the outer layers of the skin, hair and nails that affect 20-25% of the world's population, with increasing incidence. Treatment of superficial mycoses, predominantly caused by dermatophytes, is by topical and/or oral regimens. New therapeutic options with improved efficacy and/or safety profiles are desirable. There is renewed interest in natural product-based antimicrobials as alternatives to conventional treatments, including the treatment of superficial mycoses. We investigated the potential of coumarins as dermatophyte-specific antifungal agents and describe for the first time their potential utility as topical antifungals for superficial mycoses using a prodrug approach. Here we demonstrate that an inactive coumarin glycone, esculin, is hydrolysed to the antifungal coumarin aglycone, esculetin by dermatophytes. Esculin is hydrolysed to esculetin β-glucosidases. We demonstrate that β-glucosidases are produced by dermatophytes as well as members of the dermal microbiota, and that this activity is sufficient to hydrolyse esculin to esculetin with concomitant antifungal activity. A β-glucosidase inhibitor (conduritol B epoxide), inhibited antifungal activity by preventing esculin hydrolysis. Esculin demonstrates good aqueous solubility (<6 g/l) and could be readily formulated and delivered topically as an inactive prodrug in a water-based gel or cream. This work demonstrates proof-of-principle for a therapeutic application of glycosylated coumarins as inactive prodrugs that could be converted to an active antifungal in situ. It is anticipated that this approach will be applicable to other coumarin glycones.

A targeted approach for direct topical antimicrobial delivery involving the formulation of impregnated freeze-dried wafers prepared from a natural polymer has been assessed to consider potential for treatment of wounded skin. The synthetic cationic antimicrobial peptides (CAPs) NP101 and NP108 were found to have modest in vitro activity against bacterial species commonly associated with wound infections. Minimum inhibitory concentration/minimum bactericidal concentrations against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were found to be 0.31 mg/ml for NP101 and 0.25-0.5 mg/ml for NP108. Rapid, substantial cytoplasmic potassium loss was induced by NP108 in E. coli, but not the other species. Through scanning electron microscopy, both CAPs were observed to alter cell morphology, prevent normal septation, promote cell aggregation and trigger release or formation of extracellular filaments. Wafers harbouring these agents displayed substantial antibacterial activity when assessed by standard diffusion assay. These data confirm that topical delivery of CAPs, through their incorporation within freeze-dried wafer formulations prepared from natural polymers, represents a potential viable approach for treating skin infection.

Synthesis and large-scale manufacturing technologies are now available for the commercial production of even the most complex peptide anti-infectives. Married with the potential of this class of molecule as the next generation of effective, resistance-free and safe antimicrobials, and a much better understanding of their biology, pharmacology and pharmacodynamics, the first regulatory approvals and introduction into clinical practice of these promising drug candidates will likely be soon. This is a key juncture in the history/life cycle of peptide anti-infectives and, perhaps, their commercial and therapeutic potential is about to be realized. This review highlights the promise of these agents as the next generation of therapeutics and summarizes the challenges faced in, and lessons learned from, the past.

While there is an appreciable understanding of the importance of collagen breakdown in contributing to atherosclerotic plaque vulnerability and rupture, little is known about changes in collagen maturation in the atherosclerotic plaque. This is achieved through the formation of the covalent intermolecular cross-links pyridinoline (Pyd) and deoxypyridinoline (Dpd). In this study we collected carotid endarterectomy specimens from patients and undertook (i) histological assessment of collagen and inflammatory cell distribution and (ii) biochemical analysis of total collagen and cross-link content. Greater collagen deposition, increased presence of CD68 positive cells and an increased Pyd:Dpd ratio (an indicator of lysyl hydroxylase (LH-1) activity) were found in plaque versus normal vascular tissue. These findings are the first measurements of Pyd and Dpd cross-links in normal and atherosclerotic vascular tissue. The observed differences in cross-links in the plaque may adversely affect tensile strength and may have relevance to the mechanisms underlying rupture of vulnerable plaques., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme possessing lysyl hydroxylase (LH), hydroxylysyl galactosyltransferase (GT) and galactosylhydroxylysyl glucosyltransferase (GGT) activities in vitro. To investigate the in vivo importance of LH3-catalyzed lysine hydroxylation and hydroxylysine-linked glycosylations, three different LH3-manipulated mouse lines were generated. Mice with a mutation that blocked only the LH activity of LH3 developed normally, but showed defects in the structure of the basement membrane and in collagen fibril organization in newborn skin and lung. Analysis of a hypomorphic LH3 mouse line with the same mutation, however, demonstrated that the reduction of the GGT activity of LH3 disrupts the localization of type IV collagen, and thus the formation of basement membranes during mouse embryogenesis leading to lethality at embryonic day (E) 9.5-14.5. Strikingly, survival of hypomorphic embryos and the formation of the basement membrane were directly correlated with the level of GGT activity. In addition, an LH3-knockout mouse lacked GGT activity leading to lethality at E9.5. The results confirm that LH3 has LH and GGT activities in vivo, LH3 is the main molecule responsible for GGT activity and that the GGT activity, not the LH activity of LH3, is essential for the formation of the basement membrane. Together our results demonstrate for the first time the importance of hydroxylysine-linked glycosylation for collagens.

Inflammatory disorders of the bowel and colon cancer are associated with elevated indices of oxidative stress. Analogous elevations in markers of oxidative stress and loss of cell-membrane integrity are also observed in the colons of rats deficient in vitamin E (D-alpha-tocopherol), the major lipid-soluble antioxidant in biological systems. The causal relationship between colon pathologies associated with oxidative stress and dietary deficiency in antioxidant vitamins such as vitamin E is still uncertain. Investigation of potential mechanisms by which lack of dietary vitamin E may lead to clinically relevant pathological changes in colon tissue was conducted using gene expression profiling strategies on vitamin E-sufficient and -deficient rats. Morphological changes and increased indices of lipid peroxidation were linked to vitamin E deficiency. These changes in colon tissue are potentially important in disease pathogenesis of the colon linked with oxidative stress or other direct consequences of inadequate levels of vitamin E.

Lysyl hydroxylases (LH) (procollagen-lysine 2-oxoglutarate 5-dioxygenase; PLOD) catalyse the hydroxylation of lysine residues during the post-translational modification of collagenous proteins. In this paper, we describe the first identification and cloning of LH isoforms 2 and 3 from the rat, including both LH2 splice variants (LH2a and LH2b). The rat LHs are expressed in almost all tissue and cell types examined, indicating a probable lack of tissue specificity for LH function. All LH isoforms were stably transfected into CHO-K1 cells and this represents the first example of recombinant LH production in a eukaryotic cell line. Expression and production of all LH isoforms led to an increase in total collagen synthesis. LH1 and LH2a expression and production led to an increase in total pyridinium cross-link production. Evidence that LH2a possesses telopeptide lysyl hydroxylase activity, previously thought to be a novel enzyme, is presented.

The xylanase gene cluster from the rumen anaerobe Prevotella bryantii B(1)4 was found to include a gene (xynR) that encodes a multidomain regulatory protein and is downstream from the xylanase and beta-xylosidase genes xynA and xynB. Additional genes identified upstream of xynA and xynB include xynD, which encodes an integral membrane protein that has homology with Na:solute symporters; xynE, which is related to the genes encoding acylhydrolases and arylesterases; and xynF, which has homology with the genes encoding alpha-glucuronidases. XynR includes, in a single 833-amino-acid polypeptide, a putative input domain unrelated to other database sequences, a likely transmembrane domain, histidine kinase motifs, response regulator sequences, and a C-terminal AraC-type helix-turn-helix DNA binding domain. Two transcripts (3.7 and 5.8 kb) were detected with a xynA probe, and the start site of the 3.7-kb transcript encoding xynABD was mapped to a position upstream of xynD. The DNA binding domain of XynR was purified after amplification and overexpression in Escherichia coli and was found to bind to a 141-bp DNA fragment from the region immediately upstream of xynD. In vitro transcription assays demonstrated that XynR stimulates transcription of the 3.7-kb transcript. We concluded that XynR acts as a positive regulator that activates expression of xynABD in P. bryantii B(1)4. This is the first regulatory protein that demonstrates significant homology with the two-component regulatory protein superfamily and has been shown to be involved in the regulation of polysaccharidase gene expression.

A novel tetracycline resistance gene, designated tet(32), which confers a high level of tetracycline resistance, was identified in the Clostridium-related human colonic anaerobe K10, which also carries tet(W). tet(32) was transmissible in vitro to the rumen anaerobe Butyrivibrio fibrisolvens 2221(R). The predicted gene product of tet(32) has 76% amino acid identity with Tet(O). PCR amplification indicated that tet(32) is widely distributed in the ovine rumen and in porcine feces.

Transformation of Streptococcus gordonii DL1 by free DNA was studied in human saliva. Competent S. gordonii could be transformed in vitro with plasmid DNA that had been taken into the human mouth. Transformation also occurred with a plasmid that cannot replicate in S. gordonii, but that has a region of chromosomal homology, by integration into the bacterial chromosome, although linearised plasmid DNA gave no transformants. Linear chromosomal DNA fragments did however transform S. gordonii/Tn916 efficiently in saliva when regions of homology with the recipient chromosome flanked the marker gene. These findings are discussed in relation to the potential for acquisition of DNA sequences, including genetically modified DNA, by gut and oral bacteria.

pRRI2 is a small cryptic plasmid from the rumen bacterium Prevotella ruminicola 223/M2/7 which has been used for the construction of shuttle vectors (pRH3 and pRRI207) that replicate in many Bacteroides/Prevotella strains as well as in Escherichia coli. Sequence analysis of pRRI2 reveals that it is a 3240-bp plasmid carrying two clear open reading frames. Rep, encoded by ORF1, shows 48 and 47% amino acid sequence identity with RepA proteins from Bacteroides vulgatus and Bacteroides fragilis, respectively. ORF2, named Pre, shares 34% amino acid sequence identity with a putative plasmid recombination protein from the Flavobacterium spp. plasmid pFL1 and 30% amino acid sequence identity with BmpH from B. fragilis Tn5520. Disruption of ORF1 with HindIII prevents replication and maintenance in Bacteroides spp. hosts, but shuttle vectors carrying pRRI2 interrupted within ORF2, by EcoRI*, are able to replicate. pRRI2 shows no significant similarity with the only other P. ruminicola plasmid to have been studied previously, pRAM4., (Copyright 2001 Academic Press.)

An integration vector was constructed to allow introduction of the gfp gene into the chromosomes of Gram-positive bacteria. Integration depends on homologous recombination between a short 458-nt sequence of the tet(M) gene in the vector and a copy of Tn916 in the host chromosome. Strains of Lactococcus lactis IL1403, Enterococcus faecalis JH2-SS, and Streptococcus gordonii DL1 stably marked with single chromosomal copies of the gfp were readily visualised by epifluorescence microscopy. The marked L. lactis strain survived poorly in a continuous culture system inoculated with human faecal flora, while the laboratory E. faecalis strain was lost at approximately the dilution rate of the fermenter.

Natural transformation of Streptococcus bovis JB1 was demonstrated after development of competence in normal culture medium. Transformation efficiencies were not significantly increased when heat-inactivated horse serum was added to the medium before growth. This is the first time that a resident rumen bacterial species has been shown to be naturally transformable. Transformation allowed the acquisition of plasmids or integration of sequences into the chromosome. No transformation was observed in the presence of undiluted autoclaved or filter-sterilised ovine rumen fluid or filter-sterilised ovine saliva, suggesting that transformation in the ruminant digestive tract is a rare event, although transformation was observed in the presence of 1% and 0.5% filter-sterilised rumen fluid. The use of natural transformation of S. bovis should facilitate further molecular biological studies on this species.

Competitive PCR was used to monitor the survival of a 520-bp DNA target sequence from a recombinant plasmid, pVACMC1, after admixture of the plasmid with freshly sampled human saliva. The fraction of the target remaining amplifiable ranged from 40 to 65% after 10 min of exposure to saliva samples from five subjects and from 6 to 25% after 60 min of exposure. pVACMC1 plasmid DNA that had been exposed to degradation by fresh saliva was capable of transforming naturally competent Streptococcus gordonii DL1 to erythromycin resistance, although transforming activity decreased rapidly, with a half-life of approximately 50 s. S. gordonii DL1 transformants were obtained in the presence of filter-sterilized saliva and a 1-microg/ml final concentration of pVACMC1 DNA. Addition of filter-sterilized saliva instead of heat-inactivated horse serum to S. gordonii DL1 cells induced competence, although with slightly lower efficiency. These findings indicate that DNA released from bacteria or food sources within the mouth has the potential to transform naturally competent oral bacteria. However, further investigations are needed to establish whether transformation of oral bacteria can occur at significant frequencies in vivo.

A diverse collection of actinomycete strains were screened for production of extracellular peroxidase activity by adapting a chemiluminescence analysis system developed for horseradish peroxidase-based enzyme-linked immunosorbent assay. Extracellular peroxidase activity was found to be common but quantitatively variable, and this rapid and sensitive screening system permitted identification of a small group of high-producing strains. A range of spectrophotometric assays were compared for the measurement of peroxidase activity in concentrated culture supernatants of two selected thermophilic streptomycetes. Of these, the peroxide-dependent oxidation of 2,4-dichlorophenol was identified as the most robust and reproducible assay for quantitative studies.

Actinomycetes form an enormous reservoir of secondary metabolites and enzymes. The potential for exploiting rare actinomycetes is highlighted by the discovery of novel compounds from strains of Spirillospora and Nocardioides. Novel compounds of well known classes of antibiotics, such as polyenes, continue to be discovered. For compounds containing a chromophore, the analysis by high-performance liquid chromatography coupled with a diode-array detector enables the elimination of producers of known compounds and facilitates the discovery of novel compounds or derivatives. The complexity of the regulatory mechanisms is illustrated by glutamine synthetase. The characterization of thermostable amylolytic, lignolytic, peroxidase and neuramidase activities, and the isolation of novel cellulolytic actinomycetes clearly demonstrate the potential of Actinomycetes as producers of enzymes.