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R Paul Ross

Publications and source records attributed to R Paul Ross.

At least 19 recordsLinked to original sources
PubMed2007

Identification of a novel two-peptide lantibiotic, haloduracin, produced by the alkaliphile Bacillus halodurans C-125.

Complete genome sequencing of the alkaliphilic bacterium Bacillus halodurans C-125 revealed the presence of several genes homologous to those involved in the production of lantibiotic peptides. Additional bioinformatic analysis identified a total of eleven genes, spanning a 15 kbp region, potentially involved in the production, modification, immunity and transport of a two-peptide lantibiotic. Having established that strain C-125 exhibited antimicrobial activity against a wide range of Gram-positive bacteria, it was demonstrated through peptide purification, MS and site-directed mutagenesis that this activity was indeed attributable to the production of a lantibiotic encoded by these genes. This antimicrobial has been designated haloduracin and represents the first occasion wherein production of two-peptide lantibiotic has been associated with a Bacillus sp. It is also the first example of a lantibiotic of any kind to be produced by an alkaliphilic species.

Amino Acid Sequence↗
PubMed2006

Complete alanine scanning of the two-component lantibiotic lacticin 3147: generating a blueprint for rational drug design.

Lantibiotics are post-translationally modified antimicrobial peptides which are active at nanomolar concentrations. Some lantibiotics have been shown to function by targeting lipid II, the essential precursor of cell wall biosynthesis. Given that lantibiotics are ribosomally synthesized and amenable to site-directed mutagenesis, they have the potential to serve as biological templates for the production of novel peptides with improved functionalities. However, if a rational approach to novel lantibiotic design is to be adopted, an appreciation of the roles of each individual amino acid (and each domain) is required. To date no lantibiotic has been subjected to such rigorous analysis. To address this issue we have carried out complete scanning mutagenesis of each of the 59 amino acids in lacticin 3147, a two-component lantibiotic which acts through the synergistic activity of the peptides LtnA1 (30 amino acids) and LtnA2 (29 amino acids). All mutations were performed in situ in the native 60 kb plasmid, pMRC01. A number of mutations resulted in the elimination of detectable bioactivity and seem to represent an invariable core within these and related peptides. Significantly however, of the 59 amino acids, at least 36 can be changed without resulting in a complete loss of activity. Many of these are clustered to form variable domains within the peptides. The information generated in this study represents a blue-print that will be critical for the rational design of lantibiotic-based antimicrobial compounds.

Alanine↗
PubMed2006

Spontaneous resistance in Lactococcus lactis IL1403 to the lantibiotic lacticin 3147.

The ability and frequency at which target organisms can develop resistance to bacteriocins is a crucial consideration in designing and implementing bacteriocin-based biocontrol strategies. Lactococcus lactis ssp. lactis IL1403 was used as a target strain in an attempt to determine the frequency at which spontaneously resistant mutants are likely to emerge to the lantibiotic lacticin 3147. Following a single exposure to lacticin 3147, resistant mutants only emerged at a low frequency (10(-8)-10(-9)) and were only able to withstand low levels of the bacteriocin (100 AU mL(-1)). However, exposure to increasing concentrations, in a stepwise manner, resulted in the isolation of eight mutants that were resistant to moderately higher levels of lacticin 3147 (up to 600 AU mL(-1)). Interestingly, in a number of cases cross-resistance to other lantibiotics such as nisin and lacticin 481 was observed, as was cross-resistance to environmental stresses such as salt. Finally, reduced adsorption of the bacteriocin in to the cell was documented for all resistant mutants.

Adsorption↗
PubMed2006

Plasmids of lactococci - genetic accessories or genetic necessities?

Lactococci are one of the most exploited microorganisms used in the manufacture of food. These intensively used cultures are generally characterized by having a rich plasmid complement. It could be argued that it is the plasmid complement of commercially utilized cultures that gives them their technical superiority and individuality. Consequently, it is timely to reflect on the desirable characteristics encoded on lactococcal plasmids. It is argued that plasmids play a key role in the evolution of modern starter strains and are a lot more than just selfish replicosomes but more essential necessities of intensively used commercial starters. Moreover, the study of plasmid biology provides a genetic blueprint that has proved essential for the generation of molecular tools for the genetic improvement of Lactococcus lactis.

Genetic Markers↗
PubMed2006

Contribution of penicillin-binding protein homologs to antibiotic resistance, cell morphology, and virulence of Listeria monocytogenes EGDe.

Seven open reading frames, annotated as potential penicillin-binding-protein-encoding genes (lmo0441, lmo0540, lmo1438, lmo1892, lmo2039, lmo2229, and lmo2754), were targeted for insertional mutagenesis in Listeria monocytogenes EGDe. These genes were found to contribute in various degrees to beta-lactam resistance, cell morphology, or the virulence potential of this organism.

Amino Acid Sequence↗
PubMed2006

Overproduction of wild-type and bioengineered derivatives of the lantibiotic lacticin 3147.

Lacticin 3147 is a broad-spectrum two-peptide lantibiotic whose genetic determinants are located on two divergent operons on the lactococcal plasmid pMRC01. Here we introduce each of 14 subclones, containing different combinations of lacticin 3147 genes, into MG1363 (pMRC01) and determine that a number of them can facilitate overproduction of the lantibiotic. Based on these studies it is apparent that while the provision of additional copies of genes encoding the biosynthetic/production machinery and the regulator LtnR is a requirement for high-level overproduction, the presence of additional copies of the structural genes (i.e., ltnA1A2) is not.

Bacterial Proteins↗
PubMed2006

Intestinal bifidobacteria that produce trans-9, trans-11 conjugated linoleic acid: a fatty acid with antiproliferative activity against human colon SW480 and HT-29 cancer cells.

Bifidobacterium breve species of human intestinal origin have the ability to synthesize cis-9, trans-11 (c9, t11) conjugated linoleic acid (CLA) from free linoleic acid. In this study, the ability of Bifidobacterium species to isomerize C(18) polyunsaturated fatty acids was investigated, and the antiproliferative activities of the two main microbially produced CLA isomers were assessed. Linoleic acid was converted principally to c9, t11 CLA and lesser amounts of t9, t11 CLA, whereas c9, t11 CLA was converted mainly to t9, t11 CLA. Likewise, t10, c12 CLA was converted principally to t9, t11 CLA, which was incorporated into the bacterial cell membranes. To examine the antiproliferative effect of the two main CLA isomers formed, SW480 and HT-29 human colon cancer cells were cultured in the presence of c9, t11 CLA and t9, t11 CLA. The t9, t11 CLA had a more potent antiproliferative effect than c9, t11 CLA. It is tempting to suggest that the ability of Bifidobacterium to produce such bioactive metabolites may be associated with the beneficial effects of bifidobacteria present in the human gastrointestinal tract.

Antineoplastic Agents↗
PubMed2005

Posttranslational conversion of L-serines to D-alanines is vital for optimal production and activity of the lantibiotic lacticin 3147.

As a general rule, ribosomally synthesized polypeptides contain amino acids only in the L-isoform in an order dictated by the coding DNA/RNA. Two of a total of only four examples of L to D conversions in prokaryotic systems occur in posttranslationally modified antimicrobial peptides called lantibiotics. In both examples (lactocin S and lacticin 3147), ribosomally encoded L-serines are enzymatically converted to D-alanines, giving rise to an apparent mistranslation of serine codons to alanine residues. It has been suggested that this conversion results from a two-step reaction initiated by a lantibiotic synthetase converting the gene-encoded L-serine to dehydroalanine (dha). By using lacticin 3147 as a model system, we report the identification of an enzyme, LtnJ, that is responsible for the conversion of dha to D-alanine. Deletion of this enzyme results in the residues remaining as dha intermediates, leading to a dramatic reduction in the antimicrobial activity of the producing strain. The importance of the chirality of the three D-alanines present in lacticin 3147 was confirmed when these residues were systematically substituted by L-alanines. In addition, substitution with L-threonine (ultimately modified to dehydrobutyrine), glycine, or L-valine also resulted in diminished peptide production and/or relative activity, the extent of which depended on the chirality of the newly incorporated amino acid(s).

Alanine↗
PubMed2005

Genome analysis of the obligately lytic bacteriophage 4268 of Lactococcus lactis provides insight into its adaptable nature.

Analysis of the complete nucleotide sequence of the lactococcal phage 4268, which is lytic for the cheese starter Lactococcus lactis DPC4268, is presented. Phage 4268 has a linear genome of 36,596 bp, which is modularly organised and encompasses 49 open reading frames. Putative functions were assigned to approximately 45% of the predicted products of these open reading frames based on sequence similarity with known proteins, N-terminal sequence analysis and identification of conserved domains. Significantly, a segment of the genome has homology to the recently sequenced lysogenic module in lactococcal phage phi31 that contains a lytic switch but no phage integrase or attachment site. This suggests that it is derived from a prophage. A phage 4268-encoded and a host-encoded methylase were found to be highly similar, having only two nucleotide mismatches, suggesting that the phage acquired the methylase gene to protect it from a host endonuclease. Comparative genomic analysis revealed significant homology between phage 4268 and the lactococcal phage BK5-T. The comparative analysis also supported the classification of phage 4268 and other BK5-T-related phage as separate from the proposed P335 species of lactococcal phage.

Bacteriophages↗
PubMed2005

Isolation and characterization of the bovine Stearoyl-CoAdesaturase promoter and analysis of polymorphisms in the promoter region in dairy cows.

Conjugated linoleic acid (CLA) in milk arises through microbial biohydrogenation of dietary polyunsaturated fatty acids (PUFA) in the rumen, and by the action of mammary Stearoyl-CoA desaturase (Scd). A large variation (up to tenfold) in the concentration of this fatty acid in milk has been observed, even in cows receiving the same diet. The reasons for this variation are not well understood. In this study the bovine core promoter region was isolated by a genome walking strategy from genomic DNA Genome Walker libraries and then cloned and characterized. This core promoter sequence extended approximately 600 bp upstream of the translation start site. The presence of putative transcription factor binding sites conserved in bovine, human, and mouse promoters was observed. Evidence that this promoter fragment was functional in vivo was obtained from expression studies in a mammary cell line. The promoter sequence of the scd gene was compared between cows selected for the ability to produce high fatty acid methyl esters (FAME) (2.22-2.72) in their milk, with the same promoter region of low-FAME-producing cows (0.81-1.12). However, such comparisons of the sequences of the scd promoter region of cows producing high milk CLA compared with low CLA revealed no polymorphisms in this promoter segment. Furthermore, no sequence polymorphisms were observed among the scd promoter region of Holstein Friesian, Montbeliarde, Normande, Norwegian Red, Charlois, Limousin and Kerry breeds.

Animals↗
PubMed2005

Fermented functional foods based on probiotics and their biogenic metabolites.

The claimed health benefits of fermented functional foods are expressed either directly through the interaction of ingested live microorganisms, bacteria or yeast with the host (probiotic effect) or indirectly as a result of ingestion of microbial metabolites produced during the fermentation process (biogenic effect). Although still far from fully understood, several probiotic mechanisms of action have been proposed, including competitive exclusion, competition for nutrients and/or stimulation of an immune response. The biogenic properties of fermented functional foods result from the microbial production of bioactive metabolites such as certain vitamins, bioactive peptides, organic acids or fatty acids during fermentation.

Animals↗
PubMed2005

Bacteriocins: developing innate immunity for food.

Bacteriocins are bacterially produced antimicrobial peptides with narrow or broad host ranges. Many bacteriocins are produced by food-grade lactic acid bacteria, a phenomenon which offers food scientists the possibility of directing or preventing the development of specific bacterial species in food. This can be particularly useful in preservation or food safety applications, but also has implications for the development of desirable flora in fermented food. In this sense, bacteriocins can be used to confer a rudimentary form of innate immunity to foodstuffs, helping processors extend their control over the food flora long after manufacture.

Amino Acid Sequence↗
PubMed2005

Sequential actions of the two component peptides of the lantibiotic lacticin 3147 explain its antimicrobial activity at nanomolar concentrations.

Lacticin 3147 is a two-peptide (LtnA1 and LtnA2) lantibiotic produced by Lactococcus lactis subsp. lactis DPC3147 and has inhibitory activity against all gram-positive microorganisms tested. In this study the specific activities of the component peptides (alone or in combination) were determined by using L. lactis subsp. cremoris HP as the target strain. Lacticin 3147 exhibited an MIC50 of 7 nM for each component peptide (in combination), suggesting a peptide stoichiometry of 1:1. Interestingly, the LtnA1 peptide demonstrated independent inhibitory activity, with an MIC50 of 200 nM against L. lactis HP. In parallel studies, the single peptide bacteriocin nisin exhibited an MIC50 of 50 nM against the same target strain. Sequential peptide addition (with an intermediate washing step) demonstrated that LtnA1 must be added before LtnA2 rather than vice versa to observe inhibitory activity. The nanomolar activity of the lacticin peptides suggests the involvement of a docking molecule, speculated to be lipid II. Taken together with the recently determined structure of lacticin 3147 (N. I. Martin, T. Sprules, M. R. Carpenter, P. D. Cotter, C. Hill, R. P. Ross, and J. C. Vederas, Biochemistry, 43:3049-3056, 2004), these data support the hypothesis that the mode of action for lacticin 3147 involves a lipid II binding step (by the mersacidin-like LtnA1 peptide, which would explain its independent inhibitory activity), followed by insertion of the more linear LtnA2 peptide into the target membrane, resulting in pore formation and ultimate cell death.

Anti-Bacterial Agents↗
PubMed2005

Insertional inactivation of determinants for Mg2+ and Co2+ transport as a tool for screening recombinant Lactococcus species clones.

Insertional inactivation of the plasmid-encoded determinants for Mg(2+) and Co(2+) transport, orf18/corA, provides a tool for screening recombinant clones in Lactococcus, based on the observation that overexpression of orf18/corA results in cell growth inhibition on certain concentrations of CoCl(2). The lacticin 3147 immunity gene, ltnI, was used to insertionally inactivate orf18/corA. The resulting clones were capable of growth on concentrations of CoCl(2) that were inhibitory to the parent strain. Since only 3 of 17 lactococcal starters naturally harbor corA, the system has potential as a screen for selecting recombinant lactococcal clones.

Bacterial Proteins↗
PubMed2005

Bacterial lantibiotics: strategies to improve therapeutic potential.

Lantibiotics are ribosomally-synthesised antimicrobial peptides produced by Gram-positive bacteria that are characterised by the presence of lanthionine and/or methyllanthionine residues. Other unusual post-translationally modified amino acids, most frequently dehydroalanine and dehydrobutyrine, can also be present. While it has been frequently suggested that these peptides have the potential to be utilised in a wide range of medical applications, to date no actual therapeutic applications have been convincingly described. More recently, however, they have been the focus of much attention as a consequence of improved biotechnological capabilities, an improved understanding of lantibiotic biosynthesis and mode of action, and their high specific activity against multi-drug resistant bacteria. This review concerns the fundamental analyses that have revealed the importance of individual amino acids in these peptides and has permitted the implementation of rational mutagenesis strategies ('intelligenetics') to alter individual residues with a view to ultimately widening the active pH range, improve stability, and enhance binding to cell wall targets with the ultimate aim of optimising their antimicrobial activity. It is hoped that as a consequence of this improved knowledge the most suitable application of individual lantibiotics will become apparent. It should also prove possible, in the near future, to generate tailor-made lantibiotics and utilise biosynthetic enzymes to incorporate modified amino acids into non-lantibiotic peptides. In the shorter term, the extensive characterisation of lantibiotics will be instrumental in reassuring drug industry regulators of their safety and facilitate the widespread application of these novel antimicrobial agents in medicine.

Alanine↗
PubMed2004

Structural characterization of lacticin 3147, a two-peptide lantibiotic with synergistic activity.

Lantibiotics are antibacterial peptides isolated from bacterial sources that exhibit activity toward Gram-positive organisms and are usually several orders of magnitude more potent than traditional antibiotics such as penicillin. They contain a number of unique structural features including dehydro amino acid and lanthionine (thioether) residues. Introduced following ribosomal translation of the parent peptide, these moieties render conventional methods of peptide analysis ineffective. We report herein a new method using nickel boride (Ni(2)B), in the presence of deuterium gas, to reduce dehydro side chains and reductively desulfurize lanthionine bridges found in lantibiotics. Using this approach, it is possible to identify and distinguish the original locations of dehydro side chains and lanthionine bridges by traditional peptide sequencing (Edman degradation) followed by mass spectrometry. The strategy was initially verified using nisin A, a structurally well characterized lantibiotic, and subsequently extended to the novel two-component lantibiotic, lacticin 3147, produced by Lactococcus lactis subspecies lactis DPC3147. The primary structures of both lacticin 3147 peptides were then fully assigned by use of multidimensional NMR spectroscopy, showing that lacticin 3147 A1 has a specific lanthionine bridging pattern which resembles the globular type-B lantibiotic mersacidin, whereas the A2 peptide is a member of the elongated type-A lantibiotic class. Also obtained by NMR were solution conformations of both lacticin 3147 peptides, indicating that A1 may adopt a conformation similar to that of mersacidin and that the A2 peptide adopts alpha-helical structure. These results are the first of their kind for a synergistic lantibiotic pair (only four such pairs have been reported to date).

Alanine↗