The predominant lactic acid bacteria and yeasts involved in the spontaneous fermentation of millet during the production of the traditional porridge Hausa koko in Ghana.

Spontaneous fermentation of cereals like millet involves a diverse population of microbes from various sources, including raw materials, processing equipment, fermenting receptacles, and the environment. Here, we present data on the predominant microbial species and their succession at each stage of the Hausa koko production process from five regions of Ghana. The isolates were enumerated using selective media, purified, and phenotypically characterised. The LAB isolates were further characterised by 16S rRNA Sanger sequencing, typed using (GTG)5 repetitive-PCR, and whole genome sequencing, while 28S rRNA Sanger sequencing was performed for yeast identification. The pH of the millet grains ranged from mean values of 6.02-6.53 to 3.51-3.99 in the final product, depending on the processors. The mean LAB and yeast counts increased during fermentation then fell to final counts of log 2.77-3.95 CFU/g for LAB and log 2.10-2.98 CFU/g for yeast in Hausa koko samples. At the various processing stages, the counts of LAB and yeast revealed significant variations (p < 0.0001). The species of LAB identified in this study were Limosilactobacillus pontis, Pediococcus acidilactici, Limosilactobacillus fermentum, Limosilactobacillus reuteri, Pediococcus pentosaceus, Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Schleiferilactobacillus harbinensis, and Weissella confusa. The yeasts were Saccharomyces cf. cerevisiae/paradoxus, Saccharomyces cerevisiae, Pichia kudriavzevii, Clavispora lusitaniae and Candida tropicalis. The identification and sequencing of these novel isolates and how they change during the fermentation process will pave the way for future controlled fermentation, safer starter cultures, and identifying optimal stages for starter culture addition or nutritional interventions. These LAB and yeast species are linked to many indigenous African fermented foods, potentially acting as probiotics in some cases. This result serves as the basis for further studies into the technological and probiotic potential of these Hausa koko microorganisms.

Beneficial effects of GABA-producing potential probiotic Limosilactobacillus fermentum L18 of human origin on intestinal permeability and human gut microbiota.

BACKGROUND: Gamma-aminobutyric acid (GABA) is a non-protein amino acid with neuroinhibitory, antidiabetic, and antihypertensive properties and is used as a drug for treating anxiety and depression. Some strains of lactobacilli are known to produce GABA and strengthen the gut barrier function which play an important role in ameliorating the effects caused by the pathogen on the gut barrier. The probiotic bacteria are also known to modulate the human fecal microbiota, however, the role of GABA-producing strains on the gut epithelium permeability and gut microbiota is not known. RESULTS: In this study, we report the production of high levels of GABA by potential probiotic bacterium Limosilactobacillus fermentum L18 for the first time. The kinetics of the production of GABA by L18 showed that the maximum production of GABA in the culture supernatant (CS) occurred at 24 h, whereas in fermented milk it took 48 h of fermentation. The effect of L18 on the restoration of lipopolysaccharide (LPS)-disrupted intestinal cell membrane permeability in Caco-2 monolayers showed that it significantly restored the transepithelial electrical resistance (TEER) values, by significantly increasing the levels of junction proteins, occludin and E-cadherin in L18 and LPS-treated Caco-2 cells as compared to only LPS-treated cells. The effect of GABA-secreting L18 on the metataxonome of human stool samples from healthy individuals was investigated by a batch fermentor that mimics the conditions of the human colon. Although, no differences were observed in the α and ß diversities of the L18-treated and untreated samples at 24 h, the relative abundances of bacterial families Lactobacillaceae and Bifidobacteriaceae increased in the L18-treated group, but both decreased in the untreated groups. On the other hand, the relative abundance of Enterobacteriaceae decreased in the L18 samples but it increased in the untreated samples. CONCLUSION: These results indicate that Li. fermentum L18 is a promising GABA-secreting strain that strengthens the gut epithelial barrier by increasing junction protein concentrations and positively modulating the gut microbiota. It has the potential to be used as a psychobiotic or for the production of functional foods for the management of anxiety-related illnesses.

Identification of Genes Required for Glucan Exopolysaccharide Production in Lactobacillus johnsonii Suggests a Novel Biosynthesis Mechanism.

Lactobacillus johnsonii FI9785 makes two capsular exopolysaccharides-a heteropolysaccharide (EPS2) encoded by the eps operon and a branched glucan homopolysaccharide (EPS1). The homopolysaccharide is synthesized in the absence of sucrose, and there are no typical glucansucrase genes in the genome. Quantitative proteomics was used to compare the wild type to a mutant where EPS production was reduced to attempt to identify proteins associated with EPS1 biosynthesis. A putative bactoprenol glycosyltransferase, FI9785_242 (242), was less abundant in the Δeps_cluster mutant strain than in the wild type. Nuclear magnetic resonance (NMR) analysis of isolated EPS showed that deletion of the FI9785_242 gene (242) prevented the accumulation of EPS1, without affecting EPS2 synthesis, while plasmid complementation restored EPS1 production. The deletion of 242 also produced a slow-growth phenotype, which could be rescued by complementation. 242 shows amino acid homology to bactoprenol glycosyltransferase GtrB, involved in O-antigen glycosylation, while in silico analysis of the neighboring gene 241 suggested that it encodes a putative flippase with homology to the GtrA superfamily. Deletion of 241 also prevented production of EPS1 and again caused a slow-growth phenotype, while plasmid complementation reinstated EPS1 synthesis. Both genes are highly conserved in L. johnsonii strains isolated from different environments. These results suggest that there may be a novel mechanism for homopolysaccharide synthesis in the Gram-positive L. johnsoniiIMPORTANCE Exopolysaccharides are key components of the surfaces of their bacterial producers, contributing to protection, microbial and host interactions, and even virulence. They also have significant applications in industry, and understanding their biosynthetic mechanisms may allow improved production of novel and valuable polymers. Four categories of bacterial exopolysaccharide biosynthesis have been described in detail, but novel enzymes and glycosylation mechanisms are still being described. Our findings that a putative bactoprenol glycosyltransferase and flippase are essential to homopolysaccharide biosynthesis in Lactobacillus johnsonii FI9785 indicate that there may be an alternative mechanism of glucan biosynthesis to the glucansucrase pathway. Disturbance of this synthesis leads to a slow-growth phenotype. Further elucidation of this biosynthesis may give insight into exopolysaccharide production and its impact on the bacterial cell.

Lactobacillus garii sp. nov., isolated from a fermented cassava product.

A novel Gram-positive, catalase negative, rod-shaped strain, FI11369T, was isolated from gari, a traditional West African fermented food derived from cassava. Based on 16S rRNA gene sequence similarity, the closest type strains were Lactobacillus xiangfangensis LMG 26013T (99.4 % similarity), Lactobacillus plajomi NBRC 107333T (99.1 %), Lactobacillus paraplantarum DSM 10667T (99.1 %), Lactobacillus pentosus DSM 20314T (99.0 %), Lactobacillus plantarum subsp. plantarum ATCC 14917T (99.0 %), Lactobacillus modestisalitolerans NBRC 107235T (98.9 %), Lactobacillus plantarum subsp. argentoratensis DSM 16365T (98.9 %) and Lactobacillus daowaiensis NCIMB 15183T (98.8 %). The genome of strain FI11369T was sequenced and the average nucleotide identity (ANI) was compared with its closest relatives. ANI analysis showed that the closest relative, L. xiangfangensis DSM 27103T, had only a 82.4 % similarity. The main fatty acids of FI11369T were saturated C16 : 0 (18.2 %), unsaturated C18 : 1 ω9c (43.8 %) and cyclopropane C19 : 0 cyclo (ω10c and/or ω6; 22.5 %). Based on the genotypic and phenotypic data obtained in this study, a novel Lactobacillus species, Lactobacillus garii sp. nov., with the type strain FI11369T (=NCIMB 15148=DSM 108249), is proposed.

Production of multiple bacteriocins, including the novel bacteriocin gassericin M, by Lactobacillus gasseri LM19, a strain isolated from human milk.

Bacteriocins are antimicrobial peptides produced by bacteria, and their production is regarded as a desirable probiotic trait. We found that Lactobacillus gasseri LM19, a strain isolated from human milk, produces several bacteriocins, including a novel bacteriocin, gassericin M. These bacteriocins were purified from culture and synthesised to investigate their activity and potential synergy. L. gasseri LM19 was tested in a complex environment mimicking human colon conditions; it not only survived, but expressed the seven bacteriocin genes and produced short-chain fatty acids. Metagenomic analysis of these in vitro colon cultures showed that co-inoculation of L. gasseri LM19 with Clostridium perfringens gave 16S ribosomal RNA metagenomic profiles with more similarity to controls than to vessels inoculated with C. perfringens alone. These results indicate that L. gasseri LM19 could be an interesting candidate for maintaining homeostasis in the gut environment.

Use of fluorescent CTP1L endolysin cell wall-binding domain to study the evolution of Clostridium tyrobutyricum during cheese ripening.

Clostridium tyrobutyricum is a bacteria of concern in the cheese industry, capable of surviving the manufacturing process and causing butyric acid fermentation and late blowing defect of cheese. In this work, we implement a method based on the cell wall-binding domain (CBD) of endolysin CTP1L, which detects C. tyrobutyricum, to monitor its evolution in cheeses challenged with clostridial spores and in the presence or absence of reuterin, an anti-clostridial agent. For this purpose, total bacteria were extracted from cheese samples and C. tyrobutyricum cells were specifically labelled with the CBD of CTP1L attached to green fluorescent protein (GFP), and detected by fluorescence microscopy. By using this GFP-CBD, germinated spores were visualized on day 1 in all cheeses inoculated with clostridial spores. Vegetative cells of C. tyrobutyricum, responsible for butyric acid fermentation, were detected in cheeses without reuterin from 30 d onwards, when LBD symptoms also became evident. The number of fluorescent Clostridium cells increased during ripening in the blowing cheeses. However, vegetative cells of C. tyrobutyricum were not detected in cheese containing the antimicrobial reuterin, which also did not show LBD throughout ripening. This simple and fast method provides a helpful tool to study the evolution of C. tyrobutyricum during cheese ripening.

Crystal Structure of the CTP1L Endolysin Reveals How Its Activity Is Regulated by a Secondary Translation Product.

Bacteriophages produce endolysins, which lyse the bacterial host cell to release newly produced virions. The timing of lysis is regulated and is thought to involve the activation of a molecular switch. We present a crystal structure of the activated endolysin CTP1L that targets Clostridium tyrobutyricum, consisting of a complex between the full-length protein and an N-terminally truncated C-terminal cell wall binding domain (CBD). The truncated CBD is produced through an internal translation start site within the endolysin gene. Mutants affecting the internal translation site change the oligomeric state of the endolysin and reduce lytic activity. The activity can be modulated by reconstitution of the full-length endolysin-CBD complex with free CBD. The same oligomerization mechanism applies to the CD27L endolysin that targets Clostridium difficile and the CS74L endolysin that targets Clostridium sporogenes. When the CTP1L endolysin gene is introduced into the commensal bacterium Lactococcus lactis, the truncated CBD is also produced, showing that the alternative start codon can be used in other bacterial species. The identification of a translational switch affecting oligomerization presented here has implications for the design of effective endolysins for the treatment of bacterial infections.

Discovery of a novel lantibiotic nisin O from Blautia obeum A2-162, isolated from the human gastrointestinal tract.

A novel lanC-like sequence was identified from the dominant human gut bacterium Blautia obeum strain A2-162. This sequence was extended to reveal a putative lantibiotic operon with biosynthetic and transport genes, two sets of regulatory genes, immunity genes, three identical copies of a nisin-like lanA gene with an unusual leader peptide, and a fourth putative lanA gene. Comparison with other nisin clusters showed that the closest relationship was to nisin U. B. obeum A2-162 demonstrated antimicrobial activity against Clostridium perfringens when grown on solid medium in the presence of trypsin. Fusions of predicted nsoA structural sequences with the nisin A leader were expressed in Lactococcus lactis containing the nisin A operon without nisA. Expression of the nisA leader sequence fused to the predicted structural nsoA1 produced a growth defect in L. lactis that was dependent upon the presence of biosynthetic genes, but failed to produce antimicrobial activity. Insertion of the nso cluster into L. lactis MG1614 gave an increased immunity to nisin A, but this was not replicated by the expression of nsoI. Nisin A induction of L. lactis containing the nso cluster and nisRK genes allowed detection of the NsoA1 pre-peptide by Western hybridization. When this heterologous producer was grown with nisin induction on solid medium, antimicrobial activity was demonstrated in the presence of trypsin against C. perfringens, Clostridium difficile and L. lactis. This research adds to evidence that lantibiotic production may be an important trait of gut bacteria and could lead to the development of novel treatments for intestinal diseases.

The CD27L and CTP1L endolysins targeting Clostridia contain a built-in trigger and release factor.

The bacteriophage ΦCD27 is capable of lysing Clostridium difficile, a pathogenic bacterium that is a major cause for nosocomial infection. A recombinant CD27L endolysin lyses C. difficile in vitro, and represents a promising alternative as a bactericide. To better understand the lysis mechanism, we have determined the crystal structure of an autoproteolytic fragment of the CD27L endolysin. The structure covers the C-terminal domain of the endolysin, and represents a novel fold that is identified in a number of lysins that target Clostridia bacteria. The structure indicates endolysin cleavage occurs at the stem of the linker connecting the catalytic domain with the C-terminal domain. We also solved the crystal structure of the C-terminal domain of a slow cleaving mutant of the CTP1L endolysin that targets C. tyrobutyricum. Two distinct dimerization modes are observed in the crystal structures for both endolysins, despite a sequence identity of only 22% between the domains. The dimers are validated to be present for the full length protein in solution by right angle light scattering, small angle X-ray scattering and cross-linking experiments using the cross-linking amino acid p-benzoyl-L-phenylalanine (pBpa). Mutagenesis on residues contributing to the dimer interfaces indicates that there is a link between the dimerization modes and the autocleavage mechanism. We show that for the CTP1L endolysin, there is a reduction in lysis efficiency that is proportional to the cleavage efficiency. We propose a model for endolysin triggering, where the extended dimer presents the inactive state, and a switch to the side-by-side dimer triggers the cleavage of the C-terminal domain. This leads to the release of the catalytic portion of the endolysin, enabling the efficient digestion of the bacterial cell wall.

Impact of the exopolysaccharide layer on biofilms, adhesion and resistance to stress in Lactobacillus johnsonii FI9785.

BACKGROUND: The bacterial cell surface is a crucial factor in cell-cell and cell-host interactions. Lactobacillus johnsonii FI9785 produces an exopolysaccharide (EPS) layer whose quantity and composition is altered in mutants that harbour genetic changes in their eps gene clusters. We have assessed the effect of changes in EPS production on cell surface characteristics that may affect the ability of L. johnsonii to colonise the poultry host and exclude pathogens. RESULTS: Analysis of physicochemical cell surface characteristics reflected by Zeta potential and adhesion to hexadecane showed that an increase in EPS gave a less negative, more hydrophilic surface and reduced autoaggregation. Autoaggregation was significantly higher in mutants that have reduced EPS, indicating that EPS can mask surface structures responsible for cell-cell interactions. EPS also affected biofilm formation, but here the quantity of EPS produced was not the only determinant. A reduction in EPS production increased bacterial adhesion to chicken gut explants, but made the bacteria less able to survive some stresses. CONCLUSIONS: This study showed that manipulation of EPS production in L. johnsonii FI9785 can affect properties which may improve its performance as a competitive exclusion agent, but that positive changes in adhesion may be compromised by a reduction in the ability to survive stress.

Structure and biosynthesis of two exopolysaccharides produced by Lactobacillus johnsonii FI9785.

Exopolysaccharides were isolated and purified from Lactobacillus johnsonii FI9785, which has previously been shown to act as a competitive exclusion agent to control Clostridium perfringens in poultry. Structural analysis by NMR spectroscopy revealed that L. johnsonii FI9785 can produce two types of exopolysaccharide: EPS-1 is a branched dextran with the unusual feature that every backbone residue is substituted with a 2-linked glucose unit, and EPS-2 was shown to have a repeating unit with the following structure: -6)-α-Glcp-(1-3)-ß-Glcp-(1-5)-ß-Galf-(1-6)-α-Glcp-(1-4)-ß-Galp-(1-4)-ß-Glcp-(1-. Sites on both polysaccharides were partially occupied by substituent groups: 1-phosphoglycerol and O-acetyl groups in EPS-1 and a single O-acetyl group in EPS-2. Analysis of a deletion mutant (ΔepsE) lacking the putative priming glycosyltransferase gene located within a predicted eps gene cluster revealed that the mutant could produce EPS-1 but not EPS-2, indicating that epsE is essential for the biosynthesis of EPS-2. Atomic force microscopy confirmed the localization of galactose residues on the exterior of wild type cells and their absence in the ΔepsE mutant. EPS2 was found to adopt a random coil structural conformation. Deletion of the entire 14-kb eps cluster resulted in an acapsular mutant phenotype that was not able to produce either EPS-2 or EPS-1. Alterations in the cell surface properties of the EPS-specific mutants were demonstrated by differences in binding of an anti-wild type L. johnsonii antibody. These findings provide insights into the biosynthesis and structures of novel exopolysaccharides produced by L. johnsonii FI9785, which are likely to play an important role in biofilm formation, protection against harsh environment of the gut, and colonization of the host.

Expression and delivery of an endolysin to combat Clostridium perfringens.

Clostridium perfringens is a cause for increasing concern due to its responsibility for severe infections both in humans and animals, especially poultry. To find new control strategies to treat C. perfringens infection, we investigated the activity and delivery of a bacteriophage endolysin. We identified a new endolysin, designated CP25L, which shows similarity to an N-acetylmuramoyl-L-alanine amidase domain and is distinct from other C. perfringens endolysins whose activity has been demonstrated in vitro. The cp25l gene was cloned and expressed in Escherichia coli, and the gene product demonstrated lytic activity against all 25 C. perfringens strains tested. The probiotic strain Lactobacillus johnsonii FI9785 was engineered to deliver the endolysin to the gastrointestinal tract. The integration of the nisRK two-component regulatory system from the Lactococcus lactis nisin A biosynthesis operon into the chromosome of L. johnsonii allowed constitutive expression of the endolysin under the control of the nisA promoter (P nisA ), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. The high specificity and activity of the endolysin suggest that it may be developed as an effective tool to enhance the control of C. perfringens by L. johnsonii in the gastrointestinal tract.

Complete genome sequence of ΦCP51, a temperate bacteriophage of Clostridium perfringens.

During sequencing of the genome of Clostridium perfringens strain 5147-97, a putative prophage was identified, located within a gene for a proposed flavodoxin oxidoreductase. Mitomycin C induction of this strain released a bacteriophage whose morphological features examined by electron microscopy indicated it belonged to the family Siphoviridae. This phage was hence designated as vB_CpeS-CP51. The 39,108-bp genome includes 50 predicted open reading frames (ORFs), including two that may affect sporulation, and two predicted tRNAs. To determine the ends of the prophage, PCR was performed using primers facing outwards from the proposed end genes. This confirmed the presence of a circularised genome in PEG-precipitated bacteriophage particles.

Evaluation of bacteriophage therapy to control Clostridium difficile and toxin production in an in vitro human colon model system.

Clostridium difficile is a leading cause of hospital-acquired diarrhoea and represents a major challenge for healthcare providers. Due to the decreasing efficacy and associated problems of antibiotic therapy there is a need for synergistic and alternative treatments. In this study we investigated the use of a specific bacteriophage, ΦCD27, in a human colon model of C. difficile infection. Our findings demonstrate a significant reduction in the burden of C. difficile cells and toxin production with phage treatment relative to an untreated control, with no detrimental effect on commensal bacterial populations. The results demonstrate the potential of phage therapy, and highlight the limitations of using phages that have lysogenic capacity.

A representative of the dominant human colonic Firmicutes, Roseburia faecis M72/1, forms a novel bacteriocin-like substance.

During screening of human gut isolates in search of novel antimicrobials, the butyrate-producing strain Roseburia faecis M72/1 was found to produce an inhibitory substance active against Bacillus subtilis. Partial purification of the antimicrobial was achieved and activity found to be heat labile. Our findings suggest that R. faecis M72/1 produces a proteinaceous inhibitor whose production may be triggered by trypsin in the gastrointestinal tract.

Isolation and characterization of new bacteriophages active against Clostridium tyrobutyricum and their role in preventing the late blowing defect of cheese.

Lytic bacteriophages (phages) offer a great potential as biocontrol agents for spoilage Clostridium tyrobutyricum, responsible for butyric acid fermentation in semi-hard and hard ripened cheeses, resulting in late gas blowing defect. With this aim, we have isolated, identified and characterized new lytic phages of C. tyrobutyricum, and have evaluated their efficacy to control cheese late blowing by adding them to manufacture milk. Silage, soil, milk and cheese from dairy farms were screened for anti-clostridial phages, obtaining 96 isolates active against C. tyrobutyricum. According to host range, source and plaque morphology, we obtained 20 phage profiles, 8 of them (represented by phages FA3, FA21, FA29, FA52, FA58, FA67, FA70 and FA88) showing a wider host range and high quality lysis, which were further characterized. Selected isolates showed a non-contractile tail, belonging to the Siphoviridae family, and were grouped into 3 restriction profiles. Viable phages were detected after storage in sodium-magnesium buffer (SM buffer), skim milk and acidified skim milk (pH 5) for 7 d at 4 °C, 12 °C and 37 °C, although a decline in infectivity was observed in some cases. Good phage survival was also detected during semi-hard cheese manufacture and ripening (60 d), and cheese lactococci counts, pH, dry matter values, and volatile compounds were not affected by phage addition. In semi-hard cheese, phage FA67 impaired the early germination of C. tyrobutyricum spores and caused a significant decrease in clostridial vegetative cells counts at 14 d of ripening, delaying by 2 weeks the consumption of lactic acid, formation of butyric acid and appearance of late blowing symptoms, compared to the spoilt control cheese without the phage. This is the first report on the application of phage to control C. tyrobutyricum in cheese.

Genomic sequence of bacteriophage ATCC 8074-B1 and activity of its endolysin and engineered variants against Clostridium sporogenes.

Lytic bacteriophage ATCC 8074-B1 produces large plaques on its host Clostridium sporogenes. Sequencing of the 47,595-bp genome allowed the identification of 82 putative open reading frames, including those encoding proteins for head and tail morphogenesis and lysis. However, sequences commonly associated with lysogeny were absent. ORF 22 encodes an endolysin, CS74L, that shows homology to N-acetylmuramoyl-L-alanine amidases, and when expressed in Escherichia coli, the protein causes effective lysis of C. sporogenes cells when added externally. CS74L was also active on Clostridium tyrobutyricum and Clostridium acetobutylicum. The catalytic domain expressed alone (CS74L(1-177)) exhibited a similar activity and the same host range as the full-length endolysin. A chimeric endolysin consisting of the CS74L catalytic domain fused to the C-terminal domain of endolysin CD27L, derived from Clostridium difficile bacteriophage ΦCD27, was produced. This chimera (CSCD) lysed C. sporogenes cells with an activity equivalent to that of the catalytic domain alone. In contrast, the CD27L C-terminal domain reduced the efficacy of the CS74L catalytic domain when tested against C. tyrobutyricum. The addition of the CD27L C-terminal domain did not enable the lysin to target C. difficile or other CD27L-sensitive bacteria.

Expression of a bacterial, phenylpropanoid-metabolizing enzyme in tobacco reveals essential roles of phenolic precursors in normal leaf development and growth.

Tobacco plants (Nicotiana tabacum cv XHFD 8) were genetically modified to express a bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) enzyme which is active with intermediates of the phenylpropanoid pathway. We have previously shown that HCHL expression in tobacco stem resulted in various pleiotropic effects, indicative of a reduction in the carbon flux through the phenylpropanoid pathway, accompanied by an abnormal phenotype. Here, we report that in addition to the reduction in lignin and phenolic biosynthesis, HCHL expression also resulted in several gross morphological changes in poorly lignified tissue, such as abnormal mesophyll and palisade. The effect of HCHL expression was also noted in lignin-free single cells, with suspension cultures displaying an altered shape and different growth patterns. Poorly/non-lignified cell walls also exhibited a greater ease of alkaline extractability of simple phenolics and increased levels of incorporation of vanillin and vanillic acid. However, HCHL expression had no significant effect on the cell wall carbohydrate chemistry of these tissues. Evidence from this study suggests that changes in the transgenic lines result from a reduction in phenolic intermediates which have an essential role in maintaining structural integrity of low-lignin or lignin-deprived cell walls. These results emphasize the importance of the intermediates and products of phenylpropanoid pathway in modulating aspects of normal growth and development of tobacco. Analysis of these transgenic plants also shows the plasticity of the lignification process and reveals the potential to bioengineer plants with reduced phenolics (without deleterious effects) which could enhance the bioconversion of lignocellulose for industrial applications.

Molecular Cloning, Expression and Characterisation of a Bacterial Myrosinase from Citrobacter Wye1.

Glucosinolates are plant natural products which on degradation by myrosinases give rise to the beneficial bioactive isothiocyanates. Recently, a myrosinase activity was detected in a Citrobacter strain isolated from soil. This enzyme was purified enabling its amino acid sequence and gene sequence (cmyr) to be determined. In order to study this myrosinase it was necessary to establish an expression system that would enable future work such as a structural determination of the protein to be carried out. The myrosinase gene was amplified, cloned and expressed in Escherichia coli with a 6XHis-tag. The heterologous expression of cmyr enabled relatively large amounts of myrosinase to be produced (3.4 mg cmyr/100 ml culture). Myrosinase activity was determined by mixing substrate and enzyme and determining glucose release. Optimum pH and temperature were determined to be pH 6.0 and 25 °C for the Ni-NTA purified protein. The kinetic parameters of the purified myrosinase were determined using sinigrin as a substrate. Km and Vmax were estimated as 0.18 mM and 0.033 mmol/min/mg respectively for sinigrin under optimum conditions and compared to other kinetic data for myrosinases. The substrate specificity of myrosinase was determined having the highest affinity for sinigrin followed by glucoiberin, progoitrin, glucoerucin, glucoraphanin and glucotropaeolin.

Structure-based modification of a Clostridium difficile-targeting endolysin affects activity and host range.

Endolysin CD27L causes cell lysis of the pathogen Clostridium difficile, a major cause of nosocomial infection. We report a structural and functional analysis of the catalytic activity of CD27L against C. difficile and other bacterial strains. We show that truncation of the endolysin to the N-terminal domain, CD27L1-179, gave an increased lytic activity against cells of C. difficile, while the C-terminal region, CD27L180-270, failed to produce lysis. CD27L1-179 also has increased activity against other bacterial species that are targeted by the full-length protein and in addition was able to lyse some CD27L-insensitive strains. However, CD27L1-179 retained a measure of specificity, failing to lyse a wide range of bacteria. The use of green fluorescent protein (GFP)-labeled proteins demonstrated that both CD27L and CD27L1-179 bound to C. difficile cell walls. The crystal structure of CD27L1-179 confirms that the enzyme is a zinc-dependent N-acetylmuramoyl-l-alanine amidase. A structure-based sequence analysis allowed us to identify four catalytic residues, a proton relay cascade, and a substrate binding pocket. A BLAST search shows that the closest-related amidases almost exclusively target Clostridia. This implied that the catalytic domain alone contained features that target a specific bacterial species. To test this hypothesis, we modified Leu 98 to a Trp residue which is found in an endolysin from a bacteriophage of Listeria monocytogenes (PlyPSA). This mutation in CD27L resulted in an increased activity against selected serotypes of L. monocytogenes, demonstrating the potential to tune the species specificity of the catalytic domain of an endolysin.