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.
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.
•The potential of phage to prevent Clostridium difficile infection has been studied in vitro.•Phage treatment reduced vegetative C. difficile cells in 2 of 3 experiments.•Phage treatment consistently reduced C. difficile toxin production.•Lysogenisation of the phage was demonstrated.•Commensal flora was not negatively affected by phage treatment.
As a competitive exclusion agent, Lactobacillus johnsonii FI9785 has been shown to prevent the colonization of selected pathogenic bacteria from the chicken gastrointestinal tract. During growth of ...the bacterium a rare but consistent emergence of an altered phenotype was noted, generating smooth colonies in contrast to the wild type rough form. A smooth colony variant was isolated and two-dimensional gel analysis of both strains revealed a protein spot with different migration properties in the two phenotypes. The spot in both gels was identified as a putative tyrosine kinase (EpsC), associated with a predicted exopolysaccharide gene cluster. Sequencing of the epsC gene from the smooth mutant revealed a single substitution (G to A) in the coding strand, resulting in the amino acid change D88N in the corresponding gene product. A native plasmid of L. johnsonii was engineered to produce a novel vector for constitutive expression and this was used to demonstrate that expression of the wild type epsC gene in the smooth mutant produced a reversion to the rough colony phenotype. Both the mutant and epsC complemented strains had increased levels of exopolysaccharides compared to the wild type strain, indicating that the rough phenotype is not solely associated with the quantity of exopolysaccharide. Another gene in the cluster, epsE, that encoded a putative undecaprenyl-phosphate galactosephosphotransferase, was deleted in order to investigate its role in exopolysaccharide biosynthesis. The ΔepsE strain exhibited a large increase in cell aggregation and a reduction in exopolysaccharide content, while plasmid complementation of epsE restored the wild type phenotype. Flow cytometry showed that the wild type and derivative strains exhibited clear differences in their adhesive ability to HT29 monolayers in tissue culture, demonstrating an impact of EPS on surface properties and bacteria-host interactions.
A novel Gram-positive, catalase negative, rod-shaped strain, FI11369
, was isolated from
, a traditional West African fermented food derived from cassava. Based on 16S rRNA gene sequence similarity, ...the closest type strains were
LMG 26013
(99.4 % similarity),
NBRC 107333
(99.1 %),
DSM 10667
(99.1 %),
DSM 20314
(99.0 %),
subsp.
ATCC 14917
(99.0 %),
NBRC 107235
(98.9 %),
subsp.
DSM 16365
(98.9 %) and
NCIMB 15183
(98.8 %). The genome of strain FI11369
was sequenced and the average nucleotide identity (ANI) was compared with its closest relatives. ANI analysis showed that the closest relative,
DSM 27103
, had only a 82.4 % similarity. The main fatty acids of FI11369
were saturated C
(18.2 %), unsaturated C
ω9
(43.8 %) and cyclopropane C
cyclo (
10
and/or
6; 22.5 %). Based on the genotypic and phenotypic data obtained in this study, a novel
species,
sp. nov., with the type strain FI11369
(=NCIMB 15148=DSM 108249), is proposed.
FI9785 makes two capsular exopolysaccharides-a heteropolysaccharide (EPS2) encoded by the
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 Δ
mutant strain than in the wild type. Nuclear magnetic resonance (NMR) analysis of isolated EPS showed that deletion of the
gene (
) prevented the accumulation of EPS1, without affecting EPS2 synthesis, while plasmid complementation restored EPS1 production. The deletion of
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
analysis of the neighboring gene
suggested that it encodes a putative flippase with homology to the GtrA superfamily. Deletion of
also prevented production of EPS1 and again caused a slow-growth phenotype, while plasmid complementation reinstated EPS1 synthesis. Both genes are highly conserved in
strains isolated from different environments. These results suggest that there may be a novel mechanism for homopolysaccharide synthesis in the Gram-positive
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
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.
Clostridium tyrobutyricum has been identified as a major species associated with the late blowing defect (LBD) of semi-hard and hard cheeses, due to undesirable butyric acid fermentation. To find new ...strategies to control this spoilage bacterium, we investigated the delivery of a bacteriophage endolysin by a cheese starter culture. The nisin producer Lactococcus lactis subsp. lactis INIA 415 was engineered to produce the CTP1L endolysin, encoded by the virulent bacteriophage ΦCTP1 of C. tyrobutyricum and with a demonstrated lytic activity in vitro, to the cheese matrix. The presence of the nisRK two-component regulatory system in the host strain allowed constitutive expression of the endolysin under the control of the nisA promoter (PnisA), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. Engineered lysins with a second cell wall binding domain were also tested and shown to have improved lytic activity. Transformation of L. lactis subsp. lactis INIA 415 with endolysin delivery plasmids had a detrimental effect on its ability to produce nisin in milk, but did not affect its acidifying capacity. Transformed L. lactis subsp. lactis INIA 415 were evaluated as starters in cheeses contaminated with spores of C. tyrobutyricum. Evolution of microbiological parameters, pH and dry matter of cheeses were studied, and Clostridium metabolism and LBD in cheeses were monitored by sensory and instrumental analyses during ripening. Cheese made with the parental strain L. lactis subsp. lactis INIA 415 delayed LBD by one month, attributable to the activity of the nisin, but it was not sufficient to arrest the growth of C. tyrobutyricum during ripening completely. The use of the endolysin-producing strains in cheese manufacture as single cultures also delayed the appearance of LBD by one month, attributable to the activity of the endolysin produced in situ during ripening, because nisin activity in these cheeses was very low at day 1 and undetectable from 15 days onwards. Endolysin was more effective than nisin in inhibiting Clostridium growth, since cheeses made with the CTP1L or the chimeric derivative producers only as starters showed lower LBD symptoms, higher lactic acid levels and lower concentrations of propionic and butyric acids (associated with off-flavours) than cheese made with the parental strain. Investigation of different promoters to maximise endolysin production may help to implement CTP1L as a tool to control C. tyrobutyricum by L. lactis cheese starter and reduce LBD even further.
•Nisin-producing Lactococcus lactis INIA 415 was engineered to yield CTP1L endolysin.•A second cell-wall binding domain in tandem facilitated secreted lysin activity.•The lysin delivery plasmid had a detrimental effect on nisin production.•Transformed strains delayed the appearance of the late blowing defect of cheese.•Good lysin expression gave less late blowing defect symptoms than nisin production.
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.
•GFP-cell wall-binding domain (CBD) of endolysin CTP1L binds specifically to Clostridium.•Cheeses were made with C. tyrobutyricum spores and with or without reuterin.•Cheese bacteria were extracted and C. tyrobutyricum labelled with GFP-CBD-CTP1L.•Clostridial germinated spores and vegetative cells were detected in cheeses made with spores.•C. tyrobutyricum cells were not detected in cheeses with the antimicrobial reuterin.
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•96 phages active against Clostridium tyrobutyricum were isolated and characterized.•Phages survived in cheese without changing starter, pH, DM or volatile compounds.•Cheese late ...blowing caused by C. tyrobutyricum was delayed 2 weeks by phage FA67.
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.
Summary
Lactobacillus johnsonii FI9785 has an eps gene cluster which is required for the biosynthesis of homopolymeric exopolysaccharides (EPS)‐1 and heteropolymeric EPS‐2 as a capsular layer. The ...first gene of the cluster, epsA, is the putative transcriptional regulator. In this study we showed the crucial role of epsA in EPS biosynthesis by demonstrating that deletion of epsA resulted in complete loss of both EPS‐1 and EPS‐2 on the cell surface. Plasmid complementation of the epsA gene fully restored EPS production, as confirmed by transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, this complementation resulted in a twofold increase in the expression levels of this gene, which almost doubled amounts of EPS production in comparison with the wild‐type strain. Analysis of EPS by NMR showed an increased ratio of the heteropolysaccharide to homopolysaccharide in the complemented strain and allowed identification of the acetylated residue in EPS‐2 as the (1,4)‐linked βGlcp unit, with the acetyl group located at O‐6. These findings indicate that epsA is a positive regulator of EPS production and that EPS production can be manipulated by altering its expression.
The epsA gene of Lactobacillus johnsonii FI9785 is essential for the production of both heteropolysaccharide EPS‐2 and homopolysaccharide EPS‐1. Overexpression of this gene can improve EPS production and affect EPS composition, EPS acetylation and surface characteristics of mutant strains.
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