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.

High yields of 2,3-butanediol and mannitol in Lactococcus lactis through engineering of NAD⁺ cofactor recycling.

Manipulation of NADH-dependent steps, and particularly disruption of the las-located lactate dehydrogenase (ldh) gene in Lactococcus lactis, is common to engineering strategies envisaging the accumulation of reduced end products other than lactate. Reverse transcription-PCR experiments revealed that three out of the four genes assigned to lactate dehydrogenase in the genome of L. lactis, i.e., the ldh, ldhB, and ldhX genes, were expressed in the parental strain MG1363. Given that genetic redundancy is often a major cause of metabolic instability in engineered strains, we set out to develop a genetically stable lactococcal host tuned for the production of reduced compounds. Therefore, the ldhB and ldhX genes were sequentially deleted in L. lactis FI10089, a strain with a deletion of the ldh gene. The single, double, and triple mutants, FI10089, FI10089ΔldhB, and FI10089ΔldhBΔldhX, showed similar growth profiles and displayed mixed-acid fermentation, ethanol being the main reduced end product. Hence, the alcohol dehydrogenase-encoding gene, the adhE gene, was inactivated in FI10089, but the resulting strain reverted to homolactic fermentation due to induction of the ldhB gene. The three lactate dehydrogenase-deficient mutants were selected as a background for the production of mannitol and 2,3-butanediol. Pathways for the biosynthesis of these compounds were overexpressed under the control of a nisin promoter, and the constructs were analyzed with respect to growth parameters and product yields under anaerobiosis. Glucose was efficiently channeled to mannitol (maximal yield, 42%) or to 2,3-butanediol (maximal yield, 67%). The theoretical yield for 2,3-butanediol was achieved. We show that FI10089ΔldhB is a valuable basis for engineering strategies aiming at the production of reduced compounds.

Analysis of bacterial communities of traditional fermented West African cereal foods using culture independent methods.

In this study, the microbial composition of kunu-zaki and ogi, two popular foods in Nigeria produced after natural, uncontrolled fermentation of cereals, was assessed by culture-independent molecular profiling methods. In particular, PCR-denaturing gradient gel electrophoresis and construction of 16S rRNA gene clone libraries revealed the presence of diverse bacterial communities. DNA sequencing of the highly variable V3 region of the 16S rRNA genes obtained from PCR-DGGE fingerprints identified species related to Weissella confusa, Lactobacillus fermentum, Lactobacillus amylolyticus, Lactobacillus delbrueckii subsp. bulgaricus, Bacillus spp. and Lactococcus lactis spp lactis from food samples obtained from northern and southern geographical locations. A more comprehensive analysis of 272 full-length 16S rRNA gene inserts revealed that 70% of them were assigned to the Lactobacillaceae family and 19% to the Streptococcaceae family. Interestingly, sequences associated with a particular food type were also identified. For example, L. plantarum, L. pantheris and L. vaccinostercus were found in ogi but not in kunu-zaki while W. confusa, Streptococcus lutetiensis and Streptococcus gallolyticus subsp. macedonicus were found in kunu-zaki but not in ogi. Phylotypes corresponding to potentially pathogenic bacteria, such as Clostridium perfringens and Bacillus cereus were also detected highlighting the need for controlled fermentation processes.

Bacteriophage treatment significantly reduces viable Clostridium difficile and prevents toxin production in an in vitro model system.

Clostridium difficile is primarily a nosocomial pathogen, causing thousands of cases of antibiotic-associated diarrhoea in the UK each year. In this study, we used a batch fermentation model of a C. difficile colonised system to evaluate the potential of a prophylactic and a remedial bacteriophage treatment regime to control the pathogen. It is shown that the prophylaxis regime was effective at preventing the growth of C. difficile (p = <0.001) and precluded the production of detectable levels of toxins A and B. The remedial treatment regime caused a less profound and somewhat transient decrease in the number of viable C. difficile cells (p = <0.0001), but still resulted in a lower level of toxin production relative to the control. The numbers of commensal bacteria including total aerobes and anaerobes, Bifidobacterium sp., Bacteroides sp., Lactobacillus sp., total Clostridium sp., and Enterobacteriaceae were not significantly decreased by this therapy, whereas significant detrimental effects were observed with metronidazole treatment. Our study indicates that phage therapy has potential to be used for the control of C. difficile; it highlights the main benefits of this approach, and some future challenges.

Genomic sequence and characterization of the virulent bacteriophage phiCTP1 from Clostridium tyrobutyricum and heterologous expression of its endolysin.

The growth of Clostridium tyrobutyricum in developing cheese leads to spoilage and cheese blowing. Bacteriophages or their specific lytic enzymes may provide a biological control method for eliminating such undesirable organisms without affecting other microflora. We isolated the virulent bacteriophage phiCTP1 belonging to the Siphoviridae and have shown that it is effective in causing lysis of sensitive strains. The double-stranded DNA genome of phiCTP1 is 59,199 bp, and sequence analysis indicated that it has 86 open reading frames. orf29 was identified as the gene coding for the phage endolysin responsible for cell wall degradation prior to virion release. We cloned and expressed the ctp1l gene in E. coli and demonstrated that the partially purified protein induced lysis of C. tyrobutyricum cells and reduced viable counts both in buffer and in milk. The endolysin was inactive against a range of clostridial species but did show lysis of Clostridium sporogenes, another potential spoilage organism. Removal of the C-terminal portion of the endolysin completely abolished lytic activity.

Complete genome sequence of Lactobacillus johnsonii FI9785, a competitive exclusion agent against pathogens in poultry.

Lactobacillus johnsonii is a member of the acidophilus group of lactobacilli. Because of their probiotic properties, including attachment to epithelial cells, immunomodulation, and competitive exclusion of pathogens, representatives of this group are being intensively studied. Here we report the complete annotated genome sequence of Lactobacillus johnsonii FI9785, a strain which prevents the colonization of specific-pathogen-free chicks by Clostridium perfringens.

Isolation and characterization of a theta-type cryptic plasmid from Bifidobacterium longum FI10564.

A number of bifidobacterial species of human origin were screened for the presence of cryptic plasmids. One strain, Bifidobacterium longum FI10564, harbored plasmids of approximately 2.2 kb, 3.6 kb, and 4.9 kb in size. The smallest plasmid, pFI2576 (2,197 bp), was studied in detail and its complete nucleotide sequence was determined. Computer-assisted analysis of this novel plasmid (G+C content 62%) identified 9 putative open reading frames (orfs), 3 of which were shown to be probable genes. These putative genes are arranged in an operon-like structure, in which the overlapping orfs 1 and 2 encode putative Rep proteins and are highly homologous to the rep genes of the B. longum plasmid pMB1 (1,847 bp). The mechanism of replication of pFI2576 was investigated using Southern blot analysis of whole cell lysates, with and without S1 nuclease treatment, and atomic force microscopy (AFM). The results indicate that pFI2576 is likely to use the theta mode of replication.

Heterologous production of methionine-gamma-lyase from Brevibacterium linens in Lactococcus lactis and formation of volatile sulfur compounds.

The conversion of methionine to volatile sulfur compounds (VSCs) is of great importance in flavor formation during cheese ripening and is the focus of biotechnological approaches toward flavor improvement. A synthetic mgl gene encoding methionine-gamma-lyase (MGL) from Brevibacterium linens BL2 was cloned into a Lactococcus lactis expression plasmid under the control of the nisin-inducible promoter PnisA. When expressed in L. lactis and purified as a recombinant protein, MGL was shown to degrade L-methionine as well as other sulfur-containing compounds such as L-cysteine, L-cystathionine, and L-cystine. Overproduction of MGL in recombinant L. lactis also resulted in an increase in the degradation of these compounds compared to the wild-type strain. Importantly, gas chromatography-mass spectrometry analysis identified considerably higher formation of methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) in reactions containing either purified protein, whole cells, or cell extracts from the heterologous L. lactis strain. This is the first report of production of MGL from B. linens in L. lactis. Given their significance in cheese flavor development, the use of lactic acid bacteria with enhanced VSC-producing abilities could be an efficient way to enhance cheese flavor development.

Enhanced secretion of biologically active murine interleukin-12 by Lactococcus lactis.

The novel signal peptide SLPmod was used for the secretion of murine interleukin-12 (mIL-12) by Lactococcus lactis. A >4-fold increase in secretion was observed when SLPmod was used instead of the Usp45-derived secretion signal. Oral delivery of this cytokine using the autoinducible host L. lactis FI5876 utilizing SLPmod resulted in a significant increase in mIL-12 plasma levels in mice.

A short-oligonucleotide microarray that allows improved detection of gastrointestinal tract microbial communities.

BACKGROUND: The human gastrointestinal (GI) tract contains a diverse collection of bacteria, most of which are unculturable by conventional microbiological methods. Increasingly molecular profiling techniques are being employed to examine this complex microbial community. The purpose of this study was to develop a microarray technique based on 16S ribosomal gene sequences for rapidly monitoring the microbial population of the GI tract. RESULTS: We have developed a culture-independent, semi-quantitative, rapid method for detection of gut bacterial populations based on 16S rDNA probes using a DNA microarray. We compared the performance of microarrays based on long (40- and 50-mer) and short (16-21-mer) oligonucleotides. Short oligonucleotides consistently gave higher specificity. Optimal DNA amplification and labelling, hybridisation and washing conditions were determined using a probe with an increasing number of nucleotide mismatches, identifying the minimum number of nucleotides needed to distinguish between perfect and mismatch probes. An independent PCR-based control was used to normalise different hybridisation results, and to make comparisons between different samples, greatly improving the detection of changes in the gut bacterial population. The sensitivity of the microarray was determined to be 8.8 x 104 bacterial cells g-1 faecal sample, which is more sensitive than a number of existing profiling methods. The short oligonucleotide microarray was used to compare the faecal flora from healthy individuals and a patient suffering from Ulcerative Colitis (UC) during the active and remission states. Differences were identified in the bacterial profiles between healthy individuals and a UC patient. These variations were verified by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing. CONCLUSION: In this study we demonstrate the design, testing and application of a highly sensitive, short oligonucleotide community microarray. Our approach allows the rapid discrimination of bacteria inhabiting the human GI tract, at taxonomic levels ranging from species to the superkingdom bacteria. The optimised protocol is available at: http://www.ifr.ac.uk/safety/microarrays/#protocols. It offers a high throughput method for studying the dynamics of the bacterial population over time and between individuals.

Molecular characterization of a Clostridium difficile bacteriophage and its cloned biologically active endolysin.

Clostridium difficile infection is increasing in both frequency and severity, with the emergence of new highly virulent strains highlighting the need for more rapid and effective methods of control. Here, we show that bacteriophage endolysin can be used to inhibit and kill C. difficile. The genome sequence of a novel bacteriophage that is active against C. difficile was determined, and the bacteriophage endolysin gene was subcloned and expressed in Escherichia coli. The partially purified endolysin was active against 30 diverse strains of C. difficile, and importantly, this group included strains of the major epidemic ribotype 027 (B1/NAP1). In contrast, a range of commensal species that inhabit the gastrointestinal tract, including several representatives of the clostridium-like Firmicutes, were insensitive to the endolysin. This endolysin provides a platform for the generation of both therapeutic and detection systems to combat the C. difficile problem. To investigate a method for the protected delivery and production of the lysin in the gastrointestinal tract, we demonstrated the expression of active CD27L endolysin in the lactic acid bacterium Lactococcus lactis MG1363.

The lactate dehydrogenases encoded by the ldh and ldhB genes in Lactococcus lactis exhibit distinct regulation and catalytic properties - comparative modeling to probe the molecular basis.

Lactococcus lactis FI9078, a construct carrying a disruption of the ldh gene, converted approximately 90% of glucose into lactic acid, like the parental strain MG1363. This unexpected lactate dehydrogenase activity was purified, and ldhB was identified as the gene encoding this protein. The activation of ldhB was explained by the insertion of an IS905-like element that created a hybrid promoter in the intergenic region upstream of ldhB. The biochemical and kinetic properties of this alternative lactate dehydrogenase (LDHB) were compared to those of the ldh-encoded enzyme (LDH), purified from the parental strain. In contrast to LDH, the affinity of LDHB for NADH and the activation constant for fructose 1,6-bisphosphate were strongly dependent on pH. The activation constant increased 700-fold, whereas the K(m) for NADH increased more than 10-fold, in the pH range 5.5-7.2. The two enzymes also exhibited different pH profiles for maximal activity. Moreover, inorganic phosphate acted as a strong activator of LDHB. The impact of replacing LDH by LDHB on the physiology of L. lactis was assessed by monitoring the evolution of the pools of glycolytic intermediates and cofactors during the metabolism of glucose by in vivo NMR. Structural analysis by comparative modeling of the two proteins showed that LDH has a slightly larger negative charge than LDHB and a greater concentration of positive charges at the interface between monomers. The calculated pH titration curves of the catalytic histidine residues explain why LDH maintains its activity at low pH as compared to LDHB, the histidines in LDH showing larger pH titration ranges.

Nisin-controlled extracellular production of interleukin-2 in Lactococcus lactis strains, without the requirement for a signal peptide sequence.

Secretion of the cytokine interleukin-2 (IL-2) was investigated in Lactococcus lactis using the secretory machinery of the bacteriocin lactococcin A. Surprisingly, the lcnCD transport genes were not essential for mouse IL-2 secretion. Furthermore, expression of a mature mouse IL-2 gene resulted in interleukin secretion without the requirement for a leader sequence.

Comparative analyses of prophage-like elements present in two Lactococcus lactis strains.

In this study, we describe the genetic organizations of six and five apparent prophage-like elements present in the genomes of the Lactococcus lactis subsp. cremoris strains MG1363 and SK11, respectively. Phylogenetic investigation as well bioinformatic analyses indicates that all 11 prophages belong to subdivisions of the lactococcal P335 group of temperate bacteriophages.

Complete genome sequence of the prototype lactic acid bacterium Lactococcus lactis subsp. cremoris MG1363.

Lactococcus lactis is of great importance for the nutrition of hundreds of millions of people worldwide. This paper describes the genome sequence of Lactococcus lactis subsp. cremoris MG1363, the lactococcal strain most intensively studied throughout the world. The 2,529,478-bp genome contains 81 pseudogenes and encodes 2,436 proteins. Of the 530 unique proteins, 47 belong to the COG (clusters of orthologous groups) functional category "carbohydrate metabolism and transport," by far the largest category of novel proteins in comparison with L. lactis subsp. lactis IL1403. Nearly one-fifth of the 71 insertion elements are concentrated in a specific 56-kb region. This integration hot-spot region carries genes that are typically associated with lactococcal plasmids and a repeat sequence specifically found on plasmids and in the "lateral gene transfer hot spot" in the genome of Streptococcus thermophilus. Although the parent of L. lactis MG1363 was used to demonstrate lysogeny in Lactococcus, L. lactis MG1363 carries four remnant/satellite phages and two apparently complete prophages. The availability of the L. lactis MG1363 genome sequence will reinforce its status as the prototype among lactic acid bacteria through facilitation of further applied and fundamental research.

YtjE from Lactococcus lactis IL1403 Is a C-S lyase with alpha, gamma-elimination activity toward methionine.

Cheese microbiota and the enzymatic conversion of methionine to volatile sulfur compounds (VSCs) are important factors in flavor formation during cheese ripening and the foci in biotechnological approaches to flavor improvement. The product of ytjE of Lactococcus lactis IL1403, suggested to be a methionine-specific aminotransferase based on genome sequence analysis, was therefore investigated for its role in methionine catabolism. The ytjE gene from Lactococcus lactis IL1403 was cloned in Escherichia coli and overexpressed and purified as a recombinant protein. When tested, the YtjE protein did not exhibit a specific methionine aminotransferase activity. Instead, YtjE exhibited C-S lyase activity and shared homology with the MalY/PatC family of enzymes involved in the degradation of L-cysteine, L-cystine, and L-cystathionine. YtjE was also shown to exhibit alpha,gamma-elimination activity toward L-methionine. In addition, gas chromatographic-mass spectrometry analysis showed that YtjE activity resulted in the formation of H2S from L-cysteine and methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) from L-methionine. Given their significance in cheese flavor development, VSC production by YtjE could offer an additional approach for the development of cultures with optimized aromatic properties.

The crystal structures of Lactococcus lactis MG1363 Dps proteins reveal the presence of an N-terminal helix that is required for DNA binding.

Dps proteins play a major role in the protection of bacterial DNA from damage by reactive oxygen species. Previous studies have implicated the extended lysine-containing N-terminal regions of Dps subunits in DNA binding, but this part of the structure has not previously been observed crystallographically. Here the structures of two Dps proteins (DpsA and DpsB) from Lactococcus lactis MG1363 reveal for the first time the presence of an N-terminal alpha helix that extends from the core of the Dps subunit. Consequently, the N-terminal helices are displayed in parallel pairs on the exterior of the dodecameric Dps assemblies. Both DpsA and DpsB bind DNA. Deletion of the DpsA N-terminal helix impaired DNA binding. The N-terminal Lys residues of Escherichia coli Dps have been implicated in DNA binding. Replacement of the lactococcal DpsA Lys residues 9, 15 and 16 by Glu did not inhibit DNA binding. However, DNA binding was inhibited by EDTA, suggesting a role for cations in DNA binding. In contrast to E. coli, Bacillus brevis and Mycobacterium smegmatis Dps:DNA complexes, in which DNA interacts with crystalline Dps phases, L. lactis DNA:Dps complexes appeared as non-crystalline aggregates of protein and DNA in electron micrographs.

Characterisation of a novel plasmid p9785S from Lactobacillus johnsonii FI9785.

Lactobacillus johnsonii FI9785, a strain originally isolated from poultry gastrointestinal tract for its probiotic function as a competitive excluder of pathogens, was found to contain two cryptic plasmids of 3.5 and 25.6 kb. Nucleotide sequence analysis of the entire small plasmid, designated p9785S (3471 bp), indicated a G+C content of 35.8%, and revealed two open reading frames (orfs). The product of orf1 exhibited similarity to the relaxases of mobilizable plasmids, whereas the product of orf2 displayed significant homology to replication proteins of plasmids which use the rolling circle mode of replication. A conserved double-strand origin of replication was also present in p9785S. A definite minus origin was not identified although a region with extensive intrastrand base pairing potential was revealed. A 1.4 kb fragment encoding the chloramphenicol resistance gene was cloned into p9785S and the resulting vector, pFI2431, was stably maintained when introduced into the parent Lactobacillus cells.

Screening of virulence determinants in Enterococcus faecium strains isolated from breast milk.

In a previous study, the authors isolated lactic acid bacteria from breast milk of healthy mothers. Since some of the identified isolates belonged to the species Enterococcus faecium, the objective of this work was to evaluate their safety. The enterococcal strains were screened by polymerase chain reaction (PCR) and Southern hybridization for the presence of virulence determinants. The potential of the strains to acquire plasmids by conjugation was investigated by screening for genes involved in conjugation processes. Parallel, phenotypic assays were performed. Presence of genes conferring resistance to vancomycin was assessed by PCR. PCR amplifications and Southern hybridizations revealed that all the strains were clear of the majority of potential virulence determinants. None of the strains showed gelatinase activity, hemolysin production, or aggregation phenotype, and none carried the vanA or vanB genes. These findings suggest that milk of healthy mothers may be a source of avirulent E faecium isolates to the newborns.

Structure-function analysis of the vanillin molecule and its antifungal properties.

The aim of the present study was to evaluate which structural elements of the vanillin molecule are responsible for its observed antifungal activity. MICs of vanillin, its six direct structural analogues, and several other related compounds were determined in yeast extract peptone dextrose broth against a total of 18 different food spoilage molds and yeasts. Using total mean MICs after 4 days of incubation at 25 degrees C, the antifungal activity order was 3-anisaldehyde (1.97 mM) > benzaldehyde (3.30 mM) > vanillin (5.71 mM) > anisole (6.59 mM) > 4-hydroxybenzaldehyde (9.09 mM) > phenol (10.59 mM) > guaiacol (11.66 mM). No correlation was observed between the relative antifungal activity of the test compounds and log P(o/w). Furthermore, phenol (10.6 mM) was found to exhibit a greater activity than cyclohexanol (25.3 mM), whereas cyclohexanecarboxaldehyde (2.13 mM) was more active than benzaldehyde (3.30 mM). Finally, the antifungal order of isomers of hydroxybenzaldehyde and anisaldehyde was found to be 2- > 3- > 4- and 3- > 2- > 4-, respectively. In conclusion, the aldehyde moeity of vanillin plays a key role in its antifungal activity, but side-group position on the benzene ring also influences this activity. Understanding how the structure of natural compounds relates to their antimicrobial function is fundamentally important and may help facilitate their application as novel food preservatives.