Characterization and site-directed mutagenesis of Wzb, an O-phosphatase from Lactobacillus rhamnosus

<p>Abstract</p> <p>Background</p> <p>Reversible phosphorylation events within a polymerisation complex have been proposed to modulate capsular polysaccharide synthesis in <it>Streptococcus pneumoniae</it>. Similar phosphatase and kinase genes are present in the exopolysaccharide (EPS) biosynthesis loci of numerous lactic acid bacteria genomes.</p> <p>Results</p> <p>The protein sequence deduced from the <it>wzb </it>gene in <it>Lactobacillus rhamnosus </it>ATCC 9595 reveals four motifs of the polymerase and histidinol phosphatase (PHP) superfamily of prokaryotic O-phosphatases. Native and modified His-tag fusion Wzb proteins were purified from <it>Escherichia coli </it>cultures. Extracts showed phosphatase activity towards tyrosine-containing peptides. The purified fusion protein Wzb was active on <it>p</it>-nitrophenyl-phosphate (<it>p</it>NPP), with an optimal activity in presence of bovine serum albumin (BSA 1%) at pH 7.3 and a temperature of 75°C. At 50°C, residual activity decreased to 10 %. Copper ions were essential for phosphatase activity, which was significantly increased by addition of cobalt. Mutated fusion Wzb proteins exhibited reduced phosphatase activity on <it>p</it>-nitrophenyl-phosphate. However, one variant (C6S) showed close to 20% increase in phosphatase activity.</p> <p>Conclusion</p> <p>These characteristics reveal significant differences with the manganese-dependent CpsB protein tyrosine phosphatase described for <it>Streptococcus pneumoniae </it>as well as with the polysaccharide-related phosphatases of Gram negative bacteria.</p

Production, purification, sequencing and activity spectra of mutacins D-123.1 and F-59.1

<p>Abstract</p> <p>Background</p> <p>The increase in bacterial resistance to antibiotics impels the development of new anti-bacterial substances. Mutacins (bacteriocins) are small antibacterial peptides produced by <it>Streptococcus mutans </it>showing activity against bacterial pathogens. The objective of the study was to produce and characterise additional mutacins in order to find new useful antibacterial substances.</p> <p>Results</p> <p>Mutacin F-59.1 was produced in liquid media by <it>S. mutans </it>59.1 while production of mutacin D-123.1 by <it>S. mutans </it>123.1 was obtained in semi-solid media. Mutacins were purified by hydrophobic chromatography. The amino acid sequences of the mutacins were obtained by Edman degradation and their molecular mass was determined by mass spectrometry. Mutacin F-59.1 consists of 25 amino acids, containing the YGNGV consensus sequence of pediocin-like bacteriocins with a molecular mass calculated at 2719 Da. Mutacin D-123.1 has an identical molecular mass (2364 Da) with the same first 9 amino acids as mutacin I. Mutacins D-123.1 and F-59.1 have wide activity spectra inhibiting human and food-borne pathogens. The lantibiotic mutacin D-123.1 possesses a broader activity spectrum than mutacin F-59.1 against the bacterial strains tested.</p> <p>Conclusion</p> <p>Mutacin F-59.1 is the first pediocin-like bacteriocin identified and characterised that is produced by <it>Streptococcus mutans</it>. Mutacin D-123.1 appears to be identical to mutacin I previously identified in different strains of <it>S. mutans</it>.</p

Mutacin H-29B is identical to mutacin II (J-T8)

BACKGROUND: Streptococcus mutans produces bacteriocins named mutacins. Studies of mutacins have always been hampered by the difficulties in obtaining active liquid preparations of these substances. Some of them were found to be lantibiotics, defined as bacterial ribosomally synthesised lanthionine-containing peptides with antimicrobial activity. The goal of this study was to produce and characterize a new mutacin from S. mutans strain 29B, as it shows a promising activity spectrum against current human pathogens. RESULTS: Mutacin H-29B, produced by S. mutans strain 29B, was purified by successive hydrophobic chromatography from a liquid preparation consisting of cheese whey permeate (6% w/v) supplemented with yeast extract (2%) and CaCO(3 )(1%). Edman degradation revealed 24 amino acids identical to those of mutacin II (also known as J-T8). The molecular mass of the purified peptide was evaluated at 3246.08 ± 0.1 Da by MALDI-TOF MS. CONCLUSION: A simple procedure for production and purification of mutacins along with its characterization is presented. Our results show that the amino acid sequence of mutacin H-29B is identical to the already known mutacin II (J-T8) over the first 24 residues. S. mutans strains of widely different origins may thus produce very similar bacteriocins

Optimization of preservation methods allows deeper insights into changes of raw milk microbiota

The temporal instability of raw milk microbiota drastically affects the reliability of microbiome studies. However, little is known about the microbial integrity in preserved samples. Raw cow milk samples were preserved with azidiol or bronopol and stored at 4 ◦C, or with dimethyl sulfoxide (DMSO) or a mixture of azidiol and DMSO and stored at −20 ◦C for up to 30 days. Aliquots of 5-, 10-, and 30-day post-storage were treated with propidium monoazide (PMA), then analyzed by sequencing the 16S rRNA gene V3-V4 and V6-V8 regions. The V6-V8 gave a higher richness and lower diversity than the V3-V4 region. After 5-day storage at 4 ◦C, the microbiota of unpreserved samples was characterized by a drastic decrease in diversity, and a significant shift in community structure. The treatment with azidiol and DMSO conferred the best community stabilization in preserved raw milk. Interestingly, the azidiol treatment performed as well for up to 10 days, thus appearing as a suitable alternative. However, neither azidiol nor bronopol could minimize fungal proliferation as revealed by PMA-qPCR assays. This study demonstrates the preservative ability of a mixture of azidiol and DMSO and provides deeper insights into the microbial changes occurring during the cold storage of preserved raw milk

Prevalence and abundance of lactic acid bacteria in raw milk associated with forage types in dairy cow feeding

Lactic acid bacteria (LAB) found in milk can be responsible for organoleptic defects in cheese. In order to identify the source of LAB thatcouldpotentiallydevelop during cheesemaking, we evaluated theirprevalence and abundanceinmilk according to thetype of forage usedin dairy cow feeding. Foragesand bulk tank milk were sampled three times on 24farms using either hay alone (control), or grass or legume silagesupplemented or not with corn silage. Both types of silageswere either noninoculated, orinoculated with commercial preparations containing at least a Lactobacillus buchneristrainalong with Lactobacillus casei, Lactobacillus plantarum, Enterococcus faecium,or Pediococcus pentosaceus. Our resultsindicate that LAB viable counts in milksamples (2.56log cfu/mL) did not differaccording to the type of forage used. A total of 1239 LAB were isolated and identified by partial 16S rRNA gene sequencing. Although inoculation increased lactobacilli abundance in grass silage by 35%, we did not observe an effect on the LAB profile of milk. Indeed, there was no significant difference in milk LAB prevalence and abundanceaccording tothe type of forage(P >0.05). Moreover, isolates belonging to the L.buchnerigroup wererarely found in bulk tank milk (3/481isolates). Random amplified polymorphic DNA typing of 406LAB isolates revealed theplausibletransfer of some strains from silage to milk (~6%). Thus, forage is only a minor contributor to LAB contamination of milk

Bioaccessible Antioxidants in Milk Fermented by Bifidobacterium longum subsp. longum Strains

Bifidobacterium longum subsp. longum is among the dominant species of the human gastrointestinal microbiota and could thus have potential as probiotics. New targets such as antioxidant properties have interest for beneficial effects on health. The objective of this study was to evaluate the bioaccessibility of antioxidants in milk fermented by selected B. longum subsp. longum strains during in vitro dynamic digestion. The antioxidant capacity of cell extracts from 38 strains, of which 32 belong to B. longum subsp. longum, was evaluated with the ORAC (oxygen radical absorbance capacity) method. On the basis of screening and gene sequence typing by multilocus locus sequence analysis (MLSA), five strains were chosen for fermenting reconstituted skim milk. Antioxidant capacity varied among the strains tested ( = 0.0009). Two strains of B. longum subsp. longum (CUETM 172 and 171) showed significantly higher ORAC values than the other bifidobacteria strains. However, there does not appear to be a relationship between gene sequence types and antioxidant capacity. The milk fermented by each of the five strains selected (CUETM 268, 172, 245, 247, did not have higher initial ORAC values compared to the nonfermented milk samples. However, higher bioaccessibility of antioxidants in fermented milk (175-358%) was observed during digestion

Enhanced Exopolysaccharide Production by Lactobacillus rhamnosus in Co-Culture with Saccharomyces cerevisiae

Lactobacillus strains are known to produce exopolysaccharides (EPS) with recognized health benefits (i.e. prebiotic and immunomodulation) but production is limited by low yields. Co-culture has been shown to improve metabolite productivity, particularly bacteriocins and EPS. Although lactic acid bacteria (LAB) and yeasts are found in several fermented products, the molecular mechanisms linked to the microbial interactions and their influences on EPS biosynthesis are unclear. The aim of the present study was to investigate the effect of co-culture on EPS production by three Lactobacillus rhamnosus strains (ATCC 9595, R0011, and RW-9595M) in association with Saccharomyces cerevisiae. Fermentation, in both mono and co-culture, was carried out and the expression of key LAB genes was monitored. After 48 h, results revealed that EPS production was enhanced by 39%, 49%, and 42% in co-culture for R0011, ATCC 9595, and RW-9595M, respectively. Each strain showed distinctive gene expression profiles. For a higher EPS production, higher EPS operon expression levels were observed for RW-9595M in co-culture. The construction of gene co-expression networks revealed common correlations between the expression of genes related to the EPS operons, sugar metabolism, and stress during EPS production and microbial growth for the three strains. Our findings provide insight into the positive influence of inter-kingdom interactions in stimulating EPS biosynthesis, representing progress toward the development of a bio-ingredient with broad industrial applications

Invited review: Starter lactic acid bacteria survival in cheese: new perspectives on cheese microbiology

The importance of starter cultures to cheese manufacture and ripening is well known. Starters are inoculated into cheese milk at a level of ~106 cfu/mL either from a bulk culture or using commercial direct-to-vat cultures. Before ripening, starters grow in the milk to reach populations of 107 to 109 cfu/g of curd depending on processing variables such as cook temperature, inclusion of washing steps, degree of partitioning with curds and whey, and importantly salt addition rate. Inherent strain-related properties also determine final populations in the curd following manufacture and include temperature sensitivity, salt sensitivity, presence of prophage, autolytic and permeabilization properties (which are influenced by processing steps), presence and type of cell envelope proteinase, and metabolic activity. Ripening of important industrial cheese varieties such as Cheddar, Dutch, Swiss, and Italian-type cheese varieties is characterized by extended storage under temperature-controlled conditions enabling characteristic flavor and texture development to occur. Over ripening, microbiological, biochemical and enzymatic changes occur with a decline in starter viability, release of intracellular enzymes, hydrolysis of proteins, carbohydrates and lipids, and formation of a range of volatile and nonvolatile flavor components. Recent reports suggest that starter strains may be present during the later stages of ripening and therefore their potential role needs to be reconsidered. This review will focus on our current understanding of starter viability and vitality during cheese ripening and will also review the area of starter permeabilization, autolysis, and enzyme release