Identification of genes required for glucan exopolysaccharide production in Lactobacillus johnsonii suggests a novel mechanism of biosynthesis

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 Δ eps_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. johnsonii IMPORTANCE 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

Processing and Structure of the Lantibiotic Peptide Nso From the Human Gut Bacterium Blautia obeum A2-162 analysed by Mass Spectrometry

A previously reported gene cluster encoding four nisin-like peptides, three with the same sequence (NsoA1-3) and the unique NsoA4, produced antimicrobial activity in the presence of trypsin after heterologous expression in Lactococcus lactis. Protein extracts were separated by SDS gel electrophoresis or immunoprecipitation using an antibody to the NsoA2 leader. Tryptic peptides observed by LC-MS/MS covered the complete sequence of preNsoA1-3 and part of the leader sequence of preNsoA4 and confirmed the expression and the predicted sequences of the preNsoA peptides. Further, the data revealed that the preNsoA1-3 peptides were partly modified with dehydrations and formation of lanthionine rings. A certain amount of fully modified preNsoA1-3 was observed. Details of modifications of the core peptide and the C-terminal tryptic peptide TATCGCHITGK covering rings D and E indicated that 22% of these preNsoA1-3 peptides were completely modified. A lower amount of ring formation is estimated for rings A-C. Intact masses of immunoprecipitation-derived peptides determined by LC-MS accurately matched the expected preNsoA precursor peptides. The most abundant peptides detected were preNsoA2-3-8H2O followed by preNsoA1-8H2O and other states of dehydration. The results confirm incomplete processing of preNsoA peptides in the heterologous system, with the formation of a certain amount of fully modified peptides

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 Ochrobactrum haematophilum FI11154, Isolated from Kunu-Zaki, a Nigerian Millet-Based Fermented Food

Ochrobactrum haematophilum FI11154 was isolated from kunu-zaki, a Nigerian traditional fermented millet-based food. Here, we present the first complete genome sequence of this species. The genome consists of five replicons and contains genes related to iron uptake and phosphatase activities

Gut microbiota as a source of novel antimicrobials

peer reviewedBacteria, Archaea, Eukarya and viruses coexist in the human gut, and this coexistence is functionally balanced by symbiotic or antagonistic relationships. Antagonism is often characterized by the production of antimicrobials against other organisms occupying the same environmental niche. Indeed, close co-evolution in the gut has led to the development of specialized antimicrobials, which is attracting increased attention as these may serve as novel alternatives to antibiotics and thereby help to address the global problem of antimicrobial resistance. The gastrointestinal (GI) tract is especially suitable for finding novel antimicrobials due to the vast array of microbes that inhabit it, and a considerable number of antimicrobial producers of both wide and narrow spectrum have been described. In this review, we summarize some of the antimicrobial compounds that are produced by bacteria isolated from the gut environment, with a special focus on bacteriocins. We also evaluate the potential therapeutic application of these compounds to maintain homeostasis in the gut and the biocontrol of pathogenic bacteria.Teagas

Discovery of a novel lantibiotic nisin O from Blautia obeum A2-162, isolated from the human 2 gastrointestinal tract

The Institute of Food Research is funded by the BBSRC (strategic core grants IFR/08/1 BB/J004529/1); SHD and HJF acknowledge support from the Scottish Government Food Land and People programme. DH and CGF received BBSRC PhD studentship grants. Acknowledgements We are grateful to Kathryn Cross for the EM analysis and Neil Rigby for helpful advice.Peer reviewedPostprin

Identification of a Phosphorylation Site for Calcium/Calmodulindependent Protein Kinase II in the NR2B Subunit of the N-Methyl-D-aspartate Receptor

The N-methyl-D-aspartate (NMDA) subtype of excitatory glutamate receptors plays critical roles in embryonic and adult synaptic plasticity in the central nervous system. The receptor is a heteromultimer of core subunits, NR1, and one or more regulatory subunits, NR2A-D. Protein phosphorylation can regulate NMDA receptor function (Lieberman, D. N., and Mody, I. (1994) Nature 369, 235-239; Wang, Y. T., and Salter, M. W. (1994) Nature 369, 233-235; Wang, L.-Y., Orser, B. A., Brautigan, D. L., and MacDonald, J. F. (1994) Nature 369, 230-232). Here we identify a major phosphorylation site on subunit NR2B that is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), an abundant protein kinase located at postsynaptic sites in glutamatergic synapses. For the initial identification of the site, we constructed a recombinant fusion protein containing 334 amino acids of the C terminus of the NR2B subunit and phosphorylated it with CaM kinase II in vitro. By peptide mapping, automated sequencing, and mass spectrometry, we identified the major site of phosphorylation on the fusion protein as Ser-383, corresponding to Ser-1303 of full-length NR2B. The Km for phosphorylation of this site in the fusion protein was ~50 nM, much lower than that of other known substrates for CaM kinase II, suggesting that the receptor is a high affinity substrate. We show that serine 1303 in the full-length NR2B and/or the cognate site in NR2A is a major site of phosphorylation of the receptor both in the postsynaptic density fraction and in living hippocampal neurons

First evidence of production of the lantibiotic nisin P

peer reviewedNisin P is a natural nisin variant, the genetic determinants for which were previously identified in the genomes of two Streptococcus species, albeit with no confirmed evidence of production. Here we describe Streptococcus agalactiae DPC7040, a human faecal isolate, which exhibits antimicrobial activity against a panel of gut and food isolates by virtue of producing nisin P. Nisin P was purified, and its predicted structure was confirmed by nanoLC-MS/MS, with both the fully modified peptide and a variant without rings B and E being identified. Additionally, we compared its spectrum of inhibition and minimum inhibitory concentration (MIC) with that of nisin A and its antimicrobial effect in a faecal fermentation in comparison with nisin A and H. We found that its antimicrobial activity was less potent than nisin A and H, and we propose a link between this reduced activity and the peptide structure

Lactobacillus plantarum-mediated regulation of dietary aluminum induces changes in the human gut microbiota: An in vitro colonic fermentation study

The gut microbiota has been identified as a target of toxic metals and a potentially crucial mediator of the bioavailability and toxicity of these metals. In this study, we show that aluminum (Al) exposure, even at low dose, affected the growth of representative strains from the human intestine via pure culture experiments. In vitro, Lactobacillus plantarum CCFM639 could bind Al on its cell surface as shown by electron microscopy and energy dispersive X-ray analysis. The potential of L. plantarum CCFM639 to reverse changes in human intestine microbiota induced by low-dose dietary Al exposure was investigated using an in vitro colonic fermentation model. Batch fermenters were inoculated with fresh stool samples from healthy adult donors and supplemented with 86 mg/L Al and/or 109 CFU of L. plantarum CCFM639. Al exposure significantly increased the relative abundances of Bacteroidetes (Prevotella), Proteobacteria (Escherichia), Actinobacteria (Collinsella), Euryarchaeota (Methanobrevibacter), and Verrucomicrobiaceae and decreased Firmicutes (Streptococcus, Roseburia, Ruminococcus, Dialister, Coprobacillus). Some changes were reversed by the inclusion of L. plantarum CCFM639. Alterations in gut microbiota induced by Al and L. plantarum CCFM639 inevitably led to changes in metabolite levels. The short-chain fatty acid (SCFAs) contents were reduced after Al exposure, but L. plantarum CCFM639 could elevate their levels. SCFAs had positive correlations with beneficial bacteria, such as Dialister, Streptococcus, Roseburia, and negative correlations with Erwinia, Escherichia, and Serratia. Therefore, dietary Al exposure altered the composition and structure of the human gut microbiota, and this was partially mitigated by L. plantarum CCFM639. This probiotic supplementation is potentially a promising and safe approach to alleviate the harmful effects of dietary Al exposure

Probiotic and Functional Properties of Limosilactobacillus reuteri INIA P572

Limosilactobacillus reuteri INIA P572 is a strain able to produce the antimicrobial compound reuterin in dairy products, exhibiting a protective effect against some food-borne pathogens. In this study, we investigated some probiotic properties of this strain such as resistance to gastrointestinal passage or to colonic conditions, reuterin production in a colonic environment, and immunomodulatory activity, using different in vitro and in vivo models. The results showed a high resistance of this strain to gastrointestinal conditions, as well as capacity to grow and produce reuterin in a human colonic model. Although the in vitro assays using the RAW 264.7 macrophage cell line did not demonstrate direct immunomodulatory properties, the in vivo assays using a Dextran Sulphate Sodium (DSS)-induced colitic mice model showed clear immunomodulatory and protective effects of this strain.This work was supported by project no. RTA2017-00002-00-00 from the Spanish Ministry of Science and Innovation, by the Junta de Andalucía (CTS 164) and Instituto de Salud Carlos III (PI19/01058) with funds from the European Union.Ye