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Applied and Environmental Microbiology Jul 2019Recent papers have reported dipeptides containing d-amino acids to have novel effects that cannot be observed with ll-dipeptides, and such dipeptides are expected to be...
Recent papers have reported dipeptides containing d-amino acids to have novel effects that cannot be observed with ll-dipeptides, and such dipeptides are expected to be novel functional compounds for pharmaceuticals and food additives. Although the functions of d-amino acid-containing dipeptides are gaining more attention, there are few reports on the synthetic enzymes that can accept d-amino acids as substrates, and synthetic methods for d-amino acid-containing dipeptides have not yet been constructed. Previously, we developed a chemoenzymatic system for amide synthesis that comprised enzymatic activation and a subsequent nucleophilic substitution reaction. In this study, we demonstrated the application of the system for d-amino acid-containing-dipeptide synthesis. We chose six adenylation domains as targets according to our newly constructed hypothesis, i.e., an adenylation domain located upstream from the epimerization domain may activate d-amino acid as well as l-amino acid. We successfully synthesized over 40 kinds of d-amino acid-containing dipeptides, including ld-, dl-, and dd-dipeptides, using only two adenylation domains, TycA-A from tyrocidine synthetase and BacB2-A from bacitracin synthetase. Furthermore, this study offered the possibility that the epimerization domain could be a clue to the activity of the adenylation domains toward d-amino acid. This paper provides additional information regarding d-amino acid-containing-dipeptide synthesis through the combination of enzymatic adenylation and chemical nucleophilic reaction, and this system will be a useful tool for dipeptide synthesis. Because almost all amino acids in nature are l-amino acids, the functioning of d-amino acids has received little attention. Thus, there is little information available on the activity of enzymes toward d-amino acids or synthetic methods for d-amino acid-containing dipeptides. Recently, d-amino acids and d-amino acid-containing peptides have attracted attention as novel functional compounds, and d-amino acid-activating enzymes and synthetic methods are required for the development of the d-amino acid-containing-peptide industry. This study provides additional knowledge regarding d-amino acid-activating enzymes and proposes a unique synthetic method for d-amino acid-containing peptides, including ld-, dl-, and dd-dipeptides.
Topics: Amino Acids; Bacillus licheniformis; Bacterial Proteins; Biocatalysis; Dipeptides; Paenibacillus; Peptide Synthases; Protein Domains; Substrate Specificity
PubMed: 31003981
DOI: 10.1128/AEM.00120-19 -
Probiotics and Antimicrobial Proteins Mar 2020Paenibacillus alvei NP75, a Gram-positive bacterium, produces two different antimicrobial peptides, paenibacillin N and P, which has potent antimicrobial activity...
Paenibacillus alvei NP75, a Gram-positive bacterium, produces two different antimicrobial peptides, paenibacillin N and P, which has potent antimicrobial activity against many clinical pathogens. The synthesis pattern of these antimicrobial peptides by P. alvei NP75 was studied extensively. The results were outstanding in a way that the paenibacillin N was synthesized irrespective of the growth of bacteria (non-ribosomal mediated), whereas paenibacillin P production was carried out by ribosomal mediated. In addition to the antimicrobial peptides, P. alvei NP75 also produces an immunogenic extracellular protease to defend itself from its own antimicrobial peptide, paenibacillin P. Furthermore, this immunogenic protease production was impaired by the addition of protease inhibitor, phenylmethylsulfonyl fluoride (PMSF). The sodium dodecyl sulfate (SDS) treated strain (mutant) failed to produce paenibacillin P, whereas the production of neither paenibacillin N nor the protease was affected by the plasmid curing. The plasmid curing studies that divulge the genes responsible for the synthesis of paenibacillin N and protease were found to be genome encoded, and paenibacillin P was plasmid encoded. We are reporting, first of its kind, the co-production of two different antimicrobial peptides from P. alvei NP75 through non-ribosomal and ribosomal pathways that could be used as effective antibiotics.
Topics: Anti-Infective Agents; Antimicrobial Cationic Peptides; Paenibacillus; Plasmids; Protein Biosynthesis; Ribosomes
PubMed: 31001787
DOI: 10.1007/s12602-019-09541-w -
Nature Communications Aug 2018Self-assembling protein surface (S-) layers are common cell envelope structures of prokaryotes and have critical roles from structural maintenance to virulence. S-layers...
Self-assembling protein surface (S-) layers are common cell envelope structures of prokaryotes and have critical roles from structural maintenance to virulence. S-layers of Gram-positive bacteria are often attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Here we present an in-depth characterization of this interaction, with co-crystal structures of the three consecutive SLH domains from the Paenibacillus alvei S-layer protein SpaA with defined SCWP ligands. The most highly conserved SLH domain residue SLH-Gly29 is shown to enable a peptide backbone flip essential for SCWP binding in both biophysical and cellular experiments. Furthermore, we find that a significant domain movement mediates binding by two different sites in the SLH domain trimer, which may allow anchoring readjustment to relieve S-layer strain caused by cell growth and division.
Topics: Amino Acid Motifs; Bacillus anthracis; Cell Proliferation; Cell Wall; Circular Dichroism; Crystallization; Ligands; Mutagenesis; Mutagenesis, Site-Directed; Paenibacillus; Peptidoglycan; Protein Binding; Protein Domains; Recombinant Proteins
PubMed: 30087354
DOI: 10.1038/s41467-018-05471-3 -
Scientific Reports Aug 2018Although Bacillus cereus is of particular concern in food safety and public health, the role of other Bacillus species was overlooked. Therefore, we investigated the...
Poultry and beef meat as potential seedbeds for antimicrobial resistant enterotoxigenic Bacillus species: a materializing epidemiological and potential severe health hazard.
Although Bacillus cereus is of particular concern in food safety and public health, the role of other Bacillus species was overlooked. Therefore, we investigated the presence of eight enterotoxigenic genes, a hemolytic gene and phenotypic antibiotic resistance profiles of Bacillus species in retail meat samples. From 255 samples, 124 Bacillus isolates were recovered, 27 belonged to B. cereus and 97 were non-B. cereus species. Interestingly, the non-B. cereus isolates carried the virulence genes and exhibited phenotypic virulence characteristics as the B. cereus. However, correlation matrix analysis revealed the B. cereus group positively correlates with the presence of the genes hblA, hblC, and plc, and the detection of hemolysis (p < 0.05), while the other Bacillus sp. groups are negatively correlated. Tests for antimicrobial resistance against ten antibiotics revealed extensive drug and multi-drug resistant isolates. Statistical analyses didn't support a correlation of antibiotic resistance to tested virulence factors suggesting independence of these phenotypic markers and virulence genes. Of special interest was the isolation of Paenibacillus alvei and Geobacillus stearothermophilus from the imported meat samples being the first recorded. The isolation of non-B. cereus species carrying enterotoxigenic genes in meat within Egypt, suggests their impact on food safety and public health and should therefore not be minimised, posing an area that requires further research.
Topics: Bacillus cereus; Bacterial Proteins; Drug Resistance, Bacterial; Food Microbiology; Meat; Paenibacillus; Poultry Products; Virulence Factors
PubMed: 30072706
DOI: 10.1038/s41598-018-29932-3 -
Frontiers in Microbiology 2018Various mechanisms of protein cell surface display have evolved during bacterial evolution. Several Gram-positive bacteria employ S-layer homology (SLH) domain-mediated...
Various mechanisms of protein cell surface display have evolved during bacterial evolution. Several Gram-positive bacteria employ S-layer homology (SLH) domain-mediated sorting of cell-surface proteins and concomitantly engage a pyruvylated secondary cell-wall polymer as a cell-wall ligand. Specifically, pyruvate ketal linked to β-D-ManNAc is regarded as an indispensable epitope in this cell-surface display mechanism. That secondary cell wall polymer (SCWP) pyruvylation and SLH domain-containing proteins are functionally coupled is supported by the presence of an ortholog of the predicted pyruvyltransferase CsaB in bacterial genomes, such as those of and . The SCWP, consisting of pyruvylated disaccharide repeats [→4)-β-D-GlcNAc-(1→3)-4,6-Pyr-β-D-ManNAc-(1→] serves as a model to investigate the widely unexplored pyruvylation reaction. Here, we reconstituted the underlying enzymatic pathway in combination with synthesized compounds, used mass spectrometry, and nuclear magnetic resonance spectroscopy for product characterization, and found that CsaB-catalyzed pyruvylation of β-D-ManNAc occurs at the stage of the lipid-linked repeat. We produced the TagA (PAV_RS07420) and CsaB (PAV_RS07425) enzymes as recombinant, tagged proteins, and using a synthetic 11-phenoxyundecyl-diphosphoryl-α-GlcNAc acceptor, we uncovered that TagA is an inverting UDP-α-D-ManNAc:GlcNAc-lipid carrier transferase, and that CsaB is a pyruvyltransferase, with synthetic UDP-α-D-ManNAc and phosphoenolpyruvate serving as donor substrates. Next, to substitute for the UDP-α-D-ManNAc substrate, the recombinant UDP-GlcNAc-2-epimerase MnaA (PAV_RS07610) of was included in this reconstitution system. When all three enzymes, their substrates and the lipid-linked GlcNAc primer were combined in a one-pot reaction, a lipid-linked SCWP repeat precursor analog was obtained. This work highlights the biochemical basis of SCWP biosynthesis and bacterial pyruvyl transfer.
PubMed: 29997588
DOI: 10.3389/fmicb.2018.01356 -
MicrobiologyOpen Mar 2019European foulbrood is a globally distributed brood disease affecting honey bees. It may lead to lethal infections of larvae and, in severe cases, even to colony...
European foulbrood is a globally distributed brood disease affecting honey bees. It may lead to lethal infections of larvae and, in severe cases, even to colony collapse. Lately, a profound genetic and phenotypic diversity was documented for the causative agent Melissococcus plutonius. However, experimental work on the impact of diverse M. plutonius strains on hosts with different genetic background is completely lacking and the role of secondary invaders is poorly understood. Here, we address these issues and elucidate the impact and interaction of both host and pathogen on one another. Moreover, we try to unravel the role of secondary bacterial invasions in foulbrood-diseased larvae. We employed in vitro infections with honey bee larvae from queens with different genetic background and three different M. plutonius strains. Larvae infection experiments showed host-dependent survival dynamics although M. plutonius strain 49.3 consistently had the highest virulence. This pattern was also reflected in significantly reduced weights of 49.3 strain-infected larvae compared to the other treatments. No difference was found in groups additionally inoculated with a secondary invader (Enterococcus faecalis or Paenibacillus alvei) neither in terms of larval survival nor weight. These results suggest that host background contributes markedly to the course of the disease but virulence is mainly dependent on pathogen genotype. Secondary invaders following a M. plutonius infection do not increase disease lethality and therefore may just be a colonization of weakened and immunodeficient, or dead larvae.
Topics: Animals; Bees; Enterococcaceae; Gram-Positive Bacterial Infections; Host-Pathogen Interactions; Larva; Paenibacillus; Survival Analysis
PubMed: 29799173
DOI: 10.1002/mbo3.649 -
The Journal of Biological Chemistry Jul 2018Pro-Pro endopeptidases (PPEPs) belong to a recently discovered family of proteases capable of hydrolyzing a Pro-Pro bond. The first member from the bacterial pathogen...
Pro-Pro endopeptidases (PPEPs) belong to a recently discovered family of proteases capable of hydrolyzing a Pro-Pro bond. The first member from the bacterial pathogen (PPEP-1) cleaves two cell-surface proteins involved in adhesion, one of which is encoded by the gene adjacent to the gene. However, related PPEPs may exist in other bacteria and may shed light on substrate specificity in this enzyme family. Here, we report on the homolog of PPEP-1 in , which we denoted PPEP-2. We found that PPEP-2 is a secreted metalloprotease, which likewise cleaved a cell-surface protein encoded by an adjacent gene. However, the cleavage motif of PPEP-2, PLP↓PVP, is distinct from that of PPEP-1 (VNP↓PVP). As a result, an optimal substrate peptide for PPEP-2 was not cleaved by PPEP-1 and vice versa. To gain insight into the specificity mechanism of PPEP-2, we determined its crystal structure at 1.75 Å resolution and further confirmed the structure in solution using small-angle X-ray scattering (SAXS). We show that a four-amino-acid loop, which is distinct in PPEP-1 and -2 (GGST in PPEP-1 and SERV in PPEP-2), plays a crucial role in substrate specificity. A PPEP-2 variant, in which the four loop residues had been swapped for those from PPEP-1, displayed a shift in substrate specificity toward PPEP-1 substrates. Our results provide detailed insights into the PPEP-2 structure and the structural determinants of substrate specificity in this new family of PPEP proteases.
Topics: Amino Acid Sequence; Bacterial Proteins; Crystallography, X-Ray; Dipeptides; Endopeptidases; Models, Molecular; Paenibacillus; Protein Conformation; Sequence Homology; Substrate Specificity
PubMed: 29794027
DOI: 10.1074/jbc.RA118.003244 -
The Journal of Antibiotics Sep 2018Four peptide antibiotics, named paenialvin A-D, were isolated from Paenibacillus alvei DSM 29. Mass spectrum analysis determined the molecular masses of paenialvin A-D...
Four peptide antibiotics, named paenialvin A-D, were isolated from Paenibacillus alvei DSM 29. Mass spectrum analysis determined the molecular masses of paenialvin A-D to be 1891, 1875, 1877, and 1923 Da, respectively. Tandem mass spectra and nuclear magnetic resonance (NMR) were used to elucidate their chemical structures. Paenialvin A-D showed antimicrobial activity against most strains that were tested, including methicillin-resistant Staphalococcus aureus, Staphylococcus aureus, Bacillus subtilis, Loktanella hongkongensis, Escherichia coli, and Pseudomonas aeruginosa. In particular, the minimum inhibitory concentration of paenialvins against Staphalococcus aureus reached 0.8-3.2 μg/mL. Although they were cytotoxic against HeLa cells at a concentration of 50 μg/mL, the lack of hemolysis by paenialvins confirmed that they are potential candidates for anti-tumor drugs.
Topics: Anti-Bacterial Agents; Antineoplastic Agents; Bacillus subtilis; Cell Line, Tumor; Drug Resistance, Multiple, Bacterial; Escherichia coli; HeLa Cells; Humans; Mass Spectrometry; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Nuclear Magnetic Resonance, Biomolecular; Paenibacillus; Pseudomonas aeruginosa; Rhodobacteraceae
PubMed: 29760411
DOI: 10.1038/s41429-017-0001-3 -
Biometals : An International Journal on... Dec 2017Bee disease caused by spore-forming Paenibacillus larvae and Paenibacillus alvei is a serious problem for honey production. Thus, there is an ongoing effort to find an...
Bee disease caused by spore-forming Paenibacillus larvae and Paenibacillus alvei is a serious problem for honey production. Thus, there is an ongoing effort to find an effective agent that shows broad biocidal activity with minimal environmental hazard. In this study, the biocidal effect of maltose reduced silver nanoparticles (AgNPs) is evaluated against American foulbrood and European foulbrood pathogens. The results demonstrate that the maltose reduced AgNPs are excellent short and long-term biocides against P. larvae isolates. The long-term effect suggests that the Ag ions are released from the AgNPs with increasing time in a controlled manner.
Topics: Animals; Bacillus; Bees; Disinfectants; Dynamic Light Scattering; Green Chemistry Technology; Larva; Maltose; Metal Nanoparticles; Microbial Sensitivity Tests; Micrococcus; Microscopy, Electron, Transmission; Paenibacillus; Silver; Spectroscopy, Fourier Transform Infrared
PubMed: 28986750
DOI: 10.1007/s10534-017-0055-5 -
Genomics Data Dec 2017Here we report the draft genome sequence of an endophytic strain isolated from the Universiti Kebangsaan Malaysia reserve forest, Malaysia. The genome size was...
Here we report the draft genome sequence of an endophytic strain isolated from the Universiti Kebangsaan Malaysia reserve forest, Malaysia. The genome size was approximately 8.04 Mb, and the assembly consisted of 107 scaffolds with 168 contigs, and had a G + C content of 53%. Phylogenetic analysis of strain SUK123 using the 16S rRNA gene revealed that it belonged to the family with the highest similarity to SD (99%). Whole genome comparison of SUK123 with related species using average nucleotide identity (ANI) analysis revealed a similarity of 98% to Mst1, 94% to B69, 91% to A2, 68% to SC2 and 69% to DMS29. The draft genome was deposited at the European Nucleotide Archive (PRJEB21373).
PubMed: 28856101
DOI: 10.1016/j.gdata.2017.08.005