Analysis of non-polar deletion mutations in the genes of the spo0K (opp) operon of Bacillus subtilis

The spo0K (opp) operon of Bacillus subtilis encodes an oligopeptide permease that is required for uptake of oligopeptides, development of genetic competence, and initiation of sporulation. We made in-frame, non-polar deletion mutations in each of the first four genes of the five-gene spo0K operon and tested effects on oligopeptide transport, sporulation, and expression of competence genes. spo0KA, B, C, and D were required for sporulation, competence development, and oligopeptide transport. Disruption of spo0KE caused a less severe phenotype than did disruption of any of the other genes of the operon.     

Identification and Functional Characterization of a Xenorhabdus nematophila Oligopeptide Permease

The bacterium Xenorhabdus nematophila is a mutualist of Steinernema carpocapsae nematodes and a pathogen of insects. Presently, it is not known what nutrients the bacterium uses to thrive in these host environments. In other symbiotic bacteria, oligopeptide permeases have been shown to be important in host interactions, and we therefore sought to determine if oligopeptide uptake is essential for growth or symbiotic functions of X. nematophila in laboratory or host environments. We identified an X. nematophila oligopeptide permease (opp) operon of two sequential oppA genes, predicted to encode oligopeptide-binding proteins, and putative permease-encoding genes oppB, oppC, oppD, and oppF. Peptide-feeding studies indicated that this opp operon encodes a functional oligopeptide permease. We constructed strains with mutations in oppA₁, oppA₂, or oppB and examined the ability of each mutant strain to grow in a peptide-rich laboratory medium and to interact with the two hosts. We found that the opp mutant strains had altered growth phenotypes in the laboratory medium and in hemolymph isolated from larval insects. However, the opp mutant strains were capable of initiating and maintaining both mutualistic and pathogenic host interactions. These data demonstrate that the opp genes allow X. nematophila to utilize peptides as a nutrient source but that this function is not essential for the existence of X. nematophila in either of its host niches. To our knowledge, this study represents the first experimental analysis of the role of oligopeptide transport in mediating a mutualistic invertebrate-bacterium interaction.     

Targeted delivery of siRNA to differentiated murine myotubes in culture by a conjugate of cationic oligopeptide with FS2 venom

A conjugate of the ligand of FS2 venom dihydropyridine receptors with a cationic arginine-containing oligopeptide was synthesized. It was found that the conjugate provides siRNA delivery to murine myotubes differentiated in vitro. The effect of RNA interference with the use of siRNA complexes with the conjugate was observed when siRNA concentrations were an order of magnitude lower than those used in the case of siRNA complexes with a non-conjugated oligopeptide.     

Non-competitive inhibitioof ornithine decarboxylase by a phosphopeptide and phosphoamino acids

In Tetrahymena pyriformis the cytosolic ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity is considerably inhibited by the presence of polyamines in the growth medium, while the nuclear ornithine decarboxylase is only slightly affected. Experimental evidence suggests that the presence of putrescine and/or spermidine elicits the appearance of non-competitive inhibitors of ornithine decarboxylase. One pf the inhibitors has a molecular weight of 25 000 and properties of antizume. In addition, two other low molecular weight inhibitors are extracted, one which is a phosphoserine oligopeptide, and other which is phosphotyrosine. All inhibit non-competitively the homologous and heterologous (Escherichia coli and rat liver) ornithine decarboxylases. Similarly, non-competitive inhibition was obtained when the commercially available phosphoamino acids wre tested against the already mentioned ornithine decarboxylases.     

Utilization of Oligopeptides by Listeria monocytogenes Scott A

For effective utilization of peptides, Listeria monocytogenes possesses two different peptide transport systems. The first one is the previously described proton motive force (PMF)-driven di- and tripeptide transport system (A. Verheul, A. Hagting, M.-R. Amezaga, I. R. Booth, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 61:226–233, 1995). The present results reveal that L. monocytogenes possesses an oligopeptide transport system, presumably requiring ATP rather than the PMF as the driving force for translocation. Experiments to determine growth in a defined medium containing peptides of various lengths suggested that the oligopeptide permease transports peptides of up to 8 amino acid residues. Peptidase activities towards several oligopeptides were demonstrated in cell extract from L. monocytogenes, which indicates that upon internalization, the oligopeptides are hydrolyzed to serve as sources of amino acids for growth. The peptide transporters of the nonproteolytic L. monocytogenes might play an important role in foods that harbor indigenous proteinases and/or proteolytic microorganisms, since Pseudomonas fragi as well as Bacillus cereus was found to enhance the growth of L. monocytogenes to a large extent in a medium in which the milk protein casein was the sole source of nitrogen. In addition, growth stimulation was elicited in this medium when casein was hydrolyzed by using purified protease from Bacillus licheniformis. The possible contribution of the oligopeptide transport system in the establishment of high numbers of L. monocytogenes cells in fermented milk products is discussed.     

Amino acid sequencing by gas chromatography-mass spectrometry using trifluoro-dideuteroalkylated peptide derivatives: C. The primary structure of the carboxypeptidase inhibitor from potatoes

The carboxypeptidase inhibitor from potatoes has been used to demonstrate the utility of gas chromatography-mass spectrometry for the determination of the primary structure of such large polypeptides. Two mixtures of oligopeptide fragments, obtained by limited acid hydrolysis and enzymatic digestion of this polypeptide, were transformed into the corresponding mixtures of O-trimethyl-silylated trifluoro-dideuteroethyl polyamino alcohols which were then analyzed by gas chromatography-mass spectrometry. The resulting mass spectral and retention index data allowed the identification of 61 oligopeptide fragments which were assembled by the computer by positioning all 39 amino acid residues in a unique sequence (with the exception of the assignment of the primary amide groups of Asn and Gln).     

霞水母胶原蛋白活性肽和寡肽胶原抗疲劳作用的实验研究

本文对霞水母胶原蛋白活性肽和寡肽胶原的抗疲劳作用进行了研究。试验中分别以25、50、100mg/kg剂量的霞水母胶原蛋白活性肽和寡肽胶原给小鼠连续灌胃30d,然后进行小鼠负重游泳试验、血清尿素氮、肝糖原及血乳酸含量测定。结果显示,两种受试物各剂量组小鼠的游泳时间明显长于对照组(P0.05或P0.01),各剂量组小鼠运动后血清尿素氮含量及乳酸曲线下面积均低于对照组(P0.01);各剂量组的小鼠游泳后肝糖原含量和空白对照组比较均有不同程度的提高。从而表明,霞水母胶原蛋白活性肽和寡肽胶原均具有抗疲劳作用。     

Specificity of Milk Peptide Utilization by Lactococcus lactis

To study the substrate specificity of the oligopeptide transport system of Lactococcus lactis for its natural substrates, the growth of L. lactis MG1363 was studied in a chemically defined medium containing milk peptides or a tryptic digest of α_s2-casein as the source of amino acids. Peptides were separated into acidic, neutral, and basic pools by solid-phase extraction or by cation-exchange liquid chromatography. Their ability to sustain growth and the time course of their utilization demonstrated the preferential use of hydrophobic basic peptides with molecular masses ranging between 600 and 1,100 Da by L. lactis MG1363 and the inability to use large, acidic peptides. These peptide utilization preferences reflect the substrate specificity of the oligopeptide transport system of the strain, since no significant cell lysis was inferred. Considering the free amino acid content of milk and these findings on peptide utilization, it was demonstrated that the cessation of growth of L. lactis MG1363 in milk was due to deprivation of leucine and methionine.     

Identification of a Vibrio furnissii oligopeptide permease and characterization of its in vitro hemolytic activity

We describe purification and characterization of an oligopeptide permease protein (Hly-OppA) from Vibrio furnissii that has multifaceted functions in solute binding, in in vitro hemolysis, in antibiotic resistance, and as a virulence factor in bacterial pathogenesis. The solute-binding function was revealed by N-terminal and internal peptide sequences of the purified protein and was confirmed by discernible effects on oligopeptide binding, by accumulation of fluorescent substrates, and by fluorescent substrate-antibiotic competition assay experiments. The purified protein exhibited host-specific in vitro hemolytic activity against various mammalian erythrocytes and apparent cytotoxicity in CHO-K1 cells. Recombinant Hly-OppA protein and an anti-Hly-OppA monoclonal antibody exhibited and neutralized the in vitro hemolytic activity, respectively, which further confirmed the hemolytic activity of the gene product. In addition, a V. furnissii hly-oppA knockout mutant caused less mortality than the wild-type strain when it was inoculated into BALB/c mice, indicating the virulence function of this protein. Finally, the in vitro hemolytic activity was also confirmed with homologous ATP-binding cassette-type transporter proteins from other Vibrio species.     

Specificity and regulation of peptide transport on Neurospora crassa.

The tripeptide, glycyl-d,l-leucyl-l-tyrosine was chemically synthesized in radioactive form and used to directly study the specificity, regulation, and properties of an oligopeptide transport system in Neurospora. Transport activity is sensitive to azide but does not result in the accumulation of the intact peptide; rather, the radioactive label is accumulated as free tyrosine. Inhibition studies suggest that the transport system probably has a relatively wide range of specificity and is responsible for uptake of short oligopeptides of quite distinct sequences. However, free amino acids and dipeptides are not transported significantly, if at all, by the oligopeptide transport system. A free amino group appears to be a requirement for peptide transport. A mutant strain that is unable to use various peptides for growth is further described and shown to be reduced greater than 90% in transport of the tripeptide.     

Purification of an Extracellular Signaling Molecule Involved in Production of Aerial Mycelium by Streptomyces coelicolor

We have extensively purified a factor from conditioned medium that restores aerial mycelium formation to a mutant of Streptomyces coelicolor that is defective in morphological differentiation. Response to this factor is shown to depend on the presence of the BldK oligopeptide import system. We suggest that this substance acts at the first step in a putative cascade of developmental regulatory signals.     

Relationship between Spontaneous Aminoglycoside Resistance in Escherichia coli and a Decrease in Oligopeptide Binding Protein

Changes in the amount of oligopeptide binding protein (OppA) in spontaneous kanamycin-resistant mutants of Escherichia coli were investigated. Among 20 colonies obtained from 10⁸ cells cultured in the presence of 20 μg of kanamycin/ml, 1 colony had no detectable OppA and 7 colonies were mutants with reduced amounts of OppA. Sensitivity of wild-type cells to kanamycin increased slightly by transformation of the oppA gene, but the sensitivity of the mutants increased greatly by the transformation. A mutant with no OppA was found to be a nonsense mutant of the oppA gene at amino acid position 166. In a mutant having a reduced level of OppA, the reduction was due to the decrease in OppA synthesis at the translational level. These mutants were also resistant to other aminoglycoside antibiotics, including streptomycin, neomycin, and isepamicin. Isepamicin uptake activities decreased greatly in these two kinds of mutants. The results support the proposition that aminoglycoside antibiotics are transported into cells by the oligopeptide transport system, and that transport is an important factor for spontaneous resistance to aminoglycoside antibiotics.     

Indirect selection for auxotrophic mutants of saccharomyces cerevisiae using the antibiotic netropsin

The small basic oligopeptide antibiotic, netropsin, can be successfully employed as an effective counterselecting agent in Saccharomyces cerevisiae. The use of the drug results in approximately a 35-fold enrichment of auxotrophic mutants in a mutagenized culture of yeast. The experimental procedure is quite simple and less time consuming than other presently used methods for indirect mutant selection in yeast.     

Integrating Terminal Truncation and Oligopeptide Fusion for a Novel Protein Engineering Strategy To Improve Specific Activity and Catalytic Efficiency: Alkaline α-Amylase as a Case Study

In this work, we integrated terminal truncation and N-terminal oligopeptide fusion as a novel protein engineering strategy to improve specific activity and catalytic efficiency of alkaline α-amylase (AmyK) from Alkalimonas amylolytica. First, the C terminus or N terminus of AmyK was partially truncated, yielding 12 truncated mutants, and then an oligopeptide (AEAEAKAKAEAEAKAK) was fused at the N terminus of the truncated AmyK, yielding another 12 truncation-fusion mutants. The specific activities of the truncation-fusion mutants AmyKΔC500-587::OP and AmyKΔC492-587::OP were 25.5- and 18.5-fold that of AmyK, respectively. The k_cat/K_m was increased from 1.0 × 10⁵ liters · mol⁻¹ · s⁻¹ for AmyK to 30.6 × and 23.2 × 10⁵ liters · mol⁻¹ · s⁻¹ for AmyKΔC500-587::OP and AmyKΔC492-587::OP, respectively. Comparative analysis of structure models indicated that the higher flexibility around the active site may be the main reason for the improved catalytic efficiency. The proposed terminal truncation and oligopeptide fusion strategy may be effective to engineer other enzymes to improve specific activity and catalytic efficiency.     

Staphylococcus aureus NorD,a Putative Efflux Pump Coregulated with the Opp1 Oligopeptide Permease,Contributes Selectively to Fitness In Vivo

Staphylococcus aureus readily infects humans, causing infections from mild superficial skin infections to lethal bacteremia and endocarditis. Transporters produced by S. aureus allow the pathogen to adapt to a variety of settings, including survival at sites of infection and in the presence of antibiotics. The native functions of many transporters are unknown, but their potential dual contribution to fitness and antimicrobial resistance highlights their importance in staphylococcal infections. Here, we show that S. aureus NorD, a newly recognized efflux pump of the major facilitator superfamily, contributes to fitness in a murine subcutaneous abscess model. In community-associated methicillin-resistant S. aureus (CA-MRSA) strain MW2, norD was selectively upregulated 36-fold at the infection site relative to growth in vitro, and the norD mutant demonstrated significant fitness impairment in abscesses, with fitness 20- to 40-fold lower than that of the parent MW2 strain. Plasmid-encoded NorD could complement the fitness defect of the MW2 norD mutant. Chromosomal norD expression is polycistronic with the upstream oligopeptide permease genes (opp1ABCDF), which encode an ABC oligopeptide transporter. Both norD and opp1 were upregulated in abscesses and iron-restricted culture medium and negatively regulated by Fur, but only NorD contributed to fitness in the murine abscess model.     

Spatial-temporal survey of Microcystis oligopeptide chemotypes in reservoirs with dissimilar waterbody features and their relation to genetic variation

The ability of cyanobacteria to produce toxins and other secondary metabolites is patchily distributed in natural populations, enabling the use of cellular oligopeptide compositions as markers to classify strains into ecologically-relevant chemotypical subpopulations. The composition and spatiotemporal distribution of Microcystis chemotypes within and among waterbodies was studied at different time scales by analyzing (i) Microcystis strains isolated between 1998 and 2007 from different Spanish reservoirs and (ii) individual Microcystis aeruginosa colonies collected from pelagic and littoral habitats in Valmayor reservoir (Spain) during a bloom. No agreement between chemotypes and both morphotypes and genotypes (based on cpcBA-IGS, 16S–23S rRNA ITS and mcyB genes) was found, suggesting that oligopeptide profiles in individual strains evolve independently across morphospecies and phylogenetic genotypes, and that the diversity of microcystin variants produced cannot be explained by mcyB gene variations alone. The presence of identical chemotypes in spatially-distant reservoirs with dissimilar trophic state, lithology or depth indicate that waterbody characteristics and geographical boundaries weakly affect chemotype composition and distribution. At smaller spatiotemporal scales (i.e. during bloom), M. aeruginosa populations showed high number of chemotypes, as well as marked differences in chemotype composition and relative abundance among the littoral and pelagic habitats. This indicates that the factors influencing chemotype composition, relative abundance and dynamics operate at short spatial and temporal scales, and supports emerging hypotheses about interactions with antagonistic microorganisms as possible drivers for widespread chemical polymorphisms in cyanobacteria.     

Distinct Amino Acid Compositional Requirements for Formation and Maintenance of the [PSI+] Prion in Yeast

Multiple yeast prions have been identified that result from the structural conversion of proteins into a self-propagating amyloid form. Amyloid-based prion activity in yeast requires a series of discrete steps. First, the prion protein must form an amyloid nucleus that can recruit and structurally convert additional soluble proteins. Subsequently, maintenance of the prion during cell division requires fragmentation of these aggregates to create new heritable propagons. For the Saccharomyces cerevisiae prion protein Sup35, these different activities are encoded by different regions of the Sup35 prion domain. An N-terminal glutamine/asparagine-rich nucleation domain is required for nucleation and fiber growth, while an adjacent oligopeptide repeat domain is largely dispensable for prion nucleation and fiber growth but is required for chaperone-dependent prion maintenance. Although prion activity of glutamine/asparagine-rich proteins is predominantly determined by amino acid composition, the nucleation and oligopeptide repeat domains of Sup35 have distinct compositional requirements. Here, we quantitatively define these compositional requirements in vivo. We show that aromatic residues strongly promote both prion formation and chaperone-dependent prion maintenance. In contrast, nonaromatic hydrophobic residues strongly promote prion formation but inhibit prion propagation. These results provide insight into why some aggregation-prone proteins are unable to propagate as prions.     

RBP differentiation contributes to selective transmissibility of OPT3 mRNAs

Long-distance mobile mRNAs play key roles in gene regulatory networks that control plant development and stress tolerance. However, the mechanisms underlying species-specific delivery of mRNA still need to be elucidated. Here, the use of grafts involving highly heterozygous apple (Malus) genotypes allowed us to demonstrate that apple (Malus domestica) oligopeptide transporter3 (MdOPT3) mRNA can be transported over a long distance, from the leaf to the root, to regulate iron uptake; however, the mRNA of Arabidopsis (Arabidopsis thaliana) oligopeptide transporter 3 (AtOPT3), the MdOPT3 homolog from A. thaliana, does not move from shoot to root. Reciprocal heterologous expression of the two types of mRNAs showed that the immobile AtOPT3 became mobile and moved from the shoot to the root in two woody species, Malus and Populus, while the mobile MdOPT3 became immobile in two herbaceous species, A. thaliana and tomato (Solanum lycopersicum). Furthermore, we demonstrated that the different transmissibility of OPT3 in A. thaliana and Malus might be caused by divergence in RNA-binding proteins between herbaceous and woody plants. This study provides insights into mechanisms underlying differences in mRNA mobility and validates the important physiological functions associated with this process.

The long-distance movement of OPT3 is selective between herbaceous and woody plants as shown using Malus and Arabidopsis thaliana plants.     

Role of the tryptophan residue in the vicinity of the catalytic center of exonuclease III family AP endonucleases: AP site recognition mechanism

The mechanisms by which AP endonucleases recognize AP sites have not yet been determined. Based on our previous study with Escherichia coli exonuclease III (ExoIII), the ExoIII family AP endonucleases probably recognize the DNA-pocket formed at an AP site. The indole ring of a conserved tryptophan residue in the vicinity of the catalytic site presumably intercalates into this pocket. To test this hypothesis, we constructed a series of mutants of ExoIII and human APE1. Trp-212 of ExoIII and Trp-280 of APE1 were critical to the AP endonuclease activity and binding to DNA containing an AP site. To confirm the ability of the tryptophan residue to intercalate with the AP site, we examined the interaction between an oligopeptide containing a tryptophan residue and an oligonucleotide containing AP sites, using spectrofluorimetry and surface plasmon resonance (SPR) technology. The tryptophan residue of the oligopeptide specifically intercalated into an AP site of DNA. The tryptophan residue in the vicinity of the catalytic site of the ExoIII family AP endonucleases plays a key role in the recognition of AP sites.