Keystone Taxa Lactiplantibacillus and Lacticaseibacillus Directly Improve the Ensiling Performance and Microflora Profile in Co-Ensiling Cabbage Byproduct and Rice Straw

Ensiling has been widely applied to cope with agricultural solid waste to achieve organic waste valorization and relieve environmental pressure and feedstuff shortage. In this study, co-ensiling of cabbage leaf byproduct and rice straw was performed with inoculation of Lactiplantibacillusplantarum (LP) to investigate the effects of inoculation on ensiling performance and microflora profiles. Compared to the control, LP inoculation preserved more dry matter (DM) content (283.4 versus 270.9 g·kg−1 fresh matter (FM) on day 30), increased lactic acid (LA) content (52.1 versus 35.8 g·kg−1 dry matter on day 15), decreased pH (3.55 versus 3.79 on day 15), and caused accumulation of acetic acid (AA), butyric acid (BA), and ammonia. The investigation showed that LP inoculation modified microflora composition, especially resisting potential pathogens and enriching more lactic acid bacteria (LAB) (p < 0.05). Moreover, Lactiplantibacillus and Lacticaseibacillus were identified as the keystone taxa that influenced physicochemical properties and interactions in microflora. They were also the main functional species that directly restrained undesirable microorganisms (p < 0.05), rather than indirectly working via metabolite inhibition and substrate competition (p > 0.05). The results of this present study improve the understanding of the underlying effect of LP inoculation on improving silage quality and facilitate the bio-transformation of cabbage byproduct and rice straw as animal feed.

Competing Substrates for the Bifunctional Diaminopimelic Acid Epimerase/Glutamate Racemase Modulate Peptidoglycan Synthesis in Chlamydia trachomatis

ABSTRACTThe Chlamydia trachomatis genome encodes multiple bifunctional enzymes, such as DapF, which is capable of both diaminopimelic acid (DAP) epimerase and glutamate racemase activity. Our previous work demonstrated the bifunctional activity of chlamydial DapF in vitro and in a heterologous system (Escherichia coli). In the present study, we employed a substrate competition strategy to demonstrate DapFCt function in vivo in C. trachomatis. We reasoned that, because DapFCt utilizes a shared substrate-binding site for both racemase and epimerase activities, only one activity can occur at a time. Therefore, an excess of one substrate relative to another must determine which activity is favored. We show that the addition of excess l-glutamate or meso-DAP (mDAP) to C. trachomatis resulted in 90% reduction in bacterial titers, compared to untreated controls. Excess l-glutamate reduced in vivo synthesis of mDAP by C. trachomatis to undetectable levels, thus confirming that excess racemase substrate led to inhibition of DapFCt DAP epimerase activity. We previously showed that expression of dapFCt in a murI (racemase) ΔdapF (epimerase) double mutant of E. coli rescues the d-glutamate auxotrophic defect. Addition of excess mDAP inhibited growth of this strain, but overexpression of dapFCt allowed the mutant to overcome growth inhibition. These results confirm that DapFCt is the primary target of these mDAP and l-glutamate treatments. Our findings demonstrate that suppression of either the glutamate racemase or epimerase activity of DapF compromises the growth of C. trachomatis. Thus, a substrate competition strategy can be a useful tool for in vivo validation of an essential bifunctional enzyme.

Macrocyclic Peptides as Allosteric Inhibitors of Nicotinamide N-Methyltransferase (NNMT)

<p>Nicotinamide <i>N</i>-methyltransferase (NNMT) methylates nicotinamide to form 1-methylnicotinamide using <i>S</i>-adenosyl-l-methionine (SAM) as the methyl donor. The complexity of the role of NNMT in healthy and disease states is slowly being elucidated and provides indication that NNMT may be an interesting therapeutic target for a variety of diseases including cancer, diabetes, and obesity. Most inhibitors of NNMT described to date are structurally related to one or both of its substrates. In search of structurally diverse NNMT inhibitors, an mRNA display screening technique was used to identify macrocyclic peptides which bind to NNMT. Several of the cyclic peptides identified in this manner show potent inhibition of NNMT with IC₅₀ values as low as 229 nM. Interestingly, substrate competition experiments reveal that these cyclic peptide inhibitors are noncompetitive with either SAM or NA indicating they may be the first allosteric inhibitors reported for NNMT.</p>

Macrocyclic Peptides as Allosteric Inhibitors of Nicotinamide N-Methyltransferase (NNMT)

<p>Nicotinamide <i>N</i>-methyltransferase (NNMT) methylates nicotinamide to form 1-methylnicotinamide using <i>S</i>-adenosyl-l-methionine (SAM) as the methyl donor. The complexity of the role of NNMT in healthy and disease states is slowly being elucidated and provides indication that NNMT may be an interesting therapeutic target for a variety of diseases including cancer, diabetes, and obesity. Most inhibitors of NNMT described to date are structurally related to one or both of its substrates. In search of structurally diverse NNMT inhibitors, an mRNA display screening technique was used to identify macrocyclic peptides which bind to NNMT. Several of the cyclic peptides identified in this manner show potent inhibition of NNMT with IC₅₀ values as low as 229 nM. Interestingly, substrate competition experiments reveal that these cyclic peptide inhibitors are noncompetitive with either SAM or NA indicating they may be the first allosteric inhibitors reported for NNMT.</p>

Two Forms of Tyrosyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Discovery of Inhibitory Compounds

Pseudomonas aeruginosa is a multidrug-resistant (MDR) pathogen and a causative agent of both nosocomial and community-acquired infections. The genes ( tyrS and tyrZ) encoding both forms of P. aeruginosa tyrosyl-tRNA synthetase (TyrRS-S and TyrRS-Z) were cloned and the resulting proteins purified. TyrRS-S and TyrRS-Z were kinetically evaluated and the Km values for interaction with Tyr, ATP, and tRNATyr were 172, 204, and 1.5 μM and 29, 496, and 1.9 μM, respectively. The kcatobs values for interaction with Tyr, ATP, and tRNATyr were calculated to be 3.8, 1.0, and 0.2 s−1 and 3.1, 3.8, and 1.9 s−1, respectively. Using scintillation proximity assay (SPA) technology, a druglike 2000-compound library was screened to identify inhibitors of the enzymes. Four compounds (BCD37H06, BCD38C11, BCD49D09, and BCD54B04) were identified with inhibitory activity against TyrRS-S. BCD38C11 also inhibited TyrRS-Z. The IC50 values for BCD37H06, BCD38C11, BCD49D09, and BCD54B04 against TyrRS-S were 24, 71, 65, and 50 μM, respectively, while the IC50 value for BCD38C11 against TyrRS-Z was 241 μM. Minimum inhibitory concentrations (MICs) were determined against a panel of clinically important pathogens. All four compounds were observed to inhibit the growth of cultures of both Gram-positive and Gram-negative bacteria organisms with a bacteriostatic mode of action. When tested against human cell cultures, none of the compounds were toxic at concentrations up to 400 μg/mL. In mechanism of inhibition studies, BCD38C11 and BCD49D09 selectively inhibited TyrRS activity by competing with ATP for binding. BCD37H06 and BCD54B04 inhibited TyrRS activity by a mechanism other than substrate competition.

Taxonomy and diversity of coelobite bryozoans from drift coral cobbles on Co To Island, northern Vietnam

There has been no previous report detailing the taxonomy of marine bryozoans along the coasts of Vietnam. Here we report on the taxonomy and diversity of bryozoans collected among drift coral cobbles from a beach in the tropical Co To archipelago, Gulf of Tonkin, northern Vietnam. We detected 27 bryozoan species (23 cheilostomes, four cyclostomes) in a coelobite assemblage inhabiting crevices in the cobbles, and holes made by boring molluscs. The degree of bryozoan preservation varied greatly, suggesting that the cobbles had accumulated on the beach over a period of months to years, or even decades. Coral reefs in the Co To archipelago underwent a catastrophic decline in 2003–2008, and it is unclear whether the bryozoan assemblage reflects past diversity, present diversity remaining in the coral rubble, or both. We describe six new species: Parasmittina acondylata n. sp., Metroperiella cotoensis n. sp., Microporella tonkinensis n. sp., Rhynchozoon setiavicularium n. sp., R. latiavicularium n. sp., and Disporella phaohoa n. sp. All but two of the previously described species were already known from the Central Indo-Pacific coastal biogeographical realm of Spalding et al. (2007), which includes Vietnam. We report the third Recent record of the thalamoporellid Dibunostoma reversum (Harmer, 1926), which is quite similar to and might be conspecific with the lower Miocene species Thalamoporella transversa Guha & Krishna, 2004; while it is premature to synonymize the two, we transfer T. transversa to Dibunostoma, as D. transversum. The calcareous, sheet-like, encrusting foraminiferan Planorbulina larvata was prominent in the coelobite assemblage and was often observed in substrate competition with bryozoans. A limited analysis of competitive interactions indicated that the encrusting bryozoans in the coelobite assemblage encountered P. larvata more often than they encountered other bryozoans, and that P. larvata out-competed bryozoans for substrate, reinforcing a growing sense of the importance of encrusting foraminifera in tropical and subtropical hard-substrate communities.

Modulation of peptidoglycan synthesis by recycled cell wall tetrapeptides

ABSTRACTThe bacterial cell wall is made of peptidoglycan (PG), a polymer that is essential for maintenance of cell shape and survival. During growth, bacteria remodel their PG, releasing fragments that are predominantly re-internalized by the cell, where they are recycled for synthesis of new PG. Although the PG recycling pathway is widely conserved, its components are not essential and its roles in cell wall homeostasis are not well-understood. Here, we identified LdcV, a Vibrio cholerae L,D-carboxypeptidase that cleaves the terminal D-Alanine from recycled murotetrapeptides. In the absence of ldcV, recycled tetrapeptides accumulated in the cytosol, leading to two toxic consequences for the cell wall. First, incorporation of tetrapeptide-containing PG precursors into the cell wall led to reduction in D,D-cross-linkage between stem peptides, diminishing PG integrity. Second, tetrapeptide accumulation led to a decrease in canonical UDP-pentapeptide precursors, reducing PG synthesis. Thus, LdcV and the recycling pathway promote optimal cell wall assembly and composition. Furthermore, Ldc substrate preference for murotetrapeptides containing canonical (D-Alanine) vs. non-canonical (D-Methionine) D-amino acids is conserved, suggesting that accumulation of tetrapeptide recycling intermediates may modulate PG homeostasis in environments enriched in non-canonical-muropeptides via substrate competition.