Hijacking the Peptidoglycan Recycling Pathway of Escherichia coli to Produce Muropeptides.

Soluble fragments of peptidoglycan called muropeptides are released from the cell wall of bacteria as part of their metabolism or as a result of biological stresses. These compounds trigger immune responses in mammals and plants. In bacteria, they play a major role in the induction of antibiotic resistance. The development of efficient methods to produce muropeptides is, therefore, desirable both to address their mechanism of action and to design new antibacterial and immunostimulant agents. Herein, we engineered the peptidoglycan recycling pathway of Escherichia coli to produce N-acetyl-ß-D-glucosaminyl-(1→4)-1,6-anhydro-N-acetyl-ß-D-muramic acid (GlcNAc-anhMurNAc), a common precursor of Gram-negative and Gram-positive muropeptides. Inactivation of the hexosaminidase nagZ gene allowed the efficient production of this key disaccharide, providing access to Gram-positive muropeptides through subsequent chemical peptide conjugation. E. coli strains deficient in both NagZ hexosaminidase and amidase activities further enabled the in vivo production of Gram-negative muropeptides containing meso-diaminopimelic acid, a rarely available amino acid.

Biosynthesis of meso-lanthionine in Fusobacterium nucleatum.

The opportunistic oral pathogen, Fusobacterium nucleatum contains meso-lanthionine as the diaminodicarboxylic acid in the pentapeptide crosslink of the peptidoglycan layer. The diastereomer, l,l-lanthionine is formed by lanthionine synthase, a PLP-dependent enzyme that catalyzes the ß-replacement of l-cysteine with a second equivalent of l-cysteine. In this study, we explored possible enzymatic mechanisms for the formation of meso-lanthionine. Our inhibition studies with lanthionine synthase, described herein, revealed that meso-diaminopimelate, a bioisostere of meso-lanthionine, is a more potent inhibitor of lanthionine synthase compared to the diastereomer, l,l-diaminopimelate. These results suggested that lanthionine synthase could also form meso-lanthionine by the ß-replacement of l-cysteine with d-cysteine. Through steady-state and pre-steady state kinetic analysis, we confirm that d-cysteine reacts with the ⍺-aminoacylate intermediate with a kon that was 2-3-fold faster and Kd value that was 2-3fold lower compared to l-cysteine. However, given that intracellular levels of d-cysteine levels are assumed to be significantly lower than that of l-cysteine, we also determined if the gene product, FN1732, with low sequence identity to diaminopimelate epimerase could convert l,l-lanthionine to meso-lanthionine. Using diaminopimelate dehydrogenase in a coupled spectrophotometric assay, we show that FN1732 can convert l,l-lanthionine to meso-lanthionine with a kcat of 0.07 ± 0.001 s-1 and a KM of 1.9 ± 0.1 mM. In summary, our results provide two possible enzymatic mechanisms for the biosynthesis of meso-lanthionine in F. nucleatum.

Inhibition of Bacterial Peptidoglycan Cytopathy by Retina Pigment Epithelial PGRP2 Amidase.

Peptidoglycan (PGN) recognition protein 2 (PGRP2; N-acetylmuramyl-L-alanine amidase (NAMAA)) activity in corneal epithelial cells is thought to inhibit corneal inflammation by reducing the PGN-induced cytokines. PGRP2 has not been reported in human retinal pigment epithelial (RPE) cells. RPE cell lysate NAMAA activity was measured densitometrically via cleavage of FITC-tagged muramyl dipeptide (FITCMDP). RPE lysate degradation of the cytopathic activity of nucleotide-binding oligomerization domain (NOD) receptor agonists was assessed by caspase-3 activation and DNA ladder detection and quantitation. PGRP2/NAMAA protein was detected in RPE cells by immunofluorescent antibody assay. RPE lysate NAMAA cleaved FITCMDP in a dose- and time-dependent manner. RPE lysate selectively inhibited PGN cytopathic activity of NOD1 agonists containing D-γ-glutamyl-meso-diaminopimelic acid and NOD2 containing L-alanyl-D-isoglutamine. The results suggest RPE PGRP2 amidase selectively degrades PGN that stimulate NOD-mediated cytopathic activity. The failure of RPE NAMAA to degrade pro-inflammatory PGN may play a role in bacterial retinopathies.

Luteolibacter luteus sp. nov., isolated from stream bank soil.

A non-motile, Gram-stain-negative, rod-shaped and yellow-colored bacterium, designated G-1-1-1T was obtained from soil sampled at Gwanggyo stream bank, Gyeonggi-do, Republic of Korea. Cells were aerobic, catalase positive, grew optimally at 25-30 °C and hydrolysed aesculin and casein. A phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain G-1-1-1T formed a lineage within the genus Luteolibacter. The closest members were Luteolibacter flavescens GKXT (97.7% sequence similarity) and Luteolibacter arcticus MC 3726T (97.3%). The sequence similarities with other members of the genus Luteolibacter were ≤ 93.9%. The genome of strain G-1-1-1T was 6,412,079 bp long with 5176 protein-coding genes. The diagnostic amino acid of cell-wall peptidoglycan of strain G-1-1-1T was meso-diaminopimelic acid. The only respiratory quinone was menaquinone-9 and the principal polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and unidentified phospholipids. The predominant cellular fatty acids were iso-C14:0, C16:1 ω9c, C16:0, C14:0 and anteiso-C15:0. The DNA G + C content was 61.0 mol%. The anti-SMASH analysis of whole genome showed eight putative biosynthetic gene clusters responsible for various secondary metabolites. Based on genomic, chemotaxonomic, phenotypic and phylogenetic analyses, strain G-1-1-1T represents a novel species in the genus Luteobacter, for which the name Luteolibacter luteus sp. nov. is proposed. The type strain is G-1-1-1T (= KACC 21614T = NBRC 114341T).

Reconstruction of the Diaminopimelic Acid Pathway to Promote L-lysine Production in Corynebacterium glutamicum.

The dehydrogenase pathway and the succinylase pathway are involved in the synthesis of L-lysine in Corynebacterium glutamicum. Despite the low contribution rate to L-lysine production, the dehydrogenase pathway is favorable for its simple steps and potential to increase the production of L-lysine. The effect of ammonium (NH4+) concentration on L-lysine biosynthesis was investigated, and the results indicated that the biosynthesis of L-lysine can be promoted in a high NH4+ environment. In order to reduce the requirement of NH4+, the nitrogen source regulatory protein AmtR was knocked out, resulting in an 8.5% increase in L-lysine production (i.e., 52.3 ± 4.31 g/L). Subsequently, the dehydrogenase pathway was upregulated by blocking or weakening the tetrahydrodipicolinate succinylase (DapD)-coding gene dapD and overexpressing the ddh gene to further enhance L-lysine biosynthesis. The final strain XQ-5-W4 could produce 189 ± 8.7 g/L L-lysine with the maximum specific rate (qLys,max.) of 0.35 ± 0.05 g/(g·h) in a 5-L jar fermenter. The L-lysine titer and qLys,max achieved in this study is about 25.2% and 59.1% higher than that of the original strain without enhancement of dehydrogenase pathway, respectively. The results indicated that the dehydrogenase pathway could serve as a breakthrough point to reconstruct the diaminopimelic acid (DAP) pathway and promote L-lysine production.

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

The 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 DapF Ct function in vivo in C. trachomatis We reasoned that, because DapF Ct 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 DapF Ct 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 DapF Ct 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.

Peptides Containing meso-Oxa-Diaminopimelic Acid as Substrates for the Cell-Shape-Determining Proteases Csd6 and Pgp2.

The enzymes Csd6 and Pgp2 are peptidoglycan (PG) proteases found in the pathogenic bacteria Helicobacter pylori and Campylobacter jejuni, respectively. These enzymes are involved in the trimming of non-crosslinked PG sidechains and catalyze the cleavage of the bond between meso-diaminopimelic acid (meso-Dap) and d-alanine, thus converting a PG tetrapeptide into a PG tripeptide. They are known to be cell-shape-determining enzymes, because deletion of the corresponding genes results in mutant strains that have lost the normal helical phenotype and instead possess a straight-rod morphology. In this work, we report two approaches directed towards the synthesis of the tripeptide substrate Ac-iso-d-Glu-meso-oxa-Dap-d-Ala, which serves as a mimic of the terminus of an non-crosslinked PG tetrapeptide substrate. The isosteric analogue meso-oxa-Dap was utilized in place of meso-Dap to simplify the synthetic procedure. The more efficient synthesis involved ring opening of a peptide-embedded aziridine by a serine-based nucleophile. A branched tetrapeptide was also prepared as a mimic of the terminus of a crosslinked PG tetrapeptide. We used MS analysis to demonstrate that the tripeptide serves as a substrate for both Csd6 and Pgp2 and that the branched tetrapeptide serves as a substrate for Pgp2, albeit at a significantly slower rate.

Peptidoglycan-type analysis of the N-acetylmuramic acid auxotrophic oral pathogen Tannerella forsythia and reclassification of the peptidoglycan-type of Porphyromonas gingivalis.

BACKGROUND: Tannerella forsythia is a Gram-negative oral pathogen. Together with Porphyromonas gingivalis and Treponema denticola it constitutes the "red complex" of bacteria, which is crucially associated with periodontitis, an inflammatory disease of the tooth supporting tissues that poses a health burden worldwide. Due to the absence of common peptidoglycan biosynthesis genes, the unique bacterial cell wall sugar N-acetylmuramic acid (MurNAc) is an essential growth factor of T. forsythia to build up its peptidoglycan cell wall. Peptidoglycan is typically composed of a glycan backbone of alternating N-acetylglucosamine (GlcNAc) and MurNAc residues that terminates with anhydroMurNAc (anhMurNAc), and short peptides via which the sugar backbones are cross-linked to build up a bag-shaped network. RESULTS: We investigated T. forsythia's peptidoglycan structure, which is an essential step towards anti-infective strategies against this pathogen. A new sensitive radioassay was developed which verified the presence of MurNAc and anhMurNAc in the cell wall of the bacterium. Upon digest of isolated peptidoglycan with endo-N-acetylmuramidase, exo-N-acetylglucosaminidase and muramyl-L-alanine amidase, respectively, peptidoglycan fragments were obtained. HPLC and mass spectrometry (MS) analyses revealed the presence of GlcNAc-MurNAc-peptides and the cross-linked dimer with retention-times and masses, respectively, equalling those of control digests of Escherichia coli and P. gingivalis peptidoglycan. Data were confirmed by tandem mass spectrometry (MS2) analysis, revealing the GlcNAc-MurNAc-tetra-tetra-MurNAc-GlcNAc dimer to contain the sequence of the amino acids alanine, glutamic acid, diaminopimelic acid (DAP) and alanine, as well as a direct cross-link between DAP on the third and alanine on the fourth position of the two opposite stem peptides. The stereochemistry of DAP was determined by reversed-phase HPLC after dabsylation of hydrolysed peptidoglycan to be of the meso-type. CONCLUSION: T. forsythia peptidoglycan is of the A1γ-type like that of E. coli. Additionally, the classification of P. gingivalis peptidoglycan as A3γ needs to be revised to A1γ, due to the presence of meso-DAP instead of LL-DAP, as reported previously.

Serinicoccus sediminis sp. nov., isolated from tidal flat sediment.

A novel Gram-strain-positive, non-spore-forming bacterial strain, designated GP-T3-3T, was isolated from sediment sampled at a tidal flat in Gopado, Republic of Korea. Cells were aerobic, catalase-negative, oxidase-positive, non-motile cocci that occurred singly, in pairs or in clusters. Strain GP-T3-3T grew at 4-45 °C (optimum, 28-37 °C), at pH 4.0-12.0 (pH 8.0-9.0) and in the presence of 0-15 % (w/v) NaCl (3-5 %). Colonies of strain GP-T3-3T were deep-yellow, circular, smooth and pulvinate. The results of the phylogenetic analyses based on 16S rRNA gene sequences indicated that strain GP-T3-3T was closely related to Serinicoccus profundi MCCC 1A05965T (99.1 %), Serinicoccus chungangensis CAU 9536T (99.0 %) and Serinicoccus marinus JC1078T (98.0 %). The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The predominant respiratory quinone was MK-8(H4) and the major fatty acids were anteiso-C17 : 0, iso-C16 : 0, iso-C15 : 0 and anteiso-C15 : 0. The polar lipid profile consisted of diphosphadidylglycerol, phosphadidylglycerol, phosphatidylcholine, phosphatidylinositol and two unidentified phospholipids. The DNA G+C content was 72.9 mol%. DNA-DNA relatedness values between strain GP-T3-3T and type strains of the genus Serinicoccus ranged from 28.9 to 50.5 %. On the basis of the phenotypic differences and DNA-DNA relatedness data, the isolate represents a new species of the genus Serinicoccus, for which the name Serinicoccussediminis sp. nov. is proposed. The type strain is GP-T3-3T (=KCTC 49173T=JCM 32825T=KCCM 43309T=KACC 19850T).

Structural basis for substrate specificity of meso-diaminopimelic acid decarboxylase from Corynebacterium glutamicum.

l-lysine is an essential amino acid that is widely used as a food supplement for humans and animals. meso-Diaminopimelic acid decarboxylase (DAPDC) catalyzes the final step in the de novol-lysine biosynthetic pathway by converting meso-diaminopimelic acid (meso-DAP) into l-lysine by decarboxylation reaction. To elucidate its molecular mechanisms, we determined the crystal structure of DAPDC from Corynebacterium glutamicum (CgDAPDC). The PLP cofactor is bound at the center of the barrel domain and forms a Schiff base with the catalytic Lys75 residue. We also determined the CgDAPDC structure in complex with both pyridoxal 5'-phosphate (PLP) and the l-lysine product and revealed that the protein has an optimal substrate binding pocket to accommodate meso-DAP as a substrate. Structural comparison of CgDAPDC with other amino acid decarboxylases with different substrate specificities revealed that the position of the α15 helix in CgDAPDC and the residues located on the helix are crucial for determining the substrate specificities of the amino acid decarboxylases.

Aestuariimicrobium soli sp. nov., isolated from farmland soil, and emended description of the genus Aestuariimicrobium.

A Gram-reaction-positive, catalase-positive, non-spore-forming and short rod- or oval-shaped bacterial strain, designated D6T, was isolated from farmland soil in Xuancheng, Anhui Province, China. Growth occurred at 4-37 °C (optimum, 30 °C), at pH 6.5-8.5 (optimum, 7.0) and with 0-7 % (w/v) NaCl (optimum, 0.5 % NaCl). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain D6T was most closely related to Aestuariimicrobium kwangyangense DSM 21549T (98.47 %), followed by Tessaracoccus rhinocerotis YIM 101269T (94.46 %). Strain D6T had a cell-wall peptidoglycan based on ll-diaminopimelic acid. MK-9(H4) was the predominant menaquinone. The major fatty acids of strain D6T were anteiso-C15 : 0, iso-C15 : 0 and summed feature 4 (iso-C17 : 1 I and/or anteiso-C17 : 1 B). The major polar lipids were a lipid, glycolipid and phospholipid. The DNA G+C content was 69.2 mol% and strain D6T showed low DNA-DNA relatedness to A. kwangyangense DSM 21549T (36.45±0.42 %). Based on these genotypic and phenotypic data, strain D6T represents a novel species in the genus Aestuariimicrobium, for which the name Aestuariimicrobium soli sp. nov. is proposed. The type strain is D6T (=KCTC 39995T=DSM 105824T). An emended description of the genus Aestuariimicrobium is presented.

Diaminopimelic acid (DAP) analogs bearing isoxazoline moiety as selective inhibitors against meso-diaminopimelate dehydrogenase (m-Ddh) from Porphyromonas gingivalis.

Two diastereomeric analogs (1 and 2) of diaminopimelic acid (DAP) bearing an isoxazoline moiety were synthesized and evaluated for their inhibitory activities against meso-diaminopimelate dehydrogenase (m-Ddh) from the periodontal pathogen, Porphyromonas gingivalis. Compound 2 showed promising inhibitory activity against m-Ddh with an IC50 value of 14.9µM at pH 7.8. The two compounds were further tested for their antibacterial activities against a panel of periodontal pathogens, and compound 2 was shown to be selectively potent to P. gingivalis strains W83 and ATCC 33277 with minimum inhibitory concentration (MIC) values of 773µM and 1.875mM, respectively. Molecular modeling studies revealed that the inversion of chirality at the C-5 position of these compounds was the primary reason for their different biological profiles. Based on these preliminary results, we believe that compound 2 has properties consistent with it being a lead compound for developing novel pathogen selective antibiotics to treat periodontal diseases.

Bacterial peptidoglycan with amidated meso-diaminopimelic acid evades NOD1 recognition: an insight into NOD1 structure-recognition.

Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) is an intracellular pattern recognition receptor that recognizes bacterial peptidoglycan (PG) containing meso-diaminopimelic acid (mesoDAP) and activates the innate immune system. Interestingly, a few pathogenic and commensal bacteria modify their PG stem peptide by amidation of mesoDAP (mesoDAPNH2). In the present study, NOD1 stimulation assays were performed using bacterial PG containing mesoDAP (PGDAP) and mesoDAPNH2 (PGDAPNH2) to understand the differences in their biomolecular recognition mechanism. PGDAP was effectively recognized, whereas PGDAPNH2 showed reduced recognition by the NOD1 receptor. Restimulation of the NOD1 receptor, which was initially stimulated with PGDAP using PGDAPNH2, did not show any further NOD1 activation levels than with PGDAP alone. But the NOD1 receptor initially stimulated with PGDAPNH2 responded effectively to restimulation with PGDAP The biomolecular structure-recognition relationship of the ligand-sensing leucine-rich repeat (LRR) domain of human NOD1 (NOD1-LRR) with PGDAP and PGDAPNH2 was studied by different computational techniques to further understand the molecular basis of our experimental observations. The d-Glu-mesoDAP motif of GMTPDAP, which is the minimum essential motif for NOD1 activation, was found involved in specific interactions at the recognition site, but the interactions of the corresponding d-Glu-mesoDAP motif of PGDAPNH2 occur away from the recognition site of the NOD1 receptor. Hot-spot residues identified for effective PG recognition by NOD1-LRR include W820, G821, D826 and N850, which are evolutionarily conserved across different host species. These integrated results thus successfully provided the atomic level and biochemical insights on how PGs containing mesoDAPNH2 evade NOD1-LRR receptor recognition.

Helical shape of Helicobacter pylori requires an atypical glutamine as a zinc ligand in the carboxypeptidase Csd4.

Peptidoglycan modifying carboxypeptidases (CPs) are important determinants of bacterial cell shape. Here, we report crystal structures of Csd4, a three-domain protein from the human gastric pathogen Helicobacter pylori. The catalytic zinc in Csd4 is coordinated by a rare His-Glu-Gln configuration that is conserved among most Csd4 homologs, which form a distinct subfamily of CPs. Substitution of the glutamine to histidine, the residue found in prototypical zinc carboxypeptidases, resulted in decreased enzyme activity and inhibition by phosphate. Expression of the histidine variant at the native locus in a H. pylori csd4 deletion strain did not restore the wild-type helical morphology. Biochemical assays show that Csd4 can cleave a tripeptide peptidoglycan substrate analog to release m-DAP. Structures of Csd4 with this substrate analog or product bound at the active site reveal determinants of peptidoglycan specificity and the mechanism to cleave an isopeptide bond to release m-DAP. Our data suggest that Csd4 is the archetype of a new CP subfamily with a domain scheme that differs from this large family of peptide-cleaving enzymes.

Nocardioides flava sp. nov., isolated from rhizosphere of poppy plant, Republic of Korea.

Phylogenetic and taxonomic characterization was performed for bacterium, designated strain THG-DN5.4(T), isolated from the rhizosphere of poppy plant collected from Gyeryongsan, Republic of Korea. Strain THG-DN5.4(T) consists of Gram-stain-positive, aerobic, non-motile rods. The bacteria grow optimally at 18-30 °C, at pH 7.0 and in the presence of 0.5-1.0 % NaCl. Based on 16S rRNA gene sequence analysis, strain THG-DN5.4(T) was found to be most closely related to Nocardioides nitrophenolicus KCTC 047BP(T), followed by Nocardioides ginsengisoli KCTC 19135(T), Nocardioides kongjuensis KCTC 19054(T), Nocardioides simplex KACC 20620(T), Nocardioides aromaticivorans KACC 20613(T), Nocardioides daeguensis KCTC 19799(T) and Nocardioides caeni KCTC 19600(T). The DNA-DNA relatedness between strain THG-DN5.4(T) and closely related phylogenetic neighbors was below 45.0 %, and the DNA G+C content of strain THG-DN5.4(T) was 70.8 mol%. An isoprenoid quinone was identified as MK-8(H4). Strain THG-DN5.4(T) was characterized chemotaxonomically as having LL-diaminopimelic acid in the cell-wall peptidoglycan. The polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, some unidentified aminolipids and some unidentified polar lipids. iso-C16:0 and C18:1 ω9c were identified as the major fatty acids present in THG-DN5.4(T). On the basis of a polyphasic taxonomic study, strain THG-DN5.4(T) is considered to represent a novel species of the genus Nocardioides, for which the name Nocardioides flava sp. nov. is proposed. The type strain is THG-DN5.4(T) (=KCTC 39606(T)=CCTCC AB 2015298(T)).

Intestinal barrier dysfunction: implications for chronic inflammatory conditions of the bowel.

The intestinal epithelium of adult humans acts as a differentially permeable barrier that separates the potentially harmful contents of the lumen from the underlying tissues. Any dysfunction of this boundary layer that disturbs the homeostatic equilibrium between the internal and external environments may initiate and sustain a biochemical cascade that results in inflammation of the intestine. Key to such dysfunction are genetic, microbial and other environmental factors that, singularly or in combination, result in chronic inflammation that is symptomatic of inflammatory bowel disease (IBD). The aim of the present review is to assess the scientific evidence to support the hypothesis that defective transepithelial transport mechanisms and the heightened absorption of intact antigenic proinflammatory oligopeptides are important contributing factors in the pathogenesis of IBD.

A diaminopimelic acid auxotrophic Escherichia coli donor provides improved counterselection following intergeneric conjugation with actinomycetes.

Considering the medical, biotechnological, and economical importance of actinobacteria, there is a continuous need to improve the tools for genetic engineering of a broad range of these microorganisms. Intergeneric conjugation has proven to be a valuable yet imperfect tool for this purpose. The natural resistance of many actinomycetes to nalidixic acid (Nal) is generally exploited to eliminate the sensitive Escherichia coli donor strain following conjugation. Nevertheless, Nal can delay growth and have other unexpected effects on the recipient strain. To provide an improved alternative to antibiotics, we propose a postconjugational counterselection using a diaminopimelic acid (DAP) auxotrophic donor strain. The DAP-negative phenotype was obtained by introducing a dapA deletion into the popular methylase-negative donor strain E. coli ET12567/pUZ8002. The viability of ET12567 and its ΔdapA mutant exposed to DAP deprivation or Nal selection were compared in liquid pure culture and after mating with Streptomyces coelicolor. Results showed that death of the E. coli ΔdapA Nal-sensitive donor strain occurred more efficiently when subjected to DAP deprivation than when exposed to Nal. Our study shows that postconjugational counterselection based on DAP deprivation circumvents the use of antibiotics and will facilitate the transfer of plasmids into actinomycetes with high biotechnological potential, yet currently not accessible to conjugative techniques.

Mono-N-acyl-2,6-diaminopimelic acid derivatives: analysis by electromigration and spectroscopic methods and examination of enzyme inhibitory activity.

Thirteen mono-N-acyl derivatives of 2,6-diaminopimelic acid (DAP)-new potential inhibitors of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18)-were analyzed and characterized by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopies and two capillary electromigration methods: capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Structural features of DAP derivatives were characterized by IR and NMR spectroscopies, whereas CZE and MEKC were applied to evaluate their purity and to investigate their electromigration properties. Effective electrophoretic mobilities of these compounds were determined by CZE in acidic and alkaline background electrolytes (BGEs) and by MEKC in acidic and alkaline BGEs containing a pseudostationary phase of anionic detergent sodium dodecyl sulfate (SDS) or cationic detergent cetyltrimethylammonium bromide (CTAB). The best separation of DAP derivatives, including diastereomers of some of them, was achieved by MEKC in an acidic BGE (500 mM acetic acid [pH 2.54] and 60mM SDS). All DAP derivatives were examined for their ability to inhibit catalytic activity of DapE from Haemophilus influenzae (HiDapE) and ArgE from Escherichia coli (EcArgE). None of these DAP derivatives worked as an effective inhibitor of HiDapE, but one derivative-N-fumaryl, Me-ester-DAP-was found to be a moderate inhibitor of EcArgE, thereby providing a promising lead structure for further studies on ArgE inhibitors.

Convergence of innate immunity and insulin resistance as evidenced by increased nucleotide oligomerization domain (NOD) expression and signaling in monocytes from patients with type 2 diabetes.

Despite the well known role of nucleotide oligomerization domain (NOD) receptor proteins in innate immunity, their association with diabetes is less explored. Here we report the transcriptional level of NODs and their downstream molecular signatures in CD14(+) monocytes from subjects with different grades of glucose tolerance. NOD1 and NOD2 mRNA expression were significantly up-regulated in monocytes from patients with type 2 diabetes (T2DM) and positively correlated with HOMA-IR and poor glycemic control. Patients with T2DM also exhibited increased monocyte activation markers (CD11b and CD36) and proinflammatory signals downstream of NOD (RIPK2 and NFκB) along with the increased circulatory levels of TNF-α and IL-6. In vitro stimulation of monocytes with NOD specific ligands-i-EDAP and MDP significantly up regulated the mRNA expression of NOD1 and NOD2 respectively in T2DM. Our study exposes up regulation of NODs in monocytes as an important component of inflammation and insulin resistance in patients with T2DM.

Diaminopimelic Acid Metabolism by Pseudomonadota in the Ocean.

Diaminopimelic acid (DAP) is a unique component of the cell wall of Gram-negative bacteria. It is also an important component of organic matter and is widely utilized by microbes in the world's oceans. However, neither DAP concentrations nor marine DAP-utilizing microbes have been investigated. Here, DAP concentrations in seawater were measured and the diversity of marine DAP-utilizing bacteria and the mechanisms for their DAP metabolism were investigated. Free DAP concentrations in seawater, from surface to a 5,000 m depth, were found to be between 0.61 µM and 0.96 µM in the western Pacific Ocean. DAP-utilizing bacteria from 20 families in 4 phyla were recovered from the western Pacific seawater and 14 strains were further isolated, in which Pseudomonadota bacteria were dominant. Based on genomic and transcriptomic analyses combined with gene deletion and in vitro activity detection, DAP decarboxylase (LysA), which catalyzes the decarboxylation of DAP to form lysine, was found to be a key and specific enzyme involved in DAP metabolism in the isolated Pseudomonadota strains. Interrogation of the Tara Oceans database found that most LysA-like sequences (92%) are from Pseudomonadota, which are widely distributed in multiple habitats. This study provides an insight into DAP metabolism by marine bacteria in the ocean and contributes to our understanding of the mineralization and recycling of DAP by marine bacteria. IMPORTANCE DAP is a unique component of peptidoglycan in Gram-negative bacterial cell walls. Due to the large number of marine Gram-negative bacteria, DAP is an important component of marine organic matter. However, it remains unclear how DAP is metabolized by marine microbes. This study investigated marine DAP-utilizing bacteria by cultivation and bioinformational analysis and examined the mechanism of DAP metabolism used by marine bacteria. The results demonstrate that Pseudomonadota bacteria are likely to be an important DAP-utilizing group in the ocean and that DAP decarboxylase is a key enzyme involved in DAP metabolism. This study also sheds light on the mineralization and recycling of DAP driven by bacteria.