Effect of naturally occurring variations of the F-type lectin sequence motif on glycan binding: studies on F-type lectin domains with typical and atypical sequence motifs.

The typical F-type lectin domain (FLD) has an L-fucose-binding motif [HX(26)RXDX(4)R/K] with conserved basic residues that mediate hydrogen bonding with alpha-L-fucose. About one-third of the nonredundant FLD sequences in the publicly available databases are "atypical"; they have motifs with substitutions of these critical residues and/or variations in motif length. We addressed the question if atypical FLDs with substitutions of the critical residues retain lectin activity by performing site-directed mutagenesis and assessing the glycan-binding functions of typical and atypical FLDs. Site directed mutagenesis of an L-fucose-binding FLD from Streptosporangium roseum indicated that the critical His residue could be replaced by Ser and the second Arg by Lys without complete loss of lectin activity. Mutagenesis of His to other naturally substituting residues and mutagenesis of the first Arg to the naturally substituting residues, Lys, Ile, Ser, or Cys, resulted in loss of lectin activity. Glycan binding analysis and site-directed mutagenesis of atypical FLDs from Actinomyces turicensis, and Saccharomonospora cyanea confirmed that Ser and Thr can assume the L-fucose-binding role of the critical His, and further suggested that the residue in this position is dispensable in certain FLDs. We identified, by sequence and structural analysis of atypical FLDs, a Glu residue in the complementarity determining region, CDR5 that compensates for a lack of the critical His or other appropriate polar residue in this position. We propose that FLDs lacking a typical FLD sequence motif might nevertheless retain lectin activity through the recruitment of other strategically positioned polar residues in the CDR loops. © 2018 IUBMB Life, 71(3):385-397, 2019.

Actinomadura alkaliterrae sp. nov., isolated from an alkaline soil.

A polyphasic study was undertaken to establish the taxonomic status of an Actinomadura strain isolated from the margin of a saline, alkaline lake in Central Anatolia, Turkey. Strain D310ATT was shown to have chemotaxonomic, cultural and morphological properties consistent with its classification in the genus Actinomadura such as hooked or irregular spiral spore chains, meso-diaminopimelic acid as the major cell wall diaminopimelic acid, and diphosphatidylglycerol and phosphatidylinositol as major polar lipids. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain D310ATT is closely, albeit loosely, associated with Actinomadura darangshiensis DLS-70T with 97.2% sequence similarity, but was readily separated from the latter using diverse phenotypic properties. Consequently, the isolate is considered to represent a new species of Actinomadura for which the name Actinomadura alkaliterrae sp. nov. is proposed, with the type strain D310ATT (=DSM 101185T = KCTC 39657T).

Actinopolyspora biskrensis sp. nov., a novel halophilic actinomycete isolated from Northern Sahara.

A novel halophilic, filamentous actinomycete, designated H254(T), was isolated from a Saharan soil sample collected from Biskra (Northern Sahara), and subjected to a polyphasic taxonomic characterization. The strain is Gram-positive, aerobic, and halophilic, and the optimum NaCl concentration for growth is 15-20 % (w/v). The cell-wall hydrolysate contained meso-diaminopimelic acid, and the diagnostic whole-cell sugars were arabinose and galactose. The diagnostic phospholipid detected was phosphatidylcholine, and MK-9(H4) was the predominant menaquinone. The major fatty acid profiles were anteiso-C17:0 (32.8 %), C15:0 (28 %), and iso-C17:0 (12.3 %). Comparative analysis of the 16S rRNA gene sequences revealed that the strain H254(T) formed a well-separated sub-branch within the radiation of the genus Actinopolyspora, and the microorganism was most closely related to Actinopolyspora saharensis DSM 45459(T) (99.2 %), Actinopolyspora halophila DSM 43834(T) (99.1 %), and Actinopolyspora algeriensis DSM 45476(T) (99.0 %). Nevertheless, the strain had relatively lower mean values for DNA-DNA relatedness with the above strains (57.2, 68.4, and 45.6 %, respectively). Based on phenotypic features and phylogenetic position, we propose that strain H254(T) represents a novel species of the genus Actinopolyspora, for which the name Actinopolyspora biskrensis sp. nov. is proposed. The type strain of A. biskrensis is strain H254(T) (=DSM 46684(T) =CECT 8576(T)).

Simulations of the alternating access mechanism of the sodium symporter Mhp1.

Sodium coupled cotransporters of the five-helix inverted repeat (5HIR) superfamily use an alternating access mechanism to transport a myriad of small molecules across the cell membrane. One of the primary steps in this mechanism is the conformational transition from a state poised to bind extracellular substrates to a state that is competent to deliver substrate to the cytoplasm. Here, we construct a coarse-grained model of the 5HIR benzylhydantoin transporter Mhp1 that incorporates experimental structures of the outward- and inward-open states to investigate the mechanism of this conformational change. Using the weighted ensemble path-sampling method, we rigorously sample the outward- to inward-facing transition path ensemble. The transition path ensemble reveals a heterogeneous set of pathways connecting the two states and identifies two modes of transport: one consistent with a strict alternating access mechanism and another where decoupling of the inner and outer gates causes the transient formation of a continuous permeation pathway through the transporter. We also show that the conformational switch between the outward- and inward-open states results from rigid body motions of the hash motif relative to the substrate bundle, supporting the rocking bundle hypothesis. Finally, our methodology provides the groundwork for more chemically detailed investigations of the alternating mechanism.

Two genes, rif15 and rif16, of the rifamycin biosynthetic gene cluster in Amycolatopsis mediterranei likely encode a transketolase and a P450 monooxygenase, respectively, both essential for the conversion of rifamycin SV into B.

Amycolatopsis mediterranei produces an important antibiotic rifamycin, the biosynthesis of which involves many unusual modifications. Previous work suggested a putative P450 enzyme encoded by rif16 within the rifamycin biosynthetic gene cluster (rif) was required for the conversion of the intermediate rifamycin SV into the end product rifamycin B. In this study, we genetically proved that a putative transketolase encoded by rif15 is another essential enzyme for this conversion. Expression of merely rif15 and rif16 in a rif cluster null mutant of A. mediterranei U32 was able to convert rifamycin SV into B. However, this Rif15- and Rif16-mediated conversion was only detected in intact cells of A. meidterranei, but not in Streptomyce coelicolor or Mycobacterium smegmatis, suggesting that yet-characterized gene(s) in A. mediterranei other than those encoded by the rif cluster should be involved in this process.

Adapted Protocol for Saccharibacteria Cocultivation: Two New Members Join the Club of Candidate Phyla Radiation.

The growing application of metagenomics to different ecological and microbiome niches in recent years has enhanced our knowledge of global microbial biodiversity. Among these abundant and widespread microbes, the candidate phyla radiation (CPR) group has been recognized as representing a large proportion of the microbial kingdom (>26%). CPR are characterized by their obligate symbiotic or exoparasitic activity with other microbial hosts, mainly bacteria. Currently, isolating CPR is still considered challenging for microbiologists. The idea of this study was to develop an adapted protocol for the coculture of CPR with a suitable bacterial host. Based on various sputum samples, we tried to enrich CPR (Saccharibacteria members) and to cocultivate them with pure hosts (Schaalia odontolytica). This protocol was monitored by TaqMan real-time quantitative PCR (qPCR) using a system specific for Saccharibacteria designed in this study, as well as by electron microscopy and sequencing. We succeeded in coculturing and sequencing the complete genomes of two new Saccharibacteria species, "Candidatus Minimicrobia naudis" and "Candidatus Minimicrobia vallesae." In addition, we noticed a decrease in the CT values of Saccharibacteria and a significant multiplication through their physical association with Schaalia odontolytica strains in the enriched medium that we developed. This work may help bridge gaps in the genomic database by providing new CPR members, and in the future, their currently unknown characteristics may be revealed. IMPORTANCE In this study, the first TaqMan real-time quantitative PCR (qPCR) system, targeting Saccharibacteria phylum, has been developed. This technique can specifically quantify Saccharibacteria members in any sample of interest in order to investigate their prevalence. In addition, another easy, specific, and sensitive protocol has been developed to maintain the viability of Saccharibacteria cells in an enriched medium with their bacterial host. The use of this protocol facilitates subsequent studies of the phenotypic characteristics of CPR and their physical interactions with bacterial species, as well as the sequencing of new genomes to improve the current database.

Nitrite-producing oral microbiome in adults and children.

Recently, it was suggested that the nitrite (NO2-) produced from NO3- by oral bacteria might contribute to oral and general health. Therefore, we aimed to clarify the detailed information about the bacterial NO2-production in the oral biofilm. Dental plaque and tongue-coating samples were collected, then the NO2-producing activity was measured. Furthermore, the composition of the NO2--producing bacterial population were identified using the Griess reagent-containing agar overlay method and molecular biological method. NO2--producing activity per mg wet weight varied among individuals but was higher in dental plaque. Additionally, anaerobic bacteria exhibited higher numbers of NO2--producing bacteria, except in the adults' dental plaque. The proportion of NO2--producing bacteria also varied among individuals, but a positive correlation was found between NO2--producing activity and the number of NO2--producing bacteria, especially in dental plaque. Overall, the major NO2--producing bacteria were identified as Actinomyces, Schaalia, Veillonella and Neisseria. Furthermore, Rothia was specifically detected in the tongue coatings of children. These results suggest that dental plaque has higher NO2--producing activity and that this activity depends not on the presence of specific bacteria or the bacterial compositions, but on the number of NO2--producing bacteria, although interindividual differences were detected.

Differential Proteomics Based on 2D-Difference In-Gel Electrophoresis and Tandem Mass Spectrometry for the Elucidation of Biological Processes in Antibiotic-Producer Bacterial Strains.

Proteomics based on 2D-Difference In Gel Electrophoresis (2D-DIGE) coupled with mass spectrometry (MS) procedures can be considered a "gold standard" to determine quantitatively and comparatively protein abundances in cell extracts from different biological sources/conditions according to a gel-based approach. In particular, 2D-DIGE is used for protein specie separation, detection, and relative quantification, whenever tandem MS is used to obtain peptide sequence information that is managed according to bioinformatic procedures to identify the differentially represented protein species. The proteomic results consist of a dynamic portray of over- and down-represented protein species that, with the integration of gene ontology resources, allow obtaining a comprehensive understanding of the complex network of molecular signaling, regulatory circuits, and biochemical reactions occurring in cellular contexts. For this reason, proteomics has been widely used for studying molecular physiology of Gram-positive bacterial strains producing bioactive metabolites and belonging to actinomycete family. This highlighted the complex relationships linking overall regulatory processes and metabolic pathways to the biosynthesis of interesting bioactive molecules. In this chapter, we provide a detailed description of the procedures adopted to perform a differential proteomic analysis of the actinomycete Microbispora ATCC-PTA-5024, producing the promising NAI-107 lantibiotic. Although each experimental proteomic procedure has to be optimized to face the specific molecular characteristics of the organism under investigation, the protocols here described have also been used with minor modifications for proteomic studies on other bacterial strains, including the actinomycetes Streptomyces coelicolor, S. ambofaciens, Amycolatopsis balhimycina, and the Gram-negative proteobacteria Klebsiella oxytoca and Pseudoalteromonas haloplanktis.

Hamuramicins A and B, 22-membered macrolides, produced by an endophytic actinomycete Allostreptomyces sp. K12-0794.

Two new compounds, designated as hamuramicins A (1) and B (2), were isolated from the cultured broth of an endophytic actinomycete Allostreptomyces sp. K12-0794 by silica gel column chromatography and HPLC. The structures of 1 and 2 were elucidated as 22-membered macrolide containing triene and trienone with an alkyl side chain by spectroscopic analyses including NMR experiments. Both compounds showed growth inhibition activity against Kocuria rhizophia and Xanthomonas oryzae pv. oryzae as well as human cell line toxicity.

Identification of novel human high-density lipoprotein receptor Up-regulators using a cell-based high-throughput screening assay.

Scavenger receptor class B type I (SR-BI) is the high-affinity high-density lipoprotein (HDL) receptor, and CLA-1 is the human homologue of the murine SR-BI. CLA-1/SR-BI receptor has been suggested as a new preventative and/or therapeutic target for atherosclerosis due to its pivotal role in overall HDL cholesterol (HDL-C) metabolism and its antiatherogenic activity in vivo. To search for active compounds that can increase CLA-1 transcription, a novel cell-based assay was developed for application in high-throughput screening (HTS). Human hepatoma HepG2 cells were transfected with a CLA-1-promoter-luciferase reporter gene construct, and the stable transfected cell line was selected and named CLAp-LUC HepG2. With rosiglitazone as a positive control, this stable cell line was used to establish a specific CLA-1 gene expression assay in a 96-well microplate format. The evaluating parameter Z' value of 0.64 showed that this cell-based HTS assay was robust and reliable. Screening of 6000 microbial secondary metabolite crude extracts identified 8 positive strains. Between 2 identified CLA-1 up-regulators produced by actinomycete strain 04-4776, 4776B may stimulate not only the expression of CLA-1 on the transcriptional and translational levels but also the activity of CLA-1 to uptake the HDL-C in HepG2 cells. The active compounds originated from this HTS assay may be developed to drug candidates or lead compounds for new antiatherosclerosis agents.

Evolution of substrate specificity in a retained enzyme driven by gene loss.

The connection between gene loss and the functional adaptation of retained proteins is still poorly understood. We apply phylogenomics and metabolic modeling to detect bacterial species that are evolving by gene loss, with the finding that Actinomycetaceae genomes from human cavities are undergoing sizable reductions, including loss of L-histidine and L-tryptophan biosynthesis. We observe that the dual-substrate phosphoribosyl isomerase A or priA gene, at which these pathways converge, appears to coevolve with the occurrence of trp and his genes. Characterization of a dozen PriA homologs shows that these enzymes adapt from bifunctionality in the largest genomes, to a monofunctional, yet not necessarily specialized, inefficient form in genomes undergoing reduction. These functional changes are accomplished via mutations, which result from relaxation of purifying selection, in residues structurally mapped after sequence and X-ray structural analyses. Our results show how gene loss can drive the evolution of substrate specificity from retained enzymes.

Draft genome sequence of Actinotignum schaalii DSM 15541T: Genetic insights into the lifestyle, cell fitness and virulence.

The permanent draft genome sequence of Actinotignum schaalii DSM 15541T is presented. The annotated genome includes 2,130,987 bp, with 1777 protein-coding and 58 rRNA-coding genes. Genome sequence analysis revealed absence of genes encoding for: components of the PTS systems, enzymes of the TCA cycle, glyoxylate shunt and gluconeogensis. Genomic data revealed that A. schaalii is able to oxidize carbohydrates via glycolysis, the nonoxidative pentose phosphate and the Entner-Doudoroff pathways. Besides, the genome harbors genes encoding for enzymes involved in the conversion of pyruvate to lactate, acetate and ethanol, which are found to be the end products of carbohydrate fermentation. The genome contained the gene encoding Type I fatty acid synthase required for de novo FAS biosynthesis. The plsY and plsX genes encoding the acyltransferases necessary for phosphatidic acid biosynthesis were absent from the genome. The genome harbors genes encoding enzymes responsible for isoprene biosynthesis via the mevalonate (MVA) pathway. Genes encoding enzymes that confer resistance to reactive oxygen species (ROS) were identified. In addition, A. schaalii harbors genes that protect the genome against viral infections. These include restriction-modification (RM) systems, type II toxin-antitoxin (TA), CRISPR-Cas and abortive infection system. A. schaalii genome also encodes several virulence factors that contribute to adhesion and internalization of this pathogen such as the tad genes encoding proteins required for pili assembly, the nanI gene encoding exo-alpha-sialidase, genes encoding heat shock proteins and genes encoding type VII secretion system. These features are consistent with anaerobic and pathogenic lifestyles. Finally, resistance to ciprofloxacin occurs by mutation in chromosomal genes that encode the subunits of DNA-gyrase (GyrA) and topisomerase IV (ParC) enzymes, while resistant to metronidazole was due to the frxA gene, which encodes NADPH-flavin oxidoreductase.

The role of ABC transporters in antibiotic-producing organisms: drug secretion and resistance mechanisms.

Knowledge about biosynthetic gene clusters from antibiotic-producing actinomycetes is continuously increasing and the presence of an ABC transporter system is a fairly general phenomenon in most of these clusters. These transporters are involved in the secretion of the antibiotic through the cell membrane and also contribute to self resistance to the produced antibiotic.

Production of trehalose phosphorylase by Catellatospora ferruginea.

A range of microorganisms was screened for new and high producer strains of trehalose phosphorylase (EC 2.4.1.64). Trehalose phosphorylase activity was found in cells of actinomyctes of the genera Actinomadura, Amycolata, Catellatospora, Kineosporia, and Nocardia. Among them, Catellatospora ferruginea showed the highest enzyme activity. Trehalose phosphorylase from C. ferruginea was able to catalyse both the phosphorolysis of trehalose into beta-glucose 1-phosphate and D-glucose and the synthesis of trehalose from beta-glucose 1-phosphate and D-glucose.

ABC transporters in antibiotic-producing actinomycetes.

Many antibiotic-producing actinomycetes possess at least one ABC (ATP-binding cassette) transporter which forms part of the antibiotic biosynthetic pathway and in most cases confers resistance to the drug in an heterologous host. Three types of antibiotic ABC transporters have been so far described in producer organisms. In Type I two genes are involved, one encoding a hydrophilic ATP-binding protein with one nucleotide-binding domain and the other encoding a hydrophobic membrane protein. In Type II transporters only a gene encoding the hydrophilic ATP-binding protein with two nucleotide-binding domains is present and no gene encoding a hydrophobic membrane protein has been found. In Type III only one gene is involved which encodes both the hydrophilic and hydrophobic components. Possibly these ABC transporters are responsible for secretion of the antibiotics outside the cells. A comparative analysis of the ATP-binding components of the different antibiotic ABC transporters and analysis of the amino acid distances between the so-called Walker motifs suggests that the three types of transporters have probably evolved from a common ancestor containing a single nucleotide-binding domain.

Bifidobacteria in the intestinal tract of infants: an in-vitro study.

In-vitro studies showed that a number of factors are likely to influence the production and maintenance of a bifidobacillary flora and low pH in the faeces of newborn infants. Considerable importance is attached to the nature of the end products of bacterial metabolism in the large intestine. Thus, there is evidence to suggest that acetic acid and other metabolites of intraluminal bacterial growth suppress the growth of gram-negative organisms, but are without effect upon that of bifidobacteria. This mechanism in turn is controlled by the nature of the feed; important factors in breast milk include high lactose, low protein and low phosphate content.

Calcification of membranes isolated from Bacterionema matruchotii.

Lytic cultures of Bacterionema matruchotii were found to release vesicular membranes into the medium which could be obtained virtually free of other cell structures by differential centrifugation. Suspension of the membrane fraction in a metastable calcium phosphate solution resulted in the formation of both amorphous mineral and hydroxyapatite. Examination by electron microscopy showed that mineralization was associated with the membrane bilayers. The results provide further evidence that calcification of B matruchotii is related to intracytoplasmic membranes.

Comparison of calcification between Bacterionema matruchotii and Actinomyces naeslundii.

The purpose of this research was to examine the requirements for proteolipid initiation of calcification in culture. Proteolipid from a calcifiable microorganism, Bacterionema matruchotii, was compared with proteolipid isolated from a non-calcifiable microorganism, Actinomyces naeslundii. Although A. naeslundii does not calcify in culture, lyophilized cells and proteolipid-containing extracts do initiate apatite formation. A. naeslundii proteolipid (ANN) differs from B. matruchotii (BMN) in concentration, apoprotein polarity, and phospholipid composition. These differences may alter the ability of ANN to nucleate apatite in the intact cell.

Resolution of ion translocating proteolipid subclasses active in bacterial calcification.

Formation of calcium hydroxyapatite occurs on membrane surfaces via interaction of calcium, inorganic phosphate, phospholipids, calcifiable proteolipids, and ion flux to and from the nucleating site. Recently, this laboratory reported that proteolipids from the calcifying bacterium, Bacterionema matruchotti, act as an ionophore when reconstituted into bacteriorhodopsin-proteoliposomes. This ionophoric activity is blocked by [14C]dicyclohexylcarbodiimide ([14C]DCCD). SDS-PAGE shows that [14C]DCCD binds to a single band of Mr 8500. To determine whether proteins other than the [14C]DCCD-binding protein are involved, we examined the function of proteolipid species extracted by solvents of differing polarity. Proteolipids were isolated independently from chloroform:methanol (2:1) and chloroform:methanol:HCl (200:100:1) extracts of the bacteria by Sephadex LH-20 chromatography and were electrophoresed on 12.5% acrylamide gels. The chloroform:methanol extract contained a major hand at Mr 10,000 that was not present in gels of proteolipid isolated by acidified solvent. Proteolipids extracted in chloroform:methanol:HCl included a broad band at Mr 8500, which co-migrated with the [14C] DCCD-binding protein. The rate and extent of proton translocation were not altered when either proteolipid extract was added individually to bacteriorhodopsin proteoliposomes. However, when proteolipids isolated from the chloroform:methanol and chloroform:methanol:HCl extracts were combined, the rate and extent of translocation were increased. These data demonstrate that at least two proteolipid proteins are necessary for ionophoric activity, the Mr 10,000 protein isolated by chloroform:methanol 2:1 and the [14C]DCCD-binding protein requiring acidified solvent for extraction.