Exploitation of a Bacterium-Encoded Lytic Transglycosylase by a Human Oral Lytic Phage To Facilitate Infection.

Bacteriophages (phages) are an integral part of the human oral microbiome. Their roles in modulating bacterial physiology and shaping microbial communities have been discussed but remain understudied due to limited isolation and characterization of oral phage. Here, we report the isolation of LC001, a lytic phage targeting human oral Schaalia odontolytica (formerly known as Actinomyces odontolyticus) strain XH001. We showed that LC001 attached to and infected surface-grown, but not planktonic, XH001 cells, and it displayed remarkable host specificity at the strain level. Whole-genome sequencing of spontaneous LC001-resistant, surface-grown XH001 mutants revealed that the majority of the mutants carry nonsense or frameshift mutations in XH001 gene APY09_05145 (renamed ltg-1), which encodes a putative lytic transglycosylase (LT). The mutants are defective in LC001 binding, as revealed by direct visualization of the significantly reduced attachment of phage particles to the XH001 spontaneous mutants compared that to the wild type. Meanwhile, targeted deletion of ltg-1 produced a mutant that is defective in LC001 binding and resistant to LC001 infection even as surface-grown cells, while complementation of ltg-1 in the mutant background restored the LC001-sensitive phenotype. Intriguingly, similar expression levels of ltg-1 were observed in surface-grown and planktonic XH001, which displayed LC001-binding and nonbinding phenotypes, respectively. Furthermore, the overexpression of ltg-1 failed to confer an LC001-binding and -sensitive phenotype to planktonic XH001. Thus, our data suggested that rather than directly serving as a phage receptor, ltg-1-encoded LT may increase the accessibility of phage receptor, possibly via its enzymatic activity, by cleaving the peptidoglycan structure for better receptor exposure during peptidoglycan remodeling, a function that can be exploited by LC001 to facilitate infection. IMPORTANCE The evidence for the presence of a diverse and abundant phage population in the host-associated oral microbiome came largely from metagenomic analysis or the observation of virus-like particles within saliva/plaque samples, while the isolation of oral phage and investigation of their interaction with bacterial hosts are limited. Here, we report the isolation of LC001, the first lytic phage targeting oral Schaalia odontolytica. Our study suggested that LC001 may exploit the host bacterium-encoded lytic transglycosylase function to gain access to the receptor, thus facilitating its infection.

Aminoglycoside resistance of Trueperella pyogenes isolated from pigs in China.

We aimed to determine the resistance mechanisms of 27 T. pyogenes isolates from swine in the Jilin province of China. Drug sensitivity analysis indicated that most of the isolated strains were resistant to aminoglycosides. We investigated the genes involved in target alteration, drug inactivation, and increased efflux as potential resistance mechanisms. Two known aminoglycoside resistance genes (aphA1 and strB) were not found in the genomic DNA of any isolate. A 3-bp (CCC) deletion in one 16S rRNA operon was detected in all isolates, and efflux pumps were not active in the resistant group. Ultimately, genes encoding aminoglycoside-modifying enzymes carried by class 1 integrons were identified as the main cause of resistance to aminoglycosides in T. pyogenes.

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.

In vitro susceptibility of Actinobaculum schaalii to 12 antimicrobial agents and molecular analysis of fluoroquinolone resistance.

OBJECTIVES: To assess the in vitro susceptibility of Actinobaculum schaalii to 12 antimicrobial agents as well as to dissect the genetic basis of fluoroquinolone resistance. METHODS: Forty-eight human clinical isolates of A. schaalii collected in Switzerland and France were studied. Each isolate was identified by 16S rRNA sequencing. MICs of amoxicillin, ceftriaxone, gentamicin, vancomycin, clindamycin, linezolid, ciprofloxacin, levofloxacin, moxifloxacin, co-trimoxazole, nitrofurantoin and metronidazole were determined using the Etest method. Interpretation of results was made according to EUCAST clinical breakpoints. The quinolone-resistance-determining regions (QRDRs) of gyrA and parC genes were also identified and sequence analysis was performed for all 48 strains. RESULTS: All isolates were susceptible to amoxicillin, ceftriaxone, gentamicin, clindamycin (except three), vancomycin, linezolid and nitrofurantoin, whereas 100% and 85% were resistant to ciprofloxacin/metronidazole and co-trimoxazole, respectively. Greater than or equal to 90% of isolates were susceptible to the other tested fluoroquinolones, and only one strain was highly resistant to levofloxacin (MIC ≥32 mg/L) and moxifloxacin (MIC 8 mg/L). All isolates that were susceptible or low-level resistant to levofloxacin/moxifloxacin (n = 47) showed identical GyrA and ParC amino acid QRDR sequences. In contrast, the isolate exhibiting high-level resistance to levofloxacin and moxifloxacin possessed a unique mutation in GyrA, Ala83Val (Escherichia coli numbering), whereas no mutation was present in ParC. CONCLUSIONS: When an infection caused by A. schaalii is suspected, there is a risk of clinical failure by treating with ciprofloxacin or co-trimoxazole, and ß-lactams should be preferred. In addition, acquired resistance to fluoroquinolones more active against Gram-positive bacteria is possible.

Exo-beta-D-glucosaminidase from Amycolatopsis orientalis: catalytic residues, sugar recognition specificity, kinetics, and synergism.

Catalytic residues and the mode of action of the exo-beta-D-glucosaminidase (GlcNase) from Amycolatopsis orientalis were investigated using the wild-type and mutated enzymes. Mutations were introduced into the putative catalytic residues resulting in five mutated enzymes (D469A, D469E, E541D, E541Q, and S468N/D469E) that were successfully produced. The four single mutants were devoid of enzymatic activity, indicating that Asp469 and Glu541 are essential for catalysis as predicted by sequence alignments of enzymes belonging to GH-2 family. When mono-N-acetylated chitotetraose [(GlcN)3-GlcNAc] was hydrolyzed by the enzyme, the nonreducing-end glucosamine unit was produced together with the transglycosylation products. The rate of hydrolysis of the disaccharide, 2-amino-2-deoxy-D-glucopyranosyl 2-acetamido-2-deoxy-D-glucopyranose (GlcN-GlcNAc), was slightly lower than that of (GlcN)2, suggesting that N-acetyl group of the sugar residue located at (+1) site partly interferes with the catalytic reaction. The time-course of the enzymatic hydrolysis of the completely deacetylated chitotetraose [(GlcN)4] was quantitatively determined by high-performance liquid chromatography (HPLC) and used for in silico modeling of the enzymatic hydrolysis. The modeling study provided the values of binding free energy changes of +7.0, -2.9, -1.8, -0.9, -1.0, and -0.5 kcal/mol corresponding, respectively, to subsites (-2), (-1), (+1), (+2), (+3), and (+4). When chitosan polysaccharide was hydrolyzed by a binary enzyme system consisting of A. orientalis GlcNase and Streptomyces sp. N174 endochitosanase, the highest synergy in the rate of product formation was observed at the molar ratio 2:1. Thus, GlcNase would be an efficient tool for industrial production of glucosamine monosaccharide.

Cloning, expression, and characterization of a neuraminidase gene from Arcanobacterium pyogenes.

Arcanobacterium pyogenes is an opportunistic pathogen, associated with suppurative infections in domestic animals. In addition to pyolysin, a pore-forming, cholesterol-binding toxin, A. pyogenes expresses a number of putative virulence factors, including several proteases and neuraminidase activity. A 3,009-bp gene, nanH, was cloned and sequenced and conferred neuraminidase activity on an Escherichia coli host strain. The predicted 107-kDa NanH protein displayed similarity to a number of bacterial neuraminidases and contained the RIP/RLP motif and five copies of the Asp box motif found in all bacterial neuraminidases. Recombinant His-tagged NanH was found to have pH and temperature optima of 5.5 to 6.0 and 55 degrees C, respectively. Insertional deletion of the nanH gene resulted in the reduction, but not absence, of neuraminidase activity, indicating the presence of a second neuraminidase gene in A. pyogenes. NanH was localized to the A. pyogenes cell wall. A. pyogenes adhered to HeLa, CHO, and MDBK cells in a washing-resistant manner. However, the nanH mutant was not defective for adherence to epithelial cells. The role of NanH in host epithelial cell adherence may be masked by the presence of a second neuraminidase in A. pyogenes.

[Isolation and fermentation conditions of strains producing 1-phenyl-2-amino-ethanol alcohol dehydrogenase].

A Arachnia sp. P163 producing alcohol dehydrogenase which is able to reduce aminoacetophenone to R-1-phenyl-2-aminoethanol was obtained from soil and cultures. The maximum activity of enzyme was produced by the LB medium containing 1% sodium citrate and peptone, 0.1% phenylaminoethanol as inducer at 30 degrees C for 48 hs.

Characterization and cloning of celR, a transcriptional regulator of cellulase genes from Thermomonospora fusca.

CelR, a protein that regulates transcription of cellulase genes in Thermomonospora fusca (Actinomycetaceae) was purified to homogeneity. A 6-kilobase NotI-SacI fragment of T. fusca DNA containing the celR gene was cloned into Esherichia coli and sequenced. The celR gene encodes a 340-residue polypeptide that is highly homologous to members of the GalR-LacI family of bacterial transcriptional regulators. CelR specifically binds to a 14-base pair inverted repeat, which has sequence similarity to the binding sites of other family members. This site is present in regions upstream of all six cellulase genes in T. fusca. The binding of CelR to the celE promoter is inhibited specifically by low concentrations of cellobiose (0.2-0.5 mM), the major end product of cellulases. The other sugars tested did not affect binding at equivalent or 50-fold higher concentrations. The results suggest that CelR may act as a repressor, and that the mechanism of induction involves a direct interaction of CelR with cellobiose.

DdlN from vancomycin-producing Amycolatopsis orientalis C329.2 is a VanA homologue with D-alanyl-D-lactate ligase activity.

Vancomycin-resistant enterococci acquire high-level resistance to glycopeptide antibiotics through the synthesis of peptidoglycan terminating in D-alanyl-D-lactate. A key enzyme in this process is a D-alanyl-D-alanine ligase homologue, VanA or VanB, which preferentially catalyzes the synthesis of the depsipeptide D-alanyl-D-lactate. We report the overexpression, purification, and enzymatic characterization of DdlN, a VanA and VanB homologue encoded by a gene of the vancomycin-producing organism Amycolatopsis orientalis C329.2. Evaluation of kinetic parameters for the synthesis of peptides and depsipeptides revealed a close relationship between VanA and DdlN in that depsipeptide formation was kinetically preferred at physiologic pH; however, the DdlN enzyme demonstrated a narrower substrate specificity and commensurately increased affinity for D-lactate in the C-terminal position over VanA. The results of these functional experiments also reinforce the results of previous studies that demonstrated that glycopeptide resistance enzymes from glycopeptide-producing bacteria are potential sources of resistance enzymes in clinically relevant bacteria.

Regulation of biosynthesis of individual cellulases in Thermomonospora fusca.

Regulation of the biosynthesis of the six cellulases comprising the cellulolytic system of the thermophilic soil bacterium Thermomonospora fusca ER1 was studied. The levels of the individual enzymes produced on different noninducing and inducing carbon sources were determined. The lowest level of cellulase synthesis (3 nM) was observed with xylose as a carbon source, and the highest level (247 to 1,670 nM for different enzymes) was found in cultures grown on microcrystalline cellulose. Endocellulases and exocellulases showed distinctly different regulation patterns. Differences in the regulation of individual enzymes appear to be determined by the specific structural organization of the upstream regulatory sequences of their genes.

Biodegradation of the herbicide Diuron in soil by indigenous actinomycetes.

Three actinomycete strains isolated from soil treated with 2,4-D were able to degrade the herbicide Diuron in vitro. Strain CCT 4916 was the most efficient, degrading up to 37% of applied Diuron (100 mg Kg-1 soil) in 7 days, as measured by HPLC and UV/VIS spectroscopy. All strains showed protease and urease activity; intracellular activity of metapyrocatechase and pyrocatechase were not found. Actinomycete strain CCT 4916 produced manganese peroxidase, which could be potentially related to degradation of Diuron.

Purification and characterization of trehalose phosphorylase from Catellatospora ferruginea.

Trehalose phosphorylase was purified from the cell extracts of Catellatospora ferruginea. The enzyme had an apparent molecular weight of 400,000 by gel filtration and 98,000 by SDS-PAGE, suggesting that the enzyme was a tetramer. The enzyme was specific for trehalose in phosphorolysis and specific for beta-D-glucose 1-phosphate in synthesis. In addition to D-glucose, D-xylose and D-fucose were also possible sugar acceptors during synthesis. Phosphate ions were a key to the activity and stability of the enzyme, controlling the equilibrium of the reversible reaction and the heat stability of the enzyme. The enzyme was strongly inhibited by p-chloromercuribenzoate and pyridoxal phosphate. The enzyme was inactivated by heat or by storage frozen with ammonium chloride and lithium chloride.

Molecular cloning and nucleotide sequence of the pyruvate kinase gene of an actinomycete Microbispora thermodiastatica.

The gene for the thermostable pyruvate kinase of Microbispora thermodiastatica IFO 14046, a moderate thermophilic actinomycete, was cloned in Escherichia coli. This gene consists of an open reading frame of 1422 nucleotides and encodes a protein of 474 amino acids with molecular mass of 50,805 Da. The open reading frame was confirmed as the pyruvate kinase gene by comparison with the N-terminal amino acid sequence of the purified pyruvate kinase from M. thermodiastatica.

Toxic phospholipases D of Corynebacterium pseudotuberculosis, C. ulcerans and Arcanobacterium haemolyticum: cloning and sequence homology.

The genes encoding toxic phospholipases D (PLD) from Corynebacterium pseudotuberculosis (Cp)biovar equi and C. ulcerans (Cu) have been cloned and sequenced. The deduced proteins are 307 amino acids (aa) in length and include a putative signal sequences of 26-aa. A molecular mass of 31.2 and 31.0 kDa and pI values of 8.84 and 6.73 are predicted for the secreted (mature) proteins from Cp and Cu, respectively. Comparison of the deduced primary structure of the two proteins to those of the PLD produced by Cp biovar ovis and Arcanobacterium haemolyticum (Ah) revealed that the four enzymes share 64-97% identity. The aa sequences of this group of proteins were unique when compared to the sequences of other phospholipases in GenBank and were found to share only small regions of homology with other proteins, including two conserved domains of glyceraldehyde-3-phosphate dehydrogenase (G3PD). The similarity of PLD from Cp biovar equi, Cu and Ah to the PLD of Cp biovar ovis suggests that these enzymes may act as virulence determinants.

Wild-type and mutant D-xylose isomerase from Actinoplanes missouriensis: metal-ion dissociation constants, kinetic parameters of deuterated and non-deuterated substrates and solvent-isotope effects.

The metal-ion dissociation constants (Mg2+, Mn2+) of wild-type and mutant D-xylose isomerases from Actinoplanes missouriensis have been determined by titrating the metal-ion-free enzymes with Mg2+ and Mn2+ respectively. Substitution of amino acids co-ordinated to metal-ion 1 (E181D, D245N) dramatically affects the dissociation constants, pH-activity profiles and apparent substrate binding. Mutagenesis of groups ligated to metal-ion 2 is less drastic except for that of Asp-255: a decrease in metal-ion affinity, a change in metal-ion preference and an improved apparent substrate binding (at pH values above the optimum), especially in the presence of Mn2+, are observed for the D255N enzyme. Similar effects, except for a slightly increased metal-ion affinity, are obtained by mutagenesis of the adjacent Glu-186 to Gln and the unconserved Ala-25 to Lys. Moreover, the striking acidic-pH shifts observed for the D255N and E186Q enzymes support the crucial role of the water molecule, Wa-690, Asp-255 and the adjacent Glu-186 in proton transfer from 2-OH to O-1 of the open and extended aldose substrate. Mutations of other important groups scarcely affect the metal-ion dissociation constants and pH-activity profiles, although pronounced effects on the kinetic parameters may be observed.

Alpha-mannosidase: a rapid test for identification of Arcanobacterium haemolyticum.

A 4-h alpha-mannosidase test for identification of Arcanobacterium haemolyticum strains (n = 139) and differentiation of A. haemolyticum from Actinomyces pyogenes strains (n = 30) and other gram-positive rods was evaluated. Practically all A. haemolyticum strains (138 of 139) and the Listeria monocytogenes type strain were alpha-mannosidase positive, while all A. pyogenes strains and Corynebacterium (n = .25) strains as well as the Rhodococcus equi and Erysipelothrix rhusiopathiae type strains were negative. The rapid alpha-mannosidase test, in conjunction with a Gram stain and catalase and reverse CAMP tests, was useful in identification of A. haemolyticum and in differentiation of A. haemolyticum from A. pyogenes and Corynebacterium spp.

Some ecological and physiological studies on bacteria isolated from salt-affected soils of Egypt.

Members of Bacillaceae, Rhizobiaceae, actinomycetes and others were isolated from cultivated and non-cultivated saline soils. The high population of bacteria and actinomycetes were almost coincided with the relatively high levels of organic matter whatever the degree of soil salinity. Bacillus stearothermophilus and B. subtilis were more frequently isolated than other Bacillus species. Most of Rhizobium isolates were salt tolerant being able to grow in media containing 3% and 6% NaCl. The abilities of different bacterial isolates to attack citrus pectin, soluble and insoluble forms of cellulose were also tested.

Cloning of a thermostable alpha-amylase gene from Thermomonospora curvata and its expression in Streptomyces lividans.

The gene from Thermomonospora curvata CCM 3312 coding for thermostable alpha-amylase (tam) has been cloned in Streptomyces lividans TK 24 and localized to a 2.6 kb HindIII-BamHI fragment of DNA. The data presented here show that the tam gene is expressed at a high level in S. lividans and that the protein is efficiently excreted.

[Isolation of Actinomycetes synthesizing proteases with thrombolytic activity]. / Vydelenie aktinomitsetov, sinteziruiushchikh proteazy s tromboliticheskim deistviem.

Proteases with the thrombolytic activity were studied in 212 strains of actinomycetes isolated from different soils of the Soviet Union. The cultures belonged to the genera Micromonospora, Nocardia and Streptomyces. Proteases were synthesized by 41% of the studied actinomycetes and some of their strains completely dissolved in vitro artificially obtained blood thrombi within 120-240 min. In the Streptomyces genus, more active strains were found in the groups Flavus, Fradia and Globisporus. The groups Olivaceus, Violaceus and Viridis had less active strains.