Ornibactins—a new family of siderophores from Pseudomonas

Novel linear hydroxamate/hydroxycarboxylate siderophores from strains of Pseudomonas cepacia were isolated and named ornibactins. The ornibactins represent modified tetrapeptide siderophores, possessing the sequence l-Orn¹(N -OH, N -acyl)-d-threo-Asp(-OH)-l-Ser-l-Orn⁴(N -OH, N -formyl)-1,4-diaminobutane. The N -acyl groups of Orn¹(N -OH, N -acyl) may vary and represent the three acids 3-hydroxybutanoic acid, 3-hydroxyhexanoic acid and 3-hydroxyoctanoic acid, leading to a mixture of three different ornibactins, designated according to their acyl chain length as ornibactin-C4, ornibactin-C6 and ornibactin-C8. Each of the siderophores is accompanied by a small amount of a more hydrophilic component with a 16 a.m.u. higher mass. The structure elucidation was based on results from gas chromatography amino acid analysis, electrospray mass spectrometry, and one- and two-dimensional nuclear magnetic resonance techniques.     

Ilyocryptus smirnovi n. sp. (Branchiopoda,Anomopoda) – a new member of the species group Ilyocryptus brevidentatus s. lat. from Australia

All representatives of the genus Ilyocryptus with short lateral setae at the postanal margin of the postabdomen may be separated into the compact species group I. brevidentatus s. lat. A new species of Ilyocryptus from this group is described from the Lake Numalla, Queensland, Australia. I. smirnovi n. sp. shows clear morphological differences in the armature of the valve, postabdomen, and several other structures, from all other species of the group. Differences between members of the above-mentioned species group are analogous with those between European species, listed by Štifter (1991). This revised version was published online in July 2006 with corrections to the Cover Date.     

Cloning and characterization of a new β-mannosidase from Streptomyces sp. S27

A new β-mannosidase gene, designated as man2S27, was cloned from Streptomyces sp. S27 using the colony PCR method and expressed in Escherichia coli BL21 (DE3). The full-length gene consists of 2499 bp and encodes 832 amino acids with a calculated molecular mass of 92.6 kDa. The amino acid sequence shares highest identity of 62.6% with the mannosidase Man2A from Cellulomonas fimi which belongs to the glycoside hydrolase family 2. Purified recombinant Man2S27 showed optimal activity at pH 7.0 and 50 °C. The specific activity, K_m, and k_cat values for p-nitrophenyl-β-d-mannopyranoside (p-NP-β-MP) were 35.3 U mg^-1, 0.23 mM, and 305 s^-1, respectively. Low transglycosylation activity was observed when Man2S27 was incubated with p-NP-β-MP (glycosyl donor) and methyl-α-d-mannopyranoside (p-NP-α-MP) (acceptor) at 50 °C and pH 7.0, and a small amount of methylmannobioside was synthesized. Using locust bean gum as the substrate, more reducing sugars were liberated by the synergistic action of Man2S27 and β-mannanase (Man5S27), and the synergy degree in sequential reactions with Man5S27 firstly and Man2S27 secondly was higher than that in the simultaneous reactions.     

Arnoldiellina fluorescens gen. et sp. nov. – A new green autofluorescent foraminifer from the Gulf of Eilat (Israel)

A new monothalamous (single-chambered) soft-walled foraminiferal species, Arnoldiellina fluorescens gen. et sp. nov., was isolated from samples collected in the Gulf of Eilat, Israel. The species is characterized by a small elongate organic theca with a single aperture of allogromiids. It is characterized by the emission of green autofluorescence (GAF) that has so far not been reported from foraminifera. Phylogenetic analysis of a fragment of the 18S rDNA indicates that the species is related to a group of monothalamous foraminiferans classified as clade I. Although the morphology of the new species is very different compared to the other members of this clade, a specific helix in 18S rRNA secondary structure strongly supports this position.     

Characterization of α-maltotetraohydrolase produced by Pseudomonas sp. MS300 isolated from the deepest site of the Mariana Trench

We have isolated a Pseudomonas-like amylase producer, the strain MS300, which displayed a large halo on starch medium, from the deepest site of the Mariana Trench. The strain MS300 produced two major and two minor α-maltotetraohydrolases (G4-amylase). The two major G4-amylases share the same molecular weight of 55 000 but had different pI values, 5.0 and 4.7, respectively. The optimum temperature for activity of both major G4-amylases is 40°C, and the optimum pH is 6.8 for one and 8.9 for the other. MS300 produced more amylase under high hydrostatic pressure than under atmospheric pressure. Strain MS300 may be active in the deep sea at a depth of 10 897 m. Received: December 11, 1997 / Accepted: April 16, 1998     

l-Alanine-α-Ketobutyrate Aminotransferase of Pseudomonas sp.

Modified fungal product 4-O-methylascochlorin (MAC) is an experimental agent affecting lipid and carbohydrate metabolism in mammals. The hypocholesterolemic properties of MAC were studied using rats fed on a standard laboratory diet. Because of the insolubility in water, reproducibility of the hypocholesterolemic activity had usually been poor for rats fed ad libitum. The difficulty was overcome by controlled reverse-phase feeding; MAC significantly lowered serum total cholesterol (s-TC) in rats only when given by gastric intubation soon after diet intake.

MAC increased fecal excretion of neutral and acidic sterols and also increased biliary flow accompanying increments in biliary cholesterol, bile acids and phospholipids. A much larger increase in neutral sterols was characteristic for MAC. However, intestinal absorption of cholesterol and cholic acid was unaffected by MAC. Three mechanisms therefore seemed to be working in hypocholesterolemic activity: (a) withdrawal of hepatic cholesterol into bile, (b) a larger fecal loss of sterols following increment of biliary sterols and (c) enhanced bile acid synthesis compensating the larger fecal loss. A negative sterol balance often leads to an increase in hepatic cholesterogenesis. However, cholesterogenesis, as judged from incorporation of the precursors, was unchanged by MAC.     

Cloning, sequencing, characterization, and expression of a new α-amylase isozyme gene (amy3) from Pseudomonas sp.

A new gene encoding an -amylase has been cloned, sequenced and expressed in E. coli from an alkaliphilic Pseudomonas sp. KFCC10818. The structural gene is 1356 base pairs long and encodes a protein of 452 amino acids. The recombinant -amylase has been purified and biochemically characterized. Molecular mass of the protein deduced from SDS-PAGE was 50 kDa. The enzyme showed an activity optimum at pH 8 and at 40 °C with complete stability at pH 13 for 3 h. The enzyme released maltose and maltotriose on hydrolysis of soluble starch. Amylose was hydrolysed over 5 times faster than amylopectin by the enzyme while the hydrolysis of cyclodextrin or pullulan was negligible.     

Exosporodiella gen. nov. – a new coelomycetous fungus on leaves of Phoenix from India

A new coelomycetous fungus Exosporodiella phoenixesa gen. et spec, nov., has been described and illustrated from the leaves of Phoenix from India.     

Cloning and characterization of a new broadspecific β-glucosidase from Lactococcus sp. FSJ4

A β-glucosidase gene bglX was cloned from Lactococcus sp. FSJ4 by the method of shotgun. The bglX open reading frame consisted of 1,437 bp, encoding 478 amino acids. SDS-PAGE showed a recombinant bglX monomer of 54 kDa. Substrate specificity study revealed that the enzyme exhibited multifunctional catalysis activity against pNPG, pNPX and pNPGal. This enzyme shows higher activity against aryl glycosides of xylose than those of glucose or galactose. The enzyme exhibited the maximal activity at 40 °C, and the optimal pH was 6.0 with pNPG and 6.5 with pNPX as the substrates. Molecular modeling and substrate docking showed that there should be one active center responsible for the mutifuntional activity in this enzyme, since the active site pocket was substantially wide to allow the entry of pNPG, pNPX and pNPGal, which elucidated the structure–function relationship in substrate specificities. Substrate docking results indicated that Glu180 and Glu377 were the essential catalytic residues of the enzyme. The CDOCKER_ENERGY values obtained by substrate docking indicated that the enzyme has higher activity against pNPX than those of pNPG and pNPGal. These observations are in conformity with the results obtained from experimental investigation. Therefore, such substrate specificity makes this β-glucosidase of great interest for further study on physiological and catalytic reaction processes.     

ε-PolyLysine production from sugar cane molasses by a new isolates of Bacillus sp. and optimization of the fermentation condition

An attempt was made to use cane molasses as a culture medium for ε-PolyLysine (ε-PL) production by a natural bacterial isolate. The bacterium was identified as Bacillus sp., as confirmed by 16S rDNA sequence analysis. A BLAST result of the sequence indicated that the closest relative of this Bacillus BHU strain was B. thuringiensis, with 97 % homology. The molasses was found to be a better culture medium compared to commonly used culture media comprised of either glucose or glycerol as a carbon source. The various physicochemical parameters were studied for culture growth and polymer production, and were further optimized using response surface methodology (RSM). The correlation coefficient of the resulting model was found to be R ²?=?0.9828. The RSM predicted optimum conditions for ε-PL production (2.46 g/l) by the Bacillus strain was achieved by using molasses, 59.7 g/l; yeast extract, 15.2 mg/l; pH, 6.8 and fermentation time, 42 h at 30 °C. This study represents the first report on the potential application of cane molasses (a byproduct of sugarcane industries) as a culture medium for ε-PL production by Bacillus species. The specific Bacillus strain used in the present study can be exploited for developing a novel technology using inexpensive renewable resources for ε-PL production, a polymer of commercial interest.     

Cloning, expression and characterization of a new ι-carrageenase from marine bacterium, Cellulophaga sp.

Purpose of work

The purpose of this study is to report a ι-carrageenase which degrades ι-carrageenan yielding neo-ι-carratetraose as the main product in the absence of NaCl. The gene for a new ι-carrageenase, CgiB_Ce, from Cellulophaga sp. QY3 was cloned and sequenced. It comprised an ORF of 1,386 bp encoding for a protein of 461 amino acid residues. From its sequence analysis, CgiB_Ce is a new member of GH family 82 and shared the highest identity of 32 % in amino acids with ι-carrageenase CgiA2 from Zobellia galactanovorans indicating that it is a hitherto uncharacterized protein. The recombinant CgiB_Ce had maximum specific activity (1,870 U/mg) at 45 °C and pH 6.5. It was stable between pH 6.0–9.6 and below 40 °C. Although its activity was enhanced by NaCl, the enzyme was active in the absence of NaCl. CgiB_Ce is an endo-type ι-carrageenase that hydrolyzes β-1,4-linkages of ι-carrageenan, yielding neo-ι-carratetraose as the main product (more than 80 % of the total product).     

Polychaos centronucleolus n. sp. – a new terrestrial species of the genus Polychaos (Amoebozoa,Tubulinea) with nontypical nuclear structure

A new species of the “proteus-type” naked amoebae (large cells with discrete tubular pseudopodia) was isolated from tree bark sample of a birch tree in the surrounding of Kislovodsk town, Russia and named Polychaos centronucleolus n. sp. (Amoebozoa, Tubulinea). Amoebae of this species have a filamentous cell coat and a nucleus with a central compact nucleolus. This type of nucleolar organization has not been previously known for the genus Polychaos. A sequence of the 18S rRNA gene of this strain was obtained using whole genome amplification of DNA from the single amoeba cell, followed by NGS sequencing. The analysis of molecular data robustly groups this species with Polychaos annulatum within the family Hartmannellidae. Our results, together with the results of our previous studies, show that the taxonomic assignment of “proteus-type” amoebae species is becoming increasingly complex, and the taxonomic characters that can be used to classify these organisms are becoming more shadowed.     

Monochoria vaginalis var. angustifolia，a new variety of the Pontederiaceae from Thailand

A new variety of Monochoria C. Presl from Thailand, M. vaginalis （N. L. Burman）Kunth var.angustifolia G. X. Wang, is described. This variety can be distinguished from the typical one, M. vaginalis var. vaginalis, by having mature leaves narrowly lanceolat     

Purification and Characterization of a Novel α-Agarase from a Thalassomonas sp.

An agar-degrading Thalassomonas bacterium, strain JAMB-A33, was isolated from the sediment off Noma Point, Japan, at a depth of 230 m. A novel -agarase from the isolate was purified to homogeneity from cultures containing agar as a carbon source. The molecular mass of the purified enzyme, designated as agaraseA33, was 85 kDa on both SDS-PAGE and gel-filtration chromatography, suggesting that it is a monomer. The optimal pH and temperature for activity were about 8.5 and 45°C, respectively. The enzyme had a specific activity of 40.7 U/mg protein. The pattern of agarose hydrolysis showed that the enzyme is an endo-type -agarase, and the final main product was agarotetraose. The enzyme degraded not only agarose but also agarohexaose, neoagarohexaose, and porphyran.     

BioF is a novel B2 metallo-β-lactamase from Pseudomonas sp. isolated from an on-farm biopurification system

Antimicrobial resistance represents a major global health concern and environmental bacteria are considered a source of resistance genes. Carbapenems are often used as the last antibiotic option to treat multidrug-resistant bacteria. Metallo-β-lactamases (MBLs) are able to render resistance to almost all β-lactam antibiotics, including carbapenems. Unfortunately, there are no inhibitors against MBLs for clinical use. Subclass B2 MBLs are the only enzymes working as strict carbapenemases, under-represented, encoded in chromosome genes and only functional as mono-zinc enzymes. Despite current efforts in MBLs inhibitor development, B2 carbapenemase activity is especially difficult to suppress, even in vitro. In this study we characterized BioF, a novel subclass B2 MBL identified in a new environmental Pseudomonas sp. strain isolated from an on-farm biopurification system (BPS). Although bla_BioF is most likely a chromosomal gene, it is found in a genomic island and may represent a step previous to the horizontal transmission of B2 genes. The new B2 MBL is active as a mono-zinc enzyme and is a potent carbapenemase with incipient activity against some cephalosporins. BioF activity is not affected by excess zinc and is only inhibited at high metal chelator concentrations. The discovery and characterization of B2 MBL BioF as a potent carbapenemase in a BPS bacterial isolate emphasizes the importance of exploring antibiotic resistances existing in the environmental microbiota under the influence of human activities before they could emerge clinically.     

Purification and characterization of d(+)-carnitine dehydrogenase from Agrobacterium sp. — a new enzyme of carnitine metabolism

d(+)-Carnitine dehydrogenase from Agrobacterium sp. catalyzes the oxidation of d(+)-carnitine to 3-dehydrocarnitine as initial step of d(+)-carnitine degradation. The NAD⁺-specific, cytosolic enzyme was purified 126-fold to apparent electrophoretic homogeneity by 4 chromatographic steps. The molecular mass of the native enzyme was estimated to be 88 kDa by size-exclusion chromatography. It seems to be composed of 3 identical subunits with a relative molecular mass of 28 kDa as found by sodium dodecyl sulfate polyacrylamide gel electrophoresis and laser-induced mass spectrometry. The isoelectric point was found to be 4.7–5.0. The optimum temperature is 37°C and the optimum pH for the oxidation and the reduction reaction are 9.0–9.5 and 5.5–6.5, respectively. The purified enzyme was further characterized with respect to substrate specificity, kinetic parameters and amino terminal sequence. Analogues of d(+)-carnitine (l(−)-carnitine, crotonobetaine, γ-butyrobetaine, carnitine amide, glycine betaine, choline) are competitive inhibitors of d(+)-carnitine oxidation. The equilibrium constant of the reaction of d(+)-carnitine dehydrogenase was determined to be 2.2 × 10⁻¹². The purified d(+)-carnitine dehydrogenase has similar kinetic properties to the l(−)-carnitine dehydrogenase from the same microorganism as well as to l(−)-carnitine dehydrogenases of other bacteria.     

Cobitis pontica sp. nova—a new spined loach species (Cobitidae) from the Bulgarian waters

Spined loaches from the Veleka River (Black Sea coast of Bulgaria), earlier confirmed to represent a separate evolution lineage within a 50-chromosome morph occurring in the Black Sea basin, are described as a new species. This species has the karyotype (2n = 50, NF = 90) similar to one from earlier described C. taurica occurring in the Crimean Peninsula, but differs from it by larger and less numerous spots in the fourth Gambetta’s zone of pigmentation and more anterior position of suborbital spine.     

Ancylobacter dichloromethanicus sp. nov. – a new aerobic facultatively methylotrophic bacterium utilizing dichloromethane

A novel aerobic facultative methylotroph was isolated from contaminated soil. The organism (strain DM16) is a Gram-negative asporogenous non-motile curved rod multiplying by binary fission. Cells are neutrophilic and mesophilic. This strain utilized dichloromethane, methanol, formate and formaldehyde along with a variety of polycarbon compounds. Strain DM16 employs the ribulosebisphosphate pathway for C₁ assimilation. The DNA G+C content is 64.5 mol%. The major ubiquinone is Q-10. The dominant cellular fatty acids are 18:1ω7c (58.6%), cyclo-19:0ω8c (34.8%) and 16:0 (3.2%). Sequencing of the 16S rRNA gene and DNA–DNA hybridization experiments clearly indicated that this methylotroph should be classified as a new species within genus Ancylobacter – Ancylobacter dichloromethanicus sp. nov. with the type strain DM16^T (DSM 21507^T=VKM B-2484^T).     

Biochemical Properties and Atomic Resolution Structure of a Proteolytically Processed β-Mannanase from Cellulolytic Streptomyces sp. SirexAA-E

β-mannanase SACTE_2347 from cellulolytic Streptomyces sp. SirexAA-E is abundantly secreted into the culture medium during growth on cellulosic materials. The enzyme is composed of domains from the glycoside hydrolase family 5 (GH5), fibronectin type-III (Fn3), and carbohydrate binding module family 2 (CBM2). After secretion, the enzyme is proteolyzed into three different, catalytically active variants with masses of 53, 42 and 34 kDa corresponding to the intact protein, loss of the CBM2 domain, or loss of both the Fn3 and CBM2 domains. The three variants had identical N-termini starting with Ala51, and the positions of specific proteolytic reactions in the linker sequences separating the three domains were identified. To conduct biochemical and structural characterizations, the natural proteolytic variants were reproduced by cloning and heterologously expressed in Escherichia coli. Each SACTE_2347 variant hydrolyzed only β-1,4 mannosidic linkages, and also reacted with pure mannans containing partial galactosyl- and/or glucosyl substitutions. Examination of the X-ray crystal structure of the GH5 domain of SACTE_2347 suggests that two loops adjacent to the active site channel, which have differences in position and length relative to other closely related mannanases, play a role in producing the observed substrate selectivity.     

Four new β-lactones from the endophytic Streptomyces sp. T1B1

The detailed investigation of endophytic Streptomyces sp. T1B1 was performed during a search for new structural and active compounds. The strain T1B1 was isolated from the old bast tissue of Taxus yunnanensis and determined to be a member of Streptomyces, according to the 16S rRNA analysis. The extracts from the PDA solid fermentation media of Streptomyces sp. T1B1 were purified and four β-lactones were isolated. They were identified as 4α-(3,5-dihydroxy hexyl)-3α-methyl-2-oxetanone (1), 4α-(3-methyl-4-formyloxy-hexyl)-3α-methyl-2-oxetanone (2), 4α-(3,5-dihydroxy-heptyl)-3α-methyl-2-oxetanone (3) and 4α-(3-methyl-4-formyloxy-heptyl)-3α-methyl-2-oxetanone (4) on the basis of spectral data.