Phylogenetic diversity of isolates belonging to genera Geobacillus and Aeribacillus isolated from different geothermal regions of Turkey

The phylogenetic diversity of 31 thermophilic bacilli belonging to genera Geobacillus and Aeribacillus were investigated which were isolated from various geothermal sites of Turkey. Twenty-seven of these isolates were found to be belonged within the genus Geobacillus, whereas 4 of them were identified as Aeribacillus pallidus. The comparative 16S rRNA gene sequence analyses revealed that the A. pallidus isolates displayed sequence similarity values from 98.0 to 99.6% to their closest relative. Furthermore, Geobacillus isolates showed sequence similarity values from 88.9 to 99.8% with the reference type strains. According to the phylogenetic analysis, isolates belonging to genus Geobacillus were diverged into nine clusters and among these isolates, 19 of them were identified as strains related to G. caldoproteolyticus, G. thermodenitrificans, G. stearothermophilus, G. thermoglucosidasius and G. toebii with the most abundant 13 isolates from G. caldoproteolyticus. Four of the Geobacillus isolates were named as unidentified mix group, as they found to be genetically very homogenous like their closely related type species: G. thermoleovorans, G. vulcani, G. lituanicus, G. kaustophilus, G. caldovelox, G. caldotenax, and G. uralicus. Moreover, the sequence comparisons of E173a, E265, C161ab and A142 isolates demonstrated that they represented novel species among genus Geobacillus as they shared lower than 96.7% sequence similarity to all the described type species. The AluI-, HaeIII- and TaqI-ARDRA results were in congruence with the 16S rRNA gene sequence analyses. By ARDRA results, the isolates were able to be differentiated and clustered, the discriminative restriction fragments of these isolates and type species were determined and the novelty of E173, E265, C161ab and A142 isolates could be displayed. Some differentiating phenotypic characters and the ability of amylase, glucosidase and protease production of these bacilli were also studied and biotechnologically valuable thermostable enzyme producing isolates were introduced in order to use in further studies.     

Detection of heavy metal ion resistance genes in Gram-positive and Gram-negative bacteria isolated from a lead-contaminated site

Resistance to a range of heavy metal ions wasdetermined for lead-resistant and other bacteria whichhad been isolated from a battery-manufacturing sitecontaminated with high concentrations of lead. Several Gram-positive (belonging to the genera Arthrobacter and Corynebacterium) andGram-negative (Alcaligenes species) isolateswere resistant to lead, mercury, cadmium, cobalt,zinc and copper, although the levels of resistance tothe different metal ions were specific for eachisolate. Polymerase chain reaction, DNA-DNAhybridization and DNA sequencing were used to explorethe nature of genetic systems responsible for themetal resistance in eight of the isolates. SpecificDNA sequences could be amplified from the genomic DNAof all the isolates using primers for sections of themer (mercury resistance determinant on thetransposon Tn501) and pco (copperresistance determinant on the plasmid pRJ1004) geneticsystems. Positive hybridizations with mer andpco probes indicated that the amplified segmentswere highly homologous to these genes. Some of thePCR products were cloned and partially sequenced, andthe regions sequenced were highly homologous to theappropriate regions of the mer and pcodeterminants. These results demonstrate the widedistribution of mercury and copper resistance genes inboth Gram-positive and Gram-negative isolates obtainedfrom this lead-contaminated soil. In contrast, theczc (cobalt, zinc and cadmium resistance) andchr (chromate resistance) genes could not beamplified from DNAs of some isolates, indicating thelimited contribution, if any, of these genetic systemsto the metal ion resistance of these isolates.     

Pseudomonas laurylsulfatovorans sp. nov., sodium dodecyl sulfate degrading bacteria,isolated from the peaty soil of a wastewater treatment plant

Pseudomonas are known from their flexible degradation capabilities and their engagement in xenobiotic biotransformation and bioremediation in habitats like soil, active sludge, plant surfaces, and freshwater or marine environments. Here we present taxonomic characterization of three efficient sodium dodecyl sulfate degrading strains: AP3_10, AP3_20 and AP3_22^T belonging to the genus Pseudomonas, recently isolated from peaty soil used in a biological wastewater treatment plant. Sequence analyses of 16S rRNA and housekeeping genes: gyrB, rpoD and rpoB showed that the three closely related isolates classify within the Pseudomonas jessenii subgroup. ANIb or dDDH genomic comparisons of AP3_22^T (type strain DSM 105098^T = PCM 2904^T) supported by biochemical tests showed that the isolates differ significantly from their closest relatives. The combined genotypic, phenotypic and chemotaxonomic data strongly support the classification of the three strains: AP3_10, AP3_20 and AP3_22^T as a novel species of Pseudomonas, for which we propose the name Pseudomonas laurylsulfatovorans sp. nov. with AP3_22^T as the type strain.     

Antibiotic and heavy metal resistance in Gram-negative bacteria isolated from the Seyhan Dam Lake and Seyhan River in Turkey

This study aimed to determine the pattern of antibiotic and heavy metal resistance in Gram-negative bacteria isolated from five different sites in the Seyhan Dam Lake and Seyhan River in Adana, Turkey. The susceptibility of 268 isolates to 16 different antibiotics and five heavy metals was investigated by agar diffusion and dilution methods, respectively. The most common species isolated from the samples were Aeromonas hydrophila (17.5 %), Aeromonas caviae (8.9 %) and Citrobacter freundii (8.9 %). There was a high incidence of resistance to ampicillin (80.2 %), streptomycin (71.6 %) and cefazolin (60.4 %). Multiple antibiotic resistance indices ranged from 0.2 to 0.81, suggesting exposure to antibiotic contamination. The isolates showed tolerance to different concentrations of heavy metals. These results indicate that antibiotic and heavy metal resistance among the Seyhan Dam Lake and Seyhan River bacteria may pose a risk to the fish population and public health. At the same time, the finding in the aquatic environments of different combinations of resistance genes suggests their involvement in the spread of multidrug-resistant strains.     

Effects of different molecular weight fractions of dissolved organic matter on the growth of bacteria,algae and protozoa from a highly humic lake

Effects of different molecular size fractions (< 1000 MW, < 10 000 MW, < 100 000 MW and <0.1 μm) of dissolved organic matter (DOM) on the growth of bacteria, algae and protozoa from a highly humic lake were investigated. DOM from catchment drainage water as well as from the lake consisted mostly (59–63%) of high molecular weight (HMW) compounds (> 10 000 MW). With excess inorganic nutrients, the growth rate and yield of bacteria were almost identical in all size fractions. However, in < 1000 MW fractions and with glucose added, a longer lag phase occurred. Without added nutrients both the growth rates and biomasses of bacteria decreased towards the smaller size fractions and the percentage of dissolved organic carbon (DOC) used during the experiment and the growth efficiency of bacteria were lower than with excess nutrients. The growth efficiency of bacteria was estimated to vary between 3–66% in different MW fractions, largely depending on the nutrient concentrations, but the highest growth efficiencies were observed in HMW fractions and with glucose. The growth of algae was clearly lowest in the < 1000 MW fraction. In dim light no net growth of algae could be found. In contrast, added nutrients substantially enhanced algal growth and in deionized water with glucose, algae achieved almost the same growth rate and biomass as in higher MW fractions of DOM. The results suggested that bacteria and some algae were favoured by DOM, but protozoans seemed to benefit only indirectly, through bacterial grazing. The utilization of DOM by bacteria and algae was strongly affected by the availability of phosphorus and nitrogen.     

Antigenic polysaccharides of bacteria. 27. Structure of the O-specific polysaccharide chain of lipopolysaccharides from Pseudomonas syringae pv. atrofaciens 2399, phaesolica 120a and Pseudomonas holci 8299 belonging to serotype VI

Lipopolysaccharides from Pseudomonas syringae pvs atrofaciens 2399. phaseolicola 120a and Pseudomonas holci 8299, belonging to serogroup VI. possess an identical polysaccharide chain composed of D-rhamnose and D-fucose. On the hasis of methylation, partial acid hydrolysis, 1H- and 13C-NMR data, it was concluded that the backbone of the polysaccharide represents D-rhamnan built up of tetrasaccharide repeating units and alpha-D-fucofuranose residues are attached to the backbone as the monosaccharide branches. The following structure of the repeating unit is established: (Formula: see text).     

Characterization of a metal resistant Pseudomonas sp. isolated from uranium mine for its potential in heavy metal (Ni2+, Co2+, Cu2+, and Cd2+) sequestration

Heavy metal sequestration by a multimetal resistant Pseudomonas strain isolated from a uranium mine was characterized for its potential application in metal bioremediation. 16S rRNA gene analysis revealed phylogenetic relatedness of this isolate to Pseudomonas fluorescens. Metal uptake by this bacterium was monophasic, fast saturating, concentration and pH dependent with maximum loading of 1048 nmol Ni²⁺ followed by 845 nmol Co²⁺, 828 nmol Cu²⁺ and 700 nmol Cd²⁺ mg^?1 dry wt. Preferential metal deposition in cell envelope was confirmed by TEM and cell fractionation. FTIR spectroscopy and EDX analysis revealed a major role of carboxyl and phosphoryl groups along with a possible ion exchange mechanism in cation binding. Binary system demonstrated selective metal binding affinity in the order of Cu²⁺ > Ni²⁺ > Co²⁺ > Cd²⁺. A comparison with similar metal uptake reports considering live bacteria strongly indicated the superiority of this strain in metal sequestration, which could be useful for developing efficient metal removal system.     

Analysis of gut microbiota in three species belonging to different genera (Hemitragus,Pseudois, and Ovis) from the subfamily Caprinae in the absence of environmental variance

This study aimed to identify the effects of host species on the gut microbial flora in three species (Hemitragus jemlahicus, Pseudois nayaur, and Ovis orientalis) from the subfamily Caprinae, by excluding the impact of environment factors. We investigated the differences in intestinal flora of three species belonging to Caprinae, which were raised in identical conditions. Fecal samples were collected from tahr, mouflon, and bharal, and the V3–V4 region of the 16S ribosomal RNA gene was analyzed by high‐throughput sequencing. The analysis of 16S rRNA gene sequences reveals that fecal samples were mainly composed of four phyla: Firmicutes, Bacteroidetes, Spirochaetes, and Proteobacteria. The most abundant phyla included Firmicutes and Bacteroidetes accounting for >90% of the bacteria, and a higher Firmicutes/Bacteroidetes ratio was observed in tahrs. Moreover, significant differences existed at multiple levels of classifications in the relative abundance of intestinal flora, differing greatly between species. Phylogenetic analyses based on 16S rRNA gene indicated that mouflon is closely related to bharal, and it is inconsistent with previous reports in the species evolutionary relationships. In this study, we demonstrated that the gut microbiota in tahr had a stronger ability to absorb and store energy from the diet compared with mouflon and bharal, and the characteristics of host–microbiome interactions were not significant.     

Construction of a bacterial biosensor for zinc and copper and its application to the development of multifunctional heavy metal adsorption bacteria

In this study, we designed and applied molecular biosensors for heavy metals, zinc and copper, for use in bioremediation strategies. Bacteria utilize two component systems to sense changes in the environment by multiple signal components including heavy metals and control gene expression in response to changes in signal molecules. zraP and cusC promoters were selected from a genetic circuit of the ZraSR and CusSR two-component system and were fused to a dual-labeling reporter protein as an interactive biological component for zinc and copper to generate a signal from the constructed biosensor. The biosensor efficiently senses zinc and copper with a calculated detection limit of 16 μM and 26 μM, respectively, and was shown to be a sensitive and effective heavy metal monitoring bacterial system. To extend the application of the bacterial biosensor, we assembled a bioadsorption system that can trigger bacteria to sense and adsorb 13 ± 0.3 mg/L of zinc and 11.4 ± 0.42 mg/L of copper per gram of dry cell weight with induction at a concentration of 100 mg/L of the respective metal ion.     

Two new genera of cyclopinid copepods (Cyclopoida: Gyclopinidae) from anchihaline caves of the Canary and Balearic Islands, with a key to genera of the family

The presence of an endemic cyclopoid fauna in anchihaline caves is confirmed in this paper after the discovery of two new genera of cyclopinids in caves of the Canary and Balearic Islands. Oromiina fortunata sp. nov. is described from a flooded lava tube in Lanzarote; it is characterized by the extraordinary length attained by the antennae. Ginesia longi-caudata sp. nov. is described from a flooded karstic cave in Mallorca. The body is ornamented dorsally with conspicuous integumental outgrowths, and along the lateral margins of both the cephalosome and the second pedigerous somite of male, by a linear array of pores. The new taxa are most closely related to forms found in the shallow water hyperbenthos, although one trait of Ginesia suggest an affinity with deep water taxa since, for example, the linear arrays of pores were previously known only in Cyclopicina within the Cyclopinidae. A key to the 37 genera of the family Cyclopinidae is presented.     

Two new species belonging to the genus Anisomysis (Crustacea, Mysidacea) and a new record of Anisomysis bipartoculata from the Indian Ocean

This paper deals with three species of the genus Anisomysiscollected from the South West coast of Australia of which A.gracilis and A. robustispina are new to science and A. bipanocularais reported for the first time from the Indian Ocean.     

Evidence for a cyclic AMP system highly sensitive to secretin in gastric glands isolated from the rat fundus and antrum

The effects of secretin and vasointestinal peptide (VIP) on the production of cyclic AMP have been studied in gastric glands isolated by means of EDTA from rat fundic and antral mucosa. (1) In gastric fundus, secretin and VIP caused a time- and temperature-dependent stimulation of cyclic AMP production that was maximal when the test agents were incubated for 60 min at 20 degrees C in the presence of 0.5 mM 3-isobutyl-1-methylxanthine as a phosphodiesterase inhibitor. The dose-response curve was monophasic for both peptides, the production of cyclic AMP being sensitive to 10(-10) M secretin and to 5 . 10(-8) M VIP. Half-maximal stimulation was obtained with 2.9 10(-9) M secretin or 2 . 10(-7) M VIP and the maximal stimulation represented a 21-fold and a 19-fold increase above control for secretin and VIP, respectively. Histamine also stimulated cyclic AMP production, with a Km of about 5 . 10(-4) M. No additive effect on cyclic AMP production was oberved when secretin and VIP were simultaneously added at maximally active concentrations, while an additive effect was observed when secretin and histamine were added together. (2) In gastric antrum, the characteristics of the secretin- and VIP-stimulated cyclic AMP production were similar to those observed in gastric fundus. Histamine nevertheless failed to stimulate the formation of cyclic AMP in antral mucosa. (3) These data demonstrate the existence of a cyclic AMP system highly sensitive to secretin in gastric glands isolated from the rat fundus and antrum and suggest that VIP operates through this system. (4) It is proposed that the pepsinogen- and/or mucous-secreting cells are implicated in the regulation of cyclic AMP production by secretin in gastric glands of the rat.     

Evaluation of the taxonomic diversity of prosthecate bacteria belonging to the genera <Emphasis Type="Italic">Brevundimonas</Emphasis> and <Emphasis Type="Italic">Caulobacter</Emphasis> isolated from various eurasian ecosystems by analysis of the 16S rRNA genes

A taxonomic study of 35 cultures of prosthecate bacteria of the genera Brevundimonas and Caulobacter isolated from various soil and aquatic ecosystems of Eurasia was performed by amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA gene sequencing. The most widespread groups of prosthecate bacteria belonging to these genera were revealed; at least two new species belonging to the genus Brevundimonas were found. The genus Brevundimonas includes both prosthecate and non-prosthecate species; however, it is quite possible that some Brevundimonas species may exhibit heterogeneity in such an important taxonomic characteristics as the ability to form prosthecae.     

Contribution of the hydrogen-bond network involving a tyrosine triad in the active site to the structure and function of a highly proficient ketosteroid isomerase from Pseudomonas putida biotype B

Delta(5)-3-Ketosteroid isomerase from Pseudomonas putida biotype B is one of the most proficient enzymes catalyzing an allylic isomerization reaction at rates comparable to the diffusion limit. The hydrogen-bond network (Asp99... Wat504...Tyr14...Tyr55...Tyr30) which links the two catalytic residues, Tyr14 and Asp99, to Tyr30, Tyr55, and a water molecule in the highly apolar active site has been characterized in an effort to identify its roles in function and stability. The DeltaG(U)(H2O) determined from equilibrium unfolding experiments reveals that the elimination of the hydroxyl group of Tyr14 or Tyr55 or the replacement of Asp99 with leucine results in a loss of conformational stability of 3.5-4.4 kcal/mol, suggesting that the hydrogen bonds of Tyr14, Tyr55, and Asp99 contribute significantly to stability. While decreasing the stability by about 6.5-7.9 kcal/mol, the Y55F/D99L or Y30F/D99L double mutation also reduced activity significantly, exhibiting a synergistic effect on k(cat) relative to the respective single mutations. These results indicate that the hydrogen-bond network is important for both stability and function. Additionally, they suggest that Tyr14 cannot function efficiently alone without additional support from the hydrogen bonds of Tyr55 and Asp99. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30. This observation supports the role of Tyr55 OH in positioning Tyr14 properly to optimize the hydrogen bond between Tyr14 and C3-O of the steroid substrate. No significant structural changes were observed in the crystal structures of Y30F and Y30F/Y55F, which allowed us to estimate approximately the interaction energies mediated by the hydrogen bonds Tyr30...Tyr55 and Tyr14...Tyr55. Taken together, our results demonstrate that the hydrogen-bond network provides the structural support that is needed for the enzyme to maintain the active-site geometry optimized for both function and stability.     

Two new species of Tovlinius Zaitzev, from China, with a key to the genera of Bombyliinae from China and a second key to the world species (Diptera, Bombyliidae, Bombyliinae, Bombyliini)

The genus Tovlinius Zaitzev is a Palaearctic genus with just one previously described species, Tovlinius albissimus Zaitzev. Tovlinius is here recorded from China for the first time, and two new species Tovlinius pyramidatussp. n. and Tovlinius turriformissp. n. are described and illustrated. A key to the genera of Bombyliinae from China and a second key to the World species of Tovlinius are also presented herein.     

Genomic analysis of a Kpi (pilus system)-positive and CTX-M-15-producing Klebsiella pneumoniae belonging to the high-risk clone ST15 isolated from an impacted river in Brazil

Convergence of resistance and virulence in Klebsiella pneumoniae is a critical public health issue worldwide. A multidrug-resistant CTX-M-15-producing K. pneumoniae (TIES-4900 strain) was isolated from a highly impacted urban river, in Brazil. The genome was sequenced by MiSeq Illumina platform and de novo assembled using Unicycler. In silico prediction was accomplished by bioinformatics tools. The size of the genome is 5.4 Mb with 5145 protein-coding genes. TIES-4900 strain belonged to the sequence type ST15, yersiniabactin sequence type YbST10, ICEKp4, KL24 (wzi-24) and O1v1 locus. Phylogenomics confirmed genomic relatedness with ST15 clones from human and animal hosts. Convergence of broad resistome (antibiotics, heavy-metals and biocides) and virulome, including the Kpi pilus system involved in host-pathogen interaction and persistence of ST15 clone to hospital environments, were predicted. Virulent behavior was confirmed in the Galleria mellonella infection model. This study may give genomic insights on the spread of critical-priority WHO pathogens beyond hospital settings.