Biodegradation of cyclic nitramines by tropical marine sediment bacteria

Undersea deposition of unexploded ordnance (UXO) constitutes a potential source of contamination of marine environments by hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). The goal of the present study was to determine microbial degradation of RDX and HMX in a tropical marine sediment sampled from a coastal UXO field in the region of Oahu Island in Hawaii. Sediment mixed cultures growing in marine broth 2216 (21°C) anaerobically mineralized 69% or 57% (CO₂, 25 days) of the total carbon of [UL-¹⁴ C]-RDX (100 M) or [UL-¹⁴ C]-HMX (10 M), respectively. As detected by PCR-DGGE, members of -proteobacteria (Halomonas), sulfate-reducing -proteobacteria (Desulfovibrio), firmicutes (Clostridium), and fusobacterium appeared to be dominant in RDX-enrichment and/or HMX-enrichment cultures. Among 22 sediment bacterial isolates screened for RDX and HMX biodegradation activity under anaerobic conditions, 5 were positive for RDX and identified as Halomonas (HAW-OC4), Marinobacter (HAW-OC1), Pseudoalteromonas (HAW-OC2 and OC5) and Bacillus (HAW-OC6) by their 16S rRNA genes. Sediment bacteria degraded RDX to N₂O and HCHO via the intermediary formation of hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and methylenedinitramine. The present findings demonstrate that cyclic nitramine contaminants are likely to be degraded upon release from UXO into tropical marine sediment.     

Comparative genomics of DNA fragments from six Antarctic marine planktonic bacteria

Six environmental fosmid clones from Antarctic coastal water bacterioplankton were completely sequenced. The genome fragments harbored small-subunit rRNA genes that were between 85 and 91% similar to those of their nearest cultivated relatives. The six fragments span four phyla, including the Gemmatimonadetes, Proteobacteria (alpha and gamma), Bacteroidetes, and high-G+C gram-positive bacteria. Gene-finding and annotation analyses identified 244 total open reading frames. Amino acid comparisons of 123 and 113 Antarctic bacterial amino acid sequences to mesophilic homologs from G+C-specific and SwissProt/UniProt databases, respectively, revealed widespread adaptation to the cold. The most significant changes in these Antarctic bacterial protein sequences included a reduction in salt-bridge-forming residues such as arginine, glutamic acid, and aspartic acid, reduced proline contents, and a reduction in stabilizing hydrophobic clusters. Stretches of disordered amino acids were significantly longer in the Antarctic sequences than in the mesophilic sequences. These characteristics were not specific to any one phylum, COG role category, or G+C content and imply that underlying genotypic and biochemical adaptations to the cold are inherent to life in the permanently subzero Antarctic waters.     

Conspicuous veils formed by vibrioid bacteria on sulfidic marine sediment

We describe the morphology and behavior of a hitherto unknown bacterial species that forms conspicuous veils (typical dimensions, 30 by 30 mm) on sulfidic marine sediment. The new bacteria were enriched on complex sulfidic medium within a benthic gradient chamber in oxygen-sulfide countergradients, but the bacteria have so far not been isolated in pure culture, and a detailed characterization of their metabolism is still lacking. The bacteria are colorless, gram-negative, and vibrioid-shaped (1.3- to 2.5- by 4- to 10- micro m) cells that multiply by binary division and contain several spherical inclusions of poly-beta-hydroxybutyric acid. The cells have bipolar polytrichous flagella and exhibit a unique swimming pattern, rotating and translating along their short axis. Free-swimming cells showed aerotaxis and aggregated at ca. 2 micro M oxygen within opposing oxygen-sulfide gradients, where they were able to attach via a mucous stalk, forming a cohesive whitish veil at the oxic-anoxic interface. Bacteria attached to the veil kept rotating and adapted their stalk lengths dynamically to changing oxygen concentrations. The joint action of rotating bacteria on the veil induced a homogeneous water flow from the oxic water region toward the veil, whereby the oxygen uptake rate could be enhanced up to six times, as shown by model calculations. The veils showed a pronounced succession pattern. New veils were generated de novo within 24 h and had a homogeneous whitish translucent appearance. Bacterial competitors or eukaryotic predators were apparently kept away by the low oxygen concentration prevailing at the veil surface. Frequently, within 2 days the veil developed a honeycomb pattern of regularly spaced holes. After 4 days, most veils were colonized by grazing ciliates, leading to the fast disappearance of the new bacteria. Several-week-old veils finally developed into microbial mats consisting of green, purple, and colorless sulfur bacteria.     

Thermophilic sulfate-reducing bacteria in cold marine sediment

Abstract Sulfate reduction was measured with the ³⁵SO₄²⁻ -tracer technique in slurries of sediment from Aarhus Bay, Denmark, where seasonal temperatures range from 0° to 15°C. The incubations were made at temperatures from 0°C to 80°C in temperature increments of 2°C to search for presence of psychrophilic, mesophilic and thermophilic sulfate-reducing bacteria. Detectable activity was initially only in the mesophilic range, but after a lag phase sulfate reduction by thermophilic sulfate-reducing bacteria were observed. No distinct activity of psychrophilic sulfate-reducing bacteria was detected. Time course experiments showed constant sulfate reduction rates at 4°C and 30°C, whereas the activity at 60°C increased exponentially after a lag period of one day. Thermophilic, endospore-forming sulfate-reducing bacteria, designated strain P60, were isolated and characterized as D esulfotomaculum kuznetsovii . The temperature response of growth and respiration of strain P60 agreed well with the measured sulfate reduction at 50°–70°C. Bacteria similar to strain P60 could thus be responsible for the measured thermophilic activity. The viable population of thermophilic sulfate-reducing bacteria and the density of their spores was determined in most probable number (MPN) dilutions. The density was 2.8·10⁴ cells·.g⁻¹ fresh sediment, and the enumerations suggested that they were all present as spores. This result agrees well with the observed lag period in sulfate reduction above 50°C. No environment with temperatures supporting the growth of these thermophiles is known in the region around Aarhus Bay.     

Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device

Polar biofilms have become an increasingly popular biological issue because new materials and phenotypes have been discovered in microorganisms in the polar region. Various environmental factors affect the functionality and adaptation of microorganisms. Because the polar region represents an extremely cold environment, polar microorganisms have a functionality different from that of normal microorganisms. Thus, determining the effective temperature for the development of polar biofilms is crucial. Here, we present a simple, novel one-pot assay for analysis of the effect of temperature on formation of Antarctic bacterial biofilm using a microfluidic system where continuous temperature gradients are generated. We find that a specific range of temperature is required for the growth of biofilms. Thus, this microfluidic approach provides precise information regarding the effective temperature for polar biofilm development with a new high-throughput screening format.     

Succession of cable bacteria and electric currents in marine sediment

Filamentous Desulfobulbaceae have been reported to conduct electrons over centimetre-long distances, thereby coupling oxygen reduction at the surface of marine sediment to sulphide oxidation in sub-surface layers. To understand how these ‘cable bacteria'' establish and sustain electric conductivity, we followed a population for 53 days after exposing sulphidic sediment with initially no detectable filaments to oxygen. After 10 days, cable bacteria and electric currents were established throughout the top 15 mm of the sediment, and after 21 days the filament density peaked with a total length of 2 km cm⁻². Cells elongated and divided at all depths with doubling times over the first 10 days of <20 h. Active, oriented movement must have occurred to explain the separation of O₂ and H₂S by 15 mm. Filament diameters varied from 0.4–1.7 μm, with a general increase over time and depth, and yet they shared 16S rRNA sequence identity of >98%. Comparison of the increase in biovolume and electric current density suggested high cellular growth efficiency. While the vertical expansion of filaments continued over time and reached 30 mm, the electric current density and biomass declined after 13 and 21 days, respectively. This might reflect a breakdown of short filaments as their solid sulphide sources became depleted in the top layers of the anoxic zone. In conclusion, cable bacteria combine rapid and efficient growth with oriented movement to establish and exploit the spatially separated half-reactions of sulphide oxidation and oxygen consumption.     

Agar-degrading bacteria isolated from Antarctic macroalgae

This study describes the taxonomic diversity of pigmented, agar-degrading bacteria isolated from the surface of macroalgae collected in King George Island, Antarctica. A total of 30 pigmented, agarolytic bacteria were isolated from the surface of the Antarctic macroalgae Adenocystis utricularis, Monostroma hariotii, Iridaea cordata, and Pantoneura plocamioides. Based on the 16S rRNA data, the agarolytic isolates were affiliated to the genera Algibacter, Arthrobacter, Brachybacterium, Cellulophaga, Citricoccus, Labedella, Microbacterium, Micrococcus, Salinibacterium, Sanguibacter, and Zobellia. Isolates phylogenetically related to Cellulophaga algicola showed the highest agarase activity in culture supernatants when tested at 4 and 37 °C. This is the first investigation of pigmented agar-degrading bacteria, members of microbial communities associated with Antarctic macroalgae, and the results suggest that they represent a potential source of cold-adapted agarases of possible biotechnological interest.     

Psychrotrophic amylolytic bacteria from deep sea sediment of Prydz Bay, Antarctic: diversity and characterization of amylases

Seventeen psychrotrophic bacteria with cold-adaptive amylolytic, lipolytic or proteolytic activity were isolated from deep sea sediment of Prydz Bay, Antarctic. They were affiliated with γ-Proteobacteria (12 strains) and gram-positive bacteria (5 strains) as determined by 16S rDNA sequencing. The amylase-producing strains belonged to genus Pseudomonas, Rhodococcus, and Nocardiopsis. Two Pseudomonas strains, 7193 and 7197, which showed highest amylolytic activity were chosen for further study. The optimal temperatures for their growth and amylase-producing were between 15 and 20°C. Both of the purified amylases showed highest activity at 40°C and pH 9.0, and retained 50% activity at 5°C. The SDS-PAGE and zymogram activity staining showed that the molecular mass of strain 7193 and 7197 amylases were about 60 and 50 kDa respectively. The Pseudomonas sp. 7193 amylase hydrolyzed soluble starch into glucose, maltose, maltotriose, and maltotetraose, indicating that it had both activities of α-amylase and glucoamylase. The product hydrolyzed by Pseudomonas sp. 7197 amylase was meltotetraose.     

Degradation of alkanes by bacteria

Pollution of soil and water environments by crude oil has been, and is still today, an important problem. Crude oil is a complex mixture of thousands of compounds. Among them, alkanes constitute the major fraction. Alkanes are saturated hydrocarbons of different sizes and structures. Although they are chemically very inert, most of them can be efficiently degraded by several microorganisms. This review summarizes current knowledge on how microorganisms degrade alkanes, focusing on the biochemical pathways used and on how the expression of pathway genes is regulated and integrated within cell physiology.     

Degradation of p-nitrophenol by Achromobacter xylosoxidans Ns isolated from wetland sediment

Achromobacter xylosoxidans Ns strain, capable of utilizing p-nitrophenol (PNP) as the sole source of carbon, energy, and nitrogen, was isolated from wetland sediment and confirmed based on 16S rRNA gene sequence. The strain Ns could tolerate concentrations of PNP up to 1.8 mM, and degradation of PNP was achieved in 7 d at 30 °C in the dark under aerobic conditions. Biodegradation of PNP occurred quickly at an optimal pH of 7.0 and higher, and at ⩽0.5% salt (NaCl) contents. During bacterial growth on PNP, 4-nitrocatechol was observed as a key degradation intermediate using a combination of techniques, including HPLC, UV–visible spectra, and comparison with the authentic standard. In a similar way, a second degradation intermediate was identified to be 1,2,4-benzenetriol. Moreover, A. xylosoxidans Ns could also degrade 3-nitrophenol as the sole source of carbon, nitrogen, and energy, but 2-nitrophenol could not. The experimental results showed that bacteria indigenous to the wetland sediment are capable of degradading PNP and chemicals with similar structures.     

Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment

A novel lipase was isolated from a metagenomic library of Baltic Sea sediment bacteria. Prokaryotic DNA was extracted and cloned into a copy control fosmid vector (pCC1FOS) generating a library of >7000 clones with inserts of 24-39 kb. Screening for clones expressing lipolytic activity based on the hydrolysis of tributyrin and p-nitrophenyl esters, identified 1% of the fosmids as positive. An insert of 29 kb was fragmented and subcloned. Subclones with lipolytic activity were sequenced and an open reading frame of 978 bp encoding a 35.4-kDa putative lipase/esterase h1Lip1 (DQ118648) with 54% amino acid similarity to a Pseudomonas putida esterase (BAD07370) was identified. Conserved regions, including the putative active site, GDSAG, a catalytic triad (Ser148, Glu242 and His272) and a HGG motif, were identified. The h1Lip1 lipase was over expressed, (pGEX-6P-3 vector), purified and shown to hydrolyse p-nitrophenyl esters of fatty acids with chain lengths up to C14. Hydrolysis of the triglyceride derivative 1,2-di-O-lauryl-rac-glycero-3-glutaric acid 6'-methylresorufin ester (DGGR) confirmed that h1Lip1 was a lipase. The apparent optimal temperature for h1Lip1, by hydrolysis of p-nitrophenyl butyrate, was 35 degrees C. Thermal stability analysis showed that h1Lip1 was unstable at 25 degrees C and inactivated at 40 degrees C with t1/2 <5 min.     

Chitin utilization by marine bacteria. Degradation and catabolism of chitin oligosaccharides by Vibrio furnissii.

Chemotaxis of the marine bacterium Vibrio furnissii to chitin oligosaccharides has been described (Bassler, B. L., Gibbons, P. J., Yu, C., and Roseman, S. (1991) J. Biol. Chem. 266, 24268-24275). Some steps in catabolism of the oligosaccharides are reported here. GlcNAc, (GlcNAc)2, and (GlcNAc)3 are very rapidly consumed by intact cells, about 320 nmol of GlcNAc equivalents/min/mg of protein. (GlcNAc)4 is utilized somewhat more slowly. During these processes, there is virtually no release of hydrolysis products by the cells. The oligosaccharides enter the periplasmic space (via specific porins?) and are hydrolyzed by a unique membrane-bound endoenzyme (chitodextrinase) and an exoenzyme (N-acetyl-beta-glucosaminidase; beta-Glc-NAcidase). The genes encoding these enzymes have been cloned and expressed in Escherichia coli. The chitodextrinase cleaves soluble oligomers, but not chitin, to the di- and trisaccharides, while the periplasmic beta-GlcNAcidase hydrolyzes the GlcNAc termini from the oligomers. The end products in the periplasm, GlcNAc and (GlcNAc)2 (possibly (GlcNAc)3) are catabolized as follows. (a) Disaccharide pathway, A (GlcNAc)2 permease is apparently expressed by Vibrio furnissii. Translocated (GlcNAc)2 is rapidly hydrolyzed by a soluble, cytosolic beta-GlcNAcidase, and the GlcNAc is phosphorylated by an ATP-dependent, constitutive kinase to GlcNAc-6-P. (b) Monosaccharide pathway, Periplasmic GlcNAc is taken up by Enzyme IINag of the phosphoenolpyruvate:glycose phosphotransferase system, yielding GlcNAc-6-P, the common intermediate for both pathways. Finally, GlcNAc-6-P----Ac- + GlcNH2-6-P----Fru-6-P + NH3. (GlcNAc)2 is probably the "true" inducer of the chitin degradative enzymes described in this report and, depending on its concentration in the growth medium, differentially induces the periplasmic and cytosolic beta-GlcNAcidases. The disaccharide pathway appears to be the most important when the cells are confronted with low concentrations of the oligomers (e.g. in chemotaxis swarm plates). The relative activities of the induced enzymes suggest that the rate-limiting steps in oligosaccharide catabolism are the glycosidase activities in the periplasm.     

Degradation of naphthenic acids by sediment micro-organisms

AIMS: Naphthenic acids (NAs) are naturally occurring, linear and cyclic carboxylic surfactants associated with the acidic fraction of petroleum. NAs account for most of the acute aquatic toxicity of oil sands process-affected water (OSPW). The toxicity of OSPW can be reduced by microbial degradation. The aim of this research was to determine the extent of NA degradation by sediment microbial communities exposed to varying amounts of OSPW. METHODS AND RESULTS: Eleven wetlands, both natural and process-affected, and one tailings settling pond in Northern Alberta were studied. The natural wetlands and process-affected sites fell into two distinct groups based on their water chemistry. The extent of degradation of a 14C-labelled monocyclic NA surrogate [14C-cyclohexane carboxylic acid (CCA)] was relatively uniform in all sediments (approximately 30%) after 14 days. In contrast, degradation of a bicyclic NA surrogate [14C-decahydronaphthoic acid (DHNA)] was significantly lower in non process-affected sediments. Enrichment cultures, obtained from an active tailings settling pond, using commercially available NAs as the sole carbon source, resulted in the isolation of a co-culture containing Pseudomonas putida and Pseudomonas fluorescens. Quantitative GC-MS analysis showed that the co-culture removed >95% of the commercial NAs, and partially degraded the process NAs from OSPW with a resulting NA profile similar to that from 'aged wetlands'. CONCLUSIONS: Exposure to NAs induced and/or selected micro-organisms capable of more effectively degrading bicyclic NAs. Native Pseudomonas spp. extensively degraded fresh, commercial NA. The recalcitrant NAs resembled those found in process-affected wetlands. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that it may be possible to manipulate the existing environmental conditions to select for a microbial community exhibiting higher rates of NA degradation. This will have significant impact on the design of artificial wetlands for water treatment.     

Isolation of denitrifying bacteria from hydrocarbon-contaminated Antarctic soil

In this study, we report the isolation of denitrifiers from hydrocarbon-contaminated Antarctic soils. Seventy-two isolates were obtained from soils that had received a fertilizer treatment to stimulate hydrocarbon degradation. All isolates, except one, belonged to the genus Pseudomonas. The one exception was a member of the Microbacteriaceae, which was also, coincidentally, the only isolate negative for the nirS gene. The diversity of the 16S rRNA and nosZ genes was assessed by denaturing gradient gel electrophoresis and sequencing. There was a slight correlation between the 16S rRNA and nosZ operational taxonomic units. Surprisingly, many isolates contained nosZ on plasmids and, to the best of our knowledge, this is the first report of nosZ being extra-chromosomally present in Pseudomonas spp.     

Production of exopolysaccharides by Antarctic marine bacterial isolates

AIMS: This study was undertaken to examine and characterize Antarctic marine bacterial isolates and the exopolysaccharides (EPS) they produce in laboratory culture. METHODS AND RESULTS: Two EPS-producing bacterial strains CAM025 and CAM036 were isolated from particulate material sampled from seawater and sea ice in the southern ocean. Analyses of 16S rDNA sequences placed these isolates in the genus Pseudoalteromonas. In batch culture, both strains produced EPS. The yield of EPS produced by CAM025 was 30-fold higher at -2 and 10 degrees C than at 20 degrees C. Crude chemical analyses showed that these EPS were composed primarily of neutral sugars and uronic acids with sulphates. Gas chromatographic analysis of monosaccharides confirmed these gross compositional findings and molar ratios of monosaccharides revealed differences between the two EPS. CONCLUSIONS: The EPS produced by Antarctic bacterial isolates examined in this study appeared to be polyanionic and, therefore, 'sticky' with respect to cations such as trace metals. SIGNIFICANCE AND IMPACT OF THE STUDY: As the availability of iron as a trace metal is of critical importance in the southern ocean where it is know to limit primary production, the role of these bacterial EPS in the Antarctic marine environment has important ecological implications.     

Degradation of substituted thiophenes by bacteria isolated from activated sludge

Actinomycetes were isolated from activated sludge acclimated to thiophene-2-carboxylic acid (T2C) or 5-methyl-thiophene-2-carboxylic acid (T5M2C). These isolates were apparently identical and were identified as strains ofRhodococcus. The strains could grow on T2C, T5M2C, or thiophene-2-acetic acid as sole sources of carbon and energy, but could not use thiophene, methyl thiophenes, several other substituted thiophenes, dibenzothiophene, dimethyl sulfide, or pyrrole-2-carboxylic acid. T2C was degraded quantitatively to sulfate, and its carbon was converted almost entirely to cell biomass and carbon dioxide. Growth yields indicated about 25% conversion of T2C-carbon to cell-carbon. Growth was not supported by thiosulfate or methionine, nor were these compounds oxidized.Rhodococcus strain TTD-1 grown on T2C oxidized both T2C and T5M2C with an apparent K_m of 1.3×10^–5 M. Sulfide was also oxidized by T2C-grown organisms. This is the first demonstration of an actinomycete capable of the complete degradation of thiophene derivatives and of their use by it as sole substrates for growth.     

A new species ofEupenicillium from marine sediment

A new species ofEupenicillium isolated from marine sediment,Eupenicillium limosum, is described and illustrated. This species is characterized by subglobose ascospores with spinulose surface ornamentation and irregular biverticillate penicilli.     

Degradation of lactoferrin by periodontitis-associated bacteria

Abstract The degradation of human lactoferrin by putative periodontopathogenic bacteria was examined. Fragments of lactoferrin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measured by densitometry. The degradation of lactoferrin was more extensive by Porphyromonas gingivalis and Capnocytophaga sputigena , slow by Capnocytophaga ochracea , Actinobacillus actinomycetemcomitans and Prevotella intermedia , and very slow or absent by Prevotella nigrescens , Campylobacter rectus, Campylobacter sputorum, Fusobacterium nucleatum ssp. nucleatum, Capnocytophaga gingivalis, Bacteroides forsythus and Peptostreptococcus micros . All strains of P. gingivalis tested degraded lactoferrin. The degradation was sensitive to protease inhibitors, cystatin C and albumin. The degradation by C. sputigena was not affected by the protease inhibitors and the detected lactoferrin fragments exhibited electrophoretic mobilities similar to those ascribed to deglycosylated forms of lactoferrin. Furthermore a weak or absent reactivity of these fragments with sialic acid-specific lectin suggested that they are desialylated. The present data indicate that certain bacteria colonizing the periodontal pocket can degrade lactoferrin. The presence of other human proteins as specific inhibitors and/or as substrate competitors may counteract this degradation process.