Nitrosococcus Oceanus Research Articles

Comparative metagenomic analysis from Sundarbans ecosystems advances our understanding of microbial communities and their functional roles

The Sundarbans mangrove, located at the mouth of the Ganges and Brahmaputra Rivers, is the world’s largest tidal mangrove forest. These mangroves are also one of the most striking sources of microbial diversity, essential in productivity, conservation, nutrient cycling, and rehabilitation. Hence, the main objective of this study was to use metagenome analysis and provide detailed insight into microbial communities and their functional roles in the Sundarbans mangrove ecosystem. A comparative analysis was also done with a non-mangrove region of the Sundarbans ecosystem to assess the capability of the environmental parameters to explain the variation in microbial community composition. The study found several dominant bacteria, viz., Alphaproteobacteria, Actinomycetota, Bacilli, Clostridia, Desulfobacterota, Gammaproteobacteria, and Nitrospira, from the mangrove region. The mangrove sampling site reports several salt-tolerant bacteria like Alkalibacillus haloalkaliphilus, Halomonas anticariensis, and Salinivibrio socompensis. We found some probiotic species, viz., Bacillus clausii, Lactobacillus curvatus, Vibrio mediterranei and Vibrio fluvialis, from the Sundarbans mangrove. Nitrifying bacteria in Sundarbans soils were Nitrococcus mobilis, Nitrosococcus oceani, Nitrosomonas halophila, Nitrospirade fluvii, and others. Methanogenic archaea, viz., Methanoculleus marisnigri, Methanobrevibacter gottschalkii, and Methanolacinia petrolearia, were highly abundant in the mangroves as compared to the non-mangrove soils. The identified methanotrophic bacterial species, viz., Methylobacter tundripaludum, Methylococcus capsulatus, Methylophaga thiooxydans, and Methylosarcina lacus are expected to play a significant role in the degradation of methane in mangrove soil. Among the bioremediation bacterial species identified, Pseudomonas alcaligenes, Pseudomonas mendocina, Paracoccus denitrificans, and Shewanella putrefaciens play a significant role in the remediation of environmental pollution. Overall, our study shows for the first time that the Sundarbans, the largest mangrove ecosystem in the world, has a wide range of methanogenic archaea, methanotrophs, pathogenic, salt-tolerant, probiotic, nitrifying, and bioremediation bacteria.

Wastewater treatment from a science faculty during the COVID-19 pandemic by using ammonium-oxidising and heterotrophic bacteria

During and after the pandemic caused by the SARS-CoV-2 virus, the use of personal care products and disinfectants increased in universities worldwide. Among these, quaternary ammonium-based products stand out; these compounds and their intermediates caused substantial changes in the chemical composition of the wastewater produced by these institutions. For this reason, improvements and environmentally sustainable biological alternatives were introduced in the existing treatment systems so that these institutions could continue their research and teaching activities. For this reason, the objective of this study was to develop an improved culture medium to cultivate ammonium oxidising bacteria (AOB) to increase the biomass and use them in the treatment of wastewater produced in a faculty of sciences in Bogotá, D.C., Colombia. A Plackett Burman Experimental Design (PBED) and growth curves served for oligotrophic culture medium, and production conditions improved for the AOB. Finally, these bacteria were used with total heterotrophic bacteria (THB) for wastewater treatment in a pilot plant. Modification of base ammonium broth and culture conditions (6607 mg L−1 of (NH4)2SO4, 84 mg L−1 CaCO3, 40 mg L−1 MgSO4·7H2O, 40 mg L−1 CaCl2·2H2O and 200 mg L−1 KH2PO4, 10% (w/v) inoculum, no copper addition, pH 7.0 ± 0.2, 200 r.p.m., 30 days) favoured the growth of Nitrosomonas europea, Nitrosococcus oceani, and Nitrosospira multiformis with values of 8.23 ± 1.9, 7.56 ± 0.7 and 4.2 ± 0.4 Log10 CFU mL−1, respectively. NO2− production was 0.396 ± 0.0264, 0.247 ± 0.013 and 0.185 ± 0.003 mg L−1 for Nitrosomonas europea, Nitrosococcus oceani and Nitrosospira multiformis. After the 5-day wastewater treatment (WW) by co-inoculating the three studied bacteria in the wastewater (with their self-microorganisms), the concentrations of AOB and THB were 5.92 and 9.3 Log10 CFU mL−1, respectively. These values were related to the oxidative decrease of Chemical Oxygen Demand (COD), (39.5 mg L−1), Ammonium ion (NH4+), (6.5 mg L−1) Nitrite (NO2−), (2.0 mg L−1) and Nitrate (NO3−), (1.5 mg L−1), respectively in the five days of treatment. It was concluded, with the improvement of a culture medium and production conditions for three AOB through biotechnological strategies at the laboratory scale, being a promising alternative to bio-augment of the biomass of the studied bacteria under controlled conditions that allow the aerobic removal of COD and nitrogen cycle intermediates present in the studied wastewater.

Oxidation of iodide to iodate by cultures of marine ammonia-oxidising bacteria

Reaction with iodide (I−) at the sea surface is an important sink for atmospheric ozone, and causes sea-air emission of reactive iodine which in turn drives further ozone destruction. To incorporate this process into chemical transport models, improved understanding of the factors controlling marine iodine speciation, and especially sea-surface iodide concentrations, is needed. The oxidation of I− to iodate (IO3−) is the main sink for oceanic I−, but the mechanism for this remains unknown. We demonstrate for the first time that marine nitrifying bacteria mediate I− oxidation to IO3−. A significant increase in IO3− concentrations compared to media-only controls was observed in cultures of the ammonia-oxidising bacteria Nitrosomonas sp. (Nm51) and Nitrosoccocus oceani (Nc10) supplied with 9–10 mM I−, indicating I− oxidation to IO3−. Cell-normalised production rates were 15.69 (±4.71) fmol IO3− cell−1 d−1 for Nitrosomonas sp., and 11.96 (±6.96) fmol IO3− cell−1 d−1 for Nitrosococcus oceani, and molar ratios of iodate-to-nitrite production were 9.2 ± 4.1 and 1.88 ± 0.91 respectively. Preliminary experiments on nitrite-oxidising bacteria showed no evidence of I− to IO3− oxidation. If the link between ammonia and I− oxidation observed here is representative, our ocean iodine cycling model predicts that future changes in marine nitrification could alter global sea surface I− fields with potential implications for atmospheric chemistry and air quality.

Marine ammonia-oxidising archaea and bacteria occupy distinct iron and copper niches

Ammonia oxidation by archaea and bacteria (AOA and AOB), is the first step of nitrification in the oceans. As AOA have an ammonium affinity 200-fold higher than AOB isolates, the chemical niche allowing AOB to persist in the oligotrophic ocean remains unclear. Here we show that marine isolates, Nitrosopumilus maritimus strain SCM1 (AOA) and Nitrosococcus oceani strain C-107 (AOB) have contrasting physiologies in response to the trace metals iron (Fe) and copper (Cu), holding potential implications for their niche separation in the oceans. A greater affinity for unchelated Fe may allow AOB to inhabit shallower, euphotic waters where ammonium supply is high, but competition for Fe is rife. In contrast to AOB, AOA isolates have a greater affinity and toxicity threshold for unchelated Cu providing additional explanation to the greater success of AOA in the marine environment where Cu availability can be highly variable. Using comparative genomics, we predict that the proteomic and metal transport basis giving rise to contrasting physiologies in isolates is widespread across phylogenetically diverse marine AOA and AOB that are not yet available in pure culture. Our results develop the testable hypothesis that ammonia oxidation may be limited by Cu in large tracts of the open ocean and suggest a relatively earlier emergence of AOB than AOA when considered in the context of evolving trace metal availabilities over geologic time.

High Synteny and Sequence Identity between Genomes of Nitrosococcus oceani Strains Isolated from Different Oceanic Gyres Reveals Genome Economization and Autochthonous Clonal Evolution.

The ammonia-oxidizing obligate aerobic chemolithoautotrophic gammaproteobacterium, Nitrosococcus oceani, is omnipresent in the world’s oceans and as such important to the global nitrogen cycle. We generated and compared high quality draft genome sequences of N. oceani strains isolated from the Northeast (AFC27) and Southeast (AFC132) Pacific Ocean and the coastal waters near Barbados at the interface between the Caribbean Sea and the North Atlantic Ocean (C-27) with the recently published Draft Genome Sequence of N. oceani Strain NS58 (West Pacific Ocean) and the complete genome sequence of N. oceani C-107, the type strain (ATCC 19707) isolated from the open North Atlantic, with the goal to identify indicators for the evolutionary origin of the species. The genomes of strains C–107, NS58, C-27, and AFC27 were highly conserved in content and synteny, and these four genomes contained one nearly sequence-identical plasmid. The genome of strain AFC132 revealed the presence of genetic inventory unknown from other marine ammonia-oxidizing bacteria such as genes encoding NiFe-hydrogenase and a non-ribosomal peptide synthetase (NRPS)-like siderophore biosynthesis module. Comparative genome analysis in context with the literature suggests that AFC132 represents a metabolically more diverse ancestral lineage to the other strains with C-107 and NS58 potentially being the youngest. The results suggest that the N. oceani species evolved by genome economization characterized by the loss of genes encoding catabolic diversity while acquiring a higher redundancy in inventory dedicated to nitrogen catabolism, both of which could have been facilitated by their rich complements of CRISPR/Cas and Restriction Modification systems.

Optimized inhibition assays reveal different inhibitory responses of hydroxylamine oxidoreductases from beta- and gamma-proteobacterial ammonium-oxidizing bacteria

Ammonia-oxidizing bacteria (AOB), ubiquitous chemoautotrophic bacteria, convert ammonia (NH3) to nitrite (NO2−) via hydroxylamine as energy source. Excessive growth of AOB, enhanced by applying large amounts of ammonium-fertilizer to the farmland, leads to nitrogen leaching and nitrous oxide gas emission. To suppress these unfavorable phenomena, nitrification inhibitors, AOB specific bactericides, are widely used in fertilized farmland. However, new nitrification inhibitors are desired because of toxicity and weak-effects of currently used inhibitors. Toward development of novel nitrification inhibitors that target hydroxylamine oxidoreductase (HAO), a key enzyme of nitrification in AOB, we established inhibitor evaluation systems that include simplified HAO purification procedure and high-throughput HAO activity assays for the purified enzymes and for the live AOB cells. The new assay systems allowed us to observe distinct inhibitory responses of HAOs from beta-proteobacterial AOB (βAOB) Nitrosomonas europaea (NeHAO) and gamma-proteobacterial AOB (γAOB) Nitrosococcus oceani (NoHAO) against phenylhydrazine, a well-known suicide inhibitor for NeHAO. Consistently, the live cells of N. europaea, Nitrosomonas sp. JPCCT2 and Nitrosospira multiformis of βAOB displayed higher responses to phenylhydrazine than those of γAOB N. oceani. Our homology modeling studies suggest that different inhibitory responses of βAOB and γAOB are originated from different local environments around the substrate-binding sites of HAOs in these two classes of bacteria due to substitutions of two residues. The results reported herein strongly recommend inhibitor screenings against both NeHAO of βAOB and NoHAO of γAOB to develop HAO-targeting nitrification inhibitors with wide anti-AOB spectra.

Differential responses of nitrifying archaea and bacteria to methylene blue toxicity.

Methylene blue, a heterocyclic aromatic chemical compound used to treat fish diseases in the ornamental fish aquaculture industry, is believed to impair nitrification as a side effect. However, very little is known about the toxicity of methylene blue to nitrifying micro-organisms. Here, we report the susceptibility of six bacterial and one archaeal ammonia-oxidizing micro-organisms to methylene blue within the range of 10ppb to 10ppm. Remarkably high susceptibility was observed in the archaeal species Nitrosopumilus maritimus compared to the bacterial species. Ammonia oxidation by Nitrosopumilus maritimus was inhibited 65% by 10ppb of methylene blue. Of the bacterial species examined, Nitrosococcus oceani was the most resistant to methylene blue toxicity. For similar inhibition of Nitrosococcusoceani (75% inhibition), one thousand times more methylene blue (10ppm) was needed. The examination of single cell viability on Nitrosomonas marina demonstrated that methylene blue is lethal to the cells rather than reducing their single cell ammonia oxidation activity. The level of susceptibility to methylene blue was related to the cell volume, intracytoplasmic membrane arrangement and the evolutionary lineage of nitrifying micro-organisms. Our findings are relevant for effectively using methylene blue in various aquaculture settings by helping minimize its impact on nitrifiers during the treatment of fish diseases. In the future, resistant nitrifiers such as Nitrosococcusoceani may be purposely added to aquaculture systems to maintain nitrification activity during treatments with methylene blue. The susceptibility of six bacterial and one archaeal nitrifying micro-organisms to methylene blue was tested. Remarkably high susceptibility was observed in the archaeal species compared to the bacterial species. The level of resistance to methylene blue was related to the cell volume, cytomembrane system and the taxonomic position of the nitrifying micro-organisms. This may be significant in the design and management of engineered nitrification systems and the stability of the nitrification process in various ecosystems if these systems are exposed to harmful chemicals or toxins.

Sheep grazing and soil bacterial diversity in shrublands of the Patagonian Monte, Argentina

Our objective was to assess whether long-term continuous sheep grazing was associated with the bacterial community of the Patagonian Monte soils. We randomly extracted soil samples from 5 plant-covered patches (PCP) and the nearest inter-canopy areas (IC) at sites with low (L), moderate (M) and heavy (H) grazing intensity. Bacterial communities were evaluated by denaturing gradient gel electrophoresis (DGGE) and dominant bands were sequenced. Dendrograms showed a different association among grazing sites in PCP compared to IC areas. In PCP, M and H sites were clustered and DGGE revealed a high similarity in the bacterial community regardless of grazing intensity. In IC areas, analyses clustered L and M sites. Band sequencing revealed that members of these communities belonged to Sphingobacteria, Solibacteres, Gammaproteobacteria, Betaproteobacteria and Bacilli. Some of the band sequences related to Nitrosococcus oceani and Nitrosococcus watsonii which are restricted to marine environments and salt lakes. Microcoleus vaginatus and diatom chloroplast-related sequences were mainly detected in IC areas from L and M. Heavy grazing could alter autotrophic bacteria/diatom composition in IC areas possibly due to physical disturbance of biological crusts by trampling, threatening the potential of crust re-formation. Grazing intensity affected soil bacterial communities differently in PCP and IC areas with soil crust, highlighting the importance of improving our understanding of microbial diversity responses to land use.

Isotopomeric characterization of nitrous oxide produced by reaction of enzymes extracted from nitrifying and denitrifying bacteria

Abstract. Nitrous oxide (N2O) is a potent greenhouse gas and produced in denitrification and nitrification by various microorganisms. Site preference (SP) of 15N in N2O, which is defined as the difference in the natural abundance of isotopomers 14N15NO and 15N14NO relative to 14N14NO, has been reported to be a useful tool to quantitatively distinguish N2O production pathways. To determine representative SP values for each microbial process, we firstly measured SP of N2O produced in the enzyme reaction of hydroxylamine oxidoreductase (HAO) purified from two species of ammonia oxidizing bacteria (AOB), Nitrosomonas europaea and Nitrosococcus oceani, and that of nitric oxide reductase (NOR) from Paracoccus denitrificans. The SP value for NOR reaction (−5.9 ± 2.1‰) showed nearly the same value as that reported for N2O produced by P. denitrificans in pure culture. In contrast, SP value for HAO reaction (36.3 ± 2.3‰) was a little higher than the values reported for N2O produced by AOB in aerobic pure culture. Using the SP values obtained by HAO and NOR reactions, we calculated relative contribution of the nitrite (NO2–) reduction (which is followed by NO reduction) to N2O production by N. oceani incubated under different O2 availability. Our calculations revealed that previous in vivo studies might have underestimated the SP value for the NH2OH oxidation pathway possibly due to a small contribution of NO2– reduction pathway. Further evaluation of isotopomer signatures of N2O using common enzymes of other processes related to N2O would improve the isotopomer analysis of N2O in various environments.

Effects of engineered silver nanoparticles on the growth and activity of ecologically important microbes.

Currently, little is known about the impact of silver nanoparticles (AgNPs) on ecologically important microorganisms such as ammonia-oxidizing bacteria (AOB). We performed a multi-analytical approach to demonstrate the effects of uncapped nanosilver (uAgNP), capped nanosilver (cAgNP) and Ag2SO4 on the activities of the AOB: Nitrosomonas europaea, Nitrosospira multiformis and Nitrosococcus oceani, and the growth of Escherichia coli and Bacillus subtilis as model bacterial systems in relation to AgNP type and concentration. All Ag treatments caused significant inhibition to the nitrification potential rates (NPRs) of Nitrosomonas europaea (decreased from 34 to < 16.7 μM NH4+ oxidized day−1), Nitrosospira multiformis (decreased from 46 to < 24.8 μM NH4+ oxidized day−1) and Nitrosococcus oceani (decreased from 26 to < 18.4 μM NH4+ oxidized day−1). Escherichia coli-Ag interactions revealed that the percentage of damaged E. coli cells was 45% greater with Ag2SO4, 39% with cAgNPs and 33% with uAgNPs compared with controls. Generally, the inhibitory effect on AOB NPRs and E. coli/B. subtilis growth was in the following order Ag2SO4 > cAgNP > uAgNP. In conclusion, AgNPs (especially cAgNPs) and Ag2SO4 adversely affected AOB activities and thus have the potential to severely impact key microbially driven processes such as nitrification in the environment.

Thermodynamic characterization of proton‐ionizable functional groups on the cell surfaces of ammonia‐oxidizing bacteria and archaea

The ammonia-oxidizing archaeon Nitrosopumilus maritimus strain SCM1 (strain SCM1), a representative of the Thaumarchaeota archaeal phylum, can sustain high specific rates of ammonia oxidation at ammonia concentrations too low to sustain metabolism by ammonia-oxidizing bacteria (AOB). One structural and biochemical difference between N.maritimus and AOB that might be related to the oligotrophic adaptation of strain SCM1 is the cell surface. A proteinaceous surface layer (S-layer) comprises the outermost boundary of the strain SCM1 cell envelope, as opposed to the lipopolysaccharide coat of Gram-negative AOB. In this work, we compared the surface reactivities of two archaea having an S-layer (strain SCM1 and Sulfolobus acidocaldarius) with those of four representative AOB (Nitrosospira briensis, Nitrosomonas europaea, Nitrosolobus multiformis, and Nitrosococcus oceani) using potentiometric and calorimetric titrations to evaluate differences in proton-ionizable surface sites. Strain SCM1 and S.acidocaldarius have a wider range of proton buffering (approximately pH 10-3.5) than the AOB (approximately pH 10-4), under the conditions investigated. Thermodynamic parameters describing proton-ionizable sites (acidity constants, enthalpies, and entropies of protonation) are consistent with these archaea having proton-ionizable amino acid side chains containing carboxyl, imidazole, thiol, hydroxyl, and amine functional groups. Phosphorous-bearing acidic functional groups, which might also be present, could be masked by imidazole and thiol functional groups. Parameters for the AOB are consistent with surface structures containing anionic oxygen ligands (carboxyl- and phosphorous-bearing acidic functional groups), thiols, and amines. In addition, our results showed that strain SCM1 has more reactive surface sites than the AOB and a high concentration of sites consistent with aspartic and/or glutamic acid. Because these alternative boundary layers mediate interaction with the local external environment, these data provide the basis for further comparisons of the thermodynamic behavior of surface reactivity toward essential nutrients.

Effect of Salinity on Hydroxylamine Oxidation in a Marine Ammonia-Oxidizing Gammaproteobacterium, &lt;i&gt;Nitrosococcus oceani&lt;/i&gt; strain NS58: Molecular and Catalytic Properties of Tetraheme Cytochrome &lt;i&gt;c&lt;/i&gt;-554

Effect of Salinity on Hydroxylamine Oxidation in a Marine Ammonia-Oxidizing Gammaproteobacterium, &lt;i&gt;Nitrosococcus oceani&lt;/i&gt; strain NS58: Molecular and Catalytic Properties of Tetraheme Cytochrome &lt;i&gt;c&lt;/i&gt;-554

Energy-mediated vs. ammonium-regulated gene expression in the obligate ammonia-oxidizing bacterium, Nitrosococcus oceani

Ammonia serves as the source of energy and reductant and as a signaling molecule that regulates gene expression in obligate ammonia-oxidizing chemolithotrophic microorganisms. The gammaproteobacterium, Nitrosococcus oceani, was the first obligate ammonia-oxidizer isolated from seawater and is one of the model systems for ammonia chemolithotrophy. We compared global transcriptional responses to ammonium and the catabolic intermediate, hydroxylamine, in ammonium-starved and non-starved cultures of N. oceani to discriminate transcriptional effects of ammonium from a change in overall energy and redox status upon catabolite availability. The most highly expressed genes from ammonium- or hydroxylamine-treated relative to starved cells are implicated in catabolic electron flow, carbon fixation, nitrogen assimilation, ribosome structure and stress tolerance. Catabolic inventory-encoding genes, including electron flow-terminating Complexes IV, FoF1 ATPase, transporters, and transcriptional regulators were among the most highly expressed genes in cells exposed only to ammonium relative to starved cells, although the differences compared to steady-state transcript levels were less pronounced. Reduction in steady-state mRNA levels from hydroxylamine-treated relative to starved-cells were less than five-fold. In contrast, several transcripts from ammonium-treated relative to starved cells were significantly less abundant including those for forward Complex I and a gene cluster of cytochrome c encoding proteins. Identified uneven steady-state transcript levels of co-expressed clustered genes support previously reported differential regulation at the levels of transcription and transcript stability. Our results differentiated between rapid regulation of core genes upon a change in cellular redox status vs. those responsive to ammonium as a signaling molecule in N. oceani, both confirming and extending our knowledge of metabolic modules involved in ammonia chemolithotrophy.

Expression, and Molecular and Enzymatic Characterization of Cu-Containing Nitrite Reductase from a Marine Ammonia-Oxidizing Gammaproteobacterium, &lt;i&gt;Nitrosococcus oceani&lt;/i&gt;

Ammonia-oxidizing bacteria (AOB) remove intracellular nitrite to prevent its toxicity by a nitrifier denitrification pathway involving two denitrifying enzymes, nitrite reductase and nitric oxide reductase. Here, a Cu-containing nitrite reductase from Nitrosococcus oceani strain NS58, a gammaproteobacterial marine AOB, was expressed in Escherichia coli and purified to homogeneity. Sequence homology analysis indicated that the nitrite reductase from N. oceani was phylogenetically closer to its counterparts from denitrifying bacteria than that of the betaproteobacterium Nitrosomonas europaea. The recombinant enzyme was a homotrimer of a 32 kDa subunit molecule. The enzyme was green in the oxidized state with absorption peaks at 455 nm and 575 nm. EPR spectroscopy indicated the presence of type 2 Cu. Molecular activities and the affinity constant for the nitrite were determined to be 1.6×103 s−1 and 52 μM, respectively.

Nitrosococcus watsonii sp. nov., a new species of marine obligate ammonia-oxidizing bacteria that is not omnipresent in the world's oceans: calls to validate the names ‘Nitrosococcus halophilus’ and ‘Nitrosomonas mobilis’

Local associations between anammox bacteria and obligate aerobic bacteria in the genus Nitrosococcus appear to be significant for ammonia oxidation in oxygen minimum zones. The literature on the genus Nitrosococcus in the Chromatiaceae family of purple sulfur bacteria (Gammaproteobacteria, Chromatiales) contains reports on four described species, Nitrosococcus nitrosus, Nitrosococcus oceani, 'Nitrosococcus halophilus' and 'Nitrosomonas mobilis', of which only N. nitrosus and N. oceani are validly published names and only N. oceani is omnipresent in the world's oceans. The species 'N. halophilus' with Nc4(T) as the type strain was proposed in 1990, but the species is not validly published. Phylogenetic analyses of signature genes, growth-physiological studies and an average nucleotide identity analysis between N. oceani ATCC19707(T) (C-107, Nc9), 'N. halophilus' strain Nc4(T) and Nitrosococcus sp. strain C-113 revealed that a proposal for a new species is warranted. Therefore, the provisional taxonomic assignment Nitrosococcus watsonii is proposed for Nitrosococcus sp. strain C-113(T) . Sequence analysis of Nitrosococcus haoAB signature genes detected in cultures enriched from Jiaozhou Bay sediments (China) identified only N. oceani-type sequences, suggesting that different patterns of distribution in the environment correlate with speciation in the genus Nitrosococcus.

Effect of Salinity on Hydroxylamine Oxidation in a Marine Ammonia-Oxidizing Gammaproteobacterium, Nitrosococcus oceani strain NS58: Molecular and Catalytic Properties of Tetraheme Cytochrome c-554

Tetraheme cytochrome c-554 is a physiological electron acceptor of hydroxylamine oxidoreductase (HAO), a core enzyme of ammonia oxidation in chemoautotrophic nitrifiers. Here we report the purification of cytochrome c-554 from Nitrosococcus oceani strain NS58, a marine gammaproteobacterial ammonia-oxidizing bacterium. The NS58 cytochrome is a 25 kDa-protein having four hemes c. The absorption spectrum of the cytochrome showed peaks at 420 nm, 523 nm, and 554 nm, with shoulders at around 430 nm and 580 nm in the reduced state. In contrast to the highly basic counterpart from the betaproteobacterium Nitrosomonas europaea, the NS58 cytochrome c-554 was an acidic protein whose isoelectric point was 4.6. HAO was also purified, and the reaction with the NS58 cytochrome was found to be salt-tolerant. Compared with the activity observed in a non-salt solution, 60% of the activity remained in a saline concentration comparable to that of seawater.

Ammonia‐dependent differential regulation of the gene cluster that encodes ammonia monooxygenase in Nitrosococcus oceani ATCC 19707

Molecular analysis of the ammonia monooxygenase-encoding gene cluster in Nitrosococcus oceani revealed that the amo genes are differentially expressed from three promoters dependent on the external ammonia concentration. Whereas a distal amoRCABD operon promoter, pC1, is active only in the presence of ammonia, identified proximal amoC (pC2) and amoA (pA) sigma-70-type promoters are constitutive. Promoter region pC2 also contains a consensus sequence for binding of RpoN, usually involved in nitrogen starvation response. Additional regulation of transcript stability by anti-termination and mRNA degradation are proposed and discussed.

Identification, Characterization and Expression of Drought Related alpha‐Crystalline Heat Shock Protein Gene (GHSP26) from Desi Cotton

In response to water deficit stress, plants show quantitative and qualitative differences in gene expression. By using differential display and RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) techniques an alpha crystalline‐type small heat shock protein gene (GHSP26) was isolated and characterized from Gossypium arboreum L. Alignments of 1108 bp genomic and 1026 bp cDNA sequences revealed that the GHSP26 gene comprises a single open reading frame of 230 amino acids and it contains a single intron. The gene product contains the highly conserved alpha crystalline region, spanning amino acid residues 133 to 217 and a Met‐rich region from 94 to 117aa at the N terminus. Predicted amino acid sequence shares 65%, 63%, 59%, 58%, 56%, 55%, 53%, and 22% identities with Petunia hybrida, Nicotiana tabacum, Arabidopsis thaliana, Zea mays, Agrostis stolonifera, Triticum aestivum, Oryza sative, and Nitrosococcus oceani, respectively. Expression profile of the gene was studied from leaf, stem, and root tissues, through reverse transcriptase polymerase chain reaction (RT‐PCR) and quantitative real‐time RT‐PCR analysis. The results indicated that the gene was expressed in all tissues tested in both fully hydrated and dehydrated plants. However, the gene was 100‐fold more abundant in dehydrated leaves, while only two‐fold abundant in stressed root and stem as compared to control tissues.

Gene Cassette Encoding a 3- N -Aminoglycoside Acetyltransferase in a Chromosomal Integron

The large number and variety of antibiotic resistance genes found in gene cassettes associated with class 1 integrons in multiply antibiotic-resistant organisms have always indicated the existence of a large pool of gene cassettes from which they are drawn (10). The discovery of integrons, sometimes harboring very large arrays of gene cassettes, in the chromosomes of many different bacteria has confirmed the existence of this pool, but to date very few cassettes containing resistance genes have been found among these cassettes. Known examples are the catB9 (12) and blaP7 (blaP9) (7, 8) cassettes found in the cassette arrays of different Vibrio cholerae strains. Though 3-N-aminoglycoside acetyltransferases [AAC(3) enzymes] have been classified into several groups based on differences in the spectrum of aminoglycosides they modify, they fall into only two groups based on the relationships between the proteins (6). The five characterized aacC genes found in gene cassettes (see Table ​Table1)1) encode proteins of 154 to 158 amino acids that are related to one another and form family A (6). They belong to the aac(3)-I phenotypic group and confer resistance to gentamicin, sisomicin, and fortimicin but not to tobramycin, amikacin, or kanamycin (1, 3, 6, 11, 13, 15). TABLE 1. aacC-A genes and gene cassettes Our recent searches have revealed further potential aacC-A-type genes (Table ​(Table1).1). One is in a cassette found in a class 1 integron, but three of them are in draft sequences of bacterial genomes. The aacC-A7 gene in the complete Saccharophagus degradans (formerly Microbulbifer degradans) genome also lies within an identifiable gene cassette. The putative AacC-A7 [AAC(3)-Ig] acetyltransferase encoded by this cassette is 51 to 60% identical to other cassette-encoded AacC-A enzymes and most closely related to AacC-A5 [AAC(3)-Ie]. Alignment of the established and putative AacC-A sequences (Fig. ​(Fig.1)1) reveals 45 completely conserved amino acids, or 62 if only AacC-A1 to AacC-A7 are considered. It therefore seems reasonable to assume that aacC-A7 also confers resistance to aminoglycoside antibiotics. FIG. 1. Alignment of proteins in the AacC-A [AAC(3)-I] family. Amino acids completely conserved in all sequences are shown as white on black and are indicated by uppercase letters below the sequence. Amino acids conserved in 9 of the 10 sequences are indicated ... This represents only the third identification of a potential antibiotic resistance gene associated with a gene cassette that is part of the genome of a microorganism that harbors an integron. Though each aacC-A cassette has an identifiable 59-be (sometimes called an attC site) made up of two simple sites and a central region as is characteristic for 59-be (14), in most cases they are not close relatives of one another and some are not closely related to ones found in any other known cassette (6, 11). The 59-be of the Saccharophagus aacC-A7 cassette is not closely related to a 59-be in any of the many cassettes in that organism's genome. Its closest relative is in a cassette from Nitrosococcus oceani. Thus, if, as is generally assumed, cassettes that arose in a particular organism have very similar 59-be, it is unlikely that the aacC-A7 cassette was originally formed in S. degradans.

Susceptibility of ammonia-oxidizing bacteria to nitrification inhibitors.

Activity of nitrification inhibitors to several typical ammonia-oxidizing bacteria isolated recently, i. e. Nitrosococcus, Nitrosolobus, Nitrosomonas, Nitrosospira and Nitrosovibrio species was assayed using 2-amino-4-methyl-trichloromethyl-1,3,5-triazine (MAST), 2-amino-4-tribromomethyl-6-trichloromethyl-1,3,5-triazine (Br-MAST), 2-chloro-6-trichloromethylpyridine (nitrapyrin) and others, and compared to confirm the adequate control of ammonia-oxidizing bacteria by the inhibitors. The order of activity of the inhibitors to 13 species of ammonia-oxidizing bacteria examined was approximately summarized as Br-MAST > or = nitrapyrin > or = MAST > other inhibitors. Two Nitrosomonas strains, N. europaea ATCC25978 and N. sp. B2, were extremely susceptible to Br-MAST, exhibiting a pI50 > or = 6.40. These values are the position logarithms of the molar half-inhibition concentration. The 16S rRNA gene sequence similarity for the highly susceptible 4 strains of genus Nitrosomonas was 94% to 100% of Nitrosomonas europaea, although those of the less susceptible 3 strains of ammonia-oxidizing bacteria, Nitrosococcus oceanus C-107 ATCC19707, Nitrosolobus sp. PJA1 and Nitrosolobus multiformis ATCC25196, were 77.85, 91.53 and 90.29, respectively. However, no clear correlation has been found yet between pI50-values and percent similarity of 16S rRNA gene sequence among ammonia-oxidizing bacteria.