Mutational Analysis of the Multicopy hao Gene Coding for Hydroxylamine Oxidoreductase in Nitrosomonas sp. Strain ENI-11

The ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 contains three copies of the hao gene (hao ₁, hao ₂, and hao ₃) coding for hydroxylamine oxidoreductase (HAO). Three single mutants (hao ₁::kan, hao ₂::kan, or hao ₃::kan) had 68 to 75% of the wild-type growth rate and 58 to 89% of the wild-type HAO activity when grown under the same conditions. A double mutant (hao ₁::kan and hao ₃::amp) also had 68% of the wild-type growth and 37% of the wild-type HAO activity.     

Mutational analysis of the multicopy hao gene coding for hydroxylamine oxidoreductase in Nitrosomonas sp. strain ENI-11

The ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 contains three copies of the hao gene (hao1, hao2, and hao3) coding for hydroxylamine oxidoreductase (HAO). Three single mutants (hao1::kan, hao2::kan, or hao3::kan) had 68 to 75% of the wild-type growth rate and 58 to 89% of the wild-type HAO activity when grown under the same conditions. A double mutant (hao1::kan and hao3::amp) also had 68% of the wild-type growth and 37% of the wild-type HAO activity.     

Physical map location of the multicopy genes coding for ammonia monooxygenase and hydroxylamine oxidoreductase in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11

Pulsed-field gel electrophoresis of PmeI digests of the Nitrosomonas sp. strain ENI-11 chromosome produced four bands ranging from 1,200 to 480 kb in size. Southern hybridizations suggested that a 487-kb PmeI fragment contained two copies of the amoCAB genes, coding for ammonia monooxygenase (designated amoCAB(1) and amoCAB(2)), and three copies of the hao gene, coding for hydroxylamine oxidoreductase (hao(1), hao(2), and hao(3)). In this DNA fragment, amoCAB(1) and amoCAB(2) were about 390 kb apart, while hao(1), hao(2), and hao(3) were separated by at least about 100 kb from each other. Interestingly, hao(1) and hao(2) were located relatively close to amoCAB(1) and amoCAB(2), respectively. DNA sequence analysis revealed that hao(1) and hao(2) shared 160 identical nucleotides immediately upstream of each translation initiation codon. However, hao(3) showed only 30% nucleotide identity in the 160-bp corresponding region.     

The roles of the three gene copies encoding hydroxylamine oxidoreductase in Nitrosomonas europaea

The nitrifying bacterium Nitrosomonas europaea contains three copies of the gene (hao) encoding hydroxylamine oxidoreductase (HAO), the second enzyme in the nitrification pathway which oxidizes NH(2)OH to NO(2)(-). The nucleotide sequences of the hao genes differ by only one nucleotide. Two of the three gene copies have identical promoter sequences, while the third promoter has a different nucleotide sequence. Mutant strains with two of the three copies of hao inactivated were created by insertional inactivation, using DNA cassettes containing kanamycin- and gentamycin-resistance genes. All three double-mutant combinations were obtained. These double mutants were phenotypically identical under the conditions tested. Two of these double mutants were similar to wild-type cells or cells having a single hao copy inactivated regarding growth rates or hydroxylamine-dependent O(2) uptake activity, but had only about 50% of the wild-type level of in vitro HAO activity and hao mRNA. The third hao double mutant had an unstable genotype, resulting in recombination of the gentamycin marker into another copy of hao. The N. europaea genomic sequence was recently completed, revealing the locations of the copies of hao and other nitrification genes. Comparison with the arrangement of hao genes in the closely related strain, Nitrosomonas sp. strain ENI-11, showed a similar organization.     

Isolation and Characterization of cbbL and cbbS Genes Encoding Form I Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large and Small Subunits in Nitrosomonas sp.…

The cbbL and cbbS genes encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large and small subunits in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 were cloned and sequenced. The deduced gene products, CbbL and CbbS, had 93 and 87% identity with Thiobacillus intermedius CbbL and Nitrobacter winogradskyi CbbS, respectively. Expression of cbbL and cbbS in Escherichia coli led to the detection of RubisCO activity in the presence of 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). To our knowledge, this is the first paper to report the genes involved in the carbon fixation reaction in chemolithotrophic ammonia-oxidizing bacteria.     

Metabolism of Inorganic N Compounds by Ammonia-Oxidizing Bacteria

ABSTRACT

Ammonia oxidizing bacteria extract energy for growth from the oxidation of ammonia to nitrite. Ammonia monooxygenase, which initiates ammonia oxidation, remains enigmatic given the lack of purified preparations. Genetic and biochemical studies support a model for the enzyme consisting of three subunits and metal centers of copper and iron. Knowledge of hydroxylamine oxidoreductase, which oxidizes hydroxylamine formed by ammonia monooxygenase to nitrite, is informed by a crystal structure and detailed spectroscopic and catalytic studies. Other inorganic nitrogen compounds, including NO, N₂O, NO₂, and N₂ can be consumed and/or produced by ammonia-oxidizing bacteria. NO and N₂O can be produced as byproducts of hydroxylamine oxidation or through nitrite reduction. NO₂ can serve as an alternative oxidant in place of O₂ in some ammonia-oxidizing strains. Our knowledge of the diversity of inorganic N metabolism by ammonia-oxidizing bacteria continues to grow. Nonetheless, many questions remain regarding the enzymes and genes involved in these processes and the role of these pathways in ammonia oxidizers.     

Isolation and Characterization of Two Cryptic Plasmids in the Ammonia-Oxidizing Bacterium Nitrosomonas sp. Strain ENI-11

Two plasmids were discovered in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11, which was isolated from activated sludge. The plasmids, designated pAYS and pAYL, were relatively small, being approximately 1.9 kb long. They were cryptic plasmids, having no detectable plasmid-linked antibiotic resistance or heavy metal resistance markers. The complete nucleotide sequences of pAYS and pAYL were determined, and their physical maps were constructed. There existed two major open reading frames, ORF1 in pAYS and ORF2 in pAYL, each of which was more than 500 bp long. The predicted product of ORF2 was 28% identical to part of the replication protein of a Bacillus plasmid, pBAA1. However, no significant similarity to any known protein sequences was detected with the predicted product of ORF1. pAYS and pAYL had a highly homologous region, designated HHR, of 262 bp. The overall identity was 98% between the two nucleotide sequences. Interestingly, HHR-homologous sequences were also detected in the genomes of ENI-11 and the plasmidless strain Nitrosomonas europaea IFO14298. Deletion analysis of pAYS and pAYL indicated that HHR, together with either ORF1 or ORF2, was essential for plasmid maintenance in ENI-11. To our knowledge, pAYS and pAYL are the first plasmids found in the ammonia-oxidizing autotrophic bacteria.     

Structure and Sequence Conservation of hao Cluster Genes of Autotrophic Ammonia-Oxidizing Bacteria: Evidence for Their Evolutionary History

Comparison of the organization and sequence of the hao (hydroxylamine oxidoreductase) gene clusters from the gammaproteobacterial autotrophic ammonia-oxidizing bacterium (aAOB) Nitrosococcus oceani and the betaproteobacterial aAOB Nitrosospira multiformis and Nitrosomonas europaea revealed a highly conserved gene cluster encoding the following proteins: hao, hydroxylamine oxidoreductase; orf2, a putative protein; cycA, cytochrome c₅₅₄; and cycB, cytochrome c_m552. The deduced protein sequences of HAO, c₅₅₄, and c_m552 were highly similar in all aAOB despite their differences in species evolution and codon usage. Phylogenetic inference revealed a broad family of multi-c-heme proteins, including HAO, the pentaheme nitrite reductase, and tetrathionate reductase. The c-hemes of this group also have a nearly identical geometry of heme orientation, which has remained conserved during divergent evolution of function. High sequence similarity is also seen within a protein family, including cytochromes c_m552, NrfH/B, and NapC/NirT. It is proposed that the hydroxylamine oxidation pathway evolved from a nitrite reduction pathway involved in anaerobic respiration (denitrification) during the radiation of the Proteobacteria. Conservation of the hydroxylamine oxidation module was maintained by functional pressure, and the module expanded into two separate narrow taxa after a lateral gene transfer event between gamma- and betaproteobacterial ancestors of extant aAOB. HAO-encoding genes were also found in six non-aAOB, either singly or tandemly arranged with an orf2 gene, whereas a c₅₅₄ gene was lacking. The conservation of the hao gene cluster in general and the uniqueness of the c₅₅₄ gene in particular make it a suitable target for the design of primers and probes useful for molecular ecology approaches to detect aAOB.     

Effects of Aeration Cycles on Nitrifying Bacterial Populations and Nitrogen Removal in Intermittently Aerated Reactors

The effects of the lengths of aeration and nonaeration periods on nitrogen removal and the nitrifying bacterial community structure were assessed in intermittently aerated (IA) reactors treating digested swine wastewater. Five IA reactors were operated in parallel with different aeration-to-nonaeration time ratios (ANA). Populations of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were monitored using 16S rRNA slot blot hybridizations. AOB species diversity was assessed using amoA gene denaturant gradient gel electrophoresis. Nitrosomonas and Nitrosococcus mobilis were the dominant AOB and Nitrospira spp. were the dominant NOB in all reactors, although Nitrosospira and Nitrobacter were also detected at lower levels. Reactors operated with the shortest aeration time (30 min) showed the highest Nitrosospira rRNA levels, and reactors operated with the longest anoxic periods (3 and 4 h) showed the lowest levels of Nitrobacter, compared to the other reactors. Nitrosomonas sp. strain Nm107 was detected in all reactors, regardless of thereactor's performance. Close relatives of Nitrosomonas europaea, Nitrosomonas sp. strain ENI-11, and Nitrosospira multiformis were occasionally detected in all reactors. Biomass fractions of AOB and effluent ammonia concentrations were not significantly different among the reactors. NOB were more sensitive than AOB to long nonaeration periods, as nitrite accumulation and lower total NOB rRNA levels were observed for an ANA of 1 h:4 h. The reactor with the longest nonaeration time of 4 h performed partial nitrification, followed by denitrification via nitrite, whereas the other reactors removed nitrogen through traditional nitrification and denitrification via nitrate. Superior ammonia removal efficiencies were not associated with levels of specific AOB species or with higher AOB species diversity.     

Effect of adhesion to particles on the survival and activity of Nitrosomonas sp. and Nitrobacter sp.

The adhesion of Nitrosomonas sp. and Nitrobacter sp. cells isolated from fishpond sediment to different solid particles was studied. Nitrosomonas and Nitrobacter cells rapidly attached to particles of bentonite, calcium carbonate, amberlite, and fishpond sediment, however they did not adhere to phenyl-sepharose beads. The nitrifying activity of attached bacteria was greater than the activity of freely suspended cells or the activity of cells which have been detached from CaCO₃ particles. The enhancement in the nitrifying activity was rapid and was already observed within the first hour after attachment (which equals only 1/24 to 1/50 of the generation time of Nitrosomonas sp. or Nitrobacter sp. In addition, the survival of the attached bacteria under both anaerobic and under aerobic incubation was extended to weeks, compared to only a few days for the free cells. The presence of substrate (ammonia or nitrite) during the anaerobic incubation period was found not to affect the survival time of the bacteria. Finally, it was found that the attachment of Nitrosomonas and Nitrobacter cells to CaCO₃ particles affected the dispersal and sinking rate of these particles.     

Enhanced 1,3-propanediol production in recombinant <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> carrying the gene <Emphasis Type="Italic">yqh</Emphasis>D encoding 1,3-propanediol oxidoreductase isoenzyme

The yqhD gene from Escherichia coli encoding 1,3-propanediol oxidoreductase isoenzyme (PDORI) and the tetracycline resistant gene (tetR) from plasmid pHY300PLK were amplified by PCR. They were inserted into vector pUC18, yielding the recombinant expression vector pUC18-yqhD-tetR. The recombinant vector was then cloned into Klebsiella pneumoniae ME-308. The overexpression of PDORI in K. pneumoniae surprisingly led to higher 1,3-propanediol production. The final 1,3-propanediol concentration of recombinant K. pneumoniae reached 67.6 g/l, which was 125.33% of that of the original strain. The maximum activity of recombinant PDORI converting 3-HPA to 1,3-PD reached 110 IU/mg after induction by IPTG at 31°C during the fermentation, while it was only 11 IU/mg under the same conditions for the wild type strain. The K _m values of the purified PDORI for 1,3-propanediol and NADP were 12.1 mM and 0.15 mM, respectively. Compared with the original strains, the concentration of the toxic intermediate 3-hydroxypropionaldehyde during the fermentation was also reduced by 22.4%. Both the increased production of 1,3-propanediol and the reduction of toxic intermediate confirmed the significant role of 1,3-propanediol oxidoreductase isoenzyme from E. coli in converting 3-hydroxypropionaldehyde to 1,3-propanediol for 1,3-PD production.     

羟胺氧化还原酶在生物脱氮中的研究进展

羟胺氧化还原酶(hydroxylamine oxidoreductase,HAO)属于多血红素蛋白酶家族,每个单体由7个电子转移血红素和1个催化血红素组成.HAO既可分别催化羟胺和肼的氧化反应,也可催化羟胺、一氧化氮及亚硝酸盐的还原反应.不同硝化细菌中,HAO的最适温度、pH、底物、产物特异性及酶抑制剂等存在差异.作为...     

Construction of a highly bioluminescent Nitrosomonas as a probe for nitrification conditions

Cloned luciferase-encoding operons were transferred by conjugation to a natural isolate of the ammonia-oxidizing bacterial strain Nitrosomonas sp. RST41–3, thereby establishing conjugation as a tool for gene transfer into Nitrosomonas strains. Luminescence was dependent on the pH of the medium and the concentration of the substrate ammonium chloride. Moreover, the luminescence of the transconjugants was reduced immediately by micromolar concentrations of nitrapyrin and allylthiourea, which are specific inhibitors of nitrification. Our results indicate that luminescent Nitrosomonas strains may be useful as a probe to detect nitrification conditions in the natural environment as well as in sewage plants.     

The impact of organic matter on nitric oxide formation by Nitrosomonas europaea

Chemolithoautotrophically growing cells of Nitrosomonas europaea quantitatively oxidized ammonia to nitrite under aerobic conditions with no loss of inorganic nitrogen. Significant inorganic nitrogen losses occurred when cells were growing mixotrophically with ammonium, pyruvate, yeast extract and peptone. Under oxygen limitation the nitrogen losses were even higher. In the absence of oxygen pyruvate was metabolized slowly while nitrite was consumed concomitantly. Nitrogen losses were due to the production of nitric oxide and nitrous oxide. In mixed cultures of Nitrosomonas and Nitrobacter, strong inhibition of nitrite oxidation was reproducibly measured. NO and ammonium were not inhibitory to Nitrobacter. First evidence is given that hydroxylamine, the intermediate of the Nitrosomonas monooxygenase-reaction, is formed. 0.2 to 1.7 M NH₂OH were produced by mixotrophically growing cells of Nitrosomonas and Nitrosovibrio. Hydroxylamine was both a selective inhibitory agent to Nitrobacter cells and a strong reductant which reduced nitrite to NO and N₂O. It is discussed whether chemodenitrification or denitrification is the most abundant process for NO and N₂O production of Nitrosomonas.     

Chemical oxidation and reduction of the O2-evolution center in Photosystem II

Treatment of Photosystem II (PS II) with low concentrations of hydroxylamine is known to cause a two-flash delay in the O₂-evolution pattern, and in the formation of the S₂-state multiline EPR signal, due to the two-electron reduction of the S₁-state by hydroxylamine to form the S_-1-state. Past work has shown that these delays are not reversed by washing out the hydroxylamine nor by adding DCBQ or ferricyanide to oxidize the residual hydroxylamine, but are reversed by illumination with two saturating flashes followed by a 30-min dark incubation. We have examined the effects of treatments aimed at restoring the normal flash-induced O₂-evolution pattern and S₂-state multiline EPR signal after treatment of PS II with 40 M hydroxylamine. In agreement with past work, we find that the two-flash delay in O₂ evolution is not reversed when the hydroxylamine is removed by three cycles of centrifugation and resuspension in hydroxylamine-free buffer nor by adding ferricyanide or DCBQ to oxidize the unreacted hydroxylamine. However, the normal flash-induced O₂-evolution pattern is restored by illumination with two saturating flashes followed by a 30-min dark incubation (after the sample was first treated with 40 M hydroxylamine and the unreacted hydroxylamine was removed); illumination with one saturating flash followed by a 30-min dark incubation is only partially effective. These results show that ferricyanide and DCBQ are not effective at oxidizing the S_-1-state to the S₁-state. In contrast, adding hypochlorite (OCl^-) after treatment with hydroxylamine restored the normal flash-induced O₂-evolution pattern and also restored the formation of the S₂-state multiline EPR signal by illumination at 200 K. We conclude that hypochlorite is capable of oxidizing the S_-1-state to the S₁-state. This is the first example of a chemical treatment that advances the delayed flash-induced O₂ evolution pattern.Abbreviations DCBQ 2,5-dichloro-p-benzoquinone - OEC O₂-evolving center