Genes encoding denitrification enzymes

Details Genes encoding denitrification enzymes Genes encoding denitrification enzymes

2000-02-15

2002-08-06

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S191000, C435S254110, C435S320100, C435S252310, C435S254200, C435S252330, C536S023100, C536S023200, C536S023700

Reexamination Certificate

active

06429003

ABSTRACT:

FIELD OF THE INVENTION
This invention is in the field of microbial denitrification. More specifically, this invention pertains to nucleic acid fragments encoding new bacterial nitric oxide reductase and bacterial periplasmic nitrate reductase enzymes.
BACKGROUND OF THE INVENTION
The complete pathway for microbial denitrification has been established as:
NO
3
−
→NO
2
−
→NO→N
2
O→N
2
(Ye et al.,
Appl. Environ. Microbiol
. 60:1053-1058 (1994); Zumft et al.,
Microbiol. Mol. Biol. Rev
. 61:533-616 (1997)). In the natural environment, denitrification plays a major role in completing the nitrogen cycle by converting nitrate (NO
3
−
) or nitrite (NO
2
−
) to nitrogen gas (N
2
). Bacteria attempt to maintain the balance necessary between fixed nitrogen and atmospheric nitrogen. In the denitrification process, the soil bacteria use nitrate, rather than oxygen, as the ultimate electron acceptor in a series of reactions to generate a transmembrane proton gradient that is used to synthesize ATP.
In practical applications, microbial denitrification has been widely used for water purification (Mateju et al.,
Enzyme Microb. Technol
. 14:172-183 (1992)). On the other hand, nitrous oxide (N
2
O) has been shown to have detrimental effect on the stratospheric ozone layer (de Boer et al.,
Eur. J. Biochem
. 242:592-600 (1996)). NOx, along with carbon monoxide and hydrocarbons can lead to an increase in the amount of stratospheric ozone. Thus, the production of N
2
O and nitric oxide (NO) due to incomplete denitrification is of concern. It will be useful therefore to devise new and better methods for denitrification of industrial waste streams to effect complete denitrification. The identification of genes encoding proteins responsible for key denitrification reactions will be essential for the development of improved denitrification methods.
Two of the essential genes in the bacterial denitrification pathway are those encoding nitric oxide reductase (nor) and nitrate reductase (nap). Genes encoding these enzymes have been identified in both denitrifying bacteria as well as non-denitrifyers. For example, Bartnikas et al.,
J. Bacteriol
. 179:3534-3540 (1997) teach the identification and sequencing of a gene cluster required for the expression of nitric oxide reductase in
Rhodobacter sphaeroidesi
and de Boer et al., (
Eur. J. Biochem
. 242:592-600 (1996)) describe a nor gene cluster isolated from
Paracoccus denitrificans
. Genes encoding periplasmic nitrate reductase have been characterized from
Thiosphaera pantotropha
(Berks et al.,
Biochem J
., (1995), 309, 983) and from
Rhodobacter sphaeroides
(Reyes et al.,
Biochem J
., (1998), 331, 897). Finally Grove et al., (
Mol. Microbiol
. 19:467-481 (1996)) describe the identification of a gene encoding a periplasmic nitrate reductase from the non-denitrifying
E. coli
K-12.
The utility in being able to manipulate the nor and nap genes to modify bacterial denitrification is clear from the interplay of these enzymes with the genes encoding nitrite reductase (nir) in the bacterial denitrification process. In bacterial denitrification, NO is produced from NO
3
−
in two consecutive reactions catalyzed by the two metalloenzymes nitrate reductase and nitrite reductase, and then is decomposed into N
2
O by nitric oxide reductase. The key step of denitrification is the reduction of NO
2
−
by nitrite reductases. These quintessential enzymes catalyze the conversion of a mineral form of nitrogen to a gaseous form. It is well recognized that gaseous forms of nitrogen compounds are no longer easily available for assimilation by the biomass.
The product of nitrite reduction, NO, is only present in trace amount due to its efficient removal by nitric oxide reductase. However, it has been observed that mutations in the NO step render the cells incapable of using nitrate and nitrite as the alternative electron acceptors due to NO toxicity. In addition, mutations in the nitric oxide reductase (nor) region have shown a negative impact on the activity or expression level of nitrite reductase (nir) in
Pseudomonas stutzeri
and
Paracoccus denitrificans
(Grove et al.,
Mol. Microbiol
. 19:467-481 (1996); Zumft et al.,
Eur. J. Biochem
. 219:481-490 (1994)). Nonetheless, in
Rhodobacter sphaeroides
, promoter activity for the nitrite reductase gene nirK was higher in nor mutants (Bartnikas et al.,
J. Bacteriol
. 179:3534-3540 (1997)). Activities of these two steps are commonly controlled by the proteins NnrU, NirQ, and NnrR (Bell et al.,
FEBS Lett
. 265:85-87 (1990); Kastrau et al.,
Eur. J. Biochem
. 222:293-303 (1994); Tyson et al.,
Arch. Microbiol
. 168:403-411 (1997)), depending on the organism studied. This evidence distinctly suggests that reduction of nitrite and nitric oxide is highly interdependent.
Prior to the identification of role of the nitrate reductases in the periplasmic space, the principal nitrate reductase activity in bacterial denitrification has been attributed to those associated with membrane (Bell et al, 1993
, J. of General Microbiology
, Vol. 139, p. 3205-3214).
Applicant has discovered new genes isolated from Pseudomonas sp. encoding a periplasmic nitrate reductase and nitric oxide reductase. The genes are may be expressed in recombinant systems effect bacterial denitrification and the genes or portions thereof may be used to identify other denitrifying bacterial strains. Further, it has been demonstrate that the instant periplasmic nitrate reductase is the penultimate nitrate reductase in the denitrification pathway as compared with the cytoplasmic variety of the enzyme in Pseudomonas sp.
SUMMARY OF THE INVENTION
This invention relates to isolated nucleic acid fragments encoding a bacterial nitric oxide reductase selected from the group consisting of: (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8; (b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8; (c) an isolated nucleic acid fragment encoding a polypeptide having at least a 73% identity with the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8; (d) an isolated nucleic acid molecule that hybridizes with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 under the following hybridization conditions: 40% formamide, with 5× or 6×SSC; and (e) an isolated nucleic acid fragment that is complementary to (a), (b), (c) or (d).
The present invention further provides isolated nucleic acid fragments encoding a bacterial periplasmic nitrate reductase selected from the group consisting of: (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14; (b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14; (c) an isolated nucleic acid fragment encoding a polypeptide having at least a 76% identity with the amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14; (d) an isolated nucleic acid molecule that hybridizes with SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13 under the following hybridization conditions: 40% formamide, with 5× or 6×SSC.; and (e) an isolated nucleic acid fragment that is complementary to (a), (b), (c) or (d).
In another embodiment, the instant invention relates to chimeric genes encoding a bacterial nitric oxide reductase or bacterial periplasmic nitrate reductase or to chimeric genes that comprise nucleic acid fragments

Affiliated with

Also associated with

Rating

Genes encoding denitrification enzymes does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Genes encoding denitrification enzymes, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Genes encoding denitrification enzymes will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2879187