Thus, to analyze whether changing the identified essential amino acids predicted to be located at the protein surface N46, Q57, Q64, and N67 or in the Q-linker K affect NifL association to the cytoplasmic membrane under nitrogen-fixing conditions, the fully reconstituted MBP-NifL derivatives were incubated with calcein-labeled liposomes, followed by ultracentrifugation in a sucrose gradient to study liposome association under reduced and oxidized conditions.
Monitoring the effects of single amino acid changes on growth under nitrogen-fixing conditions and nif gene induction in K. However, NifL derivatives with changes in amino acids essential for membrane association should not be entirely sequestered to the cytoplasmic membrane.
When FAD was simultaneously reconstituted during purification, which generally results in full reconstitution of the wild-type protein 0.
The presence of liposomes was detected by increased fluorescence intensity as a result of the release of the calcein. Single mutations were introduced into nifL of pRS and pRS by site-directed mutagenesis using Presence of nitrogen fixing klebsiella pneumoniae QuikChange XL site-directed mutagenesis kit Stratagene according to the protocol of the manufacturer and using the respective primer sets Table 2.
These servers use homology modeling and determine a score for each model it produces. Liposomes were subsequently repeatedly sonicated in an ultrasonic bath Sonorex Super RKH [Bandelin, Berlin, Germany]; W ultrasonic peak output at room temperature for 10 min, followed by a short break on ice until the liposome solution was clear.
To analyze the effects of these NifL derivatives on growth under nitrogen-fixing conditions, the respective plasmids were transformed into K. The majority of the protein was present in fractions 7 to 14 under anaerobic conditions in the presence of liposomes Fig.
Quantification of in vivo distribution of NifL or derivatives in membrane and cytoplasmic fractions of the K. The majority of the selected amino acids are predicted to be located on the surface of NifL or to bind the FAD cofactor. Under aerobic conditions, distribution patterns similar to the wild type were obtained for Q64R, N46D, and Q57L, while significant differences were obtained for N67S.
Analyzing MBP-NifL derivatives demonstrated that except for KE all derivatives showed reduced ability to bind to the liposomal membranes. Accordingly, a certain amount of the additionally expressed NifL derivative will remain in the cytoplasm and inhibit NifA activity, which consequently results in a decrease in nitrogen fixation rates and ultimately in reduced growth.
Effects of NifL derivatives carrying single amino acid changes on nif gene induction K. The path length of the cuvette was 0.
DNA sequence analysis of the inserts demonstrated that these nifL genes carried point mutations resulting in 1 to 9 amino acid changes in NifL, with an average of three to four changes.
Protein structure analysis and modeling. Preparation of calcein-labeled liposomes.
Additional nifL expression from pRS only slightly reduced growth under nitrogen-fixing conditions, indicating that the majority of additionally synthesized NifL appears to be sequestered to the cytoplasmic membrane as well and thus nif induction by NifA is not significantly affected.
E Calcein-labeled liposomes present in the respective fractions of the ultracentrifugation gradient. Strongest effects were obtained upon changing amino acids of the N-terminal domain in positions 46, 57, 62, 64, 67, 69, 80, 87, andall located within the PAS1 domain.
Primers used for PCR amplification, sequencing, and site-directed mutagenesis Site-directed mutagenesis. For experiments under anaerobic conditions, MBP-NifL was fully reduced by an excess of sodium dithionite 10 mMfollowed by a min incubation with reduced liposomes under strict anaerobic conditions within an anaerobic chamber.
To quantify direct effects of the single amino acid changes on nif gene induction, the plasmids were introduced into K. Selected single amino acid changes were introduced into nifL on pRS by site-directed mutagenesis, taking the frequency of changes within the nonfunctional NifL derivatives identified in the genetic screen and structural features of the FAD containing PAS1 domain of NifL A.
B Diagram of the domain structure of K. Growth under these conditions was generally not affected by the additional expression of NifL or derivatives. After ultracentrifugation, the various fractions 1 ml were analyzed for NifL presence by quantitative Western blot analyses as described previously 55 and for the presence of liposomes as described above.
Cultures were grown anaerobically with 4 mM glutamine as a limiting nitrogen source, conditions which allow full nif gene induction in K. The amounts of MBP-NifL and derivatives in the fractions were determined by quantitative Western blot analysis, and relative amounts of liposomes were calculated by monitoring the calcein fluorescence.
Analyzing the plasmids revealed that pRS derivatives contained small in-frame deletions of nifL, resulting in NifL derivatives carrying the complete inhibitory C-terminal domain but lacking parts of the N-terminal domain.
In a parallel approach, the derivatives were purified in the presence of 10 mM FAD to achieve simultaneous reconstitution of the FAD cofactor.
The resulting purified fractions without FAD reconstitution showed a molar ratio of 0. The most prominent effect was obtained for Q64R, which in its reduced form showed nearly no liposome association at all. Inhibition of NifA-dependent transcription under nitrogen-fixing conditions by NifL derivatives was quantified by the extent of decrease in nifHDK induction Fig.
The presented data are representatives of three independent analyses. NifL derivative R80C completely failed to grow, whereas most of the derivatives showed significantly reduced growth e.
Structural modeling of K. The initial screen of approximately 11, K. These represented two general categories: Western blot analysis of exponentially growing cultures further demonstrated that the overall amount of NifL chromosomal expressed NifL and plasmid expressed NifL or derivatives was in general about in the same range Fig.
Under nitrogen-fixing conditions, NifL expressed from the chromosomal nifLA operon is entirely sequestered to the cytoplasmic membrane, and nif genes are maximally induced by NifA. Based on the structural information available for the N-terminal domain of A.In Klebsiella pneumoniae the highly energy-consuming process of nitrogen fixation is strictly regulated in response to the environmental signals molecular oxygen and ammonium availability to avoid unnecessary consumption of energy (5, 46, 52).
Complementation of K. pneumoniae Gln(-) strains by an Escherichia coli episome (F') simultaneously restores glutamine synthetase activity and the ability to synthesize nitrogenase.
These results indicate a role for glutamine synthetase as a positive control element for nitrogen fixation in K.
pneumoniae. Download Citation on ResearchGate | Presence of nitrogen fixing Klebsiella pneumoniae in the gut of the Formosan subterranean termite (Coptotermes formosanus) | A gram-negative facultative anaerobic enteric bacterium, Klebsiella pneumoniae was isolated from the hindgut of the Formosan subterranean termite (FST).
At growth temperatures above 37°C, Klebsiella pneumoniae does not grow in a medium containing N 2 or NO 3-as nitrogen sources. However, both the growth in the presence of other nitrogen sources as well as the in vitro nitrogenase activity are not affected at.
Endophytic nitrogen-fixing Klebsiella variicola strain DXE promotes although most of the clinical Klebsiella spp. isolates accounted for the hospital infections belong to Klebsiella pneumoniae, Klebsiella Chang S, Yang L, Li Y, An Q (b) Plant growth-promoting nitrogen-fixing enterobacteria are in association with sugarcane.
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