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The signal transduction protein GlnK is required for NifL-dependent nitrogen control of nif gene expression in Klebsiella pneumoniae.

ABSTRACT: In Klebsiella pneumoniae, transcription of the nitrogen fixation (nif) genes is regulated in response to molecular oxygen or availability of fixed nitrogen by the coordinated activities of the nifA and nifL gene products. NifA is a nif-specific transcriptional activator, the activity of which is inhibited by interaction with NifL. Nitrogen control of NifL occurs at two levels: transcription of the nifLA operon is regulated by the global ntr system, and the inhibitory activity of NifL is controlled in response to fixed nitrogen by an unknown factor. K. pneumoniae synthesizes two PII-like signal transduction proteins, GlnB, which we have previously shown not to be involved in the response of NifL to fixed nitrogen, and the recently identified protein GlnK. We have now cloned the K. pneumoniae glnK gene, studied its expression, and shown that a null mutation in glnK prevents NifL from responding to the absence of fixed nitrogen, i.e., from relieving the inhibition of NifA activity. Hence, GlnK appears to be involved, directly or indirectly, in NifL-dependent regulation of nif gene expression in K. pneumoniae. Comparison of the GlnB and GlnK amino acid sequences from six species of proteobacteria identifies five residues (residues 3, 5, 52, 54, and 64) which serve to distinguish the GlnB and GlnK proteins.

SUBMITTER: Jack R

PROVIDER: S-EPMC93492 | biostudies-literature | 1999 Feb

REPOSITORIES: biostudies-literature

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The signal transduction protein GlnK is required for NifL-dependent nitrogen control of nif gene expression in Klebsiella pneumoniae.

Jack R R De Zamaroczy M M Merrick M M

Journal of bacteriology 19990201 4

In Klebsiella pneumoniae, transcription of the nitrogen fixation (nif) genes is regulated in response to molecular oxygen or availability of fixed nitrogen by the coordinated activities of the nifA and nifL gene products. NifA is a nif-specific transcriptional activator, the activity of which is inhibited by interaction with NifL. Nitrogen control of NifL occurs at two levels: transcription of the nifLA operon is regulated by the global ntr system, and the inhibitory activity of NifL is controll ...[more]

PMID: 9973341

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Project description:BackgroundKlebsiella pneumoniae is a gram-negative, non-motile, facultative anaerobe belonging to the Enterobacteriaceae family of the γ-Proteobacteria class in the phylum Proteobacteria. Multidrug resistant K. pneumoniae have caused major therapeutic problems worldwide due to emergence of extended-spectrum β-lactamase producing strains. Two-component systems serve as a basic stimulus-response coupling mechanism to allow organisms to sense and respond to changes in many different environmental conditions including antibiotic stress.Principal findingsIn the present study, we investigated the role of an uncharacterized cpxAR operon in bacterial physiology and antimicrobial resistance by generating isogenic mutant (ΔcpxAR) deficient in the CpxA/CpxR component derived from the hyper mucoidal K1 strain K. pneumoniae NTUH-K2044. The behaviour of ΔcpxAR was determined under hostile conditions, reproducing stresses encountered in the gastrointestinal environment and deletion resulted in higher sensitivity to bile, osmotic and acid stresses. The ΔcpxAR was more susceptible to β-lactams and chloramphenicol than the wild-type strain, and complementation restored the altered phenotypes. The relative change in expression of acrB, acrD, eefB efflux genes were decreased in cpxAR mutant as evidenced by qRT-PCR. Comparison of outer membrane protein profiles indicated a conspicuous difference in the knock out background. Gel shift assays demonstrated direct binding of CpxR(KP) to promoter region of ompC(KP) in a concentration dependent manner.Conclusions and significanceThe Cpx envelope stress response system is known to be activated by alterations in pH, membrane composition and misfolded proteins, and this systematic investigation reveals its direct involvement in conferring antimicrobial resistance against clinically significant antibiotics for the very first time. Overall results displayed in this report reflect the pleiotropic role of the CpxAR signaling system and diversity of the antibiotic resistome in hyper virulent K1 serotype K. pneumoniae NTUH-K2044.

Project description:PII signal transduction proteins are ubiquitous and highly conserved in bacteria, archaea, and plants and play key roles in controlling nitrogen metabolism. However, research on biological functions and regulatory targets of PII proteins remains limited. Here, we illustrated experimentally that the PII protein Corynebacterium glutamicum GlnK (CgGlnK) increased l-arginine yield when glnK was overexpressed in Corynebacterium glutamicum Data showed that CgGlnK regulated l-arginine biosynthesis by upregulating the expression of genes of the l-arginine metabolic pathway and interacting with N-acetyl-l-glutamate kinase (CgNAGK), the rate-limiting enzyme in l-arginine biosynthesis. Further assays indicated that CgGlnK contributed to alleviation of the feedback inhibition of CgNAGK caused by l-arginine. In silico analysis of the binding interface of CgGlnK-CgNAGK suggested that the B and T loops of CgGlnK mainly interacted with C and N domains of CgNAGK. Moreover, F11, R47, and K85 of CgGlnK were identified as crucial binding sites that interact with CgNAGK via hydrophobic interaction and H bonds, and these interactions probably had a positive effect on maintaining the stability of the complex. Collectively, this study reveals PII-NAGK interaction in nonphotosynthetic microorganisms and further provides insights into the regulatory mechanism of PII on amino acid biosynthesis in corynebacteria.IMPORTANCE Corynebacteria are safe industrial producers of diverse amino acids, including l-glutamic acid and l-arginine. In this study, we showed that PII protein GlnK played an important role in l-glutamic acid and l-arginine biosynthesis in C. glutamicum Through clarifying the molecular mechanism of CgGlnK in l-arginine biosynthesis, the novel interaction between CgGlnK and CgNAGK was revealed. The alleviation of l-arginine inhibition of CgNAGK reached approximately 48.21% by CgGlnK addition, and the semi-inhibition constant of CgNAGK increased 1.4-fold. Furthermore, overexpression of glnK in a high-yield l-arginine-producing strain and fermentation of the recombinant strain in a 5-liter bioreactor led to a remarkably increased production of l-arginine, 49.978 g/liter, which was about 22.61% higher than that of the initial strain. In conclusion, this study provides a new strategy for modifying amino acid biosynthesis in C. glutamicum.

Dataset Information

The signal transduction protein GlnK is required for NifL-dependent nitrogen control of nif gene expression in Klebsiella pneumoniae.

Publications

The signal transduction protein GlnK is required for NifL-dependent nitrogen control of nif gene expression in Klebsiella pneumoniae.

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