The product of the Klebsiella pneumoniae nifX gene is a negative regulator of the nitrogen fixation (nif) regulon

Gosink, Mark; Franklin, N M; Roberts, Gary P.

doi:10.1128/jb.172.3.1441-1447.1990

Cited by 50 publications

(42 citation statements)

References 27 publications

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“…The product of nifX is required for in vitro FeMo-co synthesis with purified components (34). Nevertheless, nifX is not absolutely required in vivo for FeMo-co synthesis, because strains of A. vinelandii and K. pneumoniae with mutations in nifX are Nif ϩ (35,36). The same seems to be true for VnfX.…”

Section: Figmentioning

confidence: 78%

A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase inAzotobacter vinelandii

Rüttimann-Johnson

Staples

Rangaraj

et al. 1999

Journal of Biological Chemistry

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The vnf-encoded nitrogenase from Azotobacter vinelandii contains an iron-vanadium cofactor (FeV-co) in its active site. Little is known about the synthesis pathway of FeV-co, other than that some of the gene products required are also involved in the synthesis of the ironmolybdenum cofactor (FeMo-co) of the widely studied molybdenum-dinitrogenase. We have found that VnfX, the gene product of one of the genes contained in the vnf-regulon, accumulates iron and vanadium in a novel V-Fe cluster during synthesis of FeV-co. The electron paramagnetic resonance (EPR) and metal analyses of the V-Fe cluster accumulated on VnfX are consistent with a VFe 7-8 S x precursor of FeV-co. The EPR spectrum of VnfX with the V-Fe cluster bound strongly resembles that of isolated FeV-co and a model VFe 3 S 4 compound. The V-Fe cluster accumulating on VnfX does not contain homocitrate. No accumulation of V-Fe cluster on VnfX was observed in strains with deletions in genes known to be involved in the early steps of FeV-co synthesis, suggesting that it corresponds to a precursor of FeV-co. VnfX purified from a nifB strain incapable of FeV-co synthesis has a different electrophoretic mobility in native anoxic gels than does VnfX, which has the V-Fe cluster bound. NifB-co, the Fe and S precursor of FeMo-co (and presumably FeV-co), binds to VnfX purified from the nifB strain, producing a shift in its electrophoretic mobility on anoxic native gels. The data suggest that a precursor of FeV-co that contains vanadium and iron accumulates on VnfX, and thus, VnfX is involved in the synthesis of FeV-co.

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Section: Figmentioning

confidence: 78%

A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase inAzotobacter vinelandii

Rüttimann-Johnson

Staples

Rangaraj

et al. 1999

Journal of Biological Chemistry

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“…This strain also accumulated appreciable amounts of P-galactosidase when normally repressive amounts of NH4+ were present in the medium. Since NH4' repression of nifHDK in K. pneumoniae is not as effective in VnfX-mutant strains as it is in wild-type strains (19), it is possible that polarity effects on vnfX lead to the lack of repression of the vnJENX genes by NH4+ observed in strain CA47. It is also possible that vnfENX transcripts are made in the presence of NH4+, as has been found for the vnfHorJFd and vnfDGK operons (21).…”

Section: Resultsmentioning

confidence: 99%

“…3C). NifX has been shown to be a negative regulator of the nif regulon of K. pneumoniae (19). In the presence of NH4' and 02, NifX-mutants continued to synthesize nitrogenase proteins after synthesis ceased in NifX+ strains.…”

Section: Resultsmentioning

confidence: 99%

Nucleotide sequence and mutational analysis of the vnfENX region of Azotobacter vinelandii

Wolfinger

Bishop

1991

J Bacteriol

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The nucleotide sequence (3,600 bp) of a second copy of nifENX-like genes in Azotobacter vinelandii has been determined. These genes are located immediately downstream from vnfA and have been designated vnJENX. The vnJENX genes appear to be organized as a single transcriptional unit that is preceded by a potential RpoN-dependent promoter. While the nifEN genes are thought to be evolutionarily related to nifDK, the vaJEN genes appear to be more closely related to niJEN than to either nifDK, vnJDK, or anfDK. Mutant strains (CA47 and CA48) carrying insertions in vnfE and vnfN, respectively, are able to grow diazotrophically in molybdenum (Mo)-deficient medium containing vanadium (V) (Vnf+) and in medium lacking both Mo and V (Anf).However, a double mutant (strain DJ42.48) which contains a nijEN deletion and an insertion in vnJE is unable to grow diazotrophically in Mo-sufficient medium or in Mo-deficient medium with or without V. This suggests that NifE and NifN substitute for VnfE and VnfN when the vnjEN genes are mutationally inactivated. AnfA is not required for the expression of a vnJN-lacZ transcriptional fusion, even though this fusion is expressed under Mo-and V-deficient diazotrophic growth conditions. Azotobacter vinelandii is able to grow diazotrophically using any of three genetically distinct nitrogenases depending on the presence or absence of molybdenum (Mo) or vanadium (V) in the growth medium. The well-characterized Mo-containing nitrogenase (nitrogenase-1) is synthesized under conditions of Mo sufficiency. Under conditions where V replaces Mo, an alternative V-containing nitrogenase (nitrogenase-2) is expressed, and in the absence of both Mo and V, an alternative nitrogenase (nitrogenase-3) that does not appear to contain either Mo or V is made (18, 23). Each of these nitrogenase complexes is composed of two protein components, dinitrogenase reductase and dinitrogenase. Dinitrogenase reductase-1 is a dimer of two identical subunits with an Mr of approximately 60,000 (10, 11). Dinitrogenase reductase-2 and dinitrogenase reductase-3 are also dimers of two identical subunits (13,20). Dinitrogenase-1 is a tetramer (Mr, -240,000) consisting of two pairs of nonidentical subunits (a2P2). Dinitrogenase-2 and dinitrogenase-3, on the other hand, are probably hexamers, each containing three pairs of nonidentical subunits (a2P282) (25,39).The structural genes encoding nitrogenase-1 and nitrogenase-3 are organized as single operons (nifHDK and anf HDGKorflord2, respectively), while those encoding nitrogenase-2 form two independently regulated operons, vnfHorf Fd and vn.fDGK (8,25,26,39

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“…Real-time reverse transcription-PCR (RT-PCR) was performed using a LightCycler (Roche Applied Science) with RNA extracted as described by Gosink et al (10) and treated twice with RNase free DNase I (Roche Applied Science) to remove any contaminating genomic DNA. Total RNA was reverse transcribed and amplified by using primers specific to eefXAB mRNA (eefX5Ј/eefX3Ј, eefseq1-3Ј/eefA3Ј, and eefseq3-5Ј/eefseq2-3Ј) and 16S rRNA (RNA 16S-1/ RNA 16S-2) ( Table 2).…”

Section: Methodsmentioning

confidence: 99%

A Tripartite Efflux Pump Involved in Gastrointestinal Colonization by Klebsiella pneumoniae Confers a Tolerance Response to Inorganic Acid

et al. 2008

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In vivo assays showed that a ⌬eefA isogenic mutant strain normally colonized the gastrointestinal tract in single-strain tests but was significantly impaired in competition against wild-type strain LM21. Although the cecum was the compartment with the highest number of CFU, the ⌬eefA mutant also was detected in the stomach in numbers smaller than those of the wild-type strain. The expression of this potential efflux pump could not be linked to any antimicrobial drug resistance phenotype, but it conferred on the bacteria an acid tolerance response to inorganic acid. The expression of the eef promoter region, measured via a lacZ reporter construction, was slightly induced by an acidic environment and also by hyperosmolarity but not by the presence of bile salts. These results suggest that an efflux pump can confer measurable ecological benefits on K. pneumoniae in an environment with high competition potential.Klebsiella pneumoniae is an opportunistic pathogen responsible for many nosocomial infections, and most clinical strains exhibit high levels of resistance to a wide variety of structurally unrelated antibiotics. Epidemiological studies have shown that, whatever the infection site, the first stage in nosocomial infections due to K. pneumoniae consists of the colonization of the patient's gastrointestinal (GI) tract (6,16). This pathogen therefore has to sense and respond to numerous different environments in order to survive and, consequently, to persist in the GI tract of the host. The first major barrier encountered following oral consumption is stomach acidity. The bacteria then enter the small intestine, where they encounter stresses associated with volatile fatty acids, variations in pH and osmolarity, and competition with endogenous flora. Using signature-tagged mutagenesis (STM) in a murine model, we previously identified 13 genes encoding factors required for the in vivo colonization of the GI tract by K. pneumoniae LM21 (14). All of these mutants were selected for their inability to colonize the murine intestinal tract under competitive conditions. One of the identified genes potentially encoded a protein involved in a cryptic efflux pump, EefABC, previously described in Enterobacter aerogenes (18). This pump is thought to belong to the resistance-nodulation-division (RND) family and to be composed of an inner membrane protein, EefB, a periplasmic intermediate, EefA, and an outer membrane protein, EefC (18). No specific function has been attributed to this structure so far, whose expression is repressed by the H-NS repressor in the heterologous host Escherichia coli (18). In addition to the export of antimicrobials, several recent publications have highlighted the natural physiological role of efflux pumps in exporting noxious substances out of the bacterial cell, thereby allowing the bacteria to survive in hostile environments and facilitating intestinal colonization (11,13,24,30). In this report, we characterized the eef-like operon of K. pneumoniae and investigated the role of the potentially enc...

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The product of the Klebsiella pneumoniae nifX gene is a negative regulator of the nitrogen fixation (nif) regulon

Cited by 50 publications

References 27 publications

A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase inAzotobacter vinelandii

A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase inAzotobacter vinelandii

Nucleotide sequence and mutational analysis of the vnfENX region of Azotobacter vinelandii

A Tripartite Efflux Pump Involved in Gastrointestinal Colonization by Klebsiella pneumoniae Confers a Tolerance Response to Inorganic Acid

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