Regulation of nif gene expression in Enterobacter agglomerans: nucleotide sequence of the nifLA operon and influence of temperature and ammonium on its transcription

The nucleotide sequence of a plasmid-borne 3.9 kb XhoI-SmaI fragment comprising the 3-region of the nifM gene, the nifL and nifA genes and the 5-region of nifB gene of Enterobacter agglomerans was determined. The genes were identified by their homology to the corresponding nif genes of Klebsiella pneumoniae. A typical ⁵⁴-dependent promoter and a consensus NtrC-binding motif were identified upstream of nifL. The predicted amino acid sequence of NifL showed close similarities to NifL of K. pneumoniae and Azotobacter vinelandii. However, no histidine residue was found to correspond to histidine-304 of A. vinelandii NifL, which had been proposed to be required for the repressor activity of NifL. The NifA sequence with a putative DNA binding motif (Q(X₃) A (X₃) G (X₅)I) and an ATP binding site in the C-terminal and central domains, respectively, resembles that of other known NifA proteins. The function of the nifL and nifA genes was demonstrated in vivo using a binary plasmid system by their ability to activate a nifH promoter-lacZ fusion at different temperatures and concentrations of NH ₄ ⁺ . Maximal promoter activity occurred at 25°C, and it appears that the sensitivity of NifA to elevated temperatures is independent of NifL. The expression of nifL inhibited promoter activity in the presence of NifA when the initial NH ₄ ⁺ concentration in the medium exceeded 4 mM.Communicated by H. Böhme     

The gltF gene of Klebsiella pneumoniae: cloning and initial characterization

Summary From a gene bank of Klebsiella pneumoniae M5a1, a 1.7 kb gene fragment was isolated which was able to restore the Ntr⁺ phenotype and ammonium (methylammonium) transport, but not glutamate synthase in an Escherichia coli glt mutant (glutamate synthase deficiency). The fragment strongly hybridized with the gltF regulatory gene from E. coli. After subcloning the fragment into an overexpression vector, a protein with a molecular weight of 27000 dalton was identified as the gene product. The results indicate that the fragment cloned contains the gltF gene from K. pneumoniae.     

Genetic characterization and sequence analysis of the duplicated nifA/nifB gene region of Rhodobacter capsulatus

Summary A DNA region showing homology to Klebsiella pneumoniae nifA and nifB is duplicated in Rhodobacter capsulatus. The two copies of this region are called nifA/nifB copy I and nifA/nifB copy II. Deletion mutagenesis demonstrated that either of the two copies is sufficient for growth in nitrogen-free medium. In contrast, a double deletion mutant turned out to be deficient in nitrogen fixation. The complete nucleotide sequence of a 4838 bp fragment containing nifA/nifB copy I was determined. Two open reading frames coding for a 59653 (NifA) and a 49453 (NifB) dalton protein could be detected. Comparison of the amino acid sequences revealed that the R. capsulatus nifA and nifB gene products are more closely related to the NifA and NifB proteins of Rhizobium meliloti and Rhizobium leguminosarum than to those of K. pneumoniae. A rho-independent termination signal and a typical nif promoter region containing a putative NifA binding site and a consensus nif promoter are located within the region between the R. capsulatus nifA and nifB genes. The nifB sequence is followed by an open reading frame (ORF1) coding for a 27721 dalton protein in nifA/nifB copy I. DNA sequence analysis of nifA/nifB copy II showed that both copies differ in the DNA region downstream of nifB and in the noncoding sequence in front of nifA. All other regions compared, i.e. the 5 part of nifA, the intergenic region and the 3 part of nifB, are identical in both copies.     

Klebsiella pneumoniae nif-lac fusions are expressed in Agrobacterium tumefaciens C58

Summary Plasmids containing hybrid genes, in which different Klebsiella pneumoniae nif (nitrogen-fixation) promoters were fused with the structural part of the Escherichia coli lac operon, were introduced into a double auxotrophic derivative of Agrobacterium tumefaciens C58. A study of their expression in the new host was made simple by the inherent inability of A. tumefaciens C58 to produce -galactosidase unless provided with the wild-type lac operon of E. coli. As shown by quantitative measurements of the enzyme, all K. pneumoniae promoters were expressed well in A. tumefaciens C58, even under conditions known to repress them. It also has been shown that the activity of K. pneumoniae nif A is essential for the expression of nifHDK even when introduced into A. tumefaciens. After entering the new host the plasmids, the nif genes and the fusion alleles contained in them, remained stable. Possible mechanisms responsible for the constitutive behaviour of nif promoters in A. tumefaciens are discussed.     

Deletion analysis of the nitrogen fixation regulatory gene,nifL of Klebsiella pneumoniae

A number of in-frame deletions have been constructed in the Klebsiella pneumoniae regulatory gene nifL. The effects of each nifL mutation on NifA-mediated expression from the nifH promoter of K. pneumoniae have then been assessed with respect to both nitrogen and oxygen control. These experiments indicate that, in contrast to the situation with the homologous regulatory proteins NtrB and NtrC, NifA activity is not impaired in the absence of NifL. We conclude that the only function of NifL is to inactivate NifA in response to an increase in the nitrogen or oxygen status of the cell.     

The nifHDK genes are contiguous with a nifA-like regulatory gene in Rhodobacter capsulatus

Summary A 15.2 kb DNA fragment was isolated from Rhodobacter capsulatus (ex. Rhodopseudomonas capsulata), which was able to complement mutations both in a nifA-like regulatory gene and in the nifH gene. Physical mapping of this fragment revealed that the nifA-like gene was adjacent to, and downstream from, the nifHDK operon. Hybridization experiments were carried out using a cloned Klebsiella pneumoniae DNA fragment containing nifA and the flanking portions of nifB and nifL. This fragment failed to hybridize with a 2.15 kb HindIII fragment of R. capsulatus DNA containing the nifA-like gene, but hybridized instead with a 2.6 kb EcoRI fragment adjacent to the nifA-like gene. The homologous region was found to be located within the K. pneumoniae nifB gene. The adjacent 2.6 kb and 2.15 kb fragments also hybridized with each other, indicating the presence of repeated sequences in this region.     

Positive and negative regulation of expression of the l-sorbose (sor) operon by SorC in Klebsiella pneumoniae

Summary In Klebsiella pneumoniae the gene products involved in the degradation of the ketose l-sorbose are encoded in the sor operon. It comprises, besides structural genes for uptake and catabolism, a promoter-proximal gene sorC, encoding a protein SorC of M_r 40 kDa, for which no enzymatic function has been detected. All sor genes are coordinately expressed and inducible by l-sorbose. Polar insertions and frameshift mutations in sorC cause a pleiotropic negative effect on the expression of all other sor genes. This defect is complemented in trans by the wild-type sorC ⁺ allele for frameshift mutations, but not for polar insertions. A single promoter for all sor genes, for which SorC is the activator, thus seems to be located in front of sorC. The repressor activity of SorC was demonstrated by complementation of constitutive sorC alleles with a sorC ⁺ allele leading to inducible expression of all sor genes, including sorC, which, as visualized by the use of a series of lacZ fusions, thus autoregulates its expression, both as an activator and a repressor.     

Characterization and fouling properties of exopolysaccharide produced by Klebsiella oxytoca

Klebsiella oxytoca produced a type of exopolysaccharide (EPS) with the average molecular weight (Mw) of 116,018 Da and the average size of 260 nm. The EPS monosaccharide components contained rhamnose, fucose, arabinose, xylose, mannose, galactose and glucose and the molar ratio among them was 0.033:0.0411:0.0147:0.0051:0.2393:0.0986:0.1304. Typical EPS absorption peaks in FT-IR spectrum and pseudoplastic properties were also revealed. The polyvinylidenefluoride (PVDF) membrane showed a relatively larger flux decline resulted from the EPS fouling. The EPS filtration was dominated by more than one mechanism at the beginning phase and mainly by the cake formation at the later phase for both membranes. The pore blocking resistance had a predominant contribution to the filtration resistance and the cake resistance played a secondary role for both the membranes. The EPS adsorption resulted in a weak membrane fouling. The PVDF membrane exhibited a larger adsorption resistance than the polypropylene (PP) membrane.     

Nucleotide sequence and analysis of the mgl operon of Escherichia coli K12

Summary The nucleotide sequence of the Escherichia coli K12 -methylgalactoside transport operon, mgl, was determined. Primer extension analysis indicated that the synthesis of mRNA initiates at guanine residue 145 of the determined sequence. The operon contains three open reading frames (ORF). The operator proximal ORF, mglB, encodes the galactose binding protein, a periplasmic protein of 332 amino acids including the 23 residue amino-terminal signal peptide. Following a 62 nucleotide spacer, the second ORF, mglA, is capable of encoding a protein of 506 amino acids. The amino-terminal and carboxyl-terminal halves of this protein are homologous to each other and each half contains a putative nucleotide binding site. The third ORF, mglC, is capable of encoding a hydrophobic protein of 336 amino acids which is thought to generate the transmembrane pore. The overall organization of the mglBAC operon and its potential to encode three proteins is similar to that of the ara FGH high affinity transport operon, located approximately 1 min away on the E. coli K12 chromosome.     

Nucleotide sequence of the recF gene cluster from Staphylococcus aureus and complementation analysis in Bacillus subtilis recF mutants

We have determined the nucleotide sequence of a 3.5 kb segment in the recF region of the Staphylococcus aureus chromosome. The gene order at this locus, dnaA-dnaN-recF-gyrB is similar to that found in the replication origin region of many other bacteria. S. aureus RecF protein (predicted molecular mass 42.3 kDa), has 57% amino acid sequence identity with the Bacillus subtilis RecF protein (42.2 kDa), but only 26% with the Escherichia coli RecF protein (40.5 kDa). We have shown that the S. aureus recF gene partially complements the defect of a B. subtilis recF mutant, but does not complement an E. coli recF strain. The amino acid sequence alignment of seven available RecF proteins (five of them from bacteria of gram-negative origin) allowed us to identify eight highly conserved regions ( to ) and to predict five new conserved regions within the gram-positive group (a to f). We suggest that the basic mechanism of homologous recombination is conserved among free-living bacteria.     

Cloning and nucleotide sequence of the Salmonella typhimurium LT2 gnd gene and its homology with the corresponding sequence of Escherichia coli K12

Summary The complete nucleotide sequence of the Salmonella strain LT2 gnd gene for 6-phosphogluconate dehydrogenase was determined. The gene contains 1404 bases and encodes a 468 amino acid polypeptide, which is the same as for Escherichia coli K12. The DNA sequence shows 14.8% difference between the two and the amino acid sequence 3.6% difference. Changes are mostly in the third codon base and most of the amino acid changes are conservative.     

Cloning and characterization of a lysine decarboxylase gene from Hafnia alvei

Summary A lysine decarboxylase (LDC) gene from Hafnia alvei was cloned in the Escherichia coli strain HB101. A gene bank consisting of 2,000 clones, carrying recombinant plasmids with large DNA fragments of H. alvei integrated in the BamH1 site of pBR322, was screened for LDC activity by a colony filter radioimmunoassay. The gene bank yielded clone 462 expressing high LDC activity with the presence of a plasmid carrying a 7.5 kb insert of H. alvei. Two LDC-positive subclones derived from 462 with inserts of 2.9 and 3.3 kb were sequenced by the shotgun method. An open reading frame for a 83 K protein with 739 amino acids was determined as the coding region for the LDC. The identification of this reading frame as the true reading frame of the H. alvei LDC gene and its similarities with LDC of E. coli are described. The use of the cloned gene for the transformation of plant cells is discussed.     

Nucleotide sequence of katG of Salmonella typhimurium LT2 and characterization of its product, hydroperoxidase I

Summary The nucleotide sequence of katG from Salmonella typhimurium was determined revealing an open reading frame of 2181 by that could encode a 727 amino acid protein. The predicted sequence of the encoded hydroperoxidase I (HPI) was found to be 90% similar to HPI from Escherichia coli and was one amino acid longer. The physical and enzymatic properties of HPI from both Salmonella typhimurium and Escherichia coli were found to be virtually identical despite the 10% divergence in sequence.