Novel phosphotransferase-encoding genes revealed by analysis of the Escherichia coli genome: a chimeric gene encoding an Enzyme I homologue that possesses a putative sensory transduction domain.

Two genes (ptsI and ptsA) that encode homologues of the energy coupling Enzyme I of the phosphoenolpyruvate-dependent sugar-transporting phosphotransferase system (PTS) have previously been identified on the Escherichia coli chromosome. We here report the presence of a third E. coli gene, designated ptsP, that encodes an Enzyme I homologue, here designated Enzyme INtr. Enzyme INtr possesses an N-terminal domain homologous to the N-terminal domains of NifA proteins [(127 amino acids (aa)] joined via two tandem flexible linkers to the C-terminal Enzyme I-like domain (578 aa). Structural features of the putative ptsP operon, including transcriptional regulatory signals, are characterized. We suggest that Enzyme INtr functions in transcriptional regulation of nitrogen-related operons together with previously described PTS proteins encoded within the rpoN operon. It may thereby provide a link between carbon and nitrogen assimilatory pathways.

Nitrogen control in bacteria.

Nitrogen metabolism in prokaryotes involves the coordinated expression of a large number of enzymes concerned with both utilization of extracellular nitrogen sources and intracellular biosynthesis of nitrogen-containing compounds. The control of this expression is determined by the availability of fixed nitrogen to the cell and is effected by complex regulatory networks involving regulation at both the transcriptional and posttranslational levels. While the most detailed studies to date have been carried out with enteric bacteria, there is a considerable body of evidence to show that the nitrogen regulation (ntr) systems described in the enterics extend to many other genera. Furthermore, as the range of bacteria in which the phenomenon of nitrogen control is examined is being extended, new regulatory mechanisms are also being discovered. In this review, we have attempted to summarize recent research in prokaryotic nitrogen control; to show the ubiquity of the ntr system, at least in gram-negative organisms; and to identify those areas and groups of organisms about which there is much still to learn.

Function of hisF and hisH gene products in histidine biosynthesis.

A mutant of the enterobacterium Klebsiella pneumoniae with a defect in the hisF gene (in the histidine biosynthesis pathway) was isolated, which can only grow with high but not low ammonia concentrations. The mutated hisF product can use ammonia for the formation of the imidazole ring of histidine but not glutamine provided by the hisH product. Site-directed insertional mutagenesis of hisH led to the same dependence of prototrophic growth on high ammonia levels. The nucleotide sequence of K. pneumoniae hisF is almost identical to that of hisF from other enterobacteria. Similarities of the hisF product with the hisA product and of HisH sequences with the glutamine binding domains of TrpG-type amidotransferases provide additional evidence for the functions of the hisF and hisH products in histidine biosynthesis, namely that HisF catalyzes the ammonolytic cleavage of N'-(5'-phosphoribulosyl)-formimino-5- aminoimidazole-4-carboxamide ribonucleotide either utilizing free ammonia or deriving the ammonia moiety from glutamine bound to HisH.

In a class of its own--the RNA polymerase sigma factor sigma 54 (sigma N).

Bacteria synthesize a number of different sigma factors which allow the co-ordinate expression of groups of genes owing to the ability of sigma to confer promoter-specific transcription initiation on RNA polymerase. In nearly all cases these sigmas belong to a single family of proteins which appear to be related structurally and functionally to the major Escherichia coli sigma factor, sigma 70. A clear exception is the sigma factor sigma 54 (sigma N), encoded by rpoN, which represents a second family of sigmas that is widely distributed in prokaryotes. Studies of sigma 54 (sigma N) have demonstrated that this sigma is quite distinct both structurally and functionally from the sigma 70 family and the mode of transcription initiation which it mediates may have more in common with that found in eukaryotes than that which occurs with sigma 70 and its relatives.

Cassette mutagenesis implicates a helix-turn-helix motif in promoter recognition by the novel RNA polymerase sigma factor sigma 54.

Cassette mutagenesis has been used to study the role of a helix-turn-helix (HTH) motif in the novel RNA polymerase sigma factor sigma 54 of Klebsiella pneumoniae. Of the four residues which are predicted to be solvent-exposed in the second helix, the first (Glu-378) tolerated all substitutions, and some mutations of this residue increased expression from sigma 54-dependent promoters. Certain substitutions in the third exposed residue (Ser-382) produced a promoter-specific phenotype and all substitutions in the fourth residue (Arg-383) inactivated the protein, identifying this residue as being likely to be involved in base-specific interactions with the promoter. In vivo footprinting indicated that the inactive HTH mutants of sigma 54 were defective in interaction with both the -24 and -12 regions of the glnAp2 promoter.

Mutations in genes downstream of the rpoN gene (encoding sigma 54) of Klebsiella pneumoniae affect expression from sigma 54-dependent promoters.

Two open reading frames (ORFs), designated ORF95 and ORF162, downstream of the Klebsiella pneumoniae sigma 54 structural gene (rpoN) have been sequenced and shown to encode polypeptides of 12 kD and 16 kD, respectively. ORFs homologous to ORF95 are present downstream of four out of five rpoN genes sequenced to date from a range of Gram-negative bacteria, and ORF162 is also conserved, at least in Pseudomonas putida. Chromosomal mutations have been created in each gene using a kan cassette and both have the same phenotype, i.e. they cause an increase in the level of expression from sigma 54-dependent promoters. We propose that the products of both genes function to modulate the activity of E sigma 54, although a physiological role for these proteins has not yet been identified.

The Klebsiella pneumoniae PII protein (glnB gene product) is not absolutely required for nitrogen regulation and is not involved in NifL-mediated nif gene regulation.

The role of the Klebsiella pneumoniae PII protein (encoded by glnB) in nitrogen regulation has been studied using two classes of glnB mutants. In Class I mutants PII appears not to be uridylylated in nitrogen-limiting conditions and in Class II mutants PII is not synthesised. The effects of these mutations on expression from nitrogen-regulated promoters indicate that PII is not absolutely required for nitrogen control. Furthermore the uridylylated form of PII (PII-UMP) plays a significant role in the response to changes in nitrogen status by counteracting the effect of PII on NtrB-mediated dephosphorylation of NtrC. PII is not involved in the nif-specific response to changes in nitrogen status mediated by NifL.

Construction of multicopy expression vectors for regulated over-production of proteins in Klebsiella pneumoniae and other enteric bacteria.

A number of expression vectors have been constructed to allow over-production of selected gene products in Klebsiella pneumoniae and other enteric bacteria. The plasmids use the strong hybrid trp-lac (tac) promoter for gene expression, which is regulated by the lacIQ allele of the lac repressor carried on the vector. This provides very tight regulation of gene expression, which is important for over-production of proteins which may be detrimental to cell growth. The vectors carry the standard mp18 cloning nest in which all the restriction sites are unique to the plasmid (with the exception of EcoRI in pDK7). Derivatives were constructed carrying kanamycin, chloramphenicol or ampicillin resistance as selectable markers, the first two of which are advantageous in K. pneumoniae due to the high inherent beta-lactamase activity of this organism.

Over-production and characterization of the nifA gene product of Klebsiella pneumoniae--the transcriptional activator of nif gene expression.

The nifA gene of Klebsiella pneumoniae, which encodes the transcriptional activator of nif gene expression, was cloned into a number of plasmid vectors to obtain high-level synthesis of nifA product (NifA). When over-produced, NifA was very insoluble and it precipitated with the cell debris after cell lysis. Localization of beta-galactosidase activity from a nifA-lacZ translational fusion confirmed the insoluble nature of NifA. Analysis of two translational fusions in which the last six C-terminal amino acids of NifA were deleted suggests that these residues are required for activity.

Analysis of the Klebsiella pneumoniae ntrB gene by site-directed in vitro mutagenesis.

A number of in-frame insertion and deletion mutations have been constructed in vitro in the Klebsiella pneumoniae ntrB gene and the effects of each mutant NtrB protein on NtrC activity have been assessed after reintroduction of the ntrB mutation into the glnA ntrBC operon. These experiments suggest that the phosphorylation of NtrC catalysed by NtrB not only makes NtrC competent as a transcriptional activator but also improves the DNA-binding properties and hence the negative control functions of NtrC. The variety of NtrB phenotypes obtained suggest a structure/function model for the protein.

A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae and Escherichia coli.

A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae M5a1 and Escherichia coli K12 has been developed. The method, which uses a freeze-thaw cycle in the presence of CaCl2 to facilitate DNA uptake, is substantially more efficient for K. pneumoniae M5a1 than the conventional transformation procedure for E. coli. The simplicity and speed of the method makes it very attractive for routine transformation of K. pneumoniae M5a1 and E. coli K12.

The nucleotide sequence of the nitrogen-regulation gene ntrA of Klebsiella pneumoniae and comparison with conserved features in bacterial RNA polymerase sigma factors.

The nucleotide sequence of the Klebsiella pneumoniae ntrA gene has been determined. NtrA encodes a 53,926 Dalton acidic polypeptide; a calculated molecular weight which is significantly lower than that determined by SDS polyacrylamide gel analysis. NtrA is followed by another open-reading frame (orf) of at least 75 amino acids. In the spacer region between ntrA and orf there are no apparent transcription termination or promoter sequences and therefore orf may be co-transcribed with ntrA. Previous authors have proposed that NtrA could act as an RNA polymerase sigma factor but the NtrA amino acid sequence does not show a high level of homology to any known sigma factor. However analysis of sequences of five sigma factors from E. coli and B. subtilis has identified two conserved sequences at the C-terminal end of all these polypeptides. These sequences resemble those found in known site-specific DNA-binding domains and may be involved in recognition of conserved -35 and -10 promoter sequences. A similar pair of sequences is present at the C-terminus of NtrA and could play a role in recognition of ntr-activatable promoters.

Studies on the regulation and function of the Klebsiella pneumoniae ntrA gene.

The ntrA gene from Klebsiella pneumoniae has been cloned and the product identified as a 76-kDal acidic polypeptide. An ntrA::lacZ fusion was used to demonstrate that expression of ntrA is not controlled by the nitrogen regulation (ntr) system and is independent of the nitrogen status of the cell. Studies with multicopy plasmids carrying ntrA and rpoD suggest that the ntrA product competes with the rpoD product (sigma 70 of RNA polymerase) in mediating transcription initiation by RNA polymerase at ntrA-dependent promoters. No significant homology between ntrA and rpoD was detected by Southern blotting.

Nitrogen control of the nif regulon in Klebsiella pneumoniae: involvement of the ntrA gene and analogies between ntrC and nifA.

The ntrC and nifA gene products of Klebsiella pneumoniae are transcriptional activators involved in general nitrogen control and nif-specific regulation, respectively. Multicopy plasmids expressing either ntrC or nifA from a foreign promoter were used to study the relationship between these two genes and ntrA. The nifA product substituted for ntrC product in activation of a number of genes including nifLA, hutUH and genes for arginine and proline utilisation. NtrC could not substitute for nifA in transcriptional activation of the nifHDKY operon. In ntrA- strains, neither the ntrC nor the nifA product functioned to activate transcription of nif promoters. In vitro transcription/translation studies with plasmid clones demonstrated similar levels of expression of ntrC and nifA in ntr+ and ntrA- S-30 extracts. Hence, lack of activator function in an ntrA mutant indicates that both the ntrC and nifA products require a functional ntrA for activity. When expressed from foreign promoters, both the ntrC and nifA products were active in conditions which would normally repress nif expression. Hence, the ntrA product was apparently not limiting in these conditions.

A morphological and genetic mapping study of bald colony mutants of Streptomyces coelicolor.

Twelve bld mutations of Streptomyces coelicolor resulting in a lack of visible aerial mycelium were mapped genetically. The mutants were classified into three groups on the basis of colony morphology, production of antibiotics and morphology on different carbon sources. Four map locations were found for the bld genes and three of these were very near the loci of whi genes, which are also involved in differentiation. Closely linked bld mutations had similar phenotypes.

Hybridization and selection for increased penicillin titre in wild-type isolates of Aspergillus nidulans.

Repeated hybridization and selection among wild-type isolates produced strains of Aspergillus nidulans with increased penicillin titre. Four independent selection lines were established, each originating from a sexual cross between two different heterokaryon-incompatible wild-type isolates. In each generation, two selected high-titre sister strains were crossed to produce the next generation. An initial increase in titre was obtained in each line, but after four or five generations of selection the genetic variation was considerably reduced and the rate of response to selection had decreased. From a base population of wild-type isolates with a mean titre of 8-6 units/ml the progeny mean titre was raised to between 16 and 20 units/ml in each line. The gradual nature of the response suggests that a number of genes determine penicillin titre in the wild-type isolates used. The gene action throughout the selection programme was predominantly additive.

Hybridization and selection for increased penicillin titre in wile-type isolates of Aspergillus nidulans.

Repeated hybridization and selection among wild-type isolates produced strains of Aspergillus nidulans with increased penicillin titre. Four independent selection lines were established, each originating from a sexual cross between two different heterokaryon-incompatible wild-type isolates. In each generation, two selected high-titre sister strains were crossed to produce the next generation. An initial increase in titre was obtained in each line, but after four or five generations of selection the genetic variation was considerably reduced and the rate of response to selection had decreased. From a base population of wild-type isolates with a mean titre of 8-6 units/ml the progeny mean titre was raised to between 16 and 20 units/ml in each line. The gradual nature of the response suggests that a number of genes determine penicillin titre in the wild-type isolates used. The gene action throughout the selection programme was predominantly additive.