Polyphosphate is a primordial chaperone.

Composed of up to 1,000 phospho-anhydride bond-linked phosphate monomers, inorganic polyphosphate (polyP) is one of the most ancient, conserved, and enigmatic molecules in biology. Here we demonstrate that polyP functions as a hitherto unrecognized chaperone. We show that polyP stabilizes proteins in vivo, diminishes the need for other chaperone systems to survive proteotoxic stress conditions, and protects a wide variety of proteins against stress-induced unfolding and aggregation.

Regulation of the histidine utilization (hut) system in bacteria.

The ability to degrade the amino acid histidine to ammonia, glutamate, and a one-carbon compound (formate or formamide) is a property that is widely distributed among bacteria. The four or five enzymatic steps of the pathway are highly conserved, and the chemistry of the reactions displays several unusual features, including the rearrangement of a portion of the histidase polypeptide chain to yield an unusual imidazole structure at the active site and the use of a tightly bound NAD molecule as an electrophile rather than a redox-active element in urocanase. Given the importance of this amino acid, it is not surprising that the degradation of histidine is tightly regulated.

Genetic analysis of the nitrogen assimilation control protein from Klebsiella pneumoniae.

The nitrogen assimilation control protein (NAC) from Klebsiella pneumoniae is a typical LysR-type transcriptional regulator (LTTR) in many ways. However, the lack of a physiologically relevant coeffector for NAC and the fact that NAC can carry out many of its functions as a dimer make NAC unusual among the LTTRs. In the absence of a crystal structure for NAC, we analyzed the effects of amino acid substitutions with a variety of phenotypes in an attempt to identify functionally important features of NAC.

A NAC for regulating metabolism: the nitrogen assimilation control protein (NAC) from Klebsiella pneumoniae.

The nitrogen assimilation control protein (NAC) is a LysR-type transcriptional regulator (LTTR) that is made under conditions of nitrogen-limited growth. NAC's synthesis is entirely dependent on phosphorylated NtrC from the two-component Ntr system and requires the unusual sigma factor σ54 for transcription of the nac gene. NAC activates the transcription of σ70-dependent genes whose products provide the cell with ammonia or glutamate.

Properties of the NAC (nitrogen assimilation control protein)-binding site within the ureD promoter of Klebsiella pneumoniae.

The nitrogen assimilation control protein (NAC) of Klebsiella pneumoniae is a LysR-type transcriptional regulator that activates transcription when bound to a DNA site (ATAA-N5-TnGTAT) centered at a variety of distances from the start of transcription. The NAC-binding site from the hutU promoter (NBShutU) is centered at -64 relative to the start of transcription but can activate the lacZ promoter from sites at -64, -54, -52, and -42 but not from sites at -47 or -59. However, the NBSs from the ureD promoter (ureDp) and codB promoter (codBp) are centered at -47 and -59, respectively, and NAC is fully functional at these promoters.

The LysR-type nitrogen assimilation control protein forms complexes with both long and short DNA binding sites in the absence of coeffectors.

Most LysR-type transcriptional regulators (LTTRs) function as tetramers when regulating gene expression. The nitrogen assimilation control protein (NAC) generally functions as a dimer when binding to DNA and activating transcription. However, at some sites, NAC binds as a tetramer.

Expanded role for the nitrogen assimilation control protein in the response of Klebsiella pneumoniae to nitrogen stress.

Klebsiella pneumoniae is able to utilize many nitrogen sources, and the utilization of some of these nitrogen sources is dependent on the nitrogen assimilation control (NAC) protein. Seven NAC-regulated promoters have been characterized in K. pneumoniae, and nine NAC-regulated promoters have been found by microarray analysis in Escherichia coli.

The hpx genetic system for hypoxanthine assimilation as a nitrogen source in Klebsiella pneumoniae: gene organization and transcriptional regulation.

Growth experiments showed that adenine and hypoxanthine can be used as nitrogen sources by several strains of K. pneumoniae under aerobic conditions. The assimilation of all nitrogens from these purines indicates that the catabolic pathway is complete and proceeds past allantoin.

Complex regulation of urease formation from the two promoters of the ure operon of Klebsiella pneumoniae.

Klebsiella pneumoniae can use urea as the sole source of nitrogen, thanks to a urease encoded by the ureDABCEFG operon. Expression of this operon is independent of urea and is regulated by the supply of nitrogen in the growth medium. When cells were growth rate limited for nitrogen, the specific activity of urease was about 70 times higher than that in cells grown under conditions of excess nitrogen.

Importance of tetramer formation by the nitrogen assimilation control protein for strong repression of glutamate dehydrogenase formation in Klebsiella pneumoniae.

The nitrogen assimilation control protein (NAC) from Klebsiella pneumoniae is a very versatile regulatory protein. NAC activates transcription of operons such as hut (histidine utilization) and ure (urea utilization), whose products generate ammonia. NAC also represses the transcription of genes such as gdhA, whose products use ammonia.

The Klebsiella pneumoniae O antigen contributes to bacteremia and lethality during murine pneumonia.

Bacterial surface carbohydrates are important pathogenic factors in gram-negative pneumonia infections. Among these factors, O antigen has been reported to protect pathogens against complement-mediated killing. To examine further the role of O antigen, we insertionally inactivated the gene encoding a galactosyltransferase necessary for serotype O1 O-antigen synthesis (wbbO) from Klebsiella pneumoniae 43816.

Nitrogen regulation of the codBA (cytosine deaminase) operon from Escherichia coli by the nitrogen assimilation control protein, NAC.

Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. Beta-galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required.

Isolation of a negative control mutant of the nitrogen assimilation control protein, NAC, in Klebsiella aerogenes.

A negative control mutant of the nitrogen assimilation control protein, NAC, has been isolated. Mutants with the leucine at position 111 changed to a nonhydrophobic residue activate transcription from hut and ure promoters, but fail to repress gdhA expression. This failure does not result from failure to bind to either of the two sites required for gdhA repression, but the binding at those sites is altered in the mutant.

Repression of glutamate dehydrogenase formation in Klebsiella aerogenes requires two binding sites for the nitrogen assimilation control protein, NAC.

In Klebsiella aerogenes, the gdhA gene codes for glutamate dehydrogenase, one of the enzymes responsible for assimilating ammonia into glutamate. Expression of a gdhAp-lacZ transcriptional fusion was strongly repressed by the nitrogen assimilation control protein, NAC. This strong repression (>50-fold under conditions of severe nitrogen limitation) required the presence of two separate NAC binding sites centered at -89 and +57 relative to the start of gdhA transcription.