Nitrogen-limited Medium Research Articles

Ethanol and acetate production by Clostridium ljungdahlii and Clostridium autoethanogenum using resting cells

Combined gasification and fermentation technologies can potentially produce biofuels from renewable biomass. Gasification generates synthesis gas consisting primarily of CO, CO(2), H(2), N(2), with smaller amounts of CH(4), NO(x), O(2), C(2) compounds, ash and tars. Several anaerobic bacteria species can ferment bottled mixtures of pure synthesis gas constituents. However, there are challenges to maintaining culture viability of synthesis gas exposed cells. This study was designed to enhance culture stability and improve ethanol-to-acetate ratios using resting (non-growing) cells in synthesis gas fermentation. Resting cell states were induced in autotrophic Clostridium ljungdahlii cultures with minimal ethanol and acetate production due to low metabolic activity compared to growing cell production levels of 5.2 and 40.1 mM of ethanol and acetate. Clostridium autoethanogenum cultures were not induced into true resting states but did show improvement in total ethanol production (from 5.1 mM in growing cultures to 9.4 in one nitrogen-limited medium) as well as increased shifts in ethanol-to-acetate production ratios.

Structural and physiological diversity among cystlike resting cells of bacteria of the genus Pseudomonas

Cystlike resting cells (CRC) of non-spore-forming gram-negative bacteria of the genus Pseudomonas, P. aurantiaca and P. fluorescens, were obtained and characterized for the first time; their physiological and morphological diversity was demonstrated. The following properties were common for all the revealed types of CRC as dormant forms: (1) long-term (up to 6 months or longer) maintenance of viability in the absence of culture growth and cell respiration; (2) absence of an experimentally detectable level of metabolism; (3) higher resistance to damage and autolysis under the action of provoking factors than in metabolically active vegetative cells; and (4) specific features of ultrastructural organization absent in vegetative cells: thickened and lamellar envelopes, clumpy structure of the cytoplasm, and condensed DNA in nucleoid. The differences in various types of CRC concern the thickness and lamellar structure of cell envelopes, as well as the presence and thickness of the capsular layer. In particular, forms ultrastructurally similar to typical bacterial cysts were revealed in pseudomonad populations growing on soil agar. Physiological diversity was revealed in different levels of viability preservation and thermal resistance in various types of CRC and depended on the conditions of their formation. The optimal conditions and procedures for obtaining P. aurantiaca and P. fluorescens CRC that retain the ability to form colonies on standard nutrient media are as follows: (1) a twofold decrease of nitrogen content in the growth medium; (2) an increased level of anabiosis autoinducer (C12-AHB, 10(-4) M) in stationary cultures; (3) transfer of the cells from stationary cultures to a starvation medium with silica; (4) cultivation in soil extract; and (5) development of cultures on soil agar. The CRC from the cultures grown in soil extract or starvation medium with silica proved to be resistant to heat treatment (60 degrees C, 5 min). In the CRC formed in nitrogen-limited media, the degree of heat resistance increased at longer incubation (1.5 to 6 months). CRCs on soil agar surface were resistant to desiccation. The ultrastructure of the morphologically varied types of P. aurantiaca CRC formed under simulated natural conditions is described for the first time. The data on the intraspecies diversity of pseudomonad dormant forms contribute to the concept of plasticity of the life style and adaptive reactions that ensure survival of these bacteria in unfavorable environmental conditions.

Functional Analysis of GlnE, an Essential Adenylyl Transferase in Mycobacterium tuberculosis

Glutamine synthetase (GS) plays an important role in nitrogen assimilation. The major GS of Mycobacterium tuberculosis is GlnA1, a type I GS whose activity is controlled by posttranscriptional modification by GlnE. GlnE is an adenylyl transferase comprised of an adenylylating domain and a deadenylylating domain which modulate GS activity. We previously demonstrated that GlnE is essential in M. tuberculosis in normal growth medium. In this study, we further show that GlnE is required under multiple medium conditions, including in nitrogen-limited medium. We demonstrate that adenylylation is the critical activity for M. tuberculosis survival, since we were able to delete the deadenylylation domain with no apparent effect on growth or GS activity. Furthermore, we identified a critical aspartate residue in the proposed nucleotidyltransferase motif. Temperature-sensitive mutants of GlnE were generated and shown to have a defect in growth and GS activity in nitrogen-limited medium. Finally, we were able to generate a GlnE null mutant in the presence of L-methionine sulfoximine, a GS inhibitor, and glutamine supplementation. In the presence of these supplements, the null mutant was able to grow similarly to the wild type. Surprisingly, the GlnE mutant was able to survive and grow for extended periods in liquid medium, but not on solid medium, in the absence of GS inhibition. Thus, we have confirmed that the unusual requirement of M. tuberculosis for GlnE adenylylation activity is linked to the activity of GS in the cell.

Enzymatic degradation of anthracene by the white rot fungus Phanerochaete chrysosporium immobilized on sugarcane bagasse

Bagasse is a by-product of sugar milling and important fuel resource for that industry. It is a fibrous, low density material with a very wide range of particle sizes and high moisture content. The goal of this study is the development of a system based on the use of the ligninolytic enzyme manganese peroxidase (MnP) produced by Phanerochaete chrysosporium for the degradation of polycyclic aromatic hydrocarbons (PAHs), of which anthracene was selected as an example. The white rot fungus P. chrysosporium immobilized on bagasse was grown in both stationary and agitated cultures (rotary shaker, 80 rpm) using nitrogen limited growth medium to study the ability of the fungus to degrade anthracene in aqueous media. Production of MnP occurred simultaneously in nitrogen limited culture medium with the added MnSO 4 at 40 ppm. The MnP activity was at relatively high level (76 U l −1) and in this condition, the residual anthracene concentration was 16%.

Saline-Dependent Regulation of Manganese Peroxidase Genes in the Hypersaline-Tolerant White Rot Fungus Phlebia sp. Strain MG-60

The expression pattern of manganese peroxidases (MnPs) in nitrogen-limited cultures of the saline-tolerant fungus Phlebia sp. strain MG-60 is differentially regulated under hypersaline conditions at the mRNA level. When MG-60 was cultured in nitrogen-limited medium (LNM) containing 3% (wt/vol) sea salts (LN-SSM), higher activity of MnPs was observed than that observed in normal medium (LNM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that two MnP isoenzymes were de novo synthesized in the culture of LN-SSM. Three MnP-encoding genes (MGmnp1, MGmnp2, and MGmnp3) were isolated by reverse transcription (RT)-PCR and rapid amplification of cDNA ends PCR techniques. The corresponding isozymes were identified by peptide mass fingerprinting analysis. MnP isozymes encoded by MGmnp2 and MGmnp3 were observed mainly in LN-SSM. Real-time RT-PCR analysis revealed high levels of MGmnp2 and MGmnp3 transcripts in LN-SSM 48 h after the addition of 2% NaCl. The induction of MnP production and the accumulation of gene transcripts by saline were well correlated in the presence of Mn(2+). However, in the absence of Mn(2+), there was no clear correlation between mnp transcripts levels and MnP activity, suggesting posttranscriptional regulation by Mn(2+).

Effects of culture conditions on ligninolytic enzymes and protease production by Phanerochaete chrysosporium in air

The production of ligninolytic enzymes and protease by Phanerochaete chrysosporium was investigated under different culture conditions. Different amounts of medium were employed in free and immobilized culture, together with two kinds of medium with different C/N ratios. Little lignin peroxidase (LiP) (< 2U/L) was detected in free culture with nitrogen-limited medium (C/N ratio: 56/2.2, in mmol/L), while manganese peroxidase (MnP) maximum activity was 231 and 240 U/L in 50 and 100 ml medium culture, respectively. Immobilized culture with 50 ml nitrogen-limited medium gave the highest MnP and LiP production with the maximum values of 410 and 721 U/L separately on the day 5; however, flasks containing 100 ml nitrogen-limited medium only produced less MnP with a peak value of 290 U/L. Comparatively, carbon-limited medium (C/N ratio: 28/44, in mmol/L) was adopted in culture but produced little MnP and LiP. Medium type had the greatest impact on protease production. Large amount of protease was produced due to glucose limitation. Culture type and medium volume influence protease activity corporately by affecting oxygen supply. The results implied shallow immobilized culture was a possible way to gain high production of ligninolytic enzymes.

Comparative study on using carbon or nitrogen limited medium to culture white rot fungi for reactive brilliant red dye K-2BP decolotization under non-sterile conditions

In order to explore ways for the application of white rot fungus in dye effluent treatment under non-sterile conditions, experiment on decolorization of reactive brilliant red was carried out, employing nitrogen-limited and carbon-limited medium with C/N ratio of 56/2.2 and 28/44 (in mmol/L), respectively. The results showed that the decolorization rate reached 92% while culturing white rot fungus with nitrogen-limited medium; however, the decolorization process ended in carbon-limited medium (n(C)/n(N) = 28/44) because of bacterial contamination. In addition, pH rose up to 9.31 after 4 d of decolorization, which was caused by bacterial contamination in the carbon-limited system. Therefore, it is concluded that nitrogen-limited medium can inhibit bacterial growth to some extent while carbon-limited medium is more easily contaminated by bacteria. Nitrogen-limited medium is more suitable in culture of white rot fungus for decolorization of reactive dye. Medium with the ability of inhibiting yeast growth should be developed by adjusting other components of nitrogen-limited medium.

Cloning and Expression of an α-Amylase Gene from Phanerochaete chrysosporium

Based on the genomic sequence and cDNA library screening, the cDNA sequence encoding an alpha-amylase was cloned from the filamentous white-rot fungus Phanerochaete chrysosporium and designated as pcamy1. Alignment results showed that the predicted protein has up to 43% amino acid homology to the known alpha-amylases in other organisms and is close to those from some filamentous fungi. Under nitrogen-starvation condition, the transcription of pcamy1 was accordingly upregulated or downregulated when soluble starch or glucose is sole carbon source. Addition of oxygen to nitrogen-limited media led to pcamy1 transcription and removal of glucose metabolic repression. The result indicated that the pcamy1 transcript was not only regulated by nutrients such as the carbon source but also by the cultivation environment, such as oxygen. This coordinate-regulatory model is likely common in P. chrysosporium. The expressed product of this gene in Escherichia coli could hydrolyze soluble starch, and its enzymatic activity was determined. As far as we know, this is the first report about cloning and expression study on the alpha-amylase in P. chrysosporium.

Influence of glucose feeding on the ligninolytic enzyme production of the white-rot fungus Phanerochaete chrysosporium

The present work studied the influence of glucose feeding on the ligninolytic enzyme production of Phanerochaete chrysosporium in a nitrogen-limited (C/N ratio is 56/8.8 mmol/L) medium. Several sets of shaking flask experiments were conducted. The results showed that 2 g/L glucose feeding on the first day of the culture (24 h after the inoculation) simulated both fungal biomass growth and enzyme production. The manganese peroxidase (MnP) activity was 2.5 times greater than that produced in cultures without glucose feeding. Furthermore, the glucose feeding mode in fed-batch culture was also investigated. Compared to cultures with glucose feeding every 48 h, cultures with glucose feeding of 1.5 g/L (final concentration) every 24 h produced more enzymes. The peak and total yield of MnP activity were 2.7 and 3 times greater compared to the contrast culture, respectively, and the enzyme was kept stable for 4 days with an activity of over 200 U/L.

Ammonia removal from livestock wastewater by ammonia-assimilating microorganisms immobilized in polyvinyl alcohol

We isolated ammonia-assimilating microorganisms from the livestock manure treatment systems and evaluated their ammonia-assimilating ability. Many isolates utilized ammonia at high rates when they were purely cultivated in a nitrogen-limited medium to which sterilized lagoon extract had been added. Some isolates that were immobilized in polyvinyl alcohol (PVA) utilized ammonia present in the media containing viable lagoon microorganisms. Staining with 4',6'-diamidino-2-phenylindole (DAPI) indicated that the immobilized high ammonia-assimilating isolates grew dominantly within the PVA beads. High ammonia-assimilating isolates in the mixed culture containing viable lagoon microorganisms were identified as Pseudomonas spp. and member of Rhizobiaceae species by partial sequencing of the 16S ribosomal DNA.

The impact of severe nitrogen limitation and microaerobic conditions on extended continuous cultivations of Saccharomyces cerevisiae with cell recirculation

Continuous cultivations of Saccharomyces cerevisiae ATCC 96581 with severe nitrogen limitation (C/N ratios 200 and 400 g g −1) and cell recirculation were carried out under anaerobic and microaerobic conditions for more than 300 h. With a dilution rate of 0.06 h −1 and 90% recirculation in combination with an estimated 70% biomass sedimentation rate in the bleed flow, specific growth rates of 0.002–0.006 h −1 were obtained. Under these conditions, ethanol yields of 0.46–0.48 g g −1 were achieved. The biomass yields on ATP were only 1.6–2.9 g mol −1, indicating metabolic uncoupling or high maintenance energy requirements. Viability levels, measured by FUN ® staining and fluorescence microscopy, usually varied between 100 and 80%. However, under anaerobic conditions at C/N ratio 400, a reproducible drop to 25% viability occurred between 250 and 300 h of fermentation, after which the culture recovered again. Under anaerobic conditions, an increase in the C/N ratio from 200 to 400 resulted in a three-fold higher specific glycerol production, in spite of lower biomass formation and lower cellular protein and RNA content. A low oxygen addition eliminated the large drop in viability and the increased glycerol production observed at C/N 400, and caused viability and glycerol levels similar to the anaerobic C/N 200 case. A S. cerevisiae W303-1A gpd1Δ gpd2Δ mutant, completely deficient in glycerol production, could ferment a nitrogen-limited medium under RQ-controlled microaerobic conditions with an ethanol yield of 0.45 g g −1, indicating that the increased glycerol production under nitrogen limitation is not necessary, as long as there is sufficient oxygen transferred to the culture.

Induction of pullulanase production in Bacillus cereus FDTA-13

Studies were carried out on the production of pullulanase by a newly isolated strain Bacillus cereus FDTA-13. High titres of the enzyme were obtained in a medium containing branched polysaccharides. To further enhance the yield, induction of pullulanase using conventional inducers were studied. Maltooligosaccharides (maltose to maltotetraose) when added in the medium individually, or in a 1:1 combination of maltotriose and maltotetraose resulted different levels of pullulanase compared to control. Growth under carbon limited conditions (5 g l −1) with inducers resulted remarkably enhanced pullulanase activity. Pullulanase activity was severely repressed in presence of glucose. Low levels of pullulanase was observed in nitrogen limited medium, even with combinations of several maltosaccharides.

Utilization of various industrial wastes for the production of poly-β-hydroxy butyrate (PHB) by Alcaligenes eutrophus

Considering the industrial interest of poly-β-hydroxy butyrate (PHB) and its high production cost, work has been undertaken for the production of PHB by Alcaligenes eutrophus. Different industrial wastes (malt, soya, sesame, molasses, bagasse and pharmaceutical waste) were used as a cheap substrate to minimize the production of cost and nitrogen limited minimal agar synthetic medium was also used for comparison. Accumulation of PHB granules in the organism was analyzed by sudan black method. The PHB production in various industrial waste based medium and nitrogen limited minimal agar synthetic medium was studied by crotonic acid method. The pure form of PHB was collected and qualitatively analyzed by infrared and nuclear magnetic resonance methods. Highest PHB production was found in nitrogen limited minimal agar synthetic medium. Among the various industrial wastes based media, highest yield was obtained with sesame oil waste as carbon source.

Identification of Genes Affecting Lipid Content Using Transposon Mutagenesis inSaccharomyces cerevisiae

Genes involved in lipid accumulation were identified in Saccharomyces cerevisiae using transposon insertion mutagenesis. Five ORFs, such as SNF2, IRA2, PRE9, PHO90, and SPT21 were found from the analysis of the insertion sites in transposon insertion mutants with higher lipid content. Since these ORFs are not directly involved in storage lipid biosynthesis, we speculate that they are involved in carbon fluxes into storage lipids in response to nutrient conditions. Lipid analysis of disruptants of these ORFs indicated that the Deltasnf2, and Deltaira2 disruptants had significantly higher lipid content. Cultivation in a nitrogen-limited medium increased the lipid content in all disruptants, among which the Deltapre9 disruptant was the most sensitive to nitrogen limitation. We then focused on the Deltasnf2 disruptant due to its higher lipid content and its function as a regulator of phospholipid synthesis. Lipid class analysis indicated that triacylglycerol and free fatty acids contributed to the increase in total lipids of the Deltasnf2 disruptant. The addition of exogenous fatty acids was not so effective at increasing the lipid content in the Deltasnf2 disruptant as it was in the wild type. It should be noticed that exogenous free linoleic acid was much higher in the Deltasnf2 disruptant than in the wild type, as in the case of endogenous free fatty acids. In addition, the incorporation of exogenous fatty acids into cells increased in the disruptant, suggesting that fatty acid transporters were regulated by SNF2. The results suggest that metabolic fluxes into storage lipids, which are activated in the Deltasnf2 disruptant, is repressed by the incorporation of exogenous fatty acids. They provide new insight into the biosynthesis of storage lipids in yeast.

Kinetic Studies of Polyhydroxybutyrate Granule Formation in Wautersia eutropha H16 by Transmission Electron Microscopy

Wautersia eutropha, formerly known as Ralstonia eutropha, a gram-negative bacterium, accumulates polyhydroxybutyrate (PHB) as insoluble granules inside the cell when nutrients other than carbon are limited. In this paper, we report findings from kinetic studies of granule formation and degradation in W. eutropha H16 obtained using transmission electron microscopy (TEM). In nitrogen-limited growth medium, the phenotype of the cells at the early stages of granule formation was revealed for the first time. At the center of the cells, dark-stained "mediation elements" with small granules attached were observed. These mediation elements are proposed to serve as nucleation sites for granule initiation. TEM images also revealed that when W. eutropha cells were introduced into nitrogen-limited medium from nutrient-rich medium, the cell size increased two- to threefold, and the cells underwent additional volume changes during growth. Unbiased stereology was used to analyze the two-dimensional TEM images, from which the average volume of a W. eutropha H16 cell and the total surface area of granules per cell in nutrient-rich and PHB production media were obtained. These parameters were essential in the calculation of the concentration of proteins involved in PHB formation and utilization and their changes with time. The extent of protein coverage of the granule surface area is presented in the accompanying paper.

Isolation of palm oil-utilising, polyhydroxyalkanoate (PHA)-producing bacteria by an enrichment technique

In early attempts to isolate palm oil-utilising bacteria from palm oil mill effluent (POME), diluted liquid samples of POME were spread on agar containing POME as primary nutrient. 45 purified colonies were screened for intracellular lipids by staining with Sudan Black B. Of these, 10 isolates were positively stained. The latter were grown in a nitrogen-limiting medium with palm olein (a triglyceride) or saponified palm olein (salts of fatty acids) as carbon source. None of the isolates grew in the palm olein medium but all grew well in the saponified palm olein medium. Of the latter however, only one isolate was positively stained with Nile Blue A, indicating the presence of PHA. This method did not successfully generate bacterial isolates which could metabolise palm olein to produce PHA. An enrichment technique was therefore developed whereby a selective medium was designed. The latter comprised minerals and palm olein (1% w/v) as sole carbon source to which POME (2.5% v/v) was added as the source of bacteria. The culture was incubated with shaking at 30 °C for 4 weeks. Out of seven isolates obtained from the selective medium, two isolates, FLP1 and FLP2, could utilise palm olein for growth and production of the homopolyester, poly(3-hydroxybutyrate). FLP1 is gram-negative and is identified (BIOLOG) to have 80% similarity to Burkholderia cepacia. When grown with propionate or valerate, FLP1 produced a copolyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

Phosphorylation-independent Dimer-Dimer Interactions by the Enhancer-binding Activator NtrC of Escherichia coli: A THIRD FUNCTION FOR THE C-TERMINAL DOMAIN

The response regulator NtrC transcriptionally activates genes of the nitrogen-regulated (Ntr) response. Phosphorylation of its N-terminal receiver domain stimulates an essential oligomerization of the central domain. Deletion of the central domain reduces, but does not eliminate, intermolecular interactions as assessed by cooperative binding to DNA. To analyze the structural determinants and function of this central domain-independent as well as phosphorylation-independent oligomerization, we randomly mutagenized DNA coding for an NtrC without its central domain and isolated strains containing NtrC with defective phosphorylation-independent cooperative binding. The alterations were primarily localized to helix B of the C-terminal domain. Site-specific mutagenesis that altered surface residues of helix B confirmed this localization. The purified NtrC variants, with or without the central domain, were specifically defective in phosphorylation-independent cooperative DNA binding and had little defect, if any, on other functions, such as non-cooperative DNA binding. We propose that this region forms an oligomerization interface. Full-length NtrC variants did not efficiently repress the glnA-ntrBC operon when NtrC was not phosphorylated, which suggests that phosphorylation-independent cooperative binding sets the basal level for glutamine synthetase and the regulators of the Ntr response. The NtrC variants in these cells generally, but not always, supported wild-type growth in nitrogen-limited media and wild-type activation of a variety of Ntr genes. We discuss the differences and similarities between the NtrC C-terminal domain and the homologous Fis, which is also capable of intermolecular interactions.

Pyrolytic descriptors responsive to changes in humic acid characteristics after long-term sustainable management of dryland farming systems in Central Spain

Structural changes in humic acids from a semiarid soil of an experimental farm in Central Spain have been studied by Curie-point pyrolysis. Soil has received periodic inputs of farmyard manure or crop wastes for the last 16 years, and mineral nitrogen fertilisation in 50% of the plots. Analytical pyrolysis suggested substantial differences in terms of the effect of soil management on the accumulation mechanisms of the humic acid fraction. When compared with control plots, humic acids from plots amended with crop wastes displayed well-defined methoxyphenol assemblages, indicating that the diagenetic transformation of lignin could be a dominant mechanism of organic matter stabilisation. The greatest yields of methoxyphenols after organic inputs were obtained in plots receiving nitrogen fertilisation, what agrees with the expected higher performance of the lignin biodegradation in nitrogen-limited media. Increased yields of lignin-derived methoxyphenols were also observed in plots treated with manure, which released in addition a conspicuous series of alkyl compounds suggesting recalcitrant wax-derived lipids incorporated to the humic acids. Highest yields of pyrolytic fatty acids were observed in humic acids from manure-amended plots without nitrogen fertilisation. Alkylbenzene proportions were also pyrolytic descriptors responsive to mineral nitrogen inputs, showing very significant differences ( P<0.01) in both control and amended plots. Results suggested that continued organic inputs can not be considered to increase soil organic matter quality, since microbial reworking of lignins became the dominant mechanism of accumulation of humic acid-type substances, even when nitrogen inputs were applied.

Overexpression of the ASN1 gene enhances nitrogen status in seeds of Arabidopsis.

In wild-type Arabidopsis, levels of ASN1 mRNA and asparagine (Asn) are tightly regulated by environmental factors and metabolites. Because Asn serves as an important nitrogen storage and transport compound used to allocate nitrogen resources between source and sink organs, we tested whether overexpression of the major expressed gene for Asn synthetase, ASN1, would lead to changes in nitrogen status in the ultimate storage organ for metabolites-seeds. Transgenic Arabidopsis constitutively overexpressing ASN1 under the cauliflower mosaic virus 35S promoter were constructed (35S-ASN1). In seeds of the 35S-ASN1 lines, three observations support the notion that the nitrogen status was enhanced: (a) elevations of soluble seed protein contents, (b) elevations of total protein contents from acid-hydrolyzed seeds, and (c) higher tolerance of young seedlings when grown on nitrogen-limiting media. Besides quantitative differences, changes in the relative composition of the seed amino acid were also observed. The change in seed nitrogen status was accompanied by an increase of total free amino acids (mainly Asn) allocated to flowers and developing siliques. In 35S-ASN1 lines, sink tissues such as flowers and developing siliques exhibit a higher level of free Asn than source tissues such as leaves and stems, despite significantly higher levels of ASN1 mRNA observed in the source tissues. This was at least partially due to an enhanced transport of Asn from source to sink via the phloem, as demonstrated by the increased levels of Asn in phloem exudates of the 35S-ASN1 plants.

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. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position -59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions -120 to +67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions -83 to +67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.