Genetic Modifications in Bacteria for the Degradation of Synthetic Polymers: A Review.

Synthetic polymers, commonly known as plastics, are currently present in all aspects of our lives. Although they are useful, they present the problem of what to do with them after their lifespan. There are currently mechanical and chemical methods to treat plastics, but these are methods that, among other disadvantages, can be expensive in terms of energy or produce polluting gases. A more environmentally friendly alternative is recycling, although this practice is not widespread. Based on the practice of the so-called circular economy, many studies are focused on the biodegradation of these polymers by enzymes. Using enzymes is a harmless method that can also generate substances with high added value. Novel and enhanced plastic-degrading enzymes have been obtained by modifying the amino acid sequence of existing ones, especially on their active site, using a wide variety of genetic approaches. Currently, many studies focus on the common aim of achieving strains with greater hydrolytic activity toward a different range of plastic polymers. Although in most cases the depolymerization rate is improved, more research is required to develop effective biodegradation strategies for plastic recycling or upcycling. This review focuses on a compilation and discussion of the most important research outcomes carried out on microbial biotechnology to degrade and recycle plastics.

Genome sequence analysis and characterization of Bacillus altitudinis B12, a polylactic acid- and keratin-degrading bacterium.

Keratin-rich wastes, mainly in the form of feathers, are recalcitrant residues generated in high amounts as by-products in chicken farms and food industry. Polylactic acid (PLA) is the second most common biodegradable polymer found in commercial plastics, which is not easily degraded by microbial activity. This work reports the 3.8-Mb genome of Bacillus altitudinis B12, a highly efficient PLA- and keratin-degrading bacterium, with potential for environmental friendly biotechnological applications in the feed, fertilizer, detergent, leather, and pharmaceutical industries. The whole genome sequence of B. altitudinis B12 revealed that this strain (which had been previously misclassified as Bacillus pumilus B12) is closely related to the B. altitudinis strains ER5, W3, and GR-8. A total of 4056 coding sequences were annotated using the RAST server, of which 2484 are core genes of the pan genome of B. altitudinis and 171 are unique to this strain. According to the sequence analysis, B. pumilus B12 has a predicted secretome of 353 proteins, among which a keratinase and a PLA depolymerase were identified by sequence analysis. The presence of these two enzymes could explain the characterized PLA and keratin biodegradation capability of the strain.

Genome sequencing analysis of Streptomyces coelicolor mutants that overcome the phosphate-depending vancomycin lethal effect.

BACKGROUND: Glycopeptide antibiotics inhibit bacterial cell-wall synthesis, and are important for the treatment of infections caused by multi drug-resistant strains of enterococci, streptococci and staphylococci. The main mechanism by which bacteria resist the action of glycopeptides is by producing a modified cell-wall in which the dipeptide D-Alanine-D-Alanine is substituted by D-Alanine-D-Lactate or D-Alanine-D-Serine. Recently, it has been shown that inorganic phosphate (Pi) induces hypersensitivity to vancomycin in Streptomyces coelicolor (which is highly resistant to the antibiotic in low-Pi media). This finding was surprising because the bacterium possesses the entire set of genes responsible for vancomycin resistance (VR); including those coding for the histidine kinase/response regulator pair VanS/VanR that activates the system. RESULTS: This work shows that high Pi amounts in the medium hamper the activation of the van promoters and consequently inhibit VR in S. coelicolor; i.e. the repression effect being stronger when basic or acidic forms of the nutrient are used. In addition, this work shows that lysozyme resistance is also highly regulated by the Pi concentration in the medium. At least five different mutations contribute to the overcoming of this repression effect over VR (but not over lysozyme resistance). Therefore, the interconnection of VR and lysozyme resistance mechanisms might be inexistent or complex. In particular, two kinds of mutant in which Pi control of VR has been lost (one class expresses the van genes in a constitutive manner; the other retains inducibility by vancomycin) have been isolated and further characterized in this study. Sequencing revealed that the first class of mutation conferred a single amino acid substitution in the second transmembrane helix of the VanS protein; whereas the other class hampered the expression or activity of a putative homolog (SCO1213) to the staphylococcal GatD protein. Complementation, phenotypic and bioinformatics analyses identified SCO1213, and its upstream gene (i.e. murT), as relevant genetic determinants involved with VR in S. coelicolor. CONCLUSION: The genomic approach of this study together with other genetic and phenotypic analyses has allowed the identification of the uncharacterized murT-gatD Streptomyces genes and the characterization of their involvement with the Pi control of VR in S. coelicolor.

Green fluorescent protein as a reporter for the spatial and temporal expression of actIII in Streptomyces coelicolor.

Polyketides constitute a large group of structurally diverse natural products with useful biological activities. Insights into their biosynthetic mechanisms are crucial for developing new structures. One of the most studied model polyketide is the blue-pigmented antibiotic actinorhodin, produced by Streptomyces coelicolor. This aromatic polyketide is synthesized by minimal type II polyketide synthases and tailoring enzymes. The ActIII actinorhodin ketoreductase is a key tailoring enzyme in actinorhodin biosynthesis. Previous papers have reported contradictory findings for localization of the protein in the cytoplasmic fraction or associated with the cell wall. We have now used green fluorescent protein as a reporter to analyse the spatial and temporal expression of actIII (SCO5086) in S. coelicolor under actinorhodin producing and non-producing conditions. We provide evidence in support of ActIII being a cytosolic protein, with limited if any association with the membrane or cell wall.

Streptomyces tsukubaensis as a new model for carbon repression: transcriptomic response to tacrolimus repressing carbon sources.

In this work, we identified glucose and glycerol as tacrolimus repressing carbon sources in the important species Streptomyces tsukubaensis. A genome-wide analysis of the transcriptomic response to glucose and glycerol additions was performed using microarray technology. The transcriptional time series obtained allowed us to compare the transcriptomic profiling of S. tsukubaensis growing under tacrolimus producing and non-producing conditions. The analysis revealed important and different metabolic changes after the additions and a lack of transcriptional activation of the fkb cluster. In addition, we detected important differences in the transcriptional response to glucose between S. tsukubaensis and the model species Streptomyces coelicolor. A number of genes encoding key players of morphological and biochemical differentiation were strongly and permanently downregulated by the carbon sources. Finally, we identified several genes showing transcriptional profiles highly correlated to that of the tacrolimus biosynthetic pathway regulator FkbN that might be potential candidates for the improvement of tacrolimus production.

Genome-wide analysis of the regulation of pimaricin production in Streptomyces natalensis by reactive oxygen species.

To investigate the molecular mechanisms that interplay between oxygen metabolism and secondary metabolism in Streptomyces natalensis, we compared the transcriptomes of the strains CAM.02 (ΔsodF), pimaricin under-producer phenotype, and CAM.04 (ΔahpCD), pimaricin over-producer phenotype, with that of the wild type at late exponential and stationary growth phases. Microarray data interpretation was supported by characterization of the mutant strains regarding enzymatic activities, phosphate uptake, oxygen consumption and pimaricin production.Both mutant strains presented a delay in the transcription activation of the PhoRP system and pimaricin biosynthetic gene cluster that correlated with the delayed inorganic phosphate (Pi) depletion in the medium and late onset of pimaricin production, respectively. The carbon flux of both mutants was also altered: a re-direction from glycolysis to the pentose phosphate pathway (PPP) in early exponential phase followed by a transcriptional activation of both pathways in subsequent growth phases was observed. Mutant behavior diverged at the respiratory chain/tricarboxylic acid cycle (TCA) and the branched chain amino acid (BCAA) metabolism. CAM.02 (ΔsodF) presented an impaired TCA cycle and an inhibition of the BCAA biosynthesis and degradation pathways. Conversely, CAM.04 (ΔahpCD) presented a global activation of BCAA metabolism.The results highlight the cellular NADPH/NADH ratio and the availability of biosynthetic precursors via the BCAA metabolism as the main pimaricin biosynthetic bottlenecks under oxidative stress conditions. Furthermore, new evidences are provided regarding a crosstalk between phosphate metabolism and oxidative stress in Streptomyces.

What is the role of microbial biotechnology and genetic engineering in medicine?

Microbial products are essential for developing various therapeutic agents, including antibiotics, anticancer drugs, vaccines, and therapeutic enzymes. Genetic engineering techniques, functional genomics, and synthetic biology unlock previously uncharacterized natural products. This review highlights major advances in microbial biotechnology, focusing on gene-based technologies for medical applications.

Genome-scale metabolic model of the versatile bacterium Paracoccus denitrificans Pd1222.

A genome scale metabolic model of the bacterium Paracoccus denitrificans has been constructed. The model containing 972 metabolic genes, 1,371 reactions, and 1,388 unique metabolites has been reconstructed. The model was used to carry out quantitative predictions of biomass yields on 10 different carbon sources under aerobic conditions. Yields on C1 compounds suggest that formate is oxidized by a formate dehydrogenase O, which uses ubiquinone as redox co-factor. The model also predicted the threshold methanol/mannitol uptake ratio, above which ribulose biphosphate carboxylase has to be expressed in order to optimize biomass yields. Biomass yields on acetate, formate, and succinate, when NO3- is used as electron acceptor, were also predicted correctly. The model reconstruction revealed the capability of P. denitrificans to grow on several non-conventional substrates such as adipic acid, 1,4-butanediol, 1,3-butanediol, and ethylene glycol. The capacity to grow on these substrates was tested experimentally, and the experimental biomass yields on these substrates were accurately predicted by the model.IMPORTANCEParacoccus denitrificans has been broadly used as a model denitrifying organism. It grows on a large portfolio of carbon sources, under aerobic and anoxic conditions. These characteristics, together with its amenability to genetic manipulations, make P. denitrificans a promising cell factory for industrial biotechnology. This paper presents and validates the first functional genome-scale metabolic model for P. denitrificans, which is a key tool to enable P. denitrificans as a platform for metabolic engineering and industrial biotechnology. Optimization of the biomass yield led to accurate predictions in a broad scope of substrates.

Identification of different promoters in the absA1-absA2 two-component system, a negative regulator of antibiotic production in Streptomyces coelicolor.

The absA1-absA2 genes encode a two-component system that negatively regulates the transcription of multiple antibiotic gene clusters of Streptomyces coelicolor. The microarray dataset time series of a S. coelicolor M145 bioreactor culture indicated that the transcription values of absA2 were approximately four times higher than those of absA1 throughout the time course of the culture. The co-transcription of absA1 and absA2 genes has been previously shown, although an independent absA2 promoter was not detected. In this study, we show by different technical approaches that the absA1-absA2 operon is transcribed from at least two promoters, the first producing a read-through transcript that includes both absA1 and absA2 genes and the second including only the absA2 gene. An absA2 mRNA 5' end was mapped by primer extension and confirmed as TSS by deep sequencing in combination with TEX. Promoter-probe analyses detected promoter activity in both the absA1 and absA2 upstream regions. The absA2 upstream region showed a higher promoter activity, at least sevenfold higher than that of absA1. Furthermore, the absA2 gene may contain at least two additional promoters as shown by deep sequencing analyses. All together this work contributes to the understanding of the complex transcriptional regulation of these antibiotic regulators genes in S. coelicolor.

Characterization of the Keratinolytic Activity of Three Streptomyces Strains and Impact of Their Co-Cultivation on This Activity.

In this study, we describe the characterization of three efficient chicken feather-degrading Streptomyces bacteria isolated from honeybee samples and assess the impact of their co-cultivation on this activity and antistaphylococcal activity. Streptomyces griseoaurantiacus AD2 was the strain showing the highest keratinolytic activity (4000 U × mL-1), followed by Streptomyces albidoflavus AN1 and Streptomyces drozdowiczii AD1, which both generated approximately 3000 U × mL-1. Moreover, a consortium constituted of these three strains was able to use chicken feathers as its sole nutrient source and growth in such conditions led to a significant increase in antibiotic production. S. griseoaurantiacus AD2 was the only strain that exhibited weak antimicrobial activity against Staphylococcus aureus. UPLC analyses revealed that a significant number of peaks detected in the extracts of co-cultures of the three strains were missing in the extracts of individual cultures. In addition, the production of specialized metabolites, such as undecylprodigiosin and manumycin A, was clearly enhanced in co-culture conditions, in agreement with the results of the antimicrobial bioassays against S. aureus. Our results revealed the benefits of co-cultivation of these bacterial species in terms of metabolic wealth and antibiotic production. Our work could thus contribute to the development of novel microbial-based strategies to valorize keratin waste.

Characterization of Bacillus Strains from Natural Honeybee Products with High Keratinolytic Activity and Antimicrobial Potential.

Two efficient feather-degrading bacteria were isolated from honeybee samples and identified as Bacillus sonorensis and Bacillus licheniformis based on 16S rRNA and genome sequencing. The strains were able to grow on chicken feathers as the sole carbon and nitrogen sources and degraded the feathers in a few days. The highest keratinase activity was detected by the B. licheniformis CG1 strain (3800 U × mL-1), followed by B. sonorensis AB7 (1450 U × mL-1). Keratinase from B. licheniformis CG1 was shown to be active across a wide range of pH, potentially making this strain advantageous for further industrial applications. All isolates displayed antimicrobial activity against Micrococcus luteus; however, only B. licheniformis CG1 was able to inhibit the growth of Mycobacterium smegmatis. In silico analysis using BAGEL and antiSMASH identified gene clusters associated with the synthesis of non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKSs) and/or ribosomally synthesized and post-translationally modified peptides (RiPPs) in most of the Bacillus isolates. B. licheniformis CG1, the only strain that inhibited the growth of the mycobacterial strain, contained sequences with 100% similarity to lichenysin (also present in the other isolates) and lichenicidin (only present in the CG1 strain). Both compounds have been described to display antimicrobial activity against distinct bacteria. In summary, in this work, we have isolated a strain (B. licheniformis CG1) with promising potential for use in different industrial applications, including animal nutrition, leather processing, detergent formulation and feather degradation.

Screening Enzymes That Can Depolymerize Commercial Biodegradable Polymers: Heterologous Expression of Fusarium solani Cutinase in Escherichia coli.

In recent years, a number of microbial enzymes capable of degrading plastics have been identified. Biocatalytic depolymerization mediated by enzymes has emerged as a potentially more efficient and environmentally friendly alternative to the currently employed methods for plastic treatment and recycling. However, the functional and systematic study of depolymerase enzymes with respect to the degradation of a series of plastic polymers in a single work has not been widely addressed at present. In this study, the ability of a set of enzymes (esterase, arylesterase and cutinase) to degrade commercial biodegradable polymers (PBS, PBAT, PHB, PHBH, PHBV, PCL, PLA and PLA/PCL) and the effect of pre-treatment methods on their degradation rate was assessed. The degradation products were identified and quantified by HPLC and LC-HRMS analysis. Out of the three enzymes, Fusarium solani cutinase (FsCut) showed the highest activity on grinded PBAT, PBS and PCL after 7 days of incubation. FsCut was engineered and heterologous expressed in Escherichia coli, which conferred the bacterium the capability of degrading solid discs of PBAT and to grow in PBS as the sole carbon source of the medium.

Is PhoR-PhoP partner fidelity strict? PhoR is required for the activation of the pho regulon in Streptomyces coelicolor.

Two-component regulatory systems play a key role in the cell metabolism adaptation to changing nutritional and environmental conditions. The fidelity between the two cognate proteins of a two-component system is important since it determines whether a specific response regulator integrates the signals transmitted by different sensor kinases. Phosphate regulation in Streptomyces coelicolor is mostly mediated by the PhoR-PhoP two-component system. Previous studies elucidated the mechanisms that control phosphate regulation as well as the genes directly regulated by the response regulator PhoP (pho regulon) in this organism. However, the role of the histidine kinase PhoR in Streptomyces coelicolor had not been unveiled so far. In this work, we report the characterization of a non-polar ΔphoR deletion mutant in S. coelicolor that keeps its native promoter. Induction of the phoRP operon was dependent upon phosphorylation of PhoP, but the ΔphoR mutant expressed phoP at a basal level. RT-PCR and reporter luciferase assays demonstrated that PhoR plays a key role in the activation of the pho regulon in this organism. Our results point towards a strict cognate partner specificity in terms of the phosphorylation of PhoP by PhoR thus corroborating the tight interaction between the two-components of this system.

Transcriptomic studies of phosphate control of primary and secondary metabolism in Streptomyces coelicolor.

Phosphate controls the biosynthesis of many classes of secondary metabolites that belong to different biosynthetic groups, indicating that phosphate control is a general mechanism governing secondary metabolism. We refer in this article to the molecular mechanisms of regulation, mediated by the two-component system PhoR-PhoP, of the primary metabolism and the biosynthesis of antibiotics. The two-component PhoR-PhoP system is conserved in all Streptomyces and related actinobacteria sequenced so far, and involves a third component PhoU that modulates the signal transduction cascade. The PhoP DNA-binding sequence is well characterized in Streptomyces coelicolor. It comprises at least two direct repeat units of 11 nt, the first seven of which are highly conserved. Other less conserved direct repeats located adjacent to the core ones can also be bound by PhoP through cooperative protein-protein interactions. The phoR-phoP operon is self-activated and requires phosphorylated PhoP to mediate the full response. About 50 up-regulated PhoP-dependent genes have been identified by comparative transcriptomic studies between the parental S. coelicolor M145 and the ΔphoP mutant strains. The PhoP regulation of several of these genes has been studied in detail using EMSA and DNase I footprinting studies as well as in vivo expression studies with reporter genes and RT-PCR transcriptomic analyses.

Identification of Antimicrobial Compounds in Two Streptomyces sp. Strains Isolated From Beehives.

The World Health Organization warns that the alarming increase in antibiotic resistant bacteria will lead to 2.7 million deaths annually due to the lack of effective antibiotic therapies. Clearly, there is an urgent need for short-term alternatives that help to alleviate these alarming figures. In this respect, the scientific community is exploring neglected ecological niches from which the prototypical antibiotic-producing bacteria Streptomycetes are expected to be present. Recent studies have reported that honeybees and their products carry Streptomyces species that possess strong antibacterial activity. In this study, we have investigated the antibiotic profile of two Streptomycetes strains that were isolated from beehives. One of the isolates is the strain Streptomyces albus AN1, which derives from pollen, and shows potent antimicrobial activity against Candida albicans. The other isolate is the strain Streptomyces griseoaurantiacus AD2, which was isolated from honey, and displays a broad range of antimicrobial activity against different Gram-positive bacteria, including pathogens such as Staphylococcus aureus and Enterococus faecalis. Cultures of S. griseoaurantiacus AD2 have the capacity to produce the antibacterial compounds undecylprodigiosin and manumycin, while those of S. albus AN1 accumulate antifungal compounds such as candicidins and antimycins. Furthermore, genome and dereplication analyses suggest that the number of putative bioactive metabolites produced by AD2 and AN1 is considerably high, including compounds with anti-microbial and anti-cancer properties. Our results postulate that beehives are a promising source for the discovery of novel bioactive compounds that might be of interest to the agri-food sector and healthcare pharmaceuticals.

Modulation of Multiple Gene Clusters' Expression by the PAS-LuxR Transcriptional Regulator PteF.

PAS-LuxR transcriptional regulators are conserved proteins governing polyene antifungal biosynthesis. PteF is the regulator of filipin biosynthesis from Streptomyces avermitilis. Its mutation drastically abates filipin, but also oligomycin production, a macrolide ATP-synthase inhibitor, and delays sporulation; thus, it has been considered a transcriptional activator. Transcriptomic analyses were performed in S. avermitilis DpteF and its parental strain. Both strains were grown in a YEME medium without sucrose, and the samples were taken at exponential and stationary growth phases. A total of 257 genes showed an altered expression in the mutant, most of them at the exponential growth phase. Surprisingly, despite PteF being considered an activator, most of the genes affected showed overexpression, thereby suggesting a negative modulation. The affected genes were related to various metabolic processes, including genetic information processing; DNA, energy, carbohydrate, and lipid metabolism; morphological differentiation; and transcriptional regulation, among others, but were particularly related to secondary metabolite biosynthesis. Notably, 10 secondary metabolite gene clusters out of the 38 encoded by the genome showed altered expression profiles in the mutant, suggesting a regulatory role for PteF that is wider than expected. The transcriptomic results were validated by quantitative reverse-transcription polymerase chain reaction. These findings provide important clues to understanding the intertwined regulatory machinery that modulates antibiotic biosynthesis in Streptomyces.

Biodegradation of bioplastics under aerobic and anaerobic aqueous conditions: Kinetics, carbon fate and particle size effect.

The biodegradation of PHB, PHBV, PBS, PBAT, PCL, PLA, and a PLA-PCL blend was compared under aerobic and anaerobic aqueous conditions assessing biodegradation kinetics, extent, carbon fate and particle size influence (in the range of 100-1000 µm). Under standard test conditions, PHB and PBHV were biodegraded anaerobically (83.9 ± 1.3% and 81.2 ± 1.7%, respectively) in 77 days or aerobically (83.0 ± 1.6% and 87.4 ± 7.5%) in 117 days, while PCL was only biodegraded (77.6 ± 2.4%) aerobically in 177 days. Apparent biomass growth accounted for 10 to 30.5% of the total initial carbon depending on the bioplastic and condition. Maximum aerobic and anaerobic biodegradation rates were improved up to 331 and 405%, respectively, at the lowest particle size tested (100-250 µm). This study highlights the usefulness of analysing biodegradation kinetics and carbon fate to improve both the development and testing of biodegradable materials, and waste treatments in the context of a circular bioeconomy.

Complex transcriptional control of the antibiotic regulator afsS in Streptomyces: PhoP and AfsR are overlapping, competitive activators.

The afsS gene of several Streptomyces species encodes a small sigma factor-like protein that acts as an activator of several pathway-specific regulatory genes (e.g., actII-ORF4 and redD in Streptomyces coelicolor). The two pleiotropic regulators AfsR and PhoP bind to overlapping sequences in the -35 region of the afsS promoter and control its expression. Using mutated afsS promoters containing specific point mutations in the AfsR and PhoP binding sequences, we proved that the overlapping recognition sequences for AfsR and PhoP are displaced by 1 nucleotide. Different nucleotide positions are important for binding of AfsR or PhoP, as shown by electrophoretic mobility shift assays and by reporter studies using the luxAB gene coupled to the different promoters. Mutant promoter M5 (with a nucleotide change at position 5 of the consensus box) binds AfsR but not PhoP with high affinity (named "superAfsR"). Expression of the afsS gene from this promoter led to overproduction of actinorhodin. Mutant promoter M16 binds PhoP with extremely high affinity ("superPhoP"). Studies with ΔafsR and ΔphoP mutants (lacking AfsR and PhoP, respectively) showed that both global regulators are competitive transcriptional activators of afsS. AfsR has greater influence on expression of afsS than PhoP, as shown by reverse transcriptase PCR (RT-PCR) and promoter reporter (luciferase) studies. These two high-level regulators appear to integrate different nutritional signals (particularly phosphate limitation sensed by PhoR), S-adenosylmethionine, and other still unknown environmental signals (leading to AfsR phosphorylation) for the AfsS-mediated control of biosynthesis of secondary metabolites.

The RNA polymerase omega factor RpoZ is regulated by PhoP and has an important role in antibiotic biosynthesis and morphological differentiation in Streptomyces coelicolor.

The RNA polymerase (RNAP) omega factor (ω) forms a complex with the α2ßß' core of this enzyme in bacteria. We have characterized the rpoZ gene of Streptomyces coelicolor, which encodes a small protein (90 amino acids) identified as the omega factor. Deletion of the rpoZ gene resulted in strains with a slightly reduced growth rate, although they were still able to sporulate. The biosynthesis of actinorhodin and, particularly, that of undecylprodigiosin were drastically reduced in the ΔrpoZ strain, suggesting that expression of these secondary metabolite biosynthetic genes is dependent upon the presence of RpoZ in the RNAP complex. Complementation of the ΔrpoZ mutant with the wild-type rpoZ allele restored both phenotype and antibiotic production. Interestingly, the rpoZ gene contains a PHO box in its promoter region. DNA binding assays showed that the phosphate response regulator PhoP binds to such a region. Since luciferase reporter studies showed that rpoZ promoter activity was increased in a ΔphoP background, it can be concluded that rpoZ is controlled negatively by PhoP, thus connecting phosphate depletion regulation with antibiotic production and morphological differentiation in Streptomyces.

Phosphate control over nitrogen metabolism in Streptomyces coelicolor: direct and indirect negative control of glnR, glnA, glnII and amtB expression by the response regulator PhoP.

Bacterial growth requires equilibrated concentration of C, N and P sources. This work shows a phosphate control over the nitrogen metabolism in the model actinomycete Streptomyces coelicolor. Phosphate control of metabolism in Streptomyces is exerted by the two component system PhoR-PhoP. The response regulator PhoP binds to well-known PHO boxes composed of direct repeat units (DRus). PhoP binds to the glnR promoter, encoding the major nitrogen regulator as shown by EMSA studies, but not to the glnRII promoter under identical experimental conditions. PhoP also binds to the promoters of glnA and glnII encoding two glutamine synthetases, and to the promoter of the amtB-glnK-glnD operon, encoding an ammonium transporter and two putative nitrogen sensing/regulatory proteins. Footprinting analyses revealed that the PhoP-binding sequence overlaps the GlnR boxes in both glnA and glnII. 'Information theory' quantitative analyses of base conservation allowed us to establish the structure of the PhoP-binding regions in the glnR, glnA, glnII and amtB genes. Expression studies using luxAB as reporter showed that PhoP represses the above mentioned nitrogen metabolism genes. A mutant deleted in PhoP showed increased expression of the nitrogen metabolism genes. The possible conservation of phosphate control over nitrogen metabolism in other microorganisms is discussed.