Search

One of the emerging biopolymers that are currently under active investigation is bacterial poly(γ-glutamic acid) (γ-PGA). However, before its full industrial exploitation, a substantial increase in microbial productivity is required. γ-PGA obtained from the Bacillus subtilis laboratory strain 168 offers the advantage of a producer characterized by a well defined genetic framework and simple manipulation techniques. In this strain, the knockout of genes for the major γ-PGA degrading enzymes, pgdS and ggt, leads to a considerable improvement in polymer yield, which attains levels analogous to the top wild γ-PGA producer strains. This study highlights the convenience of using the laboratory strain of B. subtilis over wild isolates in designing strain improvement strategies aimed at increasing γ-PGA productivity.

OBJECTIVE: Red cell adenylate kinase (AK) deficiency is a rare hereditary erythroenzymopathy associated with moderate to severe nonspherocytic hemolytic anemia and, in some cases, with mental retardation and psychomotor impairment. To date, diagnosis of AK deficiency depends upon demonstration of low enzyme activity in red blood cells and detection of mutations in AK1 gene. To investigate the molecular bases of the AK deficiency, we characterized five variants of AK1 isoenzyme-bearing mutations (118G>A, 190G>A, 382C>T, 418-420del, and 491A>G) found in AK-deficient patients with chronic hemolytic anemia. MATERIALS AND METHODS: The complete AK1 cDNA was obtained by standard procedures and using as template the reticulocyte RNA. The cDNA was cloned in a plasmid vector and the enzyme was expressed in Escherichia coli BL21(DE3)pLysS, and purified by standard protocols to homogeneity. DNA mutants bearing point mutations were obtained from the cloned wild-type cDNA using standard methods of site-directed mutagenesis, whereas the DNA mutant with deletion of codon 140 was obtained by a two-step method. RESULTS: Four mutant enzymes (Gly40Arg, Gly64Arg, Arg128Trp, Asp140del) were severely affected in activity, displaying a catalytic efficiency of four orders of magnitude lower than the wild-type; one (Tyr164Cys) was grossly perturbed in protein stability. CONCLUSIONS: The altered properties displayed by the mutant enzymes support the cause-effect relationship between AK1 mutations and hemolytic anemia.

The impact of transgenic white poplars (Populus alba L. cv. 'Villafranca') was assessed on the soil aerobic spore-forming bacteria (SFB). The genetically modified poplars, expressing either the StSy gene for resveratrol production or the bar gene for herbicide tolerance, were cultivated in greenhouse. The occurrence of SFB was monitored in soil samples collected at eight different timepoints over a two-year period. The total culturable bacterial population of the StSy and bar trials underwent significant seasonal fluctuations in the range of 10(6)-2.5 x 10(8) CFU/g dry soil and of 10(4)-5 x 10(8) CFU/g dry soil, respectively. Changes occurred also within the culturable SFB population with size varying at 10(3)-5 x 10(4) CFU/g dry soil and 10(2)-2 x 10(5) CFU/g dry soil in the StSy and bar trials, respectively. No significant differences in the size of the total and SFB culturable populations were observed when comparing each transgenic line with the nontransformed control line while seasonal shifts of soil bacterial populations were evident in both trials. The culturable SFB fraction included three isolates (SFB-1, SFB-2 and SFB-3) classified by 16S rDNA sequence analysis as members of the Bacillus genus. According to the reported data, cultivation of both herbicide-resistant and resveratrol-producing GM white poplars did not affect the culturable SFB population at the soil level.

Soil microcosms seeded with spores of a tracer organism (Bacillus subtilis strain PB5332) were used to test five different DNA extraction protocols hereby indicated as A, B, C, D and E. The representativity of DNA samples obtained from each procedure was evaluated by PCR amplification of theswrAA gene, unique to PB5332 strain, followed by Southern hybridization with a gene-specific probe. A significant improvement of DNA extraction from spores was obtained using grinding under liquid N2 associated with sodium-dodecyl sulphate (SDS)-based lysis in presence of 1% hexadecyltrimethylammonium bromide (CTAB; protocol C). The same procedure was tested on soil samples from two distinct greenhouse trials carried out with genetically modified white poplars (Populus alba L) expressing theStSy gene for resveratrol production and thebar gene for Basta® tolerance, respectively. The representativity of DNA samples recovered from the greenhouse soil was assessed using three spore-forming bacteria (SFB) as tracer organisms. The tracers (SFB-1, SFB-2 and SFB-3) were previously isolated from the same trials classified as members of the genusBacillus. All the tested DNA samples produced the expected amplification products, indicating the presence at the soil level of the tracers and confirming the reliability of the optimized DNA extraction protocol.

Hereditary pyrimidine 5'-nucleotidase deficiency is the most frequent enzymopathy of red blood cell nucleotide metabolism that causes hereditary non-spherocytic hemolytic anemia. The disease is usually characterized by mild-to-moderate hemolytic anemia, reticulocytosis and hyperbilirubinemia. To date, diagnosis ultimately depends upon demonstration of a reduced level of pyrimidine 5'-nucleotidase type-I (P5'N-1) activity in red cells and detection of mutations in the P5'N-1 gene. To unravel the causes of the P5'N deficiency and to obtain data for a definitive diagnosis three newly described missense mutations (c.187T>C, c.469G>C and c.740T>C) identified in patients with hemolytic anemia have been characterized at protein level. The mutant enzymes (C63R, G157R and I247T) were obtained as recombinant forms and purified to homogeneity. The enzymes were altered, although to a different extent, in both thermal stability and catalytic efficiency. The catalytic efficiency of all mutants was reduced especially towards UMP (up to more than 200 times), owing to the increased Km values (approximately, 10-25 times higher). The G157R enzyme was severely heat unstable and lost half of its activity after about 23 min of incubation at 37 degrees C. At higher temperature C63R and I247T mutants as well were less stable than the wild-type enzyme. Therefore, although the mutations targeted different regions of the P5'N-1 structure, they produced similar effects on the molecular properties of the enzyme. Thus, all affected amino acids are functionally and structurally important for preserving the enzyme activity during the red cell life span.

FlgM proteins, also known as Anti-sigma-28 factor (sigma28), are negative regulators of flagellin synthesis. Recently, a three-dimensional structure of the Aquifex aeolicus sigma28/FlgM complex (PDB code: 1rp3) was determined by X-ray crystallography at 2.3 A resolution. Furthermore, experimental data on bacterial FlgM, including site-directed mutagenesis and structural characterization by NMR are also available. However, an interpretation of the sequence-structure-function relationships combining X-ray and NMR data with the evolutionary information extracted from the increasing number of FlgM-related sequences annotated in databases is not available. In the present study, we combined database sequence searches and sequence-analysis tools to update the multiple sequence alignment of a previously characterized cluster of orthologs (COG2747) and the PFAM classification of protein domains (PF04316) for the FlgM family. A phylogenetic analysis of 77 protein sequences revealed the presence of at least three major sequence clades within the FlgM family. Besides, we predicted functional residues using a SequenceSpace method. We also generated homology models for Bacillus subtilis and Salmonella typhimurium FlgM proteins, for which sequence-structure-function relationship data are available, and used the docking program ClusPro to hypothesize about the dimer association between FlgM proteins. In conclusion, the analysis presented in this work will be useful in designing new experiments to understand better protein-protein interactions between FglM, sigma factors, and putative molecules from the flagellar export apparatus. Electronic Supplementary Material is available in the online version of this article at http://link.springer.de/

Bacillus subtilis implements several adaptive strategies to cope with nutrient limitation experienced at the end of exponential growth. The DegS-DegU two-component system is part of the network involved in the regulation of postexponential responses, such as competence development, the production of exoenzymes, and motility. The degU32 (Hy) mutation extends the half-life of the phosphorylated form of DegU (DegU-P); this in turn increases the production of alkaline protease, levan-sucrase, and other exoenzymes and inhibits motility and the production of flagella. The expression of the flagellum-specific sigma factor SigD, of the flagellin gene hag , and of the fla-che operon is strongly reduced in a degU32 (Hy) genetic background. To investigate the mechanism of action of DegU-P on motility, we isolated mutants of degU32 (Hy) that completely suppressed the motility deficiency. The mutations were genetically mapped and characterized by PCR and sequencing. Most of the mutations were found to delete a transcriptional termination signal upstream of the main flagellar operon, fla-che , thus allowing transcriptional readthrough from the cod operon. Two additional mutations improved the σ A -dependent promoter sequence of the fla-che operon. Using an electrophoretic mobility shift assay, we have demonstrated that purified DegU binds specifically to the P A promoter region of the fla-che operon. The data suggest that DegU represses transcription of the fla-che operon, and they indicate a central role of the operon in regulating the synthesis and assembly of flagella.

Deficiency of human erythrocyte isozyme (RPK) is, together with glucose-6-phosphate dehydrogenase deficiency, the most common cause of the nonspherocytic hemolytic anemia. To provide a molecular framework to the disease, we have solved the 2.7 A resolution crystal structure of human RPK in complex with fructose 1,6-bisphosphate, the allosteric activator, and phosphoglycolate, a substrate analogue, and we have functionally and structurally characterized eight mutants (G332S, G364D, T384M, D390N, R479H, R486W, R504L, and R532W) found in RPK-deficient patients. The mutations target distinct regions of RPK structure, including domain interfaces and catalytic and allosteric sites. The mutations affect to a different extent thermostability, catalytic efficiency, and regulatory properties. These studies are the first to correlate the clinical symptoms with the molecular properties of the mutant enzymes. Mutations greatly impairing thermostability and/or activity are associated with severe anemia. Some mutant proteins exhibit moderate changes in the kinetic parameters, which are sufficient to cause mild to severe anemia, underlining the crucial role of RPK for erythrocyte metabolism. Prediction of the effects of mutations is difficult because there is no relation between the nature and location of the replaced amino acid and the type of molecular perturbation. Characterization of mutant proteins may serve as a valuable tool to assist with diagnosis and genetic counseling.

Human erythrocyte pyruvate kinase plays an important role in erythrocyte metabolism. Mutation on the gene results in pyruvate kinase deficiency and is an important cause of hereditary nonspherocytic hemolytic anemia. Because of difficulties in isolating the mutant enzymes from patients, these mutations have not been fully studied. In this study, a complementary DNA (cDNA) encoding the human erythrocyte pyruvate kinase was generated. The cDNA was cloned into several expression vectors, and the protein was expressed and purified. The tetrameric protein exhibited properties characteristic of authentic human erythrocyte pyruvate kinase, including response to substrate, phosphoenolpyruvate, activation by fructose 1,6-bisphosphate, and inhibition by adenosine triphosphate (ATP). The N-terminal segment of the protein was highly susceptible to proteolysis, but only 2 of the 4 subunits were cleaved and lacked 47 N-terminal amino acid residues. A mutant protein, R510Q, which is the most frequently occurring mutation among Northern European population, was also generated and purified. The mutant protein retained its binding capacity to and could be activated by fructose 1,6-bisphosphate and showed similar kinetics toward phosphoenolpyruvate and adenosine diphosphate as for the wild-type enzyme. Conversely, the mutant protein has a dramatically decreased stability toward heat and is more susceptible to ATP inhibition. The enzyme instability decreases the enzyme level in the cell, accounting for the clinically observed “pyruvate kinase deficiency” of patients who are homozygous for this mutation. This study provides the first detailed functional characterization of human erythrocyte pyruvate kinase. These findings will allow the establishment of a fine correlation between molecular abnormalities and the clinical expression of the disease.

Pyruvate kinase (PK) is critical for the regulation of the glycolytic pathway. The regulatory properties of Escherichia coli were investigated by mutating six charged residues involved in interdomain salt bridges (Arg(271), Arg(292), Asp(297), and Lys(413)) and in the binding of the allosteric activator (Lys(382) and Arg(431)). Arg(271) and Lys(413) are located at the interface between A and C domains within one subunit. The R271L and K413Q mutant enzymes exhibit altered kinetic properties. In K413Q, there is partial enzyme activation, whereas R271L is characterized by a bias toward the T-state in the allosteric equilibrium. In the T-state, Arg(292) and Asp(297) form an intersubunit salt bridge. The mutants R292D and D297R are totally inactive. The crystal structure of R292D reveals that the mutant enzyme retains the T-state quaternary structure. However, the mutation induces a reorganization of the interface with the creation of a network of interactions similar to that observed in the crystal structures of R-state yeast and M1 PK proteins. Furthermore, in the R292D structure, two loops that are part of the active site are disordered. The K382Q and R431E mutations were designed to probe the binding site for fructose 1, 6-bisphosphate, the allosteric activator. R431E exhibits only slight changes in the regulatory properties. Conversely, K382Q displays a highly altered responsiveness to the activator, suggesting that Lys(382) is involved in both activator binding and allosteric transition mechanism. Taken together, these results support the notion that domain interfaces are critical for the allosteric transition. They couple changes in the tertiary and quaternary structures to alterations in the geometry of the fructose 1, 6-bisphosphate and substrate binding sites. These site-directed mutagenesis data are discussed in the light of the molecular basis for the hereditary nonspherocytic hemolytic anemia, which is caused by mutations in human erythrocyte PK gene.

FlgM is an anti-sigma factor of the flagellar-specific sigma (sigma) subunit of RNA polymerase in Bacillus subtilis, and it is responsible of the coupling of late flagellar gene expression to the completion of the hook-basal body structure. We have overproduced the protein in soluble form and characterized it. FlgM forms dimers as shown by gel exclusion chromatography and native polyacrylamide gel electrophoresis and interacts in vitro with the cognate sigmaD factor. The FlgM.sigmaD complex is a stable heterodimer as demonstrated by gel exclusion chromatography, chemical cross-linking, native polyacrylamide gel electrophoresis, and isoelectric focusing. sigmaD belongs to the group of sigma factors able to bind to the promoter sequence even in the absence of core RNA polymerase. The FlgM.sigmaD complex gave a shift in a DNA mobility shift assay with a probe containing a sigmaD-dependent promoter sequence. Limited proteolysis studies indicate the presence of two structural motifs, corresponding to the N- and C-terminal regions, respectively.

In B. subtilis swarming and robust swimming motility require the positive trigger of SwrA on fla/che operon expression. Despite having an essential and specific activity, how SwrA executes this task has remained elusive thus far. We demonstrate here that SwrA acts at the main σ(A)-dependent fla/che promoter PA(fla/che) through DegU. Electrophoretic mobility shift assays (EMSA) reveal that SwrA forms a complex with the phosphorylated form of DegU (DegU~P) at PA(fla/che) while it is unable to do so with either unphosphorylated DegU or the DegU32(Hy) mutant protein. Motility assays show that a highly phosphorylated DegU is not detrimental for flagellar motility provided that SwrA is present; however, DegU~P represses PA(fla/che) in the absence of SwrA. Overall, our data support a model in which DegU~P is a dual regulator, acting either as a repressor when alone or as a positive regulator of PA(fla/che) when combined with SwrA. Finally, we demonstrate that the σ(D)-dependent PD3(fla/che) promoter plays an important role in motility, representing a contingent feedback loop necessary to maintain basal motility when swrA is switched to the non-functional swrA(-) status.

We have cloned and sequenced the nrd (nucleotide reductase) locus of Bacillus subtilis. The locus seems to be organized in an operon comprising four ORFs. The first three encode polypeptides highly similar to the product of the coding sequences characterizing the nrdEF operons of Enterobacteriaceae. The sequencing of the conditional lethal mutation ts-A13, localized in the nrdE cistron, and the lethality of insertional mutations targeted in the internal region of nrdE and nrdF, demonstrated the essential role of this locus. The fourth ORF, ymaB, part of the putative operon, which is not similar to any known protein, is also essential. The regulation of expression of the operon, monitored by lacZ transcriptional fusions, is similar to the regulation of the functionally relevant nrdAB operon of Escherichia coli. The operon was induced by thymidine starvation and its expression was directly or indirectly affected by RecA function. Genetic and functional analysis strongly indicates that in B. subtilis the class I ribonucleotide reductase encoded by this nrd operon is evolutionarily distant from the homologous class I enzyme of Enterobacteria.

The alternative sigma factor sigma D directs transcription of a number of genes involved in chemotaxis, motility, and autolysis in Bacillus subtilis (sigmaD regulon). The activity of SigD is probably in contrast to that of FlgM, which acts as an antisigma factor and is responsible for the coupling of late flagellar gene expression to the assembly of the hook-basal body complex. We have characterized the effects of an in-frame deletion mutation of flgM. By transcriptional fusions to lacZ, we have shown that in FlgM-depleted strains there is a 10-fold increase in transcription from three different sigmaD-dependent promoters, i.e., Phag, PmotAB, and PfliDST. The number of flagellar filaments was only slightly increased by the flgM mutation. Overexpression of FlgM from a multicopy plasmid under control of the isopropyl-beta-D-thiogalactopyranoside-inducible spac promoter drastically reduced the level of transcription from the hag promoter. On the basis of these results, we conclude that, as in Salmonella typhimurium, FlgM inhibits the activity of SigD, but an additional element is involved in determining the number of flagellar filaments.

The outB gene of Bacillus subtilis is involved in spore germination and outgrowth and is essential for growth. The OutB protein was obtained by expression in Escherichia coli and purified to apparent homogeneity. Here we report experiments showing that OutB is a NH3-dependent NAD synthetase, the enzyme that catalyzes the final reaction in the biosynthesis of NAD. The enzyme is composed of two identical subunits of 30,240 Da and is NH3-dependent, whereas glutamine is inefficient as an amide donor. The NAD synthetase is highly resistant to heat, with a half-time of inactivation at 100 degrees C of 13 min. A mutant NAD synthetase was purified from a B. subtilis strain temperature-sensitive during spore germination and outgrowth. The mutant enzyme was 200 times less active than the wild-type one, with a lower temperature optimum and a non-hyperbolic kinetic versus NH4+. The time course of synthesis of OutB showed that synthesis of the enzyme started during germination and outgrowth, and reached the highest level at the end of exponential growth. The enzyme could be recovered from dormant spores.

SUMMARY: The nucleotide sequence of 20 kb contiguous to the pksX locus of Bacillus subtilis was determined. Six ORFs were recognized, one of which extended for 13 341 nucleotides. Their predicted products have significant similarities to proteins with known functions involved in the synthesis of polypeptides and polyketides or in fatty acid metabolism. At the nucleotide level, three regions with a high level of sequence identity (49-54%) to the Aspergillus nidulans wA gene, responsible for the synthesis of a polyketide pigment, were recognized. The observed similarities suggest that the 20 kb region and the previously reported 13-6 kb region containing pksX are part of the same locus, possibly involved in secondary metabolism.

The nucleotide (nt) sequence of 13.6 kb of the outG locus of Bacillus subtilis, which maps at approximately 155 degrees between the genetic markers nrdA and polC, was determined. One putative coding sequence was identified corresponding to a large polypeptide of 4427 amino acids (aa). Structural organization at the nt and aa sequence level and extensive similarities of the deduced product, especially to EryA, suggest that the locus is potentially responsible for the synthesis of a polyketide molecule. The locus has been renamed pksX. Comparison of the deduced product with known fatty acid and polyketide synthases (PKS) suggested the presence of beta-ketosynthase, dehydratase, beta-ketoreductase and acyl-carrier protein domains. Preliminary data obtained with deletion mutants indicate that pksX is not an essential gene.

Poly-γ-glutamic acid (γ-PGA) is an extracellular polymer produced by various strains ofBacillus. Ιt was first described as the component of the capsule inBacillus anthracis, where it plays a relevant role in virulence.γ-PGA is also a distinctive component of ‘natto’, a traditional Japanese food consisting of soybean fermented byBacillus subtilis(natto). DomesticatedB.subtilisstrains do not synthesizeγ-PGA although they possess the functional biosyntheticpgsoperon. In the present work we explore the correlation between the genetic determinants,swrAAanddegU, which allow a derivative of the domestic strain JH642 to display a mucoid colony morphology on LB agar plates due to the production ofγ-PGA. Full activation of thepgsoperon requires the co-presence of SwrAA and the phosphorylated form of DegU (DegU∼P). The presence of either DegU∼P or SwrAA alone has only marginal effects onpgsoperon transcription andγ-PGA production. Although SwrAA was identified as necessary for swarming and full swimming motility together with DegU, we show that motility is not involved inγ-PGA production. Activation ofγ-PGA synthesis is therefore a motility-independent phenotype in which SwrAA and DegU∼P display a cooperative effect.

The parasporal crystal of Bacillus thuringiensis is composed of polypeptides highly toxic to a number of insect larvae. The structural genes (cryIA) encoding the Lepidoptera-specific toxin from different bacterial strains diverge primarily in a single hypervariable region, whereas the N-terminal and C-terminal parts of the proteins are highly conserved. In this report, we describe the generation of hybrid genes between two cryIA genes. Two truncated cryIA genes were cloned in a plasmid vector in such way as to have only the hypervariable region in common. The two truncated cryIA genes were separated by the tetracycline-resistance determinant (or part of it). In vivo recombination between the hypervariable regions of the cryIA genes reconstituted an entire hybrid cryIA gene. Direct sequence analysis of 17 recombinant plasmids identified eleven different crossover regions which did not alter the reading frame and allowed the production of eight different hybrid proteins. The recombination events were independent from the RecA function of Escherichia coli. Some of the hybrid gene products were more specific in their insecticidal action and one had acquired a new biological activity.

We demonstrate that transcription of the gene swrAA , required for swarming migration in Bacillus subtilis , is driven by two promoters: a sigD -dependent promoter and a putative sigA -dependent promoter, which is inactive during growth in liquid Luria-Bertani medium and becomes active in the presence of the phosphorylated form of the response regulator DegU or on semisolid surfaces. Since sigD transcription is enhanced by SwrAA, this finding reveals that swrA expression is controlled by a positive feedback loop. We also demonstrate that the positive action of SwrAA in swimming and swarming motility is prevented in strains carrying a deletion of the two-component system degS-degU and that this effect is independent of swrAA transcription. Therefore, both DegU and SwrAA must be present to achieve full motility in B. subtilis.

The aim of this study was to investigate the culturable bacteria living in soil cultivated with Basta®-tolerant transgenic white poplars ( Populus alba L. ‘Villafranca’). Plate Count Agar medium containing phosphinothricin, the active component of Basta®, was used to isolate the herbicide-resistant bacteria (HRB). No significant changes in the size of the soil microbial flora following herbicide treatment were observed. The characterization of HRB isolates by 16S rDNA-based taxonomy revealed a predominance of Pseudomonas and Bacillus species. The screening carried out on soil samples allowed for the recovery of isolates with useful properties for biotechnological and agronomical purposes, particularly in relation to root development. Among the tested isolates, only HRB-1b, HRB-1c, and HRB-7 showed remarkable swarming ability, a valuable trait supporting the beneficial plant–microbe interactions. HRB-1c was also characterized by consistent production of indoleacetic acid (17.8 ± 0.09 μg·mL–1· (OD600unit)–1), and it was able to stimulate the in vitro growth of Villafranca explants. Since novel tools are constantly required to enhance productivity of perennial species and to expand their use for practical purposes, the availability of bacteria that support tree growth, such as the HRB-1c isolate, represents a significant advantage.

Inherited pyrimidine 5'-nucleotidase type-1 (P5'N-1) deficiency is the most frequent abnormality of red cell nucleotide metabolism causing non-spherocytic hemolytic anemia. We describe two novel mutations in two Italian patients affected by P5'N-1 deficiency. One mutation is a two base deletion that occurs at the splice site junction between intron 7 and exon 8 (c.396-397del AG); the second is an in-frame deletion of three adjacent bases (c.427-429del CAA), leading to deletion of glutamine 143. The kinetic properties of Q143del variant were not grossly altered, but the variant was very heat unstable even at physiological temperatures.

The recombinant homodimeric hemoglobin from the strictly aerobe gram-negative bacterium Vitreoscilla stercoraria has been expressed in Escherichia coli, purified to homogeneity, and crystallized by vapor diffusion techniques, using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group P21 and diffract to HIGH resolution. The unit cell parameters are a = 62.9, b = 42.5, c = 63.2 A, β = 106.6°; the asymmetric unit contains the homodimeric hemoglobin, with a volume solvent content of 42%. Proteins 27:154–156 © 1997 Wiley-Liss, Inc.

The number and disposition of flagella harbored by eubacteria are regulated by a specific trait successfully maintained over generations. The genes governing the number of flagella in Bacillus subtilis have never been identified, although the ifm locus has long been recognized to influence the motility phenotype of this microorganism. The characterization of a spontaneous ifm mutant of B. subtilis , displaying diverse degrees of cell flagellation in both liquid and solid media, raised the question of how the ifm locus governs the number and assembly of functional flagella. The major finding of this investigation is the characterization of a newly identified dicistronic operon, named swrA , that controls both swimming motility and swarming differentiation in B. subtilis . Functional analysis of the swrA operon allowed swrAA (previously named swrA [D. B. Kearns, F. Chu, R. Rudner, and R. Losick, Mol. Microbiol. 52: 357-369, 2004]) to be the first gene identified in B. subtilis that controls the number of flagella in liquid environments and the assembly of flagella in response to cell contact with solid surfaces. Evidence is given that the second gene of the operon, swrAB , is essential for enabling the surface-adhering cells to undergo swarming differentiation. Preliminary data point to a molecular interaction between the two gene products.

NAD(+)-synthetase is a ubiquitous enzyme catalyzing the last step in the biosynthesis of NAD+. Mutants of NAD+ synthetase with impaired cellular functions have been isolated, indicating a key role for this enzyme in cellular metabolism. Crystals of the enzyme from Bacillus subtilis suitable for x-ray crystallographic investigation have been grown from polyethylene glycol solutions. Investigation on the structural organization of NAD+ synthetase, an enzyme fundamental for NAD+ biosynthesis, and belonging to the recently characterized amidotransferase enzymatic family, will provide more insight into the catalytic mechanism of deamido-NAD+-->NAD+ conversion, a biosynthetic process that is a potential target for the development of antibiotic compounds against Bacillus sp. and related bacteria.

Inherited pyrimidine 5′-nucleotidase type I (P5′N-1) deficiency is the third most common erythrocyte enzymopathy that causes hemolysis. Fourteen different mutations have been identified to date. We have investigated the molecular bases of the disease by studying the biochemical properties of the recombinant wild-type human enzyme and 4 variant proteins (D87V, L131P, N179S, and G230R) bearing missense mutations found in patients affected by nonspherocytic hemolytic anemia. P5′N-1 is a relatively stable protein and has essentially identical catalytic efficiency toward cytidine monophosphate (CMP) and uridine monophosphate (UMP). All investigated mutant proteins display impaired catalytic properties and/or reduced thermostability, providing a rationale for the pathological effects of the mutations. Despite the substantial changes in the kinetic and thermostability parameters, the enzyme activity detected in the red blood cells of patients homozygous for mutations L131P and G230R exhibits moderate alterations. This suggests that P5′N-1 deficiency is compensated, possibly by other nucleotidases or alternative pathways in nucleotide metabolism. Therefore, nucleotidase activity may not be considered a prognostic indicator in patients affected by the enzymopathy. (Blood. 2005;105:3340-3345)

Knowledge of the highly regulated processes governing the production of flagella in Bacillus subtilis is the result of several observations obtained from growing this microorganism in liquid cultures. No information is available regarding the regulation of flagellar formation in B. subtilis in response to contact with a solid surface. One of the best-characterized responses of flagellated eubacteria to surfaces is swarming motility, a coordinate cell differentiation process that allows collective movement of bacteria over solid substrates. This study describes the swarming ability of a B. subtilis hypermotile mutant harboring a mutation in the ifm locus that has long been known to affect the degree of flagellation and motility in liquid media. On solid media, the mutant produces elongated and hyperflagellated cells displaying a 10-fold increase in extracellular flagellin. In contrast to the mutant, the parental strain, as well as other laboratory strains carrying a wild-type ifm locus, fails to activate a swarm response. Furthermore, it stops to produce flagella when transferred from liquid to solid medium. Evidence is provided that the absence of flagella is due to the lack of flagellin gene expression. However, restoration of flagellin synthesis in cells overexpressing σ D or carrying a deletion of flgM does not recover the ability to assemble flagella. Thus, the ifm gene plays a determinantal role in the ability of B. subtilis to contact with solid surfaces.

NADP is essential for biosynthetic pathways, energy, and signal transduction. In living organisms, NADP biosynthesis proceeds through the phosphorylation of NAD with a reaction catalyzed by NAD kinase. We expressed, purified, and characterized Bacillus subtilis NAD kinase. This enzyme represents a new member of the inorganic polyphosphate [poly(P)]/ATP NAD kinase subfamily, as it can use poly(P), ATP, or other nucleoside triphosphates as phosphoryl donors. NAD kinase showed marked positive cooperativity for the substrates ATP and poly(P) and was inhibited by its product, NADP, suggesting that the enzyme plays a major regulatory role in NADP biosynthesis. We discovered that quinolinic acid, a central metabolite in NAD(P) biosynthesis, behaved like a strong allosteric activator for the enzyme. Therefore, we propose that NAD kinase is a key enzyme for both NADP metabolism and quinolinic acid metabolism.

The efficient production of recombinant proteins in Escherichia coli requires a proper termination of translation to ensure the synthesis of only the desired product. During the recombinant production of Bacillus subtilis flgM in E. coli, we detected an additional polypeptide of molecular mass higher than the expected, corresponding to a product of a translational readthrough of the UGA stop codon. In this paper we show that the readthrough was abolished when the synthesis of the recombinant protein was carried out at 25 °C. The possible causes that contribute to reduce the proportion of readthrough protein species against the correct terminated product are discussed.

Deficiency of human erythrocyte isozyme (RPK) is, together with glucose-6-phosphate dehydrogenase deficiency, the most common cause of the nonspherocytic hemolytic anemia. To provide a molecular framework to the disease, we have solved the 2.7 A resolution crystal structure of human RPK in complex with fructose 1,6-bisphosphate, the allosteric activator, and phosphoglycolate, a substrate analogue, and we have functionally and structurally characterized eight mutants (G332S, G364D, T384M, D390N, R479H, R486W, R504L, and R532W) found in RPK-deficient patients. The mutations target distinct regions of RPK structure, including domain interfaces and catalytic and allosteric sites. The mutations affect to a different extent thermostability, catalytic efficiency, and regulatory properties. These studies are the first to correlate the clinical symptoms with the molecular properties of the mutant enzymes. Mutations greatly impairing thermostability and/or activity are associated with severe anemia. Some mutant proteins exhibit moderate changes in the kinetic parameters, which are sufficient to cause mild to severe anemia, underlining the crucial role of RPK for erythrocyte metabolism. Prediction of the effects of mutations is difficult because there is no relation between the nature and location of the replaced amino acid and the type of molecular perturbation. Characterization of mutant proteins may serve as a valuable tool to assist with diagnosis and genetic counseling.

Quinolinate synthetase catalyzes the second step of the de novo biosynthetic pathway of pyridine nucleotide formation. In particular, quinolinate synthetase is involved in the condensation of dihydroxyacetone phosphate and iminoaspartate to form quinolinic acid. To study the mechanism of action, the specificity of the enzyme and the interaction with l-aspartate oxidase, the other component of the so-called "quinolinate synthetase complex," the cloning, the overexpression, and the purification to homogeneity of Escherichia coli quinolinate synthetase were undertaken. The results are presented in this paper. Since the overexpression of the enzyme resulted in the formation of inclusion bodies, a procedure of renaturation and refolding had to be set up. The overexpression and purification procedure reported in this paper allowed the isolation of 12 mg of electrophoretically homogeneous quinolinate synthetase from 1 liter of E. coli culture. A new, continuous, method for the evaluation of quinolinate synthetase activity was also devised and is presented. Finally, our data definitely exclude the possibility that other enzymes are involved in the biosynthesis of quinolinic acid in E. coli, since it is possible to synthesize quinolinic acid from l-aspartate, dihydroxyacetone phosphate, and O(2) by using only nadA and nadB gene overexpressed products.

The natural precipitation of calcium carbonate crystals by bacteria has been proposed for conservative interventions in monument restoration, even if the use of heterotrophic viable organisms does not always seem appropriate for this purpose. In fact, chemical reactions with stone minerals due to metabolic by-products and the growth of fungi, due to the application of organic nutrients for bacterial development, can have negative effects on stone surfaces. We have studied crystal precipitation in a laboratory strain of Bacillus subtilis. The effectiveness of this process has been analyzed on small bioclastic limestone samples (Pietra di Lecce), determining their total porosity, amount of water absorbed and superficial cohesion. To overcome some of the possible problems involved with the use of viable bacterial cells, we have investigated the genetic mechanisms that control calcite precipitation. For this reason, we have selected Bacillus subtilis mutants that do not precipitate or which form calcite crystals faster. The data obtained suggest the presence of several genes involved in crystal formation.

DNA sequences upstream (UP element) of the core promoter (−10, −35 region) of the Bacillus subtilis flagellin gene hag stimulate transcription in vivo and in vitro. We constructed a number of hybrids, placing the UP element of hagp upstream of the core of one SigD-dependent (fliDp) and two SigA-dependent (tmsp, vegp) B. subtilis promoters. The hybrid promoters were fused to a lacZ reporter gene and their activity tested in vivo. The presence of the UP module enhanced transcription at both types of promoters. We conclude that the hagp UP sequence can act as a promoter module independently of the core sequence.

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Background: The first hemoglobin identified in bacteria was isolated from Vitreoscilla stercoraria (VtHb) as a homodimeric species. The wild-type protein has been reported to display medium oxygen affinity and cooperative ligand-binding properties. Moreover, VtHb can support aerobic growth in Escherichia coli with impaired terminal oxidase function. This ability of VtHb to improve the growth properties of E. coli has important applications in fermentation technology, assisting the overexpression of recombinant proteins and antibiotics. Oxygen binding heme domains have been identified in chimeric proteins from bacteria and yeast, where they are covalently linked to FAD- and NAD(P)H-binding domains. We investigate here the fold, the distal heme site structure and the quaternary assembly of a bacterial hemoglobin which does not bear the typical flavohemoglobin domain organization. Results: The VtHb three-dimensional structure conforms to the well known globin fold. Nevertheless, the polypeptide segment connecting helices C and E is disordered, and residues E7–E10 (defined according to the standard globin fold nomenclature) do not adopt the usual α -helical conformation, thus locating Gln53(E7) out of the heme pocket. Binding of azide to the heme iron introduces substantial structural perturbations in the heme distal site residues, particularly Tyr29(B10) and Pro54(E8). The quaternary assembly of homodimeric VtHb, not observed before within the globin family, is based on a molecular interface defined by helices F and H of both subunits, the two heme iron atoms being 34 A apart. Conclusions: The unusual heme distal site structure observed shows that previously undescribed molecular mechanisms of ligand stabilization are operative in VtHb. The polypeptide chain disorder observed in the CE region indicates a potential site of interaction with the FAD/NADH reductase partner, in analogy with observations in the chimeric flavohemoglobin from Alcaligenes eutrophus .

The Bacillus subtilis mutS and mutL genes, involved in the DNA mismatch repair system, have been cloned and characterized. From sequence analysis the two genes appear to be organized in a single operon, located immediately downstream of the cotE gene (approximately 150 degrees on the genetic map). The deduced MutS protein is 49% identical to HexA and MutL is 46% identical to HexB of Streptococcus pneumoniae. Deletion of both mutS and mutL resulted in an increase in the frequency of spontaneous mutations and abolished the marker effect observed in transformation. The expression of the mut operon was studied with the use of a mutSL-lacZ transcriptional fusion. An increase in expression was observed during late exponential growth.

Flagellin is one of the most abundant proteins in motile bacteria, yet its expression requires a low abundance sigma factor (sigma 28). We show that transcription from the Bacillus subtilis flagellin promoter is stimulated 20-fold by an upstream A+T-rich region [upstream promoter (UP) element] both in vivo and in vitro. This UP element is contacted by sigma 28 holoenzyme bound at the flagellin promoter and binds the isolated alpha 2 subassembly of RNA polymerase. The UP element increases the affinity of RNA polymerase for the flagellin promoter and stimulates transcription when initiation is limited by the rate of RNA polymerase binding. Comparison with other promoters in the flagellar regulon reveals a bipartite architecture: the -35 and -10 elements confer specificity for sigma 28, while promoter strength is determined largely by upstream DNA sequences.

The regulation of flagellin gene expression in Bacillus subtilis was examined in vivo by means of a lacZ translational fusion to the flagellin structural gene (hag). We have tested the effects of two known mutations (flaA4 and flaA15) in the major flagellar operon and of three deletions. One deletion was in frame in the fliI cistron, one was out of frame in the fliK cistron, and the last spanned about 21 kb of the flaA operon. In all instances, the expression of the flagellin gene was defective. Flagellin gene expression was restored in the strain with the 21-kb deletion by overexpression of the sigD gene under control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible spac promoter. These results indicate that transcription of the flagellin gene is dependent on the formation of the flagellar basal body but that such a requirement can be bypassed by overexpression of sigD. Lack of expression of hag was observed in the presence of flaD1, flaD2, and delta sin mutations as well.

A 10-kilobase EcoRI fragment of Azospirillum brasilense genomic DNA was cloned in Escherichia coli. Two open reading frames of 4548 and 1446 base pairs (bp) were identified within the fragment as the structural genes for the alpha and beta subunits (gltB and gltD, respectively) of A. brasilense GltS. The organization of the gltBD region of A. brasilense differs from that of the corresponding region in E. coli: in A. brasilense, gltD is upstream relative to gltB, and its stop codon is separated by 141 bp from the first ATG of gltB. The deduced amino acid sequences reveal a high similarity with GltS from E. coli and with the ferredoxin-dependent GltS from maize. Binding domains for flavin cofactors and NADPH, a domain for glutamine binding and activation, and cysteine clusters for iron-sulfur centers formation were tentatively identified on the basis of sequence comparison with flavoproteins, pyridine nucleotide-dependent enzymes, amidotransferases, and iron-sulfur proteins.

Three additional alleles of the outB gene of Bacillus subtilis, whose activity is required for spore outgrowth, were identified. The nucleotide sequence of three mutant genes was determined. Analyses of dominance-recessivity showed that the wild-type allele is dominant over the mutant ones. When the outB gene was placed under the control of the inducible spac-1 promoter, the presence of IPTG was necessary to obtain normal growth. The results suggested that the outB gene is required for growth of B. subtilis. Expression of outB from the sporulation promoter spoIID negatively affected subsequent spore outgrowth, without altering vegetative growth and sporulation.

We isolated two clones of recombinant lambda bacteriophage with overlapping inserts of Bacillus subtilis chromosomal DNA corresponding to part of the flaA locus. The flaA4 and flaA15 mutations were localized on the physical map by marker rescue experiments. The flaA locus and the flaB (sigD) gene were mapped in transduction crosses, and the order glnA polC flaB flaA was determined. FlaB was linked to polC in transformation crosses.

We cloned and sequenced 8.3 kb of Bacillus subtilis DNA corresponding to the flaA locus involved in flagellar biosynthesis, motility, and chemotaxis. The DNA sequence revealed the presence of 10 complete and 2 incomplete open reading frames. Comparison of the deduced amino acid sequences to data banks showed similarities of nine of the deduced products to a number of proteins of Escherichia coli and Salmonella typhimurium for which a role in flagellar functioning has been directly demonstrated. In particular, the sequence data suggest that the flaA operon codes for the M-ring protein, components of the motor switch, and the distal part of the basal-body rod. The gene order is remarkably similar to that described for region III of the enterobacterial flagellar regulon. One of the open reading frames was translated into a protein with 48% amino acid identity to S. typhimurium FliI and 29% identity to the beta subunit of E. coli ATP synthase.

Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible transfer of a phoshoryl-group from 1,3-bisphosphoglycerate (1,3-BPG) to ADP forming 3-phosphoglycerate (3-PG) and ATP. PGK is a typical two-domain hinge-bending enzyme, with a highly conserved structure. The N-terminal domain binds 1,3-BPG/3-PG, whereas the C-terminal domain binds Mg-ADP/Mg-ATP.Humans have two PGK isozymes, PGK1 and PGK2, where PGK1 is an ubiquitous enzyme that is expressed in all somatic cells and PGK2 is a testis-specific enzyme. The PGK1 gene is located on the X-chromosome q-13.1, contains 11 exons and encodes a protein of 416 amino acids. Mutations of the PGK1 gene result in an enzyme deficiency that is for the most clinically characterized by mild-to severe hemolytic anemia and various defects in the central nervous system. To date, 19 different mutations with worldwide distribution have been reported. No correlation between the residual PGK activity and the severity of the clinical manifestations have been documented so far. To analyze the mutations at protein level and possibly to correlate the genotype to clinical phenotype, we started with the molecular characterization of the wild-type PGK1 enzyme and three mutants (I47N, D164 and S320N) obtained from E.coli as recombinant proteins. The corresponding mutations, i.e., c.140T>A, c.491A>T and c.959G>A, have been identified in patients with PGK deficiency and affected by severe hemolytic anemia and progressive mental retardation. The cDNA encoding the PGK1 was prepared starting from a blood sample of a healthy donor, with normal PGK1 activity. Site-directed mutagenesis was used to introduce the desired mutations into the PGK1 cDNA. The wild type enzyme was expressed to its maximum level (about 80–100 mg of enzyme per liter of culture) after 5 hours of induction with 0.5 mM IPTG at 37 °C. For mutant enzymes the induction temperature was lowered to 25°C. All recombinant enzymes were purified to homogeneity after a single chromatographic step on DEAE Sepharose column. The wild-type enzyme was crystallized in both free form or complexed with 3-PG. The corresponding structures were solved to high resolution (1.8 and 1.6 A, respectively) and compared. Essentially, binding 3-PG caused a 6° rotation of the N-domain in respect to the C-domain. The recombinant enzyme exhibited kinetic properties similar to those of the authentic enzyme, displaying vs 3-PG and ATP alike specific activities (about 1000 U/mg) and alike Km values (about 1mM). I47N and S320N mutant enzymes showed kcat values 3-fold lower than the wild-type enzyme. The D164V was characterized by a Km value vs 3-PG 15 times higher than that of the other enzymes studied and a catalytic efficiency 70 times lower. Finally, all mutant enzymes turned out to be highly heat unstable with respect to the wildtype enzyme, losing half of their activity after approximately 10 minutes of incubation at 37 °C. At higher temperatures, the wild-type enzyme was protected from heat inactivation by Mg-ATP or 3-PG. On the contrary, no one mutant was protect by Mg-ATP and the D164V and S320N mutants were not even protected by 3-PG. Therefore, these preliminary studies indicate that all mutations target amino acid residues located in positions primarily important for preserving the protein stability during the red cell life span.

Pyrimidine 5′-nucleotidase (P5′N-1) is a dephosphorylating enzyme that catalyzes the hydrolysis of various pyrimidine nucleoside 5′-monophosphates, particularly UMP and CMP, to produce the corresponding nucleosides. In RBC the reaction is essential for the removal of the nucleotides mainly arising from ribosomal RNA degradation during final erythroid maturation. Hereditary P5′N-1 deficiency is the third most common enzymopathy causing hereditary non-spherocytic hemolytic anemia. The disorder is transmitted as an autosomal recessive trait and is usually characterized by mild-to-moderate hemolytic anemia and accumulation of pyrimidine nucleotides within the erythrocyte. The enzyme is strongly inactivated by heavy metals; thus P5′N-1 deficiency can be acquired as a result of lead poisoning. The P5′N-1 gene is localized on 7p15-p14 and the cDNA has been cloned and sequenced. 24 different mutations have been identified so far, most of them at the homozygous level. Recently, five pathological variants of P5′N-1 have been in-depth characterized, and the molecular bases of the P5′N-1 deficiency has been elucidated. To unravel the cause of the P5′N-1 deficiency found in patients with hemolytic anemia and homozygous for 3 newly identified missense mutations (c.187T>C, c.469G>C, c.740T>C; Balta et al, Blood ASH2006, 108:3743; Manco et al, Haematologica2006, 91:266–267), we have undertaken a functional analysis of the 3 mutant enzymatic forms. The C63R, G157R and I247T proteins were produced as recombinant forms, purified and biochemically characterized. All enzymes were altered, although to a different extent, either in their catalytic efficiency or in thermal stability, the G157R being the most impaired enzyme. Catalytic efficiency of all mutants turned expecially towards UMP (about 50 to 200 times), owing to the increased Km values (about 10–25 times higher). The kinetic behaviour vs CMP was partly affected, the catalytic activity being moderately reduced (Kcat lowered to 5–20%). The G157R protein was highly heat unstable, halving the activity in about 23 min at 37°C, whereas C63R and I247T mutants at the same temperature maintained fully activity for more than 2 hours. However, at higher temperature also C63R and I247T mutants resulted less stable than the wild-type enzyme losing the activity in few minutes (t1/2 at 46°C, about 5 min vs 2 hours of the wild-type enzyme). Therefore, although mutations targeted different regions of the P5′N-1 structure, unexpectedly they produced similar aberrant effects on the molecular properties of the enzyme. Gly157 is a conserved amino acid, located close to the substrate binding site. Very likely, position 157 cannot tolerate the large and charged arginine side-chain introduced by c.469G>C mutation. Thus, it is conceivable that the drastic G157R substitution not only indirectly affects the binding of the substrate(s), but also weakens the protein stability. Cys63 and Ile247 are located far away from the catalytic site. Nevertheless, our biochemical data indicate that they are functionally and structurally important for preserving the enzyme activity. Thus, as in other cases, the decreased catalytic efficiency of C63R and I247T enzymes seems to result from secondary effects related to propagating conformational changes.

Pyrimidine 5′-nucleotidase type-I (P5′N-1) catalyzes the dephosphorylation of UMP and CMP to their respective nucleosides. In red blood cells, the enzyme has a major role in the catabolism of nucleotides formed from RNA degradation. P5′N-1 possesses also phospho-transferase activity suggesting an additional role of the enzyme in nucleotide metabolism. P5′N-1 deficiency is an autosomal recessive disorder characterized by hemolytic nonspherocytic anemia, heavy basophilic stippling in the peripheral blood smear, and accumulation of pyrimidine nucleotides within the erythrocytes. P5′N-1 deficiency is the third most common RBC enzymopathy causing hemolysis after G6PD and PK deficiency. Fourteen different mutations have been identified at the DNA level to date including four missense mutations. To increase our understanding on molecular basis of the P5′N-1 deficiency, after mutants N190S and G241R (Chiarelli et al, Blood 2003, abstract; Chiarelli et al, The Hematology Journal 2004, abstract), we have undertaken the biochemical characterization of D98V and L142P enzymes, identified respectively in a Norwegian family and in Japanese patients. The proteins were produced in E. coli cells as recombinant forms, and purified to homogeneity. The L142P protein showed a drastic reduction in the thermal stability (t1/2 at 37°C about 6 min compared to a fully stable wild-type), and kinetic properties slightly altered (kcat values nearly halved and Km 3–5 times higher). D98V exhibited reduced heat stability (t1/2 at 37° about 25 min) and catalytic efficiency turned especially versus UMP (about 25 times) owing the increased Km values. Thus, the decreased activity observed in Japanese patients homozygous for the L142P mutation is essentially due to lowered enzyme levels caused by protein instability, whereas the D98V mutation of Norwegian patients alters both stability and catalytic efficiency. We suggest that substitution D98V affects an amino acid residue involved in substrates binding site.