γ-PGA Hydrolases of Phage Origin in Bacillus subtilis and Other Microbial Genomes.

Poly-γ-glutamate (γ-PGA) is an industrially interesting polymer secreted mainly by members of the class Bacilli which forms a shield able to protect bacteria from phagocytosis and phages. Few enzymes are known to degrade γ-PGA; among them is a phage-encoded γ-PGA hydrolase, PghP. The supposed role of PghP in phages is to ensure access to the surface of bacterial cells by dismantling the γ-PGA barrier. We identified four unannotated B. subtilis genes through similarity of their encoded products to PghP; in fact these genes reside in prophage elements of B. subtilis genome. The recombinant products of two of them demonstrate efficient polymer degradation, confirming that sequence similarity reflects functional homology. Genes encoding similar γ-PGA hydrolases were identified in phages specific for the order Bacillales and in numerous microbial genomes, not only belonging to that order. The distribution of the γ-PGA biosynthesis operon was also investigated with a bioinformatics approach; it was found that the list of organisms endowed with γ-PGA biosynthetic functions is larger than expected and includes several pathogenic species. Moreover in non-Bacillales bacteria the predicted γ-PGA hydrolase genes are preferentially found in species that do not have the genetic asset for polymer production. Our findings suggest that γ-PGA hydrolase genes might have spread across microbial genomes via horizontal exchanges rather than via phage infection. We hypothesize that, in natural habitats rich in γ-PGA supplied by producer organisms, the availability of hydrolases that release glutamate oligomers from γ-PGA might be a beneficial trait under positive selection.

Knockout of pgdS and ggt genes improves γ-PGA yield in B. subtilis.

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.

The role of SwrA, DegU and P(D3) in fla/che expression in B. subtilis.

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.

Molecular insights on pathogenic effects of mutations causing phosphoglycerate kinase deficiency.

Phosphoglycerate kinase (PGK) catalyzes an important ATP-generating step in glycolysis. PGK1 deficiency is an uncommon X-linked inherited disorder, generally characterized by various combinations of non-spherocytic hemolytic anemia, neurological dysfunctions, and myopathies. Patients rarely exhibit all three clinical features. To provide a molecular framework to the different pathological manifestations, all known mutations were reviewed and 16 mutant enzymes, obtained as recombinant forms, were functionally and structurally characterized. Most mutations heavily affect thermal stability and to a different extent catalytic efficiency, in line with the remarkably low PGK activity clinically observed in the patients. Mutations grossly impairing protein stability, but moderately affecting kinetic properties (p.I47N, p.L89P, p.C316R, p.S320N, and p.A354P) present the most homogeneous correlation with the clinical phenotype. Patients carrying these mutations display hemolytic anemia and neurological disorders, and,except for p.A354P variant, no myopaty. Variants highly perturbed in both catalytic efficiency (p.G158V, p.D164V, p.K191del, D285V, p.D315N, and p.T378P) and heat stability (all, but p.T378P) result to be mainly associated with myopathy alone. Finally, mutations faintly affecting molecular properties (p.R206P, p.E252A, p.I253T, p.V266M, and p.D268N) correlate with a wide spectrum of clinical symptoms. These are the first studies that correlate the clinical symptoms with the molecular properties of the mutant enzymes. All findings indicate that the different clinical manifestations associated with PGK1 deficiency chiefly depend on the distinctive type of perturbations caused by mutations in the PGK1 gene, highlighting the need for determination of the molecular properties of PGK variants to assist in prognosis and genetic counseling. However, the clinical symptoms can not be understood only on the bases of molecular properties of the mutant enzyme. Different (environmental, metabolic, genetic and/or epigenetic) intervening factors can contribute toward the expression of PGK deficient clinical phenotypes.

SwrAA activates poly-gamma-glutamate synthesis in addition to swarming in Bacillus subtilis.

Poly-gamma-glutamic acid (gamma-PGA) is an extracellular polymer produced by various strains of Bacillus. Iotat was first described as the component of the capsule in Bacillus anthracis, where it plays a relevant role in virulence. gamma-PGA is also a distinctive component of 'natto', a traditional Japanese food consisting of soybean fermented by Bacillus subtilis (natto). Domesticated B. subtilis strains do not synthesize gamma-PGA although they possess the functional biosynthetic pgs operon. In the present work we explore the correlation between the genetic determinants, swrAA and degU, which allow a derivative of the domestic strain JH642 to display a mucoid colony morphology on LB agar plates due to the production of gamma-PGA. Full activation of the pgs operon requires the co-presence of SwrAA and the phosphorylated form of DegU (DegU approximately P). The presence of either DegU approximately P or SwrAA alone has only marginal effects on pgs operon transcription and gamma-PGA production. Although SwrAA was identified as necessary for swarming and full swimming motility together with DegU, we show that motility is not involved in gamma-PGA production. Activation of gamma-PGA synthesis is therefore a motility-independent phenotype in which SwrAA and DegU approximately P display a cooperative effect.

Autoregulation of swrAA and motility in Bacillus subtilis.

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.

Molecular basis of pyrimidine 5'-nucleotidase deficiency caused by 3 newly identified missense mutations (c.187T>C, c.469G>C and c.740T>C) and a tabulation of known mutations.

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.

Recovery of useful traits from isolates inhabiting an agricultural soil cultivated with herbicide-resistant poplars.

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 microg x mL-1 x (OD600 unit)-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.

Erythrocyte adenylate kinase deficiency: characterization of recombinant mutant forms and relationship with nonspherocytic hemolytic anemia.

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.

Bacillus subtilis gene cluster involved in calcium carbonate biomineralization.

Calcium carbonate precipitation, a widespread phenomenon among bacteria, has been investigated due to its wide range of scientific and technological implications. Nevertheless, little is known of the molecular mechanisms by which bacteria foster calcium carbonate mineralization. In our laboratory, we are studying calcite formation by Bacillus subtilis, in order to identify genes involved in the biomineralization process. A previous screening of UV mutants and of more than one thousand mutants obtained from the European B. subtilis Functional Analysis project allowed us to isolate strains altered in the precipitation phenotype. Starting from these results, we focused our attention on a cluster of five genes (lcfA, ysiA, ysiB, etfB, and etfA) called the lcfA operon. By insertional mutagenesis, mutant strains carrying each of the five genes were produced. All of them, with the exception of the strain carrying the mutated lcfA operon, were unable to form calcite crystals. By placing transcription under IPTG (isopropyl-beta-d-thiogalactopyranoside) control, the last gene, etfA, was identified as essential for the precipitation process. To verify cotranscription in the lcfA operon, reverse transcription-PCR experiments were performed and overlapping retrocotranscripts were found comprising three adjacent genes. The genes have putative functions linked to fatty acid metabolism. A link between calcium precipitation and fatty acid metabolism is suggested.

Two new mutations of the P5'N-1 gene found in Italian patients with hereditary hemolytic anemia: the molecular basis of the red cell enzyme disorder.

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.

Swarming differentiation and swimming motility in Bacillus subtilis are controlled by swrA, a newly identified dicistronic operon.

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.

Functional analysis of pyrimidine 5'-nucleotidase mutants causing nonspherocytic hemolytic anemia.

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.

DegU-P represses expression of the motility fla-che operon in Bacillus subtilis.

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 sigmaA-dependent promoter sequence of the fla-che operon. Using an electrophoretic mobility shift assay, we have demonstrated that purified DegU binds specifically to the PA 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.

Surface-associated flagellum formation and swarming differentiation in Bacillus subtilis are controlled by the ifm locus.

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 sigma(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.

Allosteric regulation of Bacillus subtilis NAD kinase by quinolinic acid.

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.

Growth at low temperature suppresses readthrough of the UGA stop codon during the expression of Bacillus subtilis flgM gene in Escherichia coli.

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 degrees C. The possible causes that contribute to reduce the proportion of readthrough protein species against the correct terminated product are discussed.

Structure and function of human erythrocyte pyruvate kinase. Molecular basis of nonspherocytic hemolytic anemia.

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.