Identification of Azospirillum strains by restriction fragment length polymorphism of the 16S rDNA and of the histidine operon

Abstract DNA fingerprints of several Azospirillum strains, belonging to the five known species A. amazonense, A. brasilense, A. halopraeferens, A. irakense and A. lipoferum , were obtained by restriction analysis of the amplified 16S rDNA and by restriction fragment length polymorphism of the histidine biosynthetic genes. Data obtained showed that amplified rDNA restriction analysis is an easy, fast, reproducible and reliable tool for identification of Azospirillum strains, mainly at the species level, whereas restriction fragment length polymorphism could, in some cases, differentiate strains belonging to the same species. Moreover, both analyses gave congruent results in grouping strains and in the assignment of new strains to a given species.     

Molecular cloning of the imidazoleglycerolphosphate dehydratase gene of Trichoderma harzianum by genetic complementation in Saccharomyces cerevisiae using a direct expression vector

Summary The Trichoderma harzianum imidazoleglycerolphosphate dehydratase gene (igh) has been isolated by complementation of a Saccharomyces cerevisiae his3 mutant using a direct expression vector. This Escherichia coli-yeast shuttle vector was developed to allow efficient cloning and expression of cDNA libraries. The cDNA is 627 nucleotides long and codes for a protein of 209 amino acids with an apparent molecular mass of 22 466 daltons. The predicted protein sequence showed 63.6%, 58.7%, and 38.4% identity respectively to the corresponding enzymes from S. cerevisiae, Pichia pastoris and E. coli. Northern analysis showed that the expression of the igh gene in T. harzianum is not inhibited by external histidine and the level of igh mRNA was about threefold higher in cells starved of histidine.     

NTA-mediated protein capturing strategy in screening experiments for small organic molecules by surface plasmon resonance

Nitrilotriacetate (NTA)-mediated capture of a histidine-tagged protein is widely used as an easy and simple method to reversibly immobilize the protein onto a sensor chip for surface plasmon resonance (SPR). However, in spite of its advantages, the NTA-capturing strategy is rarely employed for ligand screening experiments using SPR, because it was thought to cause substantial errors in binding responses, due to the inevitable protein dissociation during the monitoring period. In this study, as demonstrated in a ligand screening for the histidine-tagged SH3 domain of the human phosphatidylinositol 3-kinase p85alpha subunit, false responses after adhesion of undesirable compounds to a target protein could be minimized with the NTA strategy, while binding responses of a positive control peptide still stayed within a 1%-deviation against the theoretical binding capacity.     

Metal selectivity determinants in a family of transition metal transporters

Metal tolerance proteins (MTPs) are plant members of the cation diffusion facilitator (CDF) transporter family involved in cellular metal homeostasis. Members of the CDF family are ubiquitously found in all living entities and show principal selectivity for Zn(2+), Mn(2+), and Fe(2+). Little is known regarding metal selectivity determinants of CDFs. We identified a novel cereal member of CDFs in barley, termed HvMTP1, that localizes to the vacuolar membrane. Unlike its close relative AtMTP1, which is highly selective for Zn(2+), HvMTP1 exhibits selectivity for both Zn(2+) and Co(2+) as assessed by its ability to suppress yeast mutant phenotypes for both metals. Expression of HvMTP1/AtMTP1 chimeras in yeast revealed a five-residue sequence within the AtMTP1 N-segment of the His-rich intracytoplasmic loop that confines specificity to Zn(2+). Furthermore, mutants of AtMTP1 generated through random mutagenesis revealed residues embedded within transmembrane domain 3 that additionally specify the high degree of Zn(2+) selectivity. We propose that the His-rich loop, which might play a role as a zinc chaperone, determines the identity of the metal ions that are transported. The residues within transmembrane domain 3 can also influence metal selectivity, possibly through conformational changes induced at the cation transport site located within the membrane or at the cytoplasmic C-terminal domain.     

Glutamine in the pathogenesis of acute hepatic encephalopathy

Hepatic encephalopathy (HE) is the major neurological disorder associated with liver disease. It presents in chronic and acute forms, and astrocytes are the major neural cells involved. While the principal etiological factor in the pathogenesis of HE is increased levels of blood and brain ammonia, glutamine, a byproduct of ammonia metabolism, has also been implicated in its pathogenesis. This article reviews the current status of glutamine in the pathogenesis of HE, particularly its involvement in some of the events triggered by ammonia, including mitochondrial dysfunction, generation of oxidative stress, and alterations in signaling mechanisms, including activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-κB). Mechanisms by which glutamine contributes to astrocyte swelling/brain edema associated with acute liver failure (ALF) will also be described.     

A critical role for the histidine residues in the catalytic function of acyl-CoA:cholesterol acyltransferase catalysis: evidence for catalytic difference between ACAT1 and ACAT2

To investigate a role for histidine residues in the expression of normal acyl-CoA:cholesterol acyltransferase (ACAT) activity, the histidine residues located at five different positions in two isoenzymes were substituted by alanine, based on the sequence homology between ACAT1 and ACAT2. Among the 10 mutants generated by baculovirus expression technology, H386A-ACAT1, H460A-ACAT1, H360A-ACAT2, and H399A-ACAT2 lost their enzymatic activity completely. A reduction in catalytic activity is unlikely to result from structural changes in the substrate-binding pocket, because their substrate-binding affinities were normal. However, the enzymatic activity of H386A-ACAT1 was restored to <37% of the level of the wild-type activity when cholesterol was replaced by 25-hydroxycholesterol as substrate. H527A-ACAT1 and H501A-ACAT2, termed carboxyl end mutants, exhibit activities of ∼96% and ∼75% of that of the wild-type. Interestingly, H425A-ACAT1 showed 59% of the wild-type activity, in contrast to its equivalent mutant, H399A-ACAT2. These results demonstrate that the histidine residues located at the active site are very crucial both for the catalytic activity of the enzyme and for distinguishing ACAT1 from ACAT2 with respect to enzyme catalysis and substrate specificity.     

Histidine biosynthesis in plants

Summary. The study of histidine metabolism has never been at the forefront of interest in plant systems despite the significant role that the analysis of this pathway has played in development of the field of molecular genetics in microbes. With the advent of methods to analyze plant gene function by complementation of microbial auxotrophic mutants and the complete analysis of plant genome sequences, strides have been made in deciphering the histidine pathway in plants. The studies point to a complex evolutionary origin of genes for histidine biosynthesis. Gene regulation studies have indicated novel regulatory networks involving histidine. In addition, physiological studies have indicated novel functions for histidine in plants as chelators and transporters of metal ions. Recent investigations have revealed intriguing connections of histidine in plant reproduction. The exciting new information suggests that the study of plant histidine biosynthesis has finally begun to flower.     

Short-chain oligopeptides with copper(II) binding properties: The impact of specific structural modifications on the copper(II) coordination abilities

A series of linear tetrapeptides containing two histidyl residues in position 2 and 4, namely DHGH, DHGdH, KHGH, KHGdH, Ac-DHGH-NH₂, Ac-DHGdH-NH₂, Ac-KHGH-NH₂, and Ac-KHGdH-NH₂, were synthesized and characterised. Their copper(II) binding properties were investigated in depth through a variety of physicochemical methods. Potentiometric titrations were first carried out to establish the stoichiometry and the stability of the resulting copper(II)-peptide complexes. The copper(II) chromophores that are formed in the various cases in dependence of pH were subsequently characterised by extensive spectroscopic analysis (UV-Vis, EPR, CD) in strict correlation with potentiometric data. The effects of the nature of the first amino acid (Lys versus Asp) and of N-terminal amino group protection on copper(II) binding were specifically addressed. On turn, the careful comparison of the copper(II) coordination abilities of the linear peptides with those of their cyclic analogs provided insight into the effects of cyclization on the overall metal binding properties.     

Functional dissection of adenylate cyclase R, an inducer of spore encapsulation

Cyclic AMP acting on protein kinase A controls sporulation and encystation in social and solitary amoebas. In Dictyostelium discoideum, adenylate cyclase R (ACR), is essential for spore encapsulation. In addition to its cyclase (AC) domain, ACR harbors seven transmembrane helices, a histidine kinase domain, and two receiver domains. We investigated the role of these domains in the regulation of AC activity. Expression of an ACR-YFP fusion protein in acr(-) cells rescued their sporulation defective phenotype and revealed that ACR is associated with the nuclear envelope and endoplasmic reticulum. Loss of the transmembrane helices (ΔTM) caused a 60% reduction of AC activity, but ΔTM-ACR still rescued the acr(-) phenotype. The isolated AC domain was properly expressed but inactive. Mutation of three essential ATP-binding residues in the histidine kinase domain did not affect the AC activity or phenotypic rescue. Mutation of the essential phosphoryl-accepting aspartate in receivers 1, 2, or both had only modest effects on AC activity and did not affect phenotypic rescue, indicating that AC activity is not critically regulated by phosphorelay. Remarkably, the dimerizing histidine phosphoacceptor subdomain, which in ACR lacks the canonical histidine for autophosphorylation, was essential for AC activity. Transformation of wild-type cells with an ACR allele (ΔCRA) that is truncated after this domain inhibited AC activity of endogenous ACR and replicated the acr(-) phenotype. Combined with the observation that the isolated AC domain was inactive, the dominant-negative effect of ΔCRA strongly suggests that the defunct phosphoacceptor domain acquired a novel role in enforcing dimerization of the AC domain.     

Cross-linking Chemistry of Squid Beak

In stark contrast to most aggressive predators, Dosidicus gigas (jumbo squids) do not use minerals in their powerful mouthparts known as beaks. Their beaks instead consist of a highly sclerotized chitinous composite with incremental hydration from the tip to the base. We previously reported l-3,4-dihydroxyphenylalanine (dopa)-histidine (dopa-His) as an important covalent cross-link providing mechanical strengthening to the beak material. Here, we present a more complete characterization of the sclerotization chemistry and describe additional cross-links from D. gigas beak. All cross-links presented in this report share common building blocks, a family of di-, tri-, and tetra-histidine-catecholic adducts, that were separated by affinity chromatography and high performance liquid chromatography (HPLC) and identified by tandem mass spectroscopy and proton nuclear magnetic resonance (¹H NMR). The data provide additional insights into the unusually high cross-link density found in mature beaks. Furthermore, we propose both a low molecular weight catechol, and peptidyl-dopa, to be sclerotization agents of squid beak. This appears to represent a new strategy for forming hard tissue in animals. The interplay between covalent cross-linking and dehydration on the graded properties of the beaks is discussed.