Tn5-induced mutants of Azotobacter vinelandii affected in nitrogen fixation under Mo-deficient and Mo-sufficient conditions

Mutants of Azotobacter vinelandii affected in N2 fixation in the presence of 1 microM Na2MoO4 (conventional system), 50 nM V2O5, or under Mo deficiency (alternative system) have been isolated after Tn5 mutagenesis with the suicide plasmid pSUP1011. These mutants can be grouped into at least four broad phenotypic classes. Mutants in the first class are Nif- under Mo sufficiency but Nif+ under Mo deficiency or in the presence of V2O5. A nifk mutant and a mutant apparently affected in regulation of the conventional system belong to this class. Mutants in the second class are Nif- under all conditions. An FeMo-cofactor-negative mutant (NifB-) belongs to this class, implying an involvement of nifB in both the conventional and the alternative N2 fixation systems. The third mutant class consists of mutants incapable of N2-dependent growth under Mo deficiency. Most of the mutants in this class are also affected in N2 fixation in the presence of 1 microM Na2MoO4, with acetylene reduction rates ranging from 28 to 51% of the rates of the wild type. Strains constructed by genetic transfer of the Kanr marker of mutants from this class into nifHDK or nifK deletion mutants showed N2-dependent growth only in the presence of V2O5, suggesting that growth in the presence of V2O5 and growth under Mo deficiency are independent phenomena. The only mutant in the fourth class shows wild-type nitrogenase activity under Mo sufficiency, but only 10% of the acetylene reduction activity of the wild type in the presence of 50 nM V2O5. The acetylene reduction rates of whole cells of this mutant are identical in Mo-deficient medium and in medium containing V2O5. The conventional nitrogenase subunits are expressed in this mutant even under Mo deficiency or in the presence of V2O5; however, the NH4+- and Mo-repressible proteins normally seen under these conditions could not be detected on two-dimensional gels. The Tn5 insertion carried by this mutant makes N2 fixation dependent solely on the conventional system and consequently abolishes the vanadium effect.     

Crystallization and preliminary crystallographic studies of the precursor and mature forms of a neutral lipase from the fungus Rhizopus delemar

A neutral lipase from the filamentous fungus Rhizopus delemar has been crystallized in both its proenzyme and mature forms. Although the latter crystallizes readily and produces a variety of crystal forms, only one was found to be suitable for X-ray studies. It is monoclinic (C2, a = 92.8 Å, b = 128.9 Å, c = 78.3 Å, β = 135.8) with two molecules in the asymmetric unit related by a noncrystallographic diad. The prolipase crystals are orthorhombic (P2₁2₁2₁, with a = 79.8 Å, b = 115.2 Å, c = 73.0 Å) and also contain a pair of molecules in the asymmetric unit. Initial results of molecular replacement calculations using the refined coordinates of the related lipase from Rhizomucor miehei identified the correct orientations and positions of the protein molecules in the unit cells of crystals of both proenzyme and the mature form. © 1994 John Wiley & Sons, Inc.     

Cloning, expression, and nucleotide sequence of the Lactobacillus helveticus 481 gene encoding the bacteriocin helveticin J.

Lactobacillus helveticus 481 produces a 37-kDa bacteriocin called helveticin J. Libraries of chromosomal DNA from L. helveticus were prepared in lambda gt11 and probed for phage-producing fusion proteins that could react with polyclonal helveticin J antibody. Two recombinant phage, HJ1 and HJ4, containing homologous inserts of 350 and 600 bp, respectively, produced proteins that reacted with antibody. These two phage clones specifically hybridized to L. helveticus 481 total genomic DNA but not to DNA from strains that did not produce helveticin J or strains producing unrelated bacteriocins. HJ1 and HJ4 lysogens produced beta-galactosidase fusion proteins that shared similar epitopes with each other and helveticin J. The intact helveticin J gene (hlv) was isolated by screening a library of L. helveticus chromosomal DNA in lambda EMBL3 with the insert DNA from phage HJ4 as a probe. The DNA sequence of a contiguous 3,364-bp region was determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequenced fragment. The 3' end of another open reading frame, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. ORF2 could encode an 11,808-Da protein. The L. helveticus DNA inserts of the HJ1 and HJ4 clones reside within ORF3, which begins 30 bp downstream from the termination codon of ORF2. ORF3 could encode a 37,511-Da protein. Downstream from ORF3, the 5' end of another ORF (ORF4) was found. A Bg/II fragment containing ORF2 and ORF3 was cloned into pGK12, and the recombinant plasmid, pTRK135, was transformed into Lactobacillus acidophilus via electroporation. Transformants carrying pTRK135 produced a bacteriocin that was heat labile and exhibited an acitivity spectrum that was the same as that of helveticin J.     

Fascioloides magna (Bassi, 1875) in feral swine from southern Texas.

Between 1971 and 1975, Fascioloides magna was found in 46 of 67 (69%) feral swine (Sus scrofa) in southern Texas. Flukes were recovered from swine in areas where F. magna commonly has been recovered from white-tailed deer and cattle. One to 12 flukes were recovered from each infected animal. Their presence was indicated by black hematin pigment on the liver and various other internal organs. Eggs were not detected in the gallbladder or feces of infected animals although mature flukes and eggs were recovered in the livers suggesting that, like cattle, feral swine can be infected but are aberrant hosts for the parasite and do not disseminate eggs.     

Murine macrophage inflammatory cytokine production and immune activation in response to Vibrio parahaemolyticus infection

Vibrio parahaemolyticus is the most common cause of bacterial, seafood‐related illness in the USA. Currently, there is a dearth of published reports regarding immunity to infection with this pathogen. Here, production of both pro‐ and anti‐inflammatory cytokines by V. parahaemolyticus‐infected RAW 264.7 murine macrophages was studied. It was determined that this infection results in increased concentrations of IL‐1α, IL‐6, TNF‐α and IL‐10. Additionally, decreases in cell surface TLR2 and TLR4 and increases in T‐cell co‐stimulatory molecules CD40 and CD86 were discovered. The data presented here begin to identify the immune variables required to eliminate V. parahaemolyticus from infected host tissues.     

IL-11 and IL-11Rα immunolocalisation at primate implantation sites supports a role for IL-11 in placentation and fetal development

Embryo implantation, endometrial stromal cell decidualization and formation of a functional placenta are critical processes in the establishment and maintenance of pregnancy. Interleukin (IL)-11 signalling is essential for adequate decidualization in the mouse uterus and IL-11 promotes decidualization in the human. IL-11 action is mediated via binding to the specific IL-11 receptor α (IL-11Rα). The present study examined immunoreactive IL-11 and IL-11Rα in cycling rhesus monkey endometrium, at implantation sites in cynomolgus and rhesus monkeys and in human first trimester decidua and defined distinct spatial and temporal patterns. In cycling rhesus monkey endometrium, IL-11 and IL-11Rα increased in both basalis and functionalis regions during the secretory compared with the proliferative phase, with changing cellular locations in luminal and glandular epithelium and stroma. The patterns were similar overall to those previously described in human endometrium. Differences were seen in immunostaining during implantation in cynomologus and rhesus monkey. In the cynomolgus, very little staining for IL-11 or IL-11Rα was seen in syncytio- and cyto-trophoblast cells in the villi between days 12 and 150 of pregnancy although there was moderate staining in cytotrophoblast in the shell between days 12 and 17 and in subpopulations of cytotrophoblast cells invading the arteries at day 17. By contrast in the rhesus monkey between days 24 and 35 of pregnancy and in human first trimester placenta, cyto- and syncytio-trophoblast in the villi but not cytotrophoblast in the shell were positively stained. The most intense staining for both IL-11 and IL-11Rα was present within the decidua in the maternal component of implantation sites in all three primates but moderate staining was also present in maternal vascular smooth muscle and glands perivascular cells and epithelial plaques. These results are consistent with a role for IL-11 both during decidualization and placentation in primates.     

Bacteria as workers in the living factory: metal-accumulating bacteria and their potential for materials science

Metal micro-/nano-particles with suitable chemical modification can be organized into new ceramic-metal (cermet) or organic-metal (orgmet) composites or structured materials. These materials are attracting significant attention because of their unique structures and highly optimized properties. However, the synthesis of composite materials with inhomogeneities on the nanometer or sub-micrometer scale is a continuing challenge in materials science. Many industrial physical and chemical surface-coating processes using conventional techniques are both energy and cost inefficient and require sophisticated instrumentation. In the future, biology might offer a superior option.     

Structures of l-fuculose-1-phosphate aldolase mutants outlining motions during catalysis

The crystal structures of l-fuculose-1-phosphate aldolase (FucA) with and without a ligated analogue of dihydroxyacetone phosphate (DHAP) and of a number of active center mutants have resulted in a model of the catalytic mechanism. This model has now been confirmed by structural analyses of further mutations at the zinc coordination sphere and at the phosphate site. In addition, these mutants have revealed new aspects of the catalysis: the hydroxyl group of Tyr113' (from a neighboring subunit), which sits just outside the zinc coordination sphere, steers DHAP towards a productive binding mode at the zinc ion; Glu73 contacts zinc in between the two ligand positions intended for the DHAP oxygen atoms and thus avoids blocking of these positions by a tetrahedrally coordinated hydroxy ion; the FucA polypeptide does not assume its minimum energy state but oscillates between two states of elevated energy as demonstrated by a mutant in a minimum energy state. The back and forth motion involves a mobile loop connecting the phosphate site with intersubunit motions and thus with the Brownian motion of the solvent. The phosphate group is bound strongly at a given distance to the zinc ion, which prevents the formation of too tight a DHAP:zinc complex. This observation explains our failure to find mutants that accept phosphate-free substitutes for DHAP. The FucA zinc coordination sphere is compared with that of carbonic anhydrase.