Nitrogen fixation (acetylene reduction) associated with roots of winter wheat and sorghum in Nebraska

Root segments and root-soil cores (6.5-cm diameter) from fields and nurseries of winter wheat and sorghum were tested for N2 fixation by using the acetylene reduction assay. Wheat samples (approximately 1,200) from 109 sites generally had low or no activity (0 to 3.1 nmol of C2H4 produced per h per g [dry weight] of root segments), even after 24 h of incubation. However, a commercial field of Scout 66, located in western Nebraska, exhibited appreciable activity (290 nmol of C2H4 produced per h per g [dry weight] of root segments). Of 400 sorghum lines and crosses, grain sorghums (i.e., CK-60A, Wheatland A, B517, and NP-16) generally exhibited higher nitrogenase activity than forage sorghums or winter wheats. CK-60A, a male sterile grain sorghum, was sampled at four locations and had the most consistent activity of 24 to 1,100 nmol of C2H4 produced per h per core. The maximum rate extrapolated to 2.5 g of N per hectare per day. Numerous N2-fixing bacterial isolates were obtained from wheat and sorghum roots that exhibited high nitrogenase activity. Most isolates were members of the Enterobacteriacae, i.e., Klebsiella pneumoniae, Enterobacter cloacae, and Erwinia herbicola.     

Mammalian heterotrimeric G-protein-like proteins in mycobacteria: implications for cell signalling and survival in eukaryotic host cells

Mammalian heterotrimeric GTP-binding proteins (G proteins) are involved in transmembrane signalling that couples a number of receptors to effectors mediating various physiological processes in mammalian cells. We demonstrate that bacterial proteins such as a Ras-like protein from Pseudomonas aeruginosa or a 65 kDa protein from Mycobacterium smegmatis can form complexes with human or yeast nucleoside diphosphate kinase (Ndk) to modulate their nucleoside triphosphate synthesizing specificity to GTP or UTP. In addition, we demonstrate that bacteria such as M. smegmatis or Mycobacterium tuberculosis harbour proteins that cross react with antibodies against the α-, β- or the γ-subunits of heterotrimeric G proteins. Such antibodies also alter the GTP synthesizing ability of specific membrane fractions isolated from glycerol gradients of such cells, suggesting that a membrane-associated Ndk–G-protein homologue complex is responsible for part of GTP synthesis in these bacteria. Indeed, purified Ndk from human erythrocytes and M. tuberculosis showed extensive complex formation with the purified mammalian α and β G-protein subunits and allowed specific GTP synthesis, suggesting that such complexes may participate in transmembrane signalling in the eukaryotic host. We have purified the α-, β- and γ-subunit homologues from M. tuberculosis and we present their internal amino acid sequences as well as their putative homologies with mammalian subunits and the localization of their genes on the M. tuberculosis genome. Using oligonucleotide probes from the conserved regions of the α- and γ-subunit of M. tuberculosis G-protein homologue, we demonstrate hybridization of these probes with the genomic digest of M. tuberculosis H37Rv but not with that of M. smegmatis, suggesting that M. smegmatis might lack the genes present in M. tuberculosis H37Rv. Interestingly, the avirulent strain H37Ra showed weak hybridization with these two probes, suggesting that these genes might have been deleted in the avirulent strain or are present in limited copy numbers as opposed to those in the virulent strain H37Rv.     

Differentiation-promoting effect of 1-O (2 methoxy) hexadecyl glycerol in human colon cancer cells

Alkylglycerols are naturally occurring bioactive ether lipids found in great abundance in the livers of many marine species. In this study, we evaluated the differentiation-promoting potential of a methoxy substituted alkylglycerol--1-O (2 methoxy) hexadecyl glycerol (MHG)--to promote a more benign or differentiated phenotype in human colon cancer cells. Three cell lines with different biological and phenotypic properties were used. They were the moderately differentiated and growth factor-responsive Moser, the growth factor-unresponsive and malignant HT29, and the poorly differentiated and growth factor-unresponsive HCT116. Treatment of these cell lines with MHG resulted in a downmodulation of cellular proliferation, a reduced propensity for anchorage-independent growth, and a reduced capacity in cellular invasion. Induction of the colon-associated and differentiation-related molecule carcinoembryonic antigen was also observed in the three cell lines. Induction of the transformation-sensitive and differentiation-related glycoprotein fibronectin was observed in the HT29 cells. It is concluded that MHG was biologically active and promoted a more benign or differentiated phenotype in these colon cancer cells. Since differentiation-inducing agents may possess chemoprevention properties, the use of MHG and the alkylglycerols in inducing differentiation or in chemoprevention of malignant diseases warrants further investigation.     

Identification and characterization of EhCaBP2. A second member of the calcium-binding protein family of the protozoan parasite Entamoeba histolytica

Entamoeba histolytica, an early branching eukaryote, is the etiologic agent of amebiasis. Calcium plays a pivotal role in the pathogenesis of amebiasis by modulating the cytopathic properties of the parasite. However, the mechanistic role of Ca(2+) and calcium-binding proteins in the pathogenesis of E. histolytica remains poorly understood. We had previously characterized a novel calcium-binding protein (EhCaBP1) from E. histolytica. Here, we report the identification and partial characterization of an isoform of this protein, EhCaBP2. Both EhCaBPs have four canonical EF-hand Ca(2+) binding domains. The two isoforms are encoded by genes of the same size (402 bp). Comparison between the two genes showed an overall identity of 79% at the nucleotide sequence level. This identity dropped to 40% in the 75-nucleotide central linker region between the second and third Ca(2+) binding domains. Both of these genes are single copy, as revealed by Southern hybridization. Analysis of the available E. histolytica genome sequence data suggested that the two genes are non-allelic. Homology-based structural modeling showed that the major differences between the two EhCaBPs lie in the central linker region, normally involved in binding target molecules. A number of studies indicated that EhCaBP1 and EhCaBP2 are functionally different. They bind different sets of E. histolytica proteins in a Ca(2+)-dependent manner. Activation of endogenous kinase was also found to be unique for the two proteins and the Ca(2+) concentration required for their optimal functionality was also different. In addition, a 12-mer peptide was identified from a random peptide library that could differentially bind the two proteins. Our data suggest that EhCaBP2 is a new member of a class of E. histolytica calcium-binding proteins involved in a novel calcium signal transduction pathway.     

Probing protein-surfactant interaction by steady state and time-resolved fluorescence spectroscopy

The microenvironment of the probe coumarin 153 (C-153) in 1% bovine serum albumin (BSA) is more hydrophobic in nature compared to that in pure micelles or protein-surfactant complexes. In the native state of protein, we have not observed any solvation using C-153 as a probe but we have observed a slow dynamics on protein surface using 8-anilino-1-naphthalenesulfonic acid (ANS) as a probe. This may be due to the location of the probe (C-153) in the hydrophobic, solvent-inaccessible pocket of the BSA. Solvation dynamics in the BSA-surfactant (SDS) complexes in the solution phase is markedly different from that in pure micelles. This is may be due to the formation of 'necklace and bead' structure in the complexes. The rotational motion is also severely hindered in the surface of the protein.     

Energy-generating enzymes of Burkholderia cepacia and their interactions with macrophages

We previously demonstrated that several clinical and environmental isolates of Burkholderia cepacia secreted ATP-utilizing enzymes to the medium; the secretion of these enzymes by cystic fibrosis lung isolate strain 38 was shown to be greatly enhanced in the presence of alpha(2)-macroglobulin. Fractionation of the growth medium of cystic fibrosis isolate strain 71 belonging to genomovar I demonstrated the presence of two additional proteins, homologues of Pseudomonas aeruginosa azurin and cytochrome c(551), which are normally involved in electron transfer during denitrification. A Q-Sepharose column flowthrough fraction of the growth medium of B. cepacia strain 71 enriched with the azurin and cytochrome c(551) homologues triggered apoptosis in macrophages and mast cells, leading to their death. Incubation of the Q-Sepharose column flowthrough fraction with antiazurin and anti-cytochrome c(551) antibodies greatly reduced cell death. We cloned and hyperexpressed a gene from B. cepacia strain 71 that encodes the homologue of P. aeruginosa azurin. Such azurin homologues were detected in the growth medium of several strains belonging to genomovars I, III, and VI but not in the growth medium of strains belonging to other genomovars. The growth medium of the strains that elaborated the azurin homologue had high cytotoxicity towards macrophages. Purified azurin homologue was shown to induce apoptosis in macrophages in a caspase-dependent manner and was localized in both the cytosol and nucleus when incubated with or microinjected into macrophages. This is an interesting example of the interaction of a bacterial protein normally involved in cellular energetics with macrophages to effect their cell death.     

Cellular function of elastase in Pseudomonas aeruginosa: role in the cleavage of nucleoside diphosphate kinase and in alginate synthesis

Elastase is a major virulence factor in Pseudomonas aeruginosa that is believed to cause extensive tissue damage during infection in the human host. Elastase is secreted in non-mucoid P. aeruginosa. It is known that secretion of most virulence factors such as elastase, lipase, exotoxin A, etc., in P. aeruginosa is greatly reduced in alginate-secreting mucoid cells isolated from the lungs of cystic fibrosis (CF) patients. We have previously reported that in mucoid P. aeruginosaan intracellular protease cleaves the 16 kDa form of nucleoside diphosphate kinase (Ndk) to a truncated 12 kDa form. This smaller form is membrane associated and has been observed to form complexes with specific proteins to predominantly generate GTP, an important molecule in alginate synthesis. The main aim of this study was to purify and characterize this protease. The protease was purified by hydrophobic interaction chromatography of the crude extract of mucoid P. aeruginosa 8821, a CF isolate. Further analysis using a gelatin containing SDS–polyacrylamide gel detected the presence of a 103 kDa protease, which when boiled, migrated as a 33 kDa protein on a SDS–polyacrylamide gel. The first 10 amino acids from the N-terminus of the 33 kDa protease showed 100% identity to the mature form of elastase. An elastase-negative lasB ::Cm knock-out mutant in the mucoid 8821 background was constructed, and it showed a non-mucoid phenotype. This mutant showed the presence of only the 16 kDa form of Ndk both in the cytoplasm and membrane fractions. We present evidence for the retention of active elastase in the periplasm of mucoid P. aeruginosa and its role in the generation of the 12 kDa form of Ndk. Finally, we demonstrate that elastase, when overproduced in both mucoid and non-mucoid cells, stimulates alginate synthesis. This suggests that the genetic rearrangements that trigger mucoidy in P. aeruginosa also allow retention of elastase in the periplasm in an active oligomeric form that facilitates cleavage of 16 kDa Ndk to its 12 kDa form for the generation of GTP, required for alginate synthesis.     

Purification and characterization of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosa involved in biosynthesis of both alginate and lipopolysaccharide.

The algC gene from Pseudomonas aeruginosa has been shown to encode phosphomannomutase (PMM), an essential enzyme for biosynthesis of alginate and lipopolysaccharide (LPS). This gene was overexpressed under control of the tac promoter, and the enzyme was purified and its substrate specificity and metal ion effects were characterized. The enzyme was determined to be a monomer with a molecular mass of 50 kDa. The enzyme catalyzed the interconversion of mannose 1-phosphate (M1P) and mannose 6-phosphate, as well as that of glucose 1-phosphate (G1P) and glucose 6-phosphate. The apparent Km values for M1P and G1P were 17 and 22 microM, respectively. On the basis of Kcat/Km ratio, the catalytic efficiency for G1P was about twofold higher than that for M1P. PMM also catalyzed the conversion of ribose 1-phosphate and 2-deoxyglucose 6-phosphate to their corresponding isomers, although activities were much lower. Purified PMM/phosphoglucomutase (PGM) required Mg2+ for maximum activity; Mn2+ was the only other divalent metal that showed some activation. The presence of other divalent metals in addition to Mg2+ in the reaction inhibited the enzymatic activity. PMM and PGM activities could not be detected in nonmucoid algC mutant strain 8858 and in LPS-rough algC mutant strain AK1012, while they were present in the wild-type strains as well as in algC-complemented mutant strains. This evidence suggests that AlgC functions as PMM and PGM in vivo, converting phosphomannose and phosphoglucose in the biosynthesis of both alginate and LPS.