Escherichia coli kasugamycin dependence arising from mutation at the rpsI locus

Escherichia coli mutants with alterations in the electrophoretic mobility of ribosomal protein S9 were used to locate rpsI, the gene for this protein, on the linkage map. rpsI was located at about 70 min, roughly halfway between argG and fabE. It was very close to the gene for ribosomal protein L13, rplM. Another mutation at the rpsI locus gave rise to a phenotype of kasugamycin dependence and resistance. In this mutant, dependence on antibiotic came from kasugamycin being necessary to slow the rate of protein synthesis.     

A tropical Atlantic species of Melibe Rang, 1829 (Mollusca,Nudibranchia, Tethyiidae)

A new species of Melibe is described based on two specimens collected in Florida. This new species is well differentiated morphologically and genetically from other species of Melibe studied to date. The four residue deletions in the cytochrome c oxidase subunit 1 protein found in all previously sequenced tropical species of Melibe sequenced (and Melibe rosea) are also present in this new species. These deletions do not appear to affect important structural components of this protein but might have fitness implications. This paper provides the first confirmed record of Melibe in the tropical western Atlantic Ocean.     

The effects of Bacillus thuringiensis Cry6A on the survival, growth, reproduction, locomotion, and behavioral response of Caenorhabditis elegans

Several families of crystal proteins from Bacillus thuringiensis exhibit nematicidal activity. Cry5B protein, a pore-forming toxin, has been intensively studied yielding many insights into the mode of action of crystal protein at molecular level and pathogenesis of pore-forming toxins. However, little attention was paid to Cry6A, another representative nematicidal crystal protein. Cry6A shares very low homology with Cry5B at amino acid sequence and probably acts in a distinct pathway from Cry5B and even the other main commercial crystal proteins. In the current study, we comprehensively investigated the nematicidal properties of Cry6Aa2 against the free-living soil nematode Caenorhabditis elegans and examined the physical response of C. elegans to Cry6Aa2 attack. Our results indicate that Cry6Aa2 exhibits high lethal activity to C. elegans and could cause detrimental effects on C. elegans, including obviously suppressed growth, decreased brood size, and even abnormal motility. Meanwhile, our study additionally shows that C. elegans could defend against the Cry6Aa2 toxin harmful threat through behavioral defense responses, such as reduced oral uptake and physical avoidance. In general, this study suggests that Cry6Aa2 possesses diverse nematicidal properties, which strongly indicates that Cry6Aa2 is a promising potential candidate of nematicidal agent. Moreover, this study highlights the importance of behavioral responses in defense of C. elegans for survival and demonstrates the key role of crystal protein in the interaction of B. thuringiensis–C. elegans. These findings could shed light on understanding the interaction of C. elegans with B. thuringiensis and provide a perfect model to study the role of pathogenic factor in the interaction of pathogen–host.     

Structural proteins of Amsacta moorei,Euxoa auxiliaris, and Melanoplus sanguinipes entomopoxviruses

The structural proteins of Amsacta moorei, Euxoa auxiliaris, and Melanoplus sanguinipes entomopoxviruses (EPVs) were separated by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels. More than 35 structural proteins were detected in each virus. Based on the distribution and the variation in the molecular weights of the virus structural proteins little homology was detected between the EPVs and vaccinia virus. The molecular weight of Amsacta EPV occlusion body matrix protein (110,000) was determined by SDS-acrylamide gel electrophoresis. The occlusion body matrix protein of Amsacta EPV occluded virus isolated from infected E. acrea larvae was rapidly degraded at pH 10.6 to peptides of approximately 94,000 and 60,000 daltons. After 2 hr incubation at alkaline pH, Amsacta EPV occlusion body protein was degraded to approximately 56,000 daltons. Proteolysis of occlusion body protein was inhibited by SDS. No proteolytic degradation was detected in occlusion body matrix protein isolated from Amsacta EPV infected BTI-EAA cells. Amino acid analysis indicates that entomopoxvirus occlusion body matrix protein consists of approximately 20% acidic amino acids and 9% of the sulfur-containing amino acids cysteine and methionine.     

Cloning, Expression, and Characterization of the katG Gene, Encoding Catalase-Peroxidase, from the Polycyclic Aromatic Hydrocarbon-Degrading Bacterium Mycobacterium sp. Strain PYR-1

A 81-kDa protein from Mycobacterium sp. strain PYR-1 was expressed in response to exposure of the strain to the polycyclic aromatic hydrocarbon pyrene and recovered by two-dimensional gel electrophoresis. The N-terminal sequence of the protein indicated that it was similar to catalase-peroxidase. An oligonucleotide probe designed from this sequence was used to screen a genomic library of Mycobacterium sp. strain PYR-1, and a positive clone, containing a part of the gene encoding the 81-kDa protein, was isolated. A gene-walking technique was used to sequence the entire gene, which was identified as katG for catalase-peroxidase. The deduced KatG protein sequence showed significant homology to KatGII of Mycobacterium fortuitum and clustered with catalase-peroxidase proteins from other Mycobacterium species in a phylogenetic tree. The katG gene was expressed in Escherichia coli to produce a protein with catalase-peroxidase activity. Since the induction of this catalase-peroxidase occurred in pyrene-induced cultures and the exposure of these cultures to hydrogen peroxide reduced pyrene metabolism, our data suggest that this enzyme plays a role in polycyclic aromatic hydrocarbon metabolism by strain PYR-1.     

Characterization of calciphorin,the low molecular weight calciu,ionophore, from rat liver mitochondria

Calciphorin, the putative mitochondrial calcium ionophore from rat liver mitochondria, exhibits the inherent properties of the mitochondrial calcium transport system and is similar to the calf heart preparation reported earlier. The protein has a strong selectivity for Ca²⁺, and has a K_d for Ca²⁺ of 56.5 ± 6.6 μM and 13.9 ± 2.1 μM in organic extraction and flow dialysis experiments, respectively. Reduction of the contaminating lipids from 23 ± 6.5 to 1.73 ± 0. moles per mole protein does not alter the affinities, Ca²⁺/protein soichiometry or selectivity for Ca²⁺.     

VdNEP, an Elicitor from Verticillium dahliae, Induces Cotton Plant Wilting

Verticillium wilt is a vascular disease of cotton. The causal fungus, Verticillium dahliae, secretes elicitors in culture. We have generated ~1,000 5′-terminal expressed sequence tags (ESTs) from a cultured mycelium of V. dahliae. A number of ESTs were found to encode proteins harboring putative signal peptides for secretion, and their cDNAs were isolated. Heterologous expression led to the identification of a protein with elicitor activities. This protein, named V. dahliae necrosis- and ethylene-inducing protein (VdNEP), is composed of 233 amino acids and has high sequence identities with fungal necrosis- and ethylene-inducing proteins. Infiltration of the bacterially expressed His-VdNEP into Nicotiana benthamiana leaves resulted in necrotic lesion formation. In Arabidopsis thaliana, the fusion protein also triggered production of reactive oxygen species and induced the expression of PR genes. When added into suspension cultured cells of cotton (Gossypium arboreum), the fusion protein elicited the biosynthesis of gossypol and related sesquiterpene phytoalexins at low concentrations, and it induced cell death at higher concentrations. On cotton cotyledons and leaves, His-VdNEP induced dehydration and wilting, similar to symptoms caused by a crude preparation of V. dahliae elicitors. Northern blotting showed a low level of VdNEP expression in the mycelium during culture. These data suggest that VdNEP is a wilt-inducing factor and that it participates in cotton-V. dahliae interactions.     

Activities, occurrence, and localization of hydrogenase in free-living and symbiotic frankia

Symbiotic and free-living Frankia were investigated for correlation between hydrogenase activities (in vivo/in vitro assays) and for occurrence and localization of hydrogenase protein by Western blots and immuno-gold localization, respectively. Freshly prepared nodule homogenates from the symbiosis between Alnus incana and a local source of Frankia did not show any detectable in vivo or in vitro hydrogenase uptake activity, as also has been shown earlier. However, a free-living Frankia strain originally isolated from these nodules clearly showed both in vivo and in vitro hydrogenase activity, with the latter being approximately four times higher. Frankia strain Cpl1 showed hydrogen uptake activity both in symbiosis with Alnus incana and in a free-living state. Western blots on the different combinations of host plants and Frankia strains used in the present study revealed that all the Frankia sources contained a hydrogenase protein, even the local source where no in vivo or in vitro activity could be measured. The 72 kilodalton protein found in the symbiotic Frankia as well as in the free-living Frankia strains were immunologically related to the large subunit of a dimeric hydrogenase purified from Alcaligenes latus. Recognitions to polypeptides with molecular masses of about 41 and 19.5 kilodaltons were also observed in Frankia strain UGL011101 and in the local source of Frankia, respectively. Immunogold localization of the protein demonstrated that in both the symbiotic state and the free-living nitrogen-fixing Frankia, the protein is located in vesicles and in hyphae. The inability to measure any uptake hydrogenase activity is therefore not due to the absence of hydrogenase enzyme. However, the possibility of an inactive hydrogenase enzyme cannot be ruled out.     

Endosome to Golgi Retrieval of the Vacuolar Protein Sorting Receptor, Vps10p, Requires the Function of the VPS29, VPS30, and VPS35 Gene Products

Mutations in the S. cerevisiae VPS29 and VPS30 genes lead to a selective protein sorting defect in which the vacuolar protein carboxypeptidase Y (CPY) is missorted and secreted from the cell, while other soluble vacuolar hydrolases like proteinase A (PrA) are delivered to the vacuole. This phenotype is similar to that seen in cells with mutations in the previously characterized VPS10 and VPS35 genes. Vps10p is a late Golgi transmembrane protein that acts as the sorting receptor for soluble vacuolar hydrolases like CPY and PrA, while Vps35p is a peripheral membrane protein which cofractionates with membranes enriched in Vps10p. The sequences of the VPS29, VPS30, and VPS35 genes do not yet give any clues to the functions of their products. Each is predicted to encode a hydrophilic protein with homologues in the human and C. elegans genomes. Interestingly, mutations in the VPS29, VPS30, or VPS35 genes change the subcellular distribution of the Vps10 protein, resulting in a shift of Vps10p from the Golgi to the vacuolar membrane. The route that Vps10p takes to reach the vacuole in a vps35 mutant does not depend upon Sec1p mediated arrival at the plasma membrane but does require the activity of the pre-vacuolar endosomal t-SNARE, Pep12p. A temperature conditional allele of the VPS35 gene was generated and has been found to cause missorting/secretion of CPY and also Vps10p to mislocalize to a vacuolar membrane fraction at the nonpermissive temperature. Vps35p continues to cofractionate with Vps10p in vps29 mutants, suggesting that Vps10p and Vps35p may directly interact. Together, the data indicate that the VPS29, VPS30, and VPS35 gene products are required for the normal recycling of Vps10p from the prevacuolar endosome back to the Golgi where it can initiate additional rounds of vacuolar hydrolase sorting.     

SlyA,a regulatory protein from Salmonella typhimurium,induces a haemolytic and pore-forming protein in Escherichia coli

A chromosomal fragment from Salmonella typhimurium, when cloned in Escherichia coli, generates a haemolytic phenotype. This fragment carries two genes, termed slyA and slyB. The expression of slyA is sufficient for the haemolytic phenotype. The haemolytic activity of E. coli carrying multiple copies of slyA is found mainly in the cytoplasm, with some in the periplasm of cells grown to stationary phase, but overexpression of SlyB, a 15 kDa lipoprotein probably located in the outer membrane, may lead to enhanced, albeit unspecific, release of the haemolytic activity into the medium. Polyclonal antibodies raised against a purified SlyA-HlyA fusion protein identified the over-expressed monomeric 17 kDa SlyA protein mainly in the cytoplasm of E. coli grown to stationary phase, although smaller amounts were also found in the periplasm and even in the culture supernatant. However, the anti-SlyA antibodies reacted with the SlyA protein in a periplasmic fraction that did not contain the haemolytic activity. Conversely, the periplasmic fraction exhibiting haemolytic activity did not contain the 17 kDa SlyA protein. Furthermore, S. typhimurium transformed with multiple copies of the slyA gene did not show a haemolytic phenotype when grown in rich culture media, although the SlyA protein was expressed in amounts similar to those in the recombinant E. coli strain. These results indicate that SlyA is not itself a cytolysin but rather induces in E. coli (but not in S. typhimurium) the synthesis of an uncharacterised, haemolytically active protein which forms pores with a diameter of about 2.6 nm in an artificial lipid bilayer. The SlyA protein thus seems to represent a regulation factor in Salmonella, as is also suggested by the similarity of the SlyA protein to some other bacterial regulatory proteins. slyA- and slyB-related genes were also obtained by PCR from E. coli, Shigella sp. and Citrobacter diversus but not from several other gram-negative bacteria tested.     

Characterization, localization, essentiality, and high-resolution crystal structure of glucosamine 6-phosphate N-acetyltransferase from Trypanosoma brucei

A gene predicted to encode Trypanosoma brucei glucosamine 6-phosphate N-acetyltransferase (TbGNA1; EC 2.3.1.4) was cloned and expressed in Escherichia coli. The recombinant protein was enzymatically active, and its high-resolution crystal structure was obtained at 1.86 Å. Endogenous TbGNA1 protein was localized to the peroxisome-like microbody, the glycosome. A bloodstream-form T. brucei GNA1 conditional null mutant was constructed and shown to be unable to sustain growth in vitro under nonpermissive conditions, demonstrating that there are no metabolic or nutritional routes to UDP-GlcNAc other than via GlcNAc-6-phosphate. Analysis of the protein glycosylation phenotype of the TbGNA1 mutant under nonpermissive conditions revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite and that the glycosylation profile of the principal parasite surface coat component, the variant surface glycoprotein (VSG), was modified. The significance of results and the potential of TbGNA1 as a novel drug target for African sleeping sickness are discussed.     

GsVAMP72, a novel Glycine soja R-SNARE protein, is involved in regulating plant salt tolerance and ABA sensitivity

Abiotic stress, especially high salinity, is a major threat to agricultural production. It has been well established that SNARE proteins sustain directed vesicle traffic to underpin plant growth and development, yet little is known about the role of SNARE protein in the capacity to withstand abiotic stress, especially in wild soybeans. Here we identified and characterized a GsCBRLK interacting protein, GsVAMP72, which is a putative vesicle-associated membrane protein in Glycine soja. GsVAMP72 protein has a longin domain at its N-terminus, belonging to R-SNARE family. Quantitative real-time (RT) PCR and beta-glucuronidase (GUS) activity assays revealed that the expression of GsVAMP72 was highly and rapidly induced by both high salt and ABA treatments. Overexpression of GsVAMP72 in Arabidopsis significantly reduced salt tolerance by modifying the ionic content and down-regulating expression of stress-responsive genes, including RD29A, COR47, KIN1, COR15A and RAB18. On the other hand, GsVAMP72 overexpression increased plant ABA sensitivity and altered the expression levels of ABA-responsive genes. Subcellular localization analysis showed that eGFP–GsVAMP72 fusion protein was observed on the plasma membrane-like and endosome-like structures but eGFP alone was distributing throughout the cytoplasm in Arabidopsis protoplasts and onion epidermal cells. GsVAMP72 promoter-controlled GUS activity was detected in both vegetative and reproductive organs, and was strongly induced by salt and ABA. In summary, we demonstrated that GsVAMP72 is a novel Glycine soja vesicle-associated membrane protein and is highly involved in regulating plant responses to salt and ABA stresses.     

Molecular Cloning, Sequencing, and Expression of a Chemoreceptor Gene from Leptospirillum ferrooxidans

We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacterium Leptospirillum ferrooxidans. This DNA contained an open reading frame for a 577-amino-acid protein showing several characteristics of the bacterial chemoreceptors and, therefore, we named this gene lcrI for Leptospirillum chemotaxis receptor I. This is the first sequence reported for a gene from L. ferrooxidans encoding a protein. The lcrI gene showed both ς²⁸-like and ς⁷⁰-like putative promoters. The LcrI deduced protein contained two hydrophobic regions most likely corresponding to the two transmembrane regions present in all of the methyl-accepting chemotaxis proteins (MCPs) which make them fold with both periplasmic and cytoplasmic domains. We have proposed a cytoplasmic domain for LcrI, which also contains the highly conserved domain (HCD region), present in all of the chemotactic receptors, and two probable methylation sites. The in vitro expression of a DNA plasmid containing the 2,262-bp fragment showed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein (an MCP from Escherichia coli), confirming some degree of antigenic conservation. In addition, this 58-kDa protein was expressed in E. coli, being associated with its cytoplasmic membrane fraction. It was not possible to determine a chemotactic receptor function for LcrI expressed in E. coli. This was most likely due to the fact that the periplasmic pH of E. coli, which differs by 3 to 4 pH units from that of acidophilic chemolithotrophs, does not allow the right conformation for the LcrI periplasmic domain.     

Geometry, thermodynamics, and protein

We derive a new continuous free energy formula for protein folding. We obtain the formula first by adding hydrophobic effect to a classical free energy formula for cavities in water. We then obtain the same formula by geometrically pursuing the structure that fits best the well-known global geometric features of native structures of globular proteins: 1. high density; 2. small surface area; 3. hydrophobic core; 4. forming domains for long polypeptide chains. Conformations of a protein are presented as an all atom CPK model where each atom is a ball B(x_i,r_i). All conformations satisfy generally defined steric conditions. For each conformation P of a globular protein, there is a closed thermodynamic system Ω_P⊃P bounded by the molecular surface M_P. Both methods derive the same free energy aV(P)+bA(P)+cW(P), where a,b,c>0, V(P), A(P), and W(P) are volume of Ω_P, area of M_P, and area of the hydrophobic surface W_P⊂M_P, which quantifies hydrophobic effect.Minimizing W(P) is sufficient to produce statistically significant native like secondary structures and hydrogen bonds in the proteins we simulated.     

The maize d2003, a novel allele of VP8, is required for maize internode elongation

The d2003 is a natural dwarf mutant from maize inbred line K36 and has less than one-third of K36 plant height with severely shortened internodes. In this study, we reported the cloning of d2003 gene using positional cloning. The results showed that there was a single-base insertion in the coding region of Viviparous8 (VP8) in d2003 mutant, which resulted in a premature stop codon. Further genetic allelism tests confirmed that d2003 mutation is a novel allele of VP8. VP8 is mainly expressed in the stem apex, young leaves, and developing vascular tissues, and its expression levels in nodes are significantly higher than that in internodes at 12-leaf stage. Subcellular localization demonstrated that the VP8 protein is localized to the endoplasmic reticulum and the N-terminal 26 amino acids (aa) of VP8 protein are essential to its localization in ER. Further transgenic experiments showed that lack of the 26 aa leads to loss of VP8 function in Arabidopsis amp1 phenotype rescue. These results strongly suggested that the N-terminal 26 aa is critical for VP8 protein localization, and the correct protein localization of VP8 in ER is necessary for its function.     

Identification, sequence, and expression of Treponema denticola mcpA, a putative chemoreceptor gene

The nucleotide sequence of a methyl-accepting chemotaxis protein gene, mcpA, from Treponema denticola has been determined. The mcpA gene encodes a 729-amino acid protein whose deduced amino acid sequence has significant homology with several bacterial MCPs. T. denticola McpA contains two N-terminal transmembrane regions and two C-terminal putative methylation sequences that are separated by a highly conserved signaling domain. The organization of these structural features is characteristic of MCPs. The observed molecular mass of the in vitro synthesized McpA (76.0 kDa) correlates with the predicted molecular mass of the protein (80.1 kDa).