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PubMed1997

Phosphoprotein PII from cyanobacteria--analysis of functional conservation with the PII signal-transduction protein from Escherichia coli.

The signal transduction protein PII from Escherichia coli is modified by uridylylation, whereas its counterpart from the cyanobacterium Synechococcus PCC 7942 is phosphorylated at a seryl residue. To elucidate functional conservations between these proteins, we compared the Synechococcus PII protein with the known properties of the E. coli PII protein. Similar to the E. coli protein, Synechococcus PII binds the metabolites 2-oxoglutarate and ATP in a mutually dependent manner. The synergism of ligand binding was analyzed in detail. The ATP-binding site of Synechococcus PII could be labelled with 5'-p-fluorosulfonylbenzoyladenosine. By heterologous expression of the cyanobacterial glnB gene in E. coli we showed that Synechococcus PII can be modified by the E. coli PII uridylyltransferase. The presence of Synechococcus PII prevents signal transduction of E. coli PII to NtrB, presumably by non-functional competition. We therefore propose that the primary function of Synechococcus PII is to sense 2-oxoglutarate, the carbon skeleton required for nitrogen assimilation.

Bacterial Proteins↗
PubMed1997

Functional analysis of a conserved aspartate D218 in Cephalosporium acremonium isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is instrumental in the catalytic conversion of a tripeptide precursor delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to a bioactive intermediate isopenicillin N in the beta-lactam antibiotic biosynthetic pathway. It has recently been shown that this reaction is dependent on a conserved aspartate, D214, in a bacterial Streptomyces jumonjinensis IPNS. Thus, this study was carried out to provide the experimental evidence for the involvement of a similarly conserved aspartate residue, D218, in a fungal Cephalosporium acremonium IPNS (cIPNS). Initially, alteration of the aspartate residue to generate the mutant D218L cIPNS protein was achieved by site-directed mutagenesis. Subsequent enzyme assays indicated that the catalytic property of the mutant protein was lost, attesting to the need for the corresponding conserved aspartate to maintain IPNS functionality. It is also evident from the observed results that site-directed mutagenesis of this particular aspartate residue in cIPNS can affect its solubility. It is therefore important to take these potential changes into consideration when site-directed mutant proteins are analysed for catalytic function.

Acremonium↗
PubMed2001

Mutagenic analysis of the conserved residues in dehalogenase IVa of Burkholderia cepacia MBA4.

Amino and carboxyl terminal deletion derivatives of dehalogenase IVa (DehIVa) of Burkholderia cepacia MBA4 were constructed and analyzed for enzyme activity and for protein integrity. The results suggested that the majority of the protein is indispensable. Point mutations on 29 conserved charged and/or polar residues were generated and characterized. Derivatives D11E, D11N, D11S and D181N were totally inactive while mutant N178D was defective in catalysis. Mutations of other conserved residues displayed varying effects. Mutation that enhances DehIVa activity has been shown to be inhibitory in other dehalogenase and essential conserved residues in DehIVa have been shown to be dispensable in others. This suggests there is no general rule for the importance of these conserved residues.

Blotting, Western↗
PubMed2001

Systematic analysis of a conserved region of the aminoglycoside 6'-N-acetyltransferase type Ib.

Alanine-scanning mutagenesis was applied to the aminoglycoside 6'-N-acetyltransferase type Ib conserved motif B, and the effects of the substitutions were analyzed by measuring the MICs of kanamycin (KAN) and its semisynthetic derivative, amikacin (AMK). Several substitutions resulted in no major change in MICs. E167A and F171A resulted in derivatives that lost the ability to confer resistance to KAN and AMK. P155A, P157A, N159A, L160A, I163A, K168A, and G170A conferred intermediate levels of resistance. Y166A resulted in an enzyme derivative with a modified specificity; it conferred a high level of resistance to KAN but lost the ability to confer resistance to AMK. Although not as pronounced, the resistance profiles conferred by substitutions N159A and G170A were related to that conferred by Y166A. These phenotypes, taken together with previous results indicating that mutant F171L could not catalyze acetylation of AMK when the assays were carried out at 42 degrees C (D. Panaite and M. Tolmasky, Plasmid 39:123-133, 1998), suggest that some motif B amino acids play a direct or indirect role in acceptor substrate specificity. MICs of AMK and KAN for cells harboring the substitution C165A were high, suggesting that the active form of the enzyme may not be a dimer formed through a disulfide bond. Furthermore, this result indicated that the acetylation reaction occurs through a direct mechanism rather than a ping-pong mechanism that includes a transient transfer of the acetyl group to a cysteine residue. Deletion of fragments at the C terminus demonstrated that up to 10 amino acids could be deleted without a loss of activity.

Acetyltransferases↗
PubMed2005

Mutagenesis analysis of a conserved region involved in acetyl coenzyme A binding in the aminoglycoside 6'-N-acetyltransferase type Ib encoded by plasmid pJHCMW1.

Alanine scanning of motif A in the pJHCMW1-encoded aminoglycoside 6'-N-acetyltransferase type Ib identified amino acids important for the ability of the enzyme to confer wild-type levels of resistance to kanamycin and amikacin. The replacement of two amino acids, D117 or L120, with alanine residues resulted in complete loss of the resistance phenotype.

Acetyl Coenzyme A↗
PubMed2002

Mutagenic analysis of a conserved region of domain III in the Cry1Ac toxin of Bacillus thuringiensis.

We used site-directed mutagenesis to probe the function of four alternating arginines located at amino acid positions 525, 527, 529, and 531 in a highly conserved region of domain III in the Cry1Ac toxin of Bacillus thuringiensis. We created 10 mutants: eight single mutants, with each arginine replaced by either glycine (G) or aspartic acid (D), and two double mutants (R525G/R527G and R529G/R531G). In lawn assays of the 10 mutants with a cultured Choristoneura fumiferana insect cell line (Cf1), replacement of a single arginine by either glycine or aspartic acid at position 525 or 529 decreased toxicity 4- to 12-fold relative to native Cry1Ac toxin, whereas replacement at position 527 or 531 decreased toxicity only 3-fold. The reduction in toxicity seen with double mutants was 8-fold for R525G/R527G and 25-fold for R529G/R531G. Five of the mutants (R525G, R525D, R527G, R529D, and R525G/R527G) were tested in bioassays with Plutella xylostella larvae and ion channel formation in planar lipid bilayers. In the bioassays, R525D, R529D, and R525G/R527G showed reduced toxicity. In planar lipid bilayers, the conductance and the selectivity of the mutants were similar to those of native Cry1Ac. Toxins with alteration at position 527 or 529 tended to remain in their subconducting states rather than the maximally conducting state. Our results suggest that the primary role of this conserved region is to maintain both the structural integrity of the native toxin and the full functionality of the formed membrane pore.

Amino Acid Sequence↗
PubMed1991

Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases.

The eukaryotic translation initiation factor 4A (eIF-4A) possesses an in vitro helicase activity that allows the unwinding of double-stranded RNA. This activity is dependent on ATP hydrolysis and the presence of another translation initiation factor, eIF-4B. These two initiation factors are thought to unwind mRNA secondary structures in preparation for ribosome binding and initiation of translation. To further characterize the function of eIF-4A in cellular translation and its interaction with other elements of the translation machinery, we have isolated mutations in the TIF1 and TIF2 genes encoding eIF-4A in Saccharomyces cerevisiae. We show that three highly conserved domains of the D-E-A-D protein family, encoding eIF-4A and other RNA helicases, are essential for protein function. Only in rare cases could we make a conservative substitution without affecting cell growth. The mutants show a clear correlation between their growth and in vivo translation rates. One mutation that results in a temperature-sensitive phenotype reveals an immediate decrease in translation activity following a shift to the nonpermissive temperature. These in vivo results confirm previous in vitro data demonstrating an absolute dependence of translation on the TIF1 and TIF2 gene products.

Amino Acid Sequence↗
PubMed2005

Comparative genome analysis reveals a conserved family of actin-like proteins in apicomplexan parasites.

BACKGROUND: The phylum Apicomplexa is an early-branching eukaryotic lineage that contains a number of important human and animal pathogens. Their complex life cycles and unique cytoskeletal features distinguish them from other model eukaryotes. Apicomplexans rely on actin-based motility for cell invasion, yet the regulation of this system remains largely unknown. Consequently, we focused our efforts on identifying actin-related proteins in the recently completed genomes of Toxoplasma gondii, Plasmodium spp., Cryptosporidium spp., and Theileria spp. RESULTS: Comparative genomic and phylogenetic studies of apicomplexan genomes reveals that most contain only a single conventional actin and yet they each have 8-10 additional actin-related proteins. Among these are a highly conserved Arp1 protein (likely part of a conserved dynactin complex), and Arp4 and Arp6 homologues (subunits of the chromatin-remodeling machinery). In contrast, apicomplexans lack canonical Arp2 or Arp3 proteins, suggesting they lost the Arp2/3 actin polymerization complex on their evolutionary path towards intracellular parasitism. Seven of these actin-like proteins (ALPs) are novel to apicomplexans. They show no phylogenetic associations to the known Arp groups and likely serve functions specific to this important group of intracellular parasites. CONCLUSION: The large diversity of actin-like proteins in apicomplexans suggests that the actin protein family has diverged to fulfill various roles in the unique biology of intracellular parasites. Conserved Arps likely participate in vesicular transport and gene expression, while apicomplexan-specific ALPs may control unique biological traits such as actin-based gliding motility.

Actins↗
PubMed2006

Integrating economic costs into the analysis of flexible conservation management strategies.

Flexible conservation management, where measures (e.g., mowing of meadows, removing invasive species) are selected in each decision period depending on the current state of the ecological system, is generally perceived as superior to fixed management, where the same measure is applied in each decision period independent of the current state of the system. In past comparisons of fixed and flexible conservation strategies the additional costs that arise only in flexible strategies have usually been ignored. In this paper, we present a framework to integrate costs of flexible management into the evaluation of flexible conservation strategies. Using the example of an endangered butterfly species we demonstrate that the costs of flexible management may reverse the rank order of flexible and fixed conservation strategies, such that fixed strategies may lead to better ecological results than flexible ones for the same financial budget.

Animals↗
PubMed1977

An experimental analysis of electricity conservation procedures.

Daily electricity consumption of four families was recorded for 106 days. A reversal design, consisting of various experimental conditions interspersed between repeated baseline conditions, was used. During experimental conditions, daily prompts (written conservation slogans attached to front doors) and/or daily feedback (daily kilowatts consumed and daily cost information) were in effect. Maximum consumption occurred during the initial baseline; minimum consumption occurred during different experimental conditions for different families. The mean decrease from the maximum to the minimum for all families was 35%. Reversals in consumption were demonstrated in three families, although successive baselines tended to decrease. No clear differences in effectiveness between prompting and feedback conditions were apparent. The procedures used resulted in considerable dollar savings for the families.

Journal Article↗
PubMed1999

Molecular and functional analysis of a conserved CTL epitope in HIV-1 p24 recognized from a long-term nonprogressor: constraints on immune escape associated with targeting a sequence essential for viral replication.

It has been hypothesized that sequence variation within CTL epitopes leading to immune escape plays a role in the progression of HIV-1 infection. Only very limited data exist that address the influence of biologic characteristics of CTL epitopes on the emergence of immune escape variants and the efficiency of suppression HIV-1 by CTL. In this report, we studied the effects of HIV-1 CTL epitope sequence variation on HIV-1 replication. The highly conserved HLA-B14-restricted CTL epitope DRFYKTLRAE in HIV-1 p24 was examined, which had been defined as the immunodominant CTL epitope in a long-term nonprogressing individual. We generated a set of viral mutants on an HX10 background differing by a single conservative or nonconservative amino acid substitution at each of the P1 to P9 amino acid residues of the epitope. All of the nonconservative amino acid substitutions abolished viral infectivity and only 5 of 10 conservative changes yielded replication-competent virus. Recognition of these epitope sequence variants by CTL was tested using synthetic peptides. All mutations that abrogated CTL recognition strongly impaired viral replication, and all replication-competent viral variants were recognized by CTL, although some variants with a lower efficiency. Our data indicate that this CTL epitope is located within a viral sequence essential for viral replication. Targeting CTL epitopes within functionally important regions of the HIV-1 genome could limit the chance of immune evasion.

Animals↗
PubMed2004

Computational methods for the analysis of differential conservation in groups of similar DNA sequences.

Multiple sequence alignments are a powerful tool for identifying the regions of DNA which have been constrained in evolutionary divergence, presumably due to their functional role. However, such constraints rarely manifest themselves as perfect conservation of a site clearly standing out in its broader environment, as they reflect the species-specific differences in proteins, as well as the ability of some proteins to interact with multiple variants of their binding sequence. In this paper we explore the use of alignment column uncertainty as an aid in locating differential phylogenetic footprints, which refer to the sites in DNA where groups of related species exhibit sequence conservation, but where the pattern may vary between the groups. We use efficient, linear-time algorithms to locate such sites. We have performed a study of the mammalian CAV2-CAV1 gene region using our software, and we conclude with several observations concerning the differential conservation and the use of computational methods for its detection. The software developed for this project is available, free of charge, by contacting the author.

Animals↗
PubMed2003

[Screening of Bacillus thuringiensis strains containing vip3A genes and analysis of gene conservation].

Vip3A, a novel insecticidal protein, is secreted by Bacillus thuringiensis (Bt) during vegetative growth. Vip3A protein possesses insecticidal activity against a wild spectrum of lepidopteran insect larvae. Since the first cloning of vip3A gene from Bt, many other vip3A genes have been isolated. To investigate vip3A genes contribution to Bt and reflect the revolution relationships, the strains containing vip3A genes were screened and gene similarity was analyzed. 114 wild-type Bacillus thuringiensis (Bt) strains isolated from different regions and 41 standard Bt strains from the Institute of Pasteur were screened for the vip3A genes using PCR amplification. 39 strains including B. thuringiensis subsp. kurstaki (Btk) HD-1 were found to contain the vip3A genes. Because acrystallerous strain Cry- B derived from Btk HD-1 was proved not to contain vip3A gene, it suppose that the vip3A gene may be located at the plasmids. Vip3A proteins expressed in these strains were detected with polyclonal antibody by Western blot and 4 strains among them were shown not to express the Vip3A proteins. The vip3A genes amplified from wild-type Bacillus thuringiensis strains S101 and 611 with different levels of activity against lepidopteran insect larvae were cloned into pGEM-T Easy vector. Alignment of these 2 putative Vip3A proteins with 6 others (Vip3A (a), Vip3A(b), Vip3A-S, Vip3A-S184, Vip83 and Vip3V) in the GenBank data base and 2 reported Vip3A proteins (Vip14 and Vip15) showed that vip3A genes are highly conservative. The plasmids pOTP-S101 and pOTP-611 were constructed by in- serting 2 vip3A genes (vip3A-S101 and vip3A-611) into the expression vector pQE30 respectively and were transformed into E. coli M15. E. coli M15 cells harboring the pOTP plasmids were induced with 1 mmol/L IPTG to express 89 kDa protein. Experiments showed that the level of soluble proteins of Vip3A-S101 in E. coli M15[pOTP-S101] and Vip3A-611 in E. coli M15 [pOTP-611] were about 48% and 35% respectively. Bioassay showed that each of these Vip3A proteins had similar toxicity against neonate Spodoptera litura larvae, indicating that some amino acids change had little effect on the insecticidal activity of proteins. Although vip3A genes are conservative, the unknown insecticidal spectrum is still to be brought out. Vip3A genes can be used for the construction of the Bt engineered strains and transgenic plants. In addition, vip3A genes are excellent candidates for delay of the pest resistance due to the difference of the action model from that of Bt delta-endotoxins.

Animals↗
PubMed2005

[Comparison of the desulfurization activity among several bacteria and analysis of the conservation of their desulfurization genes].

Several bacteria, Bacillus brevis R-6, Pseudomonas delafleldii R-8, Nocardia globerula R-9, Bacillus sphaericus R-16, Rhodococcus erythropolis LSSE8-1 and Gordonia nitida LSSEJ-1, which can convert dibenzothiophene into 2-hydroxybiphenyl and sulfate, were investigated. Desulfurization products were quantitively determined by HPLC. Result revealed that each of these bacteria desulfurize DBT at a different rate. In order to obtain more information, the fragments encoding desulfurizing enzymes were studied. Desulfurization genes of R-6 and R-8 were separately amplified via PCR with specific primers based on the related sequences of Rhodococcus sp. IGTS8. Both sequences areminimally 99% related to IGTS8 sequence. Afterwards, dsz operon of LSSEJ-1 and R-9 were amplified and cloned. Sequences are also highly conservative. Data shows that identity of dszA between R-9 and IGTS8 is 99.6%, and identity of dszA between LSSEJ-1 and IGTS8 is 99.9%; dszB sequence of R-9 and LSSEJ-1 is 99.6% similarity to their counterpart sequence from IGTS8;Identity of dszC between R-9 and IGTS8 is 99.9%, and identity of dszC between LSSEJ-1 and IGTS8 is 99.1% . It may be deduced that the origins of desulfurization genes from mesophilic bacteria are the same.

Bacillus↗