An estimate of the minimum amount of fluid lipid required for the growth of Escherichia coli.

The lipid phase transition of Escherichia coli was studied by high sensitivity differential scanning calorimetry. A temperature sensitive unsaturated fatty acid auxotroph was used to obtain lipids with subnormal unsaturated fatty acid contents. From these studies it was concluded that E. coli can grow nromally with as much as 20% of its membrane lipids in the ordered state but that if more than 55% of the lipids are ordered, growth ceases. Studies with wild-type cells show that the phase transition ends more than 10 degrees C below the growth temperature when the growth temperature is either 25 degrees C or 37 degrees C.     

Comparative use patterns of plant resources in rural areas of South Africa and Zimbabwe

Documentation of use patterns of plants across national boundaries is of relevance in understanding the importance of plant resources to livelihood strategies of different ethnic groups. Plant resources have gained prominence as a natural asset through which families derive food, firewood, income, medicines and timber, enabling particularly poor communities to achieve self-sufficiency. The objective of this study was to investigate the trends in plant usage in South Africa and Zimbabwe. An ethnobotanical investigation was conducted between January 2012 and January 2013 in the Limpopo Province, South Africa and the Midlands Province, Zimbabwe. The study used questionnaire surveys and interviews with a total of 143 participants to explore plant use patterns in South Africa and Zimbabwe. A total of 98 plant species were identified, with Zimbabwe contributing 70 species and 47 species from South Africa. The uses were classified into 15 categories, major use categories were firewood, food plants, medicine and timber. Food plant was a major plant use category in Zimbabwe, contributing 55.1%, followed by medicinal plants (36.8%), firewood (35.7%) and timber (31.6%). In contrast, firewood was the major plant use category in South Africa, contributing 18.4%, followed by food plants (17.3%), medicinal (14.3%) and timber (1.0%). Comparison of the two countries demonstrated remarkable differences in plant use patterns. The results showed that rural households in Zimbabwe were more reliant on plant resources than their counterparts in South Africa. Such a trend could be attributed to a close relationship between the local people, and their natural and agricultural environment leading to a rich knowledge base on plants, plant use and related practices. This comparative analysis strengthens the firm belief that utilization of plant resources represents an important shared heritage, preserved over the centuries, which must be exploited in order to provide further new and useful body of ethnobotanical knowledge.     

Reduced proportions of natural killer T cells are present in the relatives of lupus patients and are associated with autoimmunity

Introduction

Systemic lupus erythematosus is a genetically complex disease. Currently, the precise allelic polymorphisms associated with this condition remain largely unidentified. In part this reflects the fact that multiple genes, each having a relatively minor effect, act in concert to produce disease. Given this complexity, analysis of subclinical phenotypes may aid in the identification of susceptibility alleles. Here, we used flow cytometry to investigate whether some of the immune abnormalities that are seen in the peripheral blood lymphocyte population of lupus patients are seen in their first-degree relatives.

Methods

Peripheral blood mononuclear cells were isolated from the subjects, stained with fluorochrome-conjugated monoclonal antibodies to identify various cellular subsets, and analyzed by flow cytometry.

Results

We found reduced proportions of natural killer (NK)T cells among 367 first-degree relatives of lupus patients as compared with 102 control individuals. There were also slightly increased proportions of memory B and T cells, suggesting increased chronic low-grade activation of the immune system in first-degree relatives. However, only the deficiency of NKT cells was associated with a positive anti-nuclear antibody test and clinical autoimmune disease in family members. There was a significant association between mean parental, sibling, and proband values for the proportion of NKT cells, suggesting that this is a heritable trait.

Conclusions

The findings suggest that analysis of cellular phenotypes may enhance the ability to detect subclinical lupus and that genetically determined altered immunoregulation by NKT cells predisposes first-degree relatives of lupus patients to the development of autoimmunity.     

Function of Escherichia coli biotin carboxylase requires catalytic activity of both subunits of the homodimer

Biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase that catalyzes the first committed step in fatty acid synthesis. The Escherichia coli biotin carboxylase is readily isolated from the other components of the acetyl-CoA carboxylase complex such that enzymatic activity is retained. The three-dimensional structure of biotin carboxylase, determined by x-ray crystallography, demonstrated that the enzyme is a homodimer consisting of two active sites in which each subunit contains a complete active site. To understand how each subunit contributes to the overall function of biotin carboxylase, we made hybrid molecules in which one subunit had a wild-type active site, and the other subunit contained an active site mutation known to significantly affect the activity of the enzyme. One of the two genes encoded a poly-histidine tag at its N terminus, whereas the other gene had an N-terminal FLAG epitope tag. The two genes were assembled into a mini-operon that was induced to give high level expression of both enzymes. "Hybrid" dimers composed of one subunit with a wild-type active site and a second subunit having a mutant active site were obtained by sequential chromatographic steps on columns of immobilized nickel chelate and anti-FLAG affinity matrices. In vitro kinetic studies of biotin carboxylase dimers in which both subunits were wild type revealed that the presence of the N-terminal tags did not alter the activity of the enzyme. However, kinetic assays of hybrid dimer biotin carboxylase molecules in which one subunit had an active site mutation (R292A, N290A, K238Q, or E288K) and the other subunit had a wild-type active site resulted in 39-, 28-, 94-, and 285-fold decreases in the activity of these enzymes, respectively. The dominant negative effects of these mutant subunits were also detected in vivo by monitoring the rate of fatty acid biosynthesis by [(14)C]acetate labeling of cellular lipids. Expression of the mutant biotin carboxylase genes from an inducible arabinose promoter resulted in a significantly reduced rate of fatty acid synthesis relative to the same strain that expressed the wild type gene. Thus, both the in vitro and in vivo data indicate that both subunits of biotin carboxylase are required for activity and that the two subunits must be in communication during enzyme function.     

Fatty acid biosynthesis is involved in solvent tolerance in Pseudomonas putida DOT-T1E

The unusual tolerance of Pseudomonas putida DOT-T1E to toluene is based on the extrusion of this solvent by constitutive and inducible efflux pumps and rigidification of its membranes via phospholipid alterations. Pseudomonas putida DOT-T1E-109 is a solvent-sensitive mutant. Mutant cells were less efficient in solvent extrusion than the wild-type cells, as shown by the limited efflux of 14C-1,2,4-trichlorobenzene from the cell membranes, despite the fact that the efflux pumps are overexpressed as a result of increased expression of the ttgDEF and ttgGHI efflux pump operons. This limitation could be the result of alterations in the outer membrane because the mutant cells released more beta-lactamase to the external medium than the wild-type cells. The mutant P. putida DOT-T1E-109 showed negligible synthesis of fatty acids in the presence of sublethal concentrations of toluene as revealed by analysis of 13CH3-13COOH incorporation into fatty acids. In contrast, the mutant strain in the absence of solvents, and the wild-type strain, both in the presence and in the absence of toluene, incorporated 13CH3-13COOH at a high rate into de novo synthesized lipids. The mutation in P. putida DOT-T1E-109 increases sensitivity to the solvent because of a limited efflux of the solvent from the cell membranes with the concomitant inhibition of fatty acid biosynthesis.     

A European focus on proteomics

A report on the First International Symposium of the Austrian Proteomics Platform, Seefeld, Austria, 26-29 January 2004.     

Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD(+)-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD(+)-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of (32)P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD(+)-dependent DNA ligase from B. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.