Resistance to the antimicrobial peptide polymyxin requires myristoylation of Escherichia coli and Salmonella typhimurium lipid A

Attachment of positively charged, amine-containing residues such as 4-amino-4-deoxy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN) to Escherichia coli and Salmonella typhimurium lipid A is required for resistance to the cationic antimicrobial peptide, polymyxin. In an attempt to discover additional lipid A modifications important for polymyxin resistance, we generated polymyxin-sensitive mutants of an E. coli pmrA(C) strain, WD101. A subset of polymyxin-sensitive mutants produced a lipid A that lacked both the 3'-acyloxyacyl-linked myristate (C(14)) and l-Ara4N, even though the necessary enzymatic machinery required to synthesize l-Ara4N-modified lipid A was present. Inactivation of lpxM in both E. coli and S. typhimurium resulted in the loss of l-Ara4N addition, as well as, increased sensitivity to polymyxin. However, decoration of the lipid A phosphate groups with pEtN residues was not effected in lpxM mutants. In summary, we demonstrate that attachment of l-Ara4N to the phosphate groups of lipid A and the subsequent resistance to polymyxin is dependent upon the presence of the secondary linked myristoyl group.     

The GCN2 eIF2alpha kinase is required for adaptation to amino acid deprivation in mice

The GCN2 eIF2alpha kinase is essential for activation of the general amino acid control pathway in yeast when one or more amino acids become limiting for growth. GCN2's function in mammals is unknown, but must differ, since mammals, unlike yeast, can synthesize only half of the standard 20 amino acids. To investigate the function of mammalian GCN2, we have generated a Gcn2(-/-) knockout strain of mice. Gcn2(-/-) mice are viable, fertile, and exhibit no phenotypic abnormalities under standard growth conditions. However, prenatal and neonatal mortalities are significantly increased in Gcn2(-/-) mice whose mothers were reared on leucine-, tryptophan-, or glycine-deficient diets during gestation. Leucine deprivation produced the most pronounced effect, with a 63% reduction in the expected number of viable neonatal mice. Cultured embryonic stem cells derived from Gcn2(-/-) mice failed to show the normal induction of eIF2alpha phosphorylation in cells deprived of leucine. To assess the biochemical effects of the loss of GCN2 in the whole animal, liver perfusion experiments were conducted. Histidine limitation in the presence of histidinol induced a twofold increase in the phosphorylation of eIF2alpha and a concomitant reduction in eIF2B activity in perfused livers from wild-type mice, but no changes in livers from Gcn2(-/-) mice.     

Enhanced phosphate uptake and polyphosphate accumulation in Burkholderia cepacia grown under low pH conditions

Of bacterial cells in a sample of activated sludge, 34% contained detectable intracellular polyphosphate inclusions following Neisser staining, when grown on glucose/mineral salts medium at pH 5.5; at pH 7.5 only 7% of cells visibly accumulated polyphosphate. In a sludge isolate of Burkholderia cepacia chosen for further study, maximal removal of phosphate and accumulation of polyphosphate occurred at pH 5.5; levels were up to 220% and 330% higher, respectively, than in cells grown at pH 7.5. During the early stationary phase of growth at pH 5.5 a maximum level of intracellular polyphosphate that comprised 13.6% of cellular dry weight was reached. Polyphosphate kinase activity was detected in actively growing cells only when cultured at pH 5.5. The phenomenon of acid-stimulated phosphate uptake and polyphosphate accumulation in this environmental bacterial population parallels observations previously made by us in the yeast Candida humicola and may thus represent a widespread microbial response to low external pH values.     

Comparisons of the 1995 and 1998 coral bleaching events on the patch reefs of San Salvador Island, Bahamas

Coral patch reefs around San Salvador Island, Bahamas have been monitored with the aid of Earthwatch volunteers three times a year since 1992. During that period two significant mass bleaching events occurred: autumn 1995, and late summer 1998. Elsewhere in 1995, bleaching was caused by higher-than-normal summer sea temperatures; in San Salvador, however, temperatures were normal. In 1998 a prolonged period of higher-than-normal sea temperatures preceded bleaching on San Salvador and worldwide. During the 1995 event, one of the monitored reefs had twice the percentage of coral colonies bleached as the other two. Bleaching was more evenly distributed among the reefs during the 1998 event. In 1995 Agaricia agaricites was significantly more affected than other coral species, with almost 50% of all its colonies showing bleaching. Bleaching was more evenly spread among coral species in 1998, with five species showing bleaching on more than 40% of their colonies. Bleaching began on Millepora as early as August during the 1998 event and progressed to other species through the remainder of the autumn. In 1995 bleaching was not seen until late autumn and appeared to impact all affected species at about the same time. Recovery from the 1995 event was complete: no coral death or damage above normal background levels were seen. In the 1998 event, all Acropora cervicornis on the monitored reefs died and A. palmata was severely damaged. Millepora sp. lost almost half of their live tissue, and Montastraea sp. showed significant tissue damage following this event. Phototransect analysis suggests that more than 20% of total live tissue on affected species died during the 1998 event. A. cervicornis has demonstrated no re-growth from 1998 to 2000 on monitored reefs. Monitoring has suggested significant differences in causes and courses in these two events.     

Crystal structures of fusion proteins with large-affinity tags

The fusion of a protein of interest to a large-affinity tag, such as the maltose-binding protein (MBP), thioredoxin (TRX), or glutathione-S-transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large-affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three- to five-amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest.     

The structure of the extracellular region of human hepsin reveals a serine protease domain and a novel scavenger receptor cysteine-rich (SRCR) domain

Hepsin is an integral membrane protein that may participate in cell growth and in maintaining proper cell morphology and is overexpressed in a number of primary tumors. We have determined the 1.75 A resolution structure of the extracellular component of human hepsin. This structure includes a 255-residue trypsin-like serine protease domain and a 109-residue region that forms a novel, poorly conserved, scavenger receptor cysteine-rich (SRCR) domain. The two domains are associated with each other through a single disulfide bond and an extensive network of noncovalent interactions. The structure suggests how the extracellular region of hepsin may be positioned with respect to the plasma membrane.     

Computer simulation of introductory neurophysiology

A computer-assisted learning (CAL) package, NeuroLab, developed for use by first-year university students undertaking professional programs in the health area, is described and evaluated. NeuroLab is a simulation of a laboratory, in which students are able to impale neurons to measure resting membrane potentials and subsequently undertake experiments including measuring resting membrane potentials, determining threshold potentials, measuring refractory periods, and examining effects on membrane potential through altering the membrane permeability to sodium and potassium ions. Students find the package to be a worthwhile learning experience, with 81 +/- 2.2% reporting the package increased their understanding of neuron function, and 78 +/- 2.5% expressing a desire for more CAL packages. Exposure to the package resulted in significantly higher mean scores in a multiple-choice question test on measuring neuron membrane potentials compared with those who were not exposed (mean scores out of 4 of 2.42 and 2.02, respectively, P < 0.001).