Regulation of both gene expression and protein stability provides genetically assisted target evaluation (GATE) for microbial target validation.

The attempt to develop novel antibiotics, active against organisms resistant to current therapies, has led researchers to seek and explore new drug targets. The rapid sequencing and analysis of entire microbial genomes has identified large numbers of genes that may be sufficiently different from their human counterparts to be exploited as targets for antimicrobial treatment. As a first step, the importance of the various putative targets for microbial growth and survival must be assessed. Emerging validation technologies are becoming increasingly sophisticated and, in certain cases, allow prioritisation of the best targets. In this paper, genetically assisted target evaluation (GATE) is introduced as a versatile target validation technology. GATE concomitantly manipulates both synthesis and stability of the targeted protein using copper ions as an effector. This technology allows rapid quantitation of the lethal consequences of inactivation of targeted gene products in Saccharomyces cerevisiae. Additional tools can then be applied to extend these results into pathogenic organisms, such as Candida albicans.

Molecular analysis of CaMnt1p, a mannosyl transferase important for adhesion and virulence of Candida albicans.

There is an immediate need for identification of new antifungal targets in opportunistic pathogenic fungi like Candida albicans. In the past, efforts have focused on synthesis of chitin and glucan, which confer mechanical strength and rigidity upon the cell wall. This paper describes the molecular analysis of CaMNT1, a gene involved in synthesis of mannoproteins, the third major class of macromolecule found in the cell wall. CaMNT1 encodes an alpha-1, 2-mannosyl transferase, which adds the second mannose residue in a tri-mannose oligosaccharide structure which represents O-linked mannan in C. albicans. The deduced amino acid sequence suggests that CaMnt1p is a type II membrane protein residing in a medial Golgi compartment. The absence of CaMnt1p reduced the ability of C. albicans cells to adhere to each other, to human buccal epithelial cells, and to rat vaginal epithelial cells. Both heterozygous and homozygous Camnt1 null mutants of C. albicans showed strong attenuation of virulence in guinea pig and mouse models of systemic candidosis, which, in guinea pigs, could be attributed to a decreased ability to reach and/or adhere internal organs. Therefore, correct CaMnt1p-mediated O-linked mannosylation of proteins is critical for adhesion and virulence of C. albicans.

Molecular cloning and sequencing of a chitin synthase gene (CHS2) of Paracoccidioides brasiliensis.

The nucleotide sequence of a chitin synthase gene (CHS2) of the dimorphic fungal human pathogen Paracoccidioides brasiliensis has been determined. The deduced amino acid sequence of Chs2p consists of 1043 residues and is highly homologous to other class II fungal chitin synthases. Computational structural analyses suggest very high similarity to other fungal chitin synthases with a highly variable region at the cytosolic amino-terminal region which may be related to its possible zymogenic nature, and the putative catalytic region close to seven membrane-spanning regions at the carboxyl terminus.

Genetic evidence for two sequentially occupied K+ binding sites in the Kdp transport ATPase.

Substrate binding sites in Kdp, a P-type ATPase of Escherichia coli, were identified by the isolation and characterization of mutants with reduced affinity for K+, its cation substrate. Most of the mutants have an altered KdpA subunit, a hydrophobic subunit not found in other P-type ATPases. Topological analysis of KdpA and the locations of the residues changed in the mutants suggest that KdpA has 10 membrane-spanning segments and forms two separate and distinct sites where K+ is bound. One site is formed by three periplasmic loops of the protein and is inferred to be the site of initial binding. The other site is cytoplasmic. We believe K+ moves from the periplasmic site through the membrane to the cytoplasmic site where it becomes "occluded," i.e. inexchangeable with K+ outside the membrane. Membrane-spanning parts of KdpA probably form the path for transmembrane movement of K+. The kinetics of cation transport in the mutants indicate that each of the two binding sites contributes to the observed Km for cations as well as to the marked discrimination between K+ and Rb+ characteristic of wild-type Kdp. Energy coupling in Kdp, mediated by the KdpB subunit, is performed by a different subunit from the one that mediates transport.

The physiological function of periplasmic glucose oxidation in phosphate-limited chemostat cultures of Klebsiella pneumoniae NCTC 418.

Periplasmic oxidation of glucose into gluconate and 2-ketogluconate in Klebsiella pneumoniae occurs via glucose dehydrogenase (GDH) and gluconate dehydrogenase (GaDH), respectively. Since, as is shown here, in the presence of glucose, gluconate and 2-ketogluconate are not further metabolized intracellularly the physiological function of this periplasmic route was studied. It was found that periplasmic oxidation of glucose could function as an alternative production route of ATP equivalents. Instantaneous activation of either GDH or GaDH reduced the rate of degradation of glucose via glycolysis and the tricarboxylic acid (TCA) cycle in vivo. Furthermore, aerobic, magnesium- and phosphate-limited chemostat cultures with glucose as the carbon source showed high GDH plus GaDH activities in contrast to nitrogen- and sulphate-limited cultures. However, when fructose, which is not degraded by GDH, was the carbon source, specific oxygen consumption rates under these four conditions were essentially the same. The latter observation suggests that high transmembrane phosphate gradients which are supposedly present under phosphate-limited conditions do not cause high energetic demands due to futile cycling of phosphate ions. In addition, dissipation of the transmembrane phosphate gradient of phosphate-limited cells immediately increased the rate of intracellular glucose degradation. It is concluded that under phosphate-limited conditions (i) extensive futile cycling of phosphate ions is absent and (ii) low concentrations of phosphate ions limit intracellular degradation of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Interdependence of K+ and glutamate accumulation during osmotic adaptation of Escherichia coli.

Escherichia coli responds to an increase in medium osmolarity by accumulating K+ and glutamate. At low osmolarity a large fraction of cytoplasmic K+ serves to balance charge on macromolecular anions. That fraction of K+ is here referred to as "bound," as distinguished from "free" K+ that serves to balance charge of small anions. At higher osmolarity where cytoplasmic K+ increases markedly, the bound fraction decreases but the absolute amount of bound K+ expressed per unit of dry weight increases. The increase in bound K+ can be explained largely by the reduction of cytoplasmic putrescine at high osmolarity. At high osmolarity, glutamate is the major cytoplasmic anion, equal to at least 70% of free cytoplasmic K+. A sudden increase in the osmolarity of the medium stimulates glutamate synthesis with a lag of only about a minute; glutamate synthesis is almost totally dependent on K+ uptake. The high rate of flow of nitrogen through the glutamate pool under control conditions of growth at low osmolarity indicates that glutamate accumulation immediately after shift to high osmolarity must be due to inhibition of utilization of glutamate in the synthesis of other nitrogen-containing compounds rather than stimulation of glutamate synthesis. In agreement with this reasoning we find the kinetics of glutamate accumulation to be independent of the specific path of synthesis, whether by glutamate dehydrogenase or by glutamate synthase. Synthesis of glutamate appears to be required to attain normal values of the electrical membrane potential after shift to high osmolarity.

Futile cycling of ammonium ions via the high affinity potassium uptake system (Kdp) of Escherichia coli.

Escherichia coli Frag1 was grown under various nutrient limitations in chemostat culture at a fixed temperature, dilution rate and pH both in the presence and the absence of a high concentration of ammonium ions by using either ammonium chloride or DL-alanine as the sole nitrogen source. The presence of high concentrations of ammonium ions in the extracellular fluids of potassium-limited cultures of E. coli Frag1 caused an increase of the specific rate of oxygen consumption of these cultures. In contrast, under phosphate-, sulphate- or magnesium-limited growth conditions no such increase could be observed. The presence of high concentrations of ammonium ions in potassium-limited cultures of E. coli Frag5, a mutant strain of E. coli Frag1 which lacks the high affinity potassium uptake system (Kdp), did not increase the specific rate of oxygen consumption. These results indicate that ammonium ions, very similar to potassium ions both in charge and size, are transported via the Kdp leading to a futile cycle of ammonium ions and ammonia molecules (plus protons) across the cytoplasmic membrane. Both the uptake of ammonium ions and the extrusion of protons would increase the energy requirement of the cells and therefore increase their specific rate of oxygen consumption. The involvement of a (methyl)ammonium transport system in this futile cycle could be excluded.

The role of futile cycles in the energetics of bacterial growth.

In this contribution we describe the occurrence of futile cycles in growing bacteria. These cycles are thought to be active when organisms contain two uptake systems for a particular nutrient (one with a high, the other with a low affinity for its substrate). The high-affinity system is responsible for uptake of the nutrient, some of which is subsequently lost to the medium again via leakage through the low-affinity-system. A special futile cycle is caused under some growth conditions by the extremely rapid diffusion of ammonia through bacterial membranes. When the ammonium ion is taken up via active transport, the couple NH3/NH4+ will act as an uncoupler. This is aggravated by the chemical similarity of the potassium and the ammonium ion, which leads to ammonium ion transport via the Kdp potassium transport system when the potassium concentration in the medium is low. Other examples of futile cycles, such as those caused by the production of fatty acids by fermentation, are briefly discussed.

The role of magnesium and calcium ions in the glucose dehydrogenase activity of Klebsiella pneumoniae NCTC 418.

Magnesium-limited chemostat cultures of Klebsiella pneumoniae NCTC 418 with 20 microM CaCl2 in the medium showed a low rate of gluconate plus 2-ketogluconate production relative to potassium- or phosphate-limited cultures. However, when the medium concentration of CaCl2 was increased to 1 mM, the glucose dehydrogenase (GDH) activities also increased and became similar to those observed in potassium- or phosphate limited cultures. It is concluded that this is due to Mg2+ and Ca2+ ions being involved in the binding of pyrroloquinoline quinone (PQQ) to the GDH apoenzyme. There seems to be an absolute requirement of divalent cations for proper enzyme functioning and in this respect Ca2+ ions could replace Mg2+ ions. The high GDH activity which has been found in cells grown under Mg2(+)-limited conditions in the presence of higher concentrations of Ca2+ ions, is compatible with the earlier proposal that GDH functions as an auxiliary energy generating system involved in the maintenance of high transmembrane ion gradients.

Nitrogen-limited behaviour of micro-organisms growing in the presence of large concentrations of ammonium ions.

Cells of Klebsiella pneumoniae NCTC 418 grown at low culture pH values (4.5-5) in a glucose-limited chemostat culture contained elevated levels of glutamate synthase (EC 2.6.1.53). This can be taken as an indication that these cells show the physiology of nitrogen-limited cells, in spite of the fact that high concentrations (about 80 mM) of ammonium ions were present in the culture extracellular fluids. This phenomenon can be explained by the rapid diffusion of ammonia (NH3) through the cell membrane, leading to very low cytoplasmic ammonium (NH4+) and NH3 levels in cells that possess an almost neutral cytoplasmic pH value, but are growing at low culture pH values.

Replacement of potassium ions by ammonium ions in different micro-organisms grown in potassium-limited chemostat culture.

The biomass concentration extant in potassium-limited cultures of either Klebsiella pneumoniae or Bacillus stearothermophilus (when growing at a fixed temperature and dilution rate in a glucose/ammonium salts medium) increased progressively as the medium pH value was raised step-wise from 7.0 to 8.5. Because the macromolecular composition of the organisms did not vary significantly, this increase in biomass could not be attributed to an accumulation of storage-type polymers but appeared to reflect a pH-dependent decrease in the cells' minimum K+ requirement. Significantly, this effect of pH was not evident with cultures in which no ammonium salts were present and in which either glutamate or nitrate was added as the sole nitrogen source; however, it was again manifest when various concentrations of NH4Cl were added to the glutamate-containing medium. This suggested a functional replacement of K+ by NH4+, a proposition consistent with the close similarity of the ionic radii of the potassium ion (1.33 A) and the ammonium ion (1.43 A). At pH 8.0, and with a medium containing both glutamate (30 mM) and NH4Cl (100 mM), cultures of B. stearothermophilus would grow without added potassium at a maximum rate of 0.7 h-1. Under these conditions the cells contained maximally 0.1% (w/w) potassium (derived from contaminating amounts of this element in the medium constituents), a value which should be compared with one of 1.4% (w/w) for cells growing in a potassium-limited medium containing initially 0.5 mM K+.(ABSTRACT TRUNCATED AT 250 WORDS)