- Volume 144, Issue 7, 1998

The nucleotide sequence of a 2·4 kb BamHI--Sall fragment of Streptomyces venezuelae ISP5230 DNA that complements trpE and trpG mutations in Escherichia coli contains two ORFs. The larger of these (ORF2) encodes a 624 amino acid sequence similar to the overall sequence of the two subunits of anthranilate synthase. The two-thirds nearest the amino terminus resembles the aminase subunit; the remaining one-third resembles the glutamine amidotransferase subunit. Upstream of ORF2 is a small ORF encoding 18 amino acids that include three adjacent Trp residues; in addition the ORF contains inverted repeats with sequence and positional similarity to the products of attenuator (trpL) regions that regulate tryptophan biosynthesis in other bacteria. In cultures of a trpC mutant of S. venezuelae, increasing the concentration of exogenous tryptophan decreased the formation of anthranilate synthase; similar evidence of endproduct repression was obtained in a trpCER mutant of E. coli transformed with a vector containing the cloned DNA fragment from S. venezuelae. The anthranilate synthase activity in S. venezuelae cell extracts was inhibited by tryptophan, although only at high concentrations of the amino acid. A two-base deletion introduced into the cloned S. venezuelae DNA fragment prevented complementation of a trpE mutation in E. coli. However, S. venezuelae transformants in which the two-base deletion had been introduced by replacement of homologous chromosomal DNA did not exhibit a Trp- phenotype. The result implies that S. venezuelae has one or more additional genes for anthranilate synthase. In alignments with anthranilate synthase genes from other organisms, ORF2 from S. venezuelae most closely resembled genes for phenazine biosynthesis in Pseudomonas. The results bear on the function of the gene in S. venezuelae.

Act in is a major cytoskeletal protein of mammalian muscle and non-muscle cells. Exposure of cells to soluble factors that damage cell membranes results in the release of actin into the extracellular spaces. The α-haemolysin (HlyA) of Escherichia coli is the prototype RTX (repeat in toxin) toxin and is thought to be important in virulence because of its ability to lyse cells by formation of pores in the cell membrane. These studies were conducted to determine if actin influences growth and haemolytic activity of E. coli. Growth of E. coli in the presence of actin resulted in culture supernatant haemolytic activity that was 2.4-, 2.7- and 3.3-fold greater than that of E. coli grown in medium containing BSA, non-supplemented medium, or medium containing heat-denatured actin, respectively. The enhanced haemolytic activity occurred only when actin was present during the growth phase and there was no effect when actin was added to culture supernatants containing haemolysin. The increased haemolytic activity by actin was concentration-dependent, detectable in early-exponential-phase growth, and associated with increased concentrations of secreted HlyA by Western blotting. Actin induced a 2.9-fold increase in alkaline phosphatase activity in E. coli CC118 with a TnphoA insertion in the hlyB determinant of the recombinant haemolysin piasmid pWAM04. These results indicate that extracellular actin enhances haemolysin production by E. coli and may have implications in the pathogenesis of E. coli infections.

The mechanism of activation of Escherichia coli redox sensory protein SoxR still unclear: a [2Fe--25] cluster contained in a SoxR dimer is potentially redo sensitive, but the nature of the signal is unknown. Antioxidant vitamins C (ascorbate) and E (α-tocopherol) were used to explore the mechanism of activation of the SoxR protein in vivo. Treating E. coli cells with ascorbate o α-tocopherol increased their tolerance to paraquat (PQ, a redox-cycling compound), even in the absence of the soxRS locus, suggesting a radical-quenching activity. When using a soxS’:: lacZ fusion, whose expression is governed by activated SoxR, ascorbate and α-tocopherol also prevented the expression of α-galactosidase after PQ treatment. A secondary activity was observed in cells carrying soxR101, a mutation resulting in the constitutive expression of the sox regulon, where the overexpression of soxS’::lacZ was also reduced by ascorbate or α-tocopherol treatment. Additionally, different mechanisms of action were revealed as α-tocopherol was capable of preventing both PQ and menadione (MD) lethality, whilst ascorbate prevent PQ lethality but increased MD-mediated cell death. It is proposed that α-tocopherol, positioned in membranes, can prevent superoxide-dependent membrane damage; however, water-soluble ascorbate is unable to do so an can even increase the concentration of oxygen radicals reacting with release membrane-associated Fe(ll).

Burkholderia cepacia produces an unusual range of polar lipids, which includes two forms each of phosphatidylethanolamine (PE) and ornithine amide lipid (OL), differing in the presence or absence of 2-hydroxy fatty acids. By using chemostat cultures in chemically defined media, variations in the lipid content and the proportions of individual lipids have been studied as a function of (a) growth temperature, (b) growth rate and (c) growth-limiting nutrient (carbon, magnesium, phosphorus or oxygen). Total cellular lipid in carbon-limited cultures was lowest at high growth temperatures and low growth rates. Increases in growth temperature over the range 25--40 ° led to increases in the proportions of molecular species of PE and OL containing 2-hydroxy acids, without changing the PE: OL ratio. Growth temperature did not alter the balance between neutral and acidic lipids, but the contribution of phosphatidylglycerol to the latter increased with rising growth temperature and growth rate. Pigmentation of cells and the presence of flagella were also temperature-dependent. Change in growth rate also affected the PE: OL ratio and the extent to which monoenoic acids were replaced by their cyclopropane derivatives. Whereas similar lipid profiles were found for carbon-, magnesium-and oxygen-limited cultures, ornithine amides were the only polar lipids detected in phosphorus-limited cells.

In iron-limited medium, Azotobacter vinelandii strain UW produces three catecholate siderophores: the tricatecholate protochelin, the dicatecholate azotochelin and the monocatecholate aminochelin. Each siderophore was found to bind Fe3+ preferentially to Fe2+, in a ligand:Fe ratio of 1:1, 3:2 and 3:1, respectively. Protochelin had the highest affinity for Fe3+, with a calculated proton-independent solubility coefficient of 10439, comparable to ferrioxamine B. Iron-limited wild-type strain UW grown under N2-fixing or nitrogen-sufficient conditions hyper-produced catecholate siderophores in response to oxidative stress caused by high aeration. In addition, superoxide dismutase activity was greatly diminished in iron-limited cells, whereas catalase activity was maintained. The ferredoxin I (Fdl)-negative A. vinelandii strain LM100 also hyper-produced catecholates, especially protochelin, under oxidative stress conditions, but had decreased activities of both superoxide dismutase and catalase, and was about 10 times more sensitive to paraquat than strain UW. Protochelin and azotochelin held Fe3+ firmly enough to prevent its reduction by.O- 2 and did not promote the generation of hydroxyl radical by the Fenton reaction. Ferric-aminochelin was unable to resist reduction by O- 2 and was a Fenton catalyst. These data suggest that under iron-limited conditions, A. vinelandii suffers oxidative stress caused by.O- 2. The catecholate siderophores azotochelin, and especially protochelin, are hyper-produced to offer chemical protection from oxidative damage catalysed by.O- 2 and Fe3+. The results are also consistent with Fdl being required for oxidative stress management in A. vinelandii.

Nutrient limitation of Staphylococcus aureus induces a starvation-survival state which enables it to survive until sufficient nutrients become available to support growth. The response of starved S. aureus cells to nutritional upshift was analysed to characterize the recovery mechanism which results in the resumption of rapid growth. S. aureus 8325-4 starved for 7 d in a chemically defined medium limited for glucose was able to resume growth upon the addition of complex medium (brain heart infusion broth) or a mixture of amino acids and glucose. The addition of either glucose or amino acids alone did not lead to recovery of cells. Prior to the first cell division event, a lag period of about 120--150 min was observed, the duration of which was independent of the length of starvation survival. During this lag period, RNA synthesis increased immediately upon the addition of nutrients whilst protein synthesis was delayed by approximately 5 min. Cells rapidly enlarged within 30 min of recovery, and initiation of chromosome replication could be detected after 90 min. Changes in the profile of proteins expressed during the recovery period revealed that several starvation-specific proteins were down-regulated within 30 min, whilst other proteins were common to both starvation and recovery. Two proteins were identified which were only transiently expressed during the first 60 min of recovery. Protein synthesis could be detected during recovery even if the cells had been treated with the RNA synthesis inhibitor rifampicin for 30 min prior to the addition of recovery nutrients, demonstrating that several proteins are translated from long-lived mRNA transcripts present in starved cells.

Ralstonia eutropha strain E2 (previously Alcaligenes sp.) is a phenol-degrading bacterium expressing phenol-oxygenating activity with a low Ks (the apparent half-saturation constant in Haldane's equation) and an extremely high KSI (the apparent inhibition constant). To identify the molecular basis for these novel cellular kinetic properties, a 9.5 kb DNA fragment that allowed Pseudomonas aeruginosa PAO1c (Phl- Cat+) to grow on phenol as the sole carbon source was cloned from strain E2 into plasmid pRO1614. PAO1c harbouring this plasmid (designated pROE217) transformed phenol to catechol, indicating that this fragment contains gene(s) for phenol hydroxylase. The cloned genes consist of eight complete ORFs, designated poxRABCDEFG. The products are homologous to those of dmpRKLMNOPQ of Pseudomonas sp. CF600, sharing 30--65% identity: this suggests that the phenol hydroxylase is a multicomponent enzyme. The kinetic constants for phenol-oxygenating activity of PA01c(pROE217) were determined, and these were compared with those of strain E2. The kinetic constants of PAO1c derivatives expressing different phenol hydroxylases were also determined. A comparison of these kinetic data suggests that phenol hydroxylase, the first enzyme in the phenol-degradative pathway, determines Ks and KSI values for the cellular phenol-oxygenating activity. It is thus suggested that the phenol hydroxylase cloned from strain E2 exhibits the novel kinetic properties that were observed with intact cells of strain E2.

The growth properties of Azorhizobium caulinodans wild-type and a cytochrome aa3 mutant strain, both growing with N2 as N source at fixed dissolved partial oxygen pressures in the range 0.5--4.0 kPa, were studied by making use of continuous cultures (chemostats and pH-auxostats) and transient cultures. In succinate-limited chemostats, the wild-type exhibited a higher growth yield than the aa3 mutant at every dissolved oxygen tension tested, indicating activity of cytochrome aa3 in this entire oxygen regime. The growth yield of both the wild-type and the aa3 mutant declined when the dissolved oxygen tension was raised. In contrast, for growth on ammonia at the same dilution rate, the wild-type showed an increase in growth yield with increasing dissolved oxygen tension, whereas the growth yield of the aa3 mutant remained constant. The transient changes in growth properties observed in chemostat cultures after pulsing with succinate pointed to a negative effect of oxygen on the maximum specific growth rate. This was studied further in steady-state pH-auxostat cultures. The specific growth rate of both strains decreased with increasing dissolved oxygen tension. The less steep decline in growth rate of the wild-type compared to the aa3 mutant confirmed that cytochrome aa3 is active in the wild-type. Again, the growth yield of both strains decreased with the dissolved oxygen tension, but in contrast to the results obtained with chemostats, no difference in growth yield was observed between wild-type and mutant at any oxygen tension. In either type of continuous culture a decrease in the overall P/O ratio with increasing dissolved oxygen tension is improbable for the wild-type, and even more so for the aa3 mutant. Therefore, the adverse effects of oxygen on the growth of A. caulinodans are not readily explained by respiratory protection; alternatively, it is proposed that the catalytic oxidation of nitrogen-fixation-specific redox enzymes by oxygen (auto-protection) enables the bacterium to deal with intracellular oxygen at the expense of reducing equivalents and free energy. To compensate for the loss of free energy, respiration increases and an active cytochrome aa3 contributes to this by keeping the P/O ratio high.