Overproduction of the exopolysaccharide alginate causes mucoid conversion in Pseudomonas aeruginosa and is a poor prognosticator in cystic fibrosis. The ECF factor AlgU and its cognate antifactor MucA are two principal regulators of alginate production. Here, we report the identification of three positive regulators of alginate biosynthesis: PA4033 (designated mucE), PA3649 (designated mucP), and algW. MucE, a small protein (9.5 kDa), was identified as part of a global mariner transposon screen for new regulators of alginate production. A transposon located in its promoter caused the overexpression of MucE and mucoid conversion in P. aeruginosa strains PAO1 and PA14. Accumulation of MucE in the envelope resulted in increased AlgU activity and reduced MucA levels. Three critical amino acid residues at the C terminus of MucE (WVF) were required for mucoid conversion via two predicted proteases AlgW (DegS) and MucP (RseP/YaeL). Moreover, as in Escherichia coli, the PDZ domain of AlgW was required for signal transduction. These results suggest that AlgU is regulated similarly to E. coli E except that the amino acid triad signals from MucE and other envelope proteins that activate AlgW are slightly different from those activating DegS.alginate ͉ anti-factor ͉ PDZ domain ͉ protease cascade ͉ signal specificity

We have isolated the NILI gene, whose product is an activator of the transcription of nitrogen-regulated genes, by virtue of the homology of its zinc-finger domain to that of the previously identified activator, the product of GLN3. Disruption of the chromosomal NILI gene enabled us to compare the effects of Gln3p and of Nillp on the expression of the nitrogen-regulated genes GLN1, GDH2, and GAP), coding respectively for glutamine synthetase, NAD-linked glutamate dehydrogenase, and general amino acid permease. Our results show that the nature of GATAAG sequences that serve as the upstream activation sequence elements for these genes determines their abilities to respond to Gln3p and Nillp. The results further indicate that Gln3p is inactivated by an increase in the intracellular concentration of glutamine and that Nillp is inactivated by an increase in intracellular glutamate.The presence of glutamine in the growth medium of Saccharomyces cerevisiae prevents the expression of GLN] and of GDH2, the structural genes for glutamine synthetase and the NADI-linked glutamate dehydrogenase (1). In a medium containing glutamate as a source of nitrogen, the expression of these genes requires the product of GLN3, Gln3p, a protein containing a zinc finger domain homologous to that of GATA factors of avian and mammalian cells, capable of binding to the sequences 5'-GATAAGATAAG-3' and 5'-GATTAGAT-TAG-3' located upstream of GLN1 and GDH2, respectively (1, 2). Deletion of these sequences greatly reduces the expression of these genes. The sequence 5'-GATAA-3' is also found upstream of other nitrogen-regulated genes and is apparently responsible for the activation of the expression of these genes by Gln3p. Another protein, the product of URE2, disables Gln3p in response to an increase in the intracellular concentration of glutamine. Mutants lacking Ure2p express Gln3p-activated genes in the presence of glutamine (1).Among the genes whose transcription can be activated by Gln3p is GAP], the structural gene for the general amino acid permease. This gene is not expressed in media containing glutamine, and the lack of Gln3p greatly diminished its expression in a medium containing glutamate as the source of nitrogen. Nevertheless, the lack of Gln3p did not prevent strong expression of GAP1 in media containing ammonia or urea as source of nitrogen. Apparently, a transcription factor other than Gln3p, which we have named Nillp, is responsible for the activation of transcription of GAP] in media containing these sources of nitrogen (3). Elimination of Ure2p enabled Gln3p, but not Nillp, to activate the transcription of GAP1 in the presence of glutamine (4).Examination of the DNA sequence revealed the presence of five GATAAG sequences within the 650-bp region preceding the translational start site of GAP1. This region was analyzed by making partial deletions from the 5' and 3' ends, fusing the remainder to CYCl-lacZ, and examining the 3-galactosidase levels in wild-type and gln3 mutant cells carrying plasmids containing these fusi...

The conversion to mucoid phenotype in Pseudomonas aeruginosa during chronic infections in cystic fibrosis (CF) is due to mutations in the algU mucABCD gene cluster. This cluster encodes an extreme stress response system conserved in Gram‐negative bacteria. The system includes an ECF sigma factor, AlgU (σE), an inner membrane protein, MucA, which inhibits AlgU activity, and MucB, a periplasmic protein that negatively controls AlgU. In this work, we investigated whether and how these factor interact to transduce signals between different cellular compartments. The mutation mucAΔG440, which renders a large fraction of P. aeruginosa CF isolates mucoid, did not abrogate AlgU–MucA interactions, although it eliminated MucA–MucB interactions in the yeast two‐hybrid system. The mucAΔG440 truncation of the periplasmic C‐terminal tail of MucA destabilized the molecule resulting in low or undetectable steady‐state levels in P. aeruginosa. Somewhat reduced levels of MucA were also seen in cells with inactivated mucB or with the mucACF53 allele carrying the missense P184S mutation, which mildly affected interactions with MucB. The events downstream from MucA destabilization were also investigated. AlgU was found to associate with inner membranes in mucA+ cells. In mutants destabilizing MucA, a limited redistribution of AlgU from the membrane to the cytosol was observed. The redistribution was spontaneous in mucAΔG440 cells, while in mucB and mucACF53 mutants it required additional signals. Despite a large reduction in MucA levels in mucAΔG440 cells, only a small fraction of AlgU was redistributed to the cytosol and a significant portion of this σ factor remained membrane bound and behaved as a peripheral inner membrane protein. The fraction of AlgU that depended on MucA for association with the membrane also brought RNA polymerase into this compartment. These results are consistent with a model in which MucB–MucA–AlgU–RNA polymerase interactions at the membrane allow transduction of potentially lethal stress signals with both rapid reaction times of the preassembled complexes and efficient resupply at the membrane from the prebound components.

We have identified the product of the NIL2 gene of Saccharomyces cerevisiae which contains a zinc finger region highly homologous to those of the GATA factors Gln3p and Nil1p as an antagonist of Nil1p and to a lesser extent of Gln3p. The expression of many nitrogen-regulated genes of Saccharomyces cerevisiae requires activation by GATA factor Gln3p or Nil1p and is prevented by the presence of glutamine in the growth medium. Disruption of NIL2 results in a great increase in the expression of NIL1 and of GAP1, the structural gene for the general amino acid permease, in glutamine-grown cells in response to activation by Nil1p. The primary effect of the elimination of Nil2p appears to be an increase in the intracellular level of Nil1p, which in turn is responsible for increased expression of GAP1. Experiments using an artificial UAS (upstream activating site) consisting of three GATAAGATAAG sites revealed that Nil2p exerts its effect by competing primarily with Nil1p and less effectively with Gln3p for these sites. Apparently, the principal role of Nil2p is to prevent activation of transcription by Nil1p unless Nil1p has been converted to a more active state by the absence of glutamine and glutamate.It is now well established that the expression of many genes of Saccharomyces cerevisiae whose products are responsible for the utilization of different nitrogen compounds as sources of nitrogen is activated by two zinc finger proteins that recognize the sequence GATAAG located upstream of these genes (4,5,16,21,23,25). One of these activators is the product of the GLN3 gene, and its ability to activate transcription is opposed by the product of the URE2 gene in response to an increase in the intracellular level of glutamine (2,6,7,13,19,21). The other activator, the product of NIL1 (also called GAT1) has a zinc finger highly homologous to that of Gln3p and is capable of activating some of the same promoters as Gln3p, but its activity appears to be antagonized by an as yet unknown protein in response to the rise in the intracellular concentration of glutamate (5, 25). As a result, transcription of a susceptible gene such as GAP1, coding for the general amino acid permease, is activated almost exclusively by Gln3p during growth with glutamate as the source of nitrogen and almost exclusively by Nil1p during growth with ammonia or urea as the source of nitrogen and is not activated at all during growth in a medium containing glutamine (25).In addition to possessing homologous zinc fingers, Gln3p and Nil1p also resemble one another by possessing highly acidic amino-terminal domains, characteristic of many activators (25). Two other proteins also possess zinc fingers with high homology to those of Gln3p and Nil1p, but they lack the acidic amino-terminal portions (8,25). One of these proteins, the product of DAL80, has been identified as an antagonist of Gln3p in the case of some, but not all, Gln3p-dependent genes (10, 12). Apparently, Dal80p requires two GATAAG sequences located not more than 20 bp apart to be effective (9)....

Mucoid strains of Pseudomonas aeruginosa that overproduce the exopolysaccharide alginate are a frequent cause of chronic respiratory infections in cystic fibrosis (CF) patients. The overproduction of alginate by these strains is often caused by mutations within mucA of the algU mucABCD gene cluster. This gene cluster encodes an extreme stress response system composed of the ECF alternative sigma factor AlgU, the anti-sigma factor MucA located in the inner membrane and the negative regulator MucB located in the periplasm. Most of the mutations in mucA found in mucoid strains cause a truncation of the C-terminal, periplasmic domain of MucA. The most significant effect of these mutations appears to be to reduce the levels of MucA. PA3257 (prc) was identified as a regulator of alginate production in P. aeruginosa through the isolation and study of mutations that partially suppressed the mucoid phenotype of a mucA22 strain. The suppressor of mucoidy (som) mutants isolated produced very little alginate when grown on LB medium, but were still mucoid when grown on Pseudomonas isolation agar. These som mutations and another previously isolated suppressor mutation were complemented by cosmids or plasmids carrying PA3257. PA3257 is predicted to encode a periplasmic protease similar to Prc or Tsp of Escherichia coli. Sequencing of prc from three strains with som suppressor mutations confirmed that each had a mutation within the prc coding region. The authors propose that Prc acts to degrade mutant forms of MucA. Additional evidence in support of this hypothesis is: (1) transcription from the AlgU-regulated algD reporter was reduced in som mutants; (2) inactivation of prc affected alginate production in mucoid strains with other mucA mutations found in CF isolates; (3) inactivation or overexpression of prc did not affect alginate production in strains with wild-type MucA.

Alginate production in Pseudomonas aeruginosa and the associated mucoid phenotype of isolates from cystic fibrosis patients are under the control of the a/gU mudBCD cluster. This group of genes encodes AlgU, the P. aeruginosa equivalent of the extreme heat shock a factor aE in Gram-negative bacteria, the AlgU-cognate anti-a factor MucA, the periplasmic protein MucB and a serine protease homologue, MucD. While m u d , mucB or mucD act as negative regulators of AIgU, the function of mucC is not known. In this study the role of mucC in P. aeruginosa physiology and alginate production has been addressed. Insertional inactivation of mucC in the wild-type P. aeruginosa strain PA01 did not cause any overt effects on alginate synthesis. However, it affected growth of P. aeruginosa under conditions of combined elevated temperature and increased ionic strength or osmolarity. Inactivation of mucC in m u d or mucB mutant backgrounds resulted in a mucoid phenotype when the cells were grown under combined stress conditions of elevated temperature and osmolarity. Each of the stress factors tested separately did not cause comparable effects. The combined stress factors were not sufficient to cause phenotypically appreciable enhancement of alginate production in m u d or mucB mutants unless mucC was also inactivated. These findings support a negative regulatory role of mucC in alginate production by P. aeruginosa, indicate additive effects of muc genes in the regulation of mucoidy in this organism and suggest that multiple stress signals and recognition systems participate in the regulation of algU-dependent functions.USA

In the yeast Saccharomyces cerevisiae, glycogen serves as a major storage carbohydrate. In a previous study, mutants with altered glycogen metabolism were isolated on the basis of the altered iodine-staining properties of colonies. We found that when glycogen produced by strains carrying the glc3-lp (previously called ghal-1) mutation is stained with iodine, the absorption spectrum resembles that of starch rather than that of glycogen, suggesting that this mutation might reduce the level of branching in the glycogen particles. Indeed, glycogen branching activity was undetectable in extracts from a glc3-lp strain but was elevated in strains which expressed GLC3 from a high-copy-number plasmid. These observations suggest that GLC3 encodes the glycogen branching enzyme. In contrast to glc3-lp, the glc34 mutation greatly reduces the ability of yeast to accumulate glycogen. These mutations appear to be allelic despite the striking difference in the phenotypes which they produce. The GLC3 clone complemented both glc3-lp and gkc34. Deletions and transposon insertions in this clone had parallel effects on its ability to complement gk3-lp and glc3-4. Finally, a fragment of the cloned gene was able to direct the repair of both gk3-lp and gkc34. Disruption of GLC3 yielded the glycogen-deficient phenotype, indicating that glycogen deficiency is the null phenotype. The glc3-lp allele appears to encode a partially functional product, since it is dominant over glc34 but recessive to GLC3. These observations suggest that the ability to introduce branches into glycogen greatly increases the ability of the cell to accumulate that polysaccharide. Northern (RNA) blot analysis identified a single mRNA of 2,300 nucleotides that increased in abundance ca. 20-fold as the culture approached stationary phase. It thus appears that the expression of GLC3 is regulated, probably at the level of transcription.

SUMMARY1. The influence of urinary hyaluronidase (believed to be predominantly of renal origin) on the urinary concentrating process has been studied in rats subjected to antidiuretic stimulus.2. Antiserum against a partially purified preparation of this enzyme has been raised in rabbits. Urinary volume, solute excretion and medullary composition have been investigated in rats treated with this antiserum (0-2 ml./100 g body weight, i.v.) before water deprivation for 48 hr or infusion for up to 4 hr with arginine-vasopressin. Control rats were pre-treated with normal rabbit serum.3. Pre-treatment with antiserum against rat urinary hyaluronidase (AUase) caused water-deprived rats to excrete urine at a rate significantly greater, and of osmolality significantly lower, than that recorded in control rats.4. The increase in medullary solute gradient which typically accompanies antidiuresis was significantly reduced in water-deprived rats pre-treated with AUase.5. In rats treated with AUase and infused for 4 hr with arginine-vasopressin, there was no significant increase in the medullary solute gradient, whereas this increased markedly in control rats. 6. During the first 24 hr of water deprivation there was an increase in the rate of Ca excretion by control rats which was abolished by pre-treatment with AUase.7. The effects of antiserum against a partially purified preparation of rat testicular hyaluronidase (ATase) were studied in water-deprived rats. No evidence was obtained that this enzyme has any influence on renal function. 8. It is concluded that urinary hyaluronidase, but not testicular hyaluronidase, plays an important role in facilitating the urinary concentrating process following antidiuretic stimulus.