Effect of Salmonella typhimurium ferric uptake regulator (fur) mutations on iron- and pH-regulated protein synthesis.

Fur is an important regulatory protein known to function in the presence of iron as a repressor of iron-controlled genes. It was recently discovered that Fur is also essential to Salmonella typhimurium for mounting an adaptive acid tolerance response (J. W. Foster, J. Bacteriol 173:6896-6902, 1991). Because little is known about the effect of Fur on the physiology of this enteric pathogen, a systematic two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis was conducted to identify proteins whose synthesis is linked to iron levels. Mutations in the fur locus were identified and used to classify which proteins are controlled by Fur. Thirty-six proteins were overtly affected by iron availability, most of which were clearly under the control of Fur. Although most of the Fur-dependent proteins were under negative control, a significant portion (15 of 34) appeared to be under a form of positive control. Nine of the positively controlled proteins required Fur and iron for expression. However, Fur lacking iron was also required for the induction of six gene products. Surprisingly, not all iron-regulated proteins were controlled by Fur and not all Fur-dependent proteins were obviously regulated by iron status. Because fur mutants fail to mount an effective acid tolerance response, we made a comparative two-dimensional PAGE analysis of 100 total acid- and iron-regulated gene products. Production of most of these proteins was regulated by only one of the two stresses, yet a clear subset of seven genes were influenced by both acid and iron and were also controlled by fur. These proteins were also members of the acid tolerance response modulon. Consistent with the fur effect on pH-regulated protein synthesis, fur mutants lacked the inducible pH homeostasis system associated with the acid tolerance response. The results provide further evidence that Fur has an extensive impact on gene expression and cellular physiology and suggest an explanation for the acid-sensitive nature of fur mutants.     

Acid-sensitive mutants of Salmonella typhimurium identified through a dinitrophenol lethal screening strategy.

Salmonella typhimurium exhibits a low-pH-inducible acid tolerance response (ATR) that can protect the adapted cell from severe acid challenge (pH 3.3). It is a two-stage system, with some proteins induced at pH 5.8 (pre-acid shock) and others induced below pH 4.5 (acid shock). The genetics of acid resistance was investigated through the use of a new screening medium. The medium contained 200 microM dinitrophenol (DNP) and was adjusted to pH 4.7 to 4.8. The medium will lower the internal pH of cells to a lethal level. However, cells capable of mounting an ATR will survive longer on this medium than acid-intolerant cells. Using this DNP lethal screening strategy, we isolated several acid-sensitive insertion mutants. Some mutants were defective in the pre-acid shock ATR stage but exhibited a normal or nearly normal post-acid shock-induced acid tolerance (atrB and atrC). Others could not induce acid tolerance by using either pre- or post-acid shock strategies (atrD, atrF, and atrG). The atrB locus was found to be part of a regulon under the control of a trans-acting regulator, atbR. An insertion in atbR caused constitutive acid tolerance because of overexpression of the regulon. Mutations in atrD and atrF affected iron metabolism and, in a manner analogous to ferric uptake regulator (fur) mutations, diminished acid resistance. The atrF mutation mapped within the ent cluster, probably in a fep uptake locus. The atrD locus mapped near metC and may represent an insertion into the S. typhimurium homolog of the Escherichia coli exbB or exbD locus. The mutation in atrC caused extreme UV light sensitivity and proved to occur within the polA (DNA polymerase I) locus. The results support the concept of overlapping acid protection systems in S. typhimurium.     

Helicobacter pylori exhibits a fur-dependent acid tolerance response

Background: Helicobacter pylori colonizes the acid environment of the gastric mucosa. Like other enteric bacterial pathogens, including Salmonella enterica, which must survive a brief exposure to that environment, H. pylori displays a rapid response to subtle changes in pH, which confers an increased ability to survive at more extreme acidic pH. This two‐step acid tolerance response (ATR) requires de novo protein synthesis and is dependent on the function of the global regulatory protein Fur. Objective: We have explored the physiological bases of the ATR in H. pylori. Materials and Methods: Proteomic analysis of phenotypes of H. pylori and fur mutant strains show that subtle pH changes elicit significant changes in the pattern of proteins synthesized. Results: A loss‐of‐function mutation in the fur gene, obtained by insertion of an antibiotic resistance cassette, indicated that Fur regulates the expression of a fraction of H. pylori proteins. Conclusion: A subset of proteins is involved in the ATR and confer a negative ATR phenotype.     

The acid tolerance response of Salmonella typhimurium involves transient synthesis of key acid shock proteins.

Although Salmonella typhimurium prefers neutral-pH environments, it can adapt to survive conditions of severe low-pH stress (pH 3.3). The process, termed the acid tolerance response (ATR), includes two distinct stages. The first stage, called pre-acid shock, is induced at pH 5.8 and involves the production of an inducible pH homeostasis system functional at external pH values below 4.0. The second stage occurs following an acid shock shift to pH 4.5 or below and is called the post-acid shock stage. During this stage of the ATR, 43 acid shock proteins (ASPs) are synthesized. The present data reveal that several ASPs important for pH 3.3 acid tolerance are only transiently produced. Their disappearance after 30 to 40 min of pH 4.4 acid shock coincides with an inability to survive subsequent pH 3.3 acid challenge. Clearly, an essential feature of inducible acid tolerance is an ability to synthesize these key ASPs. The pre-acid shock stage, with its inducible pH homeostasis system, offers the cell an enhanced ability to synthesize ASPs following rapid shifts to conditions below pH 4.0, an external pH that normally prevents ASP synthesis. The data also address possible signals for ASP synthesis. The inducing signal for 22 ASPs appears to be internal acidification, while external pH serves to induce 13 others. Of the 14 transient ASPs, 10 are induced in response to changes in internal pH. Mutations in the fur (ferric uptake regulator) locus that produce an Atr- acid-sensitive phenotype also eliminate induction of six transiently induced ASPs.     

Salmonella acid shock proteins are required for the adaptive acid tolerance response.

Salmonella typhimurium, as well as other enteric bacteria, experiences significant fluctuations in H+ ion concentrations during growth in diverse ecological niches. In fact, some pH conditions which should kill cells rapidly, such as stomach acidity, are nevertheless tolerated. The complete mechanism for this tolerance is unknown. However, I have recently demonstrated that S. typhimurium has the ability to survive extreme low pH (pH 3.0 to 4.0) if first adapted to mild pH (pH 5.5 to 6.0). This phenomenon has been referred to as the acidification tolerance response (ATR). The exposure to mild acid is referred to as preshock, and the proteins involved are called preshock ATR proteins. A second type of encounter with acid, called acid shock, involves shifting cells directly from alkaline conditions (pH 7.7) to acid conditions (pH 4.5 or below). During acid shock, the organism immediately ceases reproduction and dramatically changes the expression of at least 52 proteins. All but four are distinct from the preshock ATR proteins. Surprisingly, acid shock alone did not afford significant protection against strong acid challenge in minimal medium. Furthermore, inhibiting protein synthesis prior to acid shock revealed that the acid shock proteins do not appear to contribute to acid survival in minimal medium even at pH 4.3. Constitutive cellular pH homeostatic mechanisms seem sufficient to protect cells at this pH. The data suggest that the induction of acid shock and preshock ATR proteins are separate processes requiring separate signals. However, for S. typhimurium to survive extreme acid conditions, it must induce both the preshock and acid shock systems. Preventing the expression of one or the other eliminates acid tolerance. I propose a two-stage process that allows S. typhimurium to phase in acid tolerance as the environmental pH becomes progressively more acidic.     

Vibrio cholerae fur mutations associated with loss of repressor activity: implications for the structural-functional relationships of fur.

We used the Vibrio cholerae Fur protein as a model of iron-sensitive repressor proteins in gram-negative bacteria. Utilizing manganese mutagenesis, we isolated twelve independent mutations in V. cholerae fur that resulted in partial or complete loss of Fur repressor function. The mutant fur genes were recovered by PCR and sequenced; 11 of the 12 contained point mutations (two of which were identical), and one contained a 7-bp insertion that resulted in premature truncation of Fur. All of the mutants, except that containing the prematurely truncated Fur, produced protein by Western blot (immunoblot) analysis, although several had substantially smaller amounts of Fur and two made an immunoreactive protein that migrated more rapidly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Nine of the 11 point mutations altered amino acids that are identical in all of the fur genes sequenced so far, suggesting that these amino acids may play important structural or functional roles in Fur activity. Eight of the point mutations occurred in the amino-terminal half of Fur, which is thought to mediate DNA binding; most of these mutations occurred in conserved amino acids that have been previously suggested to play a role in the interaction between adjacent alpha-helices of the protein. Three of the point mutations occurred in the carboxy-terminal half of Fur, which is thought to bind iron. One mutation at histidine-90 was associated with complete loss of Fur function; this amino acid is within a motif previously suggested as being involved in iron binding by Fur. The fur allele mutant at histidine-90 interfered with iron regulation by wild-type fur in the same cell when the mutant allele was present at higher copy number; wild-type fur was dominant over all other fur mutant alleles studied. These results are analyzed with respect to previous models of the structure and function of Fur as an iron-sensitive repressor.     

The stationary-phase sigma factor σS (RpoS) is required for a sustained acid tolerance response in virulent Salmonella typhimurium

The acid tolerance response (ATR) of log-phase Salmonella typhimurium is induced by acid exposures below pH 4.5 and will protect cells against more extreme acid. Two systems are evident: a transiently induced system dependent on the iron regulator Fur that provides a moderate degree of acid tolerance and a more effective sustained ATR that requires the alternate sigma factor σ^S encoded by rpoS. Differences between the acid responses of virulent S. typhimurium and the attenuated laboratory strain LT2 were attributed to disparate levels of RpoS caused by different translational starts. The sustained ATR includes seven newly identified acid shock proteins (ASPs) that are dependent upon σ^S for their synthesis. It is predicted that one or more of these ASPs is essential for the sustained system. The sustained ATR also provided cross-protection to a variety of other environmental stresses (heat, H₂O₂ and osmolarity); however, adaptation to the other stresses did not provide significant acid tolerance. Therefore, in addition to starvation, acid shock serves as an important signal for inducing general stress resistance. Consistent with this model, σ^S proved to be induced by acid shock. Our results also revealed a connection between the transient and sustained ATR systems. Mutations in the regulator atbR are known to cause the overproduction of ten proteins, of which one or more can suppress the acid tolerance defect of an rpoS mutant. One member of the AtbR regulon, designated atrB, was found to be co-regulated by σ^S and AtbR. Both regulators had a negative effect on atrB expression. The results suggest AtrB serves as a link between the sustained and transient ATR systems. When σ^S concentration are low, a compensatory increase in AtrB is required to engage the transiently induced, RpoS-independent system of acid tolerance. Results also suggest different acid-sensitive targets occur in log-phase versus stationary-phase cells.     

A dominant-negative fur mutation in Bradyrhizobium japonicum

In many bacteria, the ferric uptake regulator (Fur) protein plays a central role in the regulation of iron uptake genes. Because iron figures prominently in the agriculturally important symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, we wanted to assess the role of Fur in the interaction. We identified a fur mutant by selecting for manganese resistance. Manganese interacts with the Fur protein and represses iron uptake genes. In the presence of high levels of manganese, bacteria with a wild-type copy of the fur gene repress iron uptake systems and starve for iron, whereas fur mutants fail to repress iron uptake systems and survive. The B. japonicum fur mutant, as expected, fails to repress iron-regulated outer membrane proteins in the presence of iron. Unexpectedly, a wild-type copy of the fur gene cannot complement the fur mutant. Expression of the fur mutant allele in wild-type cells leads to a fur phenotype. Unlike a B. japonicum fur-null mutant, the strain carrying the dominant-negative fur mutation is unable to form functional, nitrogen-fixing nodules on soybean, mung bean, or cowpea, suggesting a role for a Fur-regulated protein or proteins in the symbiosis.     

Fur regulates acid resistance in Shigella flexneri via RyhB and ydeP

Shigella flexneri requires iron for survival, and the genes for iron uptake and homeostasis are regulated by the Fur protein. Microarrays were used to identify genes regulated by Fur and to study the physiological effects of iron availability in S. flexneri. These assays showed that the expression of genes involved in iron acquisition and acid response was induced by low-iron availability and by inactivation of fur. A fur null mutant was acid sensitive in media at pH 2.5, and acid sensitivity was also observed in the wild-type strain grown under iron-limiting conditions. Acid resistance of the fur mutant in minimal medium was restored by addition of glutamate during acid challenge, indicating that the glutamate-dependent acid resistance system was not defective. Inactivation of ryhB, a small regulatory RNA whose expression is repressed by Fur, restored acid resistance in the fur mutant, while overexpressing ryhB increased acid sensitivity in the wild-type strain. RyhB-regulated genes were identified by microarray analysis. The expression of one of the RyhB-repressed genes, ydeP, which encodes a putative oxidoreductase, suppressed acid sensitivity in the fur mutant. Furthermore, an S. flexneri ydeP mutant was defective for both glutamate-independent and glutamate-dependent acid resistance. The repression of ydeP by RyhB may be indirect, as real time polymerase chain reaction (PCR) experiments indicated that RyhB negatively regulates evgA, which encodes an activator of ydeP. These results demonstrate that the acid sensitivity defect of the S. flexneri fur mutant is due to repression of ydeP by RyhB, most likely via repression of evgA.