Low-pH-induced effects on patterns of protein synthesis and on internal pH in Escherichia coli and Salmonella typhimurium.

Escherichia coli and Salmonella typhimurium were grown in a supplemented minimal medium (SMM) at a pH of 7.0 or 5.0 or were shifted from pH 7.0 to 5.0. Two-dimensional gel electrophoretic analysis of proteins labeled with H2(35)SO4 for 20 min during the shift showed that in E. coli, 13 polypeptides were elevated 1.5- to 4-fold, whereas in S. typhimurium, 19 polypeptides were increased 2- to 14-fold over the pH 7.0 control. Upon long-term growth at pH 5.0, almost double the number of polypeptides were elevated twofold or more in S. typhimurium compared with E. coli. In E. coli, there was no apparent induction of heat shock proteins upon growth at pH 5.0 in SMM. However, growth of E. coli in a complex broth to pH 5.0, or subsequent growth of fresh E. coli cells in the filtrate from this culture, showed that a subset of five polypeptides is uniquely induced by low pH. Two of these polypeptides, D60.5, the inducible lysyl-tRNA synthetase, and C62.5, are known heat shock proteins. Measurements of the internal pH (pHi) and growth rates of both organisms were made during growth in SMM at pH 7.0, pH 5.0, and upon the pH shift. The data show that the pHi of E. coli decreases more severely than that of S. typhimurium at an external pH of 5.0; the growth rate of E. coli is about one-half that of S. typhimurium at this pH, whereas the two organisms have the same growth rate at pH 7.0. The two-dimensional gel, growth, and pHi experiments collectively suggest that, at least in SMM, S. typhimurium is more adaptive to low-pH stress than is E. coli.     

Low-pH-induced effects on patterns of protein synthesis and on internal pH in Escherichia coli and Salmonella typhimurium

Escherichia coli and Salmonella typhimurium were grown in a supplemented minimal medium (SMM) at a pH of 7.0 or 5.0 or were shifted from pH 7.0 to 5.0. Two-dimensional gel electrophoretic analysis of proteins labeled with H2(35)SO4 for 20 min during the shift showed that in E. coli, 13 polypeptides were elevated 1.5- to 4-fold, whereas in S. typhimurium, 19 polypeptides were increased 2- to 14-fold over the pH 7.0 control. Upon long-term growth at pH 5.0, almost double the number of polypeptides were elevated twofold or more in S. typhimurium compared with E. coli. In E. coli, there was no apparent induction of heat shock proteins upon growth at pH 5.0 in SMM. However, growth of E. coli in a complex broth to pH 5.0, or subsequent growth of fresh E. coli cells in the filtrate from this culture, showed that a subset of five polypeptides is uniquely induced by low pH. Two of these polypeptides, D60.5, the inducible lysyl-tRNA synthetase, and C62.5, are known heat shock proteins. Measurements of the internal pH (pHi) and growth rates of both organisms were made during growth in SMM at pH 7.0, pH 5.0, and upon the pH shift. The data show that the pHi of E. coli decreases more severely than that of S. typhimurium at an external pH of 5.0; the growth rate of E. coli is about one-half that of S. typhimurium at this pH, whereas the two organisms have the same growth rate at pH 7.0. The two-dimensional gel, growth, and pHi experiments collectively suggest that, at least in SMM, S. typhimurium is more adaptive to low-pH stress than is E. coli.     

Abnormal induction of heat shock proteins in an Escherichia coli mutant deficient in adenosylmethionine synthetase activity.

Most prototrophic strains of Escherichia coli become restricted for methionine at 44 degrees C. A mutant strain (RG62 metK) in which the level of S-adenosylmethionine synthetase activity is only 10 to 20% of normal shows constitutive expression of one of the heat shock proteins, the lysU gene product, lysyl-tRNA synthetase form II, at 37 degrees C. These findings suggested a possible linkage between methionine metabolism and heat shock. We examined the induction of heat shock polypeptides in strain RG62 (metK) and in its parent, RG (metK+), from which it was derived by spontaneous mutation. Exponential-phase cultures of the two strains were pulse-labeled with [3H]leucine shortly after a shift from 37 to 44 degrees C, and the total cellular polypeptides were examined by two-dimensional electrophoresis. The results confirmed the constitutive production of the lysU gene product previously reported for strain RG62, but also revealed that the induction of 2 of the 17 heat shock polypeptides, C14.7 and G13.5, was markedly depressed. Otherwise the heat shock induction pattern was similar in timing and magnitude in the two strains. Transformation of the mutant strain with a plasmid, pK8, containing the metK coding sequence and promoter region as a 1.8-kilobase insert into pBR322 restored normal induction of C14.7 and G13.5, but did not prevent constitutive expression of the lysU gene product in the medium required for growth of this strain. The three heat shock polypeptides abnormally controlled in strain RG62 are the three polypeptides which are not induced when rapid synthesis of the htpR gene product is induced by isopropyl-beta-D-thiogalactopyranoside at 28 degree C (R. A. VanBogelen, M. A. Acton, and F. C. Neidhardt, Genes Dev. 1:525-531, 1987). We postulate that induction of these three polypeptides involves metabolic signals in addition to the synthesis of the htpR gene product and that strain RG62 (metK) fails to produce the signals involved in induction of C14.7 and G13.5 on a shift-up in temperature and produces the signal related to lysU induction even at 37 degree C.     

Induction of heat shock proteins during initiation of solvent formation inClostridium acetobutylicum

Summary The response to stresses produced by changes in the fermentation conditions ofClostridium acetobutylicum in continuous culture was determined under acid- and solvent-producing conditions. Using a phosphate-limited chemostat it was found that specificheatshockproteins (hsp 73, hsp 72 [Dnak], hsp 67 [GroEL], hsp 17 and hsp 14) were synthesized at elevated levels during the shift from acid to solvent formation. The induction of these stress proteins was observed before acetone and butanol were detected in the medium and was therefore not a response to these solvents present in the medium. Simultaneously with the induction of hsps, changes in the synthesis rates of other cellular proteins were observed. Synthesis of proteins associated with the acid production phase decreased and of proteins correlated with the solvent production phase increased. Some hsps, including the DnaK- and GroEL-similar proteins, hsp 73 and hsp 21, were also induced by a change in the growth rate and/or the pH. The analysis of the general regulation of the heat shock response inC. acetobutylicum revealed that the induction of at least 15 hsps after a temperature up-shift was transient and that two temporal classes of hsps could be distinguished. The synthesis of one group of hsps reached a maximum after 6 min and another around 11 min after the temperature upshift and returned to steady-state levels 30 to 40 min after the shock.     

Identification and characterisation of a regulatory region in the Toxoplasma gondii hsp70 genomic locus

Toxoplasma gondii is an important human and veterinary pathogen. The induction of bradyzoite development in vitro has been linked to temperature, pH, mitochondrial inhibitors, sodium arsenite and many of the other stressors associated with heat shock protein induction. Heat shock or stress induced activation of a set of heat shock protein genes, is characteristic of almost all eukaryotic and prokaryotic cells. Studies in other organisms indicate that heat shock proteins are developmentally regulated. We have established that increases in the expression of bag1/hsp30 and hsp70 are associated with bradyzoite development. The T. gondii hsp70 gene locus was cloned and sequenced. The regulatory regions of this gene were analysed by deletion analysis using beta-galactosidase expression vectors transiently transfected into RH strain T. gondii. Expression was measured at pH 7.1 and 8.1 (i.e. pH shock) and compared to the expression obtained with similar constructs using BAG1 and SAG1 promoters. A pH-regulated region of the Tg-hsp70 gene locus was identified which has some similarities to heat shock elements described in other eukaryotic systems. Green fluorescent protein expression vectors driven by the Tg-hsp70 regulatory region were constructed and stably transfected into T. gondii. Expression of green fluorescent protein in these parasites was induced by pH shock in those lines carrying the Tg-hsp70 regulatory constructs. Gel shift analysis was carried out using oligomers corresponding to the pH-regulated region and a putative DNA binding protein was identified. These data support the identification of a pH responsive cis-regulatory element in the T. gondii hsp70 gene locus. A model of the interaction of hsp70 and small heat shock proteins (e.g. BAG1) in development is presented.     

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.     

Induction of stress proteins inLeuconostoc oenos to perform direct inoculation of wine

Summary The enhancement or induction of the protein synthesis was clearly observed in cells ofL. oenos labeled with³⁵S for five proteins during heat shock at 42°C and acid shock at pH 3. Furthermore, no stress protein was induced after exposure ofL. oenos to ethanol shock 10% (v/v). Moreover, survival ofL. oenos in wine and ability to perform alolactic fermentation was improved after direct inoculation when cells were pretreated at 42°C.     

A survey of the heat shock response in four Streptomyces species reveals two groEL-like genes and three groEL-like proteins in Streptomyces albus.

A survey of the heat shock response was carried out in a series of streptomycetes. Four major heat shock proteins (HSPs) were observed in each of four species (Streptomyces albus, S. lividans, S. parvulus, S. viridochromogenes) after pulse labeling with [35S]methionine and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three corresponded to the major procaryotic HSPs Lon, DnaK, and GroEL on the basis of their apparent molecular masses (94 to 100, 70, and 56 to 58 kDa, respectively). In addition, a smaller protein (16 to 18 kDa) was detected in all species but was most dramatically induced in S. albus. Consequently, studies focused on this species. As in other procaryotic systems, thermal induction (elicited by a shift from 30 degrees C to 41 degrees C) of the 70- and 94-kDa proteins was transient and expression returned to uninduced levels after 60 min. In contrast, the 56- to 58-kDa (GroEL) and 18-kDa proteins (HSP18) remained induced for more than 2 h. Two-dimensional gel electrophoresis allowed resolution of at least eight S. albus HSPs. HSP56-58 was composed of multiple acidic protein species, whereas HSP18 appeared to be basic. In spite of these differences in their physical characteristics, the N-terminal peptide sequence of HSP18 was similar to those of GroEL-like proteins found in other organisms and identical to one of the HSP56-58 species. In fact, N-terminal amino acid analysis of the S. albus 56- to 58-kDa species showed that it was composed of two proteins that differed in 3 of 10 positions, an observation that was supported by the detection of two groEL-like genes by Southern hybridization. The amino acid sequence of one of these proteins was identical to that of HSP18. Pulse-chase experiments did not reveal evidence of posttranslational processing of either HSP56-58 or HSP18.     

Induction of protein synthesis in response to ultraviolet light,nalidixic acid and heat shock in the cyanobacterium Phormidium laminosum

Synthesis of three polypeptides (43, 34 and 26 kDa) was enhanced or induced in the thermophilic cyanobacterium Phormidium laminosum following irradiation with UV light. Synthesis of several polypeptides was also enhanced/induced following treatment with nalidixic acid. Three had similar molecular masses to those induced by UV. One of these (43 kDa) was also apparently enhanced following prolonged heat shock, as were a number of other polypeptides with a wide range of molecular masses. The specific induction of the 34 and 26 kDa polypeptides following DNA damage and inhibition of replication suggests that they may be involved in DNA repair and, possibly, also in recombination.     

Changes in protein synthesis and acid tolerance in Clostridium perfringens type A in response to acid shock.

The induction of acid-shock proteins and the degree of acid resistance conferred on Clostridium perfringens by acid shock, and the kinetics of this resistance were determined. A sublethal acid shock at pH 4.5 for 20 min increased the acid tolerance of cells at least fifteenfold. The acquired tolerance was maintained for 3 h after acid treatment. The response of the microorganism to acid shock was also examined by analysis of pulse-labeled proteins. Five acid-shock proteins (molecular weights 120, 84, 58, 45 and 17 kDa) were identified by polyacrylamide gel electrophoresis.     

Tissue-specific variations in the induction of Hsp70 and Hsp64 by heat shock in insects

The patterns of heat-induced synthesis (37 degrees C to 45 degrees C) of heat shock proteins (Hsps) in different tissues of grasshoppers and cockroaches from natural populations and in laboratory-reared gram-pest (Heliothis armigera) were examined by 35S-methionine labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography. Whereas 45 degrees C was lethal in most cases, optimal induction of Hsp synthesis was seen between 37 degrees C and 42 degrees C. The ongoing protein synthesis was not much affected at these temperatures, except in the tissues of adult H. armigera exposed to 42 degrees C. The profiles of the Hsps induced in the tissues of the insects, however, were different. From the relative abundance of the synthesis of 70-kDa (Hsp70) and 64-kDa (Hsp64) polypeptides, three categories of heat shock response were identified: (1) induction of abundant Hsp70 but little Hsp64 (malpighian tubules, male accessory glands, and ovaries of adult grasshoppers), (2) abundant Hsp64 but little Hsp70 (testes of adult grasshoppers, testes and malpighian tubules of adult cockroaches, and testes, malpighian tubules, and fat bodies of H. armigera larvae), and (3) induction of both Hsp70 and Hsp64 in more or less equal abundance (ovaries of adult cockroaches, salivary glands of H. armigera larvae, and malpighian tubules, male accessory glands, testes, and ovaries of adult H. armigera). Cockroaches collected from storerooms showed detectable synthesis of Hsp64 and/or Hsp70 only after heat shock, but those collected from drains showed detectable synthesis of both Hsp70 and Hsp64 in different tissues without heat stress. Western blotting showed that the 64-kDa polypeptide in these insects is a member of the Hsp60 family. Grasshopper testes, which synthesized negligible Hsp70 but abundant Hsp64 after heat shock, developed thermotolerance. Thus, heat shock response is modulated by developmental and environmental factors in different tissues of insects.     

Induction of expression of the alpha v beta 1 and alpha v beta 3 integrin heterodimers during retinoic acid-induced neuronal differentiation of murine embryonal carcinoma cells

All-trans-retinoic acid, an endogenous morphogen, induced neuronal differentiation of P19 murine embryonal carcinoma cells. Peak differentiation, as judged by the elaboration of neuronal processes, occurred 8 days after exposure of the cells to 0.5 mM retinoic acid, a concentration known to induce neuronal differentiation. An examination of the expression of the extracellular matrix receptors, integrins, during this retinoic acid-induced differentiation period, demonstrated a specific and strong induction of expression of two polypeptides (130 and 115 kDa) immunoprecipitated with an anti-human vitronectin receptor antiserum. The expression of a 90-kDa polypeptide, also immunoprecipitating with this antiserum was induced as well, but to a much smaller extent. The expression of a 96-kDa polypeptide immunoprecipitated by this antiserum and present in the untreated cells was not induced by retinoic acid. The increase in the expression of these polypeptides paralleled the neuronal differentiation of the P19 embryonal carcinoma cells. The expression of these integrins was not induced in a variant of the P19 cells, P19RAC65, which are resistant to differentiation induction by retinoic acid. Utilizing integrin subunit-specific anti-cytoplasmic peptide antibodies together with immunoprecipitation and Western blot analysis, the 130- and 115-kDa polypeptides were identified as the integrin alpha v and beta 1 subunits, respectively. The 90-kDa polypeptide, also induced by retinoic acid, was identified as beta 3, whereas the identity of the uninduced 96-kDa polypeptide remains unclear as yet. Peptide map analysis of deglycosylated polypeptides demonstrated that the 90- and 96-kDa polypeptides are distinct proteins and that the 115-kDa polypeptides immunoprecipitated with either anti-alpha v or anti-beta 1 antibodies are identical, further establishing that the 115-kDa polypeptide associating with alpha v is beta 1. The retinoic acid-induced expression of beta 1 occurred at the level of mRNA expression which also paralleled neuronal differentiation, but peaked slightly ahead of the cell surface expression of beta 1. The expression of other beta 1-associated alpha subunits was not induced by retinoic acid in these cells. These data demonstrate that retinoic acid strongly induces the expression of the integrin heterodimer alpha v beta 1 and also, to a smaller extent, the expression of alpha v beta 3. The retinoic acid-induced, high level surface expression of the alpha v beta 1 heterodimer is tightly correlated with the induction of neuronal differentiation by retinoic acid. This finding suggests an important role for the alpha v beta 1 heterodimer in the neuronal differentiation process.     

pH-dependent expression of periplasmic proteins and amino acid catabolism in Escherichia coli

Escherichia coli grows over a wide range of pHs (pH 4.4 to 9.2), and its own metabolism shifts the external pH toward either extreme, depending on available nutrients and electron acceptors. Responses to pH values across the growth range were examined through two-dimensional electrophoresis (2-D gels) of the proteome and through lac gene fusions. Strain W3110 was grown to early log phase in complex broth buffered at pH 4.9, 6.0, 8.0, or 9.1. 2-D gel analysis revealed the pH dependence of 19 proteins not previously known to be pH dependent. At low pH, several acetate-induced proteins were elevated (LuxS, Tpx, and YfiD), whereas acetate-repressed proteins were lowered (Pta, TnaA, DksA, AroK, and MalE). These responses could be mediated by the reuptake of acetate driven by changes in pH. The amplified proton gradient could also be responsible for the acid induction of the tricarboxylic acid (TCA) enzymes SucB and SucC. In addition to the autoinducer LuxS, low pH induced another potential autoinducer component, the LuxH homolog RibB. pH modulated the expression of several periplasmic and outer membrane proteins: acid induced YcdO and YdiY; base induced OmpA, MalE, and YceI; and either acid or base induced OmpX relative to pH 7. Two pH-dependent periplasmic proteins were redox modulators: Tpx (acid-induced) and DsbA (base-induced). The locus alx, induced in extreme base, was identified as ygjT, whose product is a putative membrane-bound redox modulator. The cytoplasmic superoxide stress protein SodB was induced by acid, possibly in response to increased iron solubility. High pH induced amino acid metabolic enzymes (TnaA and CysK) as well as lac fusions to the genes encoding AstD and GabT. These enzymes participate in arginine and glutamate catabolic pathways that channel carbon into acids instead of producing alkaline amines. Overall, these data are consistent with a model in which E. coli modulates multiple transporters and pathways of amino acid consumption so as to minimize the shift of its external pH toward either acidic or alkaline extreme.