KatF is required for the expression of some 32 carbon starvation proteins in Escherichia coli including 6 previously identified as Pex. Mutants with the katF gene survive carbon and nitrogen starvation poorly. Many of the KatF-regulated starvation proteins are common to those induced by other stresses, and the mutant failed to develop starvation-mediated cross protection to osmotic, oxidative, and heat stresses. Furthermore, thermal resistance was not induced in the mutant by heat preadaptation, and it exhibited an altered pattern of protein synthesis at elevated temperature. Thus, KatF is a major switch that controls the starvation-mediated resistant state in E. coli.

A reverse genetics approach was used to clone a pex starvation gene that codes for an 18-kDa polypeptide, designated PexB. Single-copy pexB-lacZ operon fusions were constructed to study transcriptional regulation and the promoter region of this gene. The induction by carbon starvation or osmotic stress was transcriptional and controlled by or38 but was independent of this sigma factor by the oxidative stress; presumably, it was if70 mediated under the latter stress. During nitrogen starvation, the induction was controlled at the posttranscriptional level. The perB upstream region contained 245 nucleotides within which sequences approximating the consensus for cyclic AMP receptor protein and integration host factor binding sites were discernible. Deletion of 164 bp of the upstream region, which included these consensus sequences, did not affect starvationor osmotic stress-mediated induction ofpexB but abolished its induction by oxidative stress. The same start site was used in transcription during carbon starvation, osmotic stress, or oxidative stress, suggesting that thepexB promoter can be recognized in vivo by both if38 and cr70, depending, presumably, on the presence of appropriate transcriptional factors. The -10 and -35 regions of pexB resembled those of some but not all genes known to be controlled by if38.At the onset of carbon starvation, Escherichia coli induces some 50 polypeptides, which fall in several temporal classes. This induction is associated with the development of marked general resistance (7, 11-13, 21-23, 34). We are attempting to identify and characterize the individual polypeptides that contribute to this resistance. A core set of 15 polypeptides is induced regardless of whether the cells are starved for carbon, nitrogen, or phosphorus, and since this set includes many heat, oxidative, and osmotic shock proteins (7), we have focused on these polypeptides. This core set of proteins is regulated differently from most of the other carbon starvation proteins. First, their induction is independent of cyclic AMP (cAMP), while that of the rest requires this nucleotide. On this basis, we differentiated starvation proteins into Pex (cAMP independent) and Cst (cAMP dependent) (22,39). Second, the induction of several Pex proteins requires secondary a factors. Thus, a32 is required for the induction of at least three Pex proteins, DnaK, GroEL, and HtpG, and mutants deficient in this sigma factor survive starvation poorly (11). Similarly, c38 (also referred to as as and KatF [29]) is required for the induction of at least six Pex proteins (25), and E. coli mutants deficient in o38 (QpoS) are greatly compromised in the development of starvation-mediated resistance (18,25).This report deals with an 18-kDa membrane-associated (39) Pex protein that we identified in 1986 as spot 19 on our two-dimensional (2-D) gel electrophoresis maps (7,22,39) and subsequently named PexB (15). This protein is induced early in starvation and exhibits a sustained induction during carbon starvation: at 4 h of glucose or suc...

A novel poly-␤-hydroxybutyrate (PHB) production system in which the expression and gene dosage of the Alcaligenes eutrophus pha biosynthetic operon were effectively regulated by cultivation temperature was constructed in Escherichia coli. The pha operon was fused to the negatively regulated tac promoter and cloned into a vector in which the copy number is temperature dependent. A two-phase process was employed to produce PHB during fed-batch growth. In the growth phase, the culture was maintained at a low temperature. Under this condition, the plasmid copy number was depressed and the number of LacI proteins was sufficient to repress tac::pha transcription. The production phase was initiated by temperature upshift. At the elevated temperature, the number of plasmids surpassed the number of LacI repressors, which resulted in rapid induction of tac::pha transcription, synthesis of poly-␤-hydroxyalkanoate-specific proteins, and polymer synthesis. During the production phase, the PHB production rate was 1.07 g of PHB liter ؊1 h ؊1 under optimized conditions. This rate is comparable to that of bacteria which naturally produce this polymer.

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To determine the utility of coupling runaway replication to the expression of cloned genes under the control of strong promoters, lacZ transcriptional fusions to the trp or tac promoter (Ptrp or Ptac) were constructed using plasmids in which the copy number is thermally regulated. Cells containing these plasmids were able to produce β‐galactosidase to levels between 3700 and 46,000 Miller units when induced only by a temperature upshift. The addition of the appropriate chemical inducer, either IPTG (isopropyl‐β‐D‐thiogalactopyranoside) or IAA (3‐β‐indoleacrylic acid), did not significantly enhance the thermal induction. The Ptac‐controlled and Ptrp‐controlled lacZ induction differed slightly in that the Ptac‐controlled thermal induction exhibited a lag of approximately 1.5 h as compared to both chemical and thermal induction, whereas in the case of Ptrp‐controlled induction, an increase in β‐galactosidase expression above background occurred at approximately the same time regardless of the means of induction. The best vector, a Ptrp‐controlled lacZ fusion carried on a runaway replication vector having a basal copy number of 10, was able to mediate the expression of β‐galactosidase to approximately 40,000 Miller units of β‐galactosidase comprising 25% of the total cell protein at 17 h postinduction under optimal conditions for protein yield. In these cells, lysis occurred as lacZ was maximally expressed. Under noninducing conditions, the plasmids were stable for at least 60 generations in the absence of antibiotic in batch culture. © 1996 John Wiley & Sons, Inc.