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Mannose uptake and phosphorylation in Escherichia coli is catalyzed by the phosphoenolpyruvate:glycose phosphotransferase system (PTS). The mannose-specific complex of the PTS, designated H1MaI, comprises lipid and two membrane proteins, ll-AM and II-BMaI. The proteins are encoded by ptsM, located at "40 minutes on the E. coli chromosome. A different genetic marker, pel, maps with ptsM, and is required for X DNA penetration of the cytoplasmic membrane. Earlier studies suggested that bothpel function and ll-Bm are encoded by the same gene, while a different gene (also in ptsM) encodes II-AM". In the present studies, a ptsM done, pCS13, was isolated from an E. colU Hindml gene bank in pBR322 and restored both mannose fermentation and pelt function to ptsM mutants defective in II-BMaI. Subclones of pCS13 show that (i) two distinct genes, manY and manZ, encode thepel function and the II-BMa`protein, respectively; (ii) each gene may have its own promoter; (iii) whereas the protein encoded by manY (Pel) alone seems sufficient for X sensitivity, all three gene products are required for mannose fermentation, transport of the mannose analogue 2-deoxyglucose, and phosphorylation of the latter by cytoplasmic membranes. Thus, Pel is required for function of the HIMan complex. The efficiency of the complex may depend on the ratio of Pel to 11Man.The phosphoenolpyruvate:glycose phosphotransferase system (PTS) catalyzes the translocation of its sugar substrates across the bacterial membrane concomitant with their phosphorylation (1-3). The PTS consists of two cytoplasmic phosphorylated carrier proteins, enzyme I and HPr (which are not sugar specific) and a number of sugar-specific membrane-associated proteins, called enzyme II complexes. Phosphoryl group transfer occurs in the following sequence: phosphoenolpyruvate -* enzyme I --HPr --enzyme II sugar.Two enzyme II complexes catalyze glucose translocation across the Escherichia coli membrane (4, 5). One, designated IIGlc, is specific for glucose and methyl glucosides. The second complex, IIMan, the subject of this report, comprises two membrane-associated proteins II-AMan and II-BMan, and phosphorylates glucose, mannose, and their analogues, such as 2-deoxyglucose (4-7).The concept of two proteins in the IIMan complex was based on early biochemical data (7). By contrast, genetic mapping suggested only a single locus for ptsM (8), at =40 minutes on the E. coli map. Another mutation in this region, called pel, resulted in resistance to penetration of the bacterial inner membrane by X DNA (9, 10). pel appeared to be closely related to ptsM since strains bearing mutations in pel were unable to ferment mannose (although only 30% of the strains unable to ferment mannose were insensitive to X phage).Recent work in this and other laboratories (refs. 11 and 12; P. Saris and E. T. Palva, personal communication) has shown thatptsM contains two structural genes-one encodes II-AMan (manX), and the other encodes II-BMan (manZ). Transcription occurs from manX through manZ with a str...

Plasmid DNA purified from bacterial cells can be contaminated with endotoxin to different extents, depending on the purification method. Earlier reports indicate that endotoxin can decrease transfection efficiency in many eukaryotic cell lines; however, the amount of endotoxin required for inhibition is unclear. We determined endotoxin effects in several cell lines and observed that endotoxin levels greater than or equal to 10,000 endotoxin units (EU) were needed to significantly affect cell proliferation and viability; levels greater than 2000 EU/mu g DNA were required to significantly inhibit transfection for all but one (Huh-7) of the cell lines tested. These endotoxin levels are significantly higher than endotoxin contamination in plasmid DNA purified by anion exchange, CsCl2 gradient and endotoxin-free purification technology, but not as high as a crude alkaline lysis preparatory method. Plasmid DNA prepared using anion exchange technology was comparable to endotoxin-free technology in terms of transfection efficiency. Even Huh-7 cells, which are markedly more sensitive to endotoxins, have comparable transfection efficiencies using plasmid DNA purified by either of these two methods. We conclude that for those cell lines commonly used for transfection studies, endotoxin-free, quality DNA is not necessary because significantly higher levels of bacterial endotoxins are required to inhibit either cell proliferation or transfection.

Several potential target sites for multiple regulatory mechanisms were previously identified in the 5' flanking region of the pts operon. We have investigated the in vitro interactions of the cAMP receptor protein (CRP)'cAMP regulatory complex with two DNA binding sites, by gel mobility-shift assays, and report the analysis of the functional role of each of the binding sites in vivo. Promoter-reporter gene fusion studies identified two CRPcAMP-dependent promoters (the previously identified P1 and another promoter, PO) upstream of ptsH. The crr promoters (P2) within ptsl may be negatively regulated by CRP'cAMP.The phosphoenolpyruvate:glycose phosphotransferase system (PTS) catalyzes the phosphorylation and transport of its sugar substrates and acts as a major signal-transduction system in bacterial cells. Thus, the PTS must be responsive to multiple and diverse external signals (for a review, see ref. 1).The PTS has been extensively studied in Escherichia coli and Salmonella typhimurium and consists of a complex array of reversibly phosphorylated cytoplasmic and membrane proteins. Two general, cytoplasmic proteins are required for the phosphorylation ofall PFTS substrates: enzyme I, encoded by ptsI, and the histidine-containing phosphoprotein HPr, encoded byptsH. Specific membrane complexes are required for translocation of the individual PTS sugars, and one of these, the glucose-specific complex, comprises a soluble protein (IIIGic) and a membrane-bound enzyme (IIBGlc) encoded by the crr and ptsG genes, respectively.The ptsH, ptsl, and crr genes constitute an operon ( Fig. 1) that is located at 52 min on the E. coli chromosome; ptsG is unlinked at 25 min. The PTS, itself, is stringently regulated and the mechanisms underlying the genetic regulation ofpts expression are unclear and apparently complex. The complete DNA sequence of ptsH, ptsI, crr and their flanking regions suggested two canonical binding regions for CRP cAMP upstream of the first gene, ptsH, and a promoter for crr, the last gene, within and toward the 3' terminus of ptsl (5). Transcriptional analysis of the region (4) suggested one promoter upstream of ptsH, designated Pi, and two withinptsIthat regulated transcription of crr, designated P2-I and P2-II. The designations P1 and P2 will be employed in this paper for the previously described promoters.In the present studies, we have measured CRP binding to the two putative binding sites upstream of ptsH by gel mobility-shift assays. We also report CRP-cAMP-independent and CRP-cAMP-dependent promoter activities in the DNA regions preceding and within the pts operon, including another promoter, designated PO, upstream of P,.