The sequence of 4,362 nucleotides encompassing the proU operon of Escherichia coli was determined. Three open reading frames were identified whose orientation, order, location, and sizes were in close accord with genetic evidence for three cistrons (proV, proW, and proX) in this operon. Similarities in primary structure were observed between (i) the deduced sequence of ProV with membrane-associated components of other binding-protein-dependent transport systems, in the nucleotide-binding region of each of the latter proteins, and (ii) that of ProW with integral membrane components of the transport systems above. The DNA sequence data also conclusively established that ProX represents the periplasmic glycine betaine-binding protein. The proU locus in Escherichia coli and Salmonella typhimurium encodes a transporter for active uptake of two solutes, glycine betaine and L-proline, whose intracellular accumulation is important in the process of water stress adaptation in these organisms (3,6,8,13,(18)(19)(20)39). The expression of proU is induced approximately 200-fold, and the transporter activity is also stimulated, upon growth of these bacteria in media of elevated osmolarity (6,11,13,14,18,39). In both E. coli and S. typhimurium, a periplasmic glycine betaine-binding protein has been shown to be a product of the proU locus (3,4,14,27,39), indicating that the ProU transporter is one among the class of multicomponent binding-protein-dependent transport systems characterized in the enterobacteria (1, 19).In the accompanying paper (11), C. S. Dattananda and I adduced genetic evidence for the presence of three genes (designated proV, proW, and proX) in the proU locus, organized in a single operon; their respective gene products were shown to be 44-, 35-, and 33-kilodalton proteins, the last of which was localized in the periplasm. In this paper, I present the nucleotide sequence of the proU locus along with results from experiments directed towards characterization of its cis regulatory region. MATERIALS AND METHODSRecombinant DNA and M13 phage techniques. The methods for restriction enzyme digestion, ligation, transformation, and gel electrophoresis of DNA fragments were those described by Maniatis et al. (38). Techniques for work with recom'binant M13 phages and their host, JM101, for cloning and DNA sequence determination have been described (40).Strategy for DNA sequence determination of proU. My colleagues and I had previously established that a 5-kilobasepair (kb) segment of chromosomal DNA clockwise of a BglII site and extending up to the site of a mini-Mu phage insertion t Dedicated to Pushpa M. Bhargava on his 60th birthday.cloned in the plasmid pHYD58 (Fig. 1) encompasses the entire proU locus (20). The data of Bremer and co-workers (14, 39) and the results presented in the accompanying paper (11) suggested further that the proU operon is situated to the right of the EcoRV site shown in Fig. 1. The complete nucleotide sequence of the chromosomal region on pHYD58 to the right of the EcoRV site was, therefo...

SummaryActive mechanisms exist to prevent transcription that is uncoupled from translation in the protein-coding genes of bacteria, as exemplified by the phenomenon of nonsense polarity. Bacterial transcription-translation coupling may be viewed as one among several co-transcriptional processes, including those for mRNA processing and export in the eukaryotes, that operate in the various life forms to render the nascent transcript unavailable for formation of otherwise deleterious R-loops in the genome.

Transcription of the proU operon in Escherichia coli is induced several hundredfold upon growth of cells in media of elevated osmolarity. A low-copy-number promoter-cloning plasmid vector, with lacZ as the reporter gene, was used for assaying the osmoresponsive promoter activity of each of various lengths of proU DNA, generated by cloning of discrete restriction fragments and by an exonuclease III-mediated deletion approach. The results indicate that expression of proU in E. coli is directed from two promoters, one (P2) characterized earlier by other workers with the start site of transcription 60 nucleotides upstream of the initiation codon of the first structural gene (proV), and the other (P1) situated 250 nucleotides upstream of proV. Furthermore, a region of DNA within proV was shown to be involved in negative regulation of proU transcription; phage Mu dII1681-generated lac fusions in the early region ofproV also exhibited partial derepression ofproU regulation, in comparison with fusions further downstream in the operon. Sequences around promoter P1, sequences around P2, and the promoter-downstream negative regulatory element, respectively, conferred approximately 5-, 8-, and 25-fold osmoresponsivity on proU expression. Within the region genetically defined to encode the negative regulatory element, there is a 116-nucleotide stretch that is absolutely conserved between the proU operons of E. coli and Salmonella typhimurium and has the capability of exhibiting alternative secondary structure. Insertion of this region of DNA into each of two different plasmid vectors was associated with a marked reduction in the mean topological linking number in plasmid molecules isolated from cultures grown in high-osmolarity medium. We propose that this region of DNA undergoes reversible transition to an underwound DNA conformation under high-osmolarity growth conditions and that this transition mediates its regulatory effect on proU expression.The growth rate of Escherichia coli and Salmonella typhimurium in high-osmolarity media is promoted by the addition of small concentrations of L-proline or glycine betaine to the culture medium. The osmoprotective effect of both of these compounds is presumed to be consequent upon their intracellular accumulation under conditions of water stress and is in part dependent on the presence of a functional ProU transporter in these cells, encoded by genes of the proU locus (for a review, see reference 6).Complementation studies using a number of proU mutants, in combination with nucleotide sequence analysis of the locus, have shown that proU is an operon composed of three structural genes, proV, proW, and proX (7, 13). The product of proX is a periplasmic protein which has been purified and shown to be a glycine betaine-binding protein in vitro; furthermore, the deduced amino acid sequences of the products of proV and proW each show similarities to components of other well-characterized transport systems such as those for histidine, maltose, or arabinose, thus permitting the inference t...

The anonymous open reading frame yggA of Escherichia coli was identified in this study as a gene that is under the transcriptional control of argP (previously called iciA), which encodes a LysR-type transcriptional regulator protein. Strains with null mutations in either yggA or argP were supersensitive to the arginine analog canavanine, and yggA-lac expression in vivo exhibited argP ؉ -dependent induction by arginine. Lysine supplementation phenocopied the argP null mutation in that it virtually abolished yggA expression, even in the argP ؉ strain. The dipeptides arginylalanine and lysylalanine behaved much like arginine and lysine, respectively, to induce and to turn off yggA transcription. Dominant missense mutations in argP (argP d ) that conferred canavanine resistance and rendered yggA-lac expression constitutive were obtained. The protein deduced to be encoded by yggA shares similarity with a basic amino acid exporter (LysE) of Corynebacterium glutamicum, and we obtained evidence for increased arginine efflux from E. coli strains with either the argP d mutation or multicopy yggA ؉ . The null yggA mutation abolished the increased arginine efflux from the argP d strain. Our results suggest that yggA encodes an ArgP-regulated arginine exporter, and we have accordingly renamed it argO (for "arginine outward transport"). We propose that the physiological function of argO may be either to prevent the accumulation to toxic levels of canavanine (which is a plant-derived antimetabolite) or arginine or to maintain an appropriate balance between the intracellular lysine and arginine concentrations.

Two pathways of transcription termination, factor-independent and -dependent, exist in bacteria. The latter pathway operates on nascent transcripts that are not simultaneously translated and requires factors Rho, NusG, and NusA, each of which is essential for viability of WT Escherichia coli. NusG and NusA are also involved in antitermination of transcription at the ribosomal RNA operons, as well as in regulating the rates of transcription elongation of all genes. We have used a bisulfite-sensitivity assay to demonstrate genome-wide increase in the occurrence of RNA-DNA hybrids (R-loops), including from antisense and read-through transcripts, in a nusG missense mutant defective for Rho-dependent termination. Lethality associated with complete deficiency of Rho and NusG (but not NusA) was rescued by ectopic expression of an R-loop-helicase UvsW, especially so on defined growth media. Our results suggest that factor-dependent transcription termination subserves a surveillance function to prevent translationuncoupled transcription from generating R-loops, which would block replication fork progression and therefore be lethal, and that NusA performs additional essential functions as well in E. coli. Prevention of R-loop-mediated transcription-replication conflicts by cotranscriptional protein engagement of nascent RNA is emerging as a unifying theme among both prokaryotes and eukaryotes.A ll bacterial transcription termination occurs by one of two pathways that are referred to as factor-independent (or intrinsic) and factor-dependent (or Rho-dependent), respectively (1). The latter pathway, which requires the action of factors Rho, NusG, and NusA in Escherichia coli, serves to terminate synthesis of transcripts that are not being simultaneously translated, for example, at the ends of various genes and operons (1, 2). The same mechanism is also responsible for the classic phenomenon of nonsense polarity (3), by which a stop codon mutation within the proximal gene of an operon results in absence of transcription of the distal genes; in this manner, Rho-dependent termination provides a back-up to other mechanisms (4, 5) that act to ensure the coupling of transcription with translation in bacteria. NusG and NusA are also involved in transcription antitermination during lytic growth of the lambdoid prophages and at the ribosomal RNA operons, as well as in regulating the rates of transcription elongation of all genes (6, 7).Rho, NusG, and NusA are each essential for viability of the prototypic WT E. coli strain MG1655. Cardinale et al. (8) have reported that NusG and NusA are dispensable in strain MDS42 [which is an engineered MG1655 derivative with 14% reduced genome content because of deletions of insertion elements and cryptic prophages (9)], based on which they have proposed that the essentiality of Rho-dependent termination stems from its need for silencing of horizontally acquired genes in bacteria. Even so, the Δrho mutation in MDS42, as in MG1655, is lethal (8, 10).Several phenotypes in nusG, rho, and nusA missen...

No abstract

Salt-induced overexpression of genes cloned downstream of the phage T7 10 promoter was demonstrated in an Escherichia coli strain (GJ1158) which carries a single chromosomally integrated copy of the gene for phage T7 RNA polymerase under transcriptional control of the cis-regulatory elements of the osmoresponsive proU operon. Plasmids that have been constructed to obtain overproduction of individual target gene products in strain BL21(DE3) (by addition of isopropyl-␤-D-thiogalactopyranoside as an inducer) can directly be transformed into GJ1158. The NaCl induction regimen was also shown to be associated with a decreased propensity for sequestration of overexpressed target proteins within insoluble inclusion bodies.Escherichia coli is widely used in basic research studies as a host strain for the overproduction of proteins from cloned genes, and several proteins of pharmaceutical value are also produced on a commercial scale in E. coli. In the two most commonly used strategies, a target gene is overexpressed (directly or indirectly) by temperature induction of a phage promoter or induction with isopropyl-␤-D-thio-galactopyranoside (IPTG) of the lac promoter and its variants (reviewed in reference 13).Some problems associated with temperature-induced overexpression of recombinant gene products include (i) difficulty in achieving rapid temperature upshift, particularly when handling larger culture volumes, (ii) increased likelihood of formation of insoluble inclusion bodies at higher incubation temperatures (20), and (iii) induction of several proteases in E. coli upon heat shock (4). With the lac promoter-based systems as well, the problem of inclusion body formation may be significant, in addition to the high cost and toxicity of IPTG (13).In this study, we have developed a simple, efficient, and generally applicable method for the overproduction of recombinant proteins in E. coli that makes use of NaCl as an inducer. For this purpose, we have constructed a host strain in which synthesis of the RNA polymerase (RNAP) of bacteriophage T7 has been placed under control of the osmotically inducible proU promoter of E. coli (6). Any target gene which is cloned downstream of a phage T7 promoter (e.g., 10) and introduced into this strain will be overexpressed, through a two-tier amplification strategy, upon salt addition. We also demonstrate that the propensity for inclusion body formation may be significantly reduced with the NaCl induction protocol.Methods. Most of the genetic and recombinant DNA procedures used have been described earlier (5, 19). Induction of target genes by 1 mM IPTG in derivatives of strain BL21(DE3) was done as previously described (21,22). NaCl induction experiments were done as follows. Ten-milliliter cultures were grown at 37°C to an A 600 of around 0.8 in low-osmolarity LBON medium (Luria-Bertani medium [19] with NaCl omitted, containing [per liter] 10 g of tryptone and 5 g of yeast extract [pH adjusted to 7.0 with NaOH]) supplemented with appropriate antibiotics. Each culture was then divided...

Mu dl(Ap lac)-generated operon fusions were used in the identification of genes in Escherichia coli whose transcriptional expression is altered by changes in the osmolarity of the growth medium. One such osmoresponsive gene, designated osrA, was induced 400-fold when the osmolarity of the medium was increased with the addition of either ionic or neutral impermeable solutes but was not induced with glycerol, which is freely permeable across the cell membrane. osrA was mapped to 57.5 min and was shown to be transcribed clockwise on the E. coli chromosome. The ability of small concentrations of L-proline to promote the growth of E. coli in high-osmolar medium was shown to have been specifically lost in osrA mutants; other lines of evidence were also obtained to support the notion that osrA codes for an osmoresponsive L-proline transport system and is homologus to proU in Salmonella typhimurium. A second osmoresponsive operon identified was kdp, which codes for an inducible K+-transport system in E. coli. kdp expression was elevated 12-fold when the osmolarity of the growth medium was increased with the addition of impermeable ionic solutes but not neutral solutes; furthermore, osmoresponsivity of kdp expression was demonstrable only in K+-limiting media. kdp mutants were able to grow normally in high-osmolar media, but strains defective in both kdp and trkA (a gene for a second major K-transport system) displayed an osmosensitive phenotype. The results suggest that transport systems for L-proline and K+, specified by osrA (proU) and kdp, respectively, play independent and important roles in osmoregulation in E. coli. A third osmoresponsive gene that was identified was lamB, which codes for an outer membrane protein for maltodextrin transport and A phage adsorption; its expression was reduced fourfold with increase in the osmolarity of the growth medium. 4 6 35 15 28 B. J. Bachmann B. J. Bachmann S. K. Mahajan 43 This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study From KL14 (28), by P1 kc transduction This study From GJ11, by P1 kc transduction From GS74, in two steps From CSH57, in two steps This study From GJ11, by P1 kc transduction to Put-From MC4100, by P1 kc transduction to Put-From GJ113, by P1 kc transduction to Put-DHPF selection from Tets derivative of GJ125 DHPI selection from Tets derivative of GJ126 DHPI selection from Tets derivative of GJ121 By conjugation, KL584 x GJ134 zjd-351::TnIO By conjugation, KL584 x GJ135 zjd-351::TnlO By conjugation, KL584 x GJ157 a The nomenclature for genetic symbols follows that described by Bachmann (1), and the nomenclature for transpositional insertions follows that described by Chumley et al. (9). Allele numbers are indicated if they are known.