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The interaction between SARS-CoV PDZ-binding motifs (PBMs) and cellular PDZs is responsible for virus virulence. The PBM sequence present in the 3a and envelope (E) proteins of SARS-CoV can potentially bind to over 400 cellular proteins containing PDZ domains. The role of SARS-CoV 3a and E proteins was studied. SARS-CoVs, in which 3a-PBM and E-PMB have been deleted (3a-PBM-/E-PBM-), reduced their titer around one logarithmic unit but still were viable. In addition, the absence of the E-PBM and the replacement of 3a-PBM with that of E did not allow the rescue of SARS-CoV. E protein PBM was necessary for virulence, activating p38-MAPK through the interaction with Syntenin-1 PDZ domain. However, the presence or absence of the homologous motif in the 3a protein, which does not bind to Syntenin-1, did not affect virus pathogenicity. Mutagenesis analysis and in silico modeling were performed to study the extension of the PBM of the SARS-CoV E protein. Alanine and glycine scanning was performed revealing a pair of amino acids necessary for optimum virus replication. The binding of E protein with the PDZ2 domain of the Syntenin-1 homodimer induced conformational changes in both PDZ domains 1 and 2 of the dimer.

Both EphB2- and EphB3-deficient mice exhibit profound histological alterations in the thymic epithelial network but few changes in T-cell differentiation, suggesting that this organization would be sufficient to produce functional T lymphocytes. Also, other antigen-presenting cells involved in immunological education could substitute the thymic epithelium. Accordingly, we found an increased frequency of plasmacytoid dendritic cells but not of conventional dendritic cells, medullary fibroblasts or intrathymic B lymphocytes. In addition, there are no lymphoid infiltrates in the organs of mutant mice nor do they contain circulating autoantibodies. Furthermore, attempts to induce arthritic lesions after chicken type II collagen administration fail totally in EphB2-deficient mice whereas all WT and half of the immunized EphB3 -/- mice develop a typical collagen-induced arthritis. Our results point out that Th17 cells, IL4-producing Th2 cells and regulatory T cells are key for the induction of disease, but mutant mice appear to have deficits in T cell activation or cell migration properties. EphB2 -/- T cells show reduced in vitro proliferative responses to anti-CD3/anti-CD28 antibodies, produce low levels of anti-type II collagen antibodies, and exhibit low proportions of T follicular helper cells. On the contrary, EphB3 -/- lymph node cells respond accurately to the different immune stimuli although in lower levels than WT cells but show a significantly reduced migration in in vitro transwell assays, suggesting that no sufficient type II collagen-dependent activated lymphoid cells reached the joints, resulting in reduced arthritic lesions., (© 2024. The Author(s).)

Coronaviruses (CoVs) of genera α, β, γ, and δ encode proteins that have a PDZ-binding motif (PBM) consisting of the last four residues of the envelope (E) protein (PBM core). PBMs may bind over 400 cellular proteins containing PDZ domains (an acronym formed by the combination of the first letter of the names of the three first proteins where this domain was identified), making them relevant for the control of cell function. Three highly pathogenic human CoVs have been identified to date: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. The PBMs of the three CoVs were virulence factors. SARS-CoV mutants in which the E protein PBM core was replaced by the E protein PBM core from virulent or attenuated CoVs were constructed. These mutants showed a gradient of virulence, depending on whether the alternative PBM core introduced was derived from a virulent or an attenuated CoV. Gene expression patterns in the lungs of mice infected with SARS-CoVs encoding each of the different PBMs were analyzed by RNA sequencing of infected lung tissues. E protein PBM of SARS-CoV and SARS-CoV-2 dysregulated gene expression related to ion transport and cell homeostasis. Decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA, essential for alveolar edema resolution, was shown. Reduced CFTR mRNA levels were associated with edema accumulation in the alveoli of mice infected with SARS-CoV and SARS-CoV-2. Compounds that increased CFTR expression and activity, significantly reduced SARS-CoV-2 growth in cultured cells and protected against mouse infection, suggesting that E protein virulence is mediated by a decreased CFTR expression. IMPORTANCE Three highly pathogenic human CoVs have been identified: SARS-CoV, MERS-CoV, and SARS-CoV-2. The E protein PBMs of these three CoVs were virulence factors. Gene expression patterns associated with the different PBM motifs in the lungs of infected mice were analyzed by deep sequencing. E protein PBM motif of SARS-CoV and SARS-CoV-2 dysregulated the expression of genes related to ion transport and cell homeostasis. A decrease in the mRNA expression of the cystic fibrosis transmembrane conductance regulator (CFTR), which is essential for edema resolution, was observed. The reduction of CFTR mRNA levels was associated with edema accumulation in the lungs of mice infected with SARS-CoV-2. Compounds that increased the expression and activity of CFTR drastically reduced the production of SARS-CoV-2 and protected against its infection in a mice model. These results allowed the identification of cellular targets for the selection of antivirals.

Background: Diabetes is a major health care problem, reaching epidemic numbers worldwide. Reducing hemoglobin A 1c (HbA 1c ) levels to recommended targets is associated with a marked decrease in the risk of type 2 diabetes mellitus (T2DM)-related complications. The implementation of new technologies, particularly telemedicine, may be helpful to facilitate self-care and empower people with T2DM, leading to improved metabolic control of the disease., Objective: This study aimed to analyze the effect of a home digital patient empowerment and communication tool (DeMpower App) on metabolic control in people with inadequately controlled T2DM., Methods: The DeMpower study was multicenter with a retrospective (observational: 52 weeks of follow-up) and prospective (interventional: 52 weeks of follow-up) design that included people with T2DM, aged ≥18 and ≤80 years, with HbA 1c levels ≥7.5% to ≤9.5%, receiving treatment with noninsulin antihyperglycemic agents, and able to use a smartphone app. Individuals were randomly assigned (2:1) to the DeMpower app-empowered group or control group. We describe the effect of empowerment on the proportion of patients achieving the study glycemic target, defined as HbA 1c ≤7.5% with a ≥0.5% reduction in HbA 1c at week 24., Results: Due to the COVID-19 pandemic, the study was stopped prematurely, and 50 patients (33 in the DeMpower app-empowered group and 17 in the control group) were analyzed. There was a trend toward a higher proportion of patients achieving the study glycemic target (46% vs 18%; P=.07) in the DeMpower app group that was statistically significant when the target was HbA 1c ≤7.5% (64% vs 24%; P=.02) or HbA 1c ≤8% (85% vs 53%; P=.02). The mean HbA 1c was significantly reduced at week 24 (-0.81, SD 0.89 vs -0.15, SD 1.03; P=.03); trends for improvement in other cardiovascular risk factors, medication adherence, and satisfaction were observed., Conclusions: The results suggest that patient empowerment through home digital tools has a potential effect on metabolic control, which might be even more relevant during the COVID-19 pandemic and in a digital health scenario., (©Domingo Orozco-Beltrán, Cristóbal Morales, Sara Artola-Menéndez, Carlos Brotons, Sara Carrascosa, Cintia González, Óscar Baro, Alberto Aliaga, Karine Ferreira de Campos, María Villarejo, Carlos Hurtado, Carolina Álvarez-Ortega, Antón Gómez-García, Marta Cedenilla, Gonzalo Fernández. Originally published in JMIR Diabetes (https://diabetes.jmir.org), 03.10.2022.)

Background: In October 2013, the emergency department of our hospital started up a rheumatology and musculoskeletal emergencies clinic (RMSEC) with the participation of three specialists in Rheumatology. The purpose of this study was to describe the experience gained in the first year since the beginning of our activity., Method: A descriptive study of healthcare activity of the RMSEC throughout its first year of operation was performed., Results: 1788 assessments on 1663 patients were performed. The range of age was 7 to 67 years. 1530 (85.57%) assessments were performed in patients of the healthcare area of our hospital. Of all the assessments made, 633 (35.4%) were related to inflammatory processes and the remaining 1155 (64.6%) to mechanical or degenerative joint or soft tissue processes. According to the topography of the complaint, 435 (24.3%) patients consulted for a process related to the knee, 362 (20.3%) with axial lumbar region and 336 (18.8%) with the shoulder., Conclusion: Our results denote an intense clinical activity that could have a positive impact on the management of rheumatic and musculoskeletal general emergency., (Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Reumatología y Colegio Mexicano de Reumatología. All rights reserved.)

To explore the reasons for the dearth of minorities in Ph.D.-level biomedical research and identify opportunities to increase minority participation, we surveyed high-achieving alumni of an undergraduate biology enrichment program for underrepresented minorities. Respondents were asked to describe their career paths and to reflect on the influences that guided their career choices. We particularly probed for attitudes and experiences that influenced students to pursue a research career, as well as factors relevant to their choice between medicine (the dominant career choice) and basic science. In agreement with earlier studies, alumni strongly endorsed supplemental instruction as a mechanism for achieving excellence in basic science courses. Undergraduate research was seen as broadening by many and was transformative for half of the alumni who ultimately decided to pursue Ph.D.s in biomedical research. That group had expressed no interest in research careers at college entry and credits their undergraduate research experience with putting them on track toward a research career. A policy implication of these results is that making undergraduate research opportunities widely available to biology students (including "premed" students) in the context of a structured educational enrichment program should increase the number of minority students who choose to pursue biomedical Ph.D.s.

sigma S is an alternate sigma factor which functions with RNA polymerase to activate transcription of genes that are involved in a number of stress responses, including stationary-phase survival and osmoprotection. The similarity of the sigma S protein to sigma D (Escherichia coli's major sigma factor) in the regions thought to recognize and bind promoter sequences suggests that sigma S- and sigma D-associated RNA polymerases recognize promoter DNA in a similar manner. However, no promoter recognition sequence for sigma S holoenzyme (E sigma S) has been identified. An apparent conservation of cytosine nucleotides was noted in the -35 region of several sigma S-dependent promoters. Site-directed mutagenesis and reporter gene fusions were used to investigate the importance of the -35 cytosine nucleotides for sigma S-dependent transcription. Substitution of cytosine nucleotides for thymidine at the -35 site of the sigma D-dependent proU promoter effectively abolished transcription by E sigma D but allowed E sigma S to direct transcription from the mutant promoter. Inclusion of the sigma D consensus -10 hexamer strengthened transcription by E sigma S, demonstrating that both E sigma D and E sigma S can recognize the same -10 sequences. Conversely, replacement of -35 site cytosine nucleotides with thymidine in the sigma S-dependent osmY promoter reduced transcription by E sigma S and increased transcription by E sigma D. Our data suggest that DNA sequences in the -35 region function as part of a discriminator mechanism to shift transcription between E sigma D and E sigma S.

Transcription in vitro of two osmoregulated promoters, for the Escherichia coli osmB and osmY genes, was analysed using two species of RNA polymerase holoenzyme reconstituted from purified core enzyme and either sigma D (sigma 70, the major sigma in exponentially growing cells) or sigma S (sigma 38, the principal sigma at stationary growth phase). Under conditions of low ionic strength, the osmB and osmY promoters were transcribed by both E sigma D and E sigma S. Addition of up to 400 mM potassium glutamate (K glutamate), mimicking the intracellular ionic conditions under hyperosmotic stress, specifically enhanced transcription at these promoters by E sigma S but inhibited that by E sigma D. At similar high concentrations of potassium chloride (KCl), however, initiation at both these promoters was virtually undetectable. These data suggest that the RNA polymerase, E sigma S, itself can sense osmotic stress by responding to changes in intracellular K glutamate concentrations and altering its promoter selectivity in order to recognize certain osmoregulated promoters.

proP of Escherichia coli encodes an active transport system for proline and glycine betaine which is activated by both hyperosmolarity and amino acid-limited growth. proP DNA sequences far upstream from the translational start site are strongly homologous to the promoter of proU, an operon that specifies another osmoregulated glycine betaine transport system. Mutation and deletion analysis of proP and primer extension experiments established that this promoter, P1, was responsible for proP's strong expression in minimal medium and its response to osmotic signals. When cells were grown in complex medium, expression from a proP-lacZ fusion was induced three- to fourfold as growth slowed and cells entered stationary phase. Stationary-phase induction was dependent on rpoS, which encodes a stationary-phase sigma factor. Deletion of 158 bp of the untranslated leader sequence between P1 and the proP structural gene abolished rpoS-dependent stationary-phase regulation. Transcription initiation detected by primer extension within this region was absent in an rpoS mutant. proP is therefore a member of the growing class of sigma S-dependent genes which respond to both stationary-phase and hyperosmolarity signals.

The proU operon of Escherichia coli encodes a high-affinity glycine betaine transport system which is osmotically inducible and enables the organism to recover from the deleterious effects of hyperosmotic shock. Regulation occurs at the transcriptional level. KMnO4 footprinting showed that the preponderance of transcription initiated at a single primary promoter region and that proU transcription activation did not occur differentially at alternate promoters in response to various levels of salt shock. Mutational analysis confirmed the location of the primary promoter and identified an extended -10 region required for promoter activity. Specific nucleotides within the spacer, between position -10 and position -35, were important for maximal expression, but every mutant which retained transcriptional activity remained responsive to osmotic signals. A chromosomal 90-bp minimal promoter fragment fused to lacZ was not significantly osmotically inducible. However, transcription from this fragment was resistant to inhibition by salt shock. A mutation in osmZ, which encodes the DNA-binding protein H-NS, derepressed wild-type proU expression by sevenfold but did not alter expression from the minimal promoter. The current data support a model in which the role of the proU promoter is to function efficiently at high ionic strength while other cis-acting elements receive and respond to the osmotic signal.

Sixty-three clinical isolates identified as Escherichia coli, 30 from the human urinary tract and 33 derived from other human origins, were screened for proline/glycine betaine transporters similar to those that support proline catabolism and proline- or glycine betaine-based osmoregulation in E. coli K-12. Both molecular (DNA- and protein-based) analyses and physiological tests were performed. All tests were calibrated with E. coli K-12 derivatives from which genetic loci putP (encoding a proline transporter required for proline catabolism), proP, and (or) proU (loci encoding osmoregulatory proline/glycine betaine transporters) had been deleted. All clinical isolates showed both enhanced sensitivity to the toxic proline analogue azetidine-2-carboxylate on media of high osmolality and growth stimulation by glycine betaine in an artificial urine preparation of high osmolality. DNA sequences similar to the putP, proP, and proU loci of E. coli K-12 were detected by DNA amplification and (or) hybridization and protein specifically reactive with antibodies raised against the ProX protein of E. coli K-12 (a ProU constituent) was detected by western blotting in over 95% of the isolates. Two anomalous isolates were reclassified as non-E. coli on the basis of the API 20E series of tests. A protein immunochemically cross-reactive with the ProP protein of E. coli K-12 was also expressed by the clinical isolates. Since all three transporters were ubiquitous, no particular correlation between clinical origin and PutP, ProP, or ProU activity was observed. These data suggest that the transporters encoded in loci putP, proP, and proU perform housekeeping functions essential for the survival of E. coli cells in diverse habitats.

The osmY gene, which encodes a periplasmic protein with an apparent M(r) of 22,000, is induced by both osmotic and growth phase signals. We demonstrate here that osmY expression is regulated at the level of transcription and that transcription initiates 242 nucleotides upstream of the osmY open reading frame. Relative to the transcriptional start site, 5' deletions up to -36 did not inhibit osmY expression. 3' deletions that extended into the untranslated leader region affected the overall level of osmY::lacZ expression but did not affect inducibility. 5' and 3' deletions that extended past the transcriptional start region essentially abolished osmY expression, suggesting that there is a single promoter region. A putative promoter was identified, and its -10 region, TATATT, closely resembles the sigma 70 consensus -10 sequence, TATAAT. However, we show that osmY is not absolutely dependent on a functional sigma 70 for its expression. Since osmY expression does require rpoS (R. Hengge-Aronis, R. Lange, N. Henneberg, and D. Fischer, J. Bacteriol. 175:259-265, 1993), which encodes a stationary-phase sigma factor, sigma S (K. Tanaka, Y. Takayanagi, N. Fujita, A. Ishihama, and H. Takahashi, Proc. Natl. Acad. Sci. USA 90:3511-3515, 1993), E sigma S may be the form of RNA polymerase responsible for transcription of osmY.

Glycine betaine is a powerful osmoprotectant molecule present in the inner medulla of the kidney and excreted into urine. It may be responsible for the ability of Escherichia coli to grow in hypertonic urine. Also, strains of E. coli that cause urinary tract infections may be more salt-tolerant than strains from other sites. To explore these questions, 301 isolates from blood, urine, or stool and 12 representative enteric strains were examined. Tolerance varied from 0.1 to 0.7 M NaCl (median, 0.5) in minimal medium. There were no significant differences in salt tolerance by site of isolation. A salt-sensitive enteric strain that responded poorly to glycine betaine and mutant strains lacking the ability to synthesize or transport glycine betaine did not grow well in hypertonic urine. Accumulation of glycine betaine appears to be a mechanism by which E. coli can adapt to external osmotic forces and grow in hypertonic urine.

A new osmotically inducible gene in Escherichia coli, osmY, was induced 8- to 10-fold by hyperosmotic stress and 2- to 3-fold by growth in complex medium. The osmY gene product is a periplasmic protein which migrates with an apparent molecular mass of 22 kDa on sodium dodecyl sulfate-polyacrylamide gels. A genetic fusion to osmY was mapped to 99.3 min on the E. coli chromosome. The gene was cloned and sequenced, and an open reading frame was identified. The open reading frame encoded a precursor protein with a calculated molecular weight of 21,090 and a mature protein of 18,150 following signal peptide cleavage. Sequencing of the periplasmic OsmY protein confirmed the open reading frame and defined the signal peptide cleavage site as Ala-Glu. A mutation caused by the osmY::TnphoA genetic fusion resulted in slightly increased sensitivity to hyperosmotic stress.

proU expression has been proposed to form part of a general stress response that is regulated by increased negative DNA supercoiling brought about by environmental signals such as osmotic or anaerobic stress (N. Ni Bhriain, C. J. Dorman, and C. F. Higgins, Mol. Microbiol. 3:933-944, 1989). However, we find that although proU-containing plasmids derived from cells grown in media of elevated osmolarity were more supercoiled than plasmids from cells grown in standard media, they did not activate proU expression in vitro. The gyrA96 mutation and anaerobic conditions are known to affect DNA supercoiling but did not alter proU expression. Finally, the gyrase inhibitors coumermycin and novobiocin did not reduce in vitro proU expression. Therefore, this evidence rules out regulation by changes in DNA superhelicity for proU in Escherichia coli.

The osmB gene, which encodes an outer membrane lipoprotein, can be induced by both osmotic and growth phase signals. Construction of two transcriptional fusions, an osmB-lacZ fusion in single copy on the bacterial chromosome and an osmB-cat fusion carried on a multicopy plasmid, demonstrated that induction of osmB by hyperosmolarity and during the stationary phase of growth occurred at the level of transcription. Two transcription initiation sites were identified by RNase protection of in vivo message. The downstream P2 promoter is the primary site for regulation; the basal level of expression is initiated at P2 and transcription from P2 is induced by elevated osmolarity or upon reaching stationary phase. Transcription from the P1 promoter, 150 base pairs (bp) upstream of the P2 promoter, occurred only when both osmotic and growth phase signals were present simultaneously; that is, when cells growing in high osmolarity medium have reached stationary phase. Deletion analysis narrowed the sequences necessary for P2 regulation to the 42-bp region upstream from the transcription start site. A 7-bp sequence just upstream from the -35 region was identified as a cis-acting regulatory element essential for osmotic stimulation of osmB expression. A hexanucleotide sequence within this segment could form the left arm of a region of dyad symmetry, flanking the -35 region of the promoter. Stationary phase induction at P2 does not require the 7-bp element.

Glycine betaine, which functions as an osmoprotectant, is accumulated to high intracellular concentrations in Escherichia coli at high osmolarity. We demonstrate the presence of a high-affinity, binding protein dependent transport system for glycine betaine, which is encoded by the proU region. We show the osmotically regulated synthesis of a 32 kDa periplasmic protein that is a glycine betaine binding protein with a KD of 1.4 microM. ProU-mediated glycine betaine transport is osmotically stimulated at the level of gene expression. The osmolarity of the medium also regulates the activity of the transport system, while binding of glycine betaine to its binding protein is independent of the osmolarity. We also find a second glycine betaine transport system that is dependent on proP and exhibits a lower substrate affinity. Like ProU, this system is regulated at two levels: both gene expression and the activity of the transport system are osmotically stimulated. Using lambda plac Mu-generated lacZ operon and gene fusions, we find that expression of the proU region is osmotically regulated at the level of transcription. We cloned a part of the proU region together with the phi(proU-lacZ)hyb2 gene fusion into a multicopy plasmid and show that the DNA sequences required in cis for osmotic regulation are present on the plasmid.

A major component of the Escherichia coli response to elevated medium osmolarity is the synthesis of a periplasmic protein with an Mr of 31,000. The protein was absent in mutants with lambda placMu insertions in the proU region, a locus involved in transport of the osmoprotectant glycine betaine. This periplasmic protein has now been purified to homogeneity. Antibody directed against the purified periplasmic protein crossreacts with the fusion protein produced as a result of the lambda placMu insertion, indicating that proU is the structural gene specifying the 31-kDa protein. The purified protein binds glycine betaine with high affinity but has no affinity for either proline or choline, clarifying the role of proU in osmoprotectant transport. The amino-terminal sequence of the mature glycine betaine binding protein is Ala-Asp-Leu-Pro-Gly-Lys-Gly-Ile-Thr-Val-Asn-Pro.

Induction of the lac operon in wild type Escherichia coli strains results in synthesis of a 16-kDa inner membrane protein in addition to the known products of the lacZ, lacY, and lacA genes. Cells carrying the lacY gene on a multicopy plasmid overproduce this 16-kDa polypeptide as well as the Lac carrier, the membrane protein product of the lacY gene. However, [35S]methionine labeling of minicells carrying the lacY plasmid shows that the 16-kDa protein is not synthesized from the plasmid DNA. We have purified and partially characterized the 16-kDa protein. It is an acidic membrane protein of apparent Mr = 15,800 whose amino-terminal sequence (NH2-Met-Arg-Asn-Phe-Asp-Leu-) does not match any known lac operon DNA sequence. Using antibody prepared to the purified 16-kDa protein, we have quantitatively analyzed conditions under which this protein is made and have shown that the amount of 16-kDa protein which appears in the membrane is proportional to lac operon expression. Hybridization of a synthetic oligodeoxyribonucleotide probe complementary to the 5' end of 16-kDa protein mRNA shows that its synthesis is regulated at the level of transcription.

Osmoregulated expression of proU has been reconstituted in a cell-free system. proU encodes an osmotically inducible, high-affinity transport system for the osmoprotectant glycine betaine in Escherichia coli. Previously, a proU-lacZ fusion gene had been cloned, resulting in plasmid pOS3. In vivo osmoregulation of this extrachromosomal proU-lacZ fusion gene at low copy number showed that the plasmid-encoded fusion contained all the necessary sequences in cis for correctly receiving osmoregulatory signals during induction by osmotic stress and repression by glycine betaine. Using a cell-free (S-30) extract, plasmid pOS3 was then used to program protein synthesis in vitro. The ionic compound potassium glutamate specifically stimulated proU-lacZ expression in a concentration-dependent manner. Potassium acetate also induced some proU expression, but other salts were ineffective, thereby ruling out ionic strength as the stimulatory signal. High concentrations of sucrose, trehalose, or glycine betaine did not induce proU expression in vitro either, eliminating osmolarity per se as the stimulus. Reconstitution in a cell-free system rules out osmoregulatory mechanisms that depend on turgor, trans-membrane signaling, or trans-acting regulators synthesized after osmotic upshock.

Alkaline phosphatase, the phoA product, is synthesized constitutively in phoR mutants. This constitutive synthesis, which is independent of phosphate control, varies with changes in the osmolarity of the growth medium; phoA expression increases with increasing osmolarity. Maximum expression of the osmoregulated genes phoA, ompC, and ompF was achieved by osmotic manipulation of minimal medium; complex media repressed their expression.

Expression of a particular set of exported protein genes (ompF, ompC, phoA, and malE) can be specifically altered in three ways: variation in the osmotic strength of the growth medium, mutations in the regulatory locus envZ, or treatment with sublethal concentrations of the local anesthetic procaine. To clarify relationships among these factors in the regulation of transcription, expression of the affected genes was compared in envZ+ and envZ22(Am) mutant strains grown in media of differing osmolarities with and without procaine. Loss of the envZ product resulted in complete resistance of gene expression to procaine, supporting the hypothesis that envZ mediates procaine inhibition. The specific activity of the phoA product, alkaline phosphatase, was elevated in envZ22 mutant strains, while the amounts of both the OmpC and OmpF porins were reduced. Osmotic control of phoA and ompC was retained in the absence of envZ function, but osmoregulation of ompF was lost. Therefore, the envZ product is somehow involved in the complex regulation of all four target genes, but is not solely responsible for osmoregulation.

Adaptation to osmotic stress alters the amounts of several specific proteins in the Escherichia coli K-12 envelope. The most striking feature of the response to elevated osmolarity was the strong induction of a periplasmic protein with an Mr of 31,000. This protein was absent in mutants with lambda plac Mu insertions in an osmotically inducible locus mapping near 58 min. The insertions are likely to be in proU, a locus encoding a transport activity for the osmoprotectants glycine betaine and proline. Factors affecting the extent of proU induction were identified by direct examination of periplasmic proteins on sodium dodecyl sulfate gels and by measuring beta-galactosidase activity from proU-lac fusions. Expression was stimulated by increasing additions of salt or sucrose to minimal medium, up to a maximum at 0.5 M NaCl. Exogenous glycine betaine acted as an osmoregulatory signal; its addition to the high-osmolarity medium substantially repressed the expression of the 31,000-dalton periplasmic protein and the proU-lac+ fusions. Elevated osmolarity also caused the appearance of a second periplasmic protein (Mr = 16,000), and severe reduction in the amounts of two others. In the outer membrane, the well-characterized repression of OmpF by high osmolarity was observed and was reversed by glycine betaine. Additional changes in membrane composition were also responsive to glycine betaine regulation.

Maltose and lactose transport systems have been used to investigate the action of procaine on insertion and activity of membrane proteins and translocation of exported proteins in Escherichia coli. Procaine mildly inhibited growth on lactose. The level of inhibition was consistent with the small reduction observed in active and facilitated transport functions of the lac permease. However, procaine caused a severe reduction of growth rate on maltose, as well as an inhibition of induction of maltose regulon activities. In both constitutive and inducible strains, the synthesis of both maltose transport activity (malB operon) and amylomaltase activity (malA operon) was inhibited. Coordinate inhibition of soluble and membrane products was not observed with the lac operon. beta-Galactosidase synthesis proceeded normally during growth on procaine, whereas, the appearance of new transport activity was reduced. Regardless of carbon source, procaine specifically inhibited the appearance of ompF protein in the membrane fraction.

Parallel studies have been made of the protein coats of the temperate bacteriophage lambda and of a deletion mutant, lambda virulent. A new method for preparing ghosts of both phages by the action of Cu(++) is described. Protein ghosts of both phages can be dissolved in citrate at pH values below 3, more rapidly in the presence of 8 m urea. Both phages yielded three apparently identical protein components which can be separated by thin-layer gel filtration and thin-layer gel electrophoresis. The protein of molecular weight 47,000 +/- 1,500 represents about 55% of the protein of the ghosts and is therefore likely to be the subunit of the head. The other proteins of molecular weight 30,000 +/- 1,500 and 16,000 +/- 1,500 represent approximately 25% and 20% of the protein, respectively. Amino acid analyses of the ghosts from the two phages have been carried out and show no significant differences. The buoyant density of phage lambda virulent is 0.016 g/ml less than that of lambda. Since no differences have been found in the protein components of the two phages, this indicates that the virulent mutant contains approximately 16% less deoxyribonucleic acid than the temperate phage.

In vivo alpha-complementation of beta-galactosidase was demonstrated in 16 Z gene terminator (nonsense) mutant strains of Escherichia coli upon introduction of the episome F'M15 which specifies production of a mutant Z gene polypeptide containing a small deletion in the N-terminal region of the enzyme monomer. Genetic and biochemical analyses of the merodiploids showed that restoration of enzyme activity was due to their terminator/F'M15 genetic constitution resulting in the production of two enzymatically inactive polypeptides which associate in vivo to reconstitute active, stable beta-galactosidase. The prematurely terminated polypeptide fragments known to be rapidly degraded in haploid cells were shown by phenotypic and biochemical studies to be stabilized (i.e., protected) in merodiploids by formation of complemented enzyme complexes with the M15 protein. Phenotypic properties of complementing diploids are described and are discussed in relation to in vitro determination of beta-galactosidase activity.