Broad-spectrum biofilm inhibition by a secreted bacterial polysaccharide

The development of surface-attached biofilm bacterial communities is considered an important source of nosocomial infections. Recently, bacterial interference via signaling molecules and surface active compounds was shown to antagonize biofilm formation, suggesting that nonantibiotic molecules produced during competitive interactions between bacteria could be used for biofilm reduction. Hence, a better understanding of commensal/pathogen interactions within bacterial community could lead to an improved control of exogenous pathogens. To reveal adhesion or growth-related bacterial interference, we investigated interactions between uropathogenic and commensal Escherichia coli in mixed in vitro biofilms. We demonstrate here that the uropathogenic strain CFT073 and all E. coli expressing group II capsules release into their environment a soluble polysaccharide that induces physicochemical surface alterations, which prevent biofilm formation by a wide range of Gram-positive and Gram-negative bacteria. We show that the treatment of abiotic surfaces with group II capsular polysaccharides drastically reduces both initial adhesion and biofilm development by important nosocomial pathogens. These findings identify capsular polymers as antiadhesion bacterial interference molecules, which may prove to be of significance in the design of new strategies to limit biofilm formation on medical in dwelling devices.     

A novel mechanism for the acquisition of virulence by a human influenza A virus

Cleavage of the hemagglutinin (HA) molecule by proteases is a prerequisite for the infectivity of influenza A viruses. Here, we describe a novel mechanism of HA cleavage for a descendant of the 1918 pandemic strain of human influenza virus. We demonstrate that neuraminidase, the second major protein on the virion surface, binds and sequesters plasminogen, leading to higher local concentrations of this ubiquitous protease precursor and thus to increased cleavage of the HA. The structural basis of this unusual function of the neuraminidase molecule appears to be the presence of a carboxyl-terminal lysine and the absence of an oligosaccharide side chain at position 146 (N2 numbering). These findings suggest a means by which influenza A viruses, and perhaps other viruses as well, could become highly pathogenic in humans.     

Bactericidal group IIA phospholipase A2 in serum of patients with bacterial infections

Group IIA phospholipase A2 (PLA2-IIA) is a newly recognized antibacterial acute phase protein. The concentration of PLA2-IIA increases up to 500-fold in the blood plasma of patients with severe acute diseases, compared with healthy persons. Despite numerous studies, the exact roles of this enzyme in human diseases are unknown. This study investigated the antibacterial properties of PLA2-IIA in human acute phase serum. PLA2-IIA in serum samples of patients with bacterial infections was capable of killing 90% of Staphylococcus aureus and 99% of Listeria monocytogenes in vitro after incubation for 2 h. At concentrations found in normal human serum, PLA2-IIA killed 90% of L. monocytogenes but did not kill S. aureus or Escherichia coli. The bactericidal effects of acute phase and normal human serum were abolished after depletion of PLA2-IIA by immunoadsorption.     

Partial purification and characterization of a colony-stimulating factor secreted by a T lymphocyte clone

The mouse T lymphocyte clone L2 secretes a minimum of 10 lymphokine activities affecting at least 5 different target cells. Large amounts of colony-stimulating factor (CSF) (8.6 X 10(6) U/ml) can be obtained by stimulating L2 cells with concanavalin A. The major CSF activity secreted by L2 cells has been enriched to a specific activity of approximately 2-4 X 10(8) colonies/mg of protein using hydrophobic-interaction, gel-permeation, ion-exchange, and lectin-affinity chromatography. This preparation of CSF contains no detectable interleukin 2, interleukin 3, or interferon. The major L2-cell CSF induces granulocyte/macrophage colonies from bone marrow cells. This GM CSF has an apparent Mr of 22,000 as determined by gel-permeation chromatography. Treatment of L2-cell CSF with proteolytic enzymes abrogates biologic activity.     

A bacterial mutation blocking P2 phage late gene expression.

A mutant of Escherichia coli strain C has been isolated, called gro109, that blocks bacteriophage P2 propagation by interfering with late gene expression. DNA replication proceeds normally in P2+-infected gro109 cells, but late phage proteins are not made. Early P2 mRNA is made in normal amounts, but very little late mRNA can be detected. P2 mutants (P2 ogr) able to overcome the gro109 block have been isolated in which synthesis of late P2 mRNA and phage proteins Is restored. The gro109 mutation is closely linked to the cluster of ribosomal genes at 64 min and is recessive to the wild-type (gro+) allele. A P2 ogr mutation has been mapped on the left arm of the P2 genome, between the right-most known late gene (D) and the phage attachment site. P2 ogr can complement P2+ in gro109 cells, indicating that ogr codes for a diffusible product.     

A cDNA clone encoding a peptide highly specific for hepatitis C infection

A random primed lambda gtll-cDNA library was constructed from donors plasma presumably infected by blood-borne non-A, non-B hepatitis (hepatitis C:HC) agent and immunoscreened with serum pooled from patients with acute or chronic HC. Twelve lambda gtll-cDNA clones encoding antigens associated with HC infection in Japan as well as in the USA were isolated. Of these one clone consisting of 114 nucleotides and showing a discrete band on an immunoblot analysis, was extensively studied. The clone is not derived from the host DNA encoding one polypeptide specific and highly sensitive for serum from patients with HC and has no homology to the nucleotide sequences of known human viruses including hepatitis A, B and D viruses, Ebstein-Barr virus, coxsackievirus, immunodeficiency virus type 1 or Japanese encephalitis virus. These results suggest that this clone is derived from the genome of HC agent.     

Isolation of a cDNA clone encoding pancreatic polypeptide.

We have isolated a cDNA clone encoding pancreatic polypeptide from a cDNA library constructed with RNA from an endocrine neoplasm of the human pancreas. The cDNA was inserted into plasmid pBR322 and the plasmid was cloned in Escherichia coli. Oligonucleotides (sequence in text) specific for the amino acid sequence (sequence in text) of pancreatic polypeptide were used as hybridization probes. The pancreatic polypeptide cDNA isolated was 465 base pairs long and encoded a peptide of 95 amino acids in the coding region. The 36-amino acid sequence of pancreatic polypeptide was flanked by a 29-amino acid putative leader sequence at the amino terminus and a connecting tripeptide (Gly-Lys-Arg) followed by a 27-amino acid peptide at the carboxyl terminus. The first 20 of the amino acids in the carboxyl-terminal heptacosapeptide were identical to the structure of human pancreatic icosapeptide with the single exception of an isoleucine substitution for valine in the 18th position. This alteration results from an A----G substitution in the nucleotide sequence of the cDNA and may represent a genetic variation or a point mutation in the pancreatic polypeptide cDNA.     

Fabry disease: isolation of a cDNA clone encoding human alpha-galactosidase A.

Fabry disease is an X-linked inborn error of metabolism resulting from the deficient activity of the lysosomal hydrolase, alpha-galactosidase A (alpha-Gal A; alpha-D-galactoside galactohydrolase, EC 3.2.1.22). To investigate the structure, organization, and expression of alpha-Gal A, as well as the nature of mutations in Fabry disease, a clone encoding human alpha-Gal A was isolated from a lambda gt11 human liver cDNA expression library. To facilitate screening, an improved affinity purification procedure was used to obtain sufficient homogeneous enzyme for production of monospecific antibodies and for amino-terminal and peptide microsequencing. On the basis of an amino-terminal sequence of 24 residues, two sets of oligonucleotide mixtures were synthesized corresponding to adjacent, but not overlapping, amino acid sequences. In addition, an oligonucleotide mixture was synthesized based on a sequence derived from an alpha-Gal A internal tryptic peptide isolated by reversed-phase HPLC. Four positive clones were initially identified by antibody screening of 1.4 X 10(7) plaques. Of these, only one clone (designated lambda AG18) demonstrated both antibody binding specificity by competition studies using homogeneous enzyme and specific hybridization to synthetic oligonucleotide mixtures corresponding to amino-terminal and internal amino acid sequences. Nucleotide sequencing of the 5' end of the 1250-base-pair EcoRI insert of clone lambda AG18 revealed an exact correspondence between the predicted and known amino-terminal amino acid sequence. The insert of clone lambda AG18 appears to contain the full-length coding region of the processed, enzymatically active alpha-Gal A, as well as sequences coding for five amino acids of the amino-terminal propeptide, which is posttranslationally cleaved during enzyme maturation.     

A cDNA clone encoding a peptide highly specific for hepatitis C infection.

A random primed lambda gt11-cDNA library was constructed from donors plasma presumably infected by blood-borne non-A, non-B hepatitis (hepatitis C:HC) agent and immunoscreened with serum pooled from patients with acute or chronic HC. Twelve lambda gt11-cDNA clones encoding antigens associated with HC infection in Japan as well as in the USA were isolated. Of these one clone consisting of 114 nucleotides and showing a discrete band on an immunoblot analysis, was extensively studied. The clone is not derived from the host DNA encoding one polypeptide specific and highly sensitive for serum from patients with HC and has no homology to the nucleotide sequences of known human viruses including hepatitis A,B and D viruses, Ebstein-Barr virus, coxsackievirus, immunodeficiency virus type 1 or Japanese encephalitis virus. These results suggest that this clone is derived from the genome of HC agent.     

Dendritic-cell activation by secretory phospholipase A2

Dendritic cells (DCs), also referred to as the sentinels of the immune system, induce and coordinate important functions of immune surveillance. DCs acquire immunity-initiating capacity only after a process of maturation usually induced by ligands that bind to members of the tumor necrosis factor (TNF) or toll-like receptor families. Secretory phospholipase A2 (sPLA2), which hydrolyzes the sn-2 ester bond of glycerophospholipids, regulates a variety of cellular functions including migration of endothelial cells and neurite outgrowth. In the present study we investigated the role of sPLA2 in DC biology. We report that human monocyte-derived DC cultures lack sPLA2 activity but respond to exogenous sPLA2. sPLA2 alone and in cooperation with TNF-alpha and interleukin 1 beta (IL-1beta) induced fatty acid release from DC membranes, which was accompanied by upregulation of surface markers and by an increase in the migratory and immunostimulatory capacity of the DCs. Our findings indicate that secreted enzymes such as sPLA2 can contribute to DC maturation and emphasize the role of lipid mediators in the regulation of immune responses. This observation may also have implications for DC-based vaccine development.     

Human group X secreted phospholipase A2 induces dendritic cell maturation through lipoprotein-dependent and -independent mechanisms

ObjectiveIncreased secreted phospholipase A₂ (sPLA₂) activity has been documented in several inflammatory disorders. Among sPLA₂s, the human group X (hGX)-sPLA₂ has the highest catalytic activity towards phosphatidylcholine (PC), the major phospholipid of cell membranes and blood lipoproteins. hGX-sPLA₂ has been detected in human atherosclerotic lesions, indicating that sPLA₂s are an important link between lipids and inflammation, both involved in atherosclerosis. The presence of dendritic cells (DC), the most potent antigen presenting cells, in atherosclerotic lesions has raised the question about their role in disease progression.Methods and resultsIn this study, we show that hGX-sPLA₂-treated LDL induces human monocyte-derived DC maturation, resulting in a characteristic mature DC phenotype and enhanced DC ability to activate IFNγ secretion from T cells. hGX-sPLA₂ phospholipolysis of LDL produces high levels of lipid mediators, such as lysophosphatidylcholine (LPC) and free fatty acids (FFAs), which also modulate DC maturation. The major molecular species of LPC containing a palmitic or stearic acid esterified in the sn-1 position induce DC maturation, whereas the FFAs can positively or negatively modulate DC maturation depending on their nature. hGX-sPLA₂ added alone can also activate DC in vitro through the hydrolysis of the DC membrane phospholipids leading, however, to a different cytokine profile secretion pattern than the one observed with hGX-sPLA₂-phospholipolysed LDL.ConclusionhGX-sPLA₂ secreted in inflamed tissues can contribute to local DC maturation, resulting in pro-Th1 cells, through the production of various lipid mediators from hydrolysis of either LDL and/or cell plasma membrane.     

Defective signal transduction induced by thromboxane A2 in a patient with a mild bleeding disorder: impaired phospholipase C activation despite normal phospholipase A2 activation

A patient with a mild bleeding disorder whose platelets responded defectively to thromboxane A2 (TXA2) was identified, and the mechanism of this dysfunction was analyzed. The platelets were defective in shape change, aggregation, and release reaction in response to synthetic TXA2 mimetic (STA2). When the platelet TXA2 receptor was examined with both a 125I-labeled derivative of a TXA2 receptor antagonist ([125I]-PTAOH) and [3H]-labeled TXA2 agonist ([3H]U-46619), the equilibrium dissociation rate constants (kd) and the maximal concentrations of binding sites (Bmax) of the platelets to both ligands were within normal ranges, suggesting that the binding capacity of their TXA2 receptor was normal. STA2 could not induce IP3 formation and intracellular Ca2+ mobilization, whereas these responses to thrombin were within normal ranges. GTPase activity was also decreased when the patient's platelet membrane was challenged with STA2. On the other hand, lysophosphatidylinositol formation, which is a direct indicator of phospholipase A2 (PLA2) activation, was found to be normal when the [3H]-inositol-labeled platelets were challenged with STA2. Thromboxane B2 (TXB2) was also produced in response to STA2. These results suggested that the abnormality in these platelets was impaired coupling between TXA2 receptor and phospholipase C (PLC) activation. Furthermore, it is also suggested that the activation of PLA2 and PLC are separable events in thromboxane-induced platelet activation.     

A cDNA clone encoding a photosystem I protein with homology to photosystem II chlorophyll a/b-binding polypeptides.

We report here the isolation and nucleotide sequence of a complete cDNA clone encoding a photosystem I (PS I) polypeptide that is recognized by a monoclonal antibody made against photosystem II (PS II) chlorophyll a/b-binding (CAB) proteins. The deduced sequence of this PS I protein shows 30% overall identity to PS II CAB sequences, and two long segments within this protein show 50% and 65% identity to the corresponding segments in the PS II CAB polypeptides. Even though the sequence of this PS I CAB protein is substantially divergent from PS II CAB sequences, their hydropathy plots are very similar and suggest they all traverse the thylakoid membrane three times. A segment of the PS I CAB polypeptide shows similarity to the functionally analogous beta subunits of the antenna proteins of purple bacteria. In contrast, no homology was observed between these bacterial proteins and PS II CAB polypeptides.     

Isolation and characterization of a cDNA clone encoding wheat germ agglutinin

Two sets of synthetic oligonucleotides coding for amino acids in the amino- and carboxyl-terminal portions of wheat germ agglutinin were synthesized and used as hybridization probes to screen cDNA libraries derived from developing embryos of tetraploid wheat. The nucleotide sequence for a cDNA clone recovered from the cDNA library was determined by dideoxynucleotide chain-termination sequencing in vector M13. The amino acid sequence deduced from the DNA sequence indicated that this cDNA clone (pNVR1) encodes isolectin 3 of wheat germ agglutinin. Comparison of the deduced amino acid sequence of clone pNVR1 with published sequences indicates isolectin 3 differs from isolectins 1 and 2 by 10 and 8 amino acid changes, respectively. In addition, the protein encoded by pNVR1 extends 15 amino acids beyond the carboxyl terminus of the published amino acid sequence for isolectins 1 and 2 and includes a potential site for N-linked glycosylation. Utilizing the insert of pNVR1 as a hybridization probe, we have demonstrated that the expression of genes for wheat germ agglutinin is modulated by exogenous abscisic acid. Striking homology is observed between wheat germ agglutinin and chitinase, both of which are proteins that bind chitin.     

Isolation of a genomic clone partially encoding human hypoxanthine phosphoribosyltransferase.

Mouse cells deficient in the enzyme hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) have been transfected with total human DNA, and cells producing human enzyme were isolated by growth in selective medium. DNA from several such cell lines has been used to generate secondary transfectants that make human HPRT. Blots of the DNA of these secondary cells have been hybridized with total human DNA probes or with cloned human Alu sequences, and one of several common bands has been cloned in pBR322. Colonies of transformed Escherichia coli containing human sequences were detected by their homology with human DNA, and subclones of resulting recombinant plasmids were prepared. Two subclones free of Alu sequences were found to contain human sequences that hybridized to human X chromosome DNA. One of these, pBR1.5, also hybridized to a single RNA band on gel blots of human and secondary transfectant cytoplasmic poly(A)+RNA but not to RNA from the parent mouse cell line. These results indicate that these clones represent human HPRT gene fragments. This has been confirmed by using pBR1.5 as a probe to isolate an authentic and expressible human HPRT cDNA clone from a library prepared by H. Okayama and P. Berg.     

Human alpha-galactosidase A: nucleotide sequence of a cDNA clone encoding the mature enzyme.

The complete nucleotide sequence has been determined for a lambda gt11 cDNA clone (lambda AG18) containing the full-length coding region for the mature lysosomal form of human alpha-galactosidase A (alpha-Gal A; EC 3.2.1.22). The lambda AG18 insert contained a 1226-base-pair sequence with an open reading frame encoding 398 amino acids of the mature polypeptide (predicted Mr = 45,356) and the last 5 amino acids of the propeptide sequence. The poly(A) signals AATACA and ATTAAA occurred 28 and 11 nucleotides prior to the TAA stop codon, respectively. There was no 3' untranslated region as the poly(A) sequence immediately followed the TAA termination codon; a second independently cloned cDNA confirmed this finding. The predicted amino acid sequence was colinear with 86 nonoverlapping residues (22% of the mature subunit) determined by microsequencing amino-terminal, tryptic, and cyanogen bromide peptides of the purified mature enzyme. Four potential N-glycosylation sites were identified, all of which occurred at predicted beta turns in hydrophilic regions of secondary structure. RNA transfer hybridization analysis of HeLa poly(A)+ RNA demonstrated a single 1.45-kilobase band whose signal was decreased by prior immunoabsorption of polysomes with monospecific alpha-Gal A antibodies. Searches of nucleic acid and protein data bases did not reveal significant homology even with the limited sequences available for mammalian lysosomal enzymes.     

Identification of secreted bacterial proteins by noncanonical amino acid tagging

Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.Many bacterial pathogens use elaborate secretion systems to transfer effector proteins into target cells (1). The injected proteins disrupt host cell functions, including cytoskeletal assembly and cytokine production, to promote infection (2). An important step in understanding virulence mechanisms is the identification of injected and secreted bacterial proteins. Traditional methods have included genetic screens and candidate protein approaches, which can be laborious and noncomprehensive. Proteome-wide labeling strategies offer the potential to rapidly identify secreted pathogen proteins without bias and with limited previous knowledge of host–pathogen interactions (3).We have developed a method, termed bio-orthogonal noncanonical amino acid tagging (BONCAT), for incorporating azide functional groups into proteins as a general strategy for the enrichment of newly synthesized cellular proteins, making it possible to elucidate the spatial and temporal character of proteomic changes (4, 5). Our initial studies used the noncanonical amino acid azidohomoalanine (Aha) (structure 2, Fig. 1A), a methionine (Met) surrogate, to label newly synthesized proteins (4, 5). The azide side chain of Aha allows newly synthesized proteins to be tagged with alkyne-functionalized affinity reagents and separated from preexisting proteins by affinity chromatography. After separation, proteins are identified by tandem MS. Enrichment of newly synthesized proteins reduces the complexity of the sample and facilitates identification of the proteins of interest.Open in a separate windowFig. 1.Incorporation of Anl into injected pathogen proteins and enrichment of labeled proteins. (A) Structures of amino acids Met (1) and Met analogs Aha (2) and Anl (3). The alkyne-functionalized biotin affinity probe (4) contains an acid-cleavable silane linker. The probe can be appended to the azide side chain of Anl. Cleavage with formic acid transfers a small mass tag to each modified Anl residue. (B) NLL-MetRS charges the tRNA^Met with Anl; its expression in the pathogen allows cell-selective labeling of pathogen proteins during infection. Injected pathogen proteins are enriched after labeling with 4 and identified by tandem MS. (C) Copper-catalyzed azide-alkyne cycloaddition yields a triazole linkage.We recently showed that introduction of a mutant form of the methionyl-tRNA synthetase (designated NLL-MetRS) into Escherichia coli enables incorporation of the noncanonical amino acid azidonorleucine (Anl) (structure 3, Fig. 1A) into the bacterial proteome (6). Because Anl is not activated to any significant extent by any of the WT synthetases (6), labeling is restricted to cells in which NLL-MetRS is expressed (7). This approach has prompted recent efforts to study proteomic changes in pathogens during infection (8). At the same time, there has been considerable interest in protein labeling strategies to study secreted pathogen proteins, most notably through the use of stable-isotope labeling of amino acids in cell culture (SILAC) (9, 10). Isotopic labeling does not allow enrichment of secreted pathogen proteins, however, and enrichment is important for identification of virulence factors that otherwise would go undetected among abundant host proteins. Here, using a shotgun, bottom-up proteomics approach, we show that noncanonical amino acid labeling enables enrichment of secreted virulence factors and identification of injected proteins from host cell lysates.Pathogenic bacteria secrete proteins through various mechanisms. Secretion via type III, type IV, and type VI systems occurs by direct injection of proteins into host cells, whereas type II and type V secretion systems use a two-step passage through the inner and outer membranes of the pathogen. Secreted outer membrane vesicles also mediate export of a complex array of proteins (11). We focus here on the well-characterized type III secretion system (T3SS) of Yersinia enterocolitica, a Gram-negative bacterium. In Yersinia, the majority of secreted proteins, designated Yersinia outer proteins (Yops), are encoded on the 70-kb virulence plasmid pYV (2, 12). In addition to encoding Yops, the plasmid encodes machinery consisting of needle-shaped structures that assemble on the bacterial surface and inject proteins into the cytoplasm of host cells. The T3SS is activated by a temperature shift from 26 °C to the host temperature (37 °C); injection is initiated on surface contact with target cells (13, 14). The pYV virulence plasmid also encodes a low calcium response (LCR) that enables secretion of T3SS substrates into the medium in the absence of host cells (15). As a control for type III secretion, we used a YscU mutant strain (designated T3SS-Mut), which is unable to secrete Yops (16). YscU is an inner membrane protein required for T3SS assembly and recruitment of substrates (17).In this study, NLL-MetRS was introduced to both WT and mutant Yersinia strains to enable selective Anl labeling of bacterial proteins (Fig. 1B). Because host cells do not express NLL-MetRS, host cell proteins are not labeled with Anl. After Anl was added to the infection medium, Anl-labeled proteins were tagged by copper-catalyzed cycloaddition (18) (Fig. 1C) with alkyne-functionalized dyes and detected by in-gel fluorescence or by confocal fluorescence imaging of infected host cells. Similarly, enrichment of Anl-labeled proteins was performed after attachment of a cleavable affinity tag (structure 4, Fig. 1A) that permits binding of labeled proteins to immobilized streptavidin resin and removal of unlabeled proteins. The small mass modification resulting from tagging of Anl residues is readily detected by MS, thereby facilitating identification of enriched proteins (SI Appendix, Figs. S1 and S2).In a HeLa cell infection model, we identified the Yersinia proteins that were secreted into the medium and injected into HeLa cells. In addition to identifying known Yops, we identified secreted proteins that may play important roles in Yersinia infection. An extension of this approach allowed us to selectively label proteins secreted by Yersinia that had invaded HeLa cells and to reveal secretion of distinct subsets of virulence factors. Pulse-labeling with Anl was used to investigate the order of injection of type III substrates into HeLa cells, providing a simple method to determine the hierarchy of injection of virulence factors. The approach described here is not limited to the study of T3SS substrates, but can be used to examine the many different secretion systems of microbial pathogens.     

Symbiosis-specific expression of Rhizobium etli casA encoding a secreted calmodulin-related protein

Symbiosis between Rhizobium and its leguminous host requires elaborate communication between the partners throughout the interaction process. A calmodulin-like protein, termed calsymin, was identified in Rhizobium etli; a calmodulin-related protein in a Gram-negative bacterium had not been described previously. Calsymin possesses three repeated homologous domains. Each domain contains two predicted EF-hand Ca(2+)-binding motifs. Ca(2+)-binding activity of calsymin was demonstrated on purified protein. R. etli efficiently secretes calsymin without N-terminal cleavage of the protein. The gene encoding calsymin, casA, is exclusively expressed during colonization and infection of R. etli with the host. Expression of casA is controlled by a repressor protein, termed CasR, belonging to the TetR family of regulatory proteins. Mutation of the casA gene affects the development of bacteroids during symbiosis and symbiotic nitrogen fixation.     

Isolation of a cDNA clone encoding a biologically active thyroid hormone receptor.

We have isolated a c-erbA cDNA clone from a GH3 cell library. The clone, denoted erb62, is 4.5 kilobases long and encodes a 461-amino acid beta-type c-erbA protein. This c-erbA protein binds 3,5,3'-triiodothyronine (T3) and T3 analogs with affinities similar to those of the authentic T3 receptor. By RNA gel blot analysis, erb62 hybridizes to a 6-kilobase RNA found in organs that express T3 receptors--e.g., heart, kidney, and brain. A COS-cell transient cotransfection system was used to show that erb62 encodes a biologically active T3 receptor. An oligonucleotide, corresponding to a portion of the rat growth hormone gene 5'-flanking region that contains a T3 response element, was inserted on the 5' side of the herpes simplex virus thymidine kinase promoter in a chloramphenicol acetyltransferase-expressing plasmid. Reporter gene expression directed by this hybrid promoter was T3 inducible only if this plasmid was cotransfected with an erb62-expressing plasmid.