Minus Phenotype Research Articles

The ColRS-Regulated Membrane Protein Gene XAC1347 Is Involved in Copper Homeostasis and hrp Gene Expression in Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (Xcc) is the major causal agent of citrus canker disease. The XAC1347 gene, which encodes a conserved membrane protein in Xcc, is required for virulence during infection. However, the molecular events mediated by XAC1347 remain unclear. In this study, we reported that XAC1347 gene is positively regulated by two component regulatory system ColRS and required for type III secretion system function. A non-polar deletion mutant of the XAC1347 gene resulted in a Hrp minus phenotype in plants and reduced copper homeostasis. Real-time PCR experiments indicated that XAC1347 gene is induced by copper ions. The expression levels of representative genes from four hrp operons, including hrpB1, hrcV, hrpF, and hrpD6, were reduced in XAC1347 mutant, indicating that XAC1347 is involved hrp gene expression.

Mutant generation by allelic exchange and genome resequencing of the biobutanol organism Clostridium acetobutylicum ATCC 824.

BackgroundClostridium acetobutylicum represents a paradigm chassis for the industrial production of the biofuel biobutanol and a focus for metabolic engineering. We have previously developed procedures for the creation of in-frame, marker-less deletion mutants in the pathogen Clostridium difficile based on the use of pyrE and codA genes as counter selection markers. In the current study we sought to test their suitability for use in C. acetobutylicum.ResultsBoth systems readily allowed the isolation of in-frame deletions of the C. acetobutylicum ATCC 824 spo0A and the cac824I genes, leading to a sporulation minus phenotype and improved transformation, respectively. The pyrE-based system was additionally used to inactivate a putative glycogen synthase (CA_C2239, glgA) and the pSOL1 amylase gene (CA_P0168, amyP), leading to lack of production of granulose and amylase, respectively. Their isolation provided the opportunity to make use of one of the key pyrE system advantages, the ability to rapidly complement mutations at appropriate gene dosages in the genome. In both cases, their phenotypes were restored in terms of production of granulose (glgA) and amylase (amyP). Genome re-sequencing of the ATCC 824 COSMIC consortium laboratory strain used revealed the presence of 177 SNVs and 49 Indels, including a 4916-bp deletion in the pSOL1 megaplasmid. A total of 175 SNVs and 48 Indels were subsequently shown to be present in an 824 strain re-acquired (Nov 2011) from the ATCC and are, therefore, most likely errors in the published genome sequence, NC_003030 (chromosome) and NC_001988 (pSOL1).ConclusionsThe codA or pyrE counter selection markers appear equally effective in isolating deletion mutants, but there is considerable merit in using a pyrE mutant as the host as, through the use of ACE (Allele-Coupled Exchange) vectors, mutants created (by whatever means) can be rapidly complemented concomitant with restoration of the pyrE allele. This avoids the phenotypic effects frequently observed with high copy number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention. Our study also revealed a surprising number of errors in the ATCC 824 genome sequence, while at the same time emphasising the need to re-sequence commonly used laboratory strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0410-0) contains supplementary material, which is available to authorized users.

MicroRNA Transgene Overexpression Complements Deficiency-Based Modifier Screens in Drosophila

Dosage-sensitive modifier screening is a powerful tool for linking genes to biological processes. Use of chromosomal deletions permits sampling the effects of removing groups of genes related by position on the chromosome. Here, we explore the use of inducible microRNA transgenes as a complement to deficiency-based modifier screens. miRNAs are predicted to have hundreds of targets. miRNA overexpression provides an efficient means to reduces expression of large gene sets. A collection of transgenes was prepared to allow overexpression of 89 miRNAs or miRNA clusters. These transgenes and a set of genomic deficiencies were screened for their ability to modify the bristle phenotype of the cell-cycle regulator minus. Sixteen miRNAs were identified as dominant suppressors, while the deficiency screen uncovered four genomic regions that contain a dominant suppressor. Comparing the genes uncovered by the deletions with predicted miRNA targets uncovered a small set of candidate suppressors. Two candidates were identified as suppressors of the minus phenotype, Cullin-4 and CG5199/Cut8. Additionally, we show that Cullin-4 acts through its substrate receptor Cdt2 to suppress the minus phenotype. We suggest that inducible microRNA transgenes are a useful complement to deficiency-based modifier screens.

The archaeal COG1901/DUF358 SPOUT-methyltransferase members, together with pseudouridine synthase Pus10, catalyze the formation of 1-methylpseudouridine at position 54 of tRNA

The methylation of pseudouridine (Ψ) at position 54 of tRNA, producing m(1)Ψ, is a hallmark of many archaeal species, but the specific methylase involved in the formation of this modification had yet to be characterized. A comparative genomics analysis had previously identified COG1901 (DUF358), part of the SPOUT superfamily, as a candidate for this missing methylase family. To test this prediction, the COG1901 encoding gene, HVO_1989, was deleted from the Haloferax volcanii genome. Analyses of modified base contents indicated that while m(1)Ψ was present in tRNA extracted from the wild-type strain, it was absent from tRNA extracted from the mutant strain. Expression of the gene encoding COG1901 from Halobacterium sp. NRC-1, VNG1980C, complemented the m(1)Ψ minus phenotype of the ΔHVO_1989 strain. This in vivo validation was extended with in vitro tests. Using the COG1901 recombinant enzyme from Methanocaldococcus jannaschii (Mj1640), purified enzyme Pus10 from M. jannaschii and full-size tRNA transcripts or TΨ-arm (17-mer) fragments as substrates, the sequential pathway of m(1)Ψ54 formation in Archaea was reconstituted. The methylation reaction is AdoMet dependent. The efficiency of the methylase reaction depended on the identity of the residue at position 55 of the TΨ-loop. The presence of Ψ55 allowed the efficient conversion of Ψ54 to m(1)Ψ54, whereas in the presence of C55, the reaction was rather inefficient and no methylation reaction occurred if a purine was present at this position. These results led to renaming the Archaeal COG1901 members as TrmY proteins.

Mutations in lipopolysaccharide biosynthetic genes impair maize rhizosphere and root colonization of Rhizobium tropici CIAT899

Three transposon mutants of Rhizobium tropici CIAT899 affected in lipopolysaccharide (LPS) biosynthesis were characterized and their maize rhizosphere and endophytic root colonization abilities were evaluated. The disrupted genes coded for the following putative products: the ATPase component of an O antigen ABC-2 type transporter (wzt), a nucleotide-sugar dehydratase (lpsbeta2) and a bifunctional enzyme producing GDP-mannose (noeJ). Electrophoretic analysis of affinity purified LPS showed that all mutants lacked the smooth LPS bands indicating an O antigen minus phenotype. In the noeJ mutant, the rough LPS band migrated faster than the parental band, suggesting a truncated LPS core. When inoculated individually, the wzt and noeJ mutants colonize the rhizosphere and root to a lower extent than the parental strain while no differences were observed between the lpsbeta2 mutant and the parental strain. All mutants were impaired in competitive rhizosphere and root colonization. Pleiotropic effects of the mutations on known colonization traits such as motility and growth rate were observed, but they were not sufficient to explain the colonization behaviours. It was found that the LPS mutants were sensitive to the maize antimicrobial 6-methoxy-2-benzoxazolinone (MBOA). Only the combined effects of altered growth rate and susceptibility to maize antimicrobials could account for all the observed colonization phenotypes. The results suggest an involvement of the LPS in protecting R. tropici against maize defence response during rhizosphere and root colonization.

Biological characterization of bovine herpesvirus 1 recombinants possessing the vaccine glycoprotein E negative phenotype

Intramolecular recombination is a frequent event during the replication cycle of bovine herpesvirus 1 (BoHV-1). Recombinant viruses frequently arise and survive in cattle after concomitant nasal infections with two BoHV-1 mutants. The consequences of this process, related to herpesvirus evolution, have to be assessed in the context of large use of live marker vaccines based on glycoprotein E (gE) gene deletion. In natural conditions, double nasal infections by vaccine and wild-type strains are likely to occur. This situation might generate virulent recombinant viruses inducing a serological response indistinguishable from the vaccine one. This question was addressed by generating in vitro BoHV-1 recombinants deleted in the gE gene from seven wild-type BoHV-1 strains and one mutant strain deleted in the genes encoding gC and gE. In vitro growth properties were assessed by virus production, one step growth kinetics and plaque size assay. Heterogeneity in the biological properties was shown among the investigated recombinant viruses. The results demonstrated that some recombinants, in spite of their gE minus phenotype, have biological characteristics close to wild-type BoHV-1.

Mak5p, which is required for the maintenance of the M1 dsRNA virus, is encoded by the yeast ORF YBR142w and is involved in the biogenesis of the 60S subunit of the ribosome.

In this study, we show that the Saccharomyces cerevisiae ORF YBR142w, which encodes a putative DEAD-box RNA helicase, corresponds to MAK5. The mak5-1 allele is deficient in the maintenance of the M1 dsRNA virus, resulting in a killer minus phenotype. This allele carries two mutations, G218D in the conserved ATPase A-motif and P618S in a non-conserved region. We have separated these mutations and shown that it is the G218D mutation that is responsible for the killer minus phenotype. Mak5p is an essential nucleolar protein; depletion of the protein leads to a reduction in the level of 60S ribosomal subunits, the appearance of half-mer polysomes, and a delay in production of the mature 25S and 5.8S rRNAs. Thus, Mak5p is involved in the biogenesis of 60S ribosomal subunits.

Precore Wild-Type Hepatitis B Virus with G1896 in the Resolution of Persistent Hepatitis B Virus Infection

Objective: Factors influencing the resolution of persistent hepatitis B virus (HBV) infection were sought for. Methods: The loss of hepatitis B surface antigen (HBsAg) from serum was correlated with mutations in HBV DNA for a hepatitis B e antigen (HBeAg)-minus phenotype in patients infected with HBV genotype C and positive for HBeAg at presentation. Results: HBeAg turned negative in all the 22 patients in whom HBV infection resolved, but only in 11 of the 25 patients with severe liver diseases (100 vs. 44%, p = 0.0001). The precore wild type (G1896) persisted significantly more frequently in the 22 patients in whom HBV infection resolved than in the 11 patients who developed decompensated liver cirrhosis or hepatocellular carcinoma (15/22 or 68% vs. 1/11 or 9%, p = 0.005). The double mutation in the core promoter (T1762/A1764) was comparably frequent in the two groups of patients at presentation (14/22 or 64% vs. 7/11 or 64%) and >15 years thereafter (18/22 or 82% vs. 10/11 or 91%). Conclusion: The precore wild type (G1896) would seem to facilitate the resolution of HBV infection, while the precore mutant (A1896) may induce severe liver diseases in patients with HBeAg-positive chronic hepatitis who have lost HBeAg from serum.

The linoleic acid metabolite, 13-HpODE augments the phosphorylation of EGF receptor and SHP-2 leading to their increased association

In previous studies with Syrian hamster embryo fibroblasts, we found that a specific lipoxygenase metabolite of linoleic acid, 13(S)-hydroperoxyoctadecadienoic acid (HpODE), enhanced epidermal growth factor (EGF) signal transduction in a tumor suppressor gene plus phenotype (supB+); with a diminished response to 13(S)-HpODE in a tumor suppressor gene minus phenotype (supB−). This differential response was attributed to differences in the rate of EGF receptor (EGFR) dephosphorylation. To further define the molecular basis for these observations, in this report we examine the interaction of phosphorylated EGFR with the SH2 domain-containing protein tyrosine phosphatase, SHP-2, a positive modulator of EGF dependent cell growth. SHP-2 associated with phosphorylated EGFR to a greater extent in supB+cells when compared to supB−. This differential association could not be accounted for by differences between suppressor gene phenotypes in SHP-2 protein level or mutations in the molecular sequence. The addition of 13(S)-HpODE stimulated a concentration-dependent increase in EGF-dependent phosphorylation of SHP-2 and its association with EGFR. A more dramatic response was observed in the supB+cells. Differences in SHP-2 interaction with EGFR may account, in part, for phenotypic differences in the growth rates and responsiveness to EGF between the supB+and supB−cells. EGFR-SHP-2 association appears to play an important role in the regulation of EGFR signal transduction.The abbreviations used are: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; SHE, Syrian hamster embryo fibroblasts; HpODE, hydroperoxyoctadecadienoic acid; supB+, tumor suppressor gene (+) phenotype; supB−, tumor suppressor gene (−) phenotype; MAPK, mitogen-activated protein kinase; ECL, enhanced chemiluminescence; PBS cmf, phosphate-buffered saline, calcium/magnesium free; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; GAP, GTPase activating protein; EGTA, ethyleneglycol-bis-(β-amino ethyl ether)N,N,N′,N′-tetraacetic acid; PTP, protein tyrosine phosphatase; SH2,src -homology domain 2; SHP-2, SH2 domain-containing tyrosine phosphatase-2.

Evidence for two chemosensory pathways in Rhodobacter sphaeroides.

In contrast to enteric bacteria, chemotaxis in Rhodobacter sphaeroides requires transport and partial metabolism of chemoattractants. Although a chemotaxis operon has been identified containing homologues of the enteric cheA, cheW, cheR genes and two homologues of the cheY gene, deletion of the entire chemotaxis operon had only minor effects on chemotactic behaviour under the conditions tested. Responses to sugars were enhanced in tethered cells but in all other chemotaxis assays behaviour of the operon deletion mutant was wild type. The mutant also showed wild-type responses to weak organic acids such as acetate and propionate, the dominant chemoattractants for this organism, under all conditions. This is in direct contrast to the enterics in which CheA, CheW and CheY are absolutely essential for taxis to PTS sugars, oxygen and MCP-dependent chemoeffectors. The operon deletion mutant was subjected to Tn5 transposon mutagenesis and new mutants selected using a chemotaxis and phototaxis screen. One mutant, JPA203, was non-chemotactic on swarm plates and showed inverted responses when tethered or subjected to changes in light intensity. Characterization of the Tn5 insertion in JPA203 identified a second chemotaxis operon in R. sphaeroides that contains homologues of cheY, cheA and cheR, the first homologue of cheB and two homologues of cheW. The new genes were labelled orf10, cheY(III), cheA(II) cheW(II), cheW(III), cheR(II), cheB and tlpC. When introduced into a wild-type background, deletion of cheA(II) produced a chemotaxis minus phenotype in R. sphaeroides, suggesting that cheA(II) forms part of a dominant chemotactic pathway, whereas the earlier identified operon plays only a minor role under laboratory conditions. The data presented here support the existence of two chemosensory pathways in R. sphaeroides, a feature that so far is unique in bacterial chemotaxis.

The second finger of Urbs1 is required for iron-mediated repression of sid1 in Ustilago maydis.

The urbs1 gene encodes a transcriptional regulator of siderophore biosynthesis in Ustilago maydis. Biological and DNA-binding activities of the two putative zinc-finger motifs of Urbs1 were studied by analyzing mutants containing altered finger domains. The mutated urbs1 alleles from three previously described N'-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutants were mapped and cloned by a gap-repair procedure. Sequence analyses revealed single amino acid substitutions in two of the NTG mutants. Both mutations (G-507 to D in urbs1-1 and P-491 to L in urbs1-3), which are located in the Urbs1 C-terminal finger domain, reduced DNA-binding activity by 10-fold and were sufficient to confer a urbs1-minus phenotype. The third NTG urbs1 mutant (urbs1-2) also contained a mutation in one of the conserved amino acids (P-518 to S) in the C-terminal finger domain, but this mutation alone was not sufficient to confer a urbs1-minus phenotype. A second frame shift mutation was identified in urbs1-2 and is necessary for the urbs1-minus phenotype. In an analysis of the function of the N-terminal finger of Urbs1, the conserved amino acid Arg-350 was mutated to leucine. A Urbs1 protein with this mutation complemented a urbs1 null mutant strain. By contrast, a similar mutation in the C-terminal domain abolished the ability of Urbs1 to regulate siderophore biosynthesis and greatly reduced its ability to bind target DNA.

Identification of genes encoding for peptide synthetases in the gram-negative bacterium Lysobacter sp. ATCC 53042 and the fungus Cylindrotrichum oligospermum.

Genes encoding for the multifunctional peptide synthetases lysobactin synthetase and peptolide SDZ 214-103 synthetase were identified by hybridization of genomic libraries with oligonucleotides derived from consensus motifs of various genes encoding for delta-(L-alpha amino-adipoyl)-L-cysteinyl-D-valine (ACV) synthetases and gramicidin S synthetase. The sequence of subcloned gene fragments revealed core motifs and a modular structure typical for the family of peptide synthetase genes. A fragment of 4.6 kb of the lysobactin synthetase gene was sequenced and one amino acid activating module was localized. The cloning of lysobactin synthetase was verified by marker-exchange mutagenesis and the lysobactin minus phenotype of the mutant. The sequenced 3.1 kb fragment of peptolide SDZ 214-103 synthetase contained parts of two modules and was highly homologous to corresponding regions of module 6 and 7 of cyclosporin synthetase. Therefore, the localized modules may activate the amino acids threonine and glycine.

DNA repair mechanisms and their biologic implications in mammalian cells: Report of a NATO advanced research workshop, Fontevraud, France, Oct. 2–7, 1988

Repair of Alkylation Damage in DNA.- Structure-Function Relationships in Alkylation Damage and Repair.- The Roles of Beta- and Delta-Elimination in the Repair of AP Sites in DNA.- Imidazole Ring-Opened Purines: Occurence and Repair in Escherichia coli and Mammalian Cells.- Substrate Specificity of the Formamidopyrimidine-DNA Glycosylase of E. coli: Repair of the Imidazole Ring-Opened Form of N-Hydroxy-2-Amino-Fluorene-Guanine Adducts in DNA.- Investigation of Sequence Specificity in DNA Alkylation and Repair Using Oligodeoxynucleotide Substrates.- Towards a Role for Promutagenic Lesions in Carcinogenesis.- Acquisition of Resistance to Alkylating Agents by Expression of Methyltransferase Gene in Repair-Deficient Human Cells.- The Mer Minus Phenotype, Patient Response to Nitrosoureas, and Protooncogene Activation in Human Glioblastomas.- The Molecular Basis of Alkylating Agent Resistance in Mammalian Cells.- Differential Repair of O4-Methylthymine and O6-Methylguanine in Rat and Hamster Liver.- Formation of 1, N6-Ethenodeoxyadenosine and 3, N4-Ethenodeoxycytidine in DNA from Several Organs of Rats Exposed to Vinyl Chloride.- The Role of O6-Alkylguanine in Cell Killing and Sister Chromatid Exchange Induced by Alkylating Agents.- Inducible Responses to DNA Damage.- Inducible Repair in Mammalian Cells Treated with Alkylating Agents.- An Update of the Mammalian UV Response: Gene Regulation and Induction of a Protective Function.- DNA Repair Related Events.- The Transportation of AP Endonuclease from Cytoplasm to Nucleus in Mammalian Cells.- DNA Damage and Oxygen Species.- Mutagenesis.- Molecular Studies of Mutagenesis using Plasmid Vectors in Xeroderma Pigmentosum Cells.- Molecular Analysis of Mutations at the HPRT Locus: UV Mutation Spectra in Normal and UV-Sensitive V79 Chinese Hamster Cells.- Comparison of the Mutational Specificities Exhibited by BPDE in Escherichia coli and CHO Cells.- Deoxyribonucleotide Pools, DNA Repair and Mutagenesis.- Recombination and Mismatch Repair.- Duplex-Duplex Homologous Recombination Catalysed by a Human Nuclear Extract. Involvement in Double-Strand Break Repair.- Proteins from Yeast and Human Cells Specific for Model Holliday Junctions in DNA.- Characterization of a Strand Transferase Activity from Human Cells.- Mismatch Repair in Mammalian Cells: Approaches to the In Vitro Study of DNA Mismatch Correction Reactions.- Mismatch Repair Patterns in Simian Cells Correlate with the Specificity of a Mismatch Binding Protein Isolated from Simian and HeLa Cells.- Chromatin Proteins and Chromatin Structure in DNA Repair.- DNA Excision Repair at the Nucleosome Level of Chromatin.- Modulation of Activity of Human Chromatin-Associated Endonucleases on Damaged DNA by Nucleosome Structure.- Relationship Between DNA Repair and Transcription in Defined DNA Sequences in Mammalian Cells.- Non-Random Distribution of UV-Induced Repair in Higher-Order Chromatin Loops in Human Cells and its Relationship to Preferential Repair of Active Genes.- Electron Microscopic Visualization of Chromatin Structure at Sites of DNA Excision Repair Following Ultraviolet Irradiation of Cultured Human Fibroblasts.- Poly(ADP-Ribosyl)ation Reactions and Modulation of Chromatin Structure.- Variation of Nuclear ADP-Ribosyl Transferase in Rat Liver Carcinogenesis and in Synchronized HeLa Cells.- DNA Repair Processes in Mutant Cells Deficient in Poly(ADP-Ribose) Synthesis.- Repair Defective Cell Types.- Co-Recessive Inheritance: A Model for Diseases Associated with Defective DNA Repair.- Mammalian DNA Ligases and the Molecular Defect in Bloom's Syndrome.- Detection of Unique Antigenic Lesions in the Uracil DNA Glycosylase from Bloom's Syndrome.- DNA Ligase Activity in Human Cells from Normal Donors and from Patients with Bloom's Syndrome and Fanconi's Anemia.- Processing of Photoinduced Cross-Links and Monoadducts in Human Cell DNA: Genetic and Molecular Features.- Parallels Between Nucleotide Excision Repair in Human Cells and E. coli.- Mechanisms of Resistance to Ionising Radiations: Genetic and Molecular Studies on Ataxia-Telangiectasia and Related Radiation-Sensitive Mutants.- Defective DNA Synthesis and/or Repair in Amyotrophic Lateral Sclerosis.- Genetic Analysis in Trichothiodystrophy Repair Deficient Cells Confirms the Occurrence of Xeroderma Pigmentosum Group D Mutation in Unrelated Patients.- Chinese Hamster Cell Lines Defective in DNA Repair.- DNA Repair Genes and Oncogenes in DNA Repair.- Human DNA Repair and Recombination Genes.- Molecular Genetic Dissection of Mammalian Excision Repair.- The Cloning and Characterization of a Candidate Gene for the Correction of the Xeroderma D Defect.- Characterization of Chinese Hamster Ovary Cell Mutants Hypersensitive to DNA Damaging Agents.- Oncogene Activation in Xeroderma Pigmentosum Skin Tumors.- Anticancer Drugs in DNA Repair.- Heterogeneity in the O6-Alkylguanine DNA Alkyl-Transferase (AGT) Activity of Human Peripheral Blood Lymphocytes (PBL's).- Formation and Repair of Adducts that Lead to Cross-Links in DNA Treated with Chloro-Ethylating Agents.- Induction of Futile DNA Repair Processing by Bifunctional Intercalators.- Participants.- Author Index.

A Rhodobacter sphaeroides puf L, M and X deletion mutant and its complementation in trans with a 5.3 kb puf operon shuttle fragment

A Rhodobacter sphaeroides puf L, M and X deletion mutant was constructed using interposon mutagenesis. The puf L, M and X genes were replaced with a kanamycin resistance cartridge isolated from the transposon Tn5. The deletion strain PUF big up tri, openLMX 21 did not grow photoheterotrophically and was resistant to kanamycin. Southern blot analysis of genomic DNA from the deletion strain confirmed that the kanamycin resistance was inserted specifically into the puf operon and that the L, M and X genes were deleted. A spontaneous carotenoid mutant of PUF big up tri, openLMX was selected and was found to accumulate primarily neurosporene. Spectroscopic analysis of chromatophores isolated from the deletion strain showed normal B875 and B800-850 expression providing further evidence that the photosynthetic minus phenotype was not the result of insertional inactivation of the promoter region of the puf operon, or the puf Q region. The deletion strain could be returned to the photosynthetic plus phenotype by complementation in trans with a 5.3 kb puf operon shuttle fragment, although the generation time of the complemented strain was 30% longer than wild type.

Selection for virulence in the fish pathogen Aeromonas salmonicida, using Coomassie Brilliant Blue agar.

Coomassie Brilliant Blue Agar was used to quantify the frequency of the A-layer phenotype in different isolates of Aeromonas salmonicida. Hydrophilic, non-clumping isolates of A. salmonicida consisted predominantly of the A-layer minus phenotype. These bacteria were avirulent by intraperitoneal injection into susceptible brook trout (Salvelinus fontinalis) and could not be reisolated from infected fish. By contrast, hydrophobic, clumping isolates were predominantly of the A-layer positive phenotype, highly virulent in brook trout, and easily recovered from dead or moribund fish. A-layer positive and negative clones of A. salmonicida were derived by plating bacteria on Coomassie Blue Agar. The plating showed clearly that Coomassie Blue Agar could be used as a highly selective in vitro screening method to reclaim the virulence of certain isolates of A. salmonicida having a relatively low percentage of A-layer positive phenotypes.

A new locus, pilE, required for the binding of type 1 piliated Escherichia coli to erhythrocytes

We have identified a new locus required for mediating the binding of type 1 piliated Escherichia coli cells to guinea pig erythrocytes. This locus, pilE, was discovered after restrition site mutagenesis of a cloned segment of DNA containing the chromosomal pil region from a clinical strain of E. coli. pilE Mutants failed to agglutinate guinea pig erythrocytes but expressed pili were morphologically and antigenically indistinguishable from the parental (pilE+) strain. Construction of a chromosomal pilE mutation in E. coli K-12 was accomplished by introducing a restriction fragment containing a pilE lesion into the chromosome of a recBC sbcB host strain. Mutations in pilE could be complimented in trans by the addition of a cloned segment of DNA containing the parental pilE locus. Lesions in any of the genes required for pilus assembly also produced a hemagglutination minus phenotype suggesting that both the product(s) specified by the pilE locus and pili were required for hemagglutination. Hemagglutination experiments using partially purified pili also supported this suggestion.

A new locus,pilE, required for the binding of type 1 piliatedEscherichia colito erhythrocytes

We have identified a new locus required for mediating the binding of type 1 piliated Escherichia coli cells to guinea pig erythrocytes. This locus, pilE, was discovered after restrition site mutagenesis of a cloned segment of DNA containing the chromosomal pil region from a clinical strain of E. coli. pilE Mutants failed to agglutinate guinea pig erythrocytes but expressed pili were morphologically and antigenically indistinguishable from the parental (pilE+) strain. Construction of a chromosomal pilE mutation in E. coli K-12 was accomplished by introducing a restriction fragment containing a pilE lesion into the chromosome of a recBC sbcB host strain. Mutations in pilE could be complimented in trans by the addition of a cloned segment of DNA containing the parental pilE locus. Lesions in any of the genes required for pilus assembly also produced a hemagglutination minus phenotype suggesting that both the product(s) specified by the pilE locus and pili were required for hemagglutination. Hemagglutination experiments using partially purified pili also supported this suggestion.

Transcription initiation sites within an IS2 insertion in a Gal-constitutive mutant of Escherichia coli.

Insertion of the insertion sequence Is2(I) directly before the galE gene of the galactose operon results in a Gal minus phenotype (1, 2). The Gal-constitutive allele galc200 (and its deletion derivative galc200 delta 31) arise from such a Gal minus mutant by the insertion of LS2(II) DNA within the LS2(I) sequence (3). We have transcribed in vitro a DNA template representing the IS2-galE region of galc200 delta 31. Gal-directed transcription initiates at two sites within the IS2(I) sequence, 51 and 52 bp from the IS2-galE junction. The promoter for these transcripts, Pgal200 delta 31, is composed of a novel joint between a -10 region from the IS2(I) DNA and a -35 region contributed by the IS2(II) insertion. No promoters intrinsic to the 121 bp of the IS2(II) sequence also present on the template were detected. The relevance of Pgal200 delta 31 to the Galc phenotype of galc200 and to general mechanisms for the constitutive expression of genes adjacent to IS2 is discussed.

Mutant hamster cells exhibiting a pleiotropic effect on carbohydrate metabolism.

Sixty-seven galactose negative (Gal minus) clones were established from survivors after treatment of Chinese hamster lung (V79) cells with 5-bromodeoxyuridine and black light. The mutational origin of these clonal isolates was inferred on the basis of the previously demonstrated mutagenic action of the combined treatment, persistence of the Gal minus phenotype in cell progeny in the absence of selection, conditional lethality of three isolates, interallelic complementation, and assignment of a homologous human gene to chromosome A2. In contrast to the parental cells, Gal minus mutants could not utilize exogenous galactose, mannose, fructose, galactose 1-phosphate, glucose 1-phosphate, or glucose 6-phosphate. Permeation of galactose into mutant cells appeared unimpaired. In intra- and interspecific cell hybrids, the mutation causing the Gal minus phenotype behaved like a recessive character. The pleiotropic nutritional response was due not to deficiency of any one specific enzyme in the Leloir pathway but to significant reduction in activities of phosphoglucomutase, NADP-dependent isocitrate dehydrogenase, and perhaps other enzymes. In temperature-sensitive Gal minus mutants grown at permissive temperature or in complementing intra- and interspecific cell hybrids, the activities of these enzymes were restored to normal levels, accompanied by a regained ability to use the particular hexoses or hexose monophosphates. We postulate that the change from Gal + to Gal minus phenotype in hamster cells could be due to mutations at a regulatory gene locus, or at a yet unknown locus with enzymic defect that causes secondary metabolic imbalances.

Evidence for binding protein-independent substrate translocation by the methylgalactoside transport system of Escherichia coli K12.

Three genes, mgl A, B, and C, are required for active transport of substrate by the methylgalactose permease of E. coli K12. We report here that only two of these genes are required for substrate translocation, as seen by the ability or inability of isogenic mgl mutants (referred to as Tra+ and Tra minus, respectively) to grow on methyl-beta-D-galactopyranoside, supplied as sole carbon source. Individual mutants of both the Tra+ and Tra minus classes exhibited no detectable intracellular accumulation of methyl-beta-D-galactopyranoside; thus, the Tra+ phenotype cannot be explained by the mutants' levels of residual active transport. The phosphotransferase (Pts), the beta-galactoside (LacY), and the arabinose (Ara E and Ara F) transport systems are not required for substrate translocation by Tra+ cells. The Tra+ phenotype was identified with mutants defective in the mgl B, locus of the galactose-binding protein, by genetic complementation; the Tra minus phenotype was observed with both mgl A and mgl C mutants. The conclusion that the galactose-binding protein is not required for substrate translocation was supported by direct assays of the mgl mutants' binding protein activity. Mutants capable of translocation all showed reduced galactose-binding protein activity; mutants incapable of translocation exhibited binding protein activity equal to that of the mgl+ parent.