Alexander Hollaender

Genetic Engineering of Microorganisms for Chemicals

Selection of petite strains of yeast (that is, strains unable to respire aerobically) on media containing allyl alcohol will result in enrichment for mutants at the ADCl locus. This locus codes for the constitutive alcohol dehydrogenase, ADH-I, which is primarily responsible for the production of ethanol in yeast. The mutant enzymes are functional, and confer resistance to allyl alcohol on the cell by shifting the NAD-NADH balance in the direction of NADH. These mutants exhibit altered Kms for cofactor, substrate, or both, and often have altered Vmaxs. In this paper, the methodology for obtaining these mutants and for determining the amino acid substitutions responsible for these changes is presented. Several new mutants have been at least approximately localized, and one, DB-AA3Nl5, has been shown to be due to the substitution of an arginine for a tryptophan at position 54. This substitution would be expected, by analogy with the known tertiary structure of the horse liver alcohol dehydrogenase, to decrease the hydrophobic environment of the active site pocket. The substitution has a pronounced effect on the Km for ethanol, but far less on that for acetaldehyde. The current status of investigation of other classes of functional mutants of this enzyme, and the potential both for selection of useful variants of this molecule and for an increase understanding of its function are discussed.

Plasmids in bacteria

Structure and Evolution.- Plasmids as Organisms.- Report on a Workshop: Structure and Function.- Evolutionary Relevance of Genetic Rearrangements Involving Plasmids.- Mechanisms of Transposition in Bacteria.- Insertion of Transcriptional Elements Outside the Replication Region Can Interfere with Replication, Maintenance, and Stability of ColE1-Derived Plasmids.- Studies on the Transposition of IS1.- On the Transposition and Evolution of Tn1721 and its Relatives.- Repeated DNA Sequences Recombine 1,000 Times More Frequently in a Plasmid Than in the Chromosome of BacillusSubtilis.- Mercury Resistance Transposon Tn813 Mediates Chromosome Transfer in Rhodopseudomonas sphaeroides and Intergeneric Transfer of pBR322.- Report on a Workshop: Plasmids in Unusual Systems.- Replication, Incompatibility, and Partition.- Chairmans Introduction: Replication, Incompatibility, and Partition.- Role of the ? Initiation Protein and Direct Nucleotide Sequence Repeats in the Regulation of Plasmid R6K Replication.- Initiation of Replication of the EscherichiaColi Chromosomal Origin Reconstituted with Purified Enzymes.- Origin and Initiation Sites of ?dv DNA Replication In Vitro.- Broad Host-Range Plasmid R1162: Replication, Incompatibility, and Copy-Number Control.- Control of Plasmid Replication: Theoretical Considerations and Practical Solutions.- The Partition Functions of PI, P7, and F Miniplasmids.- Genetic Interactions of Broad Host-Range Plasmid RK2: Evidence for a Complex Replication Regulon.- Replication Determinants of the Broad Host Range Plasmid RSF1010.- Regulation of Replication and Maintenance Functions of Broad Host-Range Plasmid RK2.- Control of Chromosome Replication in Bacteria.- Stable Maintenance of Plasmid CLO DF13: Structural and Functional Relationships Between Replication, Control, Partitioning, and Incompatibility.- Replication Control for PT181, An Indirectly Regulated Plasmid.- Construction of ColE1 RNA1 Mutants and Analysis of their Function In Vivo.- Incompatibility and INCFII Plasmid Replication Control.- P1 Plasmid Maintenance: A Paradigm of Precise Control.- Partitioning of the pSC101 Plasmid During Cell Division.- DNA-Protein Interaction at the Replication Origins of Plasmid Chromosomes.- Positive Regulation and Transcription Initiation of xy1 Operons on TOL Plasmid.- Plasmid Transfer.- Chairmens Introduction: Plasmid Transfer.- Linear Plasmids with Terminal Inverted Repeats Obtained from Streptomycesrochei and Kluyveromyceslactis.- Conjugal Plasmid Transfer in BacillusThuringiensis.- Mechanisms Essential for Stable Inheritance of Mini-F Plasmid.- Sex Pheromones and Plasmid Transfer in StreptococcusFaecalis: A Pheromone, cAM373, Which is Also Excreted by StaphylococcusAureus.- General Genetic Recombination of Bacterial Plasmids.- The Origin of Plasmid DNA Transfer During Bacterial Conjugation.- Genes and Gene Products Involved in the Synthesis of F-PILI.- Genetics of Clindamycin Resistance in Bacteroides.- Effect of Chromosome Homology of Plasmid Transformation and Plasmid Conjugal Transfer in Haemophilus Influenzae.- Plasmid DNA Primases and Their Role in Bacterial Conjugation.- Promoters in the Transfer Region of Plasmid F.- Conjugative Sex Plasmids of Streptomyces.- Specialized Functions, Structure, and Evolution.- The Structure and Source of Plasmid DNA Determine the Cloning Properties of Vectors for BacillusSubtilis.- Shuttle Vector for EscherichiaColi, PseudomonasPutida, and PseudomonasAeruginosa.- Report on a Workshop: Plasmid Cloning Vehicles.- Plasmids in the Degradation of Chlorinated Aromatic Compounds.- Notes on Metabolic Plasmid Organization and Expression.- The Structure of the mer Operon.- Analysis and Manipulation of Plasmid-Encoded Pathways for the Catabolism of Aromatic Compounds by Soil Bacteria.- The Plasmid-Specified Aerobactin Iron Uptake System of EscherichiaColi.- Plasmid-Mediated Iron Sequestering Systems in Pathogenic Strains of Vibrioanguillarum and EscherichiaColi.- Chairmans Introduction: Specialized Functions.- Cloning of an Enzymatically Active Segment of the Exotoxin-A Gene of PseudomonasAeruginosa Ill.- Structure, Function, and Regulation of the Plasmid-Encoded Hemolysin Determinant of EscherichiaColi.- pIAA, A Virulence Plasmid in PseudomonasSavastanoi.- The Molecular Basis of Plant Cell Transformation by AgrobacteriumTumefaciens.- Post-Transcriptional Regulation of Chloramphenicol Acetyl Transferase.- Plasmid and Larval Toxin of Bacilluslaterosporus.- Posters (Poster abstracts have been reproduced as submitted).- Replication, Incompatibility, Partition.- Specialized Functions.- Structure and Evolution.- Transfer.- Vectors.- Rosters.- Speakers.- Participants.- Index of Poster Authors.