Lactococcal 936-species phage attachment to surface of Lactococcus lactis.

The interactions of the 936-species phages sk1, jj50, and 64 with the cell surface of Lactococcus lactis LM0230 were analyzed. Cell envelopes (walls + plasma membrane), cell wall, or plasma membrane from L. lactis ssp. lactis LM0230 each inactivated the phages in vitro. However, other 936-species phages kh and P008, which do not infect strain LM0230, were not inactivated by any of the subcellular fractions. Treating cell walls or plasma membrane with the cell wall hydrolase mutanolysin eliminated inactivation of phage sk1. This suggested that intact cell wall fragments were required for inactivation. A role for plasma membrane in phage sk1 inactivation was further investigated. Boiling, washing in 2 M KCl, 8 M urea, or 0.1 M Na(2)CO(3)/pH 11, or treating the plasma membrane with proteases did not reduce adsorption or inactivation of phage. Adding lipoteichoic acid or antibodies to lipoteichoic acid did not reduce inactivation of phage in a mixture with membrane, suggesting that lipoteichoic acid was not involved. Inactivation by envelopes or cell wall correlated with ejection of DNA from the phage sk1 capsid. Although calcium is required for plaque formation, it was not required for adsorption, inactivation, or ejection of phage DNA by envelopes or cell wall. The results suggest that at least for phages sk1, jj50, and 64, adsorption and phage DNA injection into the host does not require a host membrane protein or lipoteichoic acid, and that cell wall components are sufficient for these initial steps of phage infection.

Mutations that eliminate the requirement for the vertex protein in bacteriophage T4 capsid assembly.

The capsid of bacteriophage T4 is composed of two essential structural proteins, gp23, the major constituent of the capsid, and gp24, a less prevalent protein that is located in the pentameric vertices of the capsid. gp24 is required both to stabilize the capsid and to allow it to be further matured. This requirement can be eliminated by bypass-24 (byp24) mutations within g23. We have isolated, cloned and sequenced several new byp24 mutations. These mutations are cold-sensitive in the absence of gp24, and are located in regions of g23 not known to contain any other mutations affecting capsid assembly. The cold-sensitivity of the byp24 mutations can be reduced by further mutations within g23 (trb mutations). Cloning and sequencing of these trb mutations has revealed that they lie in regions of g23 that contain clusters of mutations that cause the production of high levels of petite and giant phage (ptg mutations). Despite the proximity of the trb mutations to the ptg mutations, none of the ptg mutations has a Trb phenotype. The mutation ptE920g, which is also located near one of the ptg clusters, and which produces only petite and wild-type phage, has been shown to confer a Trb but not a Byp24 phenotype. The relevance of these observations to our understanding of capsid assembly is discussed.

Mutant alcohol dehydrogenase (ADH III) presequences that affect both in vitro mitochondrial import and in vitro processing by the matrix protease.

Point mutations in the presequence of the mitochondrial alcohol dehydrogerase isoenzyme (ADH III) have been shown to affect either the import of the precursor protein into yeast mitochondria in vivo or its processing within the organelle. In the present work, the behavior of these mutants during in vitro import into isolated mitochondria was investigated. All point mutants tested were imported with a slower initial rate than that of the wild-type precursor. This defect was corrected when the precursors were treated with urea prior to import. Once imported, the extent of processing to the mature form of mutant precursors varied greatly and correlated well with the defects observed in vivo. This result was not affected by prior urea treatment. When matrix extracts enriched for the processing protease were used, this defect was shown to be due to failure of the protease to efficiently recognize or cleave the presequence, rather than to a lack of access to the precursor. The rate of import of two ADH III precursors bearing internal deletions in the leader sequence was similar to those of the point mutants, whereas a deletion leading to the removal of the 15 amino-terminal amino acids was poorly imported. The mature amino terminus of wild-type ADH III was determined to be Gln-25. Mutant m01 (Ser-26 to Phe), which reduced the efficiency of cleavage in vitro by 80%, was cleaved at the correct site.

Genetic control of capsid length in bacteriophage T4: DNA sequence analysis of petite and petite/giant mutants.

The T4 gene 23 product (gp23) encodes the major structural protein of the mature capsid. Mutations in this gene have been described which disrupt the normal length-determining mechanism (A.H. Doermann, F.A. Eiserling, and L. Boehner, J. Virol. 12:374-385, 1973). Mutants which produce high levels of petite and giant phage (ptg) are restricted to three tight clusters in gene 23 (A.H. Doermann, A. Pao, and P. Jackson, J. Virol. 61:2823-2827, 1987). Twenty-six of these ptg mutations were cloned, and their DNA sequence alterations were determined. Each member of this set of ptg mutants arose from a single mutation, and the set defined 10 different sites at which ptg mutations can occur in gene 23. Two petite (pt) mutations in gene 23 (pt21-34 and ptE920g), which produce high frequencies of petite particles but no giants, were also sequenced. Both pt21-34 and ptE920g were shown to include multiple mutations. The phenotypes attributed to both pt and ptg mutations are discussed relative to the mechanism of capsid morphogenesis. A site-directed mutation (SD-1E) was created at the ptgNg191 site, and its phenotypic consequences were examined. Plaque morphology revertants arising from a gene 23 mutant derivative of pt21-34 and from SD-1E were isolated. A preliminary mapping of the mutation(s) responsible for their revertant phenotypes suggested that both intra- and extragenic suppressors of the petite phenotype can be isolated by this method.

Volatile factor involved in the dimorphism of Mucor racemosus.

Both hyphal and yeastlike development of Mucor racemosus and M. rouxii were demonstrated under 100% N2. Under standardized conditions in yeast extract-peptone-glucose medium, the morphology depended on the N2 flow rate and not on the glucose concentration. The effect was related to the rate of flushing of the atmosphere over the culture medium. The results indicate that a volatile compound produced by Mucor is involved in morphogenesis.