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L W Harris-Haller

Publications and source records attributed to L W Harris-Haller.

6 recordsLinked to original sources
PubMed1996

Extragenic suppression of motA missense mutations of Escherichia coli.

The MotA and MotB proteins are thought to comprise elements of the stator component of the flagellar motor of Escherichia coli. In an effort to understand interactions among proteins within the motor, we attempted to identify extragenic suppressors of 31 dominant, plasmid-borne alleles of motA. Strains containing these mutations were either nonmotile or had severely impaired motility. Four of the mutants yielded extragenic suppressors mapping to the FlaII or FlaIIIB regions of the chromosome. Two types of suppression were observed. Suppression of one type (class I) probably results from increased expression of the chromosomal motB gene due to relief of polarity. Class I suppressors were partial deletions of Mu insertion sequences in the disrupted chromosomal motA gene. Class I suppression was mimicked by expressing the wild-type MotB protein from a second, compatible plasmid. Suppression of the other type (class II) was weaker, and it was not mimicked by overproduction of wild-type MotB protein. Class II suppressors were point mutations in the chromosomal motB or fliG genes. Among 14 independent class II suppressors characterized by DNA sequencing, we identified six different amino acid substitutions in MotB and one substitution in FliG. A number of the strongest class II suppressors had alterations of residues 136 to 138 of MotB. This particular region within the large, C-terminal periplasmic domain of MotB has previously not been associated with a specific function. We suggest that residues 136 to 138 of MotB may interact directly with the periplasmic face of MotA or help position the N-terminal membrane-spanning helix of MotB properly to interact with the membrane-spanning helices of the MotA proton channel.

Alleles↗
PubMed1995

Motility protein interactions in the bacterial flagellar motor.

Five proteins (MotA, MotB, FliG, FliM, and FliN) have been implicated in energizing flagellar rotation in Escherichia coli and Salmonella typhimurium. One model for flagellar function envisions that MotA and MotB comprise the stator of a rotary motor and that FliG, FliM, and FliN are part of the rotor. MotA probably functions as a transmembrane proton channel, and MotB has been proposed to anchor MotA to the peptidoglycan of the cell wall. To study interactions between the Mot proteins themselves and between them and other components of the flagellar motor, we attempted to isolate extragenic suppressors of 13 dominant or partially dominant motB missense mutations. Four of these yielded suppressors, which exhibited widely varying efficiencies of suppression. The pattern of suppression was partially alleles-specific, but no suppressor seriously impaired motility in a motB+ strain. Of 20 suppressors from the original selection, 15 were characterized by DNA sequencing. Fourteen of these cause single amino acid changes in MotA. Thirteen alter residues in, or directly adjacent to, the putative periplasmic loops of MotA, and the remaining one alters a residue in the middle of the fourth predicted transmembrane helix of MotA. We conclude that the MotA and MotB proteins form a complex and that their interaction directly involves or is strongly influenced by the periplasmic loops of MotA. The 15th suppressor from the original selection and 2 motB suppressors identified during a subsequent search cause single amino acid substitutions in FliG. This finding suggests that the postulated Mot-protein complex may be in close proximity to FliG at the stator-rotor interface of the flagellar motor.

Alleles↗
PubMed1995

Complete sequence of the binary vector Bin 19.

Despite the widespread use of Bin 19 as a vector for plant transformation, detailed sequence information on its T-DNA region has only recently become available. We now show that the non-T-DNA region, like the T-DNA region, contains several superfluous insertions and find that some functional elements may not contain optimal sequences. Knowledge of the complete 11,777 bp sequence will aid in the construction of exceptionally efficient derivative vectors of approximately half this size. Precise knowledge of restriction sites and removal of unnecessary sequences will facilitate plasmid manipulations and plant transformation.

Base Sequence↗
PubMed1995

Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii).

We cloned, sequenced, and overexpressed cobA, the gene encoding uroporphyrinogen III methyltransferase in Propionibacterium freudenreichii, and examined the catalytic properties of the enzyme. The methyltransferase is similar in mass (27 kDa) and homologous to the one isolated from Pseudomonas denitrificans. In contrast to the much larger isoenzyme encoded by the cysG gene of Escherichia coli (52 kDa), the P. freudenreichii enzyme does not contain the additional 22-kDa peptide moiety at its N-terminal end bearing the oxidase-ferrochelatase activity responsible for the conversion of dihydrosirohydrochlorin (precorrin-2) to siroheme. Since it does not contain this moiety, it is not a likely candidate for synthesis of a cobalt-containing early intermediate that has been proposed for the vitamin B12 biosynthetic pathway in P. freudenreichii. Uroporphyrinogen III methyltransferase of P. freudenreichii not only catalyzes the addition of two methyl groups to uroporphyrinogen III to afford the early vitamin B12 intermediate, precorrin-2, but also has an overmethylation property that catalyzes the synthesis of several tri- and tetra-methylated compounds that are not part of the vitamin B12 pathway. The enzyme catalyzes the addition of three methyl groups to uroporphyrinogen I to form trimethylpyrrocorphin, the intermediate necessary for biosynthesis of the natural products, factors S1 and S3, previously isolated from this organism. A second gene found upstream from the cobA gene encodes a protein homologous to CbiO of Salmonella typhimurium, a membrane-bound, ATP-dependent transport protein thought to be part of the cobalt transport system involved in vitamin B12 synthesis. These two genes do not appear to constitute part of an extensive cobalamin operon.

Amino Acid Sequence↗
PubMed1993

Sequence of the Candida albicans erg7 gene.

The nucleotide sequence of the Candida albicans erg7 gene, which complements erg7 mutants of Saccharomyces cerevisiae and restores oxidosqualene cyclase activity, was determined. The gene encodes a 728-aa protein that displays homology with squalene-hopene cyclase, providing further evidence that erg7 is the gene encoding 2,3-oxidosqualene cyclase.

Amino Acid Sequence↗