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M Koomey

Publications and source records attributed to M Koomey.

31 records · Page 2Linked to original sources
PubMed1995

Identification and characterization of pilG, a highly conserved pilus-assembly gene in pathogenic Neisseria.

Expression of type IV pili appears to be a requisite determinant of infectivity for the strict human pathogens Neisseria gonorrhoeae and Neisseria meningitidis. The assembly of these colonization factors is a complex process. This report describes a new pilus-assembly gene, pilG, that immediately precedes the gonococcal (Gc) pilD gene encoding the pre-pilin leader peptidase. The nucleotide sequence of this region revealed a single complete open reading frame whose derived polypeptide displayed significant identities to the pilus-assembly protein PilC of Pseudomonas aeruginosa and other polytopic integral cytoplasmic membrane constituents involved in protein export and competence. A unique polypeptide of M(r) 38 kDa corresponding to the gene product was identified. A highly related gene and flanking sequences were cloned from a group B polysaccharide-producing strain of N. meningitidis (Mc). The results indicate that the pilG genes and genetic organization at these loci in Gc and Mc are extremely conserved. Hybridization studies strongly suggest that pilG-related genes exist in commensal Neisseria species and other species known to express type IV pili. Defined genetic lesions were created by using insertional and transposon mutagenesis and moved into the Gc and Mc chromosomes by allelic replacement. Chromosomal pilG insertion mutants were devoid of pili and displayed dramatically reduced competence for transformation. These findings could not be ascribed to pilin-gene alterations or to polarity exerted on pilD expression. The results indicated that PilG exerts its own independent role in neisserial pilus biogenesis.

Amino Acid Sequence↗
PubMed1995

Characterization of the pilF-pilD pilus-assembly locus of Neisseria gonorrhoeae.

Expression of Type IV pili by the bacterial pathogen Neisseria gonorrhoeae appears to be essential for colonization of the human host. Several N. gonorrhoeae gene products have been recently identified which bear homology to proteins involved in pilus assembly and protein export in other bacterial systems. We report here the isolation and characterization of transposon insertion mutants in N. gonorrhoeae whose phenotypes indicate that the N. gonorrhoeae pilF and pilD gene products are required for gonoccocal pilus biogenesis. Mutants lacking the pilD gene product, a pre-pilin peptidase, were unable to process the pre-pilin subunit into pilin and thus were non-piliated. pilF mutants processed pilin but did not assemble the mature subunit. Both classes of mutants released S-pilin, a soluble, truncated form of the pilin subunit previously correlated with defects in pilus assembly. In addition, mutants containing transposon insertions in pilD or in a downstream gene, orfX, exhibited a severely restricted growth phenotype. Deletion analysis of pilD indicated that the poor growth phenotype observed for the pilD transposon mutants was a result of polar effects of the insertions on orfX expression. orfX encodes a predicted polypeptide of 23 kDa which contains a consensus nucleotide-binding domain and has apparent homologues in Pseudomonas aeruginosa, Pseudomonas putida, Thermus thermophilus, and the eukaryote Caenorhabditis elegans. Although expression of orfX and pilD appears to be transcriptionally coupled, mutants containing transposon insertions in orfX expressed pili. Unlike either pilF or pilD mutants, orfX mutants were also competent for DNA transformation.

Amino Acid Sequence↗
PubMed1994

Identification, localization, and distribution of the PilT protein in Neisseria gonorrhoeae.

A monoclonal antibody (MAb) directed against a highly conserved protein of Neisseria gonorrhoeae with a molecular size of 40 kDa was isolated and characterized. The protein antigen detected by this MAb was detected by enzyme-linked immunosorbent assay and immunoblotting in all strains of N. gonorrhoeae tested across a wide range of serovars. The 40-kDa protein was found to be expressed at relatively low levels and localized to both the cytosolic and cytoplasmic membrane fractions. Screening of a lambda gt11 expression library derived from gonococcal genomic DNA with the anti-40-kDa MAb and DNA sequence analysis suggested that the 40-kDa protein and the product of the gonococcal pilT gene were identical. Immunoblotting analysis of gonococcal mutants carrying defined mutations in the pilT gene confirmed that the 40-kDa protein was indeed PilT. The N-terminal sequence derived by microsequencing of the protein purified from gonococci led to the correction of the previously published pilT gene sequence. Sequencing of the pilT gene from three different strains revealed an extremely high degree of conservation at both the amino acid and DNA levels.

Adenosine Triphosphatases↗
PubMed1993

Molecular cloning and characterization of a proline iminopeptidase gene from Neisseria gonorrhoeae.

Proline iminopeptidase (Pip) is a hydrolase elaborated by virtually all strains of Neisseria gonorrhoeae that selectively removes N-terminal proline residues from peptides. Escherichia coli clones expressing the gonococcal gene coding for Pip were identified in a genomic cosmid library using a synthetic colorimetric substrate. Nucleotide sequence determination and analyses of polypeptides detected by coupled in vitro transcription/translation reactions revealed that Pip is a 311-amino-acid polypeptide with a M(r) of 35 kDa and a pI of 5.4. Southern hybridization showed that the pip gene is present in a single copy on the chromosome of N. gonorrhoeae strain MS11 which maps immediately upstream of the previously identified opaA locus. The transcriptional start site of pip in E. coli, determined by primer extension analysis, was characteristic of an NtrA or sigma-54-dependent promotor. Complementation of an E. coli mutant deficient in both proline biosynthesis and dipeptide uptake confirmed that Pip is capable of releasing biologically active proline from peptides. Pip expression was found to be non-essential for in vitro growth of N. gonorrhoeae, based on the viability of a Pip- gonococcal mutant.

Amino Acid Sequence↗
PubMed1993

Conservation of genes encoding components of a type IV pilus assembly/two-step protein export pathway in Neisseria gonorrhoeae.

Three gonococcal genes have been identified which encode proteins with substantial similarities to known components of the type IV pilus biogenesis pathway in Pseudomonas aeruginosa. Two of the genes were identified based on their hybridization with a DNA probe derived from the pilB gene of P. aeruginosa under conditions of reduced stringency. The product of the gonococcal pilF gene is most closely related to the pilus assembly protein PilB of P. aeruginosa while the product of the gonococcal pilT gene is most similar to the PilT protein of P. aeruginosa which is involved in pilus-associated twitching motility and colony morphology. The products of both of these genes display canonical nucleoside triphosphate binding sites and are predicted to be to cytoplasmically localized based on their overall hydrophilicity. The gonococcal pilD gene, identified by virtue of its linkage to the pilF gene, is homologous to a family of prepilin leader peptidase genes. When expressed in Escherichia coli, the gonococcal PilD protein functions to process gonococcal prepilin in a manner consistent with its being gonococcal prepilin peptidase. These results suggest that Neisseria gonorrhoeae is capable of expressing many of the essential elements of a highly conserved protein translocation system and that these gene products are probably involved in pilus biogenesis.

Adenosine Triphosphatases↗
PubMed1992

Gene conversion in Neisseria gonorrhoeae: evidence for its role in pilus antigenic variation.

Antigenic variation of gonococcal pili results from the unidirectional transfer of genetic information from variant-encoding partial pilin genes to an active expression locus. Two potential mechanisms that may result in the observed alterations of gene linkage and organization are conversion and transformation. To determine the relative contributions of these two distinct pathways of recombination to pilus variation, gonococcal strains carrying defined frameshift, missense, and nonsense mutations within the pilin expression locus were constructed. Reversion to a piliated state required correction of the lesions and provided a simple means of scoring productive recombination and antigenic variation. Examination of the mutants revealed a lack of correspondence between the frequencies with which they could be transformed (10(-6) per recipient) and the incidence with which they gave rise to revertants (greater than 10(-4) per colony-forming unit per generation). Further, the rates of reversion demonstrated by these mutants were not altered by growth in the presence of DNase I, conditions that abolished intercellular transfer of chromosomal markers during cultivation. Through the use of a pilin mutant in which a frameshift mutation encompassed the introduction of a restriction endonuclease site, the symmetry of recombination that resulted in reversion could be scored by Southern hybridization. In all cases examined, the DNA alterations responsible for pilin variation were nonreciprocal events. The results favor the model that productive pilin gene rearrangements in gonococci arise by gene conversion.

Amino Acid Sequence↗
PubMed1991

Pilin expression and processing in pilus mutants of Neisseria gonorrhoeae: critical role of Gly-1 in assembly.

Spontaneous mutants of Neisseria gonorrheae failing to express pili or having diminished levels of piliation were studied with regard to pilin expression. All mutants displayed altered pilin processing detectable as the release of soluble, truncated pilin molecules (S-pilin). Of particular interest was the finding, in one mutant, that substitution of serine for glycine at position -1 of propilin, a highly conserved residue among N-metPhe and related pilins, abolished pilus expression but not S-pilin release. The degree of S-pilin processing and the levels of membrane-associated pilin varied among the different classes of mutants, suggesting that each was blocked at a distinct step of pilus biogenesis. The data support a model in which increased S-pilin processing is a result of a decreased rate of pilus polymerization.

Amino Acid Sequence↗
PubMed1991

Shared antigenicity and immunogenicity of type 4 pilins expressed by Pseudomonas aeruginosa, Moraxella bovis, Neisseria gonorrhoaea, Dichelobacter nodosus, and Vibrio cholerae.

Immunoblotting with polyclonal rabbit antibodies raised against pilins expressed by Pseudomonas aeruginosa, Moraxella bovis, Neisseria gonorrhoeae, Dichelobacter nodosus, and Vibrio cholerae was used to demonstrate that these polypeptides display conserved antigenic and, in most cases, immunogenic determinants. These determinants appear to be localized to the highly homologous amino-terminal domains (residues 1 to 25).

Animals↗
PubMed1989

Phase variation of gonococcal protein II: regulation of gene expression by slipped-strand mispairing of a repetitive DNA sequence.

Expression of outer membrane protein II (P.II) of Neisseria gonorrhoeae is subject to reversible phase variation at a rate of 10(-3)-10(-4)/cell/generation. The signal peptide coding regions of P.II genes contain variable numbers of tandem repeats of the sequence CTCTT. Changes in the number of CTCTT units, leading to frameshifting within the gene, are responsible for changes in P.II expression. Phase variation mediated by the CTCTT repeat also occurred in E. coli, as assayed with a P.II-alkaline phosphatase (phoA) gene fusion. Phase variation in both the gonococcus and E. coli was recA-independent, occurred at similar rates, and involved insertions or deletions of one or more repeat units. The characteristics of the phase variation process were consistent with a model in which expression of P.II genes is regulated by slipped-strand mispairing of the DNA in the CTCTT repeat region.

Bacterial Outer Membrane Proteins↗
PubMed1987

Gene conversion accounts for pilin structural changes and for reversible piliation "phase" changes in gonococci.

Pilus+ "wild-type" gonococci (Gc) frequently display gene conversion of their expressed complete pilin gene (CPG); a copy of DNA derived from one of the Gc genome's multiple silent partial pilin genes (PPG) is recombinationally-inserted into the CPG's central and 3' portions with formation of a new, chimeric CPG. Expression of that new CPG leads to either 1) retention of pilus+ phenotype but change in pilin primary structure/antigenicity, or 2) phase change to pilus- phenotype capable of reverting. This study utilizes pilus revertants of P-rp +/- Gc and P+ colony morphotype variants spawned by P++ Gc to examine pilin gene conversion in strain MS11mk Gc in greater detail. Each revertant's and variant's expressed pilin gene's sequence (as pilin mRNA) was defined to learn whether their differences are due to gene conversion by different PPGs, or by varying stretches from the same PPG, or both. Gene conversion by PPG pilS1 copy 2 has been documented in Gc recovered from a human volunteer's urethra previously inoculated with pilus Gc (strain MS11). The pilus+ Gc isolated expressed structurally/antigenically distinct pilins.

Amino Acid Sequence↗
PubMed1987

Porin protein of Neisseria gonorrhoeae: cloning and gene structure.

The outer membrane porin molecule of Neisseria gonorrhoeae is known as protein I (PI). Among different strains of gonococci there is variability of PI, and two main classes, PIA and PIB, have been recognized. A lambda gt11 bank of gonococcal DNA was screened using monoclonal antibodies directed to a PIB-type porin molecule of N. gonorrhoeae, and three immunoreactive clones were isolated. DNA sequence analysis indicated that each contained only portions of the PI structural gene, but that together they contained the complete gene, and its structure was determined. The DNA sequence predicts a protein of 348 amino acids with a typical 19 amino acid signal peptide. The PI protein resembles Escherichia coli porins in size, lack of long hydrophobic sequences, and absence of cysteine residues. Sequence homologies between PI and the E. coli porins were found, particularly in the 100 N-terminal and the 110 C-terminal amino acids. In addition to the coding sequence of PI, the complementary strand contains a large open reading frame. At the 3' end of the PI gene, immediately following an inverted repeat (probably the transcription terminator), the clone contains an unusual sequence consisting of 31 perfect repeats of the heptamer CTGTTTT. Hybridization analysis suggests that there is a single structural gene for PI and that it is homologous to the gene found in a PIA-bearing strain of gonococcus.

Amino Acid Sequence↗
PubMed1987

Effects of recA mutations on pilus antigenic variation and phase transitions in Neisseria gonorrhoeae.

Intragenic recombination between the single complete pilin gene (expression locus) and multiple, distinct, partial pilin gene copies (silent, storage loci) is thought to account for the generation of pilus antigenic diversity and piliation phase (on-off) changes exhibited by Neisseria gonorrhoeae. The mechanisms operating in the genomic rearrangements associated with these forms of pilus variation were investigated through the study of isogenic strains of gonococci bearing either wild-type or altered recA alleles. Examination of the rates of pilus phase variation and the genetic basis for changes in piliation status displayed by these strains show that recA mediated homologous recombination is required for these high frequency events and confirm that the nonpiliated state results from mutations in the expressed pilin gene. In a strain that is deficient in recA mediated homologous recombination, pilus phase variation occurs at a 100-1000-fold reduced rate and results predominantly from one class of spontaneous frameshift mutations within the pilin structural gene.

Alleles↗