Biomedical subjects

H Steven Seifert

Publications and source records attributed to H Steven Seifert.

25 records · Page 2Linked to original sources

PubMed2003

Low-level pilin expression allows for substantial DNA transformation competence in Neisseria gonorrhoeae.

The gonococcal pilus is a major virulence factor that has well-established roles in mediating epithelial cell adherence and DNA transformation. Gonococci expressing four gonococcal pilin variants with distinct piliation properties under control of the lac regulatory system were grown in different levels of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). These pilin variants expressed various levels of pilin message and pilin protein in response to the level of IPTG in the growth medium. Moreover, posttranslational modifications of the variant pilin proteins were detected, including S-pilin production and glycosylation. The ratio of the modified and unmodified pilin forms did not substantially change with different levels of pilin expression, showing that these modifications are not linked to pilin expression levels. DNA transformation competence was also influenced by IPTG levels in the growth medium. Substantial increases in transformation competence over an isogenic, nonpiliated mutant were observed when limited amounts of three of the pilin variants were expressed. Immunoelectron microscopy showed that when limited amounts of pilin are expressed, pili are rare and do not explain the pilin-dependent transformation competence. This pilin-dependent transformation competence required prepilin processing, the outer membrane secretin PilQ, and the twitching-motility-regulating protein PilT. These requirements show that a fully functional pilus assembly apparatus is required for DNA uptake when limited pilin is produced. We conclude that the pilus assembly apparatus functions to import DNA into the bacterial cell in a pilin-dependent manner but that extended pili are not required for transformation competence.

Amino Acid Sequence↗

PubMed2002

Escherichia coli RecX inhibits RecA recombinase and coprotease activities in vitro and in vivo.

In Escherichia coli the RecA protein plays a pivotal role in homologous recombination, DNA repair, and SOS repair and mutagenesis. A gene designated recX (or oraA) is present directly downstream of recA in E. coli; however, the function of RecX is unknown. In this work we demonstrated interaction of RecX and RecA in a yeast two-hybrid assay. In vitro, substoichiometric amounts of RecX strongly inhibited both RecA-mediated DNA strand exchange and RecA ATPase activity. In vivo, we showed that recX is under control of the LexA repressor and is up-regulated in response to DNA damage. A loss-of-function mutation in recX resulted in decreased resistance to UV irradiation; however, overexpression of RecX in trans resulted in a greater decrease in UV resistance. Overexpression of RecX inhibited induction of two din (damage-inducible) genes and cleavage of the UmuD and LexA repressor proteins; however, recX inactivation had no effect on any of these processes. Cells overexpressing RecX showed decreased levels of P1 transduction, whereas recX mutation had no effect on P1 transduction frequency. Our combined in vitro and in vivo data indicate that RecX can inhibit both RecA recombinase and coprotease activities.

Adenosine Triphosphate↗

PubMed2002

The outer membrane localization of the Neisseria gonorrhoeae MsrA/B is involved in survival against reactive oxygen species.

The PilB protein of Neisseria gonorrhoeae has been reported to be involved in the regulation of pilin gene transcription, but it also possesses significant homology to the peptide methionine sulfoxide reductase family of enzymes, specifically MsrA and MsrB from Escherichia coli. MsrA and MsrB in E. coli are able to reduce methionine sulfoxide residues in proteins to methionines. In addition, the gonococcal PilB protein encodes for both MsrA and MsrB activity associated with the repair of oxidative damage to proteins. In this work, we demonstrate that the PilB protein of Neisseria gonorrhoeae is not involved in pilus expression. Additionally, we show that wild-type N. gonorrhoeae produces two forms of this polypeptide, one of which contains a signal sequence and is secreted from the bacterial cytoplasm to the outer membrane; the other lacks a signal sequence and is cytoplasmic. Furthermore, we show that the secreted form of the PilB protein is involved in survival in the presence of oxidative damage.

Alkaline Phosphatase↗

PubMed2002

The misidentification of bacterial genes as human cDNAs: was the human D-1 tumor antigen gene acquired from bacteria?

The initial analysis of the draft copy of the human genome sequence revealed the presence of several genes that were proposed to have been directly transferred from bacteria. We investigated the human D-1 antigen as a potential lateral transfer event. We report that although the human D-1 antigen seems to be an excellent candidate for lateral transfer, it is a contaminating bacterial sequence present in a human cDNA library that was included in the human genome analysis. Furthermore, several other genes present in the publicly available databases that were included in the analysis of the human genome are also likely contaminating bacterial sequences present in cDNA libraries.

Antigens, Neoplasm↗

PubMed2002

Differential cross-complementation patterns of Escherichia coli and Neisseria gonorrhoeae RecA proteins.

The Escherichia coli RecA protein is one of the best-studied enzymes, but less is understood about how RecA homologues of other species are similar to or different from the E. coli RecA. In the Gram-negative pathogen Neisseria gonorrhoeae (the gonococcus; Gc), the causative agent of gonorrhoea, RecA is involved in DNA transformation, pilin antigenic variation, and DNA repair. By expressing the recA genes from Gc and E. coli under control of lac regulatory sequences in E. coli, the authors have shown that the Gc RecA fully complements an E. coli recA mutant for homologous recombination, but only partially complements for survival to DNA damage. By expressing similar constructs in Gc, it was shown that the E. coli RecA complements for pilin antigenic variation, partially complements for DNA transformation, but does not complement for survival to DNA damage, suggesting that species-specific interactions are important for DNA repair, but not for homologous recombination. Co-expression of the E. coli recA and recX genes in Gc suggests that in this heterologous system RecX modulates RecA-mediated processes.

Bacterial Proteins↗

PubMed2002

Roles of the recJ and recN genes in homologous recombination and DNA repair pathways of Neisseria gonorrhoeae.

The paradigm of homologous recombination comes from Escherichia coli, where the genes involved have been segregated into pathways. In the human pathogen Neisseria gonorrhoeae (the gonococcus), the pathways of homologous recombination are being delineated. To investigate the roles of the gonococcal recN and recJ genes in the recombination-based processes of the gonococcus, these genes were inactivated in the N. gonorrhoeae strain FA1090. We report that both recN and recJ loss-of-function mutants show decreased DNA repair ability. In addition, the recJ mutant was decreased in pilus-dependent colony morphology variation frequency but not DNA transformation efficiency, while the recN mutant was decreased in DNA transformation efficiency but not pilus-dependent variation frequency. We were able to complement all of these deficiencies by supplying an ectopic functional copy of either recJ or recN at an irrelevant locus. These results describe the role of recJ and recN in the recombination-dependent processes of the gonococcus and further define the pathways of homologous recombination in this organism.

Amino Acid Sequence↗

PubMed2001

Inverse relationship between pilus-mediated gonococcal adherence and surface expression of the pilus receptor, CD46.

Pilus-mediated adherence to mucosal epithelial cells is a critical step for Neisseria gonorrhoeae to establish an infection in the human host. CD46, the defined receptor for the gonococcal pilus, is a complement-regulatory protein that is expressed on all human nucleated cells. It was observed that a piliated, Opa(-) variant of gonococcal strain FA1090 adhered with different efficiencies to the human epithelial cell lines tested (Chang, ME180, HEC-1B and PC-3). Surprisingly, these differences in adherence levels did not correlate with levels of CD46 expressed by these cell lines. In fact, there was an inverse relationship between total surface-exposed CD46 and gonococcal adherence. Four major isoforms of CD46 are produced due to alternative RNA splicing of a surface-exposed region and the cytoplasmic tail. The relative isoform surface expression of each cell line was determined, and each was found to express different ratios of the four CD46 isoforms. No correlation could be derived between CD46 isoform surface expression and pilus-mediated gonococcal adherence, indicating that CD46 does not act as a classic receptor for gonococcal pili.

Antigens, CD↗