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H S Seifert

Publications and source records attributed to H S Seifert.

At least 37 records · Page 2Linked to original sources
PubMed1996

The size and position of heterologous insertions in a silent locus differentially affect pilin recombination in Neisseria gonorrhoeae.

Gonococcal pilus antigenic and phase variation result from unidirectional, RecA-dependent recombination of DNA sequences from a silent pilin copy (pilS) into the expressed pilin gene (pilE). To develop a quantitative assay for pilin gene recombination that is independent of phase variation, a promoterless cat gene was inserted into pilS, and recombination of "cat into pilE was detected by selection of chloramphenicol-resistant (CmR) variants expressing "cat from the pilin promoter. Although RecA-dependent CmR variants occurred, none were generated by the simple transfer of "cat into pilE. Instead, each CmR variant contained a new pilin locus that was a hybrid of sequences from the pilE and the pilS1::cat loci in addition to the two starting loci. Therefore, this system could not be used to quantify antigenic variation. However, combined studies of these hybrid loci and of recombination products generated during additional pilS mutational analyses demonstrated that both the size and position of an insertion in pilS differentially affect pilin recombination. Also, the hybrid loci appear to be intermediates of antigenic variation. This enabled the creation of molecular models for the recombination reactions that result in pilin antigenic variation.

Antigenic Variation↗
PubMed1996

Uptake-sequence-independent DNA transformation exists in Neisseria gonorrhoeae.

A DNA transformation dose-response curve of piliated (P+) gonococci with the use of cloned DNA containing a pilE2-cat fusion showed saturation at high and low levels of transforming DNA. At low DNA concentrations, transformation of the P+ strain MS11-A was effectively inhibited by a 1000-fold molar excess of the gonococcal transformation uptake sequence (GCUS). The same molar excess of the GCUS did not inhibit transformation of MS11-A at high DNA concentrations. In MS11-B2, a nonpiliated (P-), pilin-nonproducing, isogenic variant of MS11-A, the GCUS did not inhibit transformation at any level of transforming DNA. These data suggest that two mechanisms of transformation exist in P+ cells: one which utilizes the GCUS and one which does not. In MS11-B2 P- cells, no evidence was found for the presence of the GCUS-dependent mechanism, suggesting that transformation in this background occurs solely by the GCUS-independent mechanism.

Bacterial Outer Membrane Proteins↗
PubMed1996

Questions about gonococcal pilus phase- and antigenic variation.

Pathogenic organisms inhabit one of several defined locations within a host where temperature, pH, and nutrients are relatively constant. While the microorganism must adapt to different environments within the host, the host immune system is the most formidable predator that can limit the growth of a pathogen. Neisseria gonorrhoeae (the gonococcus, Gc) is the causative agent of gonorrhoea, and has evolved several systems for varying the antigenicity of different surface antigens, presumably to help evade the effects of the human immune system. The On/Off/On phase variation of surface structure expression also alters the antigenic characteristics of the bacterial cell surface. Antigenic variation of the major subunit of the pilus, pilin, occurs by unidirectional, homologous recombination between a silent locus and the expression locus. The silent loci lie from 1 to 900 kb from the expression locus in the chromosome yet all can donate their sequences to the expression locus. The genetic composition of the pilin loci of two Gc strains has been elucidated, and the types of changes that lead to altered forms of the pilus have been extensively characterized. However, little is known about the precise molecular mechanisms used to allow high-frequency, non-reciprocal, chromosomal recombination between pilin loci or about what regulates the process of maintaining chromosome fidelity.

Antigenic Variation↗
PubMed1996

Environment, incidence, aetiology, epizootiology and immunoprophylaxis of soil-borne diseases in north-east Mexico.

Within the framework of an extensive research programme, the socio-economic and environmental conditions which influence the emergence of soil-borne diseases in north-eastern Mexico were analysed. Furthermore, specimens collected from carcasses in the field were bacteriologically examined and the causal organisms of soil-borne diseases differentiated by means of gas chromatographic analysis of their metabolic products and the long-chained fatty acids contained in the cell. With experimental clostridial vaccines prepared with the Goettingen Bioreactor Technique, trials to protect cattle and guinea-pigs against gas gangrene were carried out. It was found that the farm structure and the dry climate as well as the specific soil conditions and plant cover favour the emergence of soil-borne diseases. Causal organisms B. anthracis, C. perfringens, C. sordellii, C. haemolyticum, C. chauvoei/septicum, C. novyi A, C. botulinum and site-specific field strains of clostridia were detected. Experimental site-specific vaccines proved to be highly efficient in protecting cattle and guinea pigs.

Animals↗
PubMed1996

[Oral long-term administration of probiotic B. cereus--an alternative for the the prevention of enterotoxemia?].

Aetiology and epizootiology of enterotoxaemia, especially the connection between development of the disease and the management of feeding are described and newer knowledge about the physiopathology of the disease is presented. A comparative description of prophylactic schemes which include immuno- and chemoprophylaxis follows. The principles of the application and the effect of probiotic organisms as a new approach to prevent the disease are explained. The probiotic effect of apathogenic non-haemolysing and non-toxic B. cereus strains which have proven to be able to suppress enteropathies and also enterotoxaemia is described.

Animals↗
PubMed1995

Shuttle mutagenesis: a mini-transposon for producing PhoA fusions with exported proteins in Neisseria gonorrhoeae.

Shuttle mutagenesis is a method for producing stable mini-transposon (mTn) insertions into the genome of Neisseria gonorrhoeae (gonococcus, Gc) and other microbes. Using an mTn3 derivative, we have produced an mTn (mTnCmPhoA) which contains a phoA' gene lacking its N-terminal signal sequence useful for isolating genes which encode exported proteins. mTnCmPhoA was characterized in Gc and Escherichia coli using a cloned target containing the Gc genes, opaE1, pilA and pilB. PhoA+ Gc containing pilB::mTnCmPhoA insertions confirm that PilB is an exported protein in Gc. This system will be useful for isolating and characterizing extracytoplasmic virulence factors from Gc and other bacterial pathogens.

Alkaline Phosphatase↗
PubMed1995

Direct transformation of Neisseria gonorrhoeae by gel-isolated DNA.

The naturally competent organism, Neisseria gonorrhoeae, can efficiently transform a marker carried on DNA purified in low-melting-temperature agarose without prior purification or dilution. Neither the agarose or buffer components inhibit transformation frequencies, but exposure to UV irradiation completely abrogates transformation.

Electrophoresis, Agar Gel↗
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

Multiple gonococcal opacity proteins are expressed during experimental urethral infection in the male.

The opacity (Opa) proteins of Neisseria gonorrhoeae are a family of outer membrane proteins demonstrating phase and antigenic variation. N. gonorrhoeae strain FA0190 has 11 opa loci that encode at least 8 antigenically distinct Opa proteins. To determine if expression of one Opa protein or a subset of them is favored during gonococcal infection, we inoculated Opa-negative variants of strain FA1090 intraurethrally into male volunteers. The Opa phenotype of gonococci isolated from urine and urethral swab cultures from nine infected subjects was determined. Opa proteins were expressed in a large proportion of the reisolates from the infected subjects. Gonococci cultured from urine or urethral swab samples from six of the subjects were uniformly Opa positive, with the predominant Opa variants differing among subjects. Three different Opa proteins were represented as the predominant type in at least one subject each. In three subjects, there was more heterogeneity in Opa phenotype of the reisolates, including the presence of Opa-negative variants. An increase in the proportion of isolates expressing multiple Opa proteins occurred over time in most subjects. Passage of the inoculum in vitro did not result in similar changes in Opa expression. There was no detectable difference in infectivity of an Opa-negative variant and one expressing an Opa protein (OpaF) that was highly represented in reisolates from the original nine subjects. Reisolates from three infected volunteers inoculated with the OpaF variant showed continued expression of OpaF alone or in conjunction with other Opa proteins. These results demonstrate that there is strong selection for expression of one or more Opa proteins by strain FA1090 in vivo, but that no single protein is preferentially expressed during early infection in the male urethra.

Antigens, Bacterial↗
PubMed1994

Analysis of the fimB promoter region involved in type 1 pilus phase variation in Escherichia coli.

Many strains of Escherichia coli express type 1 pili which undergo phase variation during growth in broth, but become uniformly nonpiliated when passaged on agar media. In a previous analysis of these agar phase locked strains, we demonstrated that fimB and fimE, which mediate the site-specific DNA rearrangement involved in phase variation, are differentially transcribed under the two growth conditions. In this study the fimB promoter region was sequenced and characterized in agar phase locked strain J96. Primer extension analysis of the fimB gene identified three putative transcription initiation sites. Transcription starting from two of the sites (P1 and P2) would produce an mRNA that approximates the transcript size of fimB previously detected by Northern blot hybridizations. Gel mobility shift studies revealed the presence of promoter binding activity in broth-grown cell extracts, but not in extracts from agar-grown cells, that reacted specifically with DNA fragments upstream of the P1 and P2 promoter regions. This putative binding protein may be involved in the regulation of fimB, perhaps by acting as a transcriptional activator.

Bacterial Proteins↗
PubMed1994

A conserved DNA sequence is required for efficient gonococcal pilin antigenic variation.

Antigenic variation of the Neisseria gonorrhoeae pilus occurs when a variant pilin sequence from a silent locus recombines into the expression locus by predominantly unidirectional, homologous recombination. At the 3' end of all pilin loci lies a conserved DNA sequence, called the Sma/Cla repeat, which has sequence similarity to several recombinase-binding sites, and therefore may be involved in pilin recombination. We have developed a novel reverse transcriptase/polymerase chain reaction (RT-PCR) assay for direct monitoring of pilin recombination, and both RT-PCR and phase variation were used to examine pilin recombination in a gonococcal strain that had had the pilE Sma/Cla repeat removed. Results from these experiments showed a decrease in pilin recombination when the Sma/Cla sequence was deleted from the expression locus, showing that a specialized site (Sma/Cla) is involved in efficient pilin recombination.

Antigenic Variation↗
PubMed1994

Nonrepresentative PCR amplification of variable gene sequences in clinical specimens containing dilute, complex mixtures of microorganisms.

PCR amplification and DNA sequencing of the expression locus from Neisseria gonorrhoeae contained in urine sediments collected from experimentally infected human subjects produced two observations. First, different pilin sequences were obtained when separate aliquots of the same sample were amplified and sequenced. In contrast, the same pilin sequence was obtained when repeated amplifications were performed on individual colonies grown from the clinical samples. Second, mixed sequences (i.e., more than one nucleotide at variable positions in the pilin gene sequence) were observed in both the direct clinical isolates and individual cultures grown from the isolates. These results suggest that when clinical samples are directly examined by PCR amplification and sequencing, multiple amplifications may be required to detect sequence variants in the sample and minority variant sequences will not always be detected.

Adolescent↗
PubMed1994

Multiple gonococcal pilin antigenic variants are produced during experimental human infections.

Gonococcal pilin variation is thought to allow immune evasion and change the adherence properties of the pilus. We have examined the process of pilin antigenic variation in human volunteers inoculated with strain FA1090. Our data show that pilin variation occurred throughout the process of infection, that at each time sampled after inoculation multiple pilin variants were present, and that later pilin variants appear to be recombinants between previously expressed genes and the silent storage pilin copies. Thus, during infection a large repertoire of proteins are available to the population to help avoid immune responses, to provide pili with varying functions, and to transmit to a new host.

Amino Acid Sequence↗
PubMed1994

[Continuous production of FMD virus in a two-step bioreactor system].

The so-called Goettingen two step bioreactor system has been tested in a pilot-project in Argentina to produce FMD-virus. With a 10-1-reactor a cell density of 3.5-4 x 10(6) cells/ml could be maintained and virus titres of up to 10(7.2) TCID/ml were reached. A correlation existed between inoculum titre and incubation period of virus. With lower inoculum a higher titre of harvested virus and a shorter incubation period were obtained. In spite of considerable problems with peripheral infrastructure and laboratory equipment the bioreactor system did work reliably. The project was terminated before final results of virus yield could be reached.

Animals↗
PubMed1993

Transcriptional control of gonococcal pilE expression: involvement of an alternate sigma factor.

The pilE gene encoding Neisseria gonorrhoeae (Gc) pilin contains two putative promoter sequences 5' to the transcription start point (tsp), a Pribnow box and an RpoN-binding consensus sequence. Sequence analysis shows that the nucleotide (nt) sequence of the pilE promoter region is completely conserved among eight different Gc isolates. Using a pilE::lacZ transcriptional fusion, we demonstrate that the RpoN sigma factor can function in Escherichia coli to increase pilE transcription when the NifA activator from Klebsiella is present in trans. In addition, over-production of the native pilin protein using RpoN and NifA is lethal to E. coli. Finally, we show that the RpoN sigma factor decreases the basal expression of pilE when an activator is not present. These results suggest that, in Gc, pilE transcription may be regulated by RpoN in conjunction with an activator and that sigma 70 can also act to direct transcription of pilE.

Bacterial Proteins↗
PubMed1993

Shuttle mutagenesis: two mini-transposons for gene mapping and for lacZ transcriptional fusions in Neisseria gonorrhoeae.

Shuttle mutagenesis is a system we developed for producing stable transposon insertions in Saccharomyces cerevisiae [Seifert et al., Proc. Natl. Acad. Sci. USA 83 (1986) 735-739; Hoekstra et al., Methods Enzymol. 194 (1991) 329-342] and Neisseria gonorrhoeae (Gc) [Seifert et al., J. Bacteriol. 172 (1990) 40-46] by transposition in Escherichia coli and transformation into yeast or Gc. In developing the system for use in Gc, a series of mini-transposons (mTn) were derived from mTn3 which confer resistance to chloramphenicol in Gc (mTnCm) (Seifert et al., 1990). Herein, we describe the creation of two mTnCm derivatives for use in Gc. One of these transposons, mTnCmNS, contains the infrequently occurring NheI and SpeI restriction sites to localize genes on the gonococcal macro-restriction map which was recently developed using these restriction sites [Bihlmaier et al., Mol. Microbiol. 5 (1991) 2529-2539; Dempsey et al., J. Bacteriol. 173 (1991) 5476-5486]. The mTnCmLac was developed to generate lacZ transcriptional fusions using transposition. It contains at its end a promoterless lacZ gene which is expressed once the element has transposed downstream from a promoter in a cloned gene. In adapting the use of mTnCmLac to the shuttle mutagenesis system, we have identified some factors which affect the transformation of Gc using cloned chromosomal fragments containing the large heterologous insertion, mTnCmLac. Using mTnCmLac, we have created Gc variants containing a pilE::mTnCmLac fusion to determine that pilE transcription in Gc is not auto-regulated.

Base Sequence↗