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 54
Enhancer Binding Proteins and Myxococcus xanthus Fruiting Body
Development.Jimmy S. Jakobsen, 2004.A search of the M1genome sequence, which includes 97% of the Myxococcus xanthus genes, identified 53 sequence homologs of 54-dependent
enhancer binding proteins (EBPs) . A DNA microarray was constructed
from the M1genome that includes those homologs and 318 other M .
xanthus genes for comparison . To screen the developmental program
with this array, an RNA extract from growing cells was compared with
one prepared from developing cells at 12 h . Previous reporter studies
had shown that M . xanthus has initiated development and has
begun to express many developmentally regulated genes by 12 h . The
comparison revealed substantial increases in the expression levels of
11 transcription factors that may respond to environmental stimuli .
Six of the 53 EBP homologs were expressed at significantly higher
levels at 12 h of development than during growth . Three were
previously unknown genes, and they were inactivated to look for
effects on fruiting body development . One knockout mutant produced
fruiting bodies of abnormal shape that depended on the composition of
the medium .
Analysis of DNA Regulatory Elements Required for Expression of the Legionella pneumophila icm and dot Virulence Genes. Ohad Gal-Mor, 2002.To investigate the regulation of the Legionella pneumophila icm and dot genes required for intracellular growth, a series of nine icm::lacZ fusions were constructed . These icm::lacZ fusions were found to have different levels of expression in L . pneumophila, and five of them were more highly expressed at stationary phase than at exponential phase . When the expression of these fusions in Escherichia coli was tested, all of them were found to be expressed but three of them had dramatic changes in their levels of expression in comparison to those in L . pneumophila . Site-directed and PCR random mutagenesis with these icm::lacZ fusions was used to identify DNA regulatory elements of icm genes . Four icm genes (icmT, icmP, icmQ, and icmM) that had low levels of expression in L . pneumophila were found to contain a 6-bp sequence (TATACT) essential for their expression . This sequence was shown by primer extension to serve as their -10 promoter elements . A similar sequence, which constitutes the -10 promoter elements of the icmV, icmW, and icmR genes which had high levels of expression in L . pneumophila, was also identified . In addition, regulatory elements that probably serve as binding sites for transcription regulators were found in these genes . Altogether, 12 regulatory elements, 7 of which constitute the -10 promoter elements of the icm genes, were found . Even though all the icm and dot genes are part of one system required for L . pneumophila intracellular growth and even though their promoters are probably recognized by the vegetative sigma factor, it seems that they are subjected to different regulation mediated by several regulatory factors . Transcriptional Phase Variation of a Type III Restriction-Modification System in Helicobacter pylori. Nicolette de Vries, 2002.Phase variation is important in bacterial pathogenesis, since it generates antigenic variation for the evasion of immune responses and provides a strategy for quick adaptation to environmental changes . In this study, a Helicobacter pylori clone, designated MOD525, was identified that displayed phase-variable lacZ expression . The clone contained a transcriptional lacZ fusion in a putative type III DNA methyltransferase gene (mod, a homolog of the gene JHP1296 of strain J99), organized in an operon-like structure with a putative type III restriction endonuclease gene (res, a homolog of the gene JHP1297), located directly upstream of it . This putative type III restriction-modification system was common in H . pylori, as it was present in 15 out of 16 clinical isolates . Phase variation of the mod gene occurred at the transcriptional level both in clone MOD525 and in the parental H . pylori strain 1061 . Further analysis showed that the res gene also displayed transcriptional phase variation and that it was cotranscribed with the mod gene . A homopolymeric cytosine tract (C tract) was present in the 5' coding region of the res gene . Length variation of this C tract caused the res open reading frame (ORF) to shift in and out of frame, switching the res gene on and off at the translational level . Surprisingly, the presence of an intact res ORF was positively correlated with active transcription of the downstream mod gene . Moreover, the C tract was required for the occurrence of transcriptional phase variation . Our finding that translation and transcription are linked during phase variation through slipped-strand mispairing is new for H . pylori . Degradation of Polycyclic Aromatic Hydrocarbons at Low Temperature under Aerobic and Nitrate-Reducing Conditions in Enrichment Cultures from Northern Soils. Mikael Eriksson, 2003.Thepotential for biodegradation of polycyclic aromatic hydrocarbons (PAHs)at low temperature and under anaerobic conditions is not wellunderstood, but such biodegradation would be very useful forremediation of polluted sites . Biodegradation of a mixture of 11different PAHs with two to five aromatic rings, each at a concentrationof 10 µg/ml, was studied in enrichment cultures inoculated withsamples of four northern soils . Under aerobic conditions, lowtemperature severely limited PAH biodegradation . After 90 days, aerobiccultures at 20°C removed 52 to 88% of the PAHs . The mostextensive PAH degradation under aerobic conditions at 7°C,53% removal, occurred in a culture from creosote-contaminatedsoil . Low temperature did not substantially limit PAH biodegradationunder nitrate-reducing conditions . Under nitrate-reducing conditions,naphthalene, 2-methylnaphthalene, fluorene, and phenanthrene weredegraded . The most extensive PAH degradation under nitrate-reducingconditions at 7°C, 39% removal, occurred in a culturefrom fuel-contaminated Arctic soil . In separate transfer cultures fromthe above Arctic soil, incubated anaerobically at 7°C, removalof 2-methylnaphthalene and fluorene was stoichiometrically coupled tonitrate removal . Ribosomal intergenic spacer analysis suggested thatenrichment resulted in a few predominant bacterial populations,including members of the genera Acidovorax,Bordetella, Pseudomonas, Sphingomonas, andVariovorax . Predominant populations from different soils oftenincluded phylotypes with nearly identical partial 16S rRNA genesequences (i.e., same genus) but never included phylotypes withidentical ribosomal intergenic spacers (i.e., different species orsubspecies) . The composition of the enriched communities appeared to bemore affected by presence of oxygen, than by temperature or source oftheinoculum . Mycotoxin Fumonisin B1 Increases Intestinal Colonization by Pathogenic Escherichia coli in Pigs. Isabelle P. Oswald, 2003.Fumonisin B1 (FB1) is a mycotoxin that commonly occurs in maize . FB1 causes a variety of toxic effects in different animal species and has been implicated as a contributing factor of esophageal cancers in humans . In the present study, we examined the effect of dietary exposure to FB1 on intestinal colonization by pathogenic Escherichia coli associated with extraintestinal infection . Three-week-old weaned pigs were given FB1 by gavage as a crude extract or as a purified toxin at a dose of 0.5 mg/kg of body weight daily for 6 days . On the last day of the toxin treatment, the pigs were orally inoculated with an extraintestinal pathogenic E . coli strain . All animals were euthanized 24 h later, necropsies were performed, and tissues were taken for bacterial counts and light microscopic examination . Ingestion of FB1 had only a minimal effect on animal weight gain, did not cause any macroscopic or microscopic lesions, and did not change the plasma biochemical profile . However, colonization of the small and large intestines by an extraintestinal pathogenic E . coli strain was significantly increased . Our results show that FB1 is a predisposing factor to infectious disease and that the pig can be used as a model for the study of the consequences of ingesting mycotoxin-contaminated food .
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