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Among Multiple Phosphomannomutase Gene Orthologues, Only One Gene Encodes a Protein with Phosphoglucomutase and Phosphomannomutase Activities in Thermococcus kodakaraensis. Naeem Rashid, 2004.Four orthologous genes [TK1108, TK1404, TK1777, and TK2185]that can be annotated as phosphomannomutase [PMM] genes [COG1109]have been identified in the genome of the hyperthermophilicarchaeon Thermococcus kodakaraensis KOD1 . We previously foundthat TK1777 actually encodes a phosphopentomutase . In orderto determine which of the remaining three orthologues encodesa phosphoglucomutase [PGM], we examined the PGM activity inT . kodakaraensis cells and identified the gene responsible forthis activity . Heterologous gene expression and purificationand characterization of the recombinant protein indicated thatTK1108 encoded a protein with high levels of PGM activity [690U mg–1], along with high levels of PMM activity [401 Umg–1] . Similar analyses of the remaining two orthologuesrevealed that their protein products exhibited neither PGM norPMM activity . PGM activity and transcription of TK1108 in T.kodakaraensis were found to be higher in cells grown on starchthan in cells grown on pyruvate . Our results clearly indicatethat, among the four PMM gene orthologues in T . kodakaraensis,only one gene, TK1108, actually encodes a protein with PGM andPMM activities. Iron Acquisition and Regulation in Campylobacter jejuni. Kiran Palyada, 2004.Iron affects the physiology of bacteria in two different ways: as a micronutrient for bacterial growth and as a catalyst for the formation of hydroxyl radicals . In this study, we used DNA microarrays to identify the C . jejuni genes that have their transcript abundance affected by iron availability . The transcript levels of 647 genes were affected after the addition of iron to iron-limited C . jejuni cells . Several classes of affected genes were revealed within 15 min, including immediate-early response genes as well as those specific to iron acquisition and metabolism . In contrast, only 208 genes were differentially expressed during steady-state experiments comparing iron-rich and iron-limited growth conditions . As expected, genes annotated as being involved in either iron acquisition or oxidative stress defense were downregulated during both time course and steady-state experiments, while genes encoding proteins involved in energy metabolism were upregulated . Because the level of protein glycosylation increased with iron limitation, iron may modulate the level of C . jejuni virulence by affecting the degree of protein glycosylation . Since iron homeostasis has been shown to be Fur regulated in C . jejuni, an isogenic fur mutant was used to define the Fur regulon by transcriptome profiling . A total of 53 genes were Fur regulated, including many genes not previously associated with Fur regulation . A putative Fur binding consensus sequence was identified in the promoter region of most iron-repressed and Fur-regulated genes . Interestingly, a fur mutant was found to be significantly affected in its ability to colonize the gastrointestinal tract of chicks, highlighting the importance of iron homeostasis in vivo . Directed mutagenesis of other genes identified by the microarray analyses allowed the characterization of the ferric enterobactin receptor, previously named CfrA . Chick colonization assays indicated that mutants defective in enterobactin-mediated iron acquisition were unable to colonize the gastrointestinal tract . In addition, a mutation in a receptor (Cj0178) for an uncharacterized iron source also resulted in reduced colonization potential . Overall, this work documents the complex response of C . jejuni to iron availability, describes the genetic network between the Fur and iron regulons, and provides insight regarding the role of iron in C . jejuni colonization in vivo . No Findings of Dental Defects in Children Treated with Minocycline. Antonio Cascio, 2004.Forty-one children <8 years of age treated for brucellosis with oral minocycline (2.5 mg/kg) twice daily for 3 weeks were recalled and examined to check for dental staining and defects . Dental staining and defects were found in 14 of 41 exposed children (34.1%) and in 30 of 82 matched controls (36.6%), respectively (P > 0.2) . New Type of Osmoregulated Solute Transporter Identified in Halophilic Members of the Bacteria Domain: TRAP Transporter TeaABC Mediates Uptake of Ectoine and Hydroxyectoine in Halomonas elongata DSM 2581T. Katrin Grammann, 2002.The halophilic bacterium Halomonas elongata synthesizes as its main compatible solute the aspartate derivative ectoine . We constructed a deletion mutant of H . elongata, KB1, defective in ectoine synthesis and tolerating elevated salt concentrations only in the presence of external compatible solutes . The dependency of KB1 on solute uptake for growth in high-salt medium was exploited to select insertion mutants unable to accumulate external solutes via osmoregulated transporters . One insertion mutant out of 7,200 failed to accumulate the osmoprotectants ectoine and hydroxyectoine . Genetic analysis of the insertion site proved that the mutation affected an open reading frame (ORF) of 1,281 bp (teaC) . The nucleotide sequence upstream of teaC was determined, and two further ORFs of 603 bp (teaB) and 1,023 bp (teaA) were identified . Deletion of teaA and teaB proved that all three genes are mandatory for ectoine uptake . Sequence comparison showed significant identity of TeaA, TeaB, and TeaC to the transport proteins of the recently identified tripartite ATP-independent periplasmic transporter family (TRAP-T) . The affinity of the cells for ectoines was determined (Ks = 21.7 µM), suggesting that the transporter TeaABC exhibits high affinity for ectoines . An elevation of the external osmolarity resulted in a strong increase in ectoine uptake via TeaABC, demonstrating that this transporter is osmoregulated . Deletion of teaC and teaBC in the wild-type strain led to mutants which excreted significant amounts of ectoine into the medium when cultivated at high salt concentrations . Therefore, the physiological role of TeaABC may be primarily to recover ectoine leaking through the cytoplasmic membrane . Molecular Architecture of the Regulatory Locus sae of Staphylococcus aureus and Its Impact on Expression of Virulence Factors. Andrea Steinhuber, 2003.We characterized the sae operon, a global regulator for virulence gene expression in Staphylococcus aureus . A Tn917 sae mutant was obtained by screening a Tn917 library of the agr mutant ISP479Mu for clones with altered hemolytic activity . Sequence analysis of the sae operon revealed two additional open reading frames (ORFs) (ORF3 and ORF4) upstream of the two-component regulatory genes saeR and saeS . Four overlapping sae-specific transcripts (T1 to T4) were detected by Northern blot analysis, and the transcriptional initiation points were mapped by primer extension analysis . The T1, T2, and T3 mRNAs are probably terminated at the same stem-loop sequence downstream of saeS . The T1 message (3.1 kb) initiates upstream of ORF4, T2 (2.4 kb) initiates upstream of ORF3, and T3 (2.0 kb) initiates in front of saeR . T4 (0.7 kb) represents a monocistronic mRNA encompassing ORF4 only . sae-specific transcripts were detectable in all of the 40 different clinical S . aureus isolates investigated . Transcript levels were at maximum during the post-exponential growth phase . The sae mutant showed a significantly reduced rate of invasion of human endothelial cells, consistent with diminished transcription and expression of fnbA . The expression of type 5 capsular polysaccharide is activated in the sae mutant of strain Newman, as shown by immunofluorescence and promoter-reporter fusion experiments . In summary, the sae operon constitutes a four-component regulator system which acts on virulence gene expression in S . aureus . Nitrogen Regulation of the codBA (Cytosine Deaminase) Operon from Escherichia coli by the Nitrogen Assimilation Control Protein, NAC. Wilson B. Muse, 2003.Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium . ß-Galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation . In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required . Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position -59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription . When a longer promoter region (positions -120 to +67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site . When a shorter fragment was used (positions -83 to +67), only the primary footprint was seen . Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo . Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner . K . pneumoniae differs from E . coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K . pneumoniae but decreased it in E . coli .
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