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Some Bti isolates are able to infect renal proximal tubule cells, and can potentially cause pyelonephritis. CFT, thereby promoting its in vivo fitness. Our phylogenetic analyses support that kgtP is a widely conserved locus in commensal and pathogenic E. Global anaerobic 55038 regulators Fumarate and nitrate bg FNR and ArcA induced c expression in anaerobiosis, and C played a major role in anaerobic growth on KG.
Gene c can be specifically induced by KG, whereas the expression of kgtP does not respond to KG, yet can be stimulated during growth on glycerol. In addition, c and kgtP expression were further shown to be controlled by different alternative sigma factors RpoN and RpoS, respectively. Furthermore, dual-strain competition assays in a murine model showed that c mutant but not kgtP mutant was outcompeted by the wild-type gbi during the colonization of murine bladders and kidneys, highlighting the importance of C under in vivo conditions.
Therefore, different transcriptional regulation led to distinct roles played by C and KgtP in KG utilization and fitness in vivo. This 50038 thus potentially expanded our understanding of UPEC pathobiology. Urinary tract infection UTI is one of the most common bacterial infections in humans, and represents a significant clinical issue worldwide Foxman, UPECs have evolved multiple systems and strategies to snatch needed iron, combatting extremely low iron availability within host Opal et al.
KG lies at the intersection between the carbon and nitrogen metabolic pathways, and has long been recognized to coordinate carbon and nitrogen metabolism Kim and Gadd, Fumarate and nitrate reduction FNR positively regulates c expression by directly affecting the 5083 of KguSR KG utilization sensor and regulator Barbieri et al. Alkaline phosphatase fusion study showed that KgtP contains 12 transmembrane segments Seol and Shatkin, Genome-wide analysis of ArcA binding sites and its modulon in E.
In this study, we describe KgtP and C contribute differentially to growth on KG under aerobic and anaerobic conditions. Further analyses of their expression levels in various growth conditions and bi roles played by regulators ArcA, FNR, RpoN, RpoS, and CRP catabolite receptor protein can account for the findings of 5083 contributions to growth on KG and to colonization of murine urinary tracts.
This report, therefore, should substantially improve our understanding of UPEC physiology. Strains and plasmids used in this study are listed in Supplementary Table S1.
For genetic manipulations, all E. For growth and gene expression studies, bacteria were generally grown aerobically or anaerobically in M9 minimal salts with certain carbon sources indicated, supplemented with 2 mM MgSO 40.
All reagents were purchased from Sigma-Aldrich unless otherwise noted. For growth studies, fresh colonies of wild-type WT CFT and its derivative mutant strains were picked and inoculated in 3 ml LB medium. After OD of bacterial culture reached about 1. OD value at different time points was measured and recorded using spectrophotometer Eppendorf, basic model.
ClustalW program was used to perform multiple sequences alignments. Deletion mutants were constructed using the lambda red recombinase system described by Datsenko and Wanner For complementation studies, the coding sequences of genes plus their promoter regions bp upstream of start codons were amplified from the CFT genome and independently cloned into pGEN-MCS Lane et al.
The resulting plasmid was tested to show the functionality and an undetectable basal level without inserting any promoters upstream of promoterless lacZ. P bla constitutively expressed promoter was cloned from pGEN-MCS plasmid by amplifying bp upstream of bla gene as previous described Sperandio et al. Samples were diluted 1: 0538, bacteria were grown in M9 media supplemented with glycerol and KG to log phase. Three biological replicates of each sample were prepared.
Control reactions were included according to the manual. His 6 -ArcA Jiang et al. Protein concentrations were measured using BCA protein assay kit Pierce. DNA probes bp upstream of the start codon of kgtP or c were PCR amplified using specific primers and gel-purified.
The negative control probe was amplified from the coding region of Chloramphenicol resistance gene- cat. Reaction mixtures were then mixed with the loading dye bromophenol blue, 0. The gel was stained in 0. Mouse infection studies were performed according to the methods of Johnson et al.
Overnight statically grown LB cultures of CFT and the mutant strains were pelleted and resuspended in sterile PBS, mixed in equal number, and adjusted to make challenge inocula. Chl R mutant or kanamycin c After 48 h, the mice were sacrificed and the bladder and kidneys were aseptically removed and homogenized in tubes containing PBS using a Bioprep bullet homogenizer Allsheng, Hangzhou in China.
The numbers of colonies on selective plates were subtracted from those on LB plates to obtain the number of WT bacteria.
A group of 10 or 6 for complementation studies mice for each dual-strain challenge were used to determine alterations in fitness, and assays were performed at least twice. Competitive index is defined as the ratio of mutant to the WT isolated divided by the ratio of mutant to the WT in the inoculums.
Statistical tests were performed using GraphPad Prism version 5. For other data, unpaired Student t -test was used to estimate differences between samples. Bioinformatic analysis revealed that C belongs to the Divalent Anion: The kgtP locus in K is At the amino acid level, only three differences exist between them, all of which are non-charged amino acids.
We observed no significant difference in growth kinetics among these strains in LB or M9 medium supplemented with glucose Gluc or glycerol Glyc as the sole carbon source data not shown.
Additionally, the introduction of a complementation plasmid p kgtP carrying kgtP controlled by its native promoter restored the growth of kgtP mutant Figure 1B. These results suggest that kgtP is required whereas c is dispensable for growth on KG under aerobic conditions. Various strains were grown in M9 medium containing KG as the sole carbon and energy source.
Optical density at different time points was determined. Growth curves represent the average measurement at each time point from triplicate experiments.
Student t -test was used for statistical analysis. These data indicate that under anaerobic conditions, c played a major role and kgtP a minor role in growth on KG. Of note, in both aerobiosis and anaerobiosis, the complementation strains apparently had much shorter lag phases and higher growth rates than the WT strain carrying the empty vector. Given that kgtP and c contributed differentially to growth on KG under aerobic and anaerobic conditions, we sought to unravel their transcriptional levels under these conditions.
The —10 and —35 sites of kgtP promoter were predicted by Virtual Footprint program Munch et al. The putative ribosome binding sites were also indicated.
In parallel, a control plasmid carrying constitutively expressed promoter P bla – lacZ fusion was also transformed into LMP10 strain.
As shown in Figure 2Cc expression was not detectable in aerobiosis, but was induced in anaerobiosis. The expression of kgtP was moderate under aerobic conditions, but was greatly repressed under anaerobic conditions, with a fold reduction.
It is also evident that anaerobic expression level of c was significantly higher than aerobic expression level of kgtP about fold difference. In the control strain, P bla – lacZ expression did not exhibit difference between under aerobic and anaerobic conditions. These results clearly showed that under anaerobic conditions, c was highly expressed whereas kgtP was severely repressed, thereby at least partially explaining the phenotypic difference in growth. A Promoter region of kgtP.
B Promoter region of c C kgtP and c expression under aerobic and anaerobic conditions. Bacteria were grown aerobically or anaerobically in M9 medium containing KG as the sole carbon source.
TMAO was used as an electron acceptor for anaerobic tests. P bla – lacZ constitutive expression was used as a control. Fumarate and nitrate reduction and ArcA are two master regulators mediating bacterial adaptation to environmental oxygen availability Constantinidou et al. To probe the regulatory roles of arcA and fnr in expression of c and kgtPwe examined the effects of single deletion and double deletion of fnr and arcA on expression levels of c and kgtP under aerobic and anaerobic conditions.
Under aerobic conditions, deletion of fnr did not affect kgtP expression while loss of arcA upregulated kgtP expression about fivefold. Under anaerobic conditions, deletion of either fnr or arcA caused upregulation of kgtP expression 4-fold and fold increase, respectively. Deletion of both fnr and arcA led to a greater increase in kgtP expression than individual deletions of fnr and arcA. These results indicate that both fnr and arcA negatively modulate kgtP expression.
These results clearly demonstrate that in either fnr or arcA mutant, anaerobic repression of kgtP was partially relieved compared with that in their parental strain LMP10, suggesting both fnr and arcA are involved in the anaerobic repression, with ArcA being a major player. Roles of fnr and arcA in regulating kgtP and c expression in response to varying oxygen tension. A Loss of fnr or arcA leads to different effects on kgtP and c expression.
Various strains were grown aerobically or anaerobically in M9 medium containing KG as the sole carbon source. TMAO was used as electron acceptor anaerobically. DNA fragments with sizes around bp containing the predicted sites were prepared. With the same amounts of protein, the presence of acetyl phosphate appears to not affect binding of ArcA to DNA under our testing conditions.
Surprisingly, kgtP expression in all three media is comparable Figure 4Aindicating that kgtP expression does not respond to the presence of KG. The results showed that the order of kgtP expression levels is as follows: Response of kgtP expression to growth in different media and contribution of global regulator CRP.
A kgtP expression does not respond to the presence of KG. Bacteria were grown aerobically or anaerobically in different media containing the indicated carbon and energy source. Gluc, glucose; Glyc, glycerol. Bacteria were grown in M9 containing glycerol as sole carbon source.
Gene: BGIOSGA – Summary – Oryza sativa Indica Group – Ensembl Genomes 41
The cAMP-CRP complex is a global regulatory element that induces a number of genes involved in the utilization of carbons in the absence of glucose Botsford and Harman, ; Kolb et al. We therefore examined whether CRP contributes to the regulation 5083 kgtP expression. As shown in Figure 4BkgtP expression in the strain lacking crp was downregulated, as compared to the parental strain LMP