Novel regulators of the csgD gene encoding the master regulator of biofilm formation in Escherichia coli K-12

Under stressful conditions, Escherichia coli forms biofilm for survival by sensing a variety of environmental conditions. CsgD, the master regulator of biofilm formation, controls cell aggregation by directly regulating the synthesis of Curli fimbriae. In agreement of its regulatory role, as many as 14 transcription factors (TFs) have so far been identified to participate in regulation of the csgD promoter, each monitoring a specific environmental condition or factor. In order to identify the whole set of TFs involved in this typical multi-factor promoter, we performed in this study ‘promoter-specific transcription-factor’ (PS-TF) screening in vitro using a set of 198 purified TFs (145 TFs with known functions and 53 hitherto uncharacterized TFs). A total of 48 TFs with strong binding to the csgD promoter probe were identified, including 35 known TFs and 13 uncharacterized TFs, referred to as Y-TFs. As an attempt to search for novel regulators, in this study we first analysed a total of seven Y-TFs, including YbiH, YdcI, YhjC, YiaJ, YiaU, YjgJ and YjiR. After analysis of curli fimbriae formation, LacZ-reporter assay, Northern-blot analysis and biofilm formation assay, we identified at least two novel regulators, repressor YiaJ (renamed PlaR) and activator YhjC (renamed RcdB), of the csgD promoter.

Pathogen Inducible cis-acting Elements of Synthetic Promoters in Plants-review

Synthetic pathogen inducible promoters are used for the improvement and application of transgenic techniques in research and to increase agriculture production. The promoter contains specific cis-regulatory elements (W box, GCC box, Box S and D box) which induce anti pathogen molecular cascades. Insertion of dimerized form of cis acting elements at upstream region of promoter in promoter probe vector drives the expression of resistance gene or reporter gene. The expression indicates that synthetic promoters are responded to fungal elicitors. Expression of resistance restrict to infection sites which boost disease resistance in plants.

Influence of Salmonella enterica Serovar TyphimuriumssrBon Colonization of Eastern Oysters (Crassostrea virginica) as Revealed by a Promoter Probe Screen

ABSTRACTAlthoughSalmonellahas been isolated from 7.4 to 8.6% of domestic raw oysters, representing a significant risk for food-borne illness, little is known about the factors that influence their initial colonization bySalmonella. This study tested the hypothesis that specific regulatory changes enable a portion of the invadingSalmonellapopulation to colonize oysters. Anin vivopromoter probe library screen identified 19 unique regions as regulated during colonization. The mutants in the nearest corresponding downstream genes were tested for colonization defects in oysters. Only one mutation, inssrB, resulted in a significantly reduced ability to colonize oysters compared to that of wild-typeSalmonella. BecausessrBregulatesSalmonellapathogenicity island 2 (SPI-2)-dependent infections in vertebrate macrophages, the possibility thatssrBmediated colonization of oyster hemocytes in a similar manner was examined. However, no difference in hemocyte colonization was observed. The complementary hypothesis that signal exchange betweenSalmonellaand the oyster's native microbial community aids colonization was also tested. Signals that triggered responses in quorum sensing (QS) reporters were shown to be produced by oyster-associated bacteria and present in oyster tissue. However, no evidence for signal exchange was observedin vivo. ThesdiAreporter responded to salinity, suggesting that SdiA may also have a role in environmental sensing. Overall, this study suggests the initial colonization of live oysters bySalmonellais controlled by a limited number of regulators, includingssrB.

Plant-Influenced Gene Expression in the Rice Endophyte Burkholderia kururiensis M130

Burkholderia kururiensis M130 is one of the few rice endophytic diazotrophic bacteria identified thus far which is able to enhance growth of rice. To date, very little is known of how strain M130 and other endophytes enter and colonize plants. Here, we identified genes of strain M130 that are differentially regulated in the presence of rice plant extract. A genetic screening of a promoter probe transposon mutant genome bank and RNAseq analysis were performed. The screening of 10,100 insertions of the genomic transposon reporter library resulted in the isolation of 61 insertions displaying differential expression in response to rice macerate. The RNAseq results validated this screen and indicated that this endophytic bacterium undergoes major changes in the presence of plant extract regulating 27.7% of its open reading frames. A large number of differentially expressed genes encode membrane transporters and secretion systems, indicating that the exchange of molecules is an important aspect of bacterial endophytic growth. Genes related to motility, chemotaxis, and adhesion were also overrepresented, further suggesting plant–bacteria interaction. This work highlights the potential close signaling taking place between plants and bacteria and helps us to begin to understand the adaptation of an endophyte in planta.

Directly utilizing an endogenous gene to dissect regulatory elements in the biosynthetic gene cluster of nosiheptide

Coupling the deletion of an endogenous gene and a non-integrative promoter-probe vector allowed reliable identification of biosynthetic promoters of nosiheptide.

Functional Characterization of a Cassette-Specific Promoter in the Class 1 Integron-AssociatedqnrVC1Gene

ABSTRACTIntegrons are natural expression vectors due to the presence of an intrinsic promoter (Pc). Although rare, gene cassettes can harbor their own promoter. This study determined the functionality of an internal promoter in theqnrVC1cassette whose presence was suggested by a level of transcription similar to that of the preceding cassette (aadA2) and confirmed byin silicoanalysis. Its functionality was determined by 5′ rapid amplification of cDNA ends (RACE) and cloning into promoter-probe vectors. PqnrVCwas found in theqnrVCcassette family, stressing its role in contributing to resistance manifestation.

Gene cloning, structural gene and promoter identification, and active assay of the phosphatidylcholine synthase of Pseudomonas sp. strain 593

Pseudomonas sp. strain 593, a soil bacterium, is able to use exogenous choline to synthesize phosphatidylcholine via phosphatidylcholine synthase (Pcs). A 2020 bp DNA fragment that hybridized to a Pcs probe was cloned. This fragment contained a large open reading frame (ORF) with two potential ATG start sites that would encode for 293 and 231 amino acid proteins. Fragments containing the two ORFs encoded Pcs when they were inserted into the expression vector pET23a and expressed under the control of the T7 promoter in Escherichia coli BL21(DE3) pLysS. However, when the two ORFs were inserted into the cloning vector pMD18-T and expressed without control of the plasmid promoter in E. coli DH5α, only the larger clone exhibited Pcs activity. This suggested that the larger fragment contained a native promoter driving expression of the smaller ORF. A promoter activity assay, in which DNA fragments were inserted into the promoter-probe plasmid pCB182 and β-galactosidase activity of E. coli transformants was tested, demonstrated that a promoter is indeed present in the DNA region. All results together indicate that the 696 bp ORF, not the larger 897 bp ORF, encodes the Pcs in Pseudomonas sp. strain 593 and carries a promoter in front of its 5′ terminus.