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Identification of the genetic determinants of Salmonella enterica serotype Typhimurium that may regulate the expression of the type 1 fimbriae in response to solid agar and static broth culture conditions.

Chuang YC, Wang KC, Chen YT, Yang CH, Men SC, Fan CC, Chang LH, Yeh KS - BMC Microbiol. (2008)

Bottom Line: Type 1 fimbriae are the most commonly found fimbrial appendages on the outer membrane of Salmonella enterica serotype Typhimurium.Typhimurium is the result of the interaction and cooperation of several genes in the fim gene cluster.Typhimurium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Research, Chi Mei Medical Center, 901 Chung Hwa Road, Yong Kang City, Tainan 710, Taiwan. chuangkenneth@hotmail.com

ABSTRACT

Background: Type 1 fimbriae are the most commonly found fimbrial appendages on the outer membrane of Salmonella enterica serotype Typhimurium. Previous investigations indicate that static broth culture favours S. Typhimurium to produce type 1 fimbriae, while non-fimbriate bacteria are obtained by growth on solid agar media. The phenotypic expression of type 1 fimbriae in S. Typhimurium is the result of the interaction and cooperation of several genes in the fim gene cluster. Other gene products that may also participate in the regulation of type 1 fimbrial expression remain uncharacterized.

Results: In the present study, transposon insertion mutagenesis was performed on S. Typhimurium to generate a library to screen for those mutants that would exhibit different type 1 fimbrial phenotypes than the parental strain. Eight-two mutants were obtained from 7,239 clones screened using the yeast agglutination test. Forty-four mutants produced type 1 fimbriae on both solid agar and static broth media, while none of the other 38 mutants formed type 1 fimbriae in either culture condition. The flanking sequences of the transposons from 54 mutants were cloned and sequenced. These mutants can be classified according to the functions or putative functions of the open reading frames disrupted by the transposon. Our current results indicate that the genetic determinants such as those involved in the fimbrial biogenesis and regulation, global regulators, transporter proteins, prophage-derived proteins, and enzymes of different functions, to name a few, may play a role in the regulation of type 1 fimbrial expression in response to solid agar and static broth culture conditions. A complementation test revealed that transforming a recombinant plasmid possessing the coding sequence of a NAD(P)H-flavin reductase gene ubiB restored an ubiB mutant to exhibit the type 1 fimbrial phenotype as its parental strain.

Conclusion: Genetic determinants other than the fim genes may involve in the regulation of type 1 fimbrial expression in S. Typhimurium. How each gene product may influence type 1 fimbrial expression is an interesting research topic which warrants further investigation.

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Observation of type 1 fimbriae in S. Typhimurium LB5010 by electron microscopy. Panel A. S. Typhimurium LB5010 cells produced type 1 fimbrial appendages on the outer membrane (arrow) when cultured in LB static broth at 37°C for 48 hr (20,000 ×). Panel B. S. Typhimurium LB5010 cells did not produce type 1 fimbrial appendages on the outer membrane when cultured on LB agar at 37°C for 18 hr (20,000 ×). Bacterial cells were negatively stained with 2% of phosphotungstic acid.
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Figure 1: Observation of type 1 fimbriae in S. Typhimurium LB5010 by electron microscopy. Panel A. S. Typhimurium LB5010 cells produced type 1 fimbrial appendages on the outer membrane (arrow) when cultured in LB static broth at 37°C for 48 hr (20,000 ×). Panel B. S. Typhimurium LB5010 cells did not produce type 1 fimbrial appendages on the outer membrane when cultured on LB agar at 37°C for 18 hr (20,000 ×). Bacterial cells were negatively stained with 2% of phosphotungstic acid.

Mentions: In total, 7,239 transposon mutants were screened for those which exhibited different type 1 fimbrial phenotypes as compared to the parental strain S. Typhimurium LB5010. The expression of type 1 fimbriae was determined by yeast agglutination test. S. Typhimurium LB5010, when grown in static broth culture for 36–48 h, agglutinated Candida albicans on a glass slide. These bacterial cells produced type 1 fimbriae on the outer membrane when negatively stained with phosphotungstic acid and observed under electron microscopy (Figure 1, Panel A). On the contrary, S. Typhimurium LB5010 did not agglutinate C. albicans on a glass slide when bacterial cells were prepared from solid agar medium grown for 16–18 h. The absence of agglutination correlated with the fact that no type 1 fimbriae were observed by electron microscopy (Figure 1, Panel B). Fifty-four mutants verified by Southern blot to have a single unique transposon insertion were selected for further investigation (Figure 2). For those strains with multiple transposon insertions (Figure 2, lane 6 for example) were currently excluded from the present study. This could be due to partial digestion of the genomic DNA or multiple transposons inserted into one bacterial strain. These 54 mutants no longer exhibited the same type 1 fimbrial phenotype as the parental strain, among which 31 mutants produced type 1 fimbriae in both culture conditions, while another 23 mutants produced type 1 fimbriae in neither culture condition. The genes inactivated in the mutants were identified by cloning and sequencing the DNA fragments adjacent to the transposon insertion sites. The DNA sequences were used to search against the genomic sequence of S. Typhimurium. The position of the transposon insertion site was determined along with whether or not a transposon insertion site was located in or near an open reading frame (ORF), as predicted by the annotated genomic sequences. In the present study, we only focused on those mutants that demonstrated disruptions in ORFs; these types of mutation are expected to disrupt the functions of the ORFs involved. Table 1 lists those mutants. Transposons which disrupted between ORFs were excluded in the present study.


Identification of the genetic determinants of Salmonella enterica serotype Typhimurium that may regulate the expression of the type 1 fimbriae in response to solid agar and static broth culture conditions.

Chuang YC, Wang KC, Chen YT, Yang CH, Men SC, Fan CC, Chang LH, Yeh KS - BMC Microbiol. (2008)

Observation of type 1 fimbriae in S. Typhimurium LB5010 by electron microscopy. Panel A. S. Typhimurium LB5010 cells produced type 1 fimbrial appendages on the outer membrane (arrow) when cultured in LB static broth at 37°C for 48 hr (20,000 ×). Panel B. S. Typhimurium LB5010 cells did not produce type 1 fimbrial appendages on the outer membrane when cultured on LB agar at 37°C for 18 hr (20,000 ×). Bacterial cells were negatively stained with 2% of phosphotungstic acid.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2527010&req=5

Figure 1: Observation of type 1 fimbriae in S. Typhimurium LB5010 by electron microscopy. Panel A. S. Typhimurium LB5010 cells produced type 1 fimbrial appendages on the outer membrane (arrow) when cultured in LB static broth at 37°C for 48 hr (20,000 ×). Panel B. S. Typhimurium LB5010 cells did not produce type 1 fimbrial appendages on the outer membrane when cultured on LB agar at 37°C for 18 hr (20,000 ×). Bacterial cells were negatively stained with 2% of phosphotungstic acid.
Mentions: In total, 7,239 transposon mutants were screened for those which exhibited different type 1 fimbrial phenotypes as compared to the parental strain S. Typhimurium LB5010. The expression of type 1 fimbriae was determined by yeast agglutination test. S. Typhimurium LB5010, when grown in static broth culture for 36–48 h, agglutinated Candida albicans on a glass slide. These bacterial cells produced type 1 fimbriae on the outer membrane when negatively stained with phosphotungstic acid and observed under electron microscopy (Figure 1, Panel A). On the contrary, S. Typhimurium LB5010 did not agglutinate C. albicans on a glass slide when bacterial cells were prepared from solid agar medium grown for 16–18 h. The absence of agglutination correlated with the fact that no type 1 fimbriae were observed by electron microscopy (Figure 1, Panel B). Fifty-four mutants verified by Southern blot to have a single unique transposon insertion were selected for further investigation (Figure 2). For those strains with multiple transposon insertions (Figure 2, lane 6 for example) were currently excluded from the present study. This could be due to partial digestion of the genomic DNA or multiple transposons inserted into one bacterial strain. These 54 mutants no longer exhibited the same type 1 fimbrial phenotype as the parental strain, among which 31 mutants produced type 1 fimbriae in both culture conditions, while another 23 mutants produced type 1 fimbriae in neither culture condition. The genes inactivated in the mutants were identified by cloning and sequencing the DNA fragments adjacent to the transposon insertion sites. The DNA sequences were used to search against the genomic sequence of S. Typhimurium. The position of the transposon insertion site was determined along with whether or not a transposon insertion site was located in or near an open reading frame (ORF), as predicted by the annotated genomic sequences. In the present study, we only focused on those mutants that demonstrated disruptions in ORFs; these types of mutation are expected to disrupt the functions of the ORFs involved. Table 1 lists those mutants. Transposons which disrupted between ORFs were excluded in the present study.

Bottom Line: Type 1 fimbriae are the most commonly found fimbrial appendages on the outer membrane of Salmonella enterica serotype Typhimurium.Typhimurium is the result of the interaction and cooperation of several genes in the fim gene cluster.Typhimurium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Research, Chi Mei Medical Center, 901 Chung Hwa Road, Yong Kang City, Tainan 710, Taiwan. chuangkenneth@hotmail.com

ABSTRACT

Background: Type 1 fimbriae are the most commonly found fimbrial appendages on the outer membrane of Salmonella enterica serotype Typhimurium. Previous investigations indicate that static broth culture favours S. Typhimurium to produce type 1 fimbriae, while non-fimbriate bacteria are obtained by growth on solid agar media. The phenotypic expression of type 1 fimbriae in S. Typhimurium is the result of the interaction and cooperation of several genes in the fim gene cluster. Other gene products that may also participate in the regulation of type 1 fimbrial expression remain uncharacterized.

Results: In the present study, transposon insertion mutagenesis was performed on S. Typhimurium to generate a library to screen for those mutants that would exhibit different type 1 fimbrial phenotypes than the parental strain. Eight-two mutants were obtained from 7,239 clones screened using the yeast agglutination test. Forty-four mutants produced type 1 fimbriae on both solid agar and static broth media, while none of the other 38 mutants formed type 1 fimbriae in either culture condition. The flanking sequences of the transposons from 54 mutants were cloned and sequenced. These mutants can be classified according to the functions or putative functions of the open reading frames disrupted by the transposon. Our current results indicate that the genetic determinants such as those involved in the fimbrial biogenesis and regulation, global regulators, transporter proteins, prophage-derived proteins, and enzymes of different functions, to name a few, may play a role in the regulation of type 1 fimbrial expression in response to solid agar and static broth culture conditions. A complementation test revealed that transforming a recombinant plasmid possessing the coding sequence of a NAD(P)H-flavin reductase gene ubiB restored an ubiB mutant to exhibit the type 1 fimbrial phenotype as its parental strain.

Conclusion: Genetic determinants other than the fim genes may involve in the regulation of type 1 fimbrial expression in S. Typhimurium. How each gene product may influence type 1 fimbrial expression is an interesting research topic which warrants further investigation.

Show MeSH
Related in: MedlinePlus