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Characterization of Pseudomonas aeruginosa fliO, a gene involved in flagellar biosynthesis and adherence.

ABSTRACT: Pseudomonas aeruginosa binds to eukaryotic cells via both pilus and nonpilus adhesins, while binding of P. aeruginosa to mucin is pilus independent. To characterize genes involved in non-pilus-mediated adherence, transposon mutants of the nonpiliated strain P. aeruginosa PAK-NP that are unable to bind to cells or mucins were isolated. Two such mutants, P. aeruginosa B164 and P. aeruginosa RR18, were identified previously as deficient in binding to eukaryotic cells or mucins as well as nonmotile. The transposon insertion in each of these strains was mapped to the same gene. Sequence analysis of both DNA flanking the transposons and plasmids that could complement the mutations indicated that this open reading frame encodes a putative protein homolog of both Escherichia coli FliO and Erwinia carotovora subsp. atroseptica MopB. The transposons in both of these mutants are nonpolar, since the addition of the P. aeruginosa fliO gene in trans restored adherence to both cells and mucins to these mutants. The cloned fliO gene also complemented the motility defect of both B164 and RR18. A 1.6-kb KpnI fragment from the PAK chromosome that contained the fliO gene was sequenced. The fliO gene appears to be part of an operon with a complete open reading frame upstream of the FliO homolog encoding a putative protein homolog of FliN of both E. coli and Salmonella typhimurium. The partial open reading frame downstream of fliO encodes a putative homolog of both E. coli and S. typhimurium FliP. The fliN gene is flanked on its 5'-end by the 3'-end of a homolog of a fliM gene. The P. aeruginosa FliN protein was identified with a T7 expression system, while all attempts to identify the P. aeruginosa FliO protein were unsuccessful. Homologs of P. aeruginosa FliO are involved in the biosynthesis of flagella, but the function of FliO in this biosynthetic process remains unknown. Further study should reveal the precise role of P. aeruginosa FliO in non-pilus-mediated adherence, which could include regulation of expression or localization of a nonpilus adhesin.

SUBMITTER: Simpson DA

PROVIDER: S-EPMC173401 | biostudies-other | 1995 Aug

REPOSITORIES: biostudies-other

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Characterization of Pseudomonas aeruginosa fliO, a gene involved in flagellar biosynthesis and adherence.

Simpson D A DA Ramphal R R Lory S S

Infection and immunity 19950801 8

Pseudomonas aeruginosa binds to eukaryotic cells via both pilus and nonpilus adhesins, while binding of P. aeruginosa to mucin is pilus independent. To characterize genes involved in non-pilus-mediated adherence, transposon mutants of the nonpiliated strain P. aeruginosa PAK-NP that are unable to bind to cells or mucins were isolated. Two such mutants, P. aeruginosa B164 and P. aeruginosa RR18, were identified previously as deficient in binding to eukaryotic cells or mucins as well as nonmotile. ...[more]

PMID: 7622217

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Project description:Pseudomonas aeruginosa adheres to the mucosal surfaces of the lungs. This process appears to be mediated by nonpilus adhesins which bind to mucin. To find this nonpilus adhesin(s), mutagenesis of a nonpiliated mutant of P. aeruginosa with transposon Tn5G, followed by a screen for mucin adhesion, was used to isolate a series of mutants unable to adhere to mucin. All of these mutants were also found to be defective in motility. One such mutant, PAK-RR20, is characterized here. The site of the transposon insertion in PAK-RR20 was localized to a gene which is homologous to the fliF gene of other organisms and was flanked by other motility-related genes, fliE and fliG. Both adhesion and motility defects in PAK-RR20 were complemented by providing the fliF gene in trans. Since complementation could have been due to the presence of an internal promoter in the fliF gene or in the Tn5G transposon, which allowed the transcription of the downstream genes, another chromosomal mutant of the fliF gene was constructed by insertional inactivation with an antibiotic resistance cassette. This mutant was also nonmotile and nonadhesive. However, the two defects in this new mutant could not be complemented by the fliF gene in trans, consistent with the interpretation that there is no internal fliF promoter but possibly a functional promoter in the Tn5G transposon. The complete nucleotide sequences of the fliE and fliF genes and a partial nucleotide sequence of the fliG gene of P. aeruginosa were determined. Control of the promoter upstream of the fliE gene was analyzed by construction of a fliE-lacZ fusion and the introduction of this construct into strains of P. aeruginosa with mutations in several regulatory genes. Beta-Galactosidase expression measurements indicated that the fliE promoter does not utilize RpoF (sigma(28)) or RpoN (sigma(54)) sigma factors. The characterization of this gene as being responsible for the loss of adhesion indicates that basal body structures are probably important for localization of the adhesin.

Project description:Pseudomonas aeruginosa frequently encounters microbes that produce ethanol. Low concentrations of ethanol reduced P. aeruginosa swim zone area by up to 45% in soft agar. The reduction of swimming by ethanol required the flagellar motor proteins MotAB and two PilZ domain proteins (FlgZ and PilZ). PilY1 and the type 4 pilus alignment complex (comprising PilMNOP) were previously implicated in MotAB regulation in surface-associated cells and were required for ethanol-dependent motility repression. As FlgZ requires the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) to represses motility, we screened mutants lacking genes involved in c-di-GMP metabolism and found that mutants lacking diguanylate cyclases SadC and GcbA were less responsive to ethanol. The double mutant was resistant to its effects. As published previously, ethanol also represses swarming motility, and the same genes required for ethanol effects on swimming motility were required for its regulation of swarming. Microscopic analysis of single cells in soft agar revealed that ethanol effects on swim zone area correlated with ethanol effects on the portion of cells that paused or stopped during the time interval analyzed. Ethanol increased c-di-GMP in planktonic wild-type cells but not in ΔmotAB or ΔsadC ΔgcbA mutants, suggesting c-di-GMP plays a role in the response to ethanol in planktonic cells. We propose that ethanol produced by other microbes induces a regulated decrease in P. aeruginosa motility, thereby promoting P. aeruginosa colocalization with ethanol-producing microbes. Furthermore, some of the same factors involved in the response to surface contact are involved in the response to ethanol.IMPORTANCE Ethanol is an important biologically active molecule produced by many bacteria and fungi. It has also been identified as a potential marker for disease state in cystic fibrosis. In line with previous data showing that ethanol promotes biofilm formation by Pseudomonas aeruginosa, here we report that ethanol reduces swimming motility using some of the same proteins involved in surface sensing. We propose that these data may provide insight into how microbes, via their metabolic byproducts, can influence P. aeruginosa colocalization in the context of infection and in other polymicrobial settings.

Project description:Previous work from this laboratory has shown that a 26-kb insert in cosmid clone pFV100, isolated from a Pseudomonas aeruginosa gene library, contained genes that could restore serotype-specific B-band lipopolysaccharide (LPS) expression in rough mutant ge6. In this study, subclones from pFV100 were made to identify genes responsible for B-band LPS synthesis. Transformation of Escherichia coli HB101 with cosmid clone pFV100 resulted in expression of P. aeruginosa serotype O5 B-band LPS, indicating the presence of an rfb cluster in pFV100. Expression of P. aeruginosa LPS could not be achieved in E. coli HB101 transformed with any of the subclones. Complementation studies of well-characterized rough mutants of P. aeruginosa PAO1 deficient in B-band LPS biosynthesis were performed with the various subclones. Subclone pFV110, containing a 1.4-kb XbaI-HindIII insert, restored B-band LPS biosynthesis in mutant AK44 (A+B-; complete core). Probing chromosomal DNA from the 20 International Antigenic Typing Scheme serotypes with the 1.4-kb insert from pFV110 in Southern hybridizations revealed a positive reaction to restriction fragments in serotypes O2, O5, O16, O20, and O18. LPS of serotypes O2, O5, O16, and O20 were shown earlier to have a similar backbone structure in their O antigen. The insert in pFV110 was sequenced, and the deduced amino acid sequence was compared with sequences of protein databases. No significant homology could be detected with any sequences in the database. Open reading frame analysis identified one region, ORF303, which could encode a 33-kDa protein. Using E. coli maxicells for protein expression, orf303 mediated the expression of a unique polypeptide with an apparent molecular mass of 32.5 kDa. The deficiency in the synthesis of B-band LPS biosynthesis in mutant AK44 is apparently complemented by the 33-kDa protein encoded by orf303. We have designated this ORF rfbA. This investigation is the first report on cloning and sequencing of an rfb gene involved specifically in O-antigen biosynthesis in P. aeruginosa PAO1.

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Characterization of Pseudomonas aeruginosa fliO, a gene involved in flagellar biosynthesis and adherence.

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Characterization of Pseudomonas aeruginosa fliO, a gene involved in flagellar biosynthesis and adherence.

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