Transformation of freshwater and marine caulobacters by electroporation

Gilchrist, Angus; Smit, J

doi:10.1128/jb.173.2.921-925.1991

Cited by 68 publications

(46 citation statements)

References 26 publications

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“…Media, growth conditions, antibiotic concentrations, electroporation and selection of electrotransformants have been described previously (Gilchrist and Smit, 1991;Bingle et al, 1997).…”

Section: Methodsmentioning

confidence: 99%

Cell‐surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S‐layer of Caulobacter crescentus

Bingle

Nomellini

Smit

1997

Molecular Microbiology

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SummaryThe paracrystalline surface (S)-layer of Caulobacter crescentus is composed of a single secreted protein (RsaA) that interlocks in a hexagonal pattern to completely envelop the bacterium. Using a genetic approach, we inserted a 12 amino acid peptide from Pseudomonas aeruginosa strain K pilin at numerous semirandom positions in RsaA. We then used an immunological screen to identify those sites that presented the inserted pilin peptide on the C. crescentus cell surface as a part of the S-layer. Eleven such sites (widely separated in the primary sequence) were identified, demonstrating for the first time that S-layers can be readily exploited as carrier proteins to display 'epitope-size' heterologous peptides on bacterial cell surfaces. Whereas intact RsaA molecules carrying a pilin peptide could always be found on the surface of C. crescentus regardless of the particular insertion site, introduction of the pilin peptide at 9 of the 11 sites resulted in some proteolytic cleavage of RsaA. Two types of proteolytic phenomena were observed. The first was characterized by a single cleavage within the pilin peptide insert with both fragments of the S-layer protein remaining anchored to the outer membrane. The other proteolytic phenomenon was characterized by cleavage of the S-layer protein at a point distant from the site of the pilin peptide insertion. This cleavage always occurred at the same location in RsaA regardless of the particular insertion site, yielding a surface-anchored 26 kDa proteolytic fragment bearing the RsaA N-terminus; the C-terminal cleavage product carrying the pilin peptide was released into the growth medium. When the results of this work were combined with the results of a previous study, the RsaA primary sequence could be divided into three regions with respect to the location of a peptide insertion and its effect on S-layer biogenesis: (i) insertions in the extreme N-terminus of RsaA either produce no apparent effect on S-layer biogenesis or disrupt surface-anchoring of the protein; (ii) insertions in the extreme C-terminus either produce no apparent effect on S-layer biogenesis or disrupt protein secretion; and (iii) insertions more centrally located in the protein either have no apparent effect on S-layer biogenesis or result in proteolytic cleavage of RsaA. These data are discussed in relation to our previous assignment of the RsaA N-and C-terminus as regions that are important for surface anchoring and secretion respectively.

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“…Media, growth conditions, antibiotic concentrations, electroporation and selection of electrotransformants have been described previously (Gilchrist and Smit, 1991;Bingle et al, 1997).…”

Section: Methodsmentioning

confidence: 99%

Cell‐surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S‐layer of Caulobacter crescentus

Bingle

Nomellini

Smit

1997

Molecular Microbiology

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“…Standard methods of DNA manipulation and isolation were used (Sambrook et al, 1989). Electroporation of C. crescentus was performed as described previously (Gilchrist & Smit, 1991). For cloning of chromosomal DNA adjacent to Tn5 insertions, chromosomal DNA of a Tn5 mutant was digested with BamHI, SalI or XmaI.…”

Section: Methodsmentioning

confidence: 99%

Identification of lipopolysaccharide O antigen synthesis genes required for attachment of the S-layer of Caulobacter crescentus The GenBank accession number for the NA1000 rsaADEF and gmd, per, wbqA, wbqR, wbqX and wbqZ sequences reported in this paper is AF06235.

Awram

Smit

2001

Microbiology

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The outer surface of Caulobacter crescentus consists of a two-dimensional crystalline protein lattice layer (S-layer). A fraction of the LPS has an O antigen polymer attached to the core to form a ' smooth ' LPS (S-LPS), which is required for attachment of the protein S-layer to the outer-membrane surface. A method to screen for strains defective in LPS production, based on loss of S-layer attachment, was developed and applied to libraries of transposongenerated mutants. Eighteen distinct insertions were found with transposon interruptions in genes affecting S-LPS production, 12 of which were located near the S-layer subunit protein gene, rsaA, and its transporter genes. Sequence adjacent to transposon insertion points was determined and used to search a C. crescentus genome database. Twelve ORFs likely to be involved in S-LPS synthesis were identified. Seven of the predicted ORFs were linked to rsaA. Six of the putative genes had identity with proteins involved in synthesis of sugar residues, including five predicted to make perosamine. The remaining six ORFs were similar to glycosyltransferases involved in forming linkages between sugar residues in the O antigen, while one may be a transcription repressor. Other chemical and preliminary proton NMR studies of the S-LPS O antigen indicate that it contains an N-acetylated 4,6-dideoxy-4-aminohexose, but is not assembled as a simple, uniform homopolymer, consisting of several different linkages between sugar residues. The ORFs described here include homologues of all the enzymes involved in the synthesis of N-acetylperosamine, a 4,6-dideoxy-4-aminohexose. Overall, the data are consistent with the hypothesis that the O antigen of C. crescentus S-LPS consists primarily of N-acetylperosamine residues polymerized with multiple anomeric linkages.

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“…The mutagenized fliQR fragments were then transferred from pSelect to plasmid pRK290lac (17), generating transcriptional fusions to lacZ. These constructs were transformed into E. coli S17-1 by electroporation and then conjugated into C. crescentus NA1000 (16).…”

Section: Methodsmentioning

confidence: 99%

Caulobacter FliQ and FliR membrane proteins, required for flagellar biogenesis and cell division, belong to a family of virulence factor export proteins

Zhuang

Shapiro

1995

J Bacteriol

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Each Caulobacter crescentus cell division generates distinct progeny cells: a stalked cell which is competent for DNA replication, and a motile swarmer cell which initiates DNA replication only after it sheds its flagellum and differentiates into a stalked cell later in the cell cycle. Following the initiation of DNA replication, the flagellar transcriptional hierarchy is activated, culminating in the assembly of a single flagellum at the cell pole opposite that bearing the stalk. The biogenesis of the flagellum is a landmark in the establishment of asymmetry in the predivisional cell (Fig. 1).A large number of the flagellar structural and regulatory genes have been grouped into classes (Fig. 1) that form a regulatory hierarchy (7,9,11,43,62). We have preliminary evidence that the class I genes respond to cell cycle cues (44a). The class II flagellar genes are transcribed early in the cell cycle and are required for the transcription of genes in classes III and IV. Two class II genes, rpoN and flbD, encode a sigma factor ( 54 ) (6), and a transcriptional activator, FlbD (45, 60), respectively, which are required directly for the transcription of class III and class IV genes. Other class II genes encode proteins required for flagellar assembly and function (24, 45a, 65). The order of flagellar gene expression approximates the order of assembly of the gene products into the nascent structure (11,13,17,43,62). In C. crescentus, Salmonella typhimurium, and Escherichia coli, if flagellar assembly is aborted, transcription of the remaining flagellar genes is blocked, suggesting that the two processes are coupled (19,23,24,31).We reported previously that a nonmotile mutant with a deletion in the flaS locus not only diminished or abolished the transcription of class III and class IV genes but also exhibited a cell division defect (13). To understand the role of this class II flaS locus in the assembly of the flagellum and in cell divi- At each division, a predivisional cell gives rise to two different progeny, a stalked cell and a motile swarmer cell. As the cell cycle proceeds, the swarmer cell sheds its flagellum and assembles a stalk at the previously flagellated pole. The new stalked cell begins to divide, assembling a new flagellum at the pole opposite the stalk. The flagellar genes are expressed in an order that reflects their positions in the regulatory hierarchy and the order of assembly of their protein products into the flagellum (7,42). Arrows indicate positive regulation, such that transcription of each class of genes requires the expression of the gene products of the preceding class. Both rpoN and flbD encode transcription proteins (6, 45), whereas other class II genes appear to encode proteins involved in the assembly, structure, and function of the flagellum (24, 65).

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Transformation of freshwater and marine caulobacters by electroporation

Cited by 68 publications

References 26 publications

Cell‐surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S‐layer of Caulobacter crescentus

Cell‐surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S‐layer of Caulobacter crescentus

Identification of lipopolysaccharide O antigen synthesis genes required for attachment of the S-layer of Caulobacter crescentus The GenBank accession number for the NA1000 rsaADEF and gmd, per, wbqA, wbqR, wbqX and wbqZ sequences reported in this paper is AF06235.

Caulobacter FliQ and FliR membrane proteins, required for flagellar biogenesis and cell division, belong to a family of virulence factor export proteins

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