Type 1C fimbriae of a uropathogenic Escherichia coli strain: cloning and characterization of the genes involved in the expression of the 1C antigen and nucleotide sequence of the subunit gene

The genes responsible for expression of type 1C fimbriae have been cloned from the uropathogenic Escherichia coli strain AD110 in the plasmid vector pACYC184. Analysis of deletion mutants from these plasmids showed that a 7-kb DNA fragment was required for biosynthesis of 1C fimbriae. Further analysis of this DNA fragment showed that four genes are present encoding proteins of 16, 18.5, 21 and 89 kDal. A DNA fragment encoding the 16-kDal fimbrial subunit has been cloned. The nucleotide sequence of the structural gene and of the C- and N-terminal flanking regions was determined. The structural gene codes for a polypeptide of 181 amino acids, including a 24-residue N-terminal signal sequence. The nucleotide sequence and the deduced amino acid sequence of the 1C subunit gene were compared with the sequences of the fimA gene, encoding the type 1 fimbrial subunit of E. coli K-12. The data show absolute homology at the N- and C-termini; there is less, but significant homology in the region between the N- and C-termini. Comparison of the amino acid compositions of the 1C and FimA subunit proteins with those of the F72 and PapA proteins (subunits for P-fimbriae) revealed that homology between these two sets of fimbrial subunits is also maximal at the N- and C-termini.     

The three genes lipB, lipC, and lipD involved in the extracellular secretion of the Serratia marcescens lipase which lacks an N-terminal signal peptide.

The extracellular lipase of Serratia marcescens Sr41, lacking a typical N-terminal signal sequence, is secreted via a signal peptide-independent pathway. The 20-kb SacI DNA fragment which allowed the extracellular lipase secretion was cloned from S. marcescens by selection of a phenotype conferring the extracellular lipase activity on the Escherichia coli cells. The subcloned 6.5-kb EcoRV fragment was revealed to contain three open reading frames which are composed of 588, 443, and 437 amino acid residues constituting an operon (lipBCD). Comparisons of the deduced amino acid sequences of the lipB, lipC, and lipD genes with those of the Erwinia chrysanthemi prtDEC, prtEEC, and prtFEC genes encoding the secretion apparatus of the E. chrysanthemi protease showed 55, 46, and 42% identity, respectively. The products of the lipB and lipC genes were 54 and 45% identical to the S. marcescens hasD and hasE gene products, respectively, which were secretory components for the S. marcescens heme-binding protein and metalloprotease. In the E. coli DH5 cells, all three lipBCD genes were essential for the extracellular secretion of both S. marcescens lipase and metalloprotease proteins, both of which lack an N-terminal signal sequence and are secreted via a signal-independent pathway. Although the function of the lipD gene seemed to be analogous to those of the prtFEC and tolC genes encoding third secretory components of ABC transporters, the E. coli TolC protein, which was functional for the S. marcescens Has system, could not replace LipD in the LipB-LipC-LipD transporter reconstituted in E. coli. These results indicated that these three proteins are components of the device which allows extracellular secretion of the extracellular proteins of S. marcescens and that their style is similar to that of the PrtDEF(EC) system.     

Molecular cloning of F11 fimbriae from a uropathogenic Escherichia coli and characterization of fimbriae with polyclonal and monoclonal antibodies

Abstract The genes coding for F11 fimbriae from the uropathogenic Escherichia coli C1976 were cloned by a cosmid cloning procedure. Two cosmid clones expressed F11 fimbriae and these clones possessed an identical DNA fragment of 8.9 kb. This fragment was subcloned into pBR322 and this plasmid still produced fimbriae and caused a mannose-resistant haemagglutination (MRHA). Polyclonal and monoclonal antibodies were produced against purified cloned F11 fimbriae. Both types of antibodies were used in inhibition tests of MRHA and adherence of bacteria to the uroepithelial cell line T24. After preincubation of bacteria with polyclonal antiserum the MRHA and the MR adherence were totally inhibited. Preincubation of bacteria with monoclonal antibodies did not inhibit MRHA and MR adherence.     

Proteus mirabilis MR/P fimbriae: molecular cloning, expression, and nucleotide sequence of the major fimbrial subunit gene.

Proteus mirabilis, a cause of serious urinary tract infection and acute pyelonephritis, produces several putative virulence determinants, among them, fimbriae. Principally, two fimbrial types are produced by this species: mannose-resistant/Proteus-like (MR/P) fimbriae and mannose-resistant/Klebsiella-like (MR/K) fimbriae. To isolate MR/P fimbrial gene sequences, a P. mirabilis cosmid library was screened by immunoblotting and by hybridization with an oligonucleotide probe based on the N-terminal amino acid sequence of the isolated fimbrial polypeptide, ADQGHGTVKFVGSIIDAPCS. One clone, pMRP101, reacted strongly with a monoclonal antibody specific for MR/P fimbriae and with the DNA probe. This clone hemagglutinated both tannic acid-treated and untreated chicken erythrocytes with or without 50 mM D-mannose and was shown to be fimbriated by transmission electron microscopy. A 525-bp open reading frame, designated mrpA, predicted a 175-amino-acid polypeptide including a 23-amino-acid hydrophobic leader peptide. The unprocessed and processed polypeptides are predicted to be 17,909 and 15,689 Da, respectively. The N-terminal amino acid sequence of the processed fimbrial subunit exactly matched amino acid residues 24 to 43 predicted by the mrpA nucleotide sequence. The MrpA polypeptide shares 57% amino acid sequence identity with SmfA, the major fimbrial subunit of Serratia marcescens mannose-resistant fimbriae.     

Nucleotide sequence of the gene encoding the F72 fimbrial subunit of a uropathogenic Escherichia coli strain

The cloned DNA fragment encoding the F72 fimbrial subunit from the uropathogenic Escherichia coli strain AD110 has been identified. The nucleotide sequence of the structural gene and of 196 bp of the noncoding region preceding the gene was determined. The structural gene codes for a polypeptide of 188 amino acid residues, including a 21-residue N-terminal signal sequence. The nucleotide sequence and the deduced amino acid sequence of the F72 gene were compared with the reported sequences of the papA gene (B?ga et al., 1984). Both genes code for subunits of fimbriae that are involved in mannose-resistant hemagglutination (MRHA) of human erythrocytes. The available data show that there is absolute homology between the noncoding regions preceding both genes over 129 bp. The two proteins are homologous at the N terminus and C terminus; there is less, but significant, homology in the region between the N and C termini.     

Presence of STRA-STRB linked streptomycin-resistance genes in clinical isolate of Escherichia coil 2418

The streptomycin resistance of Escherichia coli 2418 strain has been shown to be associated with a 1.2-kb DNA fragment found in the naturally occurring plasmid R2418S. Here, nucleotide sequence analysis of the 1.2-kb DNA fragment revealed the presence of the strB gene which is located immediately downstream of the strA gene. Both sequences are identical to those of strA and strB genes in plasmid RSF1010. Thus, the observed resistance in the clinical isolate is due to the presence of strA-strB genes encoding streptomycin-modifying enzymes. The sequence downstream of strB gene showed a perfect homology with that of RSF1010. In addition, it contained the right inverted repeat of the transposon Tn5393 that has been suggested to be a relic of this transposon found in DNA plasmids isolated from human- and animal-associated bacteria.     

Cloning and expression in Escherichia coli of Haemophilus influenzae fimbrial genes establishes adherence to oropharyngeal epithelial cells.

In this report the first example of functional expression of a fimbrial gene cluster of a non-enteric human pathogen in Escherichia coli is described. This is shown for Haemophilus influenzae fimbriae which mediate adherence to oropharyngeal epithelial cells. A genomic library of H.influenzae type b, strain 770235f+bo, was constructed using a cosmid vector and screened with a synthetic oligonucleotide probe derived from the N-terminal sequence of the fimbrial subunit of H.influenzae. Four cosmid clones were found which hybridized to this oligonucleotide probe. Escherichia coli strains harbouring these clones expressed the H.influenzae fimbriae at their cell surface, as was demonstrated in a whole-cell ELISA and by immunogold electron microscopy using a monoclonal antibody specific for the H.influenzae fimbriae. Surface expression could be maintained during subcloning until a minimal H.influenzae DNA insert of approximately 8.1 kb was obtained. Escherichia coli strains harbouring the 8.1 kb H. influenzae DNA were able to cause a mannose-resistant adherence to oropharyngeal epithelial cells and a mannose-resistant haemagglutination of human AnWj-positive erythrocytes. The nucleotide sequence of hifA, the gene encoding the major fimbrial subunit, was determined. The predicted amino acid sequence shows a significant homology with a number of E.coli fimbrial subunits.     

Molecular cloning and characterization of a genetic region from Serratia marcescens involved in DNA repair

We report here the molecular isolation of a DNA fragment which encodes Tag-like activity from the Gram-negative bacterium Serratia marcescens. A recombinant plasmid encoding Tag-like activity was isolated from a S. marcescens plasmid gene library by complementation of an Escherichia coli tag mutant, which is deficient in 3-methyladenine DNA glycosylase I. The clone complements E. coli tag, recA, alkA, but not alkB, mutants for resistance to the DNA-damaging agent methyl methanesulphonate (MMS). The coding region of the Tag activity, initially isolated on a 6.5kb BamHI fragment, was defined to a 1.8kb BglII-SmaI fragment. Labelling of plasmid-encoded proteins using maxicells revealed that the 1.8kb fragment encodes two proteins of molecular weights 42,000 and 16,000. Data presented here suggest that the cloned fragment encodes a DNA repair protein(s) that has similar activity to the 3-methyladenine DNA glycosylase I of E. coli.     

Aggregative adherence of uropathogenic Proteus mirabilis to cultured epithelial cells

Proteus mirabilis is an important cause of urinary tract infection (UTI) in patients with complicated urinary tracts. Thirty-five strains of P. mirabilis isolated from UTI were examined for the adherence capacity to epithelial cells. All isolates displayed the aggregative adherence (AA) to HEp-2 cells, a phenotype similarly presented in LLC-MK(2) cells. Biofilm formation on polystyrene was also observed in all strains. The mannose-resistant Proteus-like fimbriae (MR/P), Type I fimbriae and AAF/I, II and III fimbriae of enteroaggregative Escherichia coli were searched by the presence of their respective adhesin-encoding genes. Only the MR/P fimbrial subunits encoding genes mrpA and mrpH were detected in all isolates, as well as MR/P expression. A mutation in mrpA demonstrated that MR/P is involved in aggregative adherence to HEp-2 cells, as well as in biofilm formation. However, these phenotypes are multifactorial, because the mrpA mutation reduced but did not abolish both phenotypes. The present results reinforce the importance of MR/P as a virulence factor in P. mirabilis due to its association with AA and biofilm formation, which is an important step for the establishment of UTI in catheterized patients.     

Cloning, organization and functional analysis of ilvA, ilvB and ilvC genes from Corynebacterium glutamicum.

Corynebacterium glutamicum is an industrially important bacterium for the manufacture of amino acids. We constructed genomic libraries of this Gram+ bacterium and screened for clones carrying isoleucine biosynthesis genes (ilv) by complementation of Escherichia coli mutants. Clones complementing ilvA, ilvB, and ilvC were isolated. As based on the functional analysis of the corresponding plasmids in C. glutamicum, the DNA fragments isolated encode threonine dehydratase, acetohydroxy acid synthase, and isomeroreductase, catalyzing three subsequent reactions in Ile synthesis. Subcloning and transposon mutagenesis revealed that ilvB and ilvC reside on a 7-kb chromosomal fragment and that these genes are transcribed in the same direction. A shuttle vector was constructed to allow exonuclease treatment and assay subsets of plasmids for gene expression in the original C. glutamicum background. These constructs and their enzyme activity determinations revealed that despite close linkage ilvC is expressed independently from ilvB. Using Southern blots, a 15-kb fragment of chromosomal DNA carrying the ilvBC cluster was characterized. This fragment does not contain ilvA, demonstrating the entirely different organization of the isoleucine biosynthesis genes in C. glutamicum from that in enterobacteria.     

Cloning, sequencing, and expression of cscA invertase from Escherichia coli B-62.

We have isolated a 2.5-kb DNA fragment from plasmid pST5R7 encoding a sucrose utilization system from Escherichia coli B-62 which confers a sucrose-fermenting phenotype to transformed E. coli K-12 strains. DNA-sequence determination revealed one full-length open reading frame 98% identical to cscA, the sucrose-hydrolase (invertase) gene of the csc regulon from E. coli EC3132. Functional characterization indicates that high-level expression and limited periplasmic release of invertase is responsible for the sucrose-fermenting capacity of transformed E. coli K-12 strains carrying cscA.     

Juxtaposition of the genes encoding Mycoplasma pneumoniae cytadherence-accessory proteins HMW1 and HMW3.

The loss and reacquisition of high-Mr (HMW) proteins, HMW1, 2, 3, 4 and 5, by Mycoplasma pneumoniae correlates with cytadherence phase variation. We are cloning and characterizing the genes encoding HMW1-5 to understand the mechanism regulating their coordinate expression. HMW1 was purified by polyacrylamide-gel electrophoresis. Amino acid (aa) sequence data were obtained from enzymatically generated peptide fragments from HMW1. A degenerate 17-mer probe synthesized based upon the aa sequence of one peptide clearly identified a single 4.75-kb BamHI fragment of M. pneumoniae DNA under stringent hybridization conditions. This fragment was cloned into pUC19 to generate pKV16. Restriction mapping of the 4.75-kb BamHI fragment in pKV16 revealed a possible overlap with the 9.4-kb EcoRI fragment containing the gene encoding protein HMW3. Southern blotting and reciprocal hybridization studies confirmed this overlap, establishing the juxtaposition of the genes encoding HMW1 and HMW3. Finally, physical mapping analysis by probing restriction fragments of M. pneumoniae DNA resolved by pulsed-field gel electrophoresis with the cloned genes encoding HMW1 and HMW3 revealed definitively that the hmw locus maps to a 106.8-kb ApaI fragment, rather than a 117.5-kb ApaI fragment, as had been reported previously for hmw3 [Krause and Mawn, J. Bacteriol. 172 (1990) 4790-4797].     

Complementation and regulatory interaction between two cloned fimbrial gene clusters of Escherichia coli strain KS71

Summary Complementation experiments with cloned DNA fragments encoding either the KS71A, the KS71B or the KS71C fimbriae of the pyelonephritogenic Escherichia coli strain KS71 were used to localise the P-fimbrillin genes and to demonstrate regulatory interactions between the cloned genes. The structural genes of the KS71A and KS71B fimbriae were located within a common 1.1 kilobase pair ClaI-SmaI fragment, and it was shown that the gene clusters for these fimbriae could complement each other in trans. The gene cluster encoding the KS71C fimbriae did not complement for the other KS71 fimbriae. A DNA fragment, located near the KS71A fimbrillin gene, was found to enhance the production of the KS71B fimbriae in trans.     

Common evolutionary origin of chromosomal beta-lactamase genes in enterobacteria

A 32P-labeled fragment of DNA, encoding the major part of the chromosomal ampC beta-lactamase gene of Escherichia coli K-12, was used as a hybridization probe for homologous DNA sequences in colonies of Neisseria gonorrhoeae, Pseudomonas aeruginosa, and different enterobacterial species. The ampC probe detected the presence of homologous DNA sequences in clinical isolates of E. coli, Shigella flexneri, Shigella sonnei, Klebsiella pneumoniae, Salmonella typhimurium, Serratia marcescens, and P. aeruginosa. No hybridization was found with N. gonorrhoeae colonies. In Southern blotting experiments the ampC probe hybridized to chromosomal DNA fragments of the same size in all enterobacterial species tested. However, the degree of hybridization differed with DNA from different species. DNA from the Shigella species strongly hybridized to the ampC probe. Furthermore, antibodies raised against purified E. coli K-12 ampC beta-lactamase precipitated beta-lactamases from the Shigella species, suggesting extensive sequence similarities between the ampC genes of these genera. The production of chromosomal beta-lactamase in S. sonnei increased with increasing growth rate similar to E. coli K-12. This growth rate response was abolished in two beta-lactamase-hyperproducing S. sonnei mutants, which thus seem similar to E. coli K-12 attenuator mutants. We propose that both the structure and regulation of the chromosomal beta-lactamase genes are very similar in E. coli and in S. sonnei.     

Cloning and characterisation of the serC and aroA genes of Yersinia enterocolitica, and construction of an aroA mutant

A gene library of Yersinia enterocolitica 8081 was constructed in the cosmid vector pHC79. Recombinants containing the aroA gene, encoding 5-enolpyruvylshikimate 3-phosphate synthase, were identified by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829. All six recombinant plasmids which complemented aroA also complemented the serC mutation in E. coli K-12 strain KL282. Tn5 mutagenesis suggested serC encoding 3-phosphoserine aminotransferase was the proximal gene in an operon with aroA. The nucleotide sequence of a 3-kb HindII-EcoRV fragment encoding the two genes was determined. The serC and aroA open reading frames contain 362 and 428 codons, respectively, and the deduced amino acid sequences share 78% and 81% homology, respectively, with the corresponding E. coli genes. Sequence inspection revealed no obvious terminators or promoters in the intergenic region. The cloned Y. enterocolitica aroA gene was inactivated in vitro and reintroduced into the parental Y. enterocolitica 8081 strain using the suicide vector pJM703.1. Stable aroA insertion mutants of Y. enterocolitica were isolated.     

Translocation of capsular polysaccharides in pathogenic strains of Escherichia coli requires a 60-kilodalton periplasmic protein.

An 11.6-kilobase (kb) region of a 34-kb fragment of Escherichia coli DNA that encodes the K1 capsular polysaccharide genes is necessary for translocation of the K1 polysaccharide to the bacterial cell surface. This 11.6-kb region contains a gene, kpsD, encoding a 60-kilodalton protein. The kpsD gene was localized to a 2.4-kb PstI-BamHI fragment. Cells harboring a Tn1000 insertion in kpsD did not synthesize the 60-kilodalton protein and did not express polysaccharide on the cell surface. Immunodiffusion and rocket immunoelectrophoresis of cell extracts, however, demonstrated that K1 polysaccharide was synthesized by these cells. We present evidence that the kpsD gene product is synthesized as a precursor and that the processed form is located in the periplasmic space. Analysis of alkaline phosphatase activity of a kpsD-phoA fusion demonstrated that kpsD expression was under positive regulation. A 260-base-pair AluI fragment located within the kpsD coding sequence was used as a probe and was found to hybridize to chromosomal DNA from E. coli that synthesizes the K2, K5, K7, K12, and K13 capsular polysaccharides but not K3 and K100. These results suggest that the kpsD gene product may be required for export not only of K1 but for other K antigens as well.