Actinobacillus pleuropneumoniae haemolysin II is secreted from Escherichia coli by A. pleuropneumoniae pleurotoxin secretion gene products

Abstract Actinobacillus pleuropneumoniae serotype 2 secretes type II haemolysin and pleurotoxin activities. Here, the genes for type II haemolysin were cloned in Escherichia coli , but type II haemolysin antigen and haemolysin activity were only detected intracellularly and not exported to culture supernatant. It has been reported that the genes for type II haemolysin are not linked to functional secretion genes, while those for pleurotoxin are. In this report the means of secretion of type II haemolysis was examined by constructing a hybrid plasmid carrying the genes required for type II haemolysin expression, together with determinants which allow secretion of pleurotoxin and are linked to the pleurotoxin toxin genes. These genes facilitated the export of type II haemolysin from E. coli , and may perform this function in A. pleuropneumoniae .     

Mutagenesis and cloning of hly determinants of the paP20 plasmid

The data obtained indicate that pAP20 plasmid determinants for the synthesis and excretion of Escherichia coli haemolysin are located on the plasmid SalI-fragment f3. The size of f3 fragment is 12 X 10(6) dalton.     

Inactivation of the ampD gene causes semiconstitutive overproduction of the inducible Citrobacter freundii beta-lactamase.

In Citrobacter freundii and Enterobacter cloacae, synthesis of AmpC beta-lactamase is inducible by the addition of beta-lactams to the growth medium. Spontaneous mutants that constitutively overproduce the enzyme occur at a high frequency. When the C. freundii ampC beta-lactamase gene is cloned into Escherichia coli together with the regulatory gene ampR, beta-lactamase expression from the clone is inducible. Spontaneous cefotaxime-resistant mutants were selected from an E. coli strain carrying the cloned C. freundii ampC and ampR genes on a plasmid. Virtually all isolates had chromosomal mutations leading to semiconstitutive overproduction of beta-lactamase. The mutation ampD2 in one such mutant was caused by an IS1 insertion into the hitherto unknown ampD gene, located between nadC and aroP at minute 2.4 on the E. coli chromosome. The wild-type ampD allele cloned on a plasmid could fully trans-complement beta-lactamase-overproducing mutants of both E. coli and C. freundii, restoring the wild-type phenotype of highly inducible enzyme synthesis. This indicates that these E. coli and C. freundii mutants have their lesions in ampD. We hypothesize that induction of beta-lactamase synthesis is caused by blocking of the AmpD function by the beta-lactam inducer and that this leads directly or indirectly to an AmpR-mediated stimulation of ampC expression.     

Evolutionary divergence of the Citrobacter freundii tryptophan operon regulatory region: comparison with other enteric bacteria

The regulatory region of the trp operon of Citrobacter freundii was sequenced and compared with the corresponding regions of other enteric bacteria. Significant differences were noted in the promoter region. These differences are presumably responsible for the weak expression of the cloned trp operon in Escherichia coli. The presumed operator region, although nonfunctional in E. coli, has dyad symmetry, but the sequence of the symmetrical region differs appreciably from those of operators that can be regulated by the E. coli trp repressor. The sequence of the trp leader region of C. freundii resembles that of other enteric bacteria, suggesting that the C. freundii operon is also regulated by attenuation. Comparison of the sequence of the initial portion of trpE with the homologous regions of E. coli and Salmonella typhimurium indicates that the three organisms probably are evolutionary equidistant.     

A gene encoding L-methionine gamma-lyase is present in Enterobacteriaceae family genomes: identification and characterization of Citrobacter freundii L-methionine gamma-lyase

Citrobacter freundii cells produce L-methionine gamma-lyase when grown on a medium containing L-methionine. The nucleotide sequence of the hybrid plasmid with a C. freundii EcoRI insert of about 3.0 kbp contained two open reading frames, consisting of 1,194 nucleotides and 1,296 nucleotides, respectively. The first one (denoted megL) encoded L-methionine gamma-lyase. The enzyme was overexpressed in Escherichia coli and purified. The second frame encoded a protein belonging to the family of permeases. Regions of high sequence identity with the 3'-terminal part of the C. freundii megL gene located in the same regions of Salmonella enterica serovar Typhimurium, Shigella flexneri, E. coli, and Citrobacter rodentium genomes were found.     

Characterization of the Citrobacter freundii phoE gene and development of C. freundii-specific oligonucleotides

The phoE gene of Citrobacter freundii, encoding a pore-forming outer membrane protein, was cloned and its nucleotide sequence was determined. The homologies in terms of identical amino acids between the C. freundii PhoE protein and those of Escherichia coli, E. cloacae and Klebsiella pneumoniae were 90%, 86% and 84%, respectively. Two synthetic oligonucleotides, corresponding to hypervariable, cell surface-exposed regions of the protein, were tested for their specificity in polymerase chain reactions. They were specific for the species C. freundii, i.e., no reaction was detected with 35 non-C. freundii strains tested, including 17 Salmonella, two C. amalonaticus and three C. diversus strains, whereas all five C. freundii strains tested were correctly recognized.     

Evaluation of a commercial beta-glucuronidase test for the rapid and economical identification of Escherichia coli

A commercial beta-glucuronidase (beta-GUR) test for the rapid and economical identification of Escherichia coli was evaluated. A total of 762 clinical strains and 228 environmental isolates were studied. More than 95% of the E. coli strains were found to be beta-GUR positive. Thirty-one clinical isolates of Shigella sonnei, 10 of Enterobacter cloacae, eight of Enterobacter aerogenes, nine of Citrobacter freundii and one of Salmonella enteritidis also gave positive results. The enzyme beta-GUR was also detected in two environmental strains of E. cloacae and one C. freundii. A comparative study between the beta-GUR test and the conventional identification system was carried out in 233 consecutive isolates of lactose positive enterobacteria. Agreement was observed in 223 cases and 190 E. coli strains were correctly identified using this test. Discrepancies were found in 10 cases: nine E. coli were beta-GUR negative and one C. freundii was beta-GUR positive. Escherichia coli was the only species positive for both beta-GUR and indole tests. This procedure permits a rapid, easy, precise and inexpensive identification of E. coli. beta-GUR positive Enterobacter strains have not previously been described.     

Isolation of the Actinobacillus pleuropneumoniae haemolysin gene and the activation and secretion of the prohaemolysin by the HlyC, HlyB and HlyD proteins of Escherichia coli

The gene encoding the c. 105 kD secreted haemolysin protein of the porcine pathogen Actinobacillus pleuropneumoniae serotype 1 has been isolated by screening a lambda gt11 expression library in Escherichia coli with antiserum raised against the wild-type protein. A derivative recombinant DNA pJFF702 expressed the hlylA haemolysin gene from the pUC19 lac promoter but the resulting haemolysin I protein remained within the E. coli cell and was haemolytically inactive. Export of the intracellular A. pleuropneumoniae prohaemolysin out into the medium was achieved by the presence in trans of the E. coli haemolysin secretion genes hlyB and hlyD, and high levels of intracellular haemolytic activity were attained similarly by the E. coli post-translational haemolysin activator gene, hlyC. Southern hybridization of A. pleuropneumoniae parental DNA nevertheless indicated only a low degree of nucleotide sequence identity to the haemolysin structural and secretion genes hlyA and hlyB of E. coli. The data show that despite substantial nucleotide sequence divergence the A. pleuropneumoniae serotype 1 haemolysin determinant is closely related to that which is dispersed throughout other Gram-negative human and animal pathogens.     

Sequence of the Citrobacter freundii OS60 chromosomal ampC beta-lactamase gene

The Citrobacter freundii OS60 ampC beta-lactamase gene was sequenced and found to encode a 380-amino-acid-long precursor with a 19-residue signal peptide. The mature protein has a predicted molecular mass of 39781 Da. The first 60 residues of the purified enzyme, as determined by sequential Edman degradation, are identical to the amino acid sequence inferred from the gene sequence. Also, the amino acid composition determined for the purified beta-lactamase and that given by the gene sequence are in good agreement. 77% of the amino acid positions hold identical residues in the C. freundii and Escherichia coli K12 chromosomal AmpC beta-lactamases. This clearly puts the C. freundii enzyme into the class C of beta-lactamases. Of the 68 amino-terminal residues determined for the Enterobacter cloacae P99 beta-lactamase, 44 are identical to the corresponding residues of the C. freundii enzyme. All three enzymes, as well as that of Pseudomonas aeruginosa 18S/H are highly similar around the active-site serine at position 64 of the mature protein.     

Distribution and diversity of hsd genes in Escherichia coli and other enteric bacteria.

We screened Salmonella typhimurium, Citrobacter freundii, Klebsiella pneumoniae, Shigella boydii, and many isolates of Escherichia coli for DNA sequences homologous to those encoding each of two unrelated type I restriction and modification systems (EcoK and EcoA). Both K- and A-related hsd genes were identified, but never both in the same strain. S. typhimurium encodes three restriction and modification systems, but its DNA hybridized only to the K-specific probe which we know to identify the StySB system. No homology to either probe was detected in the majority of E. coli strains, but in C. freundii, we identified homology to the A-specific probe. We cloned this region of the C. freundii genome and showed that it encoded a functional, A-related restriction system whose specificity differs from those of known type I enzymes. Sequences immediately flanking the hsd K genes of E. coli K-12 and the hsd A genes of E. coli 15T- were shown to be homologous, indicating similar or even identical positions in their respective chromosomes. E. coli C has no known restriction system, and the organization of its chromosome is consistent with deletion of the three hsd genes and their neighbor, mcrB.     

Structure and serologic properties of O-specific polysaccharide from Citrobacter freundii possessing cross-reactivity with Escherichia coli O157:H7

Citrobacter freundii OCU158 is a serologically cross-reactive strain with Escherichia coli O157:H7. To explore the close relationship between two strains, we have analyzed the chemical structures of O-specific polysaccharides and antigenic properties of lipopolysaccharides (LPSs) of both strains. The structure of O-specific polysaccharides from both strains was found to be identical by chemical and nuclear magnetic resonance analyses, in which D-PerNAc was 4-acetamido-4,6-dideoxy-D-mannose: [-->4)-beta-D-Glc-(1-->3)-alpha-D-PerNAc-(1-->4)-alpha-D-GalNAc-(1 --> 3)-alpha-L-Fuc-(1-->](n). The enzyme immunoassay using LPS derived either from E. coli O157 or from C. freundii could equally detect high levels of serum antibodies against LPS in patients with enterohemorrhagic E. coli (EHEC) O157 infection. Absorption of antibodies in EHEC patient serum by LPS from E. coli O157 or C. freundii, however, showed a difference in the epitopes. This difference was attributable to the epitope specificity of the core region and/or lipid A structure in LPS.     

Evaluation of a commercial β-glucuronidase test for the rapid and economical identification of Escherichia coli

A commercial β-glucuronidase (β-GUR) test for the rapid and economical identification of Escherichia coli was evaluated. A total of 762 clinical strains and 228 environmental isolates were studied. More than 95% of the E. coli strains were found to be β-GUR positive. Thirty-one clinical isolates of Shigella sonnei , 10 of Enterobacter cloacae , eight of Enterobacter aerogenes, nine of Citrobacter freundii and one of Salmonella enteritidis also gave positive results. The enzyme β-GUR was also detected in two environmental strains of E. cloacae and one C. freundii. A comparative study between the β-GUR test and the conventional identification system was carried out in 233 consecutive isolates of lactose positive enterobacteria. Agreement was observed in 223 cases and 190 E. coli strains were correctly identified using this test. Discrepancies were found in 10 cases: nine E. coli were β-GUR negative and one C. freundii was β-GUR positive. Escherichia coli was the only species positive for both β-GUR and indole tests. This procedure permits a rapid, easy, precise and inexpensive identification of E. coli. β-GUR positive Enterobacter strains have not previously been described.     

A cryptic melibiose transporter gene possessing a frameshift from Citrobacter freundii

Wild-type Citrobacter freundii cannot grow on melibiose as a sole source of carbon. The melibiose transporter gene melB was cloned from a C. freundii mutant M4 that could utilize melibiose as a sole carbon source. Although the cloned melB gene is closely similar to the melB genes of other bacteria, it is cryptic because of a frameshift mutation. Site-directed mutagenesis was used to construct a functional melB gene by deleting one nucleotide, resulting in the production of an active melibiose transporter. The active MelB transporter could utilize Na(+) and H(+) as coupling cations to melibiose transport. The amino acid sequence of the C. freundii MelB was found to be most similar to those of Salmonella typhimurium and Escherichia coli MelB. These facts are consistent with the phylogenetic relationship of bacteria and the cation coupling properties of the melibiose transporters.     

Characterization of swarming motility in Citrobacter freundii

Bacterial swarming motility is a flagella-dependent translocation on the surface environment. It has received extensive attention as a population behavior involving numerous genes. Here, we report that Citrobacter freundii, an opportunistic pathogen, exhibits swarming movement on a solid medium surface with appropriate agar concentration. The swarming behavior of C. freundii was described in detail. Insertional mutagenesis with transposon Mini-Tn5 was carried out to discover genetic determinants related to the swarming of C. freundii. A number of swarming genes were identified, among which flhD, motA, motB, wzx, rfaL, rfaJ, rfbX, rfaG, rcsD, rcsC, gshB, fabF, dam, pgi, and rssB have been characterized previously in other species. In mutants related to lipopolysaccharide synthesis and RcsCDB signal system, a propensity to form poorly motile bacterial aggregates on the agar surface was observed. The aggregates hampered bacterial surface migration. In several mutants, the insertion sites were identified to be in the ORF of yqhC, yeeZ, CKO_03941, glgC, and ttrA, which have never been shown to be involved in swarming. Our results revealed several novel characteristics of swarming motility in C. freundii which are worthy of further study.     

L-methionine γ-lyase from Citrobacter freundii: Cloning of the gene and kinetic parameters of the enzyme

It is shown for the first time for the Enterobacteriaceae family that a gene encoding L-methionine gamma-lyase (MGL) is present in the genome of Citrobacter freundii. Homogeneous enzyme has been purified from C. freundii cells and its N-terminal sequence has been determined. The hybrid plasmid pUCmgl obtained from the C. freundii genomic library contains an EcoRI insert of about 3000 bp, which ensures the appearance of MGL activity when expressed in Escherichia coli TG1 cells. The nucleotide sequence of the EcoRI fragment contains two open reading frames. The first frame (the megL gene) encodes a protein of 398 amino acid residues that has sequence homology with MGLs from different sources. The second frame encodes a protein with sequence homology with proteins belonging to the family of permeases. To overexpress the megL gene it was cloned into pET-15b vector. Recombinant enzyme has been purified and its kinetic parameters have been determined. It is demonstrated that a presence of a hybrid plasmid pUCmgl, containing the megL gene in the E. coli K12 cells, leads to a decrease in efficiency of EcoKI-restriction. It seems likely that decomposition of L-methionine under the action of MGL leads to a decrease in the intracellular content of S-adenosylmethionine. Expression of the megL gene in the C. freundii genome occurs only upon induction by a significant amount of L-methionine.     

Crystal structure of the nucleotide-binding subunit DhaL of the Escherichia coli dihydroxyacetone kinase

Dihydroxyacetone (Dha) kinases are a family of sequence-related enzymes that utilize either ATP or phosphoenolpyruvate (PEP) as source of high energy phosphate. The PEP-dependent Dha kinase of Escherichia coli consists of three subunits. DhaK and DhaL are homologous to the Dha and nucleotide-binding domains of the ATP-dependent kinase of Citrobacter freundii. The DhaM subunit is a multiphosphorylprotein of the PEP:sugar phosphotransferase system (PTS). DhaL contains a tightly bound ADP as coenzyme that gets transiently phosphorylated in the double displacement of phosphate between DhaM and Dha. Here we report the 2.6A crystal structure of the E.coli DhaL subunit. DhaL folds into an eight-helix barrel of regular up-down topology with a hydrophobic core made up of eight interlocked aromatic residues and a molecule of ADP bound at the narrower end of the barrel. The alpha and beta phosphates of ADP are complexed by two Mg2+ and by a hydrogen bond to the imidazole ring of an invariant histidine. The Mg ions in turn are coordinated by three gamma-carboxyl groups of invariant aspartate residues. Water molecules complete the octahedral coordination sphere. The nucleotide is capped by an alpha-helical segment connecting helices 7 and 8 of the barrel. DhaL and the nucleotide-binding domain of the C.freundii kinase assume the same fold but display strongly different surface potentials. The latter observation and biochemical data indicate that the domains of the C.freundii Dha kinase constitute one cooperative unit and are not randomly interacting and independent like the subunits of the E.coli enzyme.     

Secretion of the Rhizobium leguminosarum nodulation protein NodO by haemolysin-type systems

The Rhizobium leguminosarum biovar viciae nodulation protein NodO is partially homologous to haemolysin of Escherichia coli and, like haemolysin, is secreted into the growth medium. The NodO protein can be secreted by a strain of E. coli carrying the cloned nodO gene plus the haemolysin secretion genes hlyBD, in a process that also requires the outer membrane protein encoded by tolC. The related protease secretion genes, prtDEF, from Erwinia chrysanthemi also enable E. coli to secrete NodO. The Rhizobium genes encoding the proteins required for NodO secretion are unlinked to nodO and are unlike other nod genes, since they do not require flavonoids or NodO for their expression. Although proteins similar to NodO were not found in rhizobia other than R. leguminosarum bv. viciae, several rhizobia and an Agrobacterium strain containing the cloned nodO gene were found to have the ability to secrete NodO. These observations indicate that a wide range of the Rhizobiaceae have a protein secretion mechanism analogous to that which secretes haemolysin and related toxins and proteases in the ENterobacteriaceae.     

Comparison of the overlapping frd and ampC operons of Escherichia coli with the corresponding DNA sequences in other gram-negative bacteria.

Specific DNA probes from Escherichia coli K-12 were used to analyze the sequence divergence of the frd and ampC operons in various species of gram-negative bacteria. These operons code for the fumarate reductase complex and the chromosomal beta-lactamase, respectively. We demonstrate that the two operons show the same general pattern of divergence, although the frd operon is considerably more conserved than is the ampC operon. The major exception is Salmonella typhimurium LT2, which shows a strong homology to the E. coli frd probe but none to the E. coli ampC probe. The operons from Citrobacter freundii and Shigella sonnei were cloned and characterized by physical mapping, Southern hybridization, and protein synthesis in minicells. In S. sonnei, as in E. coli K-12, the frd and ampC operons overlap (T. Grundstr?m and B. Jaurin, Proc. Natl. Acad. Sci. U.S.A. 79:1111-1115, 1982). Only minor discrepancies between the two operons were found over the entire frd-ampC region. In C. freundii, the ampC and frd operons do not overlap, being separated by about 1,100 base pairs. Presumably the inducible property of the C. freundii chromosomal beta-lactamase is encoded by this 1,100-base-pair DNA segment.     

Copper-resistant enteric bacteria from United Kingdom and Australian piggeries.

Thirty-three enteric isolates from Australian (Escherichia coli only) and United Kingdom (U.K.) (Salmonella sp., Citrobacter spp., and E. coli) piggeries were characterized with respect to their copper resistance. The copper resistance phenotypes of four new Australian E. coli isolates were comparable with that of the previously studied E. coli K-12 strain ED8739(pRJ1004), in that the resistance level in rich media was high (up to 18 mM CuSO4) and resistance was inducible. Copper resistance was transferable by conjugation from the new Australian isolates to E. coli K-12 recipients. DNA similarity between the new Australian isolates and the pco copper resistance determinant located on plasmid pRJ1004 was strong as measured by DNA-DNA hybridization; however, the copper resistance plasmids were nonidentical as indicated by the presence of restriction fragment length polymorphisms between the plasmids. DNA-DNA hybridization and polymerase chain reaction analysis demonstrated DNA homology between the pco determinant and DNA from the U.K.E. coli, Salmonella sp., and Citrobacter freundii isolates. However, the copper resistance level and inducibility were variable among the U.K. strains. Of the U.K. E. coli isolates, 1 demonstrated a high level of copper resistance, 4 exhibited intermediate resistance, and 16 showed a low level of copper resistance; all of these resistances were expressed constitutively. A single U.K. C. freundii isolate, had a high level of copper resistance, inducible by subtoxic levels of copper. Transconjugants from one E. coli and one C. freundii donor, with E. coli K-12 strain UB1637 as a recipient, showed copper resistance levels and inducibility of resistance which differed from that expressed from plasmid pRJ1004.(ABSTRACT TRUNCATED AT 250 WORDS)